Abstracts POSTER PRESENTATIONS

Abstracts POSTER PRESENTATIONS

Jennifer Lippincott-Schwartz President / Wallace Marshall Program Chair Michael Marks Local Organizer

SUNDAY-POSTER PRESENTATIONS

POSTER PRESENTATIONS- Sunday, December 7 Imaging Technologies, Single Molecule Imaging, and SuperResolution 1 P1 Board Number: B100

Development of three color variants of super-brilliant luciferase for multi-color, real time imaging of gene expression and dynamics of organelles and the cytoskeleton without external illumination. A. Takai1, R. Haruno2, T. Nagai2,3, Y. Okada1; RIKEN Quantitative Biology Center (QBiC), Osaka, Japan, 2Osaka Univ, Osaka, Japan, 3JST PRESTO, Tokyo, Japan

1

Since bioluminescence does not require excitation light, it is free from auto-fluorescence. Therefore, it has been used for quantitative analysis of gene expression and in vivo imaging. Furthermore, it is free from potential phototoxicity and is compatible with optogenetic tools. However application of bioluminescent imaging has been limited mainly by two drawbacks. Firstly, the light output from the bioluminescent protein or luciferase was much darker than fluorescent proteins (FPs), making it difficult to study fine structures such as cytoskeletal dynamics with luminescent imaging. Secondly, color variants of luciferase have been limited compared with FPs, precluding multicolor imaging. In our recent study, we addressed the first limitation by developing a super brilliant yellow luminescent protein, Nano-lantern (Saito et al., Nat. Commun. 2012). In this study, we have overcome the second barrier. We report the development of cyan and orange variants of Nano-lantern, both of which are even brighter than the original yellow Nano-lantern by 1.5-2.3 times, and bright enough for observation with the naked eye or with a smartphone camera. Fusions of these multicolor Nano-lanterns with a variety of subcellular localization tags showed correct localization in both luminescent and fluorescent imaging, demonstrating their utility as imaging probes. In addition, expansion of the color palette of Nano-lanterns enabled not only multicolor luminescent imaging of subcellular structures, but also expression analysis of multiple genes at single cell level in embryonic stem cells, which are known to be very sensitive to phototoxicity. Furthermore, by combining split luciferase complementation with Ca2+sensing peptide (CaM-M13), we demonstrated simultaneous measurement of Ca2+ dynamics in the nucleus and mitochondria. Our multicolor Nano-lanterns are expected to be excellent imaging tools for gene-expression analysis, in vivo imaging, stem cell study and combinatorial analysis with optogenetics. Considering that the expanded color palette of FPs has driven wider application of fluorescent live imaging, we believe that

SUNDAY-POSTER PRESENTATIONS our multicolor Nano-lanterns should change the world of luminescent imaging from dim and monochrome to extremely bright and colorful.

P2 Board Number: B101

Development of novel in situ and in vivo RNA detection methods based on FIT probes. I. Gaspar1, F. Hövelmann2, A. Ephrussi 1, O. Seitz2; 1 EMBL, Heidelberg, Germany, 2Humboldt University, Berlin, Germany We have been developing new tools based on fluorogenic forced intercalation (FIT) probes for RNA detection quantification and interference in biological samples. Upon duplex formation with target nucleic acids, the base surrogates TO dye increases its quantum yield and brightness substantially (>10 fold). We turned FIT probes brighter by including a second, brighter, slightly red shifted variant dye, JO, which is hardly responsive. In this setup TO acts as a light harvester that feeds JO - we showed that a single FIT probe is sufficient to allow detection of oskar mRNA in a rapid, wash free FISH setup using conventional wide-field microscopy, making it an ideal tool for RNA localization screens. Locked nucleic acids (LNA) immediately adjacent to the TO base surrogate also enhance brightness – without significantly affecting responsiveness – by shortening the distance between stacked nucleotides in duplexes. This unique behavior of FIT probes allows synthesis of nuclease resistant oligos that are bright and contrasted enough for use in live imaging: as few as two LNA-containing FIT probes targeting different segments of oskar mRNA are sufficient to monitor oskar RNP motility in living oocytes. Motility data obtained by using 3-5 different probes in wild-type oocytes recapitulate what has been reported with the oskMS2-GFP system, indicating that this is a viable, non-transgenic alternative for studying mRNP biogenesis in vivo. While normally the aim is to avoid interference with biological processes, nuclease-resistant oligos can also be targeted against RNA secondary structures and/or protein binding interfaces of interest. FIT probes, in addition, report successful hybridization, allowing the testing of the functional role of RNA segments. We find that a FIT probe targeting the 3’ of the proximal stem of the spliced oskar localization element (SOLE) that is essential for efficient kinesin-dependent transport and oskar localization, causes motility defects nearly identical to those observed when mutating the SOLE sequence in transgenic oskar mRNAs. Finally, we demonstrated that TO fluorescence reaches its maximum at stochiometric probe-to-target ratio, and equipping FIT probes with a Cy7 presence reporter we could quantify oskar mRNA concentrations in situ. Although this qFIT method lacks single molecule sensitivity at the moment, it can measure high RNA concentration - even hundreds of RNA molecules within a confocal unit - providing a complementary approach to single molecule FISH studies.

SUNDAY-POSTER PRESENTATIONS

P3 Board Number: B102

Co-opting fluorescent proteins for cell-specific gene manipulation. C. Tang1, T. Szikra2, Y. Kozorovitskiy3, M. Teixeira2, S. Lapan1, B. Sabatini3, B. Roska2, C. Cepko1; 1 Genetics and Ophthalmology, Harvard Medical School/HHMI, Boston, MA, 2Neural Circuit Laboratories, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland, 3Neurobiology, Harvard Medical School/HHMI, Boston, MA Studies of the cell biology of multicellular organisms would be greatly facilitated by the ability to manipulate gene expression in any desired cell type. However, it remains a challenge to achieve cellspecific gene manipulation in many model systems, especially the mouse nervous system. Fluorescent proteins are commonly incorporated into transgenic organisms for labeling specific cell populations, but the unmodified forms cannot control biological activities. Using GFP-binding proteins derived from Camelid antibodies, we co-opted GFP as a scaffold for inducing formation of biologically active complexes, developing a library of hybrid transcription factors that control gene expression only in the presence of GFP or its derivatives. The modular design allows for variation in key properties such as DNA specificity, transcriptional potency, and drug dependency. Production of GFP controlled cell-specific gene expression and facilitated functional perturbations in the mouse retina. Further, retrofitting existing transgenic GFP mouse and zebrafish lines for GFP-dependent transcription enabled applications such as optogenetic probing of neural circuits. Current efforts to expand GFP-regulated activities, such as for GFP to directly control Cre recombination, will be discussed. This work establishes GFP as a multifunctional scaffold and opens the door to selective manipulation of diverse GFP-labeled cells across transgenic lines. This approach may also be extended to exploit other intracellular products as cellspecific scaffolds in multicellular organisms.

P4 Board Number: B103

“Decorating” cells with genetically encoded fluorescent proteins – what color suits you best?. J. Monbo1, L. Tu2, A. Joseph2, O. Ospovat1, A. Myers1, D. Grunwald2; 1 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 2RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA A key aspect of simultaneous multicolor single molecule real-time imaging in the living cell is choosing the appropriate combinations of fluorescent proteins. While ample information is available on molecular brightness, photo-stability, pH sensitivity and monomeric nature of most fluorescent proteins, interpolating the published data into performance in mammalian live cell experiments has been challenging. This leads to situations where the effort of testing new fluorescent proteins, combined with re-cloning of genetic constructs favor the use of suboptimal fluorescent protein combinations. The price paid is that less signal is collected using higher excitation power shortening the observation time of the

SUNDAY-POSTER PRESENTATIONS fluorescent protein. Additionally, higher excitation can also mean photo-damage to the cell thereby inducing a ‘stress’ reaction, potentially compromising results. Using a list of parameters, we tested 38 different fluorescent proteins for their suitability in live cell single molecule imaging. We refer to this set as a ‘toolbox’, and we have systematically evaluated all the fluorescent proteins for labeling of mobile (diffusing fluorescent protein & diffusive fusion protein) and immobile molecules (nuclear pore marker). We pay particular attention to the amount of fluorescence signal under low excitation power imaging conditions, sub-cellular (mis)-localization, cross excitation and cross emission at non-maximal photon output. This ‘toolbox’ will be of great use to the microscopy community.

P5 Board Number: B104

A Live Cell Antifade Solution for GFP Imaging. K.M. Chambers1; 1 Thermo Fisher Scientific, Eugene, OR Time-lapse, fluorescent imaging is a powerful tool in cell biology research. Photobleaching is one of the largest barriers for longer term imaging of fluorescent proteins and dyes. This phenomenon is caused by exposure to the intense light required to excite them. The loss of intensity of the fluorescent signal can leave researchers with incomplete or unclear data. Measures taken to minimize light exposure should be designed into each time-lapse experiment (Brown et al., 2009 and Waters, 2013). Factors to consider when designing these time-lapse, fluorescent experiments are light exposure time, the intensity of the light, and frequency of sampling (Brown et al., 2009) which can decrease the effects of photobleaching in an experiment. In addition, novel, oxygen-scavenging methods can also be used to reduce the amount of photobleaching observed in these types of experiments. Here is a study of a set of compounds analyzing their effectiveness at reducing photobleaching of fluorescent proteins and fluorophores. Results from a model system using GFP, RFP, and fluorescent dyes have led to a solution which reduces photobleaching in GFP, RFP, and several other dyes.

P6 Board Number: B105

A palette of near-infrared fluorescent proteins. D.M. Shcherbakova1, M. Baloban2, V.V. Verkhusha2; Department of Anatomy and Structural Biology and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, 2Anatomy and Structural Biology and GrussLipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 1

The genetically encoded near-infrared fluorescent probes are preferable for non-invasive in vivo imaging. In the near-infrared spectral region (650–900 nm) mammalian tissues are relatively transparent to light because the combined absorption by hemoglobin and water is minimal. Previously, we have

SUNDAY-POSTER PRESENTATIONS developed five spectrally distinct fluorescent proteins, such as iRFP670, iRFP682, iRFP702, iRFP713 (aka iRFP) and iRFP720, from bacterial phytochromes (1, 2). As a chromophore, iRFPs use a heme derivative, called biliverdin, abundant in mammalian cells. All iRFPs incorporate endogenous biliverdin efficiently and autocatalytically, do not require its exogenous supply and, therefore, can be used as easily as GFPlike proteins. iRFPs are dimers and can mainly serve for labeling of organelles and whole cells. iRFPs have enabled multicolor imaging of deep tissues in living animals. Here, we report the development of monomeric iRFPs (miRFPs) suitable for protein tagging, which also do not require external biliverdin to fluoresce. To engineer miRFPs we applied a combination of the rational and random mutagenesis, followed by a high-throughput screening. As a result, we have obtained three spectrally different monomeric near-infrared proteins, named miRFP670, miRFP703 and miRFP708. miRFPs are characterized by a high effective brightness in mammalian cells, a high pH stability and a high photostability. We demonstrated that miRFPs perform well as fusion tags for cellular proteins. The set of miRFPs should enable imaging of several tagged proteins in living mammals, and thus will be useful in cell and developmental biology and biomedicine. The developed rational design should allow efficient engineering of future miRFPs with desirable spectral and biochemical properties. (1) Filonov G.S. et al., Bright and stable near infra-red fluorescent protein for in vivo imaging. Nature Biotechnology 2011, 29: 757-761. (2) Shcherbakova D.M. and Verkhusha V.V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nature Methods 2013, 10: 751-754.

P7 Board Number: B106

Two-color imaging using spectral variants of iRFP670 and iRFP682 near-infrared fluorescent proteins. M. Baloban1, D.M. Shcherbakova2, V.V. Verkhusha3; 1 Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, 2Department of Anatomy and Structural Biology and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, 3Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY The new class of fluorescent proteins (FPs) engineered from bacterial phytochromes (BphPs) [1] attracts much attention for in vivo imaging due to their near-infrared (NIR) fluorescence spectra. These FPs utilize widely available in mammalian cells biliverdin (BV), a product of heme degradation, as a chromophore and, therefore, are as easy to use as common GFP-like proteins. We recently reported five NIR FPs, called iRFPs, with different fluorescence and biochemical properties [2]. Interestingly, two of them, iRFP670 and iRFP682, exhibited the twice higher molecular brightness, as well as the blue-shifted absorbance (643 nm and 663 nm) and fluorescence (670 nm and 682 nm) compared to other iRFPs. Here we characterized the unusual properties of these NIR FPs in detail. Our biochemical and biophysical analysis showed that iRFP670 and iRFP682 incorporate the BV chromophore in two distinct confirmations. A single amino acid mutation resulted in a depletion of one BV confirmations in the protein binding pocket and, consequently, in 30 nm red-shifts of both absorbance and fluorescence. The

SUNDAY-POSTER PRESENTATIONS point mutation also caused a slight decrease in the molecular brightness and an increase in the pH stability of the obtained red-shifted variants, which we named iRFP670-red and iRFP682-red. The effective brightness of the iRFP670-red and iRFP682-red in live mammalian cells was comparable to that of the parental proteins, suggesting that the high efficiency and high specificity of the incorporation of endogenous BV chromophore was not affected. Spectrally resolvable fluorescence of the iRFP670 and iRFP670-red pair, as well as of the iRFP682 and iRFP682-red pair, allowed easy separation of two cellular populations using FACS cytometry and straightforward two-color fluorescence microscopy of live cells, thus making them the probes of choice for cell labeling in the NIR region. Our studies are also beneficial for a rational molecular design of future enhanced NIR FPs from a variety of natural BphPs. [1] Piatkevich K.D., Subach F.V. and Verkhusha V.V. Engineering of bacterial phytochromes for nearinfrared imaging, sensing, and light-control in mammals. Chem. Soc. Reviews 2013, 42: 3441-3452. [2] Shcherbakova D.M. and Verkhusha V.V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nature Methods 2013, 10: 751-754.

P8 Board Number: B107

Minimal tags for rapid dual-color live cell labeling and super-resolution microscopy. I. Nikić1, T. Plass1, O. Schraidt1, J. Szymański1, J. Briggs1, C. Schultz1, E. Lemke2; 1 European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, 2Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany The choice of appropriate labeling technique is crucial for optimal super-resolution microscopy (SRM). Labeling density, probe size and photophysical properties are particularly important and to great extent determine the power of SRM. In addition, the probe should be attached as close as possible to the target protein as large linkers can induce artifacts and lower resolution. Many organic fluorophores offer small size and good photophysical characteristics, especially when compared to widely used GFP variants, but their direct linking to the target proteins remains challenging. We present a method that permits direct coupling- with virtually no linker- of SRM compatible dyes site-specifically to the target proteins by combining Amber codon suppression and click-chemistry technologies. To achieve this, host cells are modified to incorporate unnatural amino acids (UAAs) in response to a unique codon during translation. These UAAs carry chemical groups (strained alkynes and alkenes) that can be directly attached to tetrazine functionalized dyes in a special type of a fully biocompatible click-chemistry reaction named the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). By testing different tetrazine derivatives of organic dyes, we tuned the SPIEDAC reaction in an orthogonal descendant for rapid dual-color protein labeling. These two reactions allowed labeling of distinct insulin receptor populations and Influenza VLPs (virus-like particles) with two different dyes in living mammalian cells, as shown by SRM. While we demonstrate the applicability of our tools for SRM, generic SPIEDAC ligation mechanism is transferrable to other techniques and could e.g. be used for making PET/MRI imaging tracers.

SUNDAY-POSTER PRESENTATIONS

P9 Board Number: B108

Multicolor Fluorescence Photoactivation Localization Microscopy by Spectral Measurements. M.J. Mlodzianoski1, S. Gunewardene1, N.M. Curthoys1, S. Carter1, S.T. Hess1; 1 Department of Physics and Astronomy, University of Maine, Orono, ME The localization-based super resolution microscopy techniques Fluorescence Photoactivation Localization Microscopy (FPALM) (Hess et. al. 2006), Photoactivatable Localization Microscopy (PALM) (Betzig et. al. 2006), and Stochastic Optical Reconstruction Microscopy (STORM) (Rust et. al. 2006) circumvent the diffraction limit by measuring subsets of single molecules over a period of time. Spatially separated single molecules can then be localized with an uncertainty much smaller than the diffractionlimited resolution of the microscope. Reconstruction of images using localized single molecule positions can achieve a final image resolution on the order of tens of nanometers, a roughly ten-fold improvement over the diffraction limited resolution of ~250 nm. Imaging single molecules records information that is lost in ensemble fluorescence microscopy techniques. This single molecule information has enabled additional imaging modalities and has led to three-dimensional super-resolution imaging (Huang et. al. 2008, Juette et. al. 2008), dipole orientation (Gould et. al. 2008), and imaging of multiple fluorescent species (Huang et. al. 2007, Shroff et. al. 2008, Gunewardene et. al. 2011). Multicolor techniques are especially important in biological applications because they allow for the direct comparison of the spatial distribution of two or more differently labeled structures. Here, we describe a novel multicolor imaging method that measures the emission spectrum of each single molecule directly. Fluorescence is split into two channels with a standard 50:50 beamsplitter; one channel measures the spatial distribution while the second channel is sent through a dispersive optic. Within the spectral channel, the molecular image is shifted and broadened according to its emission spectrum. Quantification of the emission spectrum allows for identification of the fluorescent species. Previously, ratiometric simultaneous multicolor images were limited to four or fewer different species if the collected number of photons was large and background noise low. Depending on the background noise, number of photons collected, and the width of the spectral window, our spectral method can distinguish a larger number of species simultaneously. Additionally, a large set of novel super-resolution imaging methodologies can be envisioned, based on changes in the emission spectra of fluorescent molecules as a function of pH, temperature, hydrophobicity, time, or any method which induces fluorescence emission spectra changes in the sample.

SUNDAY-POSTER PRESENTATIONS

P10 Board Number: B109

Nanometer Resolution with the Time-resolved Confocal Microscope MicroTime 200. V. Buschmann1, O. Schulz1, M. Koenig1, B. Kraemer1, S. Tannert1, M. Patting1, F. Koberling1, R. Erdmann1; 1 PicoQuant GmbH, Berlin, Germany Superresolution microscopy is about to evolve into a standard tool for biological research. Overcoming the diffraction limit for fluorescence imaging has been shown to be crucial for addressing various relevant biological questions. However, the resolution is practically limited to about 20 nm for STED, STORM or PALM. Optical resolution below 20 nm can be easily achieved by combining confocal detection with AFM tip induced local fluorescence modulation. With FRET, distances even below 10 nm are readily accessible. As an open confocal microscopy platform, the MicroTime 200 is the ideal tool to combine sub diffraction spatial resolution with ultrasensitive time-resolved detection. Picosecond pulsed lasers and confocal detection together with time-correlated single photon counting (TCSPC) allow this system to add the full benefit of fluorescence lifetime based experiments to address highly specific biological questions. We present combined Confocal+AFM measurements on the single molecule level with DNA Origami structures proving a resolution of even below 10 nm. We show fluorescence lifetime based FRET imaging (FLIM FRET) to be a superior approach in the case of concomitant FRET subpopulations enabling the extraction of accurate FRET efficiencies and donor acceptor distances in the nanometer range. For multi species cell labeling we developed a pattern matching based approach which can take spectral as well as fluorescence decay information into account to improve the label recognition and separation especially in complex cellular environments. Finally the time-resolved single photon analysis is also applied to diffusion based FCS measurements in live cells to identify parasitic autofluorescence contributions on the nanosecond timescale which otherwise hamper the extraction of accurate local concentrations. Keywords: confocal microscopy, TCSPC, FLIM, FCS, FRET, AFM, single molecule, pattern matching

SUNDAY-POSTER PRESENTATIONS

P11 Board Number: B110

Video-rate super resolution microscopy in living cells. F. Huang1, C.E. Laplante2, Y. Lin3, T.D. Pollard1,2,4, J. Bewersdorf1; 1 Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 2Department of Molecular, Cellular and Developmental Biology, Yale University School of Medicine, New Haven, CT, 3 Department of Biomedical Engineering, Yale University School of Medicine, New Haven, CT, 4 Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT Super resolution microscopy based on single molecule localization relies on precise and accurate localization of large numbers of single molecules. However, the necessity of accumulating large numbers of localization estimates limits the time resolution typically to seconds to minutes1,2. Two major limitations are the acquisition speed and the photon budget. An EMCCD camera is usually used in such experiments. Replacing it with a recently introduced sCMOS camera results in leaps in both acquisition speed and effective quantum efficiency. However, the intrinsic pixel-dependent Gaussian noise of the sCMOS cameras introduces localization artifacts and greatly reduces the reliability of the results. Here, we present a set of specially designed statistics-based algorithms that characterize an sCMOS camera for the first time and allow for unbiased and precise localization analysis. Using this method we demonstrate Cramer-Rao lower bound-limited single molecule localization with an sCMOS camera. Combining the novel algorithm with a recently developed multi-emitter fitting algorithm3 and optimized imaging condition, we show that this technique shortens the typical acquisition time for fixed samples by up to two orders of magnitude without compromising the field of view. Furthermore, we demonstrate localization-based super-resolution microscopy in live cells by monitoring dynamics of protein clusters, vesicles and organelles at a temporal resolution from 2 to 30 frames per second4. These methods allowed us to investigate cytokinetic apparatus in live fission yeast at 20-30 nm resolution. In general, the significantly improved temporal resolution allows super resolution imaging of a large range of dynamic events in living cells. Patterson, G., Davidson, M., Manley, S. & Lippincott-Schwartz, J. Superresolution imaging using singlemolecule localization. Annu. Rev. Phys. Chem. 61, 345–67 (2010). Gould, T. J., Hess, S. T. & Bewersdorf, J. Optical nanoscopy: from acquisition to analysis. Annu. Rev. Biomed. Eng. 14, 231–54 (2012). Huang, F., Schwartz, S. L., Byars, J. M. & Lidke, K. A. Simultaneous multiple-emitter fitting for single molecule super-resolution imaging. Biomed. Opt. Express 2, 1377–93 (2011).

SUNDAY-POSTER PRESENTATIONS Huang, F. et al. Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nat. Methods 10, 653–8 (2013).

P12 Board Number: B111

Ultrafast super-resolution fluorescence live imaging with spinning disk confocal microscope optics. Y. Okada1, S. Hayashi2; 1 RIKEN Quantitative Biology Center (QBiC), Osaka, Japan, 2Olympus, Tokyo, Japan Most current super-resolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live cell imaging. Here, we report a new SR method based on the spinning-disk confocal microscope that achieves a spatial resolution of about 120 nm, double that of conventional microscopy. Theoretically, our method is equivalent to the structured illumination microscopy (SIM), but it is 10 times faster. Conventional SIM reconstitute a SR image from 9-15 raw images. The SR signals are modulated as Moire patterns in the raw images, and are extracted by the digital processing. Our method recovers the SR signals by the optical demodulation through the stripe pattern of the disk, and only a single averaged image through the rotating disk is required for a single SR image. Thus, the frame rate of our method is only determined by the speed of the disk and the camera. Based on this theory, we have modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples such as nuclear pores or microtubules. The rapid dynamics of EB1 at the growing ends of the microtubules were observed with 10 ms image acquisition time. Since our method requires only little optical modifications, it will enable an easy upgrade from existing spinning disk confocal to a SR microscope for live cell imaging.

P13 Board Number: B112

Device for microwave-enhanced processing of cryo-substitution for light and electron microscopy. D.W. Dorward1, J. Raae-Nielsen1, B.T. Hansen1, V. Nair1, E.R. Fischer1; 1 RTB/Microscopy Unit, NIH/NIAID/Rocky Mountain Labs, Hamilton, MT Cryo-substitution of frozen hydrated samples is the premier method for preserving native structure of specimens sectioned for light and electron microscopy. Also called freeze substitution, sample processing involves replacing ice with fixatives in organic solvents at or below -80 degrees C, followed by gradual warming to permissive temperatures for chemical cross-linking, typically over a 2-5 day period. In order to facilitate substitution and fixation, we developed and tested a cryo-thermal device for regulating sample temperature within standard laboratory microwave processors. The system consists of a microwave processor modified to accommodate a cryo-thermal sample platform and an external

SUNDAY-POSTER PRESENTATIONS programmable controller. Time, temperature, transition triggers, and ramping rates are user-defined between -/+100 degrees C, and recorded in spreadsheet format during processing. Using the system, we processed high-pressure frozen Bacillus subtillis, Saccharomyces cerevisiae, Borrelia burgdorferi, mixed pond water biota, Plasmodium-infected erythrocytes, and Chlamydia-infected HeLa cells in under 90 min. Embedding in acrylic resins in the platform at -20 or -40 degrees C added about another hour. Cooling and heating rates exceeded 20 and 10 deg/min, respectively. Recorded sample temperature plateaus varied less than 0.5 degree. Structure, contrast, tomography, and immune labeling were equivalent or superior to that achieved by traditional cryo-substitution or standard ambienttemperature fixation. Membrane contrast was reversible by adjusting the temperature at which samples were transferred from fixative mixtures to solvent only. Equivalent fixative mixtures produced negative contrast of mitochondrial cristae when transferred out of fixative at -20 degrees, and positive contrast when transferred at 20 degrees C. The system also enabled sequential use of three separate fixatives, with washing steps, in approximately 3 hours. These results indicate consistent, flexible, efficient, and effective methods for preparing and preserving diverse cryo-samples in near native state for light and electron microscopy.

P14 Board Number: B113

Title: Permeabilization Activated Reduction in Fluorescence (PARF): a novel method to measure kinetics of protein interactions with intracellular structures. P.P. Singh1, J.L. Hawthorne1, O.A. Quintero1; 1 Department of Biology, University of Richmond, Richmond, VA Understanding kinetic information is fundamental in understanding biological function. Advancement in imaging technologies have allowed for the development of assays for kinetic analysis. Fluorescence recovery after photobleaching (FRAP) is one such method that can be used to determine the diffusion rate of fluorescently labeled proteins in cellular environments and the kinetics describing interactions with intracellular structures. Through examination of the recovery kinetics of a photobleached region of interest, a researcher can determine the apparent exchange rate of the bleached fluor and half-life for recovery under equilibrium conditions. Like many of the other experimental approaches using biophotonic analysis to gain kinetic insight, FRAP requires a significant investment in specific equipment. We have developed a technique to measure kinetic information that does not require such a specialized imaging setup. Permeabilization Activated Reduction in Fluorescence (PARF) is an imaging-based kinetic analysis that allows the user to determine an apparent off-rate for a fluorescently-tagged protein of interest. To create conditions where the off-rate is observable, cells expressing a fluorescently-tagged protein are permeabilized with 25µM digitonin. The plasma membrane becomes permeable to molecules up to 200kD in size, allowing for the unbound pool of labeled protein in the cytosol to diffuse into the media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium and favoring the release of bound-protein from the structure with which it is interacting. The loss of bound protein is observed as a

SUNDAY-POSTER PRESENTATIONS loss of fluorescence from the intracellular structures. Because this change from bound to unbound is a first-order reaction, PARF kinetics can be fit to a single exponential decay, providing an apparent off-rate and half-life for loss of fluorescence. To test this approach, we compared PARF analysis of GFP, GFPVASP, and GFP-Mito with previously published FRAP analyses of those constructs. FRAP exchange rates and PARF loss rates were similar. We then examined the PARF kinetics of a panel of actin-binding proteins in the presence and absence of jasplakinolide. As predicted, stabilization of the actin network resulted in slower loss of fluorescence. These results demonstrate that PARF analysis of non-equilibrium systems can reveal information about binding interactions without the infrastructure investment required for other quantitative fluorescence approaches.

P15 Board Number: B114

Quantitative screening of antifade mounting media for fluorescence microscopy. S. Sweeney1, D. Cash1, K. Jamil1, E. Welch1; 1 Thermo Fisher Scientific, Eugene, OR Despite advancements in dye chemistry, the development of brighter and more stable fluorescent proteins and advances in the sensitivity of microscopes, photobleaching remains a significant challenge in the capture and analysis of cellular images. Advanced imaging techniques such as three-dimensional Z-stacking require multiple images of the same section to be collected with intense illumination. Retention of fluorescent signal throughout the experiment is critical not only for image quality, but also quantification of the results. The mechanism of the photobleaching of fluorescent dyes is hypothesized to be via a photo-oxidative pathway, wherein the long-lived triplet excited state of a fluorescent molecule irreversibly complexes with reactive oxygen species. To mitigate the effects of photobleaching, “antifade” reagents are often added to mounting media. These reagents slow the loss of signal from fluorescent dyes during imaging experiments through several possible routes, including acting as reducing agents, reactive oxygen scavengers or triplet-state quenchers. A number of protocols exist for the preparation of mounting media, often glycerol and polyvinyl alcohol-based, containing reducing agents (e.g. para-phenylene diamine). In many cases, however, commercial mounting media provides decreased variability in performance over time required for careful cellular analysis. Choice of mounting medium is largely dependent upon the type of sample and sample preparation, the type of imaging involved and the specific fluorophores used. Often, choice of mounting media is a compromise of photobleach resistance, initial intensity, signal to background, compatibility with fluorophores, refractive index and other factors. In order to evaluate antifade mounting media we have developed methods for quantitative assessment of photobleaching, initial intensity and signal-to-background using a combination of both confocal and wide field microscopy. Using these methods, we systematically screened a series of 20 fluorescent dyes, nuclear stains and fluorescent proteins (including TagGFP, TagRFP and mCherry) in multiple cell lines (HeLa, U2OS, A541). Samples were mounted using commonly used commercially-available mounting media for comparison. Ideally, mounting media should protect the broadest range of dye classes (e.g.

SUNDAY-POSTER PRESENTATIONS cyanine- or rhodamine-based) from photobleaching, while retaining bright initial intensity. Our analyses show that the performance of mounting media is highly variable and most will protect one subset of dyes significantly better than others, diminish initial intensity or increase off-cell background.

P16 Board Number: B115

Observation of Individual Secretory Granules in Living Mast Cells Using Confocal Microscopy. S. Tanaka1, Y. Takakuwa1; 1 Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, Tokyo, Japan Mast cells, which are distributed in the mucous membranes and epithelial tissue of the body surface, cause various allergic symptoms by releasing inflammatory mediators such as histamine from their intracellular secretory granules (SGs) in response to allergic stimuli (degranulation). It is hoped that the development of treatments that inhibit degranulation will enable the mitigation of allergy symptoms. To do so, it is necessary to observe the intracellular dynamics of these SGs to elucidate the process of their maturation, intracellular transport, and degranulation. However, it has been difficult to observe individual and distinct SGs by the conventional microscopic methods (which is by tagging SG marker proteins with fluorescent proteins) since marked fluorescent dispersion makes the target outlines unclear. Here, to overcome this problem, we observed individual SGs by overexpressing green fluorescent protein (GFP) in the cytoplasm of mast cells and using confocal microscopy to photograph the “absence of GFP” representing SGs that arose as a result of GFP not penetrating the granular lumen. First, we demonstrated that the accuracy of this new method is higher than that of the conventional observation. To validate the principle, we observed polystyrene beads labeled with a red fluorescent dye in a GFP solution. When we constructed 3D images of the beads from sequential tomography images, the dispersion of light caused the images derived from the red fluorescence to be greatly distorted in the direction of the z-axis, while the images derived from the “absence of GFP” were spherical and corresponded to the theoretical volume of the beads. Next, in a similar manner, we overexpressed GFP in the cytoplasm of the rat mast cell line RBL-2H3 and constructed 3D images. From these images, we obtained information on the construction of individual SGs (number of particles, size, volume, and arrangement), as well as on the cell as a whole. We demonstrated that the cells in these images indeed showed SGs by co-localization of red fluorescent protein-fused SG marker proteins (Neuropeptide Y or VAMP7) co-expressed in the cells. Furthermore, by inducing degranulation, we obtained information on the structure of exocytotic SGs in the cells. In this presentation, based on the structural characteristics of the SGs that we have thus obtained, we will discuss the process of the maturation, intracellular transport, and degranulation of SGs.

SUNDAY-POSTER PRESENTATIONS

P17 Board Number: B116

3D nano stiffness mapping of intracellular compartment using correlative light atomic force electron microscopy (CLAFEM). M. Popoff1, N. Barois2, S. Janel2, A. bongiovanni2, E. Werkmeister2, S. Divoux3, F. Perez3, F. Lafont1; 1 CNRS UMR 8204-CMIP, Institut Pasteur de Lille, Lille, France, 2BioImaging Center Lille, Institut Pasteur de Lille, Lille, France, 3CNRS UMR144, Institut Curie, Paris, France Mechanical properties of biological material like cells and tissues are known to play a key role the cell differentiation, cell division, cancer, diseases and other processes. Atomic Force Microscopy is routinely used to gain access to the precise information on the sample’s topography and physical properties. We propose here to unravel the elasticity of different identified organelles and microorganisms inside cells, at the nanoscale, with a novel technique combining stiffness mapping, super-resolution fluorescence and electronic microscopies, named CLAFEM (Correlative Light Atomic Electron Microscopies). We report herein the first results on bacteria, Golgi, actin tail comets and autophagosomes in both fixed and living cells.

P18 Board Number: B117

Continuous throughput and long-term observation of single-molecule FRET without immobilization. S. Tyagi1, V. Vandelinder2, N. Banterle1, G. Fuertes1, S. Milles 1, M. Agez1, E. Lemke1; Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, 2Sandia National Laboratories, Albuquerque, NM 1

Single-molecule FRET (smFRET) studies through Total internal reflection fluorescence microscopy (TIRFM) is a powerful method to extract and reveal hidden heterogeneity in biological systems from long-term FRET trajectories of individual single molecules. TIRFM popularity also owes to the possibility of adding exogeneous reagent and following the biological processes over long-time. The high signal to noise (SNR) necessary for observing fluorescence from single emitters is achieved by tethering single molecules within the shallow evanescent field (less than 200 nm) generated at the interface of coverslip and buffer. This requirement of tethering molecules to coverslip restricts the applicability of TIRFM to a limited set of biomolecules as complex immobilization procedures cannot be generally applied and sometimes perturb the native state of the biological sample. Here we present a versatile technology, termed “SWIFT” (Single molecules Without Immobilization For TIRFM) that enables us to perform multisecond observation of freely diffusing single molecules with high SNR while bypassing the need for immobilization procedures. SWIFT is a microfluidic-nanofluidic hybrid platform where nanochannels are actively and reversibly generated by collapsing 1 µm deep flow channels using pressurized nitrogen gas in a control channel, which additionally increases photo stability of fluorophores by removing oxygen.

SUNDAY-POSTER PRESENTATIONS Biomolecules continuously flow through 12 parallel 100 nm deep channels, which keep them within the evanescent field. The microfluidic device is fabricated with soft lithography and molding techniques out of an elastomeric material, which enables to exploit the full potential of microfluidics including low-cost, ease of use and fast solution exchange. We show that several second long trajectories of thousands of molecules can be recorded with millisecond time resolution, illustrating the potential for long-term automated and high throughput experiments. We demonstrate the power of our method by studying a variety of complex nucleic acid and protein systems, including DNA, Holliday junctions, nucleosomes and human transglutaminase 2. Reference: Tyagi, S., Vandelinder, V., et al. Nature Methods (2014)

P19 Board Number: B118

Video-enhanced polychromatic polarized light microscope (VEP polscope): bringing colors to the colorless world. M. Shribak1, E. Iourieva1; 1 Marine Biological Laboratory, Woods Hole, MA A conventional polarized light microscope, which is sometimes called a “polscope” for short, shows a two-dimensional image of birefringence due to structural or internal anisotropy of the specimen under investigation. The simplest polarizing setup, consisting of two crossed linear polars, produces pictures whose contrast is directionaly sensitive. The image brightness varies proportionally with sin22α sin2(180º δ/λ), where α is the specimen’s slow axis azimuth relatively to the direction of polarization of the illuminating light, δ is the specimen retardance and λ is the wavelength. If the slow axis and the light polarization are parallel, the brightness is zero. The brightness is highest if the slow axis is oriented at 45º and 135º respectively. Also the brightness is zero if the retardance equals to an integer number of wavelength. When the specimen under investigation is illuminated with white light and its retardance increases, then the brightness of all the wavelengths increases as well and the specimen appears grey. After the retardance has approached 400 nm, the blue part of the spectrum is suppressed and the specimen shifts to yellow and then red. Once the retardance reaches 600 nm, the red part of the spectrum has been blocked and the specimen turns blue and then green. The specimen exhibits Newton’s interference colors. In conventional polarized light microscopy, the color is solely determined by retardance and does not depend on the slow axis azimuth. The polarized light coloring is widely used in mineralogy and petrography. But this phenomenon could not be utilized in biology because most biological specimens exhibit a retardance of less than several tens of nanometers. Therefore, they are colorless. Even adding the first order red compensator, with a retardance of 575 nm does not help much. In this case, zero retardance corresponds to purple, and a slight color variation could be observed when the retardance change is at least 50 nm. Recently, we developed a new videoenhanced polychromatic polarized light microscope that produces interference colors at retardance

SUNDAY-POSTER PRESENTATIONS level of several nm. The polychromatic polscope employs a new principle of generating interference colors from white light. Here, color is determined by the orientation of the particle, not by its retardance. Thus, the full spectrum color can be achieved at a much lower retardance. The polychromatic polscope shows an orientation-independent birefringence image without requiring any digital computation. An eye or camera can directly see the colored polarization image in real time with a brightness corresponding to the retardance level and color corresponding to the slow axis azimuth. The previously colorless birefringent cell parts become vividly colored. We applied the new polychromatic polscope for imaging to a number of biological specimens including cancer tissue, malaria samples, mouse brain slices, various diatoms, zebrafish and sea urchins embryos, rotifer, paramecium, stentor, planaria, etc. Some of examples will be shown in the report.

P20 Board Number: B119

Improving the imaging of unstained specimens in bright field microscopy. C. Hernandez Candia1, B. Gutierrez-Medina2; 1 Molecular Biology, IPICYT, San Luis Potosi, Mexico, 2Advanced Materials, IPICYT, San Luis Potosi, Mexico Bright field is the simplest and most widespread light microscopy modality. However, its use in cellular biology has been limited due to lack of contrast in the imaging of thin, transparent samples such as cells. Instead, more evolved microscopy techniques (e. g. differential interference contrast, dark field, phase contrast, among others) have been used. An alternative to increase image contrast is deconvolution processing, a powerful method often used in fluorescence microscopy. However, application of deconvolution processing to bright field images has been scarce, mainly because acquisition of the corresponding point-spread function (PSF) has been difficult. In this work, we present direct measurement of the phase PSF of a high-aperture microscope operating in bright field. Polystyrene nanoparticles of 100 nm in diameter serve as the point objects that are imaged with high contrast and low noise using conventional microscopy plus digital image processing. To our knowledge, this is the first report that describes the experimental assessment of this function. The measured PSF allows us to demonstrate conventional deconvolution on the bright field images of living, unstained Escherichia coli cells, showing improved definition of cell boundaries and sub-cellular features. In addition, we were able to integrate the same digital imaging processing technique to an optical tweezers setup designed to perform single-molecule experiments. Using this setup, we were able to follow the stepping of the motor protein kinesin on clearly-imaged individual microtubules in real-time. In the context of optical manipulation, our microscopy approach using standard bright field eliminates the disadvantages of adding extra optical components that often constrain the properties of the optical trapping beam. Altogether, our studies provide a simple method to acquire the phase PSF, make possible the extension of deconvolution processing beyond fluorescence, and show that the improvement of bright field imaging via digital image processing can benefit single-molecule techniques such as optical tweezers.

SUNDAY-POSTER PRESENTATIONS

P21 Board Number: B120

Exploring cellular ultrastructure in 3D by Array Tomography and SEM imaging: A case study on zebrafish immune cells isolated by FAC-sorting. I.U. Wacker1, C. Bartels1, M. Thaler2, C. Grabher3, R.R. Schroeder4; 1 CAM/Cryo-EM, Universitaet Heidelberg, Heidelberg, Germany, 2Carl Zeiss Microscopy GmbH, Oberkochen, Germany, 3ITG, KIT, Karlsruhe, Germany, 4Cryo Electron Microscopy, Universitaet Heidelberg, Heidelberg, Germany Array tomography (AT) has been introduced almost ten years ago, initially to characterize neurons by multiplexing antibody labeling on serial sections of brain slices adhering to glass slides (Micheva & Smith, 2007, Neuron 55, 26). Such arrays can also be imaged in a SEM when deposited on conductive substrates such as silicon wafers or metal-coated glass (Wacker & Schröder, 2013, J Microsc, 252, 93). Here AT is applied to unknown GFP-expressing populations of blood cells isolated from zebrafish by FACsorting. Contrary to human blood cells which can be identified and sorted after staining with different combinations of CD markers for zebrafish such an approach is not possible due to lack of suitable antibodies. Some blood cells, however, have typical ultrastructural features so we aimed at reconstructing whole cells in 3D. Sorted cell populations were chemically fixed and embedded in epoxide resin. Arrays of a few hundred serial sections were produced using an ultramicrotome and a custom-built handling device. Stacks of SEM images were recorded automatically with the newly developed Atlas-AT software (Carl Zeiss Microscopy), aligned and volume-rendered or segmented. We established organelle inventories of various cell populations of the lymphoid lineage isolated from three different hematopoietic organs: Whole kidney marrow (WKM), thymus, and spleen. We found two predominant phenotypes with similar organelle inventories but different surface morphologies. One phenotype, showing a ruffled surface represented the majority of cells in a sort from spleen, was present to a lesser degree in WKM and not at all in thymus. The other phenotype had a smooth surface and few longer processes, constituted the major population in the sort from thymus and proportionally smaller populations in spleen and WKM. These observations were confirmed quantitatively by classifying and counting all cell profiles present in one section – on several sections of an array far enough apart to ensure that no cell was counted twice. AT can also be used to identify rare events: In co-cultures of cell populations isolated from WKM or thymus with the human promyeloic leukemia cell line HL60 we found a few cell couples consisting of fish cells attached to HL60 cells. Due to the close contact between the plasmamembranes of both cells and a concentration of secretory organelles towards the contact zone we concluded this to be immunological synapses, indicating cytotoxic activity for our unknown cells. This was proven by time-lapse light microscopy imaging of co-cultures. Taken together our case study demonstrates that AT can be extended from brain mapping to general imaging of exceedingly large volumes, such as whole cells, tissues and even small organisms at ultrastructural resolution.

SUNDAY-POSTER PRESENTATIONS

P22 Board Number: B121

Measuring the distribution of taurine molecule inside biological tissue via intrinsic molecular vibrations using nonlinear Raman spectroscopy. M. Kawagishi1, Y. Obara2, T. Suzuki2, M. Hayashi3, K. Misawa2, S. Terada1; 1 Dept Neuroanat Cell Neurobiol, Grad Sch Med Dent Sci, Tokyo Med Dent Univ (TMDU), Bunkyo, Tokyo, Japan, 2Dept Applied Physics, Tokyo Univ Agricul Technol (TUAT), Koganei, Tokyo, Japan, 3Wired Co., Ltd., Komae, Tokyo, Japan Distributions of small organic molecule (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterize the localization of large molecules such as proteins and nucleic acids, they cannot be easily applied to small molecule compounds: Fluorescent labels are relatively large compared to the target compounds and can change the properties of them. Raman spectroscopy is a technique to study vibrational information intrinsic to and characteristic of the chemical species of compounds. We used coherent anti-Stokes Raman scattering (CARS) spectroscopy to detect and identify a small molecule compound, taurine, without labeling. Molecular species could be uniquely identified from the spectral shape of the broadband vibrational spectra of target molecule. The local distribution of target molecule could be determined from the spectral intensity. We have developed a phase-sensitive CARS spectroscopy capable of simultaneous measurement of the broadband spectrum while maintaining a high frequency resolution.We utilized this technique to measure taurine inside mouse cornea tissue soaked in solution. We detected a Raman peak of taurine near 1000 wavenumber / cm inside cornea, and successfully characterized its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualize the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines.

P23 Board Number: B122

In vivo tracking of label-free resident cells in murine tissues via third harmonic generation microscopy. D. Malide1; 1 Light microscopy core facility, NIH/NHLBI, Bethesda, MD Although fluorescent compounds and proteins have emerged as invaluable tools to mark cells and structures of interest for confocal and two-photon microscopy imaging, such probes can interfere with cellular functions. While it is possible to visualize naturally occurring signals via two-photon microscopy, imaging remains challenging and has been limited primarily to thin and transparent biological samples. We developed and validated a methodology for high resolution 3D harmonic imaging of intact murine

SUNDAY-POSTER PRESENTATIONS tissues that requires no fluorescent labeling, based on epi- third-harmonic generation (THG) imaging by pulsed near-infrared light in the 1200–1300 nm range. Visualizing THG in the epi- (backscattered) direction enabled high resolution depth-resolved imaging of whole mount, intact tissues without the need for physical sectioning. We demonstrated intrinsic THG interface signals, outlining cell membranes and tissue inhomogeneities, revealing subcellular resolution of 3D architecture in various murine tissues and organs. These included subcutaneous and perivisceral white and brown adipose tissue, with clearly demarcated lipid droplets, blood vessels and cells, and myelinated peripheral nerves. We also visually dissected intracellular lipid-rich granules in adrenal glands, mammary glands, sebaceous glands, pancreas, liver, and spleen. Characteristic patterns were also observed in the skeletal muscle, heart, bone, skin, lung, brain, and spinal cord. Using this methodology, we examined the intracellular lipid deposits in healthy or diseased mouse tissues, including fatty liver, ischemic cardiomyocytes, and in the atherosclerotic aortic wall. Moreover, multiharmonic, combined THG and second harmonic generation (SHG) images can be assessed qualitatively and quantitatively to evaluate 3D distribution of the cells in label-free tissues and organs in control and experimental conditions. In addition, we demonstrated the feasibility of multimodal imaging by combining simultaneously excited harmonic signals with fluorescence-excitation of red-shifted fluorescent proteins expressed in vivo. Finally, time-lapse 3D THG imaging, obtained with faster acquisition settings, enabled dynamic cellular tracking in intact undisturbed tissues. This methodology uses commercially available turn-key systems allowing easy implementation of a versatile tool to track resident label-free cells in their undisturbed surroundings, as a powerful alternative or complementary contrast mechanism to fluorescence-based techniques. THG imaging could especially facilitate development of noninvasive approaches for optical biopsies of various tissue-types in diagnostic medicine.

New Technologies for Cell Biology 1 P24 Board Number: B123

Molecular interrogation of single-cells using ion mobility separation mass spectrometry. B. Shrestha1, L. Zhang1, A. Vertes1; 1 The George Washington University, Washington, DC Mass spectrometry (MS) with liquid chromatography (LC) is a standard analytical tool for analyzing small molecules in biological samples. Reduction of ion suppression due chromatography separation enhances detection of molecules. It also provides retention time values to increase identification confidence of molecules. However, due to their limited volume, analysis of single-cells with traditional chromatography methods is unviable. To overcome this limitation, we performed molecular analysis of single-cells using an on-the-fly approach, ion mobility separation (IMS). In situ sampling of cellular contents was achieved by laser ablation (laser ablation electrospray ionization or LAESI)1 or capillary microsampling2 prior to electrospray ionization. For this study, pollen tubes (Lilium longiflorum) and

SUNDAY-POSTER PRESENTATIONS hepatocyte (Homo sapiens) were used as model plant and animal cells, respectively. IMS improved signal-to-noise ratios of the ions from single-cells by discriminating them from isobaric ones found in the background. This led to an increase in the total number of molecular species detected from single-cells. In addition, the rotationally-averaged collision cross-sections (CCS) of ions were obtained from their drift times during IMS. The measured CCS of ions were used as additional parameters to increase the identification confidence of molecules from single-cells in combination with accurate mass-to-charge ratios and isotope distribution patterns. The reference CCS values were primarily obtained from existing literature and experiments with standards. When experimental values were unavailable, approximate CCS values were generated in silico. The identification of molecules of interest in single-cells was further confirmed by structural elucidation using tandem MS of the respective ions in bulk cell populations. References: [1] B. Shrestha and A. Vertes, In Situ Metabolic Profiling of Single Cells by Laser Ablation Electrospray Ionization Mass Spectrometry. Analytical Chemistry, 2009, 81, (20), 8265-8271 [2] L. Zhang, D. P. Foreman, P. A. Grant, B. Shrestha, S. A. Moody, F. Villiers, J. M. Kwak, and A. Vertes, In Situ Metabolic Analysis of Single Plant Cells by Capillary Microsampling and Electrospray Ionization Mass Spectrometry with Ion Mobility Separation. Analyst, 2014, July (online)

P25 Board Number: B124

DEVELOPMENT OF CAPILLARY BASED VACUUM ASSISTED INSTRUMENTS FOR RAPID AND EFFICIENT COLLECTION OF SINGLE CELLS FROM CELL CULTURES AND MICRODISSECTION OF HETEROGENEOUS TISSUES. S. Karsten1, A. Zavala1, Z. Ma1, L.C. Kudo1; 1 NeuroInDx, Inc., Signal Hill, CA Cell specific studies are imperative for sound research in basic and translational cell biology. Collecting single or individual cells directly from cell cultures for downstream single cell analysis or recultivation purposes has remained challenging. Similarly, tissue heterogeneity poses significant challenge for retrieval of cell and region specific information. Existing approaches, such as flow sorting and laser assisted acquisition, are costly, sophisticated and often methodologically limited. We have developed two capillary-based vacuum-assisted cell and tissue acquisition instruments (KuiqpicKTM and UnipicKTM) with which individual fluorescently labeled and/or morphologically distinct cells can be acquired from any cultures grown in standard cell culture dishes. Moreover, the instruments are suitable for individual cell collection and region specific acquisition of cell clusters and subanatomical areas from complex heterogeneous tissues such as the brain. While KuiqpicKTM is integrated with a fluorescence compatible inverted microscope (Olympus CKX41), UnipicKTM is a free standing instrument that may be mounted over any inverted microscope, providing additional flexibility in the laboratory. Both instruments demonstrate a wide range of cell and tissue acquisition parameters. They permit rapid collection of

SUNDAY-POSTER PRESENTATIONS individual cells from various cell cultures for various downstream applications including single cell analyses. Because small sample volume is incremental for single cell research, KuiqpicK/UnipicK enables the collection of a single cell in as small as 12 nl. The current work demonstrates the efficiency of KuiqpicKTM for cell collection from adherent, suspended, and 3D cultures. Individual cells were collected from primary neural progenitor (NPC), SH-SY5Y, CHO and MDA-MB-435 cell cultures based on morphology or fluorescent label. Further, the collected cells were successfully recultured, demonstrating the minimal effect the process has on cellular viability (up to 95%). The clonal expansion of collected single SH-SY5Y and CHO cells was demonstrated within 6 and 25 days, respectively. In addition, applicability of the instruments for the collection of single cells grown in three-dimensional (3D) culture system was shown using MDA-MB-435 cells. To further demonstrate KuiqpicK/UnipicK’s capabilities for cell and region acquisition from complex heterogeneous tissues, single Purkinje cells and subanatomical regions (e.g. CA1-3, dentate gyrus) were efficiently collected from rat brain tissue sections. The instrument dissected brain sections with thickness from 10 to 500 µm. Native, fresh frozen, and sucrose treated tissues could be used. High quality RNA and proteins were isolated from the collected samples.

P26 Board Number: B125

Development of single strand DNA aptamer for cell surface of human umbilical vein endothelial cell. H. Terazono1,2, H. Kim3, F. Nomura1, Y. Wada4, Y. Kurihara4, H. Kurihara4, K. Yasuda2,5; 1 Tokyo Medical/Dental Univ, Tokyo, Japan, 2KAST, Kawasaki, Japan, 3Kanagawa Academy Sci/Technol, Kawasaki, Japan, 4Univ. Tokyo, Tokyo, Japan, 5Tokyo Medical and Dental Univ, Tokyo, Japan Aptamer is a stranded molecular consisted of nucleic acid or amino acid that works as a ligand binding to targets such as protein, metal and organic small molecular. Aptamer is made by “Systematic Evolution of Ligands by EXponetial enrichment” (SELEX) method from huge number of aptamers by repeating selection procedure. Aptamer is more useful than antibody for cell labeling and tissue engineering because it is easy to elaborate clones stable and be modified with genetic engineering procedures such as adding fluorescent material or biotin, or recombining the sequence to stronger affinity ligand. Furthermore, since aptamer is degradable by nuclease, it is easy to detach the aptamer labeling. Therefore, labeled cells with aptamer to perform fluorescence-activated cell sorting (FACS) or Magneticactivated cell sorting (MACS) method is undoable to intact condition with nuclease, while antibody can’t it. SELEX method can be applied to cell surface targets or tissue. This method is called Cell-SELEX and can also make aptamer against unknown targets of cell surface. Human umbilical vein endothelial cell is used for physiological, pharmacological investigations and tissue engineering. Therefore, aptamer against cell surface of HUVEC will be useful for these research fields. ssDNA aptamer library contained a central randomized sequence of 40 nucleotides flanked by primer hybridization sites. ssDNA aptamers against cell surface of HUVEC was obtained by a modified cell-SELEX method. In briefly, after aptamer library was incubated with cells, the unbound aptamer removed, and then, the bounded ssDNA was

SUNDAY-POSTER PRESENTATIONS exfoliated from cell surface by trypsinization not to leak cytosol materials of cell. After that, bound ssDNA was amplified by the PCR and ssDNA was made by alkaline treatment from dsDNA as the next round template. Totally 10 rounds of selection were performed. The selected aptamer sequences were read by a next generation sequence and classified the sequence by a bioinformatics. Each round aptamer was labeled with FITC or biotin, and binding affinity was analyzed by MACS or a fluorescence microscopy. In results, almost of HUVEC was collected by the selected aptamer-magnetic beads conjugates. Moreover, the affinity was increased with repeated selection. Moreover, FITC-labeled aptamer stained the surface of the cells. These results indicated that our modified cell-SELEX method allowed us to make ssDNA aptamer against cell surface of HUVEC as a ligand. Aptamer for HUVEC is expected to use for cell sorting, micropattern of cell, and many biotechniques. Furthermore, reading sequence of selected aptamers in full detail by next generation sequence method gives us many information and it is possible to select the sequence against the target cells even if there are a wide variety of cell types in same tube.

P27 Board Number: B126

Reconstructing alveolar epithelial development using single-cell RNA-seq. D.G. Brownfield1,2,3, B. Treutlein4, M. Krasnow5, S. Quake4, H. Espinoza1, T. Desai6, A. Wu4, N.F. Neff7, G. Mantalas4; 1 Biochemistry, Stanford University, Stanford, CA, 2Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 3Department of Bioengineering, University of California, Berkeley, Berkeley, CA, 4Bioengineering, Stanford University, Stanford, CA, 5Stanford Univ Sch Med/HHMI, Stanford, CA, 6 Internal Medicine, Stanford School of Medicine, Stanford, CA, 7Stanford Univ Sch Med, Stanford, CA Although aspects of alveolar development have been studied by marker expression analysis and fatemapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types remain incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. With single-cell transcriptome analysis we were able to directly measure the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNAseq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics

SUNDAY-POSTER PRESENTATIONS approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

P28 Board Number: B127

Application of a live-cell RNA detection technology for use in cellular High Throughput Screening assays. D. Weldon1, A. Ko1, Y. Williams1, V. Koong1; 1 EMD Millipore, Temecula, CA High-throughput screening (HTS) approaches enable the systematic generation of vast amounts of experimental data through the automated processing and analysis of large numbers of biological samples1. Many traditional biological experiments have been expanded and modified to take advantage of HTS technologies. Modern platforms contain internal systems to automatically control incubation temperature, exchange growth media, and maintain pH levels in sample plates, allowing researchers to perform large scale studies without compromising their demand for biological relevance. Gene expression analysis is an area of research that is widely pursued today using HTS approaches. Specifically, several HTS techniques exist today for the large-scale identification and analysis of RNA content, such as in the context of identifying gene responses to drug compounds. However, many of these techniques require cell lysis for subsequent PCR amplification steps, which compromises cell viability and integrity, and prohibits the use of the assayed cells for downstream characterization and functional studies. Further, these assays also lack the ability to discern gene expression differences within heterogeneous cell samples. Performing direct RNA detection in live cells would address these key issues, enabling the researcher to gain even more biologically relevant information from critical HTS experiments. Retaining overall cell viability and integrity yields more meaningful biological data. In addition, single-cell gene expression analysis is made possible when resolution of heterogeneous populations is needed. This approach also enables the use of the interrogated cells for further downstream functional assays. In this poster, we demonstrate the live-cell detection of ERBB2 mRNA using SmartFlare™ RNA detection probes in two breast cancer cell lines, which differ in their expression of this therapeutic marker and exhibit differential responses to drug treatments. These cells were cocultured in varying ratios, incubated with SmartFlare™ probes in normal culture conditions, and directly assayed for ERBB2 mRNA. We show that sensitivity in detection can be achieved at the singlecell level, enabling greater understanding of the inherent heterogeneity within mixed cell samples, while retaining the scalability and throughput commensurate with HTS approaches. Live cell RNA detection enables single-cell analysis, preserves cellular viability and integrity, and allows samples to be further utilized for downstream research. Researchers who seek to maximize data output, minimize sample preparation, and generate biologically-relevant data from HTS gene expression experiments can benefit from incorporating live cell RNA detection into their workflows.

SUNDAY-POSTER PRESENTATIONS

P29 Board Number: B128

An automatable 3-dimensional cell invasion assay compatible with high content analysis. J. Fronczak1, S.I. Montanez-Sauri1, M.S. Gajeski1, B.R. Mader1, T.G. Burland2; 1 Platypus Technologies,LLC, Madison, WI, 2Platypus Technologies LLC, Madison, WI Cell invasion is the movement of cells through extracellular matrix (ECM). Invasion is of particular concern in cancer, where invading tumor cells lead to metastasis, the deadliest aspect of the disease. High-throughput assays suitable for screening the impact of candidate anti-cancer drugs on cell invasion are therefore highly sought after. Traditionally, cell invasion is quantified through “transwell” assays in which movement of cells from one chamber to another chamber through a synthetic membrane and ECM layer is monitored. However, transwell assays do not readily facilitate automation or real-time monitoring of invasion, nor simultaneous microscopic analysis of cell phenotypes such as changes in overall morphology, nuclear structure, etc. Moreover, the use of a synthetic membrane to separate the two transwell chambers poorly simulates conditions found in vivo. Here we report development of a fully automatable cell invasion assay that is configured in an industry-standard 96-well format and that can be used to monitor cell movement in three dimensions through ECM. The new assay seeds cells around an “exclusion zone”. Following cell attachment, an overlay of collagen I is added. Cell movement into the exclusion zone is then monitored in real-time, and cell morphology and physiology may also be monitored simultaneously. We report data comparing the information content and multiplex capabilities of traditional transwell invasion assays with the new exclusion zone invasion assay. We conclude that the fully automatable invasion assay presented in this work delivers major improvements in efficiency and information content over transwell assays, and promises to facilitate high content screening of cancer drug candidates for their potential to inhibit metastasis.

SUNDAY-POSTER PRESENTATIONS

P30 Board Number: B129

HC StratoMineR: A web-based data mining tool for the rapid analysis of high content data sets. W.A. Omta1, R.G. van Heesbeen2, R.J. Pagliero1, D. Lelieveld1, M. Kramer1, M. Nellen1, M. Yeong1, P. Kemmeren3, R.H. Medema4, S. Brinkkemper5, J. Klumperman1, D.A. Egan1; 1 Center for Molecular Medicine, Department of Cell Biology, University Medical Center Utrecht, Utrecht, Netherlands, 2Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, Netherlands, 3Center for Molecular Medicine, Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht, Netherlands, 4Division of Cell Biology, the Netherlands Cancer Institute, Amsterdam, Netherlands, 5Department of Information and Computing Sciences, University of Utrecht, Utrecht, Netherlands High content screening combines high throughput screening, and automated fluorescence microscopy and image analysis. The field has long been supported by mature technologies for automated liquid handling and automated image acquisition and analysis, such that large scale high content screens are feasible. There are however, few tools available to aid in the analysis of the large multiparameter data sets that are produced by these screens. We have developed a web-based tool called HC StratoMineR to aid the mining of high content screening datasets. This is a decision supportive data pipelining tool that assists even non-expert users in the efficient analysis of high content data sets. Our goal was to generate easy-to-read visualizations that make it possible to compare cellular morphological responses. Visualizations and other information provided are useful for the selection of useful parameters that have been generated by image analysis software. HC StratoMineR assists in the quality control of the screen and allows for the generation of a hit list based on cellular morphology. These hits are then classified using multivariate analysis techniques. Critically HC StratoMineR allows for a final report, where all decisions made are saved so that the next experiment can be analysed using the same methods. We have validated HC StratoMineR using chemical and RNAi knock-down screens. Our work shows that HC StratoMineR greatly enhances and accelerates the analysis of high content data sets. We show that the software does indeed segregate samples according to the cellular morphology they induce. We demonstrate that HC StratoMineR can highlight structure-morphology relationships in compound screens and gene function-morphology relationships in RNAi screens. This allows researchers to extract the greatest benefit from high content data sets.

SUNDAY-POSTER PRESENTATIONS

P31 Board Number: B130

Lipid nanoparticle delivery of RNA for loss-of-function and gain-of-function studies in primary neurons in vitro and in vivo. D. Zwaenepoel1, A. S. Ansari1, A. Thomas1, C. Walsh1, T. Leaver1, A. Wild1, K. Ou1, J. Taylor1, E. Ramsay1; 1 Precision Nanosystems Inc., Vancouver, BC A lipid nanoparticle (LNP) technology was developed to deliver mRNA and siRNA into neurons in vitro and in vivo with high efficiency and low toxicity. LNPs encapsulating RNA were prepared using the NanoAssemblr microfluidics platform. LNPs mimic low-density-lipoproteins (LDL) which are taken up by cells through the LDL-receptor in presence of Apolipoprotein E4. In vitro - More than 95% of Primary E18 rat Cortical Neurons (13 days in vitro) mixed with glial cells (PCN) incubated 4h with siRNA-LNP (100 ng/ml) showed fluorescent LNP uptake (Flow Cytometry, Confocal Microscopy). PCN incubated with 100 ng/mL of PTEN siRNA-LNP (siPTEN-LNP) for 72h showed > 90% knockdown (RT-qPCR, Western Blot). PCN incubated with HPRT siRNA-LNP (siHPRT-LNP) showed sustained knockdown of > 70% for 21 days after a single dose treatment (100 ng/mL) on Day 0 (RTqPCR). PCN treated with 100 ng/mL siPTEN-LNP alone, or 100 ng/mL siHPRT-LNP alone, or a combination of both nanoparticle preparations showed respective knockdown of PTEN and HPRT 72 hours post treatment (RT-qPCR).Comparison of Neuro9Kit to contemporary transfection kits showed significantly higher knockdown efficiency with 100 ng/mL sPTEN-LNP 72h post treatment (qPCR). Neuro9Kit showed no toxicity at 100 ng/ml siRNA-LNP 72 hours post treatment (LDH). PCN incubated with 500 ng/mL GFP mRNA-LNP and/or 500 ng/ml mCherry mRNA-LNP for 72h showed GFP and/or mCherry expression (Flow cytometry, Confocal Microscopy). In vivo - A somatosensory injection of siPTEN-LNP (500 nL at 5 mg/mL for 10 minutes) in Spargue-Dawley rats showed > 80 % knockdown in cortical slices up to 1 mm from the injection site 5 days post-injection (Western Blot). Solid-core LNP manufactured using the NanoAssemblr demonstrated rapid uptake by neurons which mediated effective and sustained silencing/expression of a target genes in vitro and in vivo with no detectable toxicity. This technology offers a simple and flexible alternative to viral vectors, electroporation and lipofection for loss- and gain-of-function studies in neural development, injury and degeneration.

SUNDAY-POSTER PRESENTATIONS

P32 Board Number: B131

Super-active TALEN with improved stability at 37 degree Celsius enables highly efficient and homogeneous gene knockout in mammalian embryos. K. Ikeda1, Y. Terahara1, K. Sumiyama1, N. Miyashita1, Y. Okada1; 1 RIKEN Quantitative Biology Center (QBiC), Osaka, Japan Gene editing in vivo has become possible by the development of artificial nucleases that can be designed to cut the genome DNA selectively at the target site in the genome. The DNA binding domain of a plant pathogen protein called Transcription Activator-Like Effector (TALE) has been used as the designable sequence specific DNA-binding domain. The fusion protein of TALE and the nuclease domain of Fok I endonuclease, called TALE nuclease or TALEN, has been proven to be useful for the genome editing in lower vertebrates such as zebrafish and Xenopus. In mammalian cells and embyos, however, TALEN often shows poor activity, which limited its applications. We have surmised that the TALE protein might not be stable at 37 oC, because the physiological temperature for the plant pathogen is around 25 o C. Through the analysis of the crystal structure of TALE protein, we have chosen two amino acid residues that might be pivotal for the stable alignment of the linear solenoid structure of TALE. Several mutations were introduced, and some successfully showed significantly higher activity at 37 oC both in vitro and in vivo. All-atom molecular dynamics simulations confirmed the stabilization of the conformation. The mutated residues apparently suppressed the intramolecular fluctuations. We have, therefore, named this mutant TALEN as “super-active” TALEN and examined its activity in mouse embryos. We have designed TALENs, both wild type and super-active mutant, for the mouse tyrosinase gene. The mRNAs for the TALENs were introduced to the pronuclear stage mouse embryos. The wild type TALENs failed to introduce mutations, and the coat color of the babies were agouti. Contrastingly, more than half of the babies were albino with super-active TALENs, which means both alleles of the tyrosinase gene were mutated throughout the body. Interestingly, CRISPR/Cas9 mediated gene editing of tyrosinase gene in mouse pronuclear stage embryo yielded even higher rate of coat color mutants. However, the babies were mostly mosaic of agouti and white, indicating that only some population of cells got biallelic mutation. Consistently, the genome analyses detected only less than four different alleles in each baby with super-active TALEN, while more than eight alleles were often detected in a single CRISPR/Cas9 baby. These results suggest that super-active TALEN shows its activity at two-cell stage, earlier than CRISPR/Cas9, suggesting that super-active TALEN might serve as an effective tool for the genome editing in mammalian cells and embryos.

SUNDAY-POSTER PRESENTATIONS

P33 Board Number: B132

Establishment of an in vivo electroporation method into postnatal newborn neurons in the dentate gyrus. H. Ito1, R. Morishita1, I. Ikuko1, K. Nagata1; 1 Mol Neurobiol, Inst Developmental Res/Aichi Human Service Ctr, Kasugai, Japan Electroporation-mediated gene transfer has been developed for the analysis of mammalian brain development in vivo. Indeed, in utero electroporation method is widely used for the investigation of the mouse embryonic cortical development while in vivo electroporation using neonatal mouse brain is employed for the analysis of the rostral migratory stream (RMS) and postnatal olfactory neurogenesis. In the present study, we established a stable gene-transfer method to dentate gyrus (DG) neurons by carefully determining the in vivo electroporation conditions, such as position and direction of electrode, voltage for electric pulses, and interval between electroporation and sample preparation. Consequently, GFP-positive cells in DG were observed to extend branched dendrites and long axons into the molecular layer and the hilus, respectively, 21 days after electrporation. They were morphologically identified as dentate granule neurons with many protrusions on dendrites, and some of them had wide head and thin neck that resembled matured mushroom spines. Expression of GFP in dentate neurons sustained for at least 9 months after electroporation under our experimental conditions. Taken together, the method developed here could be a powerful new tool for the analysis of the postnatal DG development.

P34 Board Number: B133

Development of Transgenic Chickens for Studies on Retinoic Acid Signaling. J. Summers1; 1 Department of Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, OK Introduction. Despite the significance of retinoic acid (atRA) in the growth and development of many major organ systems, visualization of endogenous atRA has only been indirectly determined. Moreover, due to the fundamental importance of atRA in early development, studies aimed at understanding the function of atRA in late embryogenesis or postnatal development using conventional genetic approaches have been severely limited. Therefore, the goal of this project is to develop two lines of germline transgenic chickens that will allow, for the first time, visualization and modulation of endogenous atRA in embryonic and post-hatch chicks.Methods. The pminiTol2 backbone was used to generate two constructs in order to 1) visualize endogenous atRA gradients (GEPRA B) and 2) conditionally lower endogenous atRA concentrations (tet-RALDH2 shRNA). The GEPRA B construct was cloned into the pminiTol2 plasmid containing a pCAGGS promoter for optimal expression in the chicken. The tet-RALDH2 shRNA construct was modified from a third generation lentiviral vector, the pINDUCER series, that allows tetracycline-inducible knockdown of RALDH2 as well as the expression of eGFP and DS

SUNDAY-POSTER PRESENTATIONS red fluorescent proteins to monitor transfection and induction, respectively, using a single vector. The minitol2 transposon system was used to deliver each of these constructs to circulating primordial germ cells (PGCs) in chick embryos through a recently developed direct injection technique. Briefly, transfection complexes consisting of 0.6 ug of pMiniTol-GEPRA or tet-RALDH2 shRNA, 1.2 ug of a plasmid containing the transposase sequence under the control of the CMV IE promoter, and Lipfectamine 2000 were prepared. Chick embryos stage 14 – 16 HH (~72 – 74 hrs incubation) were injected with 1 – 2 ul of the transfection complex using a pulled glass capillary micropipette inserted into the caudal portion of the left dorsal aorta. Eggs were sealed with surgical tape and incubated normally. To estimate transfection efficiency, chick embryo gonads were dissected and examined for eGFP or YFP fluorescence at various stages. To confirm transfection of PGC’s, gonads were immunolabeled using antibodies against the stem cell antigen, SSEA-1.Results. The tet-RALDH2 shRNA vector significantly reduced RALDH2 protein expression in vitro. Embryos between days 7 and 14 of incubation expressed eGFP (for tet-RALDH2 shRNA injected embryos) or YFP (for GEPRA injected embryos), preferentially in the gonads. Co-localization of YFP or eGFP with SSEA-1 confirmed the presence of transgenic PGC’s in embryonic gonads. Conclusions. This approach provides a promising alternative to the use of standard transgenic model organisms, and will enable, for the first time, functional studies on retinoic acid signaling in embryonic and post-hatch chickens.

P35 Board Number: B134

Functional Annotation for TCA and DNA Repair for Insects and Plants: A Survey. D.M. Grogan1, M. Castleman1, L.E. Carson2, E.C. Regisford3, J. Hu4; 1 Department of Agriculture, Prairie View AM University, Prairie View, TX, 2Cooperative Agricultural Research Center, Prairie View AM University, Prairie View, TX, 3Department of Biology, Prairie View AM University, Prairie View, TX, 4Department of Biochemistry and Biophysics, Texas AM University, College Station, TX In the area of agriculture, plants and insects work in harmony or against each other to provide nourishment for the rest of the animal kingdom. These species are being studied by numerous scientists, hence it would of be deep interest to determine how well these works are collected, annotated and stored. Thus, a survey of functional annotations for agriculturally important plants and Insects was performed. We focused on two of the most common metabolic pathways amongst species, the Tricarboxylic Acid (TCA) Cycle and DNA Repair. The TCA cycle uses a sequence of chemical reactions to generate energy through the oxidation of acetate from carbohydrates, proteins and fats into carbon dioxide. DNA can be damaged by endogenous and exogenous agents, causing mutation and other serious conditions. DNA Repair is a series of enzymatic processes that correct the lethal changes that affect the sequence of a DNA molecule. We selected 39 Insects, 64 Plants and used QuickGO to find all annotations associated with each species using a taxonomy ID. Then, we filtered the annotations by using the GO Terms for the TCA cycle (GO:0006099) & DNA Repair (GO:0006281). Our results indicated that sixty-three percent of the selected species had annotations that were associated with the TCA cycle

SUNDAY-POSTER PRESENTATIONS or DNA Repair. Twenty-two percent of the selected species had annotations that were associated with both DNA Repair and the TCA cycle. Therefore, we conclude that more annotations of these biological processes species needs to be done.

P36 Board Number: B135

Wild and Domestic Animals Surveyed for Functional Annotations of the TCA Cycle and Apoptosis. M. Castleman1, D. Grogan1, L.E. Carson2, E.C. Regisford3, J. Hu4; 1 Department of Agriculture, Prairie View AM University, Prairie View, TX, 2Cooperative Agricultural Research Center, Prairie View AM University, Prairie View, TX, 3Department of Biology, Prairie View AM University, Prairie View, TX, 4Department of Biochemistry and Biophysics, Texas AM University, College Station, TX There is so much information from biological processes of all organisms that needs to be stored by bioinformatics databases. The European Biological Institute (EBI) hosts one of the databases that are used to store annotated information on all organisms. With this study, we focused on two very important biological processes, the Tricarboxylic Acid cycle (TCA) and the Apoptotic process. The TCA cycle uses a series of chemical reactions to generate energy through aerobic respiration. Apoptosis, described as cell suicide or programmed cell death, is essential to maintain appropriate cell numbers in organisms. Our objective was to determine the number of animals, both wild and domesticated, which the TCA Cycle and Apoptosis are annotated. It is hypothesized that all organisms studied will have annotations for these two very important biological pathways the TCA cycle and Apoptosis. On the EBI database, we used QuickGO to retrieve all annotations associated with the taxonomy ID of the selected animals. The annotation was then filtered by using the GO term for the Krebs cycle (GO:0006099) and Apoptotic process (GO:0006915). Out of 13 wild animals studied only 5 had annotations for the TCA cycle and Apoptosis. Only 4 out 19 domesticated animals had annotations for Apoptosis, while none of 19 had annotations for the TCA cycle alone. Our data indicate that more work needs to be done by biocurators for annotations of the TCA cycle and Apoptosis in wild and domesticated animals.

SUNDAY-POSTER PRESENTATIONS

P37 Board Number: B136

Microsecond-Pulsed Dielectric Barrier Discharge Plasma Treatment Promotes Murine Ear Regeneration. N. Chernets1, D.S. Kurpad1, Q. Zhang1, Y. Song1, T.A. Freeman2; 1 Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA, 2Orthopaedic Surgery, Thomas jefferson University, Philadelphia, PA The ability of mammals to regenerate damaged tissue is limited [1-3], and more commonly wound repair results in scar tissue formation. In this report, we describe the application of non-thermal atmospheric dielectric barrier discharge plasma (DBD-plasma) as a novel modality which can promote regeneration of new ear tissue in a non-regenerating mouse’s ear after injury. It has been shown that intracellular reactive oxygen species (ROS) are elevated in response to DBD-plasma [4]. In our recent study of DBD-plasma treatment of mouse autopods, we reported that DBD-plasma generated ROS to activate signaling pathways and induced an increase of wnt activation in the distal apical epidermal ridge (AER) to enhance digit growth and elongation [4]. As parallels exist between the functionality of the AER in autopod development and the blastema in regeneration, we hypothesized that DBD-plasma could be applied to the wounded ear to induce enhanced epimorphic regeneration. To determine if DBD-plasma could promote regeneration, a through-and-through circular hole was punched in the center of the cartilaginous part the ear of non-regenerative C67BL6 mice using a 2 mm biopsy punch. A single 5 sec treatment of DBD-plasma each day for 5 days after the punch to one ear, while the other punched ear remained as an untreated control. Images of the ear holes were acquired for 32 days and the area of the hole was determined using image analysis. Histological staining and immunohistochemistry of ear regeneration samples were used to evaluate the structure of the wound epithelium and to determine the extent of tissue regeneration. DBD-plasma treated mouse ears showed accelerated wound closure, formation of blastema-like structures and replacement of cartilage, hair follicles and sebaceous glands as compared to bilateral controls. DBD-plasma treated mouse ears had a significantly higher closure rate on the 32nd day as compared to control ears (n=10, p

P38 Board Number: B137

Electrostimulation of Human Neural Stem Cells to Enhance Neuronal Differentiation. F. Ghasemi Tahrir1, G. Jin1, W. Ma1, W.H. Suh1; 1 Bioengineering, Temple University, Philadelphia, PA Differentiation guiding techniques (chemical and non-chemical, all included) for directing the differentiation of stem cells (to a specific phenotype) are considered essential platform technologies that can one-day aid in the development of clinical treatments associated with nervous system diseases

SUNDAY-POSTER PRESENTATIONS (i.e., Alzheimer’s disease, Parkinson’s disease, spinal cord injury). In the present work, electrostimulation, in particular, has been utilized as an enabling technology that promoted the differentiation of neural stem cells to neurons. We will talk about a custom-designed current stimulation cell culture device that enhanced neuronal differentiation of human adult neural stem cells (hANSCs). The differentiation via electrostimulation was confirmed by live-cell (fluorescence and confocal) microscopy, immunocytochemistry (ICC) and reverse transcription quantitative PCR (RT-qPCR). Our results indicated that precisely controlled electrostimulation over few weeks significantly increased the expression of Tubulin beta-3, which is a neuronal marker.

P39 Board Number: B138

Kinetics of Intracellular Nitric Oxide Production and Cytotoxicity in Lipopolysaccharide and Inteferon-ɣ -Induced Macrophage Activation. K.T. Tran1, K. Tyagarajan1; 1 RD, EMDMillipore, Hayward, CA Nitric oxide is a multi-functional biomolecule that can function as a bactericidal or tumoricidal mediator. The generation of nitric oxide plays a key role in several processes such as cellular proliferation, metastasis and apoptosis and has been suggested to modulate both tumorigenesis and tumor killing depending on its concentration, cell type and the oxidative milieu. It is well established that bacterial lipopolysaccharides (LPS) and interferon (IFN)-ɣ are potent inducers of nitric oxide production in murine macrophages which may contribute significantly to host defense mechanism against microbial pathogens. In this study we have systematically evaluated the mechanism and kinetics of nitric oxide production and subsequent cytotoxicity on lipopolysaccharide (LPS) and IFN-ɣ treatment of murine macrophage RAW264.7 cells. The cells were analyzed after treatment with a series of novel simplified assays on a compact microcapillary based flowcytometer including assay for intracellular nitric oxide detection, apoptotic assay for annexin V based detection along with cell death, caspase-3/7 activity and mitochondrial membrane depolarization. The kinetic studies clearly demonstrate the early production of nitric oxide observed at 6 hours and sustained levels through 48 hours of treatment. While annexin V based apoptosis and cell death were increasingly observed after 12 hours of treatment, no mitochondrial depolarization was observed throughout. Evaluation of caspase-3/7 activity in LPS-IFNɣ treated cells showed a moderate caspase-3/7 activity after 6 hours and saturated at 24 hours with only 50% of the cells displaying caspase-3/7 activation which is another distinctive indicator of apoptosis. Our data suggest that nitric oxide production and subsequent NO-mediated induction of apoptosis proceeds through alternative cell death pathways that do not involve mitochondrial depolarization. These results thus provide interesting insights into the mechanism of cytotoxicity for intracellular nitric oxide production and allows for understanding characteristic of lipopolysaccharide and IFNɣ -induced apoptosis of macrophage RAW264.7 cells.

SUNDAY-POSTER PRESENTATIONS

P40 Board Number: B139

Directly observed membrane disruption and resealing during centrifugation of sea urchin eggs. K. Miyake1, M. Goda2, S. Inoue3, N. Araki1; 1 Dep.Hist.Cell.Biol., Kagawa University, Faculty of Medicine, Kita-gun, Kagawa, Japan, 2Nagoya Univ, CeSPI, Nagoya, Japan, 3Marine Biological Lab, Woods Hole, MA Large plasma membrane disruptions (PMDs) rapidly invoke a localized exocytotic reaction that adds a ‘patch’ of internal membrane to the plasma membrane at the PMD site, a calcium-dependent resealing mechanism. We have used sea urchin eggs as a model system to define the mechanistic basis of this fundamental cell survival response. Here we directly observed plasma membrane tears that occur in sea urchin eggs during centrifugation with a special centrifuge polarizing microscope (CPM). Dilute suspensions of unfertilized eggs were layered in a centrifuge chamber above an osmotically matched dense solution containing Percoll, forming a density gradient that allowed the eggs to slowly settle to an equilibrium position. Centrifugation at speeds of up to 8,000 rpm for 20 min, separated the eggs into two parts. One part was filled with yolk granules and internal vesicles, the second part was filled with clear cytoplasm. These membrane tears by shear forces did not show variously shaped surface projections involved in exocytosis at the PMDs. These cell separations depended on the presence of calcium. However, sea urchin eggs were broken by this centrifugation in the absence of calcium. The part filled with yolk granules and internal vesicles repaired the PMDs made by a two- photon laser, but the part of the eggs containing clear cytoplasm did not repair. We conclude that shear forces caused by centrifugation make small PMDs which can reseal in the presence of extracellular calcium, although large PMDs made by laser irradiation require a ‘patch’ of internal membrane by calcium-dependent exocytosis.

P41 Board Number: B140

Harness human iPS cell differentiation and reprogramming potential using an automated cell culture system. T. Guo1, M. Watson1, N. Devaraju1, L. Szpankowski1, B. Fowler1, M. Norris1, M. Thu1, M. Wong1, D. Kemp1, H. Choi1, B. Clerkson1, G. Harris1, Y. Lu1, C. Johnson1, A. Leyrat1, X. Wang1, G. Sun1, M. Unger1, R.C. Jones2, N. Li1; 1 Fluidigm Corp, South San Francisco, CA, 2Fluidigm Corporation, South San Francisco, CA In vitro studies using pluripotent stem cells provide an essential tool for understanding hard-to-study in vivo developmental processes. Human iPSCs also holds great potential for disease modeling and cellbased therapies. A major challenge in the stem cell field, however, is to define the optimal condition for cell expansion, differentiation, and reprogramming. Since multiple intra- and extra-cellular signaling

SUNDAY-POSTER PRESENTATIONS pathways are involved in each cellular process, a combinatorial approach to screen multiple factors is highly desirable. To facilitate the exploratory processes, we have developed an automated cell culture system for cell manipulation and environmental control. The system consists of an integrated fluidic circuit (IFC), an electro-pneumatic controller instrument, experimental designer software, and automated run-time control software. Each IFC has 32 culture microchambers and 16 reagent inlets. Each microchamber can be dosed separately with different combinations and ratios of the 16 reagents at various predefined time points. Using this system, we have developed a novel nonintegrating method for direct conversion of human BJ fibroblasts to neurons at high efficiency and cell viability using microRNA mimics (miR-9, 9*, 124) and synthetic mRNAs (NeuroD2, Ascl1, and Myt1L). The system is compatible with multiple extracellular matrices including Matrigel®, vitronectin, and fibronectin, It allows 3-day hands-off operation and long term cell culture (more than three weeks) . We demonstrated that human iPSCs can differentiate to neural progenitor cells or nociceptor neurons on the IFC after treatment with small molecules. Furthermore, using combinations of small molecules and signaling proteins in chemically defined media, we demonstrated directing human iPSCs into primitive lineages of all three germ layers on one IFC within four days. Immunostaining of marker proteins and single-cell gene expression analysis were used to profile the cell types. The results were consistent with published reports and confirmed in large well-dish format. Finally, we have also developed protocol for RNA transfection that allows efficient delivery of exdogenous mRNA such as nGFP into human iPSCs as well as successful siRNA knockdowns of either endogenous or exdogenous genes. Using a siRNA against the pluripotent gene OCT4, we repeatedly demonstrated more than 50% knock-down in both RNA and protein levels of the gene, providing an easy approach for gene manipulation within human iPSCs. In summary, the automated microfluidic platform employs precise control of microenvironment of cells, facilitates studies of multifactorial combinations, and enables development of robust, reproducible, and chemically defined cell culture and manipulation.

P42 Board Number: B141

Optical clearing of mouse skeletal muscle with the CLARITY method. A. Milgroom1, E. Ralston1; 1 NIAMS, NIH, Bethesda, MD Skeletal muscle has several levels of organization that may be specifically modified during development or in diseases, from the myofibrils to the whole muscle. It may be interesting, for example, to visualize the pattern of innervation or vascularization of a whole muscle. Fluorescent proteins conjugated to muscle proteins can be expressed in skeletal muscles and can be visualized in single dissociated fibers, in small bundles of fibers, or in vivo within a millimeter or less of the muscle surface. However, they cannot be localized in whole muscles, which are opaque to light. We now report that we have successfully applied the CLARITY method (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel; Chung et al., 2013, Nature 497, 332) to the clearing of the soleus and extensor digitorum longus (EDL) muscles of the mouse. The

SUNDAY-POSTER PRESENTATIONS CLARITY method we applied toward muscle tissue clarification is an adaptation of a technique originally used to clarify soft, lipid-rich brain tissue. In our application, the muscles were treated as described in the original technique, with small modifications: incubation in a solution containing a hydrogel monomer, paraformaldehyde, and a thermal initiator for 3 days at 4°C; followed by gelling at 37°C and clearing by electrophoresis at 23V for 8h. So far, attempts to label actin myofilaments in the whole muscle with fluorescent phalloidin, as well as neuromuscular junctions with Alexa-555-conjugated bungarotoxin, have failed. However, nuclei could be viewed after staining with Hoechst 33242 and myosin heavy chain bands could be imaged in 2-photon Second Harmonic Generation (SHG) imaging, albeit with reduced intensity compared to intact muscle. Thus it is possible to clarify a tissue like skeletal muscle, rich in connective tissue and filamentous proteins and low in fat. Furthermore basic muscle fiber organization and the myosin heavy chain organization responsible for SHG are maintained in the clarified muscle.

P43 Board Number: B142

Monitoring perturbations in endoplasmic reticulum calcium homeostasis using a novel secreted reporter protein. M.J. Henderson1, E.S. Wires1, K.A. Trychta1, C.T. Richie1, X. Yan1, B.K. Harvey1; 1 NIDA/NIH, Baltimore, MD Calcium in the endoplasmic reticulum (ER) is maintained at a concentration approximately 10,000-fold greater than the cytoplasm. ER calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including diabetes, cardiovascular diseases, and neurodegenerative diseases. Defining the relationship between disease progression and ER calcium imbalance, however, is challenging due to technological limitations. We have developed a tool that allows the examination of ER calcium fluctuations over extended times, which we have termed SERCaMPs, or secreted endoplasmic reticulum calcium monitoring proteins. Our studies identified a carboxy-terminal amino acid sequence (ASARTDL) that localizes a secreted protein to the ER and confers release of the protein specifically in response to ER calcium depletion. This sequence was appended to Gaussia luciferase (GLuc), a naturally secreted luciferase protein, to create GLuc-SERCaMP. Monitoring the secretion of GLuc-SERCaMP provides a simple method to assess ER calcium homeostasis in vitro or in vivo by collecting and measuring extracellular fluids such as culture medium or blood. GLuc-SERCaMP signal is robust, with detection of ER calcium depletion possible in as few as twenty cells in culture. We assessed the utility of GLuc-SERCaMPs in models of ER stress, including hyperthermia and glutamate toxicity, in rat primary neurons. Additionally, ER calcium homeostasis in rat liver was examined by measuring release of the reporter into blood. Our results suggest that SERCaMPs will have broad applications for the long term monitoring of ER calcium homeostasis and the development of therapeutic approaches to counteract ER calcium dysregulation. This work was supported by NIDA/NIH.

SUNDAY-POSTER PRESENTATIONS

Actin and Actin-Associated Proteins 1 P44 Board Number: B144

An Alternative Mechanism for Actin Arc Formation at the Immunological Synapse of Jurkat T cells. S. Murugesan1, J. Yi1, D. Li2, L. Shao2, E. Betzig2, X. Wu3, J.A. Hammer1; 1 NIH\NHLBI, Bethesda, MD, 2HHMI/Janelia Farm Research Campus, Ashburn, VA, 3NIH/NHLBI, Bethesda, MD Actin cytoskeletal organization and dynamics play fundamental roles in T cell immunological synapse (IS) formation. The T cell’s actin cytoskeleton undergoes striking rearrangements upon contact with an antigen presenting cell (APC) to form radially symmetric segregated domains named the distal, peripheral and central supramolecular activation clusters (dSMAC, pSMAC, and cSMAC). The outer dSMAC corresponds to a lamellipodial (LP) actin network composed of branched actin arrays that undergo robust, polymerization-driven actin retrograde flow. The central pSMAC corresponds to a lamellar (LM) actin network composed of concentric actin arcs that undergo actomyosin II-driven contraction. Finally, the inner cSMAC is largely devoid of actin. This bull’s-eye pattern of SMACs generates a centripetal flow of actin that drives the inward movement of signaling and adhesion molecules to create a mature IS. Here we addressed how the concentric, linear actomyosin II arcs in the pSMAC are assembled. Current thinking would have it that the branched actin network in the dSMAC, which is created by Arp2/3-dependent nucleation, is converted by debranching and crosslinking into the concentric arcs. Using structured illumination microscopy, we identified for the first time linear actin filaments that are arranged perpendicular to the plasma membrane and that are embedded in the branched actin network in the dSMAC. As these filaments exit the inner aspect of the dSMAC, they splay out and reorient into concentric arcs with an inherent antiparallel organization required for myosin II-dependent contraction. The perpendicular actin filaments in the dSMAC are highlighted and accentuated by inhibiting Arp2/3-dependent actin polymerization using CK666, which collapses the branched actin array in between the perpendicular filaments. Importantly, inhibition of myosin II using blebbistatin results in loose, disorganized filaments in the pSMAC that fail to reorient into concentric arcs. These observations suggest that arc assembly can occur independently of Arp2/3-dependent nucleation, and that myosin II contractility is required for reorienting the perpendicular filaments emanating from the dSMAC into the concentric arcs in the pSMAC. We are now attempting to define the relative contributions of debranching/rearrangement of the branched actin network versus nucleation of linear filaments, possibly by the formin INF2 present on the plasma membrane at the tips of the perpendicular filaments, to the formation of the actin arcs in the pSMAC.

SUNDAY-POSTER PRESENTATIONS

P45 Board Number: B145

A Coarse-Grained Model of Lamellipodium Protrusion and Retraction Driven by Fluctuations in Actin Polymerization. G.L. Ryan1, D. Vavylonis2, N. Watanabe3; 1 Department of Physics, Kettering University, Flint, MI, 2Department of Physics, Lehigh University, Bethlehem, PA, 3Tohoku Univ Grad Sch Life Scis, Sendai, Japan Animal cells that spread onto a surface often rely on actin-rich cell extensions called lamellipodia to execute cell protrusion. XTC cells on a two-dimensional substrate exhibit regular protrusion and retraction of their lamellipodium, even though the cell is not translating. Travelling waves of protrusion have also been observed, similar to those observed in crawling cells. These periodic fluctuations in leading edge position have been linked to excitable actin dynamics near the cell edge using a one dimensional model of actin dynamics, as a function of arc-length along the cell. In this work we extend this earlier model of actin dynamics into two-dimensions (along the arc-length and radial directions of the cell) and include a model membrane that protrudes and retracts in response to the changing number of free barbed ends of actin filaments near the membrane. We show that if the polymerization rate of these barbed ends changes depending on their local concentration at the leading edge and the opposing force from the cell membrane, the model can reproduce the patterns of membrane protrusion and retraction seen in experiment. We investigate both Brownian ratchet and switch-like force-velocity relationships between the membrane load forces and actin polymerization rate.

P46 Board Number: B146

The role of filopodia in directed cancer cell migration in 2D and 3D. A. Simon1, S. Geraldo2, P. Maiuri2, a. funfak3, j. bibette3, m. piel1, D. Vignjevic2; 1 Inst Curie-Ctr De Recherche, Paris, France, 2Institut Curie, Paris, France, 375005, ESPCI, PARIS, France When cancer cells escape the primary tumor, they invade into the surrounding tissue and migrate through the extracellular matrix to reach the circulatory system. It is, therefore, conceivable that cancer cells migrate directionally towards the blood vessels. Directional guidance of cells following gradients of growth factors and chemokines are known to exist in vivo during embryonic development and immune response. Filopodia are thought to be the guidance organelles for directional migration through gradients. However, besides studies in neuronal and endothelial cells, there is no evidence confirming this hypothesis in other cell types, such as cancer cells. Using stable CT26 intestinal cancer cell line as a model for invasive tumorigenesis, we investigated if filopodia have a role in directed cancer cell migration in 2D and 3D substrates. The expression of proteins involved in filopodia formation and/or stabilization was inhibited in order to study the ability of depleted cells to move towards a source of growth factors. The different depleted cell lines were compared for their ability to perform chemotactic movements on a planar substrate towards a source of growth factors, using the Dunn’s chamber. WT

SUNDAY-POSTER PRESENTATIONS cells were able to move directionally and persistently towards the attractants, whereas cells depleted for Fascin, mDia2 and MyosinX were not able to migrate directionally towards the gradient. These results show that filopodia are necessary for directional migration of cancer cells on 2D. To assess the role of filopodia in invasive migration of cancer cells through 3D environments, we embedded cancer cell spheroids in 3D matrices of collagen I. After 48 hours, WT cells present typical mesenchymal morphology, invading and migrating between the collagen fibers in a stellar pattern radiating from the spheroid periphery. Only Eps8 and MyoX depleted cells presented a round morphology in 3D and a dramatic decreased invasion of collagen 3D matrices. Finally, we are currently assessing if filopodia are guidance organelles for cancer cells migration in 3D matrices. For this study we are using a microfluidic 3D chemoinvasion chamber allowing the formation of long-term gradients of growth factors in a collagen matrix.

P47 Board Number: B147

Retinomotor movements and the distribution of the visual enzyme, RPE65, in fish retinal pigment epithelial cells. M. O'Donnell1, C. King-Smith2; 1 Biology, Saint Joseph's University, Philadelphia, PA, 2Department of Biology, Saint Joseph's University, Philadelphia, PA Eyes of lower vertebrates, including fish, lack a dilatable pupil to regulate light flux. Instead, dramatic retinomotor movements occur, which involve movement of melanin pigment granules in the retinal pigment epithelium (RPE) and elongation and contraction of photoreceptor inner and outer segments. When exposed to light, pigment granules create a moveable screen and disperse throughout the RPE elongated apical projections. Rods, responsible for dim light vision, elongate and are enveloped by pigment-filled RPE. Cones, which are responsible for color and bright- light vision, contract, putting them first in line to light entering the eye. In the dark, these retinomotor movements are reversed. RPE pigment granule migration and the positional rearrangements of photoreceptors are actin- and microtubule-dependent. A different type of light-dependent motility in RPE of mammalian eyes was reported in a recent study. RPE contains an isomerase, RPE65, which regenerates the all-trans retinal chromophore back to its cis-form, crucial for the visual cycle. RPE65 localizes towards the apical side of mouse RPE in the dark and redistributes to the basal side in the light. This motility appears to be an actin-myosin dependent process, requiring the motor protein, myosin VIIa (Lopes, et al. 2011, Hum Mol Gen, 20: 2560). Since pigment granule movement in fish RPE also requires actin and is much more extensive than in mammalian eyes, we are investigating whether RPE65 similarly undergoes lightdependent translocation in fish RPE. In dissociated, isolated fish RPE cells in vitro, RPE65 was identified in apical projections using immunofluorescence, however no change in RPE65 distribution was observed in cells triggered to aggregate or disperse pigment granules. To determine if RPE65 undergoes redistribution in light- and dark- adapted eyes in situ, we labeled cryosections of RPE plus retina. Although RPE65 labeling in the RPE was visible, melanin pigment quenched some fluorescence. We are

SUNDAY-POSTER PRESENTATIONS currently working with albino catfish RPE-retina to eliminate issues with pigment quenching and to characterize RPE65 distribution in fish RPE.

P48 Board Number: B148

TRIM9 Regulates Filopodia During Cortical and Retinal Development. N. Boyer1, S. Gupton2; 1 Cell and Molecular Biology, University of North Carolina, Chapel Hill, NC, 2Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC Early in neuronal development actin-rich filopodial protrusions are a critical prerequisite for neurite formation. Later, as nascent axons extend toward their postsynaptic partners, filopodia are central in growth cone response to extracellular axon guidance cues such as netrin-1. The netrin-1 receptor DCC localizes to axonal filopodia tips and mediates netrin-1 dependent filopodia formation and elongation as well as subsequent growth cone chemoattraction toward higher concentrations of netrin-1. We previously found that the E3 ubiquitin ligase TRIM9 binds DCC and is an important regulator in netrin-1 attractive signaling in murine cortical neurons. We show that TRIM9 interacts with actin regulatory proteins that promote filopodia formation, including members of the Ena/VASP family and Myo10, and that TRIM9 regulates filopodia number. Whereas the growth cones of cortical neurons typically have low basal numbers of filopodia that increase in response to netrin-1 stimulation, cortical neurons isolated from TRIM9 knockout mice have elevated growth cone filopodia number that fail to respond to netrin-1. As such, TRIM9-/- neurons morphologically resemble wild-type neurons treated with netrin-1 but lack sensitivity to netrin-1. Using high resolution time-lapse microscopy, we analyzed filopodia dynamics in cortical neurons; this revealed that loss of TRIM9 increases the lifetime of individual filopodia, as does treatment of wild-type neurons with netrin-1. To determine if TRIM9-mediated regulation of filopodia lifetime and response to netrin-1 were conserved in other neuronal types, we investigated the role of TRIM9 in retinal development. Retinal ganglion cells (RGCs) are another neuron type for which netrin-1 signaling is critical for axon guidance into the optic nerve. Cultured RGCs from TRIM9-/- embryos again exhibited increased filopodia length compared to controls and failed to appropriately respond to netrin1. Genetic loss of TRIM9 results in a thinning of the RGC nuclear and nerve fiber layers in the adult murine retina as well as reduction in the number of RGC nuclei in vivo. Our findings suggest that TRIM9 is critical in the development of many neuronal types as well as in regulating filopodial responses to netrin-1 signaling. Current experiments are aimed to determine the mechanisms of TRIM9 mediation of pathways in netrin-1 signaling, as well as its relation to other neuronal guidance cues involved in retinal development such as Slits.

SUNDAY-POSTER PRESENTATIONS

P49 Board Number: B149

Actin filaments are necessary for maintaining the normal structure of apical tubulobulbar complexes in the seminiferous epithelium. K. Lyon1, E. Bosseboeuf2, A. Vogl1; 1 Cellular and Physiological Sciences, UBC, Vancouver, BC, 2Sciences Fondamentales et Appliquées, University de Poitiers, Poitiers, France Here we explore the effects of actin perturbation on the morphology of tubulobulbar complexes in apical regions of the seminiferous epithelium. Tubulobulbar complexes are elongate subcellular machines responsible for the internalization of intercellular junctions during sperm release. Each tubulobulbar complex consists of a double membrane tubular core terminating in a clathrin coated pit. The membrane core is surrounded by a network of actin filaments and a distinct swelling or bulb develops in the distal third of the structure. Isolated testes were perfused for one hour with oxygenated Krebs Henseleit buffer (with BSA) at 33°C containing either cytochalasin D (10, 20 or 40 uM) or DMSO (control). They were then perfusion fixed either for fluorescence or electron microscopy. Quantification of fluorescently labeled actin at apical tubulobulbar complexes was done using thresholding techniques and demonstrated that the structures at stage VII of spermatogenesis were associated with lower levels of actin in experimental spermatids compared to controls. When evaluated at the electron microscopic level, the actin networks appeared patchy and expanded further away from the membrane cores than in controls. Ectopic bulb regions were also seen near the bases of the complexes in experimentally treated tissue. We conclude that the actin networks are necessary for maintaining the normal structure of apical tubulobulbar complexes. Supported by a NSERC Discovery Grant to AWV.

P50 Board Number: B150

Mutations in Drosophila crinkled/Myosin VIIA disrupt denticle morphogenesis. J.L. Sallee1, J. Crawford2, V. Singh2, D.P. Kiehart3; 1 North Central College, Naperville, IL, 2Biology, Duke University, Durham, NC, 3Duke University, Durham, NC Actin filament crosslinking, bundling and motor proteins are necessary for the assembly of epithelial projections such as microvilli, stereocilia, hairs, and bristles. Mutations in such proteins cause defects in the shape, structure, and function of these actin-based protrusions. One protein, Myosin VIIA, is an actin-based motor protein conserved throughout phylogeny. In Drosophila melanogaster, severe mutations in crinkled (ck) are “semi-lethal” with only a very small percentage of flies surviving to adulthood. Such survivors show morphological defects related to actin bundling in hairs and bristles and are deaf. To better understand ck/MyoVIIA’s function in bundled-actin structures, we used dominant female sterile approaches to analyze the loss of maternal and zygotic ck/MyoVIIA in the morphogenesis of denticles, small actin-based projections on the ventral epidermis of Drosophila embryos.

SUNDAY-POSTER PRESENTATIONS Maternal/zygotic ck/MyoVIIA mutants displayed severe defects in denticle morphology – actin filaments initiated in the correct location, but failed to elongate and bundle to form normal projections. Using deletion mutant constructs, we demonstrated that the motor domain and both of the C-terminal MyTH4 and FERM domains are necessary for proper denticle formation. Furthermore, we showed that ck/MyoVIIA interacts genetically with dusky-like (dyl), a member of the ZPD family of proteins that links the extracellular matrix to the plasma membrane. Loss of one protein does not alter the localization of the other; however, loss of the two proteins together dramatically perturbs denticle shape. Our data indicate that ck/MyoVIIA plays a key role in arranging actin filament bundles into biologically functional units, which drive proper shape of cellular projections. Supported by National Institutes of Health GM33830 and NIDCD007673 to DPK and F32GM093592 to JLS.

P51 Board Number: B151

Dystroglycan depletion inhibits the functions of differentiated HL-60 cells. A. Martínez-Zárate1, I. Martínez-Vieyra1,2, L. Alonso-Rangel1, B. Cisneros3, S.J. Winder4, D. Cerecedo1,2; laboratorio de Hematobiología, Instituto Politécnico Nacional, Mexico DF, Mexico, 2laboratorio de Hematobiología, Instituto Politécnico Nacional, Mexico, Mexico, 3Genetics and Molecular Biology, CINVESTAV, Mexico, D.F., Mexico, 4Biomedical Science, University of Sheffield, Sheffield, United Kingdom 1

Haematopoiesis involves the coordination of several signal transduction pathways, which are induced by extracellular stimuli through cell–cell and cell–extracellular matrix (ECM) interactions. Interactions with the ECM binding partners represent the critical steps by which cells initiate cytoplasmic signaling essential for the regulation of their growth, differentiation, attachment and migration, and are important factors in the development and progression of many types of cancer. Dystroglycan (Dg) is an adhesion molecule responsible for crucial interactions between ECM and the cytoplasmic compartment. It is formed by two subunits, α-Dg (extracellular) and β-Dg (transmembrane), that bind respectively to laminin in the matrix and to one of several cytolinker proteins such as dystrophin with the cytoskeleton. As an integral part of the dystrophin glycoprotein complex (DGC) of skeletal muscle, dystroglycan is essential for signaling events mediated through this complex that may have a role as a mechanochemical, force-transducing system or shock-absorber and indirectly mediating other signaling pathways associated with the DGC. Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells and so is therefore of considerable functional importance.

SUNDAY-POSTER PRESENTATIONS

P52 Board Number: B152

Discoidin domain receptor 1 controls linear invadosome formation. J. Di Martino1, A. Juin 2, B. Leitinger 3, L. Paysan1, J. Bomo4, G. Baffet 4, J. Rosenbaum1, V. Moreau1, F. Saltel1; 1 INSERM U1053, Bordeaux, France, 2Cancer Research UK Beatson Institute, Glasgow, United Kingdom, 3 Imperial college London, National Heart and Lung Institut, London, United Kingdom, 4INSERM U1085, Rennes, France Accumulation of type I collagen fibrils in tumors is associated with an increased risk ofmetastasis. Invadosomes are F-actin structures able to degrade the extracellular matrix. Wepreviously found that collagen I fibrils induced the formation of peculiar linear invadosomes inan unexpected integrinindependent manner, raising the question of the receptor involved. Here,we show that Discoidin Domain Receptor 1 (DDR1), a collagen receptor overexpressed in cancer,co-localizes with linear invadosomes in tumor cells and is required for their formation andmatrix degradation ability. Unexpectedly, DDR1 kinase activity was not required for invadosomeformation or activity, nor was Src tyrosine kinase. We show that the RhoGTPase Cdc42 localizesto linear invadosomes, is activated upon plating cells on collagen in a DDR1-dependent manner,and is required for linear invadosome formation. Finally, DDR1 depletion blocked cell invasionin a collagen gel. Altogether, our data uncover the important role of DDR1, acting through Cdc42,in proteolysis-based cell invasion in a collagen-rich environment.

P53 Board Number: B153

Metabolic filaments, the actin cytoskeleton, and orphan diseases. R.M. Broyer1, J.E. Wilhelm2; 1 Biology, University of California, San Diego, La Jolla, CA, 2University of California, San Diego, La Jolla, CA A core question in disease research is to determine how the cell is altered on a molecular level in affected individuals. In the case of diseases caused by inborn errors of metabolism, this means identifying the relationship between activity of the affected enzyme and the observed disease phenotypes. Recently, we uncovered nearly 50 previously unidentified, conserved filamentous structures including eight novel filament systems that are comprised of enzymes with previously described roles in cellular metabolism. Because these enzymes are implicated in intermediate metabolism, we have focused on answering two key questions. First, have these filaments acquired any cell biological or cytoskeletal role that is separable from the known function of the enzyme. Second, are there any inborn errors of metabolism whose phenotypes can be explained by understanding the function of these polymers. To answer these questions, we have focused our efforts on PRPP synthase, a metabolic enzyme that forms filaments that are conserved from yeast to humans. Interestingly,

SUNDAY-POSTER PRESENTATIONS Human PRPP synthase forms a novel nuclear filament. Since there are no known roles for PRPP in the nucleus, this suggested the filament might have a specific nuclear function. Furthermore, there are several classes of human disease mutations in PRPP synthase - overexpression mutations which cause gout, loss of function mutations that cause deafness and ataxia, and feedback resistance mutations that cause gout, deafness, and ataxia. The fact that feedback resistance mutations cause both gain and loss of function phenotypes also suggested that PRPP synthase filaments might have acquired a second function separable from its enzyme activity. Using fibroblasts from human patients, we have found that superactivity mutations cause PRPP synthase to polymerize in the cytoplasm where filaments are not normally found. These filaments are not nonfunctional aggregates since treatment with purine metabolites can cause reversible disassembly of the filaments. This suggests that superactivity mutations can generate dominant negative filaments in the cytoplasm leading to a loss of function phenotype. Additionally, we have identified a protein that co-localizes with PRPP synthase filaments that also appears to be required for Drosophila bristle formation, suggesting a link between purine metabolism and actin cytoskeleton regulation. This provides the first molecular explanation for how mutations in a purine biosynthetic enzyme can cause non-syndromic deafness in humans and opens the door for exploring how other metabolic polymers might explain the symptoms associated with many inborn errors of metabolism.

P54 Board Number: B154

Actin Cytoskeletal Dynamics Regulate Adhesion and Force Generation in Airway Smooth Muscle Cells. Y.M. Beckham1,2, J. Solway3, M.L. Gardel4; 1 James Frank Institute, Institute for Biophysical Dynamics and Physics Department, University of Chicago, Chicago, IL, 2Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, 3Department of Medicine, University of Chicago, Chicago, IL, 4James Franck Institute, Institute for Biophysical Dynamics and Physics Department, University of Chicago, Chicago, IL Actin dynamics in non-muscle cells have been well characterized and shown to play crucial roles in regulation of cell adhesion, migration and force generation. Despite this extensive knowledge, much less is known about the regulation of the actin cytoskeleton in smooth muscle cells. Adhesion and force generation by smooth muscle cells are crucial to many physiological functions and contribute to the pathology of numerous diseases, including asthma. Here we demonstrate that adherent human airway smooth muscle cells (hASM) have an actin cytoskeleton that is highly reminiscent to that of non-muscle cells. The cell periphery is dominated by an Arp 2/3-mediated lamellipodium while the cell center is characterized by a myosin-rich and formin-mediated lamella. Moreover, these actin-based organelles control cell adhesion and force generation in a similar fashion to their role in non-muscle cells. Arp2/3 inhibition eliminates the lamellipodium and results in abrogated nascent adhesion assembly and a reduction in hASM cell adhesion. Inhibition of formin-mediated actin assembly results in a decrease in lamellar actin by at least 25 percent. This formin-dependent decrease in lamellar actin results in a 60

SUNDAY-POSTER PRESENTATIONS percent decrease in contractility of hASM cells. This work suggests that models of non-muscle cell contractility may be useful in understanding the mechanics of airway smooth muscle cells.

P55 Board Number: B155

Distinct Actin Filament Networks Modulate Different Cell Signalling Pathways Involved in Cell Proliferation and Senescence. G. Schevzov1, B. Wang1, J.W. Hook1, C. Tonthat1, I. Pleines2, B. Kile2, E.C. Hardeman1, P.W. Gunning1; 1 School of Medical Sciences, UNSW, Sydney, Australia, 2Water and Eliza Hall Institute, Melbourne VIC, Australia The spatial and temporal control of signalling pathways that regulate cellular processes, including proliferation and senescence, is orchestrated by a complex network of interactions involving scaffolding proteins and kinases. The actin cytoskeleton is known to play a critical role in integrating such signalling pathways. Within a cell functionally diverse actin filament networks are present. Currently, the molecular mechanism(s) by which these actin networks participate in signalling pathways is not wellunderstood principally due to a lack of tools that discriminate between them. Tropomyosins (Tm), a family of actin filament-associated proteins, can impart unique cellular functions to the actin cytoskeleton. Our investigations using mouse embryonic fibroblasts (MEFs) isolated from Tm isoformspecific knockout (KO) and transgenic mice have revealed significant alterations in cell proliferation and life span. Ablation of Tm5NM1 results in reduced cell proliferation in direct contrast to its overexpression. This result is Tm5NM1-specific since ablation of Tm4 shows no impact on proliferation. We show that Tm5NM1-mediated proliferation occurs via the MEK/ERK pathway. Furthermore we demonstrate that in the absence of Tm5NM1 the formation of pERK and its nuclear shuttling protein Importin 7 is reduced resulting in the observed impaired cell proliferation. In contrast, an in vitro life span assay demonstrates that Tm4 KO MEFs can readily escape senescence unlike the Tm5NM1 KO cells. The Tm4 KO MEFs show dysregulation of the PKC and JAK-STAT pathways. These studies provide the first evidence that specific Tm-containing actin filaments can influence signalling pathways. Taken together these findings allow us to begin to understand how signalling pathways are regulated within the cell in space and time via different actin filament networks marked by specific Tm isoforms.

SUNDAY-POSTER PRESENTATIONS

P56 Board Number: B156

Zyxin mediated regulation of airway smooth muscle function may be involved in asthmatic airway hypercontractility. S.R. Rosner1, C.D. Pascoe2, E. Blankman3, C.C. Jensen3, R. Panganiban4, R. Krishnan5, C.Y. Seow2, J. Park4, Q. Lu1, M.C. Beckerle3, P.D. Pare2, J.J. Fredberg4, M.A. Smith3; 1 Department of Environmental Health, Harvard School of Public Health, Boston, MA, 2St Paul's Hospital Center for Lung Innovation, University of British Columbia, Vancouver, BC, 3Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, 4Harvard School of Public Health, Boston, MA, 5Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Boston, MA Morbidity and mortality due to asthma arise mainly from loss of airway patency as a result of airway hypercontractility. Airway hypercontractility is the result of excessive contraction of airway smooth muscle. Airway hypercontractility in the non-asthmatic lung is readily reversed by the taking of a deep breath, which results in bronchodilation. A deep breath stretches the contracted airway smooth muscle causing it to fluidize rapidly and then resolidify slowly. On the other hand, airway hypercontractility in the asthmatic lung is generally refractory to the dilative effect of a deep breath. In the asthmatic case, the airway smooth muscle enters what is called a latch state, wherein the parenchymal tissue lacks sufficient tethering force to stretch the airway smooth muscle. The molecular mechanisms of stretch induced airway smooth muscle fluidization and resolidification are unknown, as is the mechanism of its failure in asthmatics. The LIM-domain scaffolding protein zyxin has been shown to be involved in repairing, remodeling and reinforcing actin stress fibers in response to mechanical strain. Thus, zyxin is essential for generation of peak traction forces. Zyxin is rapidly recruited to sites of stress fiber strain, where it enlists actin regulatory proteins to effect repair. Due to its role in regulation of the actin cytoskeleton’s response to force, we hypothesized zyxin might be involved in the regulation of the airway smooth muscle response to stretch. In this work we show that zyxin plays a critical role in reinforcing the actin cytoskeleton of airway smooth muscle in response to stretch. We demonstrate that zyxin responds to physiologic levels of isotropic stretch through rapid recruitment to sites of acute strain on stress fibers, and in murine airway smooth muscle, loss of zyxin results in abrogated resolidification following single or multiple stretches. In precision cut lung slices from wild type or zyxin null mice, the airways from zyxin null mice dilate more rapidly in response to stretch. Furthermore, zyxin expression is increased in the lungs of humans who experienced fatal asthma attacks. Together, these data show a role for zyxin in the regulation of airway smooth muscle response to stretch, and suggest a connection to airway hypercontractility in human asthmatics.

SUNDAY-POSTER PRESENTATIONS

P57 Board Number: B157

Synthetic polyamines promote rapid lamellipodial growth by regulating actin dynamics. D. Riveline1; 1 Laboratory of Cell Physics ISIS/IGBMC, Strasbourg, France Cellular protrusions involved in motile processes are driven by site-directed assembly of actin filaments in response to Rho-GTPase signalling. So far, only chemical compounds depolymerizing actin or stabilizing filaments, inhibiting N-WASP, Arp2/3 or formins, have been used to eliminate the formation of protrusions, while Rho-GTPase-dominant positive strategies have been designed to stimulate protrusions. Here we describe the design of four polyamines (macrocyclic and branched acyclic), and show that they enter the cell and induce specific growth of actin-enriched lamellipodia within minutes. The largest increase in cell area is obtained with micromolar amounts of a branched polyamine harbouring an 8-carbon chain. These polyamines specifically target actin both in vitro and in vivo. Analysis of their effects on filament assembly dynamics and its regulation indicates that the polyamines act by slowing down filament dynamics and by enhancing actin nucleation. These compounds provide new opportunities to study the actin cytoskeleton in motile and morphogenetic processes. •

Nedeva, I., G. Koripelly, D. Caballero, L. Chieze, B. Guichard, B. Romain, E. Pencreach, J.M. Lehn, M.F. Carlier, and D. Riveline. 2013. Synthetic polyamines promote rapid lamellipodial growth by regulating actin dynamics. Nat Commun. 4:2165.

P58 Board Number: B158

Biomechanical characterization of a new cellular model for Filamin C-related myofibrillar myopathy. W.H. Goldmann1, F. Chevessier-Tuennesen2, L. Einsiedler1,2, R. Schröder2, B. Fabry1; 1 Friedrich-Alexander-Univ/Ctr Med Physics Technol, Erlangen, Germany, 2Institute of Neuropathology, University Hospital Erlangen, Erlangen, Germany Mutations on chromosome 7q32 of the human filamin C gene (FLNC) are responsible for an autosomal dominant form of myofibrillar myopathy. The expression of mutant W2710X filamin C, the most common pathogenic mutation in humans, leads to the disruption of Z-discs of striated muscle sarcomeres and abnormal protein aggregates. We tested whether the expression of mutant filamin C protein changes the biomechanical properties and the intrinsic mechanical stress response of immortalized myogenic cells derived from heterozygous and homozygous filamin C knock-in mice bearing the human homologous W2711X mutation. Compared to wildtype cells, mutant filamin C myoblasts and myotubes showed increased cell death and substrate detachment in response to cyclic stretch on flexible membranes. Moreover, 2D force microscopy of murine myoblasts showed lower

SUNDAY-POSTER PRESENTATIONS tractions in mutated compared to wildtype cells. Our findings provide first evidence that altered mechanical properties may contribute to the progressive striated muscle pathology in filaminopathies. We postulate that the expression of mutant filamin C leads to altered biophysical properties making these mutated myogenic cells more vulnerable to mechanical stress.

P59 Board Number: B159

Determining the impact of the actin cytoskeleton on the mechanical properties of cells. I. Jalilian1, G. Schevzov1, T. Fath1, C. Heu1, L. Bischof2, H. Vindin1, J.R. Stehn1, M. Knothe Tate3, E.C. Hardeman1, P.W. Gunning1; 1 School of Medical Sciences, UNSW, Sydney, Australia, 2Quantitative Imaging Group, CSIRO, Sydney, Australia, 3Graduate School of Biomedical Engineering, UNSW, Sydney, Australia The influence of the actin cytoskeleton on the shape and mechanical properties of cells has been revealed extensively using anti-actin drugs. However, due to the lack of the specificity of these compounds for different actin filament populations it has been difficult to delineate the exact role of the actin cytoskeleton in these cellular features. Actin binding proteins largely affect the organisation and dynamics of actin in cells; however, the impact of these proteins on the mechanical properties of cells is still unknown. Tropomyosins (Tms) form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organisation through their highly regulated expression and sorting to specific intracellular sites. Hence, Tm isoforms can be used as tools to identify and manipulate functionally distinct actin filament populations in a cell. In this study we investigated the impact of Tmcontaining actin filaments on the organisation of the actin cytoskeleton and the mechanical properties of cells. To investigate the role of different Tms, and hence different actin filaments, on cellular stiffness we examined rat neuroblastoma cells stably overexpressing TmBr3, Tm3, Tm4 or Tm5NM1 in comparison with control cells. Analysis of the number and length of actin cables per cell using a morphometric linear feature detection algorithm revealed significant differences in the length and number of actin cables per cell relative to that seen in the control cells. The mechanical measurements conducted by indentationtype atomic force microscopy (AFM) showed that TmBr3-, Tm4- and Tm5NM1-overexpressing cells were significantly stiffer than control cells. In contrast, cell stiffness did not change in Tm3-overexpressing cells indicating that the observed changes in cell elasticity are not a generic effect of accumulating more Tm. Moreover, siRNA knockdown of Tm5NM1 in Tm5NM1-overexpressing cells caused the elastic modulus to revert to that of control cells. The results show that cell stiffness increases in TmBr3-, Tm4- and Tm5NM1-overexpressing cells. Since, the overexpression of Tm changes the numbers and length of the actin cables in an isoform-specific manner that is not directly related to cell stiffness, our data suggest that the mechanical properties of cells are more dependent on the organisation of the actin filaments than the number and length of actin

SUNDAY-POSTER PRESENTATIONS filaments. Therefore, we conclude that it is specific aspects of actin organisation which regulate the mechanical properties of the cell.

P60 Board Number: B160

Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane. D.S. Gokhin1, R.B. Nowak1, N.E. Kim1, J.A. Khoory2, I.C. Ghiran2, V.M. Fowler1; 1 Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, 2Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA The mammalian red blood cell (RBC) contains an underlying membrane skeleton that imparts the RBC with mechanical properties optimized for efficient passage through the microvasculature. The RBC membrane skeleton consists of nodes of short (~37-nm-long) actin filaments interconnected by long (α1β1)2-spectrin tetramer strands. RBC actin filaments are stabilized along their lengths by two tropomyosin isoforms, TM5NM1 and TM5b, and capped at their pointed and barbed ends by tropomodulin1 and αβ-adducin, respectively. However, it remains unknown whether RBC actin filaments (or subsets or portions thereof) are dynamic during normal RBC homeostasis, and, if so, whether actin dynamics regulates RBC mechanical properties. To answer these questions, we first studied actin dynamics by rhodamine-actin (rho-actin) incorporation into RBC ghosts. We also treated RBCs or ghosts with three different actin-disrupting drugs: (i) cytochalasin-D (CytoD), which inhibits actin subunit association and dissociation at barbed ends; (ii) latrunculin-A (LatA), which destabilizes and depolymerizes dynamic actin filaments by binding to and sequestering actin monomers, driving the F:Gactin balance toward the G-actin state; (iii) jasplakinolide (Jasp), which stabilizes dynamic actin filaments, driving the F:G-actin balance toward the F-actin state. Results show that resealed RBC ghosts exhibit rho-actin subunit incorporation into discrete sites within the membrane skeleton, demonstrating that RBC actin filaments are indeed dynamic. Moreover, rho-actin subunit incorporation can be blocked via treatment with CytoD, demonstrating that dynamic subunit exchange occurs at barbed ends. Consistent with dynamic filaments, treatment of intact RBCs with LatA or Jasp induces a ~2-fold increase or a ~60% decrease, respectively, in soluble actin levels. Using total internal reflection fluorescence (TIRF) microscopy, we observed diffuse F-actin staining in DMSO-treated (control) RBCs, whereas LatA (but neither CytoD nor Jasp) treatment induced dramatic rearrangement of the RBC actin network, as indicated by gaps in the F-actin staining pattern. This result suggests the presence of a dynamic subpopulation of RBC actin filaments. LatA treatment and selective disassembly of this subset of RBC actin filaments had no effect on RBC osmotic fragility, but both membrane deformability and spontaneous membrane oscillations (“flickering”) were increased. Collectively, these results provide the first direct evidence for the existence of a dynamic subpopulation of RBC actin filaments, and that actin filament architecture directly controls the mechanics of the human RBC membrane.

SUNDAY-POSTER PRESENTATIONS

P61 Board Number: B161

Localization of IQGAP1 mutants to retracting versus protruding cell areas. J. Schober1, M. Reimer1; 1 Pharmaceutical Sciences, Southern Illinois University Edwardsville, Edwardsville, IL IQGAP1 interacts with numerous binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 plays a role in cell-matrix interactions and actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. Phosphorylation in the CT domain regulates intramolecular interaction and IQGAP1 cellular activity. In a recent study, we discovered that IQGAP1 localizes to actively retracting edges, instead of protruding areas, in B16F10 mouse melanoma cells and some other cells types. In these current studies we examined localization of IQGAP1 mutants to retracting versus protruding areas in B16F10 cells. Cells were cotransfected with GFP-IQGAP1 Full Length (GFP-IQGAP1-FL), as an internal control, and one of five Myc-tagged IQGAP1 constructs (FL, S1441E/S1443D, ΔCHD, ΔGRD or ΔCT). The cotransfected cells were plated onto laminin for 30 minutes, stained with anti-Myc and anti-WAVE antibodies, and normalized fluorescence measurements were made in retracting and protruding areas. Retracting cell areas were defined as GFP-IQGAP1-FL positive and WAVE negative, while protruding cell areas were defined as GFP-IQGAP1-FL negative and WAVE positive. In retracting areas there were large decreases in both ΔGRD and ΔCT localization, a slight decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion there were large increases in both ΔGRD and ΔCT localization, and normal localization of ΔCHD and S1441E/S1443D mutants. These results indicate that two domains, GRD and CT, are essential for normal localization of IQGAP1 to retracting cell areas. Furthermore, our results are consistent with a model in which IQGAP1 in the areas of cell retraction is in the open, phosphorylated, conformation.

P62 Board Number: B162

Role of Rho/Rock/Myosin in Senescence Induced Migration. I. Aifuwa1, N. Longe1, S. An2, D. Wirtz3,4; 1 Johns Hopkins University, Baltimore, MD, 2School of Public Health, Johns Hopkins University, Baltimore, MD, 3Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, MD, 4Department of Pathology and Oncology and Sydney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD Cells induced into senescence exhibit a marked increase in the secretion of pro-inflammatory cytokines termed senescence-associated secretory proteins (SASP). Here we report that SASP from senescent stromal fibroblasts promote spontaneous morphological changes accompanied by an aggressive migratory behavior in originally non-motile human breast cancer cells. This phenotypic switch is

SUNDAY-POSTER PRESENTATIONS coordinated, in space and time, by a dramatic reorganization of the actin and microtubule filament networks, a discrete polarization of EB1 comets, and an unconventional front-to-back inversion of nucleus-MTOC polarity. SASP-induced morphological/migratory changes are critically dependent on microtubule integrity and dynamics, and are largely cooperated via the inhibition of cell contractility. RhoA/ROCK inhibition reduces focal adhesions and traction forces, but promotes a gliding mode of migration.

P63 Board Number: B163

Rapid Glucose Deprivation Immobilizes Active Myosin-V on Stabilized Actin Cables. L. Xu1, A.P. Bretscher2; 1 Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, 2Cornell University, Ithaca, NY Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles. This is mediated by myosin-Vs transporting cargos along F-actin bundles known as actin cables. Actin cables are dynamic structures regulated by assembly, stabilization and disassembly. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage. Thus upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase, translation and phosphoinositide metabolism. Here we show that glucose withdrawal rapidly depletes ATP levels and the yeast myosin V, Myo2, responds by relocalizing to actin cables within 1 min, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigorlike state of a myosin-V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Additionally, glucose depletion has differential effects on the actin cytoskeleton resulting in disassembly of actin patches with concomitant inhibition of endocytosis, and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin-V in a newly described state on selectively stabilized actin cables.

SUNDAY-POSTER PRESENTATIONS

P64 Board Number: B164

Tracking old and newly synthesized β-actin protein in three dimensional MDCK cyst cultures. P. Vedula1, A.J. Rodriguez1; 1 Federated Department of Biology, Rutgers University, Newark, NJ In vivo, most simple epithelia are organized as a monolayer of cells surrounding a central lumen (e.g. intestinal epithelium, kidney tubules, mammary acini, alveolar sacs). In vitro, epithelial cells cultured in a 3-dimensional extra-cellular matrix develop into cysts and provide a model system to study epithelial morphogenesis and lumen formation. Previous studies have shown that several polarity proteins (apical and baso-lateral) along with components of the protein sorting machinery play a role in lumen initiation in MDCK cysts grown in matrigel. However the role of mRNA localization on protein synthesis and hence lumen formation and/or maintenance has yet to be determined. In this study, we use MDCK cysts in matrigel to address the role of β-actin mRNA zipcode on its protein synthesis. Utilizing the tetracysteine reporter system and biarsenical dyes – FlAsH and ReAsH, we were able to track β-actin protein synthesis temporally in the 3D cultures. This method provides valuable insight into the dynamic nature of actin based structures in a more physiologically relevant 3 dimensional culture system, thus opening the doors to how mRNA zipcode dependent protein synthesis regulates epithelial structure and function in vivo. In fact, we observe that newly synthesized β-actin protein labeled by ReAsH is localized predominantly at the lateral cell-cell junctions while the older protein labeled by FlAsH is distributed at the apical and lateral interfaces. This provides direct evidence for the long standing hypothesis that localized translation of β-actin does indeed dictate its preferential incorporation into different compartments in the cell controlling 3D epithelial morphogenesis.

P65 Board Number: B165

Investigating EB1 interactions with the actin cytoskeleton. E. Alberico1, H. Goodson1; 1 University of Notre Dame, Notre Dame, IN Many cellular processes require coordination between the actin and microtubule cytoskeletons. Cell division and migration are two very important processes that require this type of coordination. Proteins such as mDIA1 and IQGAP1 are known to regulate these interactions. Our lab has found that EB1, a well characterized microtubule binding protein, can also directly interact with actin in vitro. Through bioinformatics and mutagenesis we have found that the EB1-actin binding site on EB1 partially overlaps the well characterized EB1-MT binding interface. We are currently investigating the role EB1 has in interacting with the actin cytoskeleton through characterization of EB1 mutants in vivo. Understanding the EB1-actin interaction in vivo will enhance our knowledge of cytoskeletal coordination and its role in a variety of cellular processes.

SUNDAY-POSTER PRESENTATIONS

P66 Board Number: B166

Microtubule end-binding protein EB1 directly regulates collaborative actin assembly by APC and formins. R. Jaiswal1, H. Kim2, D. Breitsprecher2, B.L. Goode1; Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, 2Brandeis University, Waltham, MA 1

Tight coordination of the microtubule and actin cytoskeletons is essential for many biological processes, including directed cell migration, neuronal process formation, spindle rotation, and polarized cell adhesion. However, in few cases have the underlying mechanisms controlling microtubule-actin crosstalk been defined. Earlier studies identified EB1 as a binding partner of APC (adenomatous polyposis coli) and formins (Su et al., 1995; Wen et al., 2004), which prompted us here to investigate EB1’s potential effects on APC-formin-mediated actin assembly. Using both TIRF microscopy and bulk actin assembly assays, we observed that purified full-length EB1 directly inhibits the actin nucleation activity of C-terminal fragments of APC, as well as collaborative actin assembly by APC and formins. EB1 specifically inhibited the nucleation of new actin filaments without affecting the elongation rate of existing filaments. Further, multi-wavelength single molecule TIRF experiments showed that EB1 inhibits actin nucleation by blocking the interactions of actin monomers with APC. Additionally, we show that APC directly links actin filaments to microtubule seeds, and that EB1 disrupts this linkage. Together, our results suggest that EB1 may coordinate microtubule plus end dynamics with APC-mediated actin assembly. We are now dissecting EB1-APC interactions to develop the tools required for testing this model in vivo. References: Su LK, Burrell M, Hill DE, Gyuris J, Brent R, Wiltshire R, Trent J, Vogelstein B, Kinzler KW. APC binds to the novel protein EB1. Cancer Res. 1995, 55, 2972-2977. Wen Y, Eng CH, Schmoranzer J, Cabrera-Poch N, Morris EJ, Chen M, Wallar BJ, Alberts AS, Gundersen GG. EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nat Cell Biol. 2004, 6, 820-830.

SUNDAY-POSTER PRESENTATIONS

P67 Board Number: B167

The formins FHOD-1 and INFT-1 are important for ovulation and localize to the somatic gonad of C. elegans. A. Hegsted1, D.W. Pruyne1; 1 Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY Formins are actin nucleators, and many promote actin filament elongation. To determine the physiological role of each formin in the genetic model system Caenorhabditis elegans, single deletions of its non-essential formins were obtained. These deletions produced apparently healthy worms, which suggested redundant formin functions. We generated double formin mutants and found that worms doubly-mutated for the formin genes fhod-1 and inft-1 had smaller brood sizes (number of progeny) and movement defects. This project characterizes the effect of fhod-1;inft-1 double mutation on brood size and the localization of FHOD-1 and INFT-1 within the somatic gonad. The worm gonad is composed of two symmetrical arms. Germ cells are produced in the distal gonad and migrate proximally. Myoepithelial sheath cells surround the distal and proximal gonad. The proximal sheath cells have contractile actin networks that move the developing oocytes towards the spermatheca. The spermatheca, an actin rich organ, is the site of fertilization. The developing embryo resides in the uterus until it is expelled through the vulva. FHOD-1 localizes to the vulval and uterine muscles, and the proximal sheath cells. INFT-1 is expressed at the spermatheca. An ovulation defect seen in the double mutant worms is endomitotic oocytes in the gonad arm (emo). This phenotype can be the result of a contractility defect in the proximal sheath cells and/or the spermathecae. We examined ovulation in the double mutant worms and saw that the maturing oocyte follows one of three paths: normal ovulation, engulfment by the spermatheca that breaks the oocyte, or no ovulation. This abnormal ovulation could be the reason for the smaller brood size. We hypothesize that FHOD-1 and INFT-1 are important for organizing actin in the myoepithelial sheath cells and the spermatheca to allow proper ovulation.

P68 Board Number: B168

Defining the roles of the formin Diaphanous in organizing the contractile ring and midbody ring during cytokinesis. Y. Ruella Palm1, D. d'Allard1,2, G.R. Hickson1,2; 1 UHC Ste-Justine, Montreal, QC, 2Universite de Montreal, Montreal, QC Animal cells divide by the intricate process of cytokinesis. Initially, a Rho GTPase and actomyosindependent contractile ring (CR) drives the constriction at the cell equator until a stable midbody ring (MR) forms and matures to guide the separation of the two cells. The CR and MR are highly organized structures controlled by interactions among a network of Rho-dependent proteins. One such protein is Diaphanous (Dia), a formin that nucleates unbranched actin filaments, localizes to the CR and is essential

SUNDAY-POSTER PRESENTATIONS for cytokinesis. Nevertheless, it is unclear how Dia contributes to the organization of the CR and, potentially, to the subsequent formation of the MR. We have examined the requirement for Dia and actin during cytokinesis by time-lapse video microscopy of Drosophila S2 cells expressing markers tagged with fluorescent proteins (FPs). Acute administration of the inhibitor of actin polymerization, Latrunculin A (LatA), defined a period of 20 minutes from closure of the CR during which the nascent MR requires dynamic actin: before this time, furrows regressed; after this time the MR remained stable in LatA. Moreover, a functional Dia-FP localized to the CR and nascent MR but not to the mature MR and the timing of its disappearance coincided with the transition to a LatA-insensitive MR structure. Depletion of Dia by RNAi slowed furrow ingression, enhanced furrow oscillations and prevented furrow completion. Live imaging of the F-actin probe, LifeAct-GFP, in Dia depleted cells revealed a marked decrease in equatorial F-actin at the time of normal CR formation, although a cortical flow of F-actin from the poles to the equator was subsequently observed. The CR components Myosin-FP and the scaffold protein Anillin-FP were still recruited to the equator of Diadepleted cells but formed aberrant punctate structures, demonstrating a role for Dia in maintaining the normally uniform organization of the CR. Our prior truncation analysis has uncovered multiple mechanisms contributing to the localization of Anillin to the equatorial cortex (Kechad, El Amine). An Nterminal region of Anillin that contains both F-actin and myosin binding domains and that mediates the proper localization of Anillin to the CR, was no longer recruited to the cortex in Dia-depleted cells or in cells treated with LatA. Since the latter treatment did not prevent the recruitment of Dia-FP, this suggests that Dia-dependent F-actin polymerization is specifically required for the recruitment and retention of Anillin at the CR. This is likely an important role for Dia that provides clues as to the organization of the CR. Furthermore, our data are consistent with a model in which the regulated removal of Dia from the F-actin-dependent CR promotes the transition to the Anillin-dependent MR.

P69 Board Number: B169

Potent inhibition of Diaphanous formins by actin oligomers produced by the Actin Crosslinking Domain toxin of V.cholerae. D. Heisler1, E. Kudryashova1, D. Grinevich1, S. Kotha2, J. Winkelman3, C. Suarez4, P. Narasimham2, D.R. Kovar4,5, D.S. Kudryashov1,6; 1 The Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, 2 Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, The Ohio State University, Columbus, OH, 3Molecular Genetics and Cell Biology, Univeristy of Chicago, Chicago, IL, 4Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL, 5Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 6Molecular and Cellular Developmental Biology Program, The Ohio State University, Columbus, OH Actin Crosslinking Domain (ACD) is produced by pathogenic strains of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Upon delivery to the cytoplasm of host cells via Type I or Type VI Secretion systems, ACD catalyzes the formation of an amide bond between K50 and E270 of actin monomers,

SUNDAY-POSTER PRESENTATIONS resulting in the formation of actin oligomers of varying sizes. According to the current view on the ACD pathogenesis, the ACD activity leads to a slow failure of the cytoskeleton due to the gradual accumulation of non-functional oligomers leading to depletion of both monomeric and filamentous actin pools. Instead, we propose that ACD employs a hitherto unprecedented toxicity mechanism by converting cytoplasmic actin into highly toxic oligomers that specifically target key steps of actin dynamics. Particularly, we demonstrate that the oligomers inhibit with sub-nanomolar affinity actin polymerization controlled by Diaphanous formins. TIRFM experiments reveal that the formin controlled elongation is blocked in a concentration dependent manner in the presence of nanomolar concentrations of the oligomers. Profilin plays a key role in the observed inhibition and mDia1 formin constructs with fewer proline-rich regions in their FH1 domains are inhibited by the oligomers less efficiently. Accordingly, we demonstrate that at least some vital functions of the cytoskeleton (e.g. integrity of epithelial barriers) are deeply impaired when only a small fraction of cytoplasmic actin is crosslinked into oligomers. The effects are reproduced by formin-, but not ARP2/3 complex-specific small molecule inhibitors. Intriguingly, the pool-down analysis of HeLa cell lysates using double-tagged actin demonstrates that the oligomers exhibit selectivity towards certain human formins, suggesting the possibility of creating specific ACD-based and/or ACD mechanism-inspired formin inhibitors. To conclude, we propose that delivery of only a few copies of ACD into the cytoplasm of the host cell enables this enzyme to produce actin oligomers that possess the unique combination of properties that is neither observed in G- nor F-actin. Specifically, a tandem organization of the ACD-crosslinked actin oligomers and their ability to interact with G-actin binding proteins (e.g. profilin) confers them an abnormally high affinity to tandem G-actin binding proteins (e.g. tandem proline-rich and WH2 domains of formins, VASP, and NPFs) and enables them to actively interfere with the specific nucleation and/or elongation activities of these proteins. Thereby, ACD initiates a toxicity cascade that employs actin oligomers as second messengers, whose novel, “gain-of-function” inhibitory properties potently “poison” cellular functions orchestrated by formin-mediated actin polymerization.

P70 Board Number: B170

A role of RhoA activation in IGF-I-induced muscular hypertrophy model. S. Ueda1, S. Yamada2, Y. Kirimura1, A. Kushiya1, Y. Minoru1, T. Satoh3, Y. Shirai4,5; Kobe Univ, Kobe-Shi, Hyogo-Ken, Japan, 2Biomedical Engineering, Univ California-Davis, Davis, CA, 3 Grad. Sch. Science, Osaka Prefecture Univ, Osaka, Japan, 4Kobe Univ, Kobe, Japan, 5Kobe Univ Grad Sch Agricultural Science, Kobe-Shi, Hyogo-Ken, Japan 1

Skeletal muscle loses the mass during the aging process. IGF-I, insulin like growth factor-I, stimulates several signaling pathways in the differentiation and hypertrophy of muscle cells, and prevents the skeletal muscle atrophy accompanying with aging. Small GTPase RhoA has been known to be essential for muscle development, but its precise role remains unknown. Here, we analyzed the activation and role of RhoA in an IGF-I-induced muscular hypertrophy model. To construct the IGF-I-induced muscle hypertrophy model, C2C12 cells were differentiated for 48 hours followed by further 48 hours in

SUNDAY-POSTER PRESENTATIONS differentiated media with or without IGF-I. With the addition of IGF-I, C2C12 cells showed prominent hypertrophic changes, and increased 1.3-fold in the maximum diameter of myotube by facilitating the fusion of myoblast. To determine the activation level of RhoA, we performed a pull-down assay for RhoA ･GTP. After 24 hours of IGF-I treatment, RhoA activity increased 2.0-fold compared with 0 hour, and increased 62% relative to non-treatment cells. To confirm whether the activated RhoA contributes to the hypertrophy, we transfected RhoA dominant-negative mutant (RhoA-DN) into C2C12 cells to inhibit RhoA activation by IGF-I. The expression of RhoA-DN reduced the hypertrophy induced by IGF-I, and impaired the expression of differentiation marker (MHC and M-cadherin) and p38 MAP-kinase activation. These data suggests that IGF-I promotes the muscular hypertrophy via RhoA activation in this model. To identify the binding proteins of RhoA in IGF-I-induced hypertrophic signaling, we designed RhoA tagged with promiscuous biotin ligase BirA (Roux et al. 2012). This technique may provide novel insights into the role of RhoA in the signal networks during the muscular hypertrophy.

P71 Board Number: B171

PIP2 is required for the stability of cleavage furrow-associated proteins during cytokinesis. S. Yildirim1, R. . Wong2, H. Wei3, L.L. Fabian3, G. Polevoy4, J.A. Brill5; cell biology, The hospital for sick children, Toronto, ON, 2Univ Toronto, Toronto, ON, 3Hospital for Sick Children, Toronto, ON, 4Cell Biology Program, Hospital for Sick Children, toronto, ON, 5Cell Biology Program, Hospital for Sick Children, Toronto, ON

1

Meiotic division during Drosophila melanogaster spermatogenesis serves as an excellent model for studying mechanisms of cytokinesis. Our previous studies showed that phosphatidylinositol phosphates (PIPs) act as regulators of this process. To define the role of phosphatidylinositol 4,5-bisphosphate (PIP2) in meiotic cytokinesis, we studied cleavage furrow-associated proteins such as Anillin, Sep2 (a septin) and Sqh (myosin regulatory light chain) in PIP2-depleted testes. Live imaging of spermatocytes depleted of PIP2 by ectopic expression of the Salmonella PIP2 phosphatase SigD revealed that Anillin, Sep2, and Sqh initially localize to the cleavage furrow, yet lose their cortical localization during furrow regression, leading to formation of multinucleate cells. Reduction in the abundance of these proteins in the furrow prior to furrow regression, as well as oscillations of GFP-Anillin and Sqh-GFP along the cortex, indicated a perturbation of cleavage furrow integrity. To confirm that these phenotypes resulted from PIP2 depletion, we examined the Drosophila PIP 5-kinase Sktl, which synthesizes PIP2. YFP-Sktl localized to cleavage furrows of dividing spermatocytes. Male germ cells homozygous mutant for sktl were multinucleate, consistent with a role for Sktl in cytokinesis. Moreover, dividing sktl mutant spermatocytes, like those expressing SigD, exhibited reduced levels of Anillin and Pnut (a septin) in the cleavage furrow. Taken together, our data suggest that PIP2 is synthesized at the cleavage furrow, where it serves to maintain association of furrow proteins with plasma membrane for successful completion of cytokinesis.

SUNDAY-POSTER PRESENTATIONS

P72 Board Number: B172

The small GTPase Rap1 promotes cell movement rather than stabilizes adhesion in epithelial cells responding to insulin-like growth factor I. M.A. Guvakova1, I. Prabakaran1, W.S. Lee1, D.K. Furstenau1, D.C. li1, R. Hung1, N. Kushnir1; 1 University of Pennsylvania, Philadelphia, PA How the Ras-related GTPase Rap1 controls a fine balance between cell adhesion and cell migration remains puzzling. Here, we discovered a previously unidentified regulatory role of insulin-like growth factor type I receptor (IGF-IR) in CRK SH3-binding guanine nucleotide exchange factor (C3G) -Rap1– fascin-actin axis promoting cell movement. We demonstrate that a burst of Rap1 activity, rather than presumed hyperactivation is imperative for the onset of cell movement. We show that while autophosphorylated IGF-IR signals to C3G to activate Rap1, subsequent IGF-IR internalization promotes gradual inactivation of Rap1 by putative Rap1GTPase-activating protein (GAP). Additionally, IGF-IR signaling recruits active Rap1 at sites of cell motile protrusions. C3G depletion prevents IGF-I-induced accumulation of fascin at actin microspikes and blocks protrusions. In the absence of IGF-IR’s activity, the wild type Rap1 and the constitutively active V12Rap1 mutant remain in cell-cell contacts. Forced inactivation of Rap1 signaling by overexpressing dominant negative N17Rap1, Rap1GAP, or by silencing C3G has a detrimental effect on F-actin and cell adhesion irrespective of IGF-IR signaling. We conclude that the basal levels of Rap1 activity hold up cell adhesion, whereas sequential regulation of C3G and GAP by IGF-IR reverses the labile Rap1 function from supporting adhesion to promoting migration. Furthermore, our findings support a role of C3G-Rap1 signaling in the IGF-IR –mediated regulation of an actin crosslinking protein fascin.

P73 Board Number: B173

Human formin-2 generates nuclear actin filaments in response to DNA damage. B.J. Belin1, T. Lee1, R.D. Mullins2; 1 Cellular Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, 2 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA Compelling recent evidence has implicated nuclear actin filaments as regulators of cellular responses to certain environmental stresses, including serum stimulation, heat shock and cultured models of neurodegeneration (Baarlink et al., 2013; Bamburg et al. 2010). Using human tissue culture lines, we have observed that nuclear actin polymerization is stimulated by DNA damage induced by a variety of mechanisms, including telomere uncapping, UV exposure and MMS treatment. DNA damage-associated nuclear filaments can be visualized by phalloidin staining and are detected with high specificity using our nuclear actin-specific filament reporter, Utr230-EGFP-NLS. Using this tool, we identify several classes of

SUNDAY-POSTER PRESENTATIONS nuclear actin filaments after DNA damage: nucleolus-associated, nucleoplasmic and aggregate-like filament bodies. Unlike previously described nuclear actin filaments, DNA damage-induced filaments do not contain cofilin nor require mDia1/mDia2 for polymerization. Rather, we find that DNA damageassociated nuclear actin polymerization requires formin family actin nucleator formin-2 (FMN2). FMN2 accumulates in the nucleus in response to DNA damage, and FMN2 or importin-9 knockdowns implicate nuclear actin polymerization in efficient double strand break clearance and regulation of nuclear oxidation during the DNA damage response. These results suggest that regulation of nuclear actin assembly may be a common feature of cellular stress responses and reports the first characterized function for FMN2 in mammalian somatic cells.

P74 Board Number: B174

Comparative analysis of tools for in vivo detection of actin filaments. B.J. Belin1, L.M. Goins2, R.D. Mullins3; 1 Cellular Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, 2University of California, San Francisco, San Francisco, CA, 3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA Several tools are now available for in vivo imaging of actin filaments, including Lifeact, variants of the utrophin actin-binding domain (Utr230 and Utr261), F-tractin and fluorescently labeled actin. Use of these filament reporters has become widespread and, despite growing evidence to the contrary, they are often regarded as comprehensive labels for actin filaments. To systematically evaluate their suitability for detecting commonly studied actin filament networks, we generated stable cell lines for each reporter in human, Mus musculus, Xenopus laevis and Drosophila melanogaster tissue culture cells. Within each line, we quantitatively compared reporter localization to phalloidin staining and used FRAP to determine how reporter binding and dynamics relate to filament turnover kinetics. Using this approach, we identified subtle reporter binding preferences that are qualitatively similar across organisms and provide reporter recommendations for studies of multiple filament networks, including cortical actin, lamellae, lamellipodia, filopodia and stress fibers. We find that F-tractin is the most broadly suitable filament reporter yet induces organism-specific morphological defects. Poor Lifeact binding and GFP-actin incorporation is common among formin-generated filaments, whereas Utr261 is often excluded from the Arp2/3-generated lamellipod. Utr230 is restricted to highly stable actin filaments and, surprisingly, detects Golgi-associated filaments in D. melanogaster S2 cells, which have previously been detected by immunofluorescence but cannot be seen with phalloidin. Overall our results underscore the need to test multiple visualization strategies to determine the most appropriate filament visualization tool for each desired application.

SUNDAY-POSTER PRESENTATIONS

P75 Board Number: B175

Formin tails enhance processive elongation of actin filaments in addition to nucleation and filament bundling. C.L. Vizcarra1, B. Bor2, M.E. Quinlan1,2; 1 Chemistry and Biochemistry, University California-Los Angeles, Los Angeles, CA, 2Molecular Biology Institute, University California-Los Angeles, Los Angeles, CA Formins are multi-domain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well-conserved Formin Homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous Autoinhibitory Domain (DAD), which is C-terminal to the Formin Homology-2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this story, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low affinity interactions with both actin monomers and filaments. Removal of the Capu-tail reduces both actin filament binding and bundling. Further, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that enhancement of processivity is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Despite the absence of a DAD domain, the Capu-tail mimics all known functions of other DAD domains including autoinhibition. Finally, the role of formin tails in processivity was previously unrecognized.

P76 Board Number: B176

Mechanism of IRSp53 Inhibition by 14-3-3. D.J. Kast1, R. Dominguez1; 1 Physiology, Perelman School of Medicine at the University of Pennslyvania, Philadelphia, PA Filopodia are actin-rich, dynamic membrane protrusions that play essential roles in numerous cellular processes such as cell migration, phagocytosis,and axonal guidance. IRSp53 couples signaling pathways to membrane and cytoskeleton dynamics, acting as a potent inducer of filopodia. IRSp53 contains an Nterminal inverted BAR domain, followed by overlapping Cdc42/SH3-binding sites within a sequence known as the CRIB-PR domain, and an SH3 domain that recruits several cytoskeletal effectors. We have previously shown that activation of IRSp53 from an autoinhibited state requires the binding of Cdc42 to the CRIB-PR and/or effectors, such as Eps8, to the SH3 domain, which produce a conformational change in IRSp53. The sequence between the CRIB-PR and SH3 domains is rich in Ser and Thr residues, and

SUNDAY-POSTER PRESENTATIONS some of which bind 14-3-3 in a phosphorylation-dependent manner, providing an alternative route for IRSp53 regulation. Here we explore the phosphorylation-dependent mechanism of IRSp53 inhibition by 14-3-3. Using isothermal titration calorimetry we show that 14-3-3 binds weakly to single phosphorylated sites, but that binding is enhanced ~100-fold when specific pairs of sites are phosphorylated. Crystal structures show how the different phosphorylation sites of IRSp53 bind to 14-33, either as mono- or double-phosphorylated peptides. The results identify the most likely sites for inhibition by 14-3-3, and the kinases likely involved in phosphorylation of these sites. The data support a model of inhibition whereby asymmetric binding of 14-3-3 to two distinct phosphorylation sites inhibits activation by either Cdc42 or downstream effectors.

P77 Board Number: B177

Disabled regulates Abelson nonreceptor tyrosine kinase localization and kinase activity in Drosophila. L. Kotlyanskaya1, E. Giniger2, R. Kannan2; 1 Neurobiology Curriculum, UNC Chapel Hill, Bethesda, MD, 2NINDS institute, NIH, Bethesda, MD Disabled (Dab) is a putative adaptor protein that is required for lamination of the cortex. In Drosophila it interacts with the Abelson (Abl) kinase signaling pathway to regulate epithelial structure and axon guidance. Previous evidence hints at Dab regulation of both Abl localization and Abl kinase activity. Dab mutant embryos show localized inhomogeneity in Abl localization during the midcellularization stage in early embryogenesis. Disabled structure, such as its polyproline domains which are potential SH3 binding regions, and the PTB domain, suggests that Dab may act as a scaffold and can potentially recruit Abl, by binding it directly or in a complex, may regulate its ability to bind to other proteins. We initiated a structure-function analysis to determine what domains of Dab may regulate Abl localization and activity using tagged Dab deletion constructs transiently transfected into S2 cells and subsequently assayed for Abl localization by immunoflourescence and confocal microscopy. To assay Abl activity, we took advantage of a FRET based Abl kinase reporter. We found that overexpression of Dab full length or Dab constructs containing the highly conserved central domain aggregate in puncta in S2 cells and these puncta colocalize with puncta of endogenous Abl, suggesting Dab recruitment of Abl in the cytoplasm. Under these conditions upon overexpression of Dab, we found increased Abl kinase activity as assayed by FRET, suggesting that Dab stimulates Abl kinase activity. We have further confirmed this increase in Abl kinase activity via FRET in vivo in cultured photoreceptors from Drosophila larvae overexpressing Dab. Conversely, dab mutant larvae have reduced Abl kinase activity as assayed by FRET. In conclusion, we have found that in S2 cell culture Dab adaptor protein regulates Abl kinase localization and activity. In the future we will make further refined deletions of Dab to determine what protein domains are responsible for these activities.

SUNDAY-POSTER PRESENTATIONS

P78 Board Number: B178

Dendritic actin network growth in the absence of symmetry breaking. P. Jreij1, P. Bieling1,2, T. Li1, R.D. Mullins2, D.A. Fletcher1,3,4; 1 Department of Bioengineering, University of California, Berkeley, Berkeley, CA, 2Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, 3Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA, 4Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA At the leading edge of migrating cells, dendritic actin networks exert protrusive forces through the dynamic machinery of Arp2/3-mediated actin polymerization. Previous studies have shown that dendritic actin network assembly can be reconstituted on nucleation promoting factor (NPF)-coated surfaces from a minimal system of purified proteins including actin, profilin, Arp2/3, and capping protein. Variation in concentrations of these components has produced insight into their biochemical interplay and resulting network structure. However, in most studies to date, the requirement for actin networks growing around beads or bacteria to break symmetry before exhibiting steady-state growth introduces non-uniform stresses that could alter network structure and has limited the range of protein concentrations that could be explored. To investigate a broader range of protein concentrations and avoid stresses due to curved surfaces, we reconstituted dendritic actin networks on planar glass surfaces using silane-PEG-maleimide chemistry to pattern NPF APVCA domains on defined areas. In the presence of actin, profilin, Arp2/3, and capping protein, branched actin networks assembled on the patterns and grew out of plane at a constant rate. We followed the dynamic growth in real time using three-color spinning disk confocal microscopy for a range of NPF densities, Arp2/3 concentrations, and capping protein concentrations. Our findings show that the net rate of actin incorporation into the network is independent of changes in NPF density, Arp2/3 concentration, and capping protein concentration, even though network growth rates vary significantly in response to these changes. Our estimates of nucleation rates, filament lengths, and changes in number of free barbed ends across a range of Arp2/3 and capping protein concentrations are consistent with the Monomer Gating Model and help to constrain predictive models of dendritic actin network growth.

P79 Board Number: B179

Isoform-specific oligomerization of ZASP, a myofibrillar myopathy gene product, in vitro and in vivo. R. Ohman1, L. Brubaker1, S. Kawakami-Schulz1, X. Lin1, K. Subedi1, I. Chow1, J. Kaufman2, P. Wingfield2, A.K. Mankodi1; 1 NINDS-NIH, Bethesda, MD, 2NIAMS-NIH, Bethesda, MD BACKGROUND: Myofibrillar myopathies (MFM) present clinically as progressive muscle weakness that begins in early adulthood. Zaspopathy, a prototype MFM, is caused by heterozygous mutations in ZASP,

SUNDAY-POSTER PRESENTATIONS a Z-disc associated PDZ-LIM protein. The long and short ZASP isoforms are defined by the presence or absence of LIM domains, respectively. The mutated sZM domain is common to both isoforms. ZASP interacts with skeletal actin filaments via the sZM domain. The PDZ-LIM proteins are regulated by intramolecular association. However, oligomerization of ZASP has not yet been explored. OBJECTIVE: 1) To explore self-association of ZASP; 2) To identify oligomerization domains of ZASP; and 3) To investigate the role of ZASP self-association in normal skeletal muscle and in zaspopathy. DESIGN/METHODS: ZASP self-association was investigated with analytical sedimentation equilibrium assay of purified ZASP proteins as well as co-immunoprecipitation and chemical crosslinking assays in transfected cells and mouse and patient skeletal muscle. The domains involved in dimerization were identified with yeast two-hybrid assays. Actin-bundling ability of ZASP was explored using a low-speed co-sedimentation assay. Effects of different ZASP isoforms on actin stress fibers were examined in cultured mouse muscle (C2C12) cells. RESULTS: The long ZASP isoforms self-associate, whereas the short isoform does not. Wild type and mutant ZASP form homodimers and heterodimers. ZASP self-association occurs via interaction of the LIM domains. The mutated actin-binding domain is not involved in ZASP self-association. A lowspeed co-sedimentation assay showed that GST or His6-tagged long ZASP isoform does not crosslink actin filaments. Expression of the mutated ZASP long but not short isoform caused F-actin disruption in cultured muscle cells. CONCLUSIONS: Wild-type and mutant ZASP form homodimers and heterodimers in zaspopathy, an autosomal dominant disease. Self-association of ZASP and F-actin disruptive properties of mutated ZASP are specific to the long isoform that is required for the integrity of the Zdiscs in skeletal muscle. Future studies will explore the effects of post-translational modification on ZASP dimerization. Understanding ZASP associations within both the normal and disrupted cells of the Z-disc will lead to a better understanding of structural components of the Z-disc, and the contribution of disruption of ZASP-actin associations in MFM.

SUNDAY-POSTER PRESENTATIONS

Regulation of Actin Dynamics 1 P80 Board Number: B180

Counting actin molecules in cytoskeletal structures with Lifeact by quantitative fluorescence microscopy. Q. Chen1, N. Courtemanche2,3,4, T.D. Pollard5,6,7; 1 Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, 2Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, 3Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 4Department of Cell Biology, Yale University, New Haven, CT, 5Department of Molecular, Cellular and Developmental Biology, Yale University School of Medicine, New Haven, CT, 6Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, 7Department of Cell Biology, Yale University School of Medicine, New Haven, CT Quantitative measurement of actin molecules in cytoskeletal structures is essential to understand many cellular processes, but counting actin directly using fluorescence microscopy has been challenging, because actin tagged with a fluorescent protein such as mGFP is not fully functional and cannot replace native actin. Here we present a method using Lifeact-mGFP to count actin molecules in contractile rings and actin patches in live fission yeast cells. Purified Lifeact-mCherry binds actin filaments stoichiometrically with a Kd of 16 µM. To titrate actin filaments in fission yeast we drove the expression of Lifeact-mGFP with an inducible promoter 3nmt1 and obtained cells with a wide range of total fluorescence. We used quantitative fluorescence microscopy to measure the number of Lifeact-mGFP molecules in whole cells and actin patches and contractile rings in these cells. The total numbers of Lifeact-mGFP associated with both structures increased with the cytoplasmic concentration and plateaued at high concentrations giving an apparent Kd of about 20 µM. From the plateaus of these binding curves we estimated a peak number of ~5,000 actin molecules in actin patches (similar to published values with actin-mGFP) and ~150,000 actin molecules in complete cytokinetic contractile rings.

P81 Board Number: B181

In vivo system for identifying nuclear actin regulators. C.M. Groen1, T.N. Fagan1, S. Sudhir1, T.L. Tootle2; Anatomy and Cell Biology, Univ Iowa-Carver Coll Med, Iowa City, IA, 2Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, Iowa City, IA 1

Actin is not only a critical component of the cytoplasmic cytoskeleton, but plays key roles in the nucleus – including chromatin remodeling, transcription and nuclear organization. However, the structures,

SUNDAY-POSTER PRESENTATIONS functions, and how structure affects function of nuclear actin remain poorly understood. While nuclear actin does not typically form canonical filaments as observed by phalloidin staining, other unique structures have been observed. One of these structures is nuclear actin rods. Actin rods form in response to cellular stress (i.e. heat shock, starvation/re-feeding), and when cytoplasmic filamentous actin is disrupted. Nuclear actin rods are also observed during the progression of neurodegenerative diseases including Alzheimer’s and Parkinson’s disease. While nuclear actin rods have been implicated in contributing to cell survival, their functions and the mechanisms regulating their formation remain largely unclear. We have identified a new, in vivo, multicellular system to study nuclear actin rods. Ectopic expression of GFP-Actin results in nuclear actin rod formation within the nurse cells during stages 5-9 of Drosophila oogenesis. Rod formation depends on which of the six Drosophila actins is overexpressed and its level of expression. Notably, endogenous nuclear actin is observed in the same cells during these developmental stages, suggesting that nuclear actin plays a functional role during egg development. Thus, we can utilize the actin rods formed when GFP-actin is overexpressed as a system to uncover conserved means of regulating nuclear actin. Specifically, we are quantitatively assessing the prevalence and morphology of the actin rods formed by the different actins, and how those rod phenotypes are altered by cellular stress. We are also identifying novel mechanisms regulating nuclear actin rod formation and/or structure by genetically screening for phenotypic modifiers. These studies are expected to provide significant insight into the regulation and function of nuclear actin not only during development but also during neurodegeneration.

P82 Board Number: B182

Tropomyosin 5a/b actin filaments collaborate with arp2/3 actin filaments to regulate cell motility. S. Brayford1, G. Schevzov1, E.C. Hardeman1, J.E. Bear2, P.W. Gunning1; 1 School of Medical Sciences, UNSW, Sydney, Australia, 2UNC Lineberger Comprehensive Cancer Center, Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC The mammalian actin cytoskeleton is differentially regulated by over 40 different isoforms of tropomyosin. Different isoforms sort to spatially distinct actin filaments within the cell where they perform isoform-specific regulatory roles. Using isoform-specific antibodies, we have demonstrated that of all known isoforms of tropomyosin, only Tm5a and 5b are enriched in the lamellipodium of mouse fibroblasts, indicating a role in the regulation of actin filament function within this structure. This led us to further investigate the functional role of these isoforms in the regulation of plasma membrane protrusions associated with cell motility. Using live-cell imaging, kymography and cell tracking we have shown that knockdown of Tm5a/b expression through RNA interference leads to a marked decrease in the persistence of membrane protrusions but no change in the level or location of arp2/3. This indicates that arp2/3 location is not dependent on Tm5a/b. A corresponding reduction in whole cell motility is observed in Tm5a/b knockdown cells which is paralleled by a reduction in the number of substratum adhesions. These findings suggest that the actin cytoskeleton within the lamellipodium contains two

SUNDAY-POSTER PRESENTATIONS types of actin filaments with different functional roles. One is the well-characterised branched filament network driven by arp2/3 which provides the protrusive force to generate dynamic membrane protrusions. The second is Tm5a/b-containing actin filaments, most likely unbranched, which stabilise arp2/3 protrusions by promoting the formation of adhesions to the substratum to gain traction. We propose that tropomyosin behaves as a discriminating factor that allows collaboration of different filament types to achieve a specific functional outcome for the cell.

P83 Board Number: B183

Nucleation of Actin Filaments by the Arp2/3 Complex: New Mechanistic Insights from Kinetic Simulations. S.B. Padrick1,2, L.K. Doolittle1,2, M.K. Rosen2,3; 1 Dept. of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, 2Howard Hughes Medical Institute, Dallas, TX, 3Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX The actin related protein 2/actin related protein 3 (Arp2/3) complex is a widely conserved actin nucleation factor. The Arp2/3 complex controls actin dynamics by nucleating new actin filaments in specific places, times and orientations in the cell, in response to stimulatory signals from nucleation promotion factors (NPFs) such as those of the WASP family. The fundamental signaling logic has been understood for several years; coincident signals from NPFs and pre-existing filaments combine to stimulate Arp2/3 complex to produce new filaments. Given the central role that Arp2/3 complex plays in many processes, there has been great interest in the detailed mechanism by which it nucleates new actin filaments. Here we use computational simulation of the kinetics of the nucleation process to explore a recent hypothesis that NPF release from the complex is required during nucleation. While NPFs do promote high levels of activation, structures of actin in complex with a necessary actin binding NPF element, the WASP Homology 2 (WH2) region, indicate that their engagement with actin during delivery may prevent growth of the nucleated filament. This idea is consistent with recent single molecule studies. Here we incorporate this idea into a new kinetic mechanism that also enforces thermodynamic reversibility and allosteric coupling. By comparing the predicted behavior of this model to the global behavior of in vitro Arp2/3 complex nucleation activity, we find that agreement with the data is improved but requires additional kinetic steps for a complete description. Systematic exploration of parameter space for a series of successively more complex models has lead to a new kinetic model of Arp2/3 complex activation by NPFs. This new model will serve as a predictive template to guide further studies refining our understanding of this essential nucleation factor.

SUNDAY-POSTER PRESENTATIONS

P84 Board Number: B184

Collapsin Response Mediator Protein-1 as a novel factor modulating Arp2/3dependent actin structures. H. Yu-Kemp1, W.M. Brieher1; 1 Cell and Developmental Biology, University of Illinois-Urbana-Champaign, Urbana, IL Listeria monocytogenes is a parasite that uses host proteins to assemble an Arp2/3-dependent actin comet tail for its propulsion. Comet tail assembly is more efficient in cytosol than in defined systems implying that unknown factors contribute to this reaction. We fractionated bovine brain cytosol and identified Collapsin Response Mediator Protein-1 (CRMP-1) as such a factor. CRMP-1 has long been studied as a factor for microtubule dynamics. Here, we show that CRMP-1 contributes to Arp2/3dependent actin dynamics. Imuunodepletion of CRMP-1 from brain cytosol reduced Arp2/3-dependent actin assembly around Listeria, and the defect could be successfully rescued with recombinant CRMP-1. In vitro assays confirmed that CRMP-1 enhanced Arp2/3-dependent branching and stimulated ActA- as well as VCA-activated Arp2/3-dependent actin polymerization. To test the contribution of CRMP-1 to Arp2/3-dependent polymerization inside cell, we examined the localization of CRMP-1 using immunostaining and GFP-CRMP-1 overexpression in epithelial cells. CRMP-1 localized to the leading edge of lamellipodia and cadherin-mediated cell-cell contacts, both of which are known to be Arp2/3dependent structures. Knockdown and overexpression approaches revealed a positive correlation between the amount of CRMP-1 and the amount of F-actin inside the cell. In wound closure assays, CRMP-1-depleted cells had twice less protrusions at the wounded edge, with half actin intensity at the leading edge compared to control cells; while overexpression of CRMP-1 provided the opposite results. During wound closure, CRMP-1-depleted cells moved erratically and lost their directionality with unstable membrane protrusions. In contrast, GFP-CRMP-1 overexpressing cells moved unidirectionally, with the membrane at the leading edge stably protruding forward. Depletion of CRMP-1 also reduced the amount of cadherin and actin at cell-cell contacts, resulting in weaker cell adhesion for epithelial cells. Immunobloting results indicated CRMP-1 present in the membrane fraction of cadherin enriched plasma membrane. This membrane had been shown to be able to assembly actin filaments in an Arp2/3-dependent manner. An antibody against CRMP-1 blocked this Arp2/3-dependent reaction, showing the role of CRMP-1 on regulating Arp2/3-dependent actin assembly at cadherin-mediated cellcell contacts. In summary, our results identify CRMP-1 as a novel regulator of the actin cytoskeleton that specifically contributes to Arp2/3-dependent actin assembly. CRMP family proteins have been studied in regulating metastasis and neuronal growth cone guidance. Our data provide a mechanism for understating how CRMP proteins might modulate those biological events through their potential role in actin cytoskeletal organization.

SUNDAY-POSTER PRESENTATIONS

P85 Board Number: B185

Isoform diversity modulates the ability of the Arp2/3 complex to stimulate actin polymerization. J.V. Abella1, C. Galloni1, M. Howell2, M. Way1; 1 Cell Motility Group, London Research Institute - Cancer Research UK, London, UK, 2High Throughput Screening Laboratory, London Research Institute, London, United Kingdom The Arp2/3 complex is unique among actin nucleators in its ability to generate branched actin filament networks. These networks are critical in maintaining the architecture of the cell and regulating cell motility, invasion, endocytosis and phagocytosis. A number of intracellular pathogens, such as Listeria, Shigella and Vaccinia virus also use the Arp2/3 complex to stimulate actin polymerization to enhance their cell-to-cell spread. The Arp2/3 complex consists of 7 subunits (Arp2, Arp3 and ARPC1-5), which are evolutionary conserved from protozoa to mammals. Interestingly, in humans, Arp3, ArpC1 and ArpC5 have two isoforms that are 91, 67 and 67% identical respectively. This raises the possibility that Arp2/3 complexes with different properties may exist. We found that the each isoform of Arp3, ARPC1 and ARPC5 is incorporated into an Arp2/3 complex and recruited to vaccinia induced actin tails. However, specific combinations differentially regulate actin-based motility of the virus. Depletion of ARPC1A and ARPC5, individually or together, results in longer actin tails. Conversely, loss of ARPC1B and/or ARPC5L results in very short actin tails. Loss of Arp3 versus Arp3B also results in opposite actin-tail phenotypes. Importantly, we demonstrate that isoform-mediated differences in actin tail length are dependent on both the rates of actin polymerization and filament disassembly. FRAP analysis, however, reveals that recruitment and turnover of each isoform on the virus is identical. This suggests that the activity of the Arp2/3 complex depends on its subunit composition. These differences are not unique to vacciniainduced actin polymerization. For example ARPC5L but not ARPC5 is essential for invadopodia formation and gelatin degradation. Moreover, Arp2/3 complex subunit composition also impacts on the rate of cell migration. Our observations now demonstrate that the Arp2/3 complex can no longer be considered as a unique functional assembly.

SUNDAY-POSTER PRESENTATIONS

P86 Board Number: B186

Coronin 7 and its role in organizing the actin cytoskeleton. K. Bhattacharya1, K. Swaminathan2, A. Noegel1,3,4,5, R. Rastetter6; 1 Institute for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany, 2Beatson Institute for Cancer Research, University of Glasgow, Glasgow, Scotland, 3Cologne Center for Genomics (CCG), Cologne, Germany, 4Center for Molecular Medicine Cologne (CMMC), Cologne, Germany, 5Cluster of Excellence, Cellular Stress Responses in Aging-Associated Disease, Cologne, Germany, 6Department of Anatomy and Developmental Biology, Monash Univ, Melbourne, Australia Coronins are F-actin binding proteins which belong to an evolutionary conserved family of WD40-repeat proteins. They are widely expressed in cells and tissues and are involved in signal transduction, transcriptional regulation, remodeling of the cytoskeleton and regulation of vesicular trafficking. Here, we focus on mammalian Coronin 7 (CRN7), the only `long´ coronin known in mammals, having two WD40-repeat domains which form a highly symmetrical tandem β-propeller. CRN7 localizes to the Golgi apparatus where it has a role in protein trafficking and maintenance of Golgi morphology. However, the role of CRN7 in cytoskeleton remodeling has not been addressed so far. Therefore, we have generated a CRN7 knockout (KO) mouse model and established primary fibroblast cultures to address the cellular functions and regulatory mechanisms involved with respect to the dynamics of the actin cytoskeleton. Analysis of CRN7 KO cells in vitro revealed that primary CRN7 KO fibroblasts exhibited a disrupted Golgi architecture and altered Golgi-centrosome positioning compared to the wild type (WT) counterpart. Importantly, fibroblasts deficient of CRN7 exhibited a faster cell polarization, an increased cell migration and an accelerated wound closure. Additionally, we detected a higher F-actin content in the KO fibroblasts. Re-expression of CRN7 reversed the Golgi-related phenotypic deficits in the KO fibroblasts. In line with the recently published data that a Cdc42- and Rac-interactive binding (CRIB) domain mediates functions of coronin (corA) in Dictyostelium discoideum, we likewise study here the significance of the CRIB motif present in CRN7 on both β-propellers. We found that CRN7 has a higher affinity for GDP-bound GTPases than for the corresponding GTP-bound GTPase and therefore, we draw our focus on investigating the downstream effectors of CRN7-mediated regulation of small RhoGTPases. Taken together, this study will provide an insight into the role of mammalian CRN7 as an organizer of actin-dependent cellular events.

SUNDAY-POSTER PRESENTATIONS

P87 Board Number: B187

Coronin organizes actin filament polarity required for directional gliding motility in the malaria parasite. M. Olshina1,2, K. Bane3, F. Angrisano1,2, S. Lepper3, J. Kehrer3, A. Dufour4, Y. Tan1,2, D. Marapana1,2, L. Whitehead1, W. Wong1, D. Riglar1,2, B. Catimel5, D.R. Kovar6,7, F. Frischknecht3, J. Baum8; 1 Walter and Eliza Hall Institute of Medical Research, Parkville, Australia, 2Department of Medical Biology, University of Melbourne, Parkville, Australia, 3Department of Infectious Diseases, University of Heidelberg Medical School, Heidelberg, Germany, 4Quantitative Image Analysis Unit, Institut Pasteur, Paris, France, 5Royal Melbourne Hospital, Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Parkville, Australia, 6Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL, 7Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 8Department of Life Sciences, Imperial College London, London, United Kingdom Actin-dependant processes such as cell motility rely on the appropriate control and regulation of dynamic filamentous actin. Coronins are a conserved family of regulators found across all eukaryotes and are involved in the coordination of F-actin dynamics in motile cells. Different coronins display varying functions, including binding directly to F-actin and crosslinking filaments, modulating F-actin assembly and branching via interactions with the Arp2/3 complex, as well as by mediating F-actin disassembly by coordinating ADF/cofilins. Many eukaryotic genomes encode several types of coronins, while others such as Plasmodium encode only one. The entire actin regulatory network in Plasmodium is drastically reduced, consisting of only a handful of members, and strikingly no Arp2/3 complex. Here we determine the function of Plasmodium coronin in the human and murine malaria parasites Plasmodium falciparum and P. berghei. We demonstrate that Plasmodium coronin is able to bind and organize actin filaments in vitro into parallel-bundled actin cables and bind to the membrane phospholipid phosphatidylinositol (4,5) bisphosphate. In vivo Plasmodium coronin localises to the cell periphery and relocalizes during actin-dependent fast movement. Finally, knockout of coronin in P. berghei results in aberrant fast movement altering mosquito-stage sporozoite movement from circular to erratic. These combined data suggest Plasmodium coronin is a critical part of the organizing architecture of directional force generation required by malaria parasite motility.

SUNDAY-POSTER PRESENTATIONS

P88 Board Number: B188

PI(3,5)P2 controls branched actin dynamics at late endosomes by regulating cortactin-actin interactions. N. Hong1, A.M. Weaver1,2; 1 Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, 2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of the phosphoinositide (PI) PI(3,5)P2 and demonstrate that their interaction promotes turnover of late endosomal actin assemblies. In vitro biochemical studies indicate that cortactin binds PI(3,5)P2 via the actin filament-binding region of cortactin. Furthermore, actin filaments and PI(3,5)P2 compete for binding to purified cortactin, suggesting that formation of PI(3,5)P2 on endosomes may remove cortactin from endosome-associated branched actin networks. Indeed, inhibition of PI(3,5)P2 production led to accumulation of both cortactin and actin on Rab7-positive endosomes. Knockdown of cortactin reversed inhibitor-induced actin accumulation at Rab7-positive endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

P89 Board Number: B189

The small GTPase ARF1 regulates podosome assembly. N.B.M. Rafiq1,2,3, Z. Z. Lieu 1,C. Yu1, A. Krishnasamy1, G. E. Jones3, A.D. Bershadsky1,2,4; 1Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore; 2Department of Biological Sciences, National University of Singapore, Singapore; 3Randall Division of Cell & Molecular Biophysics, King's College London, London SE1 1UL, UK; 4Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel The small GTPase ARF1 regulates podosome assembly Nisha Bte Mohd Rafiq1,2,3, Zi Zhao Lieu 1,Cheng-han Yu1, Anitha Krishnasamy1, Gareth E. Jones3, Alexander D. Bershadsky1,2,4 1

Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore

2

Department of Biological Sciences, National University of Singapore, Singapore

3

Randall Division of Cell & Molecular Biophysics, King's College London, London SE1 1UL, UK

SUNDAY-POSTER PRESENTATIONS 4

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel

Podosomes are integrin-mediated adhesion structures often formed by cells of the monocytic lineage, as well as by other cell types, including Src-transformed fibroblasts, and play an important role in cell migration and matrix degradation. Podosomes are multiple micron-sized radially symmetrical structures consisting of a central actin core associated with an “adhesive ring” containing the integrins and associated plaque proteins. Actin polymerization mediated by Arp2/3 complex is critically important for podosome formation, while upstream signaling pathways involving Rho GTPases(in particular Cdc42), are known, otherwise they are poorly understood. Here, we demonstrate that small GTPase ARF1, known to regulate the trafficking of COPI vesicles, is a potent regulator of podosome dynamics. We showed that down-regulation of ARF1 by siRNA led to a striking reduction in the number of podosomes in macrophage-like THP1 cells. In parallel, treatment of cells with Brefeldin A (BFA), a drug that targets a subset of Sec7-type GTP exchange factors (GEFs) of Arf1 led not only to dissociation of the Golgi apparatus but also to loss of podosomes in a dosedependent manner. Podosome “rosettes” often observed in Src-transformed fibroblasts were also shown to undergo gradual disruption after addition of BFA. Furthermore, we found that inhibition of another subclass of ARF GEFs, cytohesins, by a small-molecule inhibitor SecinH3 led to disassembly of podosomes in both THP1 cells and Src-transformed fibroblasts. Accordingly, RNAi silencing of ARNO (Cytohesin-2), but not Cytohesin-1, led to a dramatic reduction in podosome number. Even though bulk of ARF1 is localized to Golgi apparatus, TIRF microscopy of fluorescently-tagged ARF1 revealed punctate structures localized to regions of podosome assembly; these ARF1 puncta were also co-localized with Rab11-positive vesicles. Remarkably, ARNO was found to co-localize with components of adhesive ring in individual podosomes. Finally, in immunoprecipitation experiments, ARF1 was found to interact with the Arp2/3-inhibitory protein, arpin, which in turn was shown to co-localize with the actin core of podosomes. We conclude that ARNO-ARF1 signaling is strongly involved in the regulation of podosome assembly, most probably via modulation of Arp2/3-dependent actin polymerization.

P90 Board Number: B190

Solo plays regulatory role in astrocyte migration through activation of both Rac1 and RhoA. K. Van Vlasselaer1, G.E. Plopper2; Rensselaer Polytechnic Inst, Troy, NY, 2Rensselaer Polytech Inst, Troy, NY

1

Astrocytic migration plays a crucial role both in normal fetal development as well as in abnormal astrogliosis and traumatic spinal cord injury. Rho GTPases, specifically Rac1 and RhoA, have been shown to be crucial to normal astrocyte migration though signaling nuances have yet to be elucidated. GTPase activity is spatiotemporally regulated through function of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) to activate or inactivate GTPases respectively. One such GEF, Solo, has been described as a membrane associated Trio isoform involved in neurite elongation (Aoki,

SUNDAY-POSTER PRESENTATIONS 2006) and has been shown to be upregulated during mouse astrocyte migration. While Trio, the rat and human isoform, contains both GEF1 (activates Rac1 and RhoG) and GEF2 domains (activates RhoA), Solo contains only the GEF1 domain. Preliminary data indicates that Solo preferentially co-localizes with Rac1, and that siRNA Solo knock down induces altered astrocyte migration and cellular morphology. The interaction of Solo with both Rac1 and RhoA could indicate a more complex role in astrocytic migratory signaling than previously thought.

P91 Board Number: B191

Rho GTPase effector targeting during single cell wound healing. A. Moe1; 1 University of Wisconsin-Madison, Madison, WI Wounding of single cells triggers formation and closure of an actomyosin ring around the wound. Formation of this ring reflects activation of the small GTPases, Rho and Cdc42, in concentric zones around the wound. However, the links between GTPase zones and the cytoskeleton are not well characterized. Here I have connected the Rho GTPases and cytoskeleton through analysis of the localization of Rho GTPase effectors at wounds. To determine the localization of effectors, Xenopus laevis oocytes were injected with mRNA encoding fluorescently labeled effectors and markers for active Rho/Cdc42 and were imaged using confocal microscopy and laser wounding. To analyze the localization patterns of effectors a new method was used to create circle scans, which are a modified line scan that encompasses 360 degrees of zone information. While some effectors such as P21 activated kinase 2 (Pak2) localize throughout the entire Cdc42 zone during wound healing, other effectors change localization over time towards the leading edge of the Cdc42 zone as healing progresses. Neural Wiskott-Aldrich syndrome protein (NWASP), which activates the Arp2/3 complex to nucleate branched actin filaments, and Transducer of Cdc42 dependent actin assembly 1 (Toca1), which is necessary for full activation of NWASP, show a slight change in localization over time towards the leading edge of the Cdc42 zone. Slingshot Phosphatase, which activates cofilin to sever actin filaments, localizes to the leading edge of the Cdc42 zone to a greater extent than NWASP or Toca1. Eliminating the ability of Toca1 to bind Cdc42 inverts the localization of Toca1 such that it localizes to the trailing edge and outside of the Cdc42 zone instead of the leading edge. Cofilin shows the most prominent shift in localization over time towards the leading edge of the Cdc42 zone. LIM kinase (LIMK1), a Rho and Cdc42 effector that inhibits cofilin, localizes throughout the Rho and Cdc42 zones with a peak in the Rho zone. Formin-like 1, an unbranched actin nucleator, localizes throughout the Cdc42 zone and in the Rho zone. Rho associated kinase (ROCK), a Rho effector that activates Myosin 2, localizes to the Rho zone. The fact that different effectors show different patterns of localization during wound healing suggests that there is subcompartmentalization within a Rho GTPase zone. This ultimately leads to the idea that similar to gradients of information leading to morphogenesis, a single cell is also capable of pattern formation using gradients of information to close a wound.

SUNDAY-POSTER PRESENTATIONS

P92 Board Number: B192

Mechanosensitivity of actin assembly maintains homeostasis in the stress fiber network. S. Wang1, S. Thiyagarajan1, M.A. Smith2, E. Blankman2, L.M. Chapin2, M.C. Beckerle2, B. O'Shaughnessy3; 1 Department of Physics, Columbia University, New York, NY, 2Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, 3Department of Chemical Engineering, Columbia University, New York, NY The cytoskeleton is a complex network subject to constant remodeling. A major component is the network of actomyosin stress fibers. Even in cells at rest subject to no external perturbations, the network undergoes continuous and apparently stochastic adjustments as stress fiber sarcomeres change length in an apparently random fashion. The origin of these fluctuations, and the mechanism that cells employ to continuously remodel actin to accommodate these length changes and maintain network homeostasis are unknown. Here we systematically measured the statistics of stress fiber fluctuations in mouse embryonic fibroblasts stably expressing zyxin-GFP and other fluorescently tagged stress fiber components. These statistics are a constant of the network and define its stochastic dynamics, even though the network itself is in constant flux. Using zyxin-GFP to identify sarcomere boundaries from time-lapse confocal microscopy images, we measured the temporal statistics of sarcomere length over many sarcomeres and stress fibers, following individual sarcomeres for ~1000 s. Statistical analysis revealed an early phase (less than ~50 s), when sarcomere length changed randomly in time with an effective length ‘diffusion constant’ Dearly ~ 1000 nm2/s, and a late phase (greater than ~50 s) with a smaller diffusion constant Dlate ~ 200 nm2/s. This remarkable feature was reproducible over many cells and stress fibers. To explain this behavior we developed a mathematical model attributing the stochasticity to myosin-II, the principal driving force for sarcomere length changes. Fluctuations in myosin-II force are inevitable due to turnover, variations in phosphorylation and the fraction of heads bound to actin, etc. We hypothesized that actin disassembly (assembly) is mechanosensitive, steered by elastic stresses which grow (decrease) when sarcomeres shorten (lengthen) and actin filaments are forced to overlap (pulled apart). This mechanism quantitatively reproduced the observed behavior: (i) The early phase emerged as the initial sliding of actin filaments at fixed length as a sarcomere shortens or lengthens. This resets the degree of overlap, and the net actin assembly rate that depends on this overlap. (ii) In the late phase, filaments continuously adjust length in phase with the changing sarcomere length. Both phases show linear time dependence of the mean square change in sarcomere length, as seen experimentally. Best fit model parameters were remarkably close to those obtained from our previous study of spontaneously severing stress fibers. Thus, mechanosensitivity of actin assembly allows cells to continuously remodel the stress fiber network and to maintain network homeostasis.

SUNDAY-POSTER PRESENTATIONS

P93 Board Number: B193

α-Actinin modulates actomyosin network contractile response. H. Ennomani*1, G. Letort*1, C. Guérin1, R. Boujemaa-Paterski1, F. Nédélec2, E. De La Cruz3, M. Théry4, L. Blanchoin1; 1 iRTSV, CEA, Grenoble, France, 2EMBL, Heidelberg, Germany, 3Yale University, New Haven, CT, 4iRTSV, CEA, Paris, France Cellular contractility – the internal generation of force or tension by a cell orchestrated by the actomyosin machinery – has emerged as a critical regulator of a wide range of processes during development. Tremendous efforts have deepened our understanding of the biochemical and mechanical properties of the actomyosin contractile units encountered in the complex cell environment. Yet, how the structural organization of actin filaments regulates myosin-driven contraction remains to be established. We have recently developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays (Reymann et al., 2010). These actin templates were used to evaluate the response of oriented actin structures to myosin-induced contractility in presence of regulatory proteins. We demonstrated that crosslinking level modulates actin network deformation and/or disassembly upon myosin-induced tension. We showed also that crosslinkers are necessary to sustain myosin-driven deformation and force production of dynamic actin networks in the presence of disassembly factors (ADF/cofilin). In addition, we developped numerical simulation in order to relate the observed myosin-driven actin deformation with the underlying microscopic mechanism. Because we are working with a reconstituted system for which we control the composition and the geometrical conditions, we are in a very good position to study the fundamental rules by which myosinmediated contractility produce force.

P94 Board Number: B194

Mechanisms of actomyosin contraction during embryonic wound repair in Drosophila. A. Kobb1, R. Fernandez-Gonzalez1,2,3; 1 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, 2Department of Cell and Systems Biology, University of Toronto, Toronto, ON, 3Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON The coordination of cellular behaviors is critical during embryonic development, cancer metastasis and tissue repair. Coordinated cell movement is mediated by networks of actin and the motor protein nonmuscle myosin II that span multiple cells. In particular, during embryonic wound repair, the cells

SUNDAY-POSTER PRESENTATIONS adjacent to the wound accumulate actin and myosin at the interface with the wounded cells. Actomyosin redistribution results in the assembly of a supracellular actomyosin cable around the wound whose contraction acts as a purse string and drives wound closure. However, the mechanisms of actomyosin recruitment to the wound margin and purse string contraction are not well understood. The mechanisms of actomyosin contraction have been investigated during cytokinesis, the final step of cell division. During cytokinesis, a contractile actomyosin ring separates the daughter cells, and actin depolymerization, but not polymerization, plays a central role in the contraction of the ring. To investigate actomyosin dynamics during embryonic wound repair, we used a high-power ultraviolet laser to irradiate and create wounds in the epidermis of Drosophila embryos expressing fluorescentlytagged filamentous actin or myosin. We used Fluorescence Recovery After Photobleaching (FRAP) to quantify actomyosin dynamics at the wound margin and in cells far from the wound, during purse string assembly (2 min after wounding) and contraction (4 min after wounding). Our results showed that filamentous actin fluorescence rapidly recovered to 50% of its initial value within 22.5 ± 6.5 seconds during purse string assembly, and within 16 ± 4 seconds during contraction. Using image analysis we found that total actin levels at the purse string remained relatively constant throughout wound closure. Together, these data suggest that, unlike in cytokinesis, actin polymerization takes place during wound closure. Using FRAP, we found that myosin fluorescence at the purse string had a1.5-fold greater mobile fraction compared to non-contractile myosin cables away from the wound, consistent with a mechanisms in which actomyosin filaments slide with respect to each other to promote contraction. We are currently integrating these data into a computational model to investigate actomyosin dynamics during purse string contraction. This model will allow us to make predictions about the role of different actomyosin regulators that we will then test experimentally to understand the molecular mechanisms by which cells coordinate their migration during embryonic wound repair.

P95 Board Number: B195

Myosin light chain kinase activity regulates front-back polarity during cell migration by modulating protrusion lifetime. S. Lou1, J.A. Theriot2,3; 1 Chemical and Systems Biology, Stanford University, Stanford, CA, 2Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 3Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA Rapidly motile cells such as neutrophils and keratocytes typically polarize to generate a single front and back in order to sustain productive forward movement. However, other less persistently motile cell types, such as fibroblasts and epithelial cells, frequently have multiple, apparently independent, protrusive fronts. The mechanisms that limit polarization to a single front and back for rapidly motile cells, and how those mechanisms differ for slow-moving cell types are not fully understood. Here we develop embryonic zebrafish keratocytes as a model system for keratocyte cell biology, and use these cells to investigate how a purely lamellipodial cytoskeleton self-organizes to regulate the number of

SUNDAY-POSTER PRESENTATIONS protrusive fronts and overall cell polarity. Specifically, we have observed that keratocytes isolated from 4 days post-fertilization (dpf) embryos often form multiple protrusive fronts, suggesting that the motility machinery in these cells polarizes differently from single-front keratocytes such as those found at 2dpf or in adults. Using a combination of genomic, genetic, and pharmacological approaches, we identify increased myosin light chain kinase (MLCK) expression in 4dpf keratocytes as a causative agent in enabling the formation of multiple protrusions. We show that MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which decreases protrusion lifetime, thus limiting their size. These results suggest that rapidly motile cells may be able to polarize to a single front and back because they normally have less myosin activity than many slow-moving cell types

P96 Board Number: B200

CARMIL2 localizes to vimentin filaments and leading-edge membrane-ruffles in migrating cells. M.H. Lanier1, J.A. Cooper1; 1 Cell Biology and Physiology, Washington University in St. Louis, Saint Louis, MO Although recent studies have demonstrated that vimentin can regulate lamellipodia formation, the mechanisms through which vimentin influences actin assembly are not well understood. However, CARMIL2 is a strong candidate to play a key role. Previous studies have shown that CARMIL2 localizes to vimentin filaments in cells and regulates capping protein (CP), a key regulator of actin networks, in actin polymerization assays. Additionally, we have identified a membrane-localizing region near the Cterminus of CARMIL2 that is both necessary and sufficient for localization to leading-edge membraneruffles and the membrane surrounding macropinosomes. CARMIL2 knockdown cells exhibit migration defects, diminished lamellipodial ruffling and macropinocytosis, and possess a striking multi-protrusion phenotype. How the CARMIL2 localization pattern relates to CARMIL2 function and the importance of CP regulation by CARMIL2 for cell migration are not established. We hypothesize that CARMIL2 regulates polarity during cell motility by regulating CP and coordinating actin filament dynamics with the vimentin network. Here, we report live-cell dynamics of CARMIL2 on vimentin filaments and membraneruffles. Additionally, we have created CARMIL2 vimentin-localizing mutants and CARMIL2 membranebinding mutants. In the future, we will test the ability of these mutants to rescue the CARMIL2 knockdown phenotype.

SUNDAY-POSTER PRESENTATIONS

Myosin Motors 1 P97 Board Number: B202

Structural determinants of myosin-I mechanosensing: the N-terminal region. M.J. Greenberg1,2, T. Lin1,2, H. Shuman1,2, E.M. Ostap1,2; 1 Pennsylvania Muscle Institute, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, 2Department of Physiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA Myosins are actin-based motors that are mechanically and kinetically tuned to function in a wide range of cellular processes. The myosin-I family members Myosin-IB (Myo1b) and Myosin-IC (Myo1c) have similar structures and ATPase kinetics. However, when mechanical loads are placed on the motors, they display very different behaviors. The ATP-dependent, actin-detachment kinetics of Myo1c are relatively unchanged when the myosin powerstroke is resisted by loads of less than 2 pN, so it is able to generate power and act as a molecular transporter. However, the ATP-dependent, actin-detachment kinetics of Myo1b slow dramatically under similar loads, enabling it to act as a tension-sensing anchor. We recently determined the high-resolution structure of the Myo1b motor domain and found the N-terminal region (NTR) in a conformation that has not yet been observed in other myosins. Because the NTR sequence is highly variable and alternatively spliced within the myosin-I family, we investigated whether the NTR plays a role in tuning the mechanochemical properties of myosin-I motors. We generated recombinant constructs of Myo1b and Myo1c in which their NTRs were deleted or swapped, and we characterized their kinetic and mechanochemical properties using stopped-flow techniques, single molecule optical trapping techniques, and in vitro actin-gliding assays. Our results show that the NTR does indeed play an important role in tuning the mechanochemical properties of Myo1b and Myo1c. Moreover, our results demonstrate differences in how the NTR tunes Myo1b and Myo1c, suggesting that the sequence diversity and alternative splicing of this region may play an important role in generating diversity of myosin-I function in the cell. This work was supported by a grant to EMO from NIH (GM057247).

P98 Board Number: B203

Functional analysis of disease-associated mutations in myosin I using fission yeast as a model system. J. Bi1, R.T. Carroll1, M.L. James1, J.L. Ouderkirk1, M. Krendel1, V. Sirotkin1; Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY

1

Myosin 1e (myo1e) is a mammalian non-muscle myosin, which is expressed in the renal filtration unit (the glomerulus) in humans and mice. We have previously shown that Myo1e knockout mice have defects in renal filtration and glomerular ultrastructure that are similar to those observed in patients

SUNDAY-POSTER PRESENTATIONS with kidney disease called focal segmental glomerulosclerosis (FSGS). Recent genetic studies have also identified point mutations in the MYO1E gene in patients with FSGS. However, the precise relationship between the point mutations found in FSGS patients and the functional activity of myosin I remains to be determined. Using fission yeast S. Pombe as a versatile tool for analyzing myosin functions, we were able to directly test the effects of FSGS-associated mutations on myosin activity. Fission yeast have a single myosin I, Myo1p, which is associated with yeast actin patches, which are branched actin networks assembling at the sites of endocytosis. In the absence of Myo1p, yeast exhibit defects in cell growth under high salt and high temperature conditions and severe endocytosis defects. Taking advantage of the conservation of motor domains between human Myo1e and S. pombe Myo1p, we generated mutations in the Myo1p motor domain that were equivalent to FSGS-associated mutations in Myo1e. We found that these mutations in Myo1p resulted in the defects in yeast growth and endocytosis similar to those observed in the Δmyo1p strain. Fluorescently tagged Myo1p was used to examine myosin localization relative to actin patch marker, fimbrin. Mutations in Myo1p disrupted its localization to actin patches and resulted in relocation of Myo1p to membrane-associated protein complexes known as eisosomes. Finally, to assess the stability and folding of Myo1p mutants, we compared the localization of Myo1p to that of Rng3p, a member of UCS family proteins that acts as a myosin chaperone. Earlier studies have demonstrated that mutations in the Myo1p motor domain can promote recruitment of Rng3p to Myo1p, likely due to the decreased stability of the Myo1p. We hypothesized that Myo1p mutants that are nonfunctional due to defective folding would also interact with Rng3p and recruit it to Myo1p mutant containing structures.

P99 Board Number: B204

Role of Myo1 C-terminal domains in Myo1 localization and regulation of actin patch dynamics. R.T. Carroll1, E. Oakes2, M.L. James1, V. Sirotkin1; 1 Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY, 2 LeMoyne College, Syracuse, NY Myosin-1 (Myo1), the sole type 1 myosin in Schizosaccharomyces pombe, is recruited to sites of endocytosis and its arrival is followed by Arp2/3 complex mediated branched actin assembly into structures known as actin patches. Myo1 has an N-terminal motor domain, a lever arm with two light chain binding motifs (IQ1 and IQ2), and a C-terminal cargo binding tail. The Myo1 tail consists of a phospholipid binding TH1 domain, a proline-rich TH2 domain, an SH3 protein interaction domain and a CA domain, which binds Arp2/3 complex. Previously, we found that disruption of IQ motifs led to decreased recruitment of Myo1 to endocytic sites and endocytosis defects, the severity of which correlated with the reduction in the amount of Myo1 at the endocytic site. To investigate the role of the Myo1 C-terminal domains in Myo1 localization and regulation of actin patch dynamics, we created a series of GFP-tagged and untagged myo1 mutants with increasingly long deletions starting from the Cterminus. Using genetic crosses we combined these mutants with mCherry-tagged fimbrin Fim1. We

SUNDAY-POSTER PRESENTATIONS found that deletion of CA did not have any effect on Myo1 localization and actin patch dynamics. Deletion of CA and SH3 domains resulted in a 30% reduction in peak levels of myosin compared to wild type, increased assembly time for fimbrin and a decreased efficiency of internalization. Deletion of CA, SH3 and TH2 domains further reduced myosin levels at the patch (60% reduction) with similar actin assembly and internalization defects as in SH3-CA deletion mutant. The TH2-SH3-CA deletion mutant accumulated to the same peak levels as the Myo1 mutant with an internal deletion of TH2, suggesting that the TH2 domain is a major contributor to Myo1 patch localization. Deletion of CA, SH3, TH2 and TH1 domains resulted in the loss of Myo1 localization to actin patches and endocytic internalization defects similar to those in cells with a complete myo1 deletion, suggesting that TH1 is required for Myo1 localization at the endocytic site. By crossing untagged C-terminal truncation myo1 mutants with GFPtagged ARPC5 subunit of the Arp2/3 complex, we observed trends in patch dynamics defects that were similar to defects observed with fimbrin. However, the defects were more severe since GFP-tagged Arp2/3 complex is only partially functional and is synthetically lethal with TH1-TH2-SH3-CA truncation or complete myo1 deletion. In myo1 tail truncation mutants, similar to cells depleted for Myo1 or yeast WASp homolog Wsp1, Arp2/3 complex accumulated at the slower rate but reached the same peak levels as in wild type. Since depletion of Myo1 also reduced accumulation of Wsp1 in endocytic patches, we propose that Myo1 plays a role in recruiting Wsp1, directly or indirectly, to the endocytic sites.

P100 Board Number: B205

Characterization of kidney disease associated mutations in myosin 1e. J. Bi1, C.D. Pellenz1, M. Krendel1; 1 Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY Focal segmental glomerulosclerosis (FSGS) is one of the most common primary glomerular disorders that may lead to end-stage kidney disease. One of the pathological features of FSGS is the presence of massive proteinuria (protein excretion in the urine), which results from defects in protein filtration by the renal filtration unit, the glomerulus. Pathogenesis of FSGS involves impaired function of glomerular podocytes, specialized epithelial cells that cover the surface of the glomerular capillaries. Myosin 1e (Myo1e) is a non-muscle myosin, which is expressed in kidney podocytes in humans and mice. We have previously found that Myo1e is a component of the specialized junctional complexes associated with cell-cell contacts between podocytes, and that mice lacking Myo1e develop proteinuria. Mutations in the MYO1E gene have also been found in patients with childhood familial FSGS. Based on these findings, recent sequencing studies focused on identifying FSGS-associated mutations have included MYO1E as one of the genes of interest. This resulted in identification of novel mutations in Myo1e whose effects on protein localization and function have not been determined. Using immortalized Myo1e-null cultured podocytes that were isolated from knockout mouse kidneys, we were able to examine localization and activity of the mutant Myo1e constructs. We introduced GFP-tagged Myo1e constructs into cultured podocytes using adenoviral vectors and studied their localization in live cells. We have also utilized live-cell imaging to visualize the recruitment of junctional components during cell-

SUNDAY-POSTER PRESENTATIONS cell contact assembly. Using this approach, we were able to directly demonstrate the pathogenic nature of some of these mutations, which result in mislocalization of Myo1e and defects in cell-cell junction assembly. The observed junctional defects may lead to the formation of “leaky” glomeruli in vivo.

P101 Board Number: B206

Myosin 1e role in regulation of breast cancer cell migration and invasion. J.L. Ouderkirk1, N.O. Deakin1, G.J. Goreczny1, C.E. Turner1, M. Krendel1; 1 Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY Cell migration contributes to many normal physiological processes such as embryogenesis and the immune response, but can also be pathological as seen with cancer cell invasion and metastasis. The actin cytoskeleton and its associated proteins, including myosin motors, play an important role in cancer cell migration. The aim of this study was to dissect how class I myosin 1e (Myo1e) regulates cancer cell migration. Myo1e includes a motor domain that interacts with actin filaments and a tail domain that contains membrane-binding and protein interaction motifs. Thus, it may act as a scaffold connecting actin filaments to the plasma membrane. Using an invasive breast cancer cell line MDA-MB-231 as a model, we found that shRNA-mediated knockdown of Myo1e reduced cell migration in a transwell assay compared to cells treated with scrambled shRNA. Myo1e knockdown also impaired cell invasion through Matrigel, a thin layer of matrix proteins coating a transwell filter. Observation of cells migrating towards EGF under a layer of agarose showed that control cells migrated further than Myo1e knockdown cells. Overall, these finding show that Myo1e is involved in cancer cell migration, especially in the settings where cells have to undergo complex shape changes, such as transwell migration and movement under a layer of viscous media. GFP-Myo1e localized to the outermost portion of the leading edge in migrating cells, and we are currently investigating whether it contributes to formation of protrusions at the leading edge. Based on our observations in vitro, we predict that Myo1e may also promote tumor cell migration in vivo. To test this hypothesis, we have generated Myo1e knockout mice expressing MMTV-PyMT oncogene. The MMTV-PyMT transgenic mouse is a well characterized mouse model of invasive breast cancer. We will use this model to examine how deletion of Myo1e affects breast cancer progression in vivo.

SUNDAY-POSTER PRESENTATIONS

P102 Board Number: B207

Controlling Initiation and Termination of Kinesin-1-Driven Transport with MyosinIc and Non-Muscle Tropomyosin. B.B. McIntosh1,2, E.L. Holzbaur1, E.M. Ostap1,3; 1 Department of Physiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, 2 Pennsylvania Muscle Institute, Philadelphia, PA, 3Pennsylvania Muscle Institute, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA Intracellular transport is largely driven by processive actin- and microtubule-based molecular motors. Non-processive motors have also been localized to trafficking cargos, but their respective roles during transport are not well understood. Myosin-Ic (Myo1c) is a non-processive actin motor that functions in a variety of exocytic events, yet the molecular roles of this motor during trafficking are unclear. To investigate the interplay between myosin-I motors and the canonical long distance transport motor kinesin-1, we attached both Myo1c and kinesin-1 motors to lipid-coated bead cargo. This is a more physiological cargo-motor attachment strategy that allows motors to actively reorganize within the membrane and respond to the cytoskeletal environment. We compared the motility of these cargos in the absence and presence of Myo1c motors at engineered actin filament-microtubule intersections attached to the surface of a coverslip. We found that Myo1c influences kinesin-1 run initiation by significantly increasing the frequency of microtubule-based runs that begin at actin filamentmicrotubule intersections. Myo1c also regulates run termination. Beads with both motors bound consistently pause at actin-microtubule intersections, remaining tethered for an average of 20 seconds, with pauses longer than 200 seconds also observed. Actin-binding proteins such as non-muscle tropomyosin have been proposed to positively or negatively regulate the interactions between specific myosin motors and actin filament populations in vivo. In vitro, we found that non-muscle tropomyosin-2 (Tm2) abrogates Myo1c-driven actin filament gliding. In the crossed filament assay, we found that Tm2 abolishes Myo1c-specific effects on run initiation and run termination. Together these observations suggest that within the cell, Myo1c is important for the selective initiation and termination of kinesindriven runs along microtubules at actin filament intersections. The regulation of Myo1c by tropomyosin provides a mechanism for regional specificity, preventing inappropriate pausing during long distance transport, while also enabling targeted delivery of cargo to highly exocytic, dynamic actin populations, beneath the plasma membrane.

SUNDAY-POSTER PRESENTATIONS

P103 Board Number: B208

Conserved Binding Partner of Vertebrate and Invertebrate Class III Myosins. M. Raval1, K. Mecklenburg 2, S. Freed3, O.A. Quintero4, J.E. O'Tousa2,3, C.M. Yengo5; 1 Pennstate College of Medicine, Hershey, PA, 2Indiana University, South Bend, IN, 3Biology, University of Notre Dame, Notre Dame, IN, 4Department of Biology, University of Richmond, Richmond, VA, 5 Pennsylvania State Univ Coll Med, Hershey, PA Class III myosins (MYO3A and MYO3B) are actin-based myosin motors which are proposed to function as transporters in actin rich structures such as the stereocilia of inner ear hair cells and calycal processes of photoreceptors (PRs). The first member of the MYO3 family was identified in Drosophila photoreceptors and called NINAC (Neither inactivation nor afterpotential C) based on its role in phototransduction. It was observed that NINAC null mutant flies undergo light and age dependent retinal degeneration. Previous work demonstrated that NINAC is found in a complex with a protein called retinophilin (RTP). In our current work we demonstrate that NINAC interacts with RTP via the proximal region of the tail domain, a region conserved in invertebrates. We also show that NINAC localization in the rhabdomere requires the presence of RTP. We find that the vertebrate homologue of RTP, membrane occupation nexus repeat protein (MORN4), interacts with MYO3A. We demonstrate that MORN4 gets transported to the filopodial tips of Cos7 cells in a MYO3A dependent manner. The presence of MORN4 enhances the MYO3A-induced elongation of filopodia. The domain structure of the MYO3A tail is encoded by 6 exons (30-35). Using tail exon deletion constructs we demonstrate that MYO3A tail exons 30-31 are the probable sites for MORN4 interaction. We also demonstrate that MYO3B which has a shorter tail (and lacks sequence similar to exon 30-31 of MYO3A tail) is unable to bind MORN4. We used expressed and purified GST labelled MORN4 to demonstrate that MORN4 specifically interacts with the MYO3A tail domain in GST pull down assays. The MYO3A tail domain exon 30 contains a proposed calmodulin (CaM)-binding IQ motif (IQ3) of unknown function. Thus, we hypothesized that Ca2+ may play a regulatory role in the MYO3A-MORN4 interaction. We observed a 30% increase in MORN4 tip localization in ionomycin treated Cos7 cells which were co-transfected with GFP-MYO3AΔK and mCherry-MORN4 as compared to control conditions. No significant change was observed in the MYO3A tip localization. Overall, our results suggest that the Retinophilin/NINAC interaction is conserved in vertebrate orthologs MORN4/MYO3A. In vertebrates we propose that MORN4 stabilizes MYO3A by tethering it to the membrane, thus allowing MYO3A to generate membrane tension and contribute to the process of elongation of actin rich protrusions.

SUNDAY-POSTER PRESENTATIONS

P104 Board Number: B209

The TAP-MYO5A mouse: a Tandem Affinity Purification tag knockin mouse for the isolation and identification of proteins that interact with myosin Va. C.J. Alexander*1, W. Wagner*2, N.A. Copeland3, N.A. Jenkins3, J.A. Hammer4; 1 NHLBI, NIH, Bethesda, MD, 2ZMNH/UKE, Hamburg, Germany, 3The Methodist Hospital Research Institute, Houston, TX, 4LCB, NIH/NHLBI, Bethesda, MD Myosin Va, a processive class V motor, is involved in the transport of diverse cargos, including melanosomes, endoplasmic reticulum (ER), and mRNAs. The creation of tools to identify proteins that interact with myosin Va should increase our understanding of the cellular processes supported by this myosin. Towards that end, we report here the generation of a tandem affinity purification (TAP) tag knockin mouse at the MYO5A locus. A recombineering-based approach was used to insert via homologous recombination a TAP-tag composed of the IgG binding domain of Protein A, a TEV cleavage site, and the FLAG epitope tag into MYO5A locus immediately after the initiation codon. Mice homozygous for the knockin allele, which express the TAP-tagged version of myosin Va (TAP-MyoVa) exclusively and under the control of the endogenous MYO5A promoter, exhibit normal coat color and no evidence of ataxia, arguing that TAP-MyoVa functions normally. Consistently, the dendritic spines of Purkinje neurons isolated from this mouse are fully loaded with ER, in contrast to the spines of Purkinje neurons from dilute (myosin Va null) mice, which are devoid of ER. Similarly, melanosomes are distributed normally in melanocytes from the TAP-MyoVa mouse, in contrast to melanocytes from dilute mice, where the organelles are concentrated in the cell center. Moreover, introduction of a CMV promoter-driven TAP-MyoVa construct into dilute melanocytes rescues melanosome distribution. Given this clear evidence that TAP-MyoVa is fully functional, we purified TAP-MyoVa and associated proteins directly from juvenile mouse cerebella excised from TAP-tagged mice and subjected the samples to mass spectroscopic analyses. Importantly, elutes contained several known myosin Va binding partners (Dynein light chain 1 and Neurofilament light chain), further verifying that TAP-MyoVa is fully functional. Moreover, we found numerous novel interacting proteins, including Annexin A2 and Elongation Factor 1A. The mouse model created here should facilitate the identification of novel myosin Va binding partners, which in turn should advance our understanding of the roles played by this myosin in vivo. *Authors contributed equally to this study

SUNDAY-POSTER PRESENTATIONS

P105 Board Number: B210

Filopod Extension in Dictyostelium Requires MyTH4-FERM Myosin Motor Activity. K.J. Petersen1, G.G. Luxton2, M.A. Titus2; 1 Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, 2Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN Filopodia are cellular protrusions composed of bundled parallel actin filaments that initiate from the cortex with roles in cell adhesion and signaling during directed cell migration. MyTH4-FERM myosins (including Myosin 7, 10 and 15) localize to the distal tips of filopodia, stereocilia, and microvilli. Myo10 is required for filopod growth in mammalian cells while Myo7 is essential for filopod growth in Dictyostelium. We performed live cell imaging using GFP-tagged Myo7 in null cells. Myo7 rescued filopod formation and localized to filopodia tips and actin-based pseudopods. In pseudopods, enrichment at the leading edge near the cell membrane preceded the growth of filopodia. Tip extension velocity remained constant throughout the growing phase independent of Myo7 accumulation at the tip. A construct lacking MyTH4-FERM domains but including the putative SAH domain (Myo7 Motor) failed to rescue filopodia and did not co-localize with cortical actin. Neither of the two FERM domains were essential for rescue of filopod formation. Deletion of FERM2 led to increased production of substrate-adhered filopodia suggesting FERM2 might negatively regulate Myo7 activity in vivo. We tested this by identifying conserved residues implicated in auto-inhibition of Myo10 and expressing a mutated Myo7 (KK2333,2336AA) that also showed increased production of filopodia relative to wild type Myo7. A mutant lacking the motor domain (Myo7 Tail) was enriched near the cell membrane but failed to rescue filopod formation. Neither FERM domain was essential for localization, although a mutant lacking both domains failed to localize. These results suggest that normal formation of filopodia requires membrane-localized Myo7 with precisely regulated motor activity. (This work is supported by an NSF grant to MAT)

P106 Board Number: B211

Myosin X Reveals Dynamics and Function of Basal Filopodia. D. Courson1, K. Liu1, A. Fanning1, R. Cheney1; 1 Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC Though often depicted as simple and flat, the basal surface of monolayers of polarized epithelial cells is actually seething with protrusive activity. Using live-cell, time-lapse TIRF microscopy we visualize the dynamics of the basal surface of stable and migrating mammalian epithelial monolayers. Previous work showed that epithelial cells in migrating sheets extend cryptic lamellipodia, in the direction of migration. In this report we show that polarized mammalian epithelial cells (MDCK and Caco2) also have filopodia on their basal surface. Basal filopodia are numerous during monolayer formation and maturation, and probe ahead of the lamellipodia during migration.

SUNDAY-POSTER PRESENTATIONS Basal filopodia have gone largely unnoticed, however we demonstrate that myosin X (Myo10) marks the tips of epithelial filopodia of cells in isolation and basal filopodia of cells in polarized sheets. In wound healing assays Myo10 puncta are planar polarized at the leading edge of cells found throughout the monolayer, from deep within the sheet out to the leading edge. Basal filopodia and Myo10 puncta become less prominent as the monolayer becomes quiescent but reappear within minutes of wounding. Myo10 and filopodia have both recently been shown to play roles in cancer metastasis. Our findings demonstrate that Myo10 and basal filopodia both directly affect cellular motility. We show that knocking down Myo10 results in the loss of basal filopodia and ~40% reduction in the rate of collective migration. A similar decrease in motility is observed by inhibition of filopodia via low dose cytochalasin. Interestingly, the difference in motility between wild type and Myo10 knockdown cells is lost upon treatment with low dose cytochalasin, consistent with the hypothesis that the loss of filopodia, rather than some other Myo10 function, is responsible for the observed motility defect in the knockdown cells. Ongoing work is examining the mechanism behind Myo10 recruitment, filopodia formation, and their roles in collective migration.

P107 Board Number: B212

Myosin 18A Co-assembles with Myosin II to Drive the Functional Diversity of Myosin II Bipolar Filaments. J.R. Beach1, N. Billington2, K. Remmert1, M. Barzik3, S.M. Heissler2, L. Shao4, D. Li4, T.B. Friedman5, E. Betzig4, J.R. Sellers6, J.A. Hammer1; 1 LCB, NIH/NHLBI, Bethesda, MD, 2NIH/NHLBI, Bethesda, MD, 3NIH/NIDCD, Bethesda, MD, 4HHMI/Janelia Farm Research Campus, Ashburn, VA, 5NIH/NIDCD, Rockville, MD, 6LMP, NIH/NHLBI, Bethesda, MD Bipolar filaments of nonmuscle myosin II (NMII) power myriad cellular and developmental processes. Here we provide evidence that myosin 18Aα (M18Aα) and myosin 18Aβ (M18Aβ), two M18A splice variants, co-assemble with NMII, opening the door to novel mechanisms of bipolar filament regulation. Structurally, M18Aα and M18Aβ resemble NMII with extra domains at their N- and C-termini. Specifically, both isoforms possess a non-helical tailpiece that contains interaction sites for SH3 and PDZ domain-containing proteins, while M18Aα also possesses a ~300 residue N-terminal extension containing a KE-rich region, a nucleotide-insensitive actin-binding site, and a PDZ domain. Importantly, both isoforms contain numerous substitutions in their active site and lack actin-activated ATPase activity, arguing that they are not motor proteins (Guzik-Lendrum et al., JBC, 2013). Using a combination of TIRF and structured-illumination microscopy (TIRF-SIM), which we used recently to show that NMIIA, IIB and IIC co-assemble into heterotypic bipolar filaments in live cells (Beach et al., Current Biology, 2014), we find clear evidence that M18Aα and M18Aβ (both expressed and endogenous molecules) coassemble with NMII in live cells in a variety of structures, including sub-nuclear stress fibers (SNSFs). Consistently, sedimentation, EM and single-molecule imaging show that purified M18A co-assembles with NMII in vitro. Incorporation of M18A into NMII bipolar filaments could serve to regulate filament assembly/disassembly. Indeed, excess M18A disrupts NMII filament assembly both in vitro and in live

SUNDAY-POSTER PRESENTATIONS cells. M18A levels in vivo are, however, markedly sub-stoichiometric to NMII (ranging from ~1:10 to ~1:100), and preliminary FRAP studies do not show an obvious effect of M18A depletion on NMIIA filament turnover. These results favor the idea that small numbers of M18A molecules dope NMII filaments to serve, via their extra N-and C-terminal domains, as adaptors to link the filament to cellular structures. Consistently, the targeting of M18Aα to SNSFs depends on its PDZ domain, suggesting that this domain links SNSFs to the nuclear envelope. M18A could also serve to recruit molecules to the NMII filament. Consistent with this idea, we show M18A’s non-helical tailpiece binds βPIX, a Rac GEF with roles in adhesion and protrusion, via βPIX’s SH3 domain. Finally, we identify two additional M18A splice variants (M18Aδ and M18Aγ) that possess unique N- and C-terminal domains. Collectively, our data argue that diversity of NMII function in higher eukaryotes is driven in part by co-assembly with various spliced isoforms of M18A, which serve as adaptors to link the filaments to different cellular structures and signaling molecules without interfering with NMII motor activity.

P108 Board Number: B213

Myosin 18A localizes with myosin II at cell: cell junctions in epithelial cells and tissues. K. Remmert1, J.R. Beach1, N. Porat-Shliom2, R. Weigert2, Y. Yang3, J.R. Sellers4, J.A. Hammer1; 1 LCB, NIH/NHLBI, Bethesda, MD, 2NIDCR, NIH, Bethesda, MD, 3Hunan Agricultural University, Changsha, China, 4LMP, NIH/NHLBI, Bethesda, MD Class 18A myosins (M18A) are a poorly understood subclass of myosin with a domain architecture similar to that of myosin II (MII). Specifically, both M18Aα and M18Aβ, two M18A isoforms generated by alternative splicing, consist of a “motor” domain followed by a short neck region, an extended coiledcoil domain, and a C-terminal non-helical tailpiece harboring binding sites for SH3 and PDZ domaincontaining proteins. Myosin 18Aα also possesses an N-terminal extension containing a KE-rich region, an ATP-insensitive actin-binding site, and a PDZ domain. Knockout of myosin 18A results in embryonic lethality in both mice and flies, suggesting a fundamental role in development. Despite their structural similarity to MII, M18A isoforms have no actin-activated ATPase activity and do not translocate actin filaments in vitro, suggesting that their functions do not require motor activity. Importantly, M18A and MII co-assemble via their extended coiled-coil domains into individual mixed bipolar filaments both in vitro and in vivo. This critical finding suggests that myosin 18A may serve to regulate MII filament turnover and/or act as an adaptor to link the filaments to different cellular structures/signaling molecules via its extra N- and C-terminal domains, all without interfering with MII motor activity. M18A is ubiquitously expressed across mammalian tissues, with elevated expression and isoform-specific expression in numerous cell types, including epithelia. In this study we determined the subcellular localization of M18A in polarized MDCK cell sheets and in cryo-sections of various mouse epithelia using a M18A-specific antibody. We find that M18A is concentrated at cell-cell junctions at the apical surface of polarized MDCK cells, a site where MII is known to be critical for maintaining the integrity of adherens junctions. Similarly, M18A is enriched in kidney proximal tubules and in intestinal brush border microvilli

SUNDAY-POSTER PRESENTATIONS specifically at cell: cell junctions where MII is also enriched. Finally, we find in secretory tissues such as the pancreas and salivary gland that M18A localizes along with MII onto the secretory granules after their fusion with the plasma membrane. Functional studies are underway to determine if M18A, like MII (Masedunskas et al, PNAS 2011), is required to facilitate the integration of the granular membranes into the apical plasma membrane, thereby completing the exocytic process. Together, our data suggest that M18A may be working together with MII to maintain the integrity of epithelia and to drive secretion in specialized tissues such as the pancreas and salivary gland.

P109 Board Number: B214

MYO19 ensures symmetric partitioning of mitochondria and coupling of mitochondrial segregation to cell division. J.L. Rohn1, J.V. Patel2, O.A. Quintero3, B. Baum2; 1 Centre for Clinical Science and Technology, University College London, London, United Kingdom, 2 Laboratory for Molecular Cell Biology, University College London, London, United Kingdom, 3 Department of Biology, University of Richmond, Richmond, VA During animal cell division, an actin-based ring cleaves the entire cell into two. Defects in this process can lead to chromosome mis-segregation, a hallmark of cancer, as well as to defects in cytoplasmic inheritance and the partitioning of organelles. Although a great deal is known about how chromosome segregation is coupled to the process of cell division, the way organelles coordinate their inheritance during partitioning to daughter cells is less well understood. Here, using a high-content live-imaging siRNA screen, we identify myosin-XIX (MYO19) as a novel regulator of cell division. Previously this actinbased motor was shown to control the interphase movement of mitochondria. Using live-cell imaging, our analysis shows that MYO19 is indeed localised to mitochondria, and that its silencing leads to asymmetric segregation of mitochondria during anaphase and telophase, as well as to defects in mitochondrial partitioning at cytokinesis. Moreover, MYO19 RNAi cells frequently fail to complete division. This cell division failure phenotype persists in cell lines stably expressing shRNA against MYO19, as these cell lines display a higher percentage of multinucleate cells, compared to controls. This phenotype appears to be due to the physical interference of cytokinesis machinery by mitochondria, as the phenotype can be mimicked using a treatment that blocks mitochondrial fission and rescued by decreasing mitochondrial fusion. The phenotype can also be rescued by exogenous expression of GFPMYO19 refractory to RNAi treatment. Taken together, these data support a model whereby the actinbased myosin motor, MYO19, is responsible for the faithful segregation of mitochondria during cell division, and highlights the importance of coupling organelle inheritance to cytokinesis.

SUNDAY-POSTER PRESENTATIONS

P110 Board Number: B215

Myosin XIX (Myo19) regulates actin-based mitochondrial dynamics. X. Hu1; 1 Department of biology, Institute of Molecular Cell Biology, Münster, Germany Mitochondria are highly mobile and dynamic organelles. They are involved in several important cellular functions, including energy production, metabolism, calcium buffering and apoptosis. Defects in their localization and dynamics lead to human disease. Myosin-XIX (Myo19) is a novel myosin in vertebrates that has been reported to function as an actin-based motor that associates with mitochondria. Myosins generally consist of a motor, neck and tail region. The tail region of Myo19 is necessary and sufficient for mitochondrial localization. However, at present it is not known how Myo19 associates with mitochondria and how it affects mitochondrial dynamics. We found that overexpression in HeLa cells of either the full length Myo19 or the Myo19 tail region resulted in dramatic alterations in mitochondria distribution, morphology and movement. Overexpression of Myo19 typically resulted in a collapse of mitochondria around the nucleus. Live cell imaging revealed that overexpression of Myo 19 induced in many continuously moving mitochondria a tadpole-like shape. Overexpression of the tail region of Myo19 caused a similar collapse of the mitochondria to the perinuclear region and the movement of individual mitochondria was hard to discern. The transport of mitochondria in dendrites and axons of neurons is particularly vital to meet local cellular energy demands and for buffering intracellular calcium. We found that mitochondrial length was significantly decreased in Myo19 overexpressing hippocampal neurons. No other differences were found between neurons transfected with RFP-Mito alone or RFPMito together with EGFP-Myo19 or EGFP-Myo19 tail regarding mitochondria density, velocity, run length, directionality etc. These results suggest that Myo19 is involved in regulating mitochondria morphology in neurons, but not transport. In conclusion, Myo19 regulates actin-based mitochondrial dynamics.

P111 Board Number: B216

Positively Charged Residues are Essential for Proper Localization of the MYO19 MyMOMA Domain to the Mitochondrial Outer Membrane. P.R. Mehta1, P.P. Singh1, J.L. Hawthorne1, N.Q. Wong1, O.A. Quintero1; 1 Department of Biology, University of Richmond, Richmond, VA Myosins are well characterized for their roles as molecular motors that convert the chemical energy of ATP hydrolysis into the mechanical force essential for cellular processes such as muscle contraction, cytokinesis, and intracellular transport. Class XIX myosin (MYO19) is an unconventional myosin found in organisms ranging from unicellular holozoans to mammals that contains a unique tail domain responsible for its interaction with mitochondria. The Myo19 mitochondria outer membrane association domain (MyMOMA) contains approximately 150 amino acids with a pI ~ 9, and is sufficient for

SUNDAY-POSTER PRESENTATIONS localization to the mitochondrial outer membrane. However, the minimal sequence required for mitochondrial binding and the specific amino acid residues required for mitochondrial targeting have not been identified. Here we report that a stretch of approximately 83 residues in the human MyMOMA domain is sufficient for mitochondrial outer membrane localization, and that two positively charged residues are essential for recognition of the mitochondrial outer membrane. We generated a suite of MyMOMA truncations based on sequence analysis of 13 vertebrate MYO19 orthologs, establishing the boundaries for truncations based on lack of sequence homology. The 83-amino acid minimal binding region is enriched for positively charged amino acids has a pI ~ 10.5. Endogenous MYO19 copurifies with mitochondria-enriched fractions via differential centrifugation, and can be released from those fractions by incubation in high pH buffer, suggesting that positively-charged residues may be involved in the MYO19 binding mechanism. We sequentially replaced positively charged residues in the minimal binding domain with uncharged residues via mutagenesis, identifying the specific residues required for interactions with the mitochondrial outer membrane, RK872-873. Interestingly, constructs containing the RK872-873AA mutation localized to the endoplasmic reticulum. To determine if ER-bound mutant MYO19 tail and mitochondria-bound wild type MYO19 tail display differences in strength of membrane interaction, we performed kinetic studies using permeabilization activated reduction in fluorescence (PARF) analysis. These studies indicated that both constructs display similar apparent off-rates. Taken together these data support the hypothesis that the MyMOMA domain contains a membrane-binding activity, and that membrane targeting is mediated through a yet-to-be determined mechanism requiring amino acids RK872-873.

P112 Board Number: B217

Characterizing the proximal protein interactions of Myosin-XIX (MYO19) via promiscuous biotin ligase. E.L. Schinski1, F. Yan2, M.B. Major2, O.A. Quintero1; 1 Department of Biology, University of Richmond, Richmond, VA, 22Department of Cell Biology Physiology, University of North Carolina, Chapel Hill, NC Myosin XIX (MYO19) is a motor protein that binds to mitochondria, and plays a role in the organization and dynamics of mitochondria. MYO19 consists of a motor domain, a 3 IQ motif-containing neck region, and a short tail. In conjunction with filamentous actin, the motor converts the energy released by ATP hydrolysis into useful force that is transmitted through the neck region to act upon the cargo-binding tail domain, thereby imparting force on the cargo. According to this model, there are multiple instances where interaction with other proteins is likely for MYO19 function, separate from the interaction with actin. We aim to elucidate the protein interactions of MYO19 by identifying proximal components and potential binding partners. To identify such interactors, we have created fusion constructs capable of tagging nearby proteins. By tagging MYO19 truncations with the promiscuous E. coli biotin ligase, BirA*/BioID, we are able to induce biotinylation of proximal cellular proteins. We hypothesize that the IQ motifs in the neck region bind EF-hand regulatory proteins such as calmodulin, and that the tail

SUNDAY-POSTER PRESENTATIONS domain interacts with proteins on the mitochondrial outer membrane. We first transfected HeLa cells with the BioID-MYO19 constructs, and incubated the cells in media containing biotin. We were able to observe expression of the MYO19/BioID constructs via fluorescence immunohistochemistry. Biotinylation was observed via immunostaining and anti-streptavadin blotting of HeLa cell lysates. Biotinylated proteins were isolated from lysates using magnetic strepavadin-coated beads, and the bound proteins processed via mass spectrometry. Through the analysis of these MYO19 associated proteins, we hope to better understand the cellular function of MYO19 by identifying proteins with which it may interact.

P113 Board Number: B218

Investigating the role of myosin 7b in intramicrovillar transport. M.L. Weck1, S.W. Crawley1, M.J. Tyska1; 1 Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN The intestinal brush border serves as the sole site of nutrient absorption within the body and also acts as an important barrier against luminal pathogens. The brush border is comprised of membrane protrusions called microvilli that are found on the apical surface of enterocytes. These protrusions are supported by a core bundle of 20 to 30 parallel actin filaments with the plus ends oriented towards the lumen. Recently, our lab showed that two protocadherins, protocadherin-24 and mucin-like protocadherin, play a key role in the organization of the brush border during its assembly. These protocadherins interact to form a trans-heterophilic adhesion complex that physically connects the distal tips of microvilli and regulates the tight packing of the brush border. However, the mechanism of how these complexes are targeted to the tips of microvilli remains unknown. The microvillar protocadherins interact with two cytoplasmic binding partners, harmonin, a scaffolding protein, and myosin 7b (myo7b), an unconventional myosin. We hypothesize that the motor protein myo7b is responsible for transporting these complexes to the plus end of microvillar actin bundles. To test this hypothesis, we generated an shRNA-mediated knockdown of myo7b in CACO-2BBE intestinal epithelial cells. Knockdown of myo7b results in reduced distal tip localization of the intermicrovillar adhesion complex (IMAC) components, as well as perturbations in microvillar clustering and brush border morphology. To further characterize the potential of myo7b as a transporter, we created a myo7b stable cell line using brush border-expressing LLC-PK1-CL4 kidney epithelial cells. Robust targeting of myo7b to the tips of microvilli occurs after several days of polarization. This localization is dependent on the tail domain as deletion of this region results in a loss of distal tip targeting. Previous studies of other myosins have indicated that cargo binding to the tail domain may promote motor processivity by inducing dimerization. To explore this possibility for myo7b, we engineered forced dimer constructs of the motor domain (MD) and full length myo7b using a GCN4 leucine zipper motif. The MD forced dimer targets in fully polarized LLC-PK1-CL4 cells, indicating that dimerization is sufficient for targeting and likely mediated by the tail domain through cargo binding. Additionally, we also examined how point mutations that disrupt motor domain function impact the distal tip targeting of myo7b. These data

SUNDAY-POSTER PRESENTATIONS suggest that active cycling and force generation are required for microvillar tip targeting. Taken together, these data indicate a role for myo7b in brush border formation through the localization of the IMAC to the tips of microvilli.

P114 Board Number: B219

Computational and experimental FRAP analyses of myosin XI-dependent vesicular transport show coupling with F-actin in polarized cell growth. J.P. Bibeau1, J.L. Kingsley2, F. Furt3, E. Tuzel2, L. Vidali3; 1 Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA, 2Physics, Worcester Polytechnic Institute, Worcester, MA, 3Biology/ Biotechnology, Worcester Polytechnic Institute, Worcester, MA Plant polarized cell growth is driven by the trafficking of secretory vesicles to the site of cellular expansion. Although the actin cytoskeleton and its associated motor myosin XI are heavily implicated in this process, little is known about their relative dynamics and binding properties in vivo. Here we applied fluorescent recovery after photo-bleaching (FRAP) techniques to the tip-growing cells of the moss Physcomitrella patens to investigate myosin XI dynamics and its relation to secretory vesicles. To complement our FRAP experiments, a three-dimensional Brownian motion simulation of FRAP was used to gain further insight into the dynamic behavior of myosin XI and vesicles. Our results clearly indicate that the dynamics of myosin XI at the cell apex differ significantly from those measured at the sub-apical region. Specifically, the rate of fluorescent recovery was found to be more rapid at the sub-apical region, indicting that a fraction of myosin XI is less mobile at the tip. To evaluate if the reduction on motility is dependent on F-actin, we depolymerized the actin using latrunculin B. This treatment significantly increased the mobile fraction of myosin XI at the cell apex, but not to the levels of the subapical region. To clarify this we used computer simulations that indicate that this discrepancy can be fully explained by cell boundary effects on the fluorescent recovery. In addition, our computer simulations have allowed us to estimate diffusion coefficients for myosin XI and vesicles, and at the same time estimate the fraction of myosin XI molecules associated with vesicles at the tip of the cell. Taken together our experimental results and simulation data support a model where myosin XI, secretory vesicles, and F-actin act cooperatively to drive polarized secretion and growth.

SUNDAY-POSTER PRESENTATIONS

P115 Board Number: B220

Biochemical characterization of the interaction between Rab proteins and myosin XI in the moss P. patens. S. Kaptur1, J. Garbarino1, M.L. Dubuke2, F. Furt1, E. Armstrong1, M. Munson2, L. Vidali1; 1 Biology/ Biotechnology, Worcester Polytechnic Institute, Worcester, MA, 2Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA Actin-mediated trafficking plays an essential role in intracellular transport in all studied eukaryotic cells, including the moss, Physcomitrella patens. P. patens is an excellent model organism for studying actinmediated trafficking because of its genetic tractability and simple cytology. Actin-mediated trafficking in P. patens is essential for polarized growth of its tip cells. The actin-associated motor, myosin XI, has been identified as an important protein for this process. Myosin XI is most similar to the class V myosins from yeast and animal cells. Similar to myosin V, P. patens myosin XI functions as a dimer with a head, neck, coiled-coil, and cargo binding domains. Myosin XI is hypothesized to function as a molecular motor carrying exocytotic vesicles along actin filaments to the plasma membrane. In mammalian and yeast cells, the association between myosin and its vesicle cargo is mediated by Rab-GTPases, such as Rab11 and Ypt31/32 respectively. We hypothesize that, in plants, a similar Rab-myosin interaction is necessary for vesicle transport. To evaluate this, we selected three classes of plant Rab proteins based on homology to Rab11 and Ypt31/32 and we are developing an in vitro binding assay to determine if these candidate Rab proteins have myosin XI binding activity. Previous work in our laboratory also identified mutations in the myosin XI cargo-binding domain that failed to complement the RNAi-based knockdown of myosin XI. In this study, we aim to determine the structural consequences of the most severe mutations. We used PCR mutagenesis to generate protein expression constructs for three mutants of the myosin XI cargo- binding domain. We are purifying the wild type and mutant proteins, and plan to compare their folding and stability by circular dichroism, thermal denaturation, and potential for aggregation. Finally, using the Rab proteins identified as potential binding partners, we will determine if the loss-of-complementation in the RNAi screen was due to a binding defect between myosin XI and its cognate Rab. In this study, we use biochemical methods to determine the cause of the failure-to-complement of the myosin XI mutants and to determine if a subclass of Rab proteins is important for myosin XI binding to vesicles in plant cells.

SUNDAY-POSTER PRESENTATIONS

Tubulins and Associated Proteins P116 Board Number: B222

Effect of Tyrosine 18 Phosphorylation on the Conformation and Dynamics of Tau. J. Stern1, G. Morfini2, C. Berger3; 1 Cellular, Molecular and Biomedical Sciences Program, University of Vermont, Burlington, VT, 2 Department of Cell and Molecular Biology, Univ Illinois-Chicago, Chicago, IL, 3Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT Tau is a neuronal microtubule (MT) associated protein that performs numerous functions including stabilizing MTs, participating in signaling cascades and directly inhibiting kinesin-1 motility in vitro, but the relationship between Tau structure and function has yet to be fully elucidated. Recent single molecule experiments have shown that in addition to forming a conventional static complex, Tau can diffuse in an ATP-independent manner while bound to MTs, and the equilibrium between these populations differs with both Tau isoform and MT lattice structure. Furthermore, Tau can adopt a dynamic folded conformation in solution, stabilized through interactions between the N- and C-terminal regions of the molecule. In light of these findings, we hypothesize that the folded conformation of Tau represents the diffusive state and that the equilibrium between the diffusive and static states is mediated by post translational modifications that affect the stabilizing interactions of the N- and Ctermini. We further hypothesize that phosphorylation at tyrosine 18 (Y18) in Tau’s N-terminus helps stabilize the folded conformation and promotes the diffusive behavior of Tau on the MT surface. Correspondingly, dephosphorylation of Y18 leads to a shift from the diffusive to the static state on the MT surface. To test these hypotheses, phosphomimetic and unphosphorylatable control constructs of 3RS-Tau were made by substituting glutamic acid and alanine, respectively, at and around Y18. TIRF microscopy was used to examine the dynamic behavior of these Tau constructs on the MT surface, as well as their ability to inhibit kinesin motility.

P117 Board Number: B223

Down-regulation of stathmin is required for the phenotypic changes and classical activation of macrophages. K. Xu1, R.E. Harrison2; 1 University of Toronto, Toronto, ON, 2Department of Biological Sciences, University of Toronto Scarborough, Toronto, ON Macrophages are major effector cells of the innate immune response with specialized capacity for recognition and elimination of pathogens, and present antigens to lymphocytes for adaptive immunity. Macrophages become activated upon exposure to pro-inflammatory cytokines and pathogenic stimuli.

SUNDAY-POSTER PRESENTATIONS Classical activation of macrophages with interferon-γ (IFNγ) and lipopolysaccharide (LPS) triggers a wide range of protein signalling events and morphological changes to induce the immune response. Our previous microtubule (MT) proteomic work revealed that stathmin association with MTs is considerably reduced in activated macrophages, which exhibit significantly enriched stabilized MTs. Here we show that there is a global decrease in the MT catastrophe protein stathmin level in activated macrophages using both immunoblotting and immunofluorescent microscopy. Our results suggested this is an LPSspecific response that induces proteasome-mediated degradation of stathmin. Furthermore, we explored the functions of stathmin down-regulation in activated macrophages by generating of a stable cell line overexpressing stathmin-GFP. We show that stathmin-GFP overexpression impacts cytoskeletal stability by reducing MT stabilization and acetylation, which significantly diminishes morphological changes associated with cell activation. In addition, we found that stathmin overexpression severely impaired immune functions such as complement receptor 3 (CR3) - mediated particle binding and macrophage activation. Overall, our results implicating a pivotal inhibitory role for stathmin in classically activated macrophages.

P118 Board Number: B224

Doublecortin bends growing microtubules. S. Bechstedt1, J. Hsu1, G.J. Brouhard1; 1 McGill University, Montreal, QC Microtubules are the stiffest members of the eukaryotic cytoskeleton. Despite their high persistence length, microtubules are highly bent in many cellular contexts, such as the neuronal growth cone. Here we show that Doublecortin (DCX), a microtubule-associated protein (MAP) expressed in developing neurons, bends growing microtubules in vitro. We measured the curvature, k, of microtubules bent by DCX using B-splines and show that DCX induces a characteristic bend in growing microtubules, whose curvature depends on DCX concentration. Our results demonstrate for the first time, that a MAP can directly modulate microtubule curvature.

P119 Board Number: B225

CP190-microtubule association is mediated by a novel N-terminal region that requires dimerization mediated by a flanking BTB domain. K.M. Plevock1,2, R.X. Guillen 2, B.J. Galletta2, K.C. Slep3, N.M. Rusan2; 1 University North Carolina, Chapel Hill, NC, 2National Heart Lung, and Blood Institute, NIH, Bethesda, MD, 3Department of Biology, University of North Carolina, Chapel Hill, NC CP190 is a large, multi-domain protein, first identified as a centrosome protein with striking oscillatory localization over the course of the cell cycle. During interphase it has a well-established role within the

SUNDAY-POSTER PRESENTATIONS nucleus as a chromatin insulator. Upon nuclear envelope breakdown, there is a redistribution of CP190 to centrosomes and the mitotic spindle, with a population remaining associated with chromosomes. Previous work described a central CP190 region important for centrosome localization and microtubule (MT) binding. However, a detailed analysis of the CP190 N-terminal region has not been fully described. Here, we investigate CP190 in detail by performing domain analysis in cultured Drosophila S2 cells combined with protein structure determination by x-ray crystallography and in vitro biochemical characterization. Our analysis of CP190 identified a novel centrosome targeting and microtubule (MT) interacting region. This previously uncharacterized CP190 N-terminal region robustly localizes to interphase MTs and is enriched at the growing MT plus ends. This region consists of a highly conserved BTB domain and a linker region that directly interacts with taxol-stabilized MTs in vitro. We present the 2.6 Å resolution structure of the CP190 N-terminal 126 amino acids, which adopts a canonical BTB domain fold and exists as a stable dimer in solution. The CP190 BTB domain is important for robust MT localization. The ability of the linker region to colocalize with MTs in cell culture requires BTB-domain mediated dimerization, though MT colocalization can be rescued via artificial dimerization, replacing the BTB domain with either GST or the GCN4 coiled-coil. These data suggest a possible regulatory role for the BTB domain in modulating CP190 association with the MT network. Finally, in live cells, we see that this N-terminal region and the artificially dimerized linker localize to the kinetochore.

P120 Board Number: B226

The Microtubule tip is a central node for amplification of calcium signals. M. Geyer1, F. Huang1, S. Vogel1, A.B. Malik1,2, Y. Komarova1; 1 Pharmacology, Univ Illinois-Chicago, Chicago, IL, 2Univ Illinois Coll Med, Chicago, IL Growing microtubule (MT) tips establish a transient interaction with endoplasmic reticulum (ER), the main Ca2+ store in the cell, but the functional outcome of these events are poorly understood. Here we demonstrate that growing MTs serve as the binding platform for the inositol 1,4,5-trisphosphate receptors (IP3Rs), the Ca2+ gating channel on the ER membrane. End binding protein 3 (EB3), a core element of plus end tracking protein (+TIP) complex, interacts with the IP3R channel and triggers its clustering. This interaction provides the molecular basis for spatio-temporal organization of Ca2+ signals and determines the overall outcome of the complex cellular response. Targeting EB3-IP3R interactions in endothelial cells stabilizes Vascular Endothelial (VE)-cadherin adhesions and suppresses vascular inflammation suggesting that MT-dependent amplification of Ca2+ signaling is a significant pathological component that can be targeted therapeutically. These findings establish the primacy of the EB3-IP3R complex as a central signaling node in vascular pathophysiology.

SUNDAY-POSTER PRESENTATIONS

P121 Board Number: B227

An elastic shield coupled to rigid interface provides mechanical integrity to myonuclei. S. Wang1, T. Volk1; 1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Differ from most other cell types, striated muscle cells contain high number of nuclei distributed evenly on the surface of the myotube and are subjected to variable mechanical stress during muscle contraction. The characteristic oval myonuclear shape and positioning have been linked to transcriptional regulation, as well as to correct export of mRNAs and proteins. Thus, correct nuclear shape and position are critical for proper muscle function. Consistently, numerous muscular dystrophies and (cardio-)myopathies exhibit nuclear mis-positioning and aggregation. Previously, we revealed the essential role and molecular interaction between the two Drosophila KASHdomain proteins Klar and MSP-300/Nesprin in promoting the even distribution of myonuclei and their anchorage to the acto-myosin in striated muscle fibers. Recently, by performing muscle-specific RNAi screen, we identified additional proteins involved in the core mechanism for myonuclear shaping and distribution process. We demonstrate that the Spectraplakin Shortstop (Shot) is a crucial stabilizing component of a microtubule (MT)-based network that surrounds the myonucleus. Moreover, Shot and MSP-300 interact genetically with each other and their stable attachment to the nuclear envelope requires their mutual existence. Furthermore, both Shot and EB1 display a perinuclear localization pattern like MSP-300, and their knockdown leads to severe disruption of myonuclear shape, supporting a cooperative and reciprocal activity between MSP-300, Shot and EB1 in organizing the perinuclear MT network. Additionally, knockdown of Shot or EB1 perturbs nuclear shape and meanwhile induce ectopic perinuclear F-actin accumulation, indicating a potential involvement in the negative regulation of F-actin polymerization. Finally, our experiments demonstrate that MSP-300, Shot, and EB1 are essential to protect and maintain myonuclear architecture during mechanical stress implicated during muscle contraction. Together, all these components form a perinuclear flexible shield essential for protecting myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and a subsequent abnormal distribution of factors essential to maintain proper nuclear architecture. Overall, we propose a novel mechanism for protecting myonuclear morphology and reveal its critical link to correct distribution of nuclear factors in differentiated muscle fibres. These findings may shed light on the cause of various muscular dystrophies.

SUNDAY-POSTER PRESENTATIONS

P122 Board Number: B228

Induced changes in the dynamics of EB1-mNeonGreen and microtubules in Chlamydomonas. Y. Liu1, K.F. Lechtreck2, P. Yang1; 1 Department of Biological Sciences, Marquette University, Milwaukee, WI, 2Cellular Biology, University of Georgia, Athens, GA EB1 is a master plus end tracker central to microtubule-based processes. It appears like comets near the plus end of only growing microtubules where GTP in tubulin is stochastically hydrolyzed to GDP. At this position EB1 could recruit effectors that modulate microtubule dynamics or harness cellular structures to plus ends. Yet EB1 is also localized outside plus ends, such as basal bodies and the centrosome. The molecular basis for the distinct locations remains to be resolved. To investigate the factors affecting EB1's locations, we express fluorescing EB1 as endogenous EB1 in Chlamydomonas that generate a few types of microtubules. Both EB1-GFP and EB1-mNeonGreen decorated basal bodies and microtubule plus ends at the flagellar tip. However, contrary to EB1-GFP that was entirely obscured by intense autofluorescence from photosynthesis pigments, EB1-mNeonGreen exhibited comet-like patterns typical of EB1 at the plus end of profuse growing microtubules in the cell body. Therefore like EB1-GFP, EB1-mNeonGreen could report the behaviors of EB1 and mNeonGreen is conducive for live imaging of photosynthetic cells. In some preparations - depending on media content, cell density and illumination for microscopy – the comets vanished, perhaps due to diminished growing microtubules; or, unexpectedly, EB1-mNeonGreen appeared like static fibers, some of which resembles the rootlets comprised with characteristic bundles of 2 or 4 microtubules. We are currently defining the conditions that induce changes in the dynamics and interplay of microtubules and EB1.

P123 Board Number: B229

The dynamics of EB1 in Chlamydomonas flagella. Y. Liu1, A. Harris2, K.F. Lechtreck2, P. Yang1; Department of Biological Sciences, Marquette University, Milwaukee, WI, 2Cellular Biology, University of Georgia, Athens, GA

1

The motor-driven intraflagellar transport (IFT) has been considered as the principle trafficking modality for gated lengthy flagella except for small particles that rely on passive diffusion. Although EB1 is best known for binding to the plus end of growing microtubules, immunolocalization revealed EB1 at basal bodies near the entrance and the tip of Chlamydomonas flagella regardless growing, full length or shrinking. To reveal how EB1 molecules travel to and remain at the tip, we expressed EB1-GFP as endogenous EB1 in a few Chlamydomonas strains, including temperature sensitive IFT mutant fla10, in which anterograde IFT could be switched off at 32oC. In all strains and temperatures examined, EB1-GFP is present at the tip and basal bodies as well as the flagellar shaft, albeit dimmer. Fluorescence recovery

SUNDAY-POSTER PRESENTATIONS after photobleaching (FRAP) analysis showed that EB1-GFP moves directionally but far faster than IFT. The tip population in steady-state flagella fully recovers within 2-3 min post bleaching. Further analysis suggests that EB1 forms a stable pool at the flagellar tip which slowly exchanges with the population in the shaft. EB1-GFP is expected to form a dimer of about 120 kDa. Our data suggests that IFTindependent movement is sufficient to rapidly translocate cytoplasmic proteins of more than100 kD in size to the flagellar tip. We are currently investigating the nature of IFT-independent directional rapid movement of EB1 and whether the non-canonical behavior of EB1 at the flagellar tip is linked to the dynamics of the axonemal microtubules.

P124 Board Number: B230

TIP150 regulation in cell migration and functional relevance interaction with the cytoskeleton. G. Adams1, P. Xia2, D. Cao2, Z. Wang3, T. Zhu1,2, H. Duane2, X. Yao4; 1 Morehouse School of Medicine, Atlanta, GA, 2Univ Sci/Technol China, Hefei, China, 3Univ Science/Technol-China, Hefei, China, 4Cellular Dynamics, University of Science Technology of China, Hefei, China The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic, migrating from primary-to-secondary locations. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, TIP150, that promotes cell migration. TIP150 accumulates at growing microtubule plus ends through interaction with the EB1. The EB1-dependent +TIP activity of TIP150 is required for cell migration, as well as for interactions with both actin and microtubule. Here we describe the characterization and functional identification of TIP150 and its association in cellular migration. We hypothesize that TIP150 helps regulate cell migration and links actin to microtubules. To this end, we used a series of biochemical and biophotonic experiments to show TIP150 is necessary for directional cell migration and potentially links to microtubules and actin, further offering additional evidence of the importance of microtubule dynamics in the cell migration process.

SUNDAY-POSTER PRESENTATIONS

P125 Board Number: B231

The role of +TIPs in the remodelling of microtubules during intestinal epithelial differentiation. D. Goldspink1, J. Perkins1, Z. Matthews2, B. Tyrrell1, P. Powell2, T. Wileman2, E. Lund1, N. Galjart3, M. Mogensen1; 1 School of Biological Sciences, University of East Anglia, Norwich, United Kingdom, 2School of Medicine, University of East Anglia, Norwich, United Kingdom, 3Erasmus MC, Rotterdam, Netherlands Microtubule dynamics and organisation play a key role in determining cellular function in differentiated epithelial cells. The intestine contains a hierarchy of epithelial development and provides an ideal model to study microtubule organisation. Stem cells located at the base of the crypt give rise to transitamplifying cells that divide, differentiate and migrate up the crypt and into the villus in the small intestine or the surface epithelium in the colon. Here we show that epithelial cells located at the bottom of intestinal crypts contain an umbrella-like microtubule array with minus-ends anchored at apical centrosomes. However, in the villus centrosomal anchorage is lost and a stabilised apico-basal microtubule array forms with minus-ends anchored at apical non-centrosomal sites. Interestingly, some of the microtubule plus-end tracking proteins (+TIPs) show distinct expression patterns in the intestine. For example, the end-binding protein EB2 is highly expressed at the bottom of crypts but downregulated in the transit amplifying and differentiated enterocytes. EB2 reduction in intestinal epithelial cells leads to microtubule stabilisation and crosslinking to actin filaments, suggesting that EB2 downregulation plays an important role in apico-basal array formation. EB1 is expressed throughout the intestine but a shift from comet only to microtubule lattice association is evident in differentiating cells with distinct apico-basal bundles. Loss of microtubule centrosomal anchorage coincides with the redeployment of the centrosomal anchoring protein ninein to apical junctional sites. Interestingly, the +TIPs CLIP-170 and p150 are also found at these anchoring sites. Analysis of intestinal villus and surface epithelium of CLIP-170/115 knockout mice reveal a loss of ninein from these anchoring sites. Furthermore, functional inhibition in epithelial cell lines suggests that CLIP-170 along with active Rac1 and IQGAP1 ensures efficient redeployment of ninein by facilitating microtubule plus-end cortical capture at adherens junctions. Using recent advancements in intestinal organoid cultures we have further investigated the role of these +TIP proteins in epithelial differentiation. Our data suggest tight regulation of both expression and localisation of EBs and CLIP-170 during intestinal differentiation and show that these +TIPs are critical for microtubule reorganisation and epithelial differentiation.

SUNDAY-POSTER PRESENTATIONS

P126 Board Number: B232

Microtubule +TIP protein EB1 binds to GTP and undergoes dissociation from dimer to monomers upon binding with GTP. K. K. Gireesh1, J. S. Sreeja1, S. Chakraborti2, P. Singh1, G. E. Thomas1, H. Gupta1, T. Manna1; 1 School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram, Kerala, India, 2Department of Neurobiology signalling and Cancer, Institut Curie, Orsay Cedex, France Dynamic properties of microtubules predominantly rely on the conformational status of their growing plus end. The process of conformational arrangement is tightly regulated by a wide range of proteins localized at the ends. One of the highly abundant proteins localized at the plus end is EB1, which autonomously tracks the growing end and regulates the plus end dynamics. The mechanism through which EB1 recognizes the plus ends has been under intense scrutiny in recent years. Previous studies have indicated that EB1 can recognize the GTP-bound tubulin structures at the plus ends and it localizes on the microtubule surface at a site close to the exchangeable GTP-binding site of tubulin. Furthermore, its binding has been shown to promote maturation of the plus end and accelerate the GTP hydrolysis cycle at the end. Although the GTP-dependent structural change in tubulin has been demonstrated as a critical determinant for recognition of the plus ends by EB1, the effect of GTP on the structure of EB1 has remained unclear. In this study, we have used spectroscopic, calorimetric and biochemical methods to analyze the effect of GTP on EB1 in vitro. Isothermal titration calorimetry and tryptophan fluorescence quenching experiments demonstrated that EB1 binds to GTP with a dissociation constant ~ 30 µM. Circular dichroism measurements showed that EB1 undergoes changes in its secondary structure upon binding with GTP. Size exclusion chromatography and the urea-induced unfolding analyses revealed that GTP-binding induces dissociation of the EB1 dimer to its monomers. Size exclusion chromatography followed by biochemical analysis determined that EB1-GTP binding involves association of approximately one molecule of GTP per EB1 monomer. We also found that GTP did not affect the stability of the dimer formed by the C-terminus (191-268) of EB1. Detailed analysis further revealed that the GTP specifically binds to the N-terminus (1-130) of EB1 and induces conformational changes in the N-terminus, suggesting that the GTP-induced conformational change in the N-terminus destabilizes the monomer-monomer interaction in the full length EB1 dimer. The results reveal a hitherto unknown GTPdependent mechanism of dimer to monomer transition in EB1 and further implicate its possible role in regulation of EB1 dimer vs. monomer stability and the plus end regulation in cells.

SUNDAY-POSTER PRESENTATIONS

P127 Board Number: B233

Drosophila melanogaster Msps TOG3 utilizes unique structural elements to promote domain stability and maintain an integral tubulin-binding surface. A.E. Howard1,2, K.C. Slep3; 1 Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, 2Molecular and Cellular Biophysics Program, University of North Carolina, Chapel Hill, NC, 3Department of Biology, University of North Carolina, Chapel Hill, NC XMAP215 family members are potent plus-end microtubule (MT) polymerases. Mutation or depletion of XMAP215 decreases MT polymerization rates and contributes to abnormal spindle phenotypes. The XMAP215 family utilizes an array of tumor overexpressed gene (TOG) domains, consisting of six (A-F) helix-turn-helix HEAT motifs, to bind tubulin. Recently, we showed that XMAP215 family TOG domains have differential, positionally conserved architectures across the array, highlighted by the similar architectures of TOG1 and TOG2, and the differential, comparative architecture of TOG4. Differential TOG domain architectures have the potential to contribute to unique TOG-tubulin interactions, with implications for position-dependent TOG domain tubulin-binding activities and function within the XMAP215 MT polymerization mechanism. Although structures of TOG domains 1, 2, and 4 are welldescribed, structural and mechanistic information characterizing TOG domains 3 and 5 are outstanding. Here we present the crystal structure of Drosophila melanogaster Msps TOG3 to 2.3 Å resolution. Msps TOG3 has two unique features; the first is a unique C-terminal tail that interlocks and stabilizes the ultimate four HEAT repeats, the second is a unique architecture in HEAT repeat B. The Msps TOG3 Cterminal tail has conserved determinants, including an arginine (R816), that form multiple contacts with HEAT repeats C-F. Mutational and circular dichroism studies demonstrate that the TOG3 C-terminal tail promotes TOG3 domain stability. Pairwise structural alignments of TOG3 with other TOG domain structures determined to date show that TOG3 is most similar to TOG domains 1 and 2, with rmsd values of 2.4 and 3.0 Å, respectively. In contrast, TOG3 diverges from the architecture of TOG4 with a rmsd of 4.6 Å. Docking TOG3 onto recently solved Stu2 TOG1- and TOG2-tubulin structures suggests that TOG3 uses similar, conserved tubulin-binding intra-HEAT loop residues to interact with tubulin. This suggests that TOG3 has maintained a TOG1- and TOG2-like TOG-tubulin binding mode despite structural divergence in the second HEAT repeat. The similarity of TOGs 1-3 and the divergence of TOG4 suggest that a polarized, differential TOG architecture may play a key mechanistic role in XMAP215-dependent MT polymerization activity.

SUNDAY-POSTER PRESENTATIONS

P128 Board Number: B234

STU2/XMAP215 interacts with SUMO and the sumoylation machinery. A. Alonso1, M. Greenlee1, R.K. Miller1; 1 Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK Sumoylation is a ubiquitin-like modification that regulates a wide variety of fundamental cellular processes, including DNA repair, transcription, and cell-cycle transitions. However, it is currently not known how sumoylation regulates microtubules. Microtubules perform important roles in the structure of the mitotic spindle as well as serving as tracks for transportation of cargo to various destinations in the cell. Sumoylation has only recently been shown to regulate spindle positioning. Our lab has previously shown that the LIS1 homologue Pac1 is modified in vivo by SUMO and ubiquitin. Pac1 is important for recruiting dynein to the plus end of the microtubule. Dynein is subsequently “off-loaded” to the cortex where it pulls on cytoplasmic microtubules to move the mitotic spindle across the bud neck, a key step in positioning the mitotic spindle. Using two-hybrid analysis, we show that PAC1 interacts with STU2. The C-terminus of Stu2 is sufficient for this interaction. Stu2 is the yeast homologue of XMAP215 and stabilizes microtubules by facilitating the loading of tubulin dimers onto microtubule ends. Stu2 can also de-stabilize microtubules by sterically interfering with tubulin addition. Here we show by two-hybrid analysis that Stu2 interacts with Smt3/SUMO. Stu2 also interacts with the E2 Ubc9, and the E3 Nfi1 of the sumoylation pathway. Stu2/XMAP215 also interacts with SUMO in vitro and in vivo. Using an in vitro sumoylation assay, four shifted bands of Stu2p can be observed. Using a temperature sensitive allele of the ubiquitin like protease, Ulp1, Stu2 displays a higher molecular weight band in vivo that cross reacts with SUMO by immunoblotting. In whole cell extracts, Pac1 shifts more extensively than Stu2 by western blotting. Considering that Stu2 is the eight class of MAP shown to interact with SUMO, we suggest that sumoylation may be a general mechanism for regulating microtubule-associated proteins.

P129 Board Number: B235

Stu2, the budding yeast XMAP215/DIS1 homolog, is a microtubule polymerase with higher activity on yeast tubulin than on mammalian-brain tubulin. M. Podolski1, J. Howard1; 1 Molecular Biophysics and Biochemistry, Yale University, New Haven, CT Stu2 is the budding yeast member of the XMAP215/Dis1 family of microtubule polymerases. It is essential in cell division, allowing proper spindle orientation and metaphase chromosome alignment, as well as spindle elongation during anaphase. Despite Stu2 having a phenotype that suggests it promotes microtubule growth, like the other members of XMAP215/Dis1 family, studies till now with purified Stu2 indicate only that it antagonizes microtubule growth. One potential explanation for these contradictory findings is that the assays were performed with mammalian brain tubulin, which may not be the right

SUNDAY-POSTER PRESENTATIONS substrate to test the activity of Stu2, given that yeast and brain tubulins are quite divergent in sequence, and that the vertebrate tubulins are subject to many post-translational modifications. To test this possibility, we reconstituted the activity of Stu2 with purified budding yeast tubulin. We found that Stu2 accelerates microtubule growth in yeast tubulin several fold, similar to the acceleration reported for XMAP215 in porcine brain tubulin. Furthermore, Stu2 accelerates polymerization in yeast tubulin to a much greater extent than it does in porcine brain tubulin, and the concentration of Stu2 required to reach 50% maximum activity in yeast tubulin was nearly two orders of magnitude lower than in porcine brain tubulin. We conclude that Stu2 is a microtubule polymerase, like its relatives, and that its activity is considerably higher in yeast tubulin compared to mammalian brain tubulin. These studies highlight significant differences between tubulins from different sources and the importance of using conspecific tubulin when studying microtubule associated proteins.

P130 Board Number: B236

Alcohol metabolism by Cytochrome P450 2E1 does not contribute to hepatocellular defects associated with tubulin acetylation, but does impair STAT5 nuclear translocation. E. Doody1, D. Tuma2, P. Tuma1; 1 Department of Biology, Catholic Univ America, Washington, DC, 2Department of Internal Medicine, University of Nebraska, Omaha, NE Although alcoholic liver disease has been clinically well described, the molecular mechanisms that promote hepatoxicity are not yet fully understood. The liver metabolizes alcohol using two enzymes, alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP 2E1). Both ADH and CYP 2E1 metabolize alcohol into acetaldehyde, but CYP 2E1 activity also results in the production of reactive oxygen species (ROS) that lead to oxidative stress. We have previously shown that microtubules are more highly acetylated in alcohol-treated polarized hepatic WIF-B cells and livers from alcohol fed rats. We further determined that alcohol metabolism to acetaldehyde by ADH is required for enhanced acetylation. However the role of CYP2E1-mediated metabolites and ROS in tubulin acetylation was not examined. To determine if CYP 2E1-mediated alcohol metabolism is required for enhanced acetylation, we treated cells with 100 µM diallyl sulfide (DAS), a CYP 2E1 inhibitor, or 5 mM N-Acetyl Cysteine (NAC), an antioxidant that decreases ROS, in the presence of 50 mM alcohol. Alcohol-treated cells revealed that inhibition of CYP 2E1 with DAS did not alter enhanced tubulin acetylation, supporting that its activity does not mediate the effect. Consistent with this result is the finding that DAS treatment failed to rescue the impaired ASGP-R internalization observed in alcohol-treated cells. However, inhibition of CYP 2E1 did reverse the STAT5B nuclear translocation impairment that has been previously observed in alcohol-treated cells. It is believed that this is caused by a defect in STAT5B activation by Jak2 kinase, which is independent of the microtubule network, further supporting that CYP 2E1 alcohol metabolism does not affect microtubule hyperacetylation.

SUNDAY-POSTER PRESENTATIONS

P131 Board Number: B237

Alcohol-induced tubulin acetylation sites identified by mass spectrometry. J. Groebner1, D. Tuma2, P. Tuma1; 1 Department of Biology, Catholic Univ America, Washington, DC, 2Department of Internal Medicine, University of Nebraska, Omaha, NE Although the progression of alcoholic liver disease is clinically well-described, the mechanisms leading to alcohol-induced liver damage remain elusive. Previously, we determined that microtubules are hyperacetylated and more stable in ethanol-treated WIF-B cells, liver slices and in livers from ethanolfed rats. Our focus is to determine whether tubulin hyperacetylation can explain alcohol-induced defects in microtubule-dependent protein trafficking. Previously, we determined that transport of newly synthesized proteins from the Golgi to the basolateral surface and STAT5B nuclear translocation are impaired by alcohol metabolism. Recently, we confirmed that delivery of apical proteins from the basolateral-to-apical membrane via transcytosis is also impaired in ethanol-treated WIF-B cells. Unlike in control cells, transcytosing proteins accumulated sub-apically and aligned along acetylated microtubules in ethanol-treated cells. Both dynein and dynactin colocalized with transcytosing proteins, but only dynein (not dynactin), more tightly associated with microtubules from ethanol-treated cells. Thus, we conclude that enhanced dynein binding to microtubules in ethanol-treated cells leads to decreased motor processivity resulting in vesicle stalling and impaired delivery. A common feature of these delivery routes is regulation by microtubule-based motors. The “tubulin code” hypothesis proposes that microtubule-based motor trafficking for specific cellular functions may be regulated by tubulin isotype composition and post-translation modifications. To more directly examine microtubule hyperacetylation in alcohol-treated cells, we have analyzed purified taxol-stabilized microtubules from control, trichostatin A (TSA - leads to global protein acetylation)- and ethanol-treated WIF-B cells by mass spectrometry. In preliminary studies, we have successfully recovered both α- and β-tubulin with ~60% coverage in both control, TSA- and ethanol-treated cells. As predicted, Lys40 (the known α-tubulin acetylated site) was fully acetylated in ethanol-treated cells with all recovered fragments being acetylated. In contrast, no acetylated Lys40 containing peptides were recovered from control tubulin. We also identified novel acetylated lysines in the C-terminal half of α-tubulin (in ethanol-treated cells) and β-tubulin (in both control, TSA- and ethanol-treated cells). One site was more highly acetylated in ethanol-treated cells. We are currently generating site specific lysine mutants to directly identify which residues contribute to impaired motor properties and defects in protein trafficking.

SUNDAY-POSTER PRESENTATIONS

P132 Board Number: B238

α-Tubulin Acetyltransferase αTAT1 Preferentially Targets Tapered Microtubule Ends with Exposed Lys40 Acetylation Sites. C.E. Coombes1, A. Yamamoto1, T. Reid1, M. McClellan1, G.G. Luxton2, J. Alper3, M. Gardner1; 1 Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, 2Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 3Molecular Biophysics and Biochemistry, Yale University, New Haven, CT Microtubules are structural polymers inside of cells that are subject to post-translational modifications. These post-translational modifications help to create functionally distinct subsets of microtubule networks in the cell. Acetylation is unique from other post-translational modifications in that it is the only modification which takes place in the hollow lumen of the microtubule. While it is known that the α-tubulin acetyltransferase αTAT1 is responsible for the majority of the microtubule acetylation that is observed in cells, the mechanism for how αTAT1 accesses the Lys40 acetylation site inside of the microtubule lumen is not fully understood, and remains an important topic of investigation. To examine how αTAT1 accesses the lumenal acetylation site, we performed a combination of biochemical assays, fluorescence and electron microscopy experiments, and simulations. We found that the microtubule αtubulin acetylation rate is limited by accessibility of the enzyme to the lumen, and that αTAT1 preferentially targets tapered microtubule ends with exposed Lys40 acetylation sites. This preference for tapered microtubule ends may play a role in the specific targeting of αTAT1 to subsets of cellular microtubules. These results provide important insights into the mechanism for αTAT1 microtubule acetylation and its dependence on the microtubule structure.

P133 Board Number: B239

Mouse Sperm Associate Antigen 6 Regulates Cell Growth, Cell Migration And Ciliogenesis Through Activation of Acetylated Tubulin In Mouse Embryonic Fibroblast Cells. Z. Zhang1; 1 Obstetrics/Gynecology, Virginia Commonwealth University, Richmond, VA Mammalian Spag6 is the orthologue of Chlamydomonas PF16, which encodes a protein localized in the axoneme central apparatus of flagella and regulates flagellar/cilia motility. Most Spag6-deficient mice are smaller in size than their littermates, suggesting that mouse SPAG6 might also play roles other than regulating flagellar/cilia motility. When mouse embryonic fibroblasts (MEFs) were cultured from Spag6deficient mice and their littermates, it was found that both primary and immortalized Spag6-deficient MEFs proliferated at a much slower rate than the wild-type MEFS of the same passages. Spag6-deficient MEFs also had enlarged cell size, and they appeared to be more flattened and had more cytoplasmic

SUNDAY-POSTER PRESENTATIONS vesicles than the wild-type MEFs. Transwell chambers and wound-healing assays demonstrated that the Spag6-deficient MEFs had dramatically reduced migration ability. During migration, polarized distribution of α-tubulin, a subunit of microtubule, and F-actin, another cytoskeleton component observed in the wild-type MEFs was never seen in the Spag6-deficient MEFs. Abnormally dividing cells were observed in the Spag6-deficient MEFs as demonstrated by multiple centrioles illustrated by γtubulin staining. Spag6-deficient MEFs also demonstrated ciliogenesis defects; the percentage of cells with primary cilia was significantly reduced compared to the wild-type MEFs, and some Spag6-deficient MEFs had multiple cilia. The expression level of acetylated tubulin, a marker for cilia, and also an indicator for microtubule stability, was dramatically reduced in the Spag6-deficient MEFs. In contrast, overexpressing SPAG6 in multiple cultured mammalian cells significantly increased acetylated tubulin expression level, and SPAG6 co-localized with acetylated tubulin. Our studies have revealed unexpected roles of mouse SPAG6; it not only regulates flagellar/cilia motility, it also regulates cell proliferation, cell migration, cell division, and ciliogenesis, perhaps through influencing acetylated tubulin expression and microtubule stability. Given F-actin distribution is also changed in the Spag6-deficient MEFs, SPAG6 might also influence functions of other cytoskeleton systems.

P134 Board Number: B240

Not just charged strings: binding of Tubulin's disordered tails to VDAC is regulated by small sequence changes and by posttranlational modification. K.L. Sheldon1, S.M. Bezrukov1, D.L. Sackett1; 1 Program in Physical Biology, NIH/NICHD, Bethesda, MD The tubulin alpha-beta heterodimer is the subunit of microtubules (MT) but is also a freely diffusing cytoplasmic protein in its own right which can find binding partners distinct from those for MT. One of these partners is the mitochondrial outer membrane protein VDAC (Voltage Dependent Anion Channel), which mediates small molecule traffic into and out of the mitochondria. We previously showed that tubulin binding to VDAC blocks channel traffic and reduces oxidative metabolism, and that this requires the unstructured anionic tail peptides found on both the alpha- and beta-tubulin subunits. But those studies left unclear if the tubulin body per se is required, whether the alpha- and beta-tails contribute equally to VDAC blockade, and the role, if any, of tubulin posttranslational modifications (PTM), which mostly occur on the tails. These intrinsically disordered tails contain only ~3% of the mass but ~40% of the net charge on the tubulin heterodimer. Earlier studies showed that anionic polymers could cause VDAC blockade, but gave no indication of sequence sensitivity. Here we use single molecule binding studies to show that channel closure is due to the tails alone, since grafting them to albumin gives the same VDAC closure as tubulin. We show that the beta-tail is ~100-fold more potent than the alpha-tail, and that this difference is largely due to the presence or absence of a terminal tyrosine. The activation of the alpha-tail by detyrosination is reversed by subsequent removal of the next residue. We also show that nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or PTM of the disordered tails of

SUNDAY-POSTER PRESENTATIONS tubulin result in substantial changes in VDAC closure. We expect that this pattern may be repeated when comparing the numerous isotypes of alpha- and beta-tubulin, as well as other PTMs. These results also provide a sensitive system to study biology of disordered protein sequences, and their sensitivity to small chemical changes.

P135 Board Number: B241

Zeta-tubulin Orients Cilia in Multiciliated Epithelial Cells. E. Turk1, A. Wills2, J.B. Wallingford2, T. Stearns1,3; 1 Department of Biology, Stanford University, Stanford, CA, 2Molecular Biosciences, University of Texas at Austin, Austin, TX, 3Department of Genetics, Stanford School of Medicine, Stanford, CA Tubulins are a conserved superfamily of proteins that are important in the structure and function of the microtubule cytoskeleton. Members of the alpha-tubulin, beta-tubulin, and gamma-tubulin families are found in all eukaryotes, and are the structural elements of the microtubule polymer, and the main nucleator of that polymer, respectively. Members of the delta-tubulin and epsilon-tubulin families are present in many, but not all, eukaryotes and are associated with centrioles. We have characterized in vertebrates a member of another tubulin family, which most closely resembles tubulins previously named eta-tubulin or zeta-tubulin. Based on an assessment of the entire tubulin superfamily we will refer to this as the zeta-tubulin family. Zeta-tubulin is present in many single-celled eukaryotes, chordates, and marsupial mammals, but is absent in Drosophila, C. elegans, zebrafish, and placental mammals. Analysis of genome sequences indicates that zeta-tubulin is the last remaining tubulin family member to be characterized in vertebrates. Zeta-tubulin is the least well-conserved of the tubulin families, both in terms of the number of lineages that have lost the gene and in the divergence of the protein within branches that have retained it. We have characterized Xenopus laevis zeta-tubulin in a variety of cell lines and developmental stages. In the multiciliated skin cells of the frog embryo, zetatubulin is a basal body component, with a pattern of localization consistent with it being a part of the basal foot. Remarkably, Xenopus zeta-tubulin is able to localize to basal bodies in multiciliated cells of mouse, which has lost the gene for this tubulin. This suggests that the module of function, perhaps the basal foot, is conserved and that another tubulin family member might take the place of zeta-tubulin in species in which it is lost. Depletion of zeta-tubulin results in disorganization of basal body distribution and polarity in multiciliated cells, but no apparent phenotypes in other cell types. In contrast with multiciliated cells, zeta-tubulin in cycling cells does not localize to centrioles and instead is associated with the TRiC-CCT complex, a large cytoplasmic chaperone that is responsible for folding actin, tubulin, and other proteins. We conclude that zeta-tubulin performs a specialized function in basal bodies of differentiated cells, most likely in the function of the basal foot appendage that orients basal bodies with respect to the cell and tissue axes.

SUNDAY-POSTER PRESENTATIONS

P136 Board Number: B242

Intragenic complementation reveals that γ-tubulin has at least three functions important for growth of Aspergillus nidulans. M.A. Hervás-Aguilar1, K.J. Bichsel1, B.R. Oakley1; 1 Molecular Biosciences, University of Kansas, Lawrence, KS γ-tubulin plays a key role in microtubule nucleation at microtubule-organizing centers of both fungal and animal cells, but data from a number of organisms indicate that it has additional important, although less clearly defined, functions. Previously, we created temperature-sensitive mutations through alaninescanning mutagenesis of the mipA (γ-tubulin) gene of Aspergillus nidulans and one of these mutations reveals a role for γ-tubulin in inactivating the anaphase promoting complex coupled with Cdh1 at G1/S. These mutants display a range of phenotypes and we were interested in how many distinct functions were defective in our alleles. One approach to answering this question is through intragenic complementation. The principle underlying intragenic complemetation is that if two alleles of a gene that are defective for different functions are co-expressed in the same cell, the protein products of the two alleles will, between them, carry out all the required functions of the protein. Cells in which both alleles are expressed will, thus, grow better than cells in which either of the two alleles is expressed. If the two alleles are defective for the same function, all protein molecules will be defective for that function and there will be little or no improvement in growth. We tested eight conditionally growth inhibited alleles for intragenic complementation by creating strains in which one mutant allele was present at the endogenous mipA locus and the same or another mutant allele was inserted at the wA locus. All alleles were recessive to the wild-type mipA allele. For four alleles, expression of a second copy of the same allele, surprisingly, resulted in improved growth at restrictive temperatures. Several allele pairs exhibited intragenic complementation (defined as a growth advantage compared to two copies of either of the two alleles). The alleles fell into three intragenic complementation groups indicating that γtubulin has at least three functions important for growth. To determine if the functions of γ-tubulin could correlate with functional domains in the γ-tubulin, we modeled the structure of A. nidulans γtubulin and γ-tubulin complex proteins based on the structure of human TUBG1 and GCP4. The results indicate that the two-copy-suppressed alleles cluster together as do each of the intragenic complementation groups. These data indicate that γ-tubulin has at least three functions important for growth that may correlate with functional domains of the protein. Supported by the NIH and the Irving S. Johnson Fund of the KU Endowment. Kyle J. Bichsel is supported by an IRACDA fellowship.*

SUNDAY-POSTER PRESENTATIONS

P137 Board Number: B243

Interaction of Colchicine-Site Drugs with βVI-Isotype of Tubulin. M. Loew1, D.L. Sackett2, S.L. Bane1; 1 Chemistry, SUNY-Binghamton, Binghamton, NY, 2Program in Physical Biology, NIH/NICHD, Bethesda, MD Tubulin, the main component of microtubules, exists as multiple isotypes. Although these isotypes are highly conserved, the βVI isotype is significantly divergent in amino acid sequence. This isotype is found in hematologic cells and is recognized as playing a role in platelet biogenesis. Tubulin from the erythrocytes of Gallus gallus (Chicken erythrocyte Tubulin, CeTb) contains roughly 95% βVI tubulin. This form of tubulin has been found to lack interaction with colchicine, a feature previously thought to be ubiquitous of tubulin from higher-order eukaryotes. In this study, we sought to gain a better understanding of the structure-activity relationship of the colchicine site of this divergent isotype. We developed a fluorescence-based assay to detect binding of drugs to CeTb and used it to screen known colchicine-site drugs with CeTb. Drugs that showed good affinity for CeTb include podophyllotoxin, combretastatin A2 and A4, and MDL-27048. Colchicine derivatives had poor affinity with the exception of one allocolchicine derivative. Because the colchicine site of human βVI is very similar to that of chicken βVI, these results may have relevance to the action of microtubule-targeting drugs on hematopoietic tissues in humans.

P138 Board Number: B244

Using Drosophila melanogaster as a Model Organism to Study Reproductive Toxicants. K. Hernandez1, I. Cepin1, C. Juarez1, S. Lovell1, A. Nicholas1, R. Wint1, K. . Boekelheide2, J. Rollins3; 1 Division of Natural Sciences, College of Mount Saint Vincent, Bronx, NY, 2Brown University, Providence, RI, 3College of Mount Saint Vincent, Bronx, NY There is a growing international movement to make use of the ongoing revolution in biology to revise longstanding approaches to toxicity testing and risk assessment of environmental chemicals, pharmaceuticals, and consumer products. At its core, this new approach will measure molecular changes in model organisms and cells in vitro to develop a mechanistic understanding of chemicallyinduced alterations in the function of biological pathways. Model organisms, such as Drosophila melanogaster, provide the unique advantage of “simplified complexity,” portraying complex organismal processes, such as spermatogenesis, without the many redundant proteins and pathways characteristic of mammals. Six different toxicants (pharmaceutical and environmental) were studied: 2,5-haxanedione, carbendazim, taxol, di (2-ethylhexyl) phthalate, colchicine and dibromochloropropane. Three different dosing methods were tested: 1. Adult flies laid eggs in toxicant laced food in order to mimic exposure in young animal, 2. Adult flies were starved and given the toxicant directly by dissolving the toxicant in

SUNDAY-POSTER PRESENTATIONS sugar water, 3. Embryos were permiablized and treated with the toxicant directly. Method 1 was the only method that was not lethal and the effect of the toxicant on sperm development was assessed by phase contrast squashes of wild type and β tubulin-GFP flies. Many defects, such as transition to meiosis, meiotic cytokinesis, and sperm elongation and maturation can easily be quantified using this simple assay. DEHP, di (2-ethylhexyl) phthalate showed little effect on sperm development in all threedose groups. 2,5-hexanedione was lethal at high doses and effected. Flies overexpressing β tubulin by carrying a trans gene of β tubulin-GFP were more sensitive to this toxicant. The wild type exposed to the toxicant had some motile sperm but also showed signs of degenerative spermatocyte cysts. Taxol exposure yielded less motile sperm and shorter sperm tails. Wild type flies exposed to CBZ were more sensitive to the toxicant than β tubulin-GFP flies however little or no effect on sperm development. Future work on this project will be to maximize the efficiency of dosing so lethality will not be an issue and sperm development can be properly assessed using microscopy, fertility and sperm viability assays.

P139 Board Number: B245

Intravital microscopy suggests that induction of mitotic arrest by antimitotic cancer drugs does not predict their efficacy in fibrosarcoma mouse xenograft tumors. S. Florian1, D.R. Chittajallu2, R.H. Kohler3, J.D. Orth4, R. Weissleder1,3, G. Danuser5, T.J. Mitchison1; 1 Department of Systems Biology, Harvard Medical School, Boston, MA, 2Department of Cell Biology, Harvard Medical School, Boston, MA, 3Center for Systems Biology, Massachusetts General Hospital, Boston, MA, 4MCD Biology, University of Colorado Boulder, Boulder, CO, 5Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX Despite immense progress in targeted cancer drug design, to date, the most important drugs in chemotherapy remain traditional cytotoxic drugs which are thought to kill proliferating cells, no matter if malignant of not. This "antiproliferative" hypothesis is based on results from the 1970s coming from mouse leukemia models or cell culture experiments. Microtubule binding drugs are clinically important antiproliferative drugs which arrest cultured cancer cells in mitosis through activation of the spindle assembly checkpoint (SAC). As opposed to empirically found microtubule binding drugs, newer drugs designed to more specifically induce mitotic arrest have repeatedly failed in clinical trials, suggesting that the antitumor effect of microtubule binding drugs is still not well understood. Alas, tissue culture assays are not predictive of their respective clinical success, suggesting that more realistic models are needed to understand why there are such huge differences in antitumor efficacy. Therefore, we developed an integrated in vivo microscopy and image analysis pipeline of mouse xenografts to compare the cell cycle effects of microtubule drugs to those of newer, targeted antimitotic drugs in the form Ispinesib, an inhibitor of the mitotic kinesin Eg5. Image analysis with a custom developed software for automated three dimensional segmentation and cell cycle classification revealed

SUNDAY-POSTER PRESENTATIONS that the microtubule interacting drugs Paclitaxel and Eribulin which are in wide clinical use induce similar mitotic arrest as the Eg5 inhibitor Ispinesib which failed clinical trials. Nevertheless, similar to the clinical situation, they seem to be more effective in reducing tumor cell density than Ispinesib in our system. This suggests that, rather than mitotic arrest, a different mechanism, intrinsically coupled to microtubule binding, may be responsible for the antitumor effect of microtubule drugs in vivo.

P140 Board Number: B246

Characterization of a novel microtubule-associated protein in Toxoplasma gondii, a human parasite. Y. He1, J. Liu1, K. Hu1; 1 Department of Biology, Indiana University Bloomington, Bloomington, IN Toxoplasma gondii is a widespread protozoan parasite that can infect nearly all warm-blooded animals. It can cause toxoplasmosis in humans. Infections in immunocompromised individuals and unprotected fetus have devastating consequences, including the development of lethal Toxoplasma encephalitis. T. gondii is also a model for its relatives in the phylum Apicomplexa, which includes the malarial parasites. These parasites have a highly organized cytoskeleton that is essential to their replication and infection in the host. Furthermore, the cytoskeleton of the parasite has many properties distinct from that in the host cell and therefore an ideal target for anti-parasitic measures. One prominent component of the T. gondii cytoskeleton is its set of 22 cortical microtubules. Compared with microtubules found in mammalian cells, the cortical microtubules are highly ordered and exceptionally stable. It is believed that these special properties of the cortical microtubules are due to novel microtubule associated proteins, because the major tubulin subunits in T. gondii are essentially identical with those in mammalian cells. In our effort to search for the proteins that regulate the function of the cortical microtubules, we identified a group of proteins that coat the microtubules, including TLAP3 (for TrxL associated protein 3). TLAP3 has no close homolog in the sequenced genomes of mammals, thus is a potential drug target. We have constructed a transgenic line through homologous recombination. In this line, fluorescently tagged TLAP3 is expressed from its endogenous locus. Interestingly, we found that the localization of TLAP3 is restricted to an apical region close to the presumed organizing center of the cortical microtubules. This may suggest an organization role of TLAP3 in the initial construction of the cortical microtubules. Consistent with this hypothesis, TLAP3 is found close to the apex of developing daughter cells. To further test this possibility, we are creating TLAP3 knockout parasite to probe its function using Cre-LoxP mediated excision.

SUNDAY-POSTER PRESENTATIONS

P141 Board Number: B247

What is tubulin glutamylation good for?. D. Chawla1, R. Shah1, J. Lee1, Z. Barth1, N. Peel1; 1 Department of Biology, The College of New Jersey, Ewing, NJ Microtubules are dynamic polymers that play essential structural roles in cell division, vesicle trafficking, and cilia function. Much is known about the regulation of microtubule dynamics by interacting proteins, but the post-translational modification of tubulin as a regulatory mechanism remains relatively unexplored. Tubulin glutamylation is the covalent attachment of glutamic acid to tubulin in the polymerized microtubule. This modification is enriched on long-lived microtubules, and it is proposed that tubulin glutamylation contributes to centriole stability, cilia motility and axon function. Comprehensive in vivo analyses of the function of tubulin glutamylation have proved challenging because of the existence of a large family of glutamylating enzymes. C. elegans, however, has only 5 glutamylating (tubulin tyrosine ligase like; TTLL) enzymes making their combinatorial analysis feasible. We have obtained deletion mutations in all five TTLL enzymes, non of which display overt phenotypes. To test for redundancy we have combined the mutations and are currently analysing two mutant combinations. First, a triple mutant that shows reduced male fecundity. Second, a quint mutant lacking all glutamylating enzymes. By closely examining the phenotypes of these worms we hope to definitively determine the in vivo function of tubulin glutamylation.

P142 Board Number: B248

Did acetylation of α–tubulin play a role in the evolution of microtubules from filaments?. R.F. Luduena1; 1 Biochemistry, Univ Texas Hlth Sci Ctr-San Antonio, San Antonio, TX There is considerable evidence that the first tubulin molecules formed filaments rather than cylindrical microtubules (MTs) (McIntosh et al, Cell 135, 322, 2008; Ludueña, Int. Rev. Cell Mol. Biol. 302, 41, 2013). How could this filament become a microtubule (MT)? There are three factors to consider: 1) Addition of the C-terminus could have favored MT formation; removal of the C-terminus prevents incorporation into MTs (Joe et al, J. Biol. Chem. 284, 4283, 2009). This may be too fortuitous, however. 2) Lateral joining of the protofilaments (PFs) would allow them to exert more force on heavier cargo (McIntosh et al, J. Cell Sci. 123, 3425, 2010). Although this is very likely to have been a factor, it does not explain why a cylinder rather than, say, a triple helix, was favored. 3) Perhaps a more compelling rationale, however, arises from acetylation. This is a very widespread and therefore probably ancient modification of lys40 in α-tubulin that is associated with increased MT stability and intra-luminal structures as well as binding to mitochondria and Golgi (Friedman et al, J. Cell Biol. 190, 363, 2010; Deakin & Turner, J. Cell Biol. 206, 395, 2014). The modified lys40 is in the lumen of the MT. Recent findings suggest that acetylation does

SUNDAY-POSTER PRESENTATIONS not affect overall MT structure but that it mediates binding to some intra-luminal protein (Howes et al, Mol. Biol. Cell 25, 257, 2014). It is very difficult to imagine the modifying enzyme, the modification, and the associated intra-luminal protein evolving after the MT appeared. These are more likely to have evolved when tubulin formed only PFs and the lumen did not exist. The acetylation could have mediated binding to an ancestor of a present-day intra-luminal protein, or perhaps a protein linking to mitochondria or Golgi. If so, and if that protein was itself a filamentous polymer (Linck et al, J. Biol. Chem. 289, 17427, 2014) then if adjacent tubulin PFs associated with each other at an angle, so as to form a curved rather than a flat ribbon, then it would have been a stronger structure if at least two of the tubulin PFs bound to the ancestral intra-luminal protein. For this the PFs would have had to associate laterally at an angle. At this point, it would have been easy for more PFs to have added laterally until a cylinder appeared. It is unlikely that a cylinder that evolved this way would have been symmetrical, so it is not surprising that there is a seam at the join. It is not necessary for the intraluminal protein to fill the entire lumen and indeed, once the MT evolved, there was no universal need for an intra-luminal protein at all or even for an acetylated lys40, so these features could have been retained only for specific functions involving intra-luminal proteins.

P143 Board Number: B249

Reconstitution of the human augmin complex provides insights into its architecture and function. K. Hsia1, E.M. Wilson-Kubalek2, A. Dottore1, Q. Hao1, K. Tsai2, S. Forth3, Y. Shimamoto1, R.A. Milligan2, T.M. Kapoor1; 1 Rockefeller University, New York, NY, 2Scripps Res Inst, La Jolla, CA, 3Rockefeller Univ, New York, NY Proper microtubule nucleation during cell division requires augmin, a microtubule-associated heterooctameric protein complex. In current models, augmin recruits γ-tubulin, via its hDgt6 subunit’s Cterminus, to nucleate microtubules within spindles. However, augmin’s biochemical complexity has restricted analysis of its structural organization and function. Here, we reconstitute human augmin and show it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains in vitro microtubule binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to Xenopus egg extracts promotes microtubule aster formation, an activity enhanced by Ran-GTP. This activity requires microtubule binding, but not the characterized hDgt6 γ-tubulin-recruitment domain. Tetrameric subcomplexes also induce asters, but activity and microtubule bundling within asters are reduced compared to octameric complexes. Together, our findings shed light on augmin’s structural organization, microtubule binding properties and define subunits required for its function in organizing microtubulebased structures.

SUNDAY-POSTER PRESENTATIONS

P144 Board Number: B250

Characterization of human gamma-tubulin isotypes and their differential expression during neuritogenesis. E. Draberova1, S. Vinopal1, V. Sulimenko1, T. Sulimenko1, V. Sladkova1, L. D'Agostino2, C.D. Katsetos2, L. Kren3, P. Draber1; 1 Biology of Cytoskeleton, Institute of Molecular Genetics AS CR, Prague 4, Czech Republic, 2Pathology Laboratory Medicine, Drexel University College of Medicine, Philadelphia, PA, 3Faculty Hospital Brno, Brno, Czech Republic Gamma-tubulin is a highly conserved member of the tubulin superfamily essential for microtubule nucleation. Many organisms including humans possess two genes encoding gamma-tubulin, and their coding sequences share a high sequence similarity. It is thought that in mammalian tissues gammatubulin 1 represents ubiquitous form of gamma-tubulin, while gamma-tubulin 2 is found predominantly in brain where it is neuron associated. It was suggested that gamma-tubulin 2 could also have some other functions. The molecular basis of the purported functional differences between gamma-tubulins is unknown. Here we have demonstrated that human gamma-tubulins can be discriminated according to their electrophoretic properties and have unraveled through in vitro mutagenesis that the differences originate in the C-terminal regions of the gamma-tubulin molecules. Using epitope mapping we identified an antigenic determinant recognized by an anti-peptide monoclonal antibody that is specific for human gamma-tubulin 1. We have shown both at the mRNA and protein levels, using RT-qPCR and 2D-PAGE, that gamma-tubulin 1 is the predominant isotype in primary neurons derived from human fetal cerebral cortex (ScienCell Research Laboratories). Comparable amounts of both gamma-tubulin isotypes were found in autopsy samples from non-pathologic cerebral cortex of human adults. Localization of gamma-tubulin 1 in mature cortical neurons was confirmed by immunohistochemistry performed on surgically resected clinical samples containing non-lesional cerebral neocortex. Depletion of gamma-tubulin 1 by shRNAi in the human neuroblastoma SH-SY5Y cells resulted in impaired microtubule nucleation and metaphase arrest. A wild-type phenotype in gamma-tubulin 1-depleted cells was restored by expression of exogenous human gamma-tubulin 2. During all-trans retinoic acidinduced neuritogenesis in SH-SY5Y cells, expression of human TUBG2 was upregulated, while the expression of TUBG1 was basically unchanged. Our data suggest that while both human gamma-tubulins are nucleation competent, differences in their properties and accumulation of gamma-tubulin 2 in mature neurons, in the face of a predominant gamma-tubulin 1 expression in these cells, may reflect additional gamma-tubulin 2 function(s) in neurons. This work was supported in part by grants LH12050, NT14467, and M200521203.

SUNDAY-POSTER PRESENTATIONS

Assembly and Disassembly of Cilia/Flagella 1 P145 Board Number: B252

Identification of the molecular ruler that defines the 96-nm repeats of cilia/flagella. T. Oda1, H. Yanagisawa1, R. Kamiya2, M. Kikkawa1; 1 Dept Cell Biology, The University of Tokyo, Tokyo, Japan, 2Dept Life Science, Gakushuin University, Tokyo, Japan Primary ciliary dyskinesia (PCD) is an inherited disease caused by abnormal ciliary motility. Majority of PCD cases associated with axonemal disorganization result from mutations in CCDC39 and CCDC40 genes. However, their localizations and functions in cilia/flagella are not clearly resolved. In this study, we revealed the molecular mechanism how FAP59 and FAP172, the Chlamydomonas homologues of CCDC39 and CCDC40, govern the assembly of axonemal ultrastructures. Using biochemical pull-down assay and cryo-electron tomography, we showed that FAP59 and FAP172 form a complex, and the absence of the two proteins disrupts 96-nm repeats of axonemes. Structural labeling revealed that FAP59 and FAP172 take 96-nm long extended conformations along outer doublet microtubules, suggesting their possible role as a molecular ruler. To test this hypothesis, we elongated the amino acid sequences of FAP59 and FAP172 by duplicating their coiled-coil domains. Surprisingly, the repeat length became ~128-nm long, and axonemal components such as radial spokes, inner dynein arms, and dynein regulatory complexes were duplicated. These results strongly suggest that the FAP59/172 complex is the molecular ruler that determines the 96-nm repeats in cilia/flagella by regularly recruiting axonemal components to the correct binding sites.

P146 Board Number: B253

TTC26/DYF13/IFT56 transports a potential length sensor as a cargo for controlling the ciliary length. H. Ishikawa1, W. Marshall2; 1 University of California, San Francisco, San Francisco, CA, 2Biochemistry and Biophysics Dept., University of California, San Francisco, San Francisco, CA Cilia/flagella are microtubule-based organelles that protrude from the surface of most cells, are important to sense extracellular signals and make a driving force for fluid flow. Because the length of cilia/flagella is correlated with their function, cells must have cilia/flagella of adequate length in order to perform their function properly. However, the regulation mechanism of ciliary length is mostly unknown. To control the length of cilia, cells seem to sense ciliary length and to have a feedback pathway to adjust the amount of cargo transport into cilia. The assembly and maintenance of cilia/flagella are dependent on intraflagellar transport (IFT). IFT is an active transport process within cilia

SUNDAY-POSTER PRESENTATIONS mediated by a bidirectional movement of multiprotein complexes, known as IFT particles. We identified an IFT mutant Chlamydomonas strain, dyf13, which exhibits short flagella and motility defects. To identify the causes of short flagella, we measured the accumulation of an IFT protein at basal bodies in wild-type and dyf13 mutant cells. Wild-type cells showed accumulation of IFT at basal bodies that depends on the length of cilia during regeneration. However, the IFT accumulation in dyf13 mutant cells was always higher than that of wild-type cells and was independent of ciliary length. This observation suggests that DYF13 itself, or its cargo, is related to the feedback pathway and length sensor for ciliary length control.

P147 Board Number: B254

Cilia autonomous regulation of tubulin transport by IFT. J.M. Craft1, K.F. Lechtreck1; 1 Cellular Biology, University of Georgia, Athens, GA Microtubules are the major structural elements of cilia and eukaryotic flagella. During ciliary assembly, large amounts of tubulin must be transported into the organelle. Intraflagellar transport (IFT) is the major protein transport system in cilia and IFT has been implicated in tubulin transport. The IFT proteins IFT81 and IFT72, for example, form a module that binds tubulin dimers in vitro indicating that IFT particles interact directly with tubulin [1]. Direct in vivo imaging of tubulin during transport, however, has proved challenging [2]. To analyze ciliary tubulin transport and its regulation, we expressed GFP-αtubulin in wild-type Chlamydomonas reinhardtii. GFP-α-tubulin was incorporated into cytoplasmic and ciliary microtubules [3]; it was posttranslationally modified and formed heterodimers with β-tubulin suggesting that GFP-tagged and endogenous α-tubulin behave largely similar. Total internal reflection fluorescence (TIRF) microscopy revealed that GFP-α-tubulin entered flagella by active transport and by diffusion. Two-color in vivo imaging showed that GFP-α-tubulin moved on IFT trains inside cilia; thus, IFT functions as a tubulin transporter. Without of IFT, GFP-α-tubulin continued to enter cilia by diffusion. To assess how tubulin interacts with IFT, we expressed modified α- and β- GFP-tubulins; this ongoing project revealed C-terminally truncated GFP-α-tubulin is still transported by IFT and assembled into the axoneme. During ciliary growth, IFT-based transport of GFP-α-tubulin was increased ~15x over steadystate frequencies and, in growing cilia, the occupancy rate (% of IFT particles carrying GFP-α-tubulin as a cargo) increased from ~10% to ~80% indicating that tubulin transport by IFT is regulated. Fluorescence recovery after photobleaching (FRAP) analysis and western blotting revealed a strong increase in the concentration of soluble GFP-α-tubulin in regenerating over steady-state cilia. In cells possessing both growing and non-growing cilia, tubulin transport via IFT was elevated only in the growing cilia; likewise, the concentration of tubulin was increased solely in the matrix of the growing cilia. Thus, cells can direct tubulin flux specifically into growing cilia and established distinct concentrations of soluble tubulin within different cilia of a given cell. Our data suggest that tubulin transport via IFT is regulated locally in a flagella autonomous manner, likely within the basal body-cilium entity. We propose a model in which IFT functions as a tubulin pump to increase the concentration of soluble tubulin inside cilia, which will

SUNDAY-POSTER PRESENTATIONS promote the elongation of axonemal microtubules and thereby ciliary growth. 1Bhogaraju et al. 2013. Science 41:1009. 2Hao et al. 2011. Nat Cell Biol 13:790. 3Rasala et al. 2013. Plant J 74:545.

P148 Board Number: B255

A novel Chlamydomonas IFT81 mutant reveals that the IFT81 tubulin-binding domain is not crucial for tubulin transport and flagellar assembly. T. Kubo1, J.M. Brown1, G.B. Witman1; 1 Department of Cell and Developmental Biology, Univ Massachusetts Med Sch, Worcester, MA Intraflagellar transport (IFT) is a bidirectional motility of IFT complexes A and B along cilia/flagella, driven by the anterograde motor kinesin-II and the retrograde motor cytoplasmic dynein 1b. IFT is important for the assembly and maintenance of cilia/flagella. Here, we report a novel Chlamydomonas insertional mutant ift81-1 in which the hygromycin-resistance gene used for introducing and identifying the mutation has inserted into exon 7 of IFT81, resulting in an apparently null mutant. Cells of ift81-1 fail to assemble flagella; this phenotype is similar to that of ift74-2, a null mutation for IFT74, which interacts with IFT81 in IFT complex B (Brown et al., in preparation). Transformation of ift81-1 with the wild-type IFT81 gene rescued flagellar assembly and function, confirming that the phenotype is due to loss of IFT81. Recently, it was proposed, based primarily on in vitro experiments, that the calponin-homology domain of IFT81 together with the highly basic N-terminal domain of IFT74 forms a tubulin-binding module that is the basis for tubulin transport within cilia (Bhogaraju et al., 2013). The ift81-1 mutant has provided an opportunity to test this hypothesis in vivo. Within its calponin-homology domain, IFT81 has five highly conserved residues (K73, R75, R85, K112, and R113 in Chlamydomonas) which have been implicated in tubulin binding (Bhogaraju et al., 2013). To assess their importance in tubulin transport and flagellar assembly, we rescued ift81-1 with constructs expressing versions of IFT81 with one or more of these basic residues changed to glutamate. Surprisingly, all the resulting strains, including one in which four out of five of the basic residues were substituted to glutamate (K73R75K112R113/EEEE), had normal IFT and nearly normal length flagella. Substitution of one or two residues (K73R75/EE, R85E, and K112R113/EE) had no effect on flagellar regeneration kinetics, indicating normal tubulin incorporation into the cell’s axoneme. More severe mutations (K73R75R85/EEE and K73R75K112R113/EEEE) resulted in slightly slower flagellar regeneration, suggesting a subtle reduction in tubulin incorporation. Because the effects of modifying the putative tubulin-binding residues of IFT81 were mild, we propose that tubulin binding at the IFT81/IFT74 site is mediated primarily by IFT74; alternatively, tubulin transport may be supplemented by diffusion or binding to other IFT-particle proteins.

SUNDAY-POSTER PRESENTATIONS

P149 Board Number: B256

Chlamydomonas IFT43 is essential for the integrity of IFT complex A and required for flagellar assembly. B. Zhu1, J. Pan2; 1 Tsinghua, Beijing, China, 2School of Life Sciences, Tsinghua University, Beijing, China The assembly and maintenance of flagella and cilia depends on Intraflagellar transport (IFT). Mutations in the gene encoding IFT43, a subunit of the IFT complex A , cause Sensenbrenner syndrome in humans. In our genetic screens, we find an aflagellate insertional mutant that is disrupted in IFT43, identified through RESDA based PCR cloning strategy. Most mutant cells form clumps of 4 cells within one mother cell wall. After lysin treatment to release the cells from mother cell wall, no cells with flagella could be detected under light microscope. Transformation of the mutants with HA-tagged IFT43 rescued the mutant phenotype, confirming that IFT43 is the causal gene. Thus. IFT43 is essential for flagellar assembly. Similar to other IFT proteins, IFT43 is enriched at the basal body region and present in the flagella. YFP-tagged IFT43 exhibits intraflagellar transport and IFT43 is a part of IFT A complex as evidenced by sucrose gradient analyses and co-immunoprecipitation. IFT43 affects the stability and integrity of IFT-A but not complex B in that the amount of IFT139 in the mutants was reduced and a smaller IFT-A complex was formed in the ift43 mutant. EM analysis showed that ift43 mutant has residual flagella that barely protrude out of the flagellar collar.. Surprisingly, no IFT particles were found to accumulate in the short flagella revealed by EM in the ift43 mutants. The possible function of IFT43 in IFT will be discussed.

P150 Board Number: B257

Chlamydomonas MKS1 is required for fully assembly of flagella beyond the transition zone. W. Chen1, H. Ho2, C. Tsao1; 1 Department of Biological Science and Technology, National University of Tainan, Tainan, Taiwan, 2 Department of Anatomy, Tzu-Chi University, Hualien, Taiwan A Chlamydomonas mutant strain Lu3 was obtained by insertional mutagenesis and a visual screen for motility defects. Genotyping revealed that the insertion of an exogenous marker in the Lu3 genome causes a deletion of a 60-kb region, which contains 14 genes including flagellar inner dynein arm I1 intermediate chain IC140 and Chlamydomonas MKS1 ortholog. MKS1 is a component of the transition zone complex, and the defect of human MKS1 leads to Meckel-Gruber Syndrome. The growth of Chlamydomonas Lu3 strain showed no difference comparing to the cw15 reference strain although Lu3 lost normal motility. Unlike Chlamydomonas cep290 mutant, which lacks a transition zone protein required for integrity of the microtubule-membrane connector at the flagellar base but could still

SUNDAY-POSTER PRESENTATIONS assemble axonemes (Craige et al. 2010), morphology of the transition zone in Lu3 cells appeared normal. Lu3 cells failed to assemble full length flagella, and the axonemal doublet microtubule terminated abruptly distal to the transition zone. These observations indicate that Chlamydomonas MKS1 is required for the fully assembly of axonemes beyond the transition zone but is not essential for the microtubule-membrane connector.

P151 Board Number: B258

Chlamydomonas CC2D2A localizes to the proximal flagellar transition zone and is required for normal flagellar protein content and flagellar assembly. B. Craige1, J.M. Brown1, Y. Hou1, G.B. Witman1; 1 Department of Cell and Developmental Biology, Univ Massachusetts Med Sch, Worcester, MA The transition zone (TZ), the region at the base of cilia and flagella where the triplet microtubules of the basal body transition into doublets, contains a large number of highly conserved proteins whose cognate genes are often mutated in human ciliopathies. Mutations in CC2D2A result in severe ciliopathies characterized by pleiotropic developmental defects and neonatal lethality (Meckel syndrome), or cystic kidney disease, blindness, brain malformation, and mental deficit (Joubert syndrome). In vertebrates, CC2D2A has been reported to localize to the TZ and interact with other TZ proteins; however, the specific molecular function of CC2D2A is unknown. We are using multiple Chlamydomonas TZ mutants to elucidate the functional, structural, and biochemical consequences of disruption of TZ integrity, and we have isolated a mutant harboring an insertion in an exon near the middle of Chlamydomonas CC2D2A. The mutant cells have defects in flagellar assembly and fail to hatch out of the mother cell wall following mitosis, but cells grow flagella following digestion of the mother cell wall by autolysin treatment. In each of two rounds of backcrossing to wild-type cells, the mutation segregated 100% with the phenotype, and the phenotype was completely rescued by transformation with a genomic construct encoding HA-tagged CC2D2A, indicating that the phenotype results from the mutation in CC2D2A. CC2D2A-HA localizes to the TZ, slightly proximal to and partially overlapping with the TZ protein CEP290. CEP290, which is required for normal assembly of the microtubule-membrane Y connectors within the TZ, localizes normally within the TZ of the cc2d2a mutant; consistent with this, the Y connectors are unperturbed in the cc2d2a mutant. Conversely, in the absence of CEP290, CC2D2A-HA no longer localizes to the TZ, and instead accumulates on or near the basal bodies, indicating that CEP290 and/or the Y connectors are required to localize CC2D2A to the TZ. A third TZ protein, NPHP4, which is located distal to CEP290 in the TZ, is not required to localize either CEP290 or CC2D2A to the TZ, and is also dispensable for Y connector formation. A triple-mutant strain, harboring the cep290, nphp4, and cc2d2a mutations, displays slower growth kinetics, indicating that loss of all three proteins might perturb cell cycle progression. Finally, biochemical analysis of isolated cc2d2a mutant flagella revealed protein content abnormalities in the membrane + matrix fraction of the isolated flagella, indicating that CC2D2A, like CEP290 and NPHP4, is required for establishing and/or maintaining normal flagellar protein content.

SUNDAY-POSTER PRESENTATIONS

P152 Board Number: B259

Architectural variation at the base of the cilia is required for cilia structure and function diversity. S.C. Jana1, P. Machado1,2, J. Rocha1, S. Mendonça1, S. Werner1, M. Bettencourt-Dias1; 1 Instituto Gulbenkian de Ciência, Oeiras, Portugal, 2EMBL, Heidelberg, Germany Cilia are microtubule -based membrane protrusions conserved across evolution involved in cell motility, fluid flow and sensing. The diversity in functions is generally attributed to a core conserved microtubulebased structure, the axoneme, decorated by different structures, membrane and signaling systems. Here we study four classes of cilia that represent very diverse motility and sensory functions within a single organism, the fruit fly. We uncover that the base of the cilia, the basal body and transition zone, is much more diverse than previously thought, showing large variation in number, length, ultrastructure, and connection to other cellular structures. We further demonstrate that basal body diversity is imparted by differential regulation of evolutionarily conserved core components. The tissue specific regulation of core basal body and transition zone genes suggests mechanisms that generate tissue specific phenotypes in human ciliopatic syndromes.

P153 Board Number: B260

Kinesin-2 and kinesin-9 are required for ciliogenesis during rapid development. E.J. Tomei1, S.M. Wolniak1; 1 Cell Biology and Molecular Genetics, University of Maryland, College Park, MD Spermatogenesis in the semi-aquatic fern, Marsilea vestita, is a rapid, synchronous process that is initiated when dry microspores are placed in water. Development is post-transcriptionally driven and can be divided into two phases. The first phase consists of a series of mitotic divisions that produce 7 sterile cells and 32 spermatids. During the second phase, each spermatid differentiates into a corkscrewshaped motile spermatozoid with ~144 cilia. Ciliogenesis occurs on basal bodies that are positioned in two rows at regular intervals along the dorsal face of a coiled microtubule ribbon. Kinesin motor proteins are known to participate in both mitosis and ciliogenesis and provide the unique opportunity to study the mechanisms that regulate both stages of development using one class of proteins. Through RNAseq, we assembled a transcriptome that shows the abundance of transcripts throughout development. Over 150 kinesin-like sequences were identified in this transcriptome that represent at least 57 unique kinesin transcripts. Based on alignments of the kinesin motor domain, members of the kinesin-2, -4/10, -5, -7, -8, -9, -13, -14, -15, -16, -18, and -19 families as well as several orphan kinesins were found. This complement of kinesins is similar to other plants that make ciliated spermatozoids, such as P. patens. In M. vestita most of the kinesin transcripts (84%) change in abundance during gametophyte development. Kinesin mRNAs that decrease in abundance (44%) encode proteins thought to be involved in mitosis, while those that increase in abundance during development (40%) have

SUNDAY-POSTER PRESENTATIONS proposed roles in ciliogenesis. Kinesin-2 and -9 are only found in ciliated organisms, and the abundance of these transcripts increases ~12-fold during later stages of spermatogenesis. This increase in transcript abundance is independent of transcription and is instead results from the unmasking and processing of stored pre-mRNAs. RNAi knockdowns show that kinesin-2 is required for the later divisions of spermatogenous cells and that both kinesin-2 and -9 are required for de novo basal body formation and ciliogenesis. Without kinesin-2, most spermatozoids contain two or more coiled microtubule ribbons with attached cilia and very large cell bodies. Those that emerge from the spore have cilia that are substantially longer than controls. Kinesin-9 knockdowns have basal bodies that are irregularly positioned and cilia are not localized in the anterior portion of the spermatozoid. Spermatozoid swimming behavior in kinesin-2 and -9 knockdowns is altered because of resultant defects in ciliogenesis. Kinesin-2 knockdowns can only swim or quiver in place while kinesin-9 knockdowns swim erratically compared to controls. (Supported by NSF grant MCB-0842525 to SMW).

P154 Board Number: B261

Meckelin guides orientation of basal bodies along the striated rootlet. J. Van Houten1, T. Picariello1, M. Valentine1, A. Nabi1, J. Yano1; 1 Univ Vermont, Burlington, VT Meckelin (MKS3) is a protein of the primary cilia transition zone that functions in ciliogenesis and ciliary gating. MKS3 appears to have similar functions and location in Paramecium tetraurelia since FLAGMKS3 is found associated with and slightly above each basal body and RNAi for MKS3 leads to loss of cilia. However, RNAi for MKS3 also leads to the disorganization of rows of basal bodies that normally run from anterior to posterior. In the areas of misalignments, the basal bodies with their post ciliary and transverse rootlets are found rotated and out of their rows. However, the rootlets are attached to the basal bodies at the expected angles relative to each other. We propose that MKS3 guides new basal bodies as they move toward the anterior of the cell along the striated rootlet (SR) of the parent basal body. These SRs are aligned along the rows of basal bodies; new basal bodies do not yet have their own SR. The loss of MKS3 results in loss of interactions between the new basal body and the parent basal body SR. Without a guide to maintain orientation, the new basal bodies migrate out their rows and out of line and, when they form their SRs, these rootlets do not project toward the anterior as expected. To test for interactions of MKS3 with SR components, we first identified potential SR proteins, expressed them with epitope tags and determined whether they were located in the SR. Of the 24 potential SR protein genes, 13 were selected as representative of sets of paralogs for epitope tagging. Nine of them were associated with the SR, often with non-uniform distributions. Those sequences with SF assemblin domains (similar to those in the Chlamydomonas rootlet proteins) coded for proteins that we found in the Paramecium SRs; conversely those without this domain were not in the rootlets. MKS3 interacted sufficiently (directly or indirectly) with the striated rootlet proteins to pull them down with a GST-fusion of the 252 C-terminal residues of the Paramecium MKS3. We thank J. Beisson for the anti-SR antibody. P20 GM103449; P30 GM103498; R01 GM 59988

SUNDAY-POSTER PRESENTATIONS

P155 Board Number: B262

Asymmetrically localized Fop1 stabilizes basal bodies. B.A. Bayless1, T. Giddings2, I.M. Cheeseman3, C.G. Pearson4; 1 Cells, Stem Cells and Development, University of Colorado- Anschutz Medical Center, Aurora, CO, 2 Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 3 Whitehead Inst Biomed Res, Cambridge, MA, 4Department of Cell and Developmental Biology, University of Colorado-Anschutz Medical Center, Aurora, CO Motile cilia are necessary for directed extracellular fluid flow. Basal bodies position the cilium and anchor the mechanical forces produced by ciliary beating. The basal body is made up of a set of nine triplet microtubule blades arranged in a cylinder. These microtubules are somehow stabilized to maintain the basal body during ciliary beating. The ciliary beat pattern is asymmetric, a unidirectional power stroke is followed by a refractory stroke which occurs along the same axis. The bulk of the force that the basal body experiences from ciliary beating likely occurs along this axis. The basal body assembly factor Bld10/Cep135 is also necessary to stabilize basal bodies, in bld10Δ cells, triplet microtubules are unstable and their disassembly initiates at the triplet microtubules along the axis of the cilium beat stroke. Inhibiting ciliary beating rescues the basal body disassembly phenotype suggesting that Bld10 stabilizes the triplet microtubules from the forces of ciliary beating. Poc1 and Fop1 interact with Bld10, stabilize basal bodies, and localize along the entire length of the basal body. We examined the relationship between Poc1 and Fop1, and find that Poc1 is necessary for the temporal loading of Fop1 into the basal body. Interestingly, Fop1 localizes asymmetrically to the basal body at triplet microtubules that align with the axis of the ciliary beat stroke. We hypothesize that Fop1 helps to stabilize the triplet microtubules on the sides of the basal body that experience the most force from ciliary beating. Taken together, our data suggest that Bld10, Fop1 and Poc1 are stability factors for the basal body and Poc1 helps to load Fop1 asymmetrically along the triplet microtubules.

P156 Board Number: B263

SPEF1 regulates the length of central microtubules in 9+2 motile cilia. M. Guha1; 1 Cellular Biology, The University of Georgia, Athens, GA SPEF1 is a microtubule-binding protein with a calponin homology (CH) domain, and structural similarity to the End Binding (EB) plus end tracking proteins. SPEF1 homologs are present in most ciliated organisms. The ciliate Tetrahymena thermophila has two paralogs of SPEF1. Cells lacking both of these paralogs swim very slowly and have fewer but normal length cilia. Ultrastructural studies revealed a mixture of normal 9+2 and abnormal 9+1 or 9+0 axoneme cross-sections, indicating defects in the assembly of central microtubules. Longitudinal sections revealed that frequently the proximal portions of the central microtubules (near the minus ends) were truncated. Purified SPEF1 bound to microtubules

SUNDAY-POSTER PRESENTATIONS in a sedimentation assay. Phylogenetic studies revealed that SPEF1 is absent in species with cilia naturally lacking central microtubules. Taken together, these results suggest that SPEF1 regulates the length of the central microtubules; possibly by affecting the stability of their minus ends. Interestingly, a knockout strain lacking a conserved central pair protein, PF20, also has fewer cilia. Thus, in Tetrahymena defects in the central apparatus are associated with reduced ciliogenesis.

P157 Board Number: B264

Cep164, but not EB1, is essential for centriolar localization of TTBK2 and its function in ciliogenesis. T. Nagai1, O. Toshiaki1, S. Chiba1, K. Mizuno1; 1 Tohoku Univ., Sendai, Japan Primary cilia are microtubule-based antenna-like organelles that emanates from the mother centriolederived basal body in most quiescent mammalian cells. Primary cilia transduce chemical and mechanical signals from the extracellular milieu and play critical roles in the development and homeostasis of many tissues. Defects in formation and/or function of primary cilia are associated with many human diseases, including polycystic kidney disease, retinal degeneration and neurological defects. In ciliogenesis, a centriolar protein CP110 is removed from the distal region of the mother centriole, which allows the mother centriole to move and dock to the plasma membrane via the distal appendages of the mother centriole and to nucleate the ciliary axoneme. CP110 and its interactor Cep97 suppress ciliogenesis by blocking axonemal microtubule assembly; therefore, removal of CP110 from the mother centriole is required for initiating axoneme extension and ciliogenesis. Tau tubulin kinase-2 (TTBK2) has an essential role in initiation of ciliogenesis by promoting CP110 removal and recruitment of intraflagellar transport (IFT) protein complexes. When cells are growth-arrested by serum starvation, TTBK2 is recruited to the distal region of the mother centriole. Therefore, the centriolar localization of TTBK2 is a key step in initiation of ciliogenesis. Previous studies demonstrated that TTBK2 binds to EB1 and Cep164, both of which are essential for ciliogenesis. However, the roles of the binding of these proteins in ciliogenesis and centriolar localization of TTBK2 have remained unclear. To elucidate the mechanisms underlying centriolar localization of TTBK2, we analyzed the roles of EB1 and Cep164 in TTBK2 localization. TTBK2 bound to EB1 and Cep164 through two SxIP motifs and a proline-rich motif in the C-terminal region of TTBK2, respectively. TTBK2 mutants at SxIP motifs did not localize at the plus-ends of microtubules, but localized at the mother centriole, and they rescued the defect in ciliogenesis upon TTBK2 knockdown. These results suggest that EB1 is not essential for centriolar localization of TTBK2. In contrast, depletion of Cep164 or overexpression of Cep164-N, which can bind to TTBK2 but not localize on the centriole, suppressed centriolar localization of TTBK2. Furthermore, non-Cep164-binding mutants of TTBK2 at the proline-rich motif did not rescue the defects in ciliogenesis and CP110 removal upon TTBK2 knockdown. These results suggest that Cep164, but not EB1 is essential for centriolar localization of TTBK2 in the initiation of ciliogenesis. We also showed that TTBK2 phosphorylates Cep164 and Cep97 and inhibits the interaction between Cep164 and its binding protein Dishevelled-3.

SUNDAY-POSTER PRESENTATIONS

P158 Board Number: B265

The G-domain of the Joubert syndrome protein ARL13B is required for anchoring to the axoneme and uniform distribution along the cilium. E. Revenkova1, G.L. Gusella2, T. Tedeschi1, C. Iomini1; Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY, 2Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 1

Cilia-mediated signal transduction involves precise targeting and localization of selected molecules along the ciliary membrane, but the molecular mechanism underlying these events is unclear. ARL13B is a membrane-associated small G-protein that localizes along the cilium and functions in ciliary protein transport and signaling. In human, mutations of ARL13B cause Joubert syndrome, and in model organisms ARL13B deficiencies lead to structural defects in cilia and mislocalization of ciliary proteins. To gain mechanistic insight into ARL13B activity and positioning along the ciliary membrane, we sought to identify interaction partners essential for its function in the cilium. Using a tandem affinity purification (TAP) approach and mass spectrometry, we found that the major proteins co-purifying with TAP-tagged ARL13B from ciliated RPE cells were alpha- and beta-tubulins. This interaction was confirmed by immunoprecipitation of both GST-tagged and endogenous ARL13B from cell extracts. Moreover, GST pull-down assay using a mixture of purified GST-ARL13B and purified tubulin demonstrated that the interaction with tubulin is direct. Mapping of the tubulin–interacting regions of ARL13B revealed that the G-domain and significantly truncated fragments of the G-domain, but not the coiled-coil region, interact with soluble tubulin. To address the function of the G-domain within the ciliary compartment, we obtained an ARL13B mutant lacking the G-domain (ARL13B-ΔGD) but retaning the ciliary localization. In wild-type mouse embryonic fibroblasts (MEFs), wild-type ARL13B or mutant ARL13B-ΔGD, which were expressed as GFP fusions using lentiviral vectors, followed the distribution of the endogenous ARL13B along the whole cilium. In contrast to the even distribution in wild-type cells, in Arl13b null (hnn) MEFs ARL13B-ΔGD accumulated at the distal tip and dramatically diminished along the cilium. While the overexpression of ARL13B-ΔGD caused cilium elongation in wild-type cells, it failed to rescue shortened cilia in hnn MEFs. To test whether ARL13B G-domain is required for anchoring ARL13B to the ciliary axoneme, we performed in vitro pull down of GST-ARL13B fragments using axonemes purified from Chlamydomonas flagella. The assay showed that both full-length ARL13B and the G-domain bind isolated axonemes, while the ARL13B coil-coil domain does not, indicating that ARL13B interacts with axoneme through its G-domain capable of binding to tubulin. We conclude that the G-domain of ARL13B is not required for trafficking to the cilium but it is indispensable for proper axonemal distribution of ARL13B.

SUNDAY-POSTER PRESENTATIONS

P159 Board Number: B266

Primary cilia in neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide. Q. He1, G. Wang1, M.B. Dinkins1, S. Wakada1, S. Dasgupta1, J. Kong1, S.D. Spassieva2, E. Bieberich1; Department of Neuroscience and Regenerative Medicine, Georgia Regents University, Augusta, GA, 2 Med Univ South Carolina, Charleston, SC 1

We show here that human embryonic stem (ES) and induced pluripotent stem (iPS) cell - derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC (aPKC), both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes. Neural differentiation of human ES cells to NPs is concurrent with a 3-fold elevation of ceramide, in particular saturated, long chain C16:0 ceramide (N-palmitoyl sphingosine) and non-saturated, very long chain C24:1 ceramide (Nnervonoyl sphingosine). Decreasing ceramide levels by inhibiting ceramide synthase or neutral sphingomyelinase 2 (nSMase2) leads to translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora kinase A (AurA).Inhibition of aPKC, AurA, or a downstream target of AurA, HDAC6, restores ciliogenesis in ceramide-depleted cells. Importantly, addition of exogenous C24:1 ceramide reestablishes membrane association of aPKC, restores primary cilia, and accelerates neuronal process formation. Taken together, these results suggest that ceramide prevents activation of HDAC6 by cytosolic aPKC and AurA, which promotes acetylation of tubulin in primary cilia and potentially, neuronal processes. This is the first report on the critical role for ceramide generated by nSMase2 in stem cell ciliogenesis and differentiation. (Supported by grant NSF00028 and AHASE00089)

P160 Board Number: B267

A new step in ciliogenesis: The ANO1 Nimbus. C.C. Ruppersburg1, P.G. Donlin-Asp1, G.J. Bassell1,2, C. Hartzell1; Cell Biology, Emory University School of Medicine, Atlanta, GA, 2Neurology, Emory University School of Medicine, Atlanta, GA 1

Many cells possess a single, non-motile, primary cilium that is thought to be a sensory transducer akin to a cellular antenna. Ciliogenesis involves migration of the basal body to the cell surface followed by outgrowth of the axoneme by intraflagellar transport. We have identified another early step in cilium development, formation of a novel structure - the nimbus. Prior to cilia extension, ANO1, a Ca2+activated Cl- channel, is organized into a torus-shaped structure along with other ciliary proteins including the small GTPases CDC42 and Arl13b, exocyst complex components, and acetylated α-tubulin and γ-tubulin. Furthermore, we have identified this region as an area of RNA localization. This structure we call the nimbus forms an interface between the microtubule cytoskeleton of the nascent cilium and

SUNDAY-POSTER PRESENTATIONS the surrounding cortical actin cytoskeleton. During ciliogenesis, the nimbus disassembles and ciliary components, including ANO1, move into the cilium. Our data support a model where the nimbus provides a scaffold for staging of ciliary components and cilia assembly and expands to include the potential role of RNA localization in cilium formation.

P161 Board Number: B268

Actin and microtubule-interacting protein Girdin promotes ciliogenesis by regulating basal body positioning. I. Nechipurenko1, A. Olivier-Mason1, J. Kennedy2, O.E. Blacque2, P. Sengupta1; 1 Biology, Brandeis University, Waltham, MA, 2Univ Coll Dublin, Dublin, Ireland Primary cilia are present on the surface of most cell types in metazoans and have recently emerged as key mediators of diverse signaling pathways critical for embryonic development and adult tissue homeostasis. Cilia from all studied organisms share a conserved core organization comprised of a microtubule-based axoneme surrounded by a specialized membrane. Primary cilium assembly is a multistep process that relies on mother centriole-to-basal body (BB) maturation, BB migration and anchoring to membrane, establishment of the ‘ciliary gate’, and axoneme elongation from the BB via intraflagellar transport (IFT) of ciliary components. Although actin and microtubule networks are pivotal for ciliogenesis, the molecular mechanisms regulating cytoskeletal dynamics during different steps of cilia biogenesis are not well understood. We find that the actin and microtubule-interacting protein Girdin localizes to the BB in C. elegans sensory neurons and mammalian cells. Loss of girdin function in C. elegans results in defective sensory cilia morphology, ultrastructure, and localization of BB and transition zone proteins. Similarly, Girdin knockdown in mammalian cells leads to loss of, or shortened cilia. Analysis of cytoskeletal organization shows that both actin and microtubule networks may be disrupted in mammalian cells upon Girdin knockdown, leading to altered BB positioning and decreased IFT accumulation at the ciliary base. Mammalian Girdin is known to couple extracellular cues to actin dynamics during cell migration. Our preliminary findings suggest that it may also coordinate cytoskeletal dynamics to ensure proper BB positioning and cilia assembly. We will present our ongoing efforts to further characterize a novel role for this highly conserved protein in primary cilia biogenesis and function in C.elegans sensory neurons and mammalian cells.

SUNDAY-POSTER PRESENTATIONS

Centrosome Assembly and Functions 1 P162 Board Number: B270

The centrosome is also an actin-organizing center, ATOC. F. Farina1, J. Gaillard1, C. Guérin1, L. Blanchoin1, J. Sillibourne*2, M. Théry2; 1 iRTSV, CEA, Grenoble, France, 2iRTSV, CEA, Paris, France The centrosome is the main microtubule-organizing centre. It is involved in the regulation of cell polarity, cell motility and cell division. Although centrosome functions mainly depend on microtubules, the actin network has been shown to be involved in several key centrosome activities such as centrosome positioning at the cell centre, membrane docking during ciliogenesis or immune synapse formation as well as mitotic spindle assembly and positioning. However, the centrosome-actin connection has remained elusive. Actin and actin-binding proteins have been detected at the centrosome by proteomic analyses and immuno-stainings, but experimental data demonstrating the capacity of centrosome to organize actin filaments and interact with the actin network are still missing. Here, we provide the first compelling evidence that centrosomes assemble and anchor actin filaments. Centrosomes were isolated from human T lymphocytes expressing eGFP-centrin. As expected, they induced the nucleation of microtubules in the presence of purified tubulin dimers, but they also promoted the assembly of large radial arrays of actin filaments in the presence of actin monomers. These networks appeared to stem from short filaments, stably and tightly bound to the purified centrosomes. These filaments could not be disassembled by actin depolymerizing drugs. Highly stable actin filaments were also present at the centrosome of living cells. Centrosome displayed the same capacity to promote the growth of long filaments in the presence of purified actin monomers after removal of cell membrane. The actin organizing property of centrosome in living cells was further demonstrated by time-lapse monitoring of lymphocytes, in which a cytoplasmic actin cloud permanently accompanied the moving centrosome. These results point toward a new role for the centrosome as an actin-organizing centre. They shine some light on numerous prior observations of centrosome and actin network mutually influencing each other.

SUNDAY-POSTER PRESENTATIONS

P163 Board Number: B271

Centrosomes promote efficient spindle assembly, maintain cell viability, and help orient spindles in epithelial tissue during development. J.S. Poulton1, J.C. Cuningham1, M. Peifer1; 1 Department of Biology, University North Carolina, Chapel Hill, NC Mitotic spindles are critical for accurate chromosome segregation. Centrosomes, the primary microtubule nucleating centers of animal cells, play key roles in forming and orienting mitotic spindles. However, the survival of Drosphila without centrosomes suggested they are dispensable in somatic cells, challenging the canonical view. We used fly wing disc epithelia as a model to resolve these conflicting hypotheses, revealing that centrosomes play vital roles in spindle assembly, function, and orientation. Many acentrosomal cells exhibit prolonged spindle assembly, chromosome mis-segregation, DNA damage, misoriented divisions, and eventual apoptosis. We found that multiple mechanisms buffer the effects of centrosome loss, including alternative microtubule nucleation pathways (Augmin and RanGTP) and the Spindle Assembly Checkpoint (SAC). Apoptosis of acentrosomal cells is mediated by JNK signaling, which also drives compensatory proliferation to maintain tissue integrity and viability. These data reveal the importance of centrosomes in fly epithelia, but also demonstrate the robust compensatory mechanisms at the cellular and organismal level. Interestingly, these mechanisms are insufficient when we disrupt the SAC in acentrosomal cells. We are currently exploring how this affects brain development, which is grossly perturbed in the absence of both centrosomes and the SAC, and may serve as a model for microcephaly.

P164 Board Number: B272

The centrosome as a phoenix organelle: burned by its own proteasome during fever and revived by its own molecular chaperone Hsp70. S. Doxsey1, A. Vertii2; 1 Univ Massachusetts Med Sch, Worcester, MA, 2PMM, Univ Massachusetts Med Sch, Worcester, MA The centrosome is a non-membranous organelle that performs many essential functions including microtubule nucleation/organization, ciliogenesis, cell division, membrane trafficking, and immunological synapse formation. The immune response during inflammation is often accompanied by elevated body temperature. Although the detrimental effect of heat shock on centrosome is established, the physiological relevance and mechanisms of centrosome damage remain elusive. Here we provide evidence that the febrile condition results in centrosome damage in patients’ leukocytes. We find that local proteasome machinery at the centrosome is responsible for centrosome damage and functional inactivation of centrosome substructures. This heat-induced centrosome degradation pathway appears to be unique to the centrosome, as another non-membranous organelle, the midbody,

SUNDAY-POSTER PRESENTATIONS remains unchanged following heat shock. Forced targeting of the molecular chaperone, Hsp70-GFP, to the centrosome prevents centrosome inactivation, whereas centrosome-excluded Hsp70 or chaperoneinactive mutants fail to protect the centrosome. This novel mechanism of centrosome regulation during fever is an important aspect of the previously demonstrated role of centrosomes in the immune response.

P165 Board Number: B273

Plk1 controls PCM assembly by phosphorylating SPD-2 and SPD-5 in C. elegans. S.D. Ochoa1, V. Viscardi1, E. Zanin1, S. Bahmanyar1, A. Desai1, K. Oegema1; 1 Ludwig Institute for Cancer Research, La Jolla, CA Centrosomes, the major microtubule organizing centers in animal cells, are composed of a centriole pair surrounded by a matrix of pericentriolar material (PCM) that nucleates and anchors microtubules. Centrioles direct assembly of the PCM, which expands during mitotic entry in a process termed centrosome maturation; the expanded PCM is subsequently disassembled during mitotic exit. The mechanisms that restrict PCM assembly around centrioles and regulate its cell cycle dynamics remain poorly understood. Here we use the C. elegans embryo to analyze the regulation of PCM assembly. Genome-wide screens have identified 4 C. elegans proteins that are required for PCM assembly. SPD-5 is a large coiled-coil protein that is thought to be the major component of the PCM matrix. PCM assembly also requires SPD-2, Aurora A, and the polo family kinase Plk1. We confirmed the essential role of Plk1 in PCM assembly by constructing C. elegans strains in which the only source of PLK-1 was a single copy transgene expressing either wild-type PLK-1 (PLK-1WT) or an analog-sensitive “Shokat” mutant (PLK1AS), in which the catalytic pocket was mutated to accept bulky purine analogs and enable kinase inhibition by the cell-permeable PP1 analog 1NM-PP1. Strains also included a transgene expressing the centrosomal marker GFP::γ-tubulin to monitor the PCM. Addition of 1NM-PP1 to permeabilized embryos during S-phase prior to nuclear envelope breakdown abolished mitotic PCM expansion in embryos carrying the plk-1AS, but not the plk-1WT, transgene. Addition of 1NM-PP1 to embryos arrested in metaphase with a proteasome inhibitor also led to rapid disassembly of the mitotic PCM in embryos carrying the plk-1AS, but not the plk-1WT, transgene, indicating that Plk1 activity is essential for both establishment and maintenance of mitotic PCM. To understand how Plk1 contributes to PCM assembly, we are analyzing the consequences of mutating all predicted Plk1 sites that are conserved among nematodes, either individually or in localized groups, in SPD-2 (7 sites) and SPD-5 (25 sites). We have already identified 2 Plk1 sites in SPD-2 and 2 sites in SPD-5 that are essential for PCM assembly, suggesting that Plk1 regulates SPD-5 polymerization by phosphorylating both SPD-2 and SPD-5.

SUNDAY-POSTER PRESENTATIONS

P166 Board Number: B274

Regulated assembly of a supramolecular centrosome scaffold in vitro. J. Woodruff1, O. Wueseke1, V. Viscardi2, J. Mahamid3, K. Oegema2, A. Hyman1; 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 2Ludwig Institute for Cancer Research, La Jolla, CA, 3Max Planck Institute of Biochemistry, Martinsried, Germany A centrosome comprises a pair of centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite its importance as major microtubule-organizing center, the mechanism of PCM assembly and its regulation are not understood. In a C. elegans embryo, the coiledcoil protein SPD-5 is essential for PCM assembly. We found that recombinant SPD-5 can polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Interestingly, only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Our results suggest that PCM size and binding capacity emerge from the regulated formation of a porous network from one coiled-coil protein. Furthermore, these results suggest that PCM growth is autocatalytic, such that polymerized SPD-5 can recruit additional SPD-5 and its own positive regulators, leading to rapid network expansion.

P167 Board Number: B275

Plk1 binds to the two distinct phospho-motifs on a centrosomal scaffold, Cep192, to promote bipolar spindle formation. L. Meng1, J. Park2, T. Kim2, K. Lee2; 1 National Institute of Health, Bethesda, MD, 2NIH/NCI, Bethesda, MD Serving as microtubule (MT)-organizing centers, centrosomes play a key role in bipolar spindle formation during mitosis. A centrosomal scaffold protein, Cep192, is required for proper centrosome organization and bipolar spindle assembly. However, how it promotes these events remains largely unknown. A recent study with Xenopus extracts suggested that Plx1 interacts with the p-T46 motif of Cep192 to form a Cep192-dependent AurA-Plx1 cascade critical for the establishment of bipolar spindles. Here we demonstrated that, in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner and this event is important to promote the reciprocal activation of the latter two enzymes at centrosomes. Strikingly, Plk1 interacted with human Cep192 through two distinct phospho-motifs—the p-T44 motif (which is analogous to the Xenopus p-T46 motif) and a yet uncharacterized p-S995 motif—in a manner that requires its C-terminal noncatalytic polo-box domain (PBD). Intriguingly, the interaction of Plk1 with the p-T44 motif required the presence of AurA, whereas the interaction with the p-T995 motif occurred only in the absence of AurA, suggesting that Plk1 binds to Cep192 in two biochemically distinguishable modes. Consistent with this view, loss of either one of these interactions induced a mild defect in bipolar spindle formation and mitotic progression as a result of improper recruitment of Plk1 and γ-

SUNDAY-POSTER PRESENTATIONS tubulin to centrosomes. Under the same conditions, elimination of both interactions induced a greatly enhanced defect in these processes, suggesting that the defect associated with the loss of T44- and S995-dependent interactions is additive. Taken together, we propose that Plk1 forms two functionally non-overlapping complexes with Cep192 to promote centrosome-based bipolar spindle formation and therefore proper M-phase progression.

P168 Board Number: B276

Measuring the in-vivo Activity of Separase at Centrosome. F.G. Agircan1, E. Schiebel1; 1 ZMBH, Heidelberg, Germany Separase is best known for its function in sister chromatid separation at the metaphase-anaphase transition. It also has a role in centriole disengagement in late mitosis/G1. To gain insight into the activity of separase at centrosomes, we developed two separase activity sensors: mCherry-Scc1(142-467)∆NLS -eGFP-PACT and mCherry-kendrin(2059-2398)-eGFP-PACT. Both localize to the centrosomes and enabled us to monitor local separase activity at the centrosome in real time. Both centrosomal sensors were cleaved by separase before anaphase onset, earlier than the corresponding H2B-mCherry-Scc1(142-467)eGFP sensor at chromosomes. This indicates that substrate cleavage by separase is not synchronous in the cells. Depletion of the proteins astrin or Aki1, which have been described as inhibitors of centrosomal separase, did not led to a significant activation of separase at centrosomes, emphasizing the importance of direct separase activity measurements at the centrosomes. Inhibition of polo-like kinase Plk1, on the other hand, decreased the separase activity towards the Scc1 but not the kendrin reporter. Together these findings indicate that Plk1 regulates separase activity at the level of substrate affinity at centrosomes and may explain in part the role of Plk1 in centriole disengagement.

P169 Board Number: B277

PLK1 phosphorylation is essential for the separase-dependent cleavage of pericentrin during mitotic exit. J. Kim1, K. Lee1, K. Rhee1; 1 Biological Sciences, Seoul National University, Seoul, Korea The centrosome duplication events are tightly linked to the cell cycle. In order to duplicate in the next cell cycle, mother and daughter centrioles should be disengaged at the end of mitosis. Therefore, centriole disengagement is considered a licensing step for centriole duplication. Separase is known to play an essential role in the centriole disengagement. We and others identified pericentrin as a substrate of separase. Here, we report the importance of PLK1 phosphorylation in the separasedependent cleavage of pericentrin. PLK1 phosphorylates multiple sites of pericentrin during mitotic

SUNDAY-POSTER PRESENTATIONS entry. We observed that separase could not cleave the phospho-resistant mutants of pericentrin during mitotic exit. Furthermore, centriole disengagement was inhibited in the cells rescued with the phosphoresistant mutants of pericentrin. Based on these results, we propose that PLK1 phosphorylation is a prerequisite step for separase-dependent cleavage of pericentrin and eventually for centriole disengagement.

P170 Board Number: B278

The RNA-binding Protein ATX-2 Interacts with SZY-20, and Opposes ZYG-1 to limit Centrosome Size. M. Stubenvoll1, J. Medley1, M. Irwin1, M. Song1; 1 Biological Sciences, Oakland University, Rochester, MI Centrosomes are critical sites for controlling microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules to form bipolar spindles. The szy-20 gene encodes a centrosome-associated RNA-binding protein that negatively regulates ZYG-1, a key centrosome duplication factor. It has been shown that szy-20 mutants possess enlarged centrosomes, causing abnormal microtubule processes and embryonic lethality. SZY-20 contains putative RNA-binding domains; mutating these domains perturbs RNA-binding by SZY-20 in vitro and its capacity to regulate centrosome size in vivo. To further elucidate the roles of SZY-20 and RNA-binding in regulating centrosome assembly and size, we sought to identify factors associated with SZY-20. By proteomics, we identified an RNA-binding protein ATX-2 reproducibly pulled-down with SZY20. Consistent with its physical association, depleting ATX-2 produces embryonic lethality and cell division defects seen in szy-20(bs52) embryos, including a failure of cytokinesis and polar body extrusion, and abnormal spindle positioning. Thus, ATX-2 is required for proper cell divisions in which atx-2 acts synergistically with szy-20: Knocking down ATX-2 in szy-20(bs52) mutants enhanced the embryonic lethality and cell division defects. Genetic analyses indicate that not only szy-20 but also atx2 negatively regulates centrosome assembly. atx-2(RNAi) partially restores centrosome duplication to zyg-1(it25) embryos. Knocking down both szy-20 and atx-2 almost completely restored bipolar spindle formation to zyg-1(it25) embryos, suggesting a positive interaction between atx-2 and szy-20 in regulating centrosome assembly. Intriguingly, ATX-2-depleted embryos exhibit enlarged centrosomes as shown in szy-20(bs52). Embryos partially depleted of ATX-2 possess significantly increased ZYG-1 and pericentriolar materials at centrosomes. Enlarged centrosomes are more evident in a null allele atx2(ne4297) embryos, suggesting an inverse correlation between the amount of ATX-2 and centrosome size. Quantitative western and immunostaining analyses show that ATX-2 is nearly absent in szy20(bs52), much similarly in atx-2(ne4297), but unaffected in zyg-1 mutants. In contrast, SZY-20 levels are unaffected in atx-2 or zyg-1 mutants. Thus, SZY-20 acts upstream of ATX-2 and promotes the level of ATX-2, which opposes ZYG-1 to limit centrosome size. In this study we identify ATX-2 as a negative regulator of centrosome assembly and size.

SUNDAY-POSTER PRESENTATIONS

P171 Board Number: B279

Determining the function of the protein PCID2 at the centrosome. N. Schramm1, K. Resendes1; Biology, Westminster College, New Wilmington, PA

1

Human PCID2 is a member of the TREX2 complex which associates with the nuclear pore basket to facilitate mRNA export. Multiple components of the TREX2 complex, including PCID2, serve additional roles in the cell other than in nuclear mRNA export. Specifically, PCID2 and centrin 2 co-localize outside of the nucleus at the centrosome. Recent characterization indicates that PCID2 is present at the centrosome during the G1/S and G2/M phases of the cell cycle. The localization of Centrin 2 at the centrosome in those phases is linked to its role as required factor for centriole duplication. While the relationship between PCID2 and centrin 2 at the centrosome during these periods is not yet fully understood, we have previously shown that siRNA knockdown of centrin 2 disrupts centrosomal localization of PCID2. In contrast, PCID2 knockdown does not affect the positioning of centrin 2. Because characterization of PCID2 in human cells thus far has been limited to its roles in nuclear mRNA and protein export investigating its significance outside of the nucleus is crucial to understanding its overall role in the cell and its functional relevance at the centrosome. We are performing both siRNA knockdown of PCID2 as well as overexperssion of a GFP-PCID2 recombinant plasmid to determine the effects of altering PCID2 expression levels on the centrosome. Because the PCI domain contained within PCID2 typically serves as a structurally stabilizing scaffolding unit within large complexes, it is expected that increased or decreased levels of PCID2 will produce an observable effect on positioning and assembly of the centrosome due to abnormal protein stoichiometry. We are currently testing this prediction by assessing changes in centrosome number, position and mitotic defects such as altered spindle organization via fluorescence microscopy.

P172 Board Number: B280

The role of mouse CKAP2 phosphorylation in centrosome biogenesis. B. Yoo1, C. Park1, H. Kim1, J. Park1, C. Bae1; 1 Sungkyunkwan Univ Sch Med, Suwon, Korea CKAP2, cytoskeleton-associated protein, is regulated during the cell cycle, with the protein being degraded in late mitosis in an APC/C-Cdh1 dependent manner. Our previous studies have suggested that cellular function of CKAP2 pertain to regulation of the mitotic spindle dynamics and proper chromosome segregation. Here we show that the effect of phosphorylation of the mouse CKAP during cell cycle on the centrosome biogenesis. Depleting the CKAP2 in NIH 3T3 induced the centrosome amplification and fragmentations leading to abnormal chromosome segregation, which was rescued by ectopic expression of wild type CKAP2. Phosphorylation is one of the key mechanisms which regulate centrosome

SUNDAY-POSTER PRESENTATIONS biogenesis and spindle assembly. We have hypothesized that phosphorylation status of the CKAP2 may be related with centrosome biogenesis. In order to explore the function of the phosphorylated CKAP2, we introduced phospho-deficient mutation at T603 which were known to be phosphorylated in human CKAP2 by Cdk1-cyclin B1, respectively. The expression of phosphorylation deficient mutant forms of mouse CKAP2, T603A could not restore the centrosomal abnormalities of CKAP2 depleted NIH 3T3 cells. These results indicates that phosphorylation of the CKAP2 is critical for both centrosome biogenesis and bipolar spindle formation. Further studies are required to elucidate the mechanisms maintaining centrosome integrity by mitosis specific phosphorylation of the CKAP2.

P173 Board Number: B281

WDR62 a critical target for the Aurora-A overexpression phenotype?. A. Eskat1, M. Hoveyda1, M. de Medina Redondo1, M. Gstaiger2, P. Meraldi3; 1 Cell Physiology and Metabolism, University of Geneva, Genève, Switzerland, 2Institute of Molecular Systems Biology, ETH Zürich, Zurich, Switzerland, 3Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland The Aurora-A serine/threonine kinase is frequently overexpressed in many epithelial tumors, cancers of solid organs and hematological malignancies. It has been shown that overexpression of Aurora-A is implicated in causing or maintaining the malignant phenotype and resistance to taxanes, which are effective anticancerous drugs and routinely used for the treatment of many cancers. At the physiological level Aurora-A regulates many aspects of mitosis, such as mitotic entry, bipolar formation of the mitotic spindle and centrosome regulation, including centrosome separation and centrosome maturation. However, very little is known about the molecular consequences of Aurora-A overexpression and how the overexpression is linked to tumorigenesis. In the current hypothesis Aurora-A overexpression leads to a failure in the spindle assembly checkpoint in a kinase-dependent manner, which would result in chromosome segregation errors with cytokinesis failure and chromosomally instable tetraploid cells (Anand et al., 2003). Surprisingly, we find that Aurora-A disrupts mitotic progression in a kinaseindependent way. And second, we show that exogenous Aurora-A does not impair the spindle checkpoint. It is rather that Aurora-A overexpression leads to spindle orientation defects and causes the formation of DNA bridges in anaphase. These DNA bridges are known to impair cytokinesis, which in turn could result in tetraploid cells. Searching for the causes for these mitotic defects, we set out to identify the proteins that are titrated out by Aurora-A overexpression with mass spectrometry. We found WDR62 as an interaction partner, which has been recently reported as an Aurora-A activator protein (Chen et al., 2014). Mutations in this centrosomal protein lead to microcephaly in human patients (Bilgüvar et al., 2010; Nicholas et al., 2010; Yu et al., 2010). Here, we show that the abundance of WDR62 at centrosomes is reduced in Aurora-A overexpressing cells and that depletion of WDR62 gives also rise to spindle orientation defects and DNA bridges in anaphase. Rescue experiments show that co-expression of WDR62 and Aurora-A rescues the Aurora-A overexpression phenotype (DNA bridges and spindle orientation defects). Therefore we postulate that overexpression of Aurora-A

SUNDAY-POSTER PRESENTATIONS reduces WDR62 amounts in HeLa cells, which in turn leads to cell divisions with DNA bridges that disturb cytokinesis.

P174 Board Number: B282

Localization determinants in the Mps1 amino terminus distinguish centrosomal and kinetochore targeting. J.R. Marquardt1, K. Beuoy1, H.A. Fisk1; 1 Department of Molecular Genetics, The Ohio State University, Columbus, OH Cells have evolved elaborate mechanisms to ensure the faithful segregation of chromosomes. The centrosome, which consists of two microtubule-based centrioles, must be precisely duplicated only once in S-phase to become the poles in a proper bipolar mitotic spindle. The spindle assembly checkpoint (SAC) serves to block the progression of mitosis until all chromosomes are properly attached to spindle microtubules from both poles. Failure to regulate either of these processes has the potential to cause aneuploidy, which is a hallmark of many cancers. The kinase Mps1 has previously been shown to regulate both the duplication of centrioles in S-phase and the SAC in mitosis. This implies that different localization prompts in Mps1 govern its localization throughout the cell cycle. Here we show that separate elements in the Mps1 N-terminus, which includes the known domains N-terminal extension (NTE), tetratricopeptide repeat (TPR), and centrosome localization domain (CLD), govern localization to either the kinetochore or the centrosome. Domains that have previously been shown to be responsible for kinetochore targeting were found to be dispensable for centrosomal localization and function. A separate motif is shown to be necessary for centrosomal targeting and the interaction with the centrosomal protein voltage dependent anion channel 3 (VDAC3), which has previously been shown to recruit Mps1 to the centrosome. Deletion of this element resulted in a version of Mps1 that maintains kinetochore localization and SAC function, while failing to elicit centriole reduplication. This data is consistent with a separation of function for the kinase based on separate elements of an N-terminal localization determinant.

SUNDAY-POSTER PRESENTATIONS

P175 Board Number: B283

Differential inhibition of centrosome amplification in 2-CEES-treated Saos-2 cells by antioxidants glutathione and Trolox. R.A. Bennett1, E. Behrens2, A. Zinn3, C. Duncheon2, T.J. Lamkin4; 1 Department of Biology, University of Southern Indiana, Evansville, IN, 2University of Southern Indiana, Evansville, IN, 3Department of Biological Science, University of Toledo, Toledo, OH, 4Molecular Signatures Section - Molecular Genetics, Air Force Research Laboratory, Wright-Patterson AFB, OH Mustard gas (MG) is a chemical weapon that was first used in World War I (Duchovic and Vilensky, 2007). In recent times, its mortality and morbidity combined with its simple formulation have raised concerns over its potential use by terrorist organizations and unstable governments. MG is a powerful vesicant and alkylating agent that causes DNA monoadducts and crosslinks to form (Fidder et al, 1994). Acute exposure to MG results in painful blisters and respiratory distress, whereas chronic, low-level exposure to MG, such as that which is found in facilities that manufacture it, results in higher incidences of cancer in those exposed (Smith et al, 1995; Easton et al, 1988). Although the exact mechanism of carcinogenesis is not known, it is thought that MG induces tumor formation through its ability to induce oxidative stress and DNA damage. In addition to nucleic acids as targets of MG toxicity, several proteins have been shown to either be targets of MG-induced damage or mediate its toxicity. Among these proteins are those that have been shown to regulate centrosome biology (e.g. p53, poly(ADP-ribose) polymerase, and NFκB (Ruff and Dillman, 2010). Additionally, DNA damage and oxidative stress have been shown to induce centrosome amplification (i.e. more than 2 centrosomes per cell) as well (Dodson et al, 2004; Chae et al, 2005). Centrosomes direct the segregation of chromosomes during mitosis through the formation of the bipolar mitotic spindle. Cells with amplified centrosomes during mitosis can segregate their chromosomes unequally, resulting in chromosome instability (CIN), a common phenotype of cancer cells (Fukasawa, 2005). In our studies, we utilized the widely accepted, lesshazardous surrogate of MG called 2-chloroethyl ethylsulfide (2-CEES) to determine whether or not it could induce centrosome amplification in cells and subsequent CIN. We have previously shown that 5% and 1% of untreated Saos-2 and NIH3T3 cells, respectively, exhibit centrosome amplification, while 35% and 32% of cells, respectively, treated with 250 μM 2-CEES exhibit centrosome amplification. Cells with centrosome amplification also show an increase in CIN as measured by an increase in aneuploidy in 2CEES-treated cells (Bennett et al, 2014). Here, we show that glutathione, a scavenger of 2-CEES molecules and reactive oxygen species (ROS), reduces the percent of cells with amplified centrosomes in 2-CEES-treated Saos-2 cells from 52% to 12.5%, while treatment with the antioxidant Trolox, an inhibitor of ROS, reduces centrosome amplification in 2-CEES-treated Saos-2 cells from 37% to 24%. These data indicate that 2-CEES can induce centrosome amplification and CIN in cells and that it may do so through both its ROS-inducing and DNA damaging capacities.

SUNDAY-POSTER PRESENTATIONS

P176 Board Number: B284

Understanding how aneuploidy and multipolar mitosis are formed after chrysotile and vincristine treatments. B.A. Cortez1, P.R. Teixeira2, S.D. Redick3, S. Doxsey4, G. . Machado-Santelli2; 1 Depto Genetica e Biologia Evolutiva, University of São Paulo, São Paulo, Brazil, 2Department of Cell and Developmental Biology, University of Sao Paulo, Sao Paulo, Brazil, 3Univ Massachusetts Med Sch, Worcesteer, MA, 4Univ Massachusetts Med Sch, Worcester, MA Aneuploidy is a feature of solid tumors. Aneuploid cells result from errors during mitosis, such as centrosome amplification, multipolar mitosis and cytokinesis abnormalities. The capability of aneuploidy to promote and to suppress tumorigenesis has driven efforts to characterize mitotic errors that form viable and not viable aneuploid cells. We have previously shown that chrysotile, an asbestos fiber, and vincristine, a chemotherapeutic agent, are able to induce aneuploidy and multipolar mitosis. Now we directed our focus to discover possible mechanisms involved in aneuploid cell formation after these treatments. Herein we evaluated centrosome morphology, chromosome number, location of proteins required to mitosis, and origins and fates of multipolar mitosis and multinucleated cells after chrysotile and vincristine treatment. The experiments were performed using human normal epithelial cells (RPE) and human lung cancer cells (HK2). Chrysotile fibers, in normal and cancer cells, led to mislocalization of proteins involved in intercellular bridge establishment and abscission, such as Septin 9, Anillin, Aurora B and Alix. Cytokinesis regression was observed in almost 20% of cytokinesis in the cultures after fiber treatment for 24 h, resulting in tetraploid multinucleated cells. These cells were able to enter cell cycle, giving rise to multipolar mitosis and aneuploid cells in cancer and normal lineages. Vincristine treatment led to specific and common responses in normal and cancer cells. During metaphase arrest pericentrosomal matrix was fragmented but no centriole reduplication was observed, and arrested cells could be conducted to mitotic slippage in both lineages, generating multinucleated tetraploid cells. Both cell lineages showed during recovery periods increased centriole numbers and abundant pericentriolar matrix. However, normal tetraploid cells could not progress through cell cycle and neither form multipolar mitosis, while cancer tetraploid cells showed Aurora A overexpression, centrosome abnormalities, multipolar mitosis and high levels of aneuploidy. The results showed that HK2 cancer cells could proliferate even after several mitotic errors induced by chrysotile and vincristine, while RPE normal cells could only overcome errors induced by chrysotile treatment. These observations suggest that normal cells do not display protection mechanisms against aneuploid cell formation induced by chrysotile.

SUNDAY-POSTER PRESENTATIONS

Spindle Assembly 1 P177 Board Number: B285

Feedback and Spatial Organization of the Ran Pathway in Mitosis. D. Oh1, D.J. Needleman1,2,3; 1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 2FAS Center for Systems Biology, Harvard University, Cambridge, United States, 3School of Engineering and Applied Sciences, Harvard University, Cambridge, MA During cell division, the GTPase Ran forms gradients around chromosomes. Current models of spindle assembly assume that the size of the spindle is determined by the length of these gradients. We used a new multipoint fluorescence fluctuation spectroscopy technique to study the Ran pathway in mitosis. We found that the distribution of components of the Ran pathway which regulate microtubule behaviors is determined by their interactions with microtubules, producing a feedback in which microtubule nucleators are localized by the microtubules whose formation they stimulate. Our results indicate that gradients in Ran act to trigger spindle assembly around chromosomes, but do not produce the length scales of spindle structure. Rather, the size of the spindle results from the feedback process itself, leading to robust spindle self-organization.

P178 Board Number: B286

Spatio-temporal Model for Silencing of the Mitotic Spindle Assembly Checkpoint. J. Chen1, J. Liu2; 1 NIH, Bethesda, MD, 2National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD The spindle assembly checkpoint arrests mitotic progression until each kinetochore secures a stable attachment to the spindle. Despite fluctuating noise, this checkpoint remains robust and remarkably sensitive to even a single unattached kinetochore among many attached kinetochores; moreover, the checkpoint is silenced only after the final kinetochore-spindle attachment. Experimental observations showed that checkpoint components stream from attached kinetochores along microtubules toward spindle poles. Here, we incorporate this streaming behavior into a theoretical model that accounts for the robustness of checkpoint silencing. Poleward streams are integrated at spindle poles, but are diverted by any unattached kinetochore; consequently, accumulation of checkpoint components at spindle poles increases markedly only when every kinetochore is properly attached. This step-change robustly triggers checkpoint silencing after, and only after, the final kinetochore-spindle attachment. Our model offers a conceptual framework that highlights the role of spatiotemporal regulation in mitotic spindle checkpoint signaling and fidelity of chromosome segregation.

SUNDAY-POSTER PRESENTATIONS

P179 Board Number: B287

A new role for the endocytic pathway during spindle pole formation and mitosis. H.A. Hehnly1,2, S. Doxsey3, J. Scott4; 1 Univ Massachusetts, Worcester, MA, 2Department of Pharmacology, University of Washington Medical School, Seattle, WA, 3PMM, Univ Massachusetts Med Sch, Worcester, MA, 4Department of Pharmacology, University of Washington Medical School, HHMI, Seattle, WA Rab11-GTPase-containing endosomes were recently implicated in spindle pole assembly and function, and in spindle orientation (Hehnly and Doxsey, 2014). However, whether their role in spindle assembly is due to a direct role in their spindle maturation was not characterized. During the cell cycle, specifically at growth phase to mitotic entry, the duplicated centrosomes mature into mitotic spindle poles to ultimately set up a microtubule-based spindle to separate duplicated genetic material into a mother and daughter cell. We examined Rab11-endosome tracks towards the centrosome/spindle pole in interphase cells compared to prophase cells. Surprisingly, we found that an increased number of vesicles transported at an elevated rate to spindle poles during prophase when compared to interphase. When we took a closer look, we observed that the Rab11 effector, FIP3, and Rab11-endosomes had a burst of recruitment to a single maturing spindle pole late in G2. This spindle was then characterized to be the oldest spindle pole that contained a 2-fold increase in mother centriole localized proteins (e.g. cenexin, centriolin) compared to the other. Based on our previous observation that Rab11 is localized to the appendages of the mother centriole and the appendage proteins, cenexin and centriolin, can mediate Rab11-endosomes organization and association with the centriole, we decided to examine whether cenexin- or centriolin-depletion disrupted recruitment of endosomes during spindle maturation. Interestingly, recruitment wasn’t inhibited, but the preferential recruitment to the oldest spindle pole was diminished where both spindle poles now recruit similar ratios of Rab11-endosomes. Cenexin and centriolin-depleted cells assembled γ-tubulin to the maturing spindle pole, but there was no longer an asymmetric distribution. These cells, like Rab11-depleted cells, display asymmetric spindle orientation during mitosis. One possibility is that asymmetric protein recruitment to the oldest spindle pole is required to ensure division symmetry, and under conditions of Rab11-depletion or loss of mother centriole components spindle asymmetry becomes perturbed.

SUNDAY-POSTER PRESENTATIONS

P180 Board Number: B288

The Nucleoporin ALADIN is Essential for Proper Mitotic Spindle Formation. S. Carvalhal1, K. Koehler2, A. Huebner2, E.R. Griffis1; 1 Centre for Gene Regulation and Expression, University of Dundee, Dundee, Scotland, 2Department of Pediatrics, Technische Universität Dresden, Dresden, Germany In an RNAi screen for genes essential for mitotic progression, we have identified the nucleoporin ALADIN as a factor that is essential for timely chromosome alignment and spindle morphology. When ALADIN is depleted, we see a delay in chromosome alignment, hyperstretching of paired kinetochores, and destabilized K-fibres. When we examined the molecular nature of these defects, we observed that the protein NuMA retracts from its localization on spindle poles and concentrates on spindles. Conversely, we find that after ALADIN depletion, Aurora A kinase becomes less focused at centrosomes (where the active form is reduced by 40% compared to control cells) and spreads along spindle microtubules. We find that the relocalization of NuMA seen after ALADIN depletion can be phenocopied by inhibiting Aurora A; however, the localizations of other Aurora A substrates such as Eg5 and TACC3 are not perturbed by ALADIN depletion, suggesting that the effects we see are specific to a subset of Aurora A targets. We find that ALADIN, which normally enriches near spindle poles, can be recruited to centrosomes and can bind to Aurora A after this kinase is inhibited by MLN8237. We currently favor a model in which ALADIN scavenges inactive Aurora A to promote its reactivation and maintain a high concentration of this protein near spindle poles where it can phosphorylate substrates like NuMA. Curiously, ALADIN was first identified as a gene that is mutated in patients who have triple A syndrome, which impairs lacrimal, adrenal, and neural functioning. We have investigated the localization of mutant forms of ALADIN in mitosis, and have seen that some show a gain of function localization on kinetochores, while the expression of others inhibit chromosome alignment. We are currently testing whether these mutant forms are still able to interact with Aurora A, and we are beginning to use IPS cells to explore the possibility that mitotic defects underlie some of the symptoms of triple A syndrome.

P181 Board Number: B289

RanBP1 governs spindle assembly by defining mitotic Ran-GTP production. M. Zhang1, A. Arnaoutov1, M. Dasso1; 1 National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD Proper spindle formation requires a chromatin-based gradient of the small GTPase Ran, with high RanGTP levels in the vicinity of mitotic chromosomes. This gradient is established through the activity of Ran’s nucleotide exchange factor (RCC1), which concentrates on chromatin. RanBP1 is a co-activator of Ran’s GTPase activating protein (RanGAP1). It has been known for nearly two decades that RanBP1 also forms a stable heterotrimeric complex with RCC1 and Ran (RRR complex), which inhibits RCC1’s RanGEF activity. The function of the RRR complex has remained mysterious, however, because RanBP1 is

SUNDAY-POSTER PRESENTATIONS physically separated from RCC1 during interphase. We found that the RRR complex formed readily in Mphase Xenopus egg extracts (CSF-XEEs). RCC1 binding to chromatin and RRR complex assembly were mutually exclusive, so that promoting RRR complex formation through the addition of recombinant RanBP1 sequestered RCC1 away from chromatin. Consistent with earlier reports, RRR complex assembly inhibited RCC1’s RanGEF activity. Together, these findings suggest that the RRR complex plays a key mitotic role in determining the partitioning of RCC1 between its active chromatin-bound and inactive soluble states, thereby setting both the location and magnitude of mitotic Ran-GTP production. Notably, RCC1’s association to mitotic chromatin is dynamic, and there are particularly large changes during the metaphase-to-anaphase window. However, the timing of reported RCC1 modifications suggests that they do not cause such changes. We found that RanBP1 is phosphorylated during anaphase, and that this modification disrupts the RRR complex. Further analysis showed that RanBP1 phosphorylation drove increased RCC1 binding to chromatin in cycling XEE, and thereby indirectly enhanced anaphase Ran-GTP production. This modification may also contribute to Ran pathway function in early interphase, because elevated RCC1 on anaphase chromatin should provide high levels of Ran-GTP to facilitate nuclear reassembly. Finally, separation of RCC1 and RanBP1 after RRR complex dissociation allows RCC1 sequestration to re-forming nuclei while excluding RanBP1 into the early interphase cytosol. In summary, we have documented a novel role of the RanBP1 protein in controlling the localization and activity of Ran’s nucleotide exchange factor, RCC1. We have shown that phosphorylation of RanBP1 during anaphase drives changes in RCC1 dynamics and allows increased Ran-GTP production. These findings resolve important and long-standing questions within the Ran field regarding the function of the RanBP1/Ran/RCC1 complex and its dynamics.

P182 Board Number: B290

The budding yeast Polo-like kinase Cdc5 is spatially regulated for timely mitotic exit and adaptation to DNA damage. V.V. Botchkarev1, V.V. Eapen1, V. Rossio1, A. Matthews1, S. Yoshida1, J.E. Haber1; 1 Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA Polo-like kinases regulate many events including mitotic entry, chromosome segregation, mitotic exit, cytokinesis, and adaptation to DNA damage; however, how Polo activity is regulated remains poorly understood. The budding yeast Polo-like kinase Cdc5 is regulated spatially during the cell cycle. Cdc5 accumulates in the nucleus upon mitotic entry, consistent with the fact that many known Cdc5 substrates are nuclear. Interestingly, in anaphase, we found that Cdc5 translocates from the nucleus to the cytoplasm. We took genetic and biochemical approaches to show that this nuclear-cytoplasmic translocation of Cdc5 in anaphase is required for activation of the Mitotic Exit Network (MEN). We found that the Cdc14 phosphatase, a downstream target of MEN, is required for this nuclear export of Cdc5. This finding suggests the existence of a positive feedback loop between Cdc5 and Cdc14 to regulate timely mitotic exit. Cdc5 also accumulates in the nucleus after DNA damage checkpoint-induced G2/M

SUNDAY-POSTER PRESENTATIONS arrest. However, by restricting Cdc5 localization either to the nucleus or cyctoplasm, we demonstrated the requirement of both nuclear and cytoplasmic pools of Cdc5 for adaptation to DNA damage. Collectively, our data demonstrate that spatial regulation of Cdc5 is required for timely mitotic exit and adaptation to DNA damage.

P183 Board Number: B291

Novel mechanisms of APC regulation in mouse oocyte meiosis. I. Nabti1, J. Carroll2; 1 cell and developmental biology, Univ Coll-London, London, England, 2Monash Univ, Melbourne, Australia The anaphase-promoting complex (APC) is an E3 ubiquitin ligase responsible for regulated destruction of substrates at specific stages of the cell cycle. Two APC co-activators, Cdc20 and Cdh1, mediate the timing and selectivity of substrates recognition. Cdc20 recognizes substrates containing the D-box motif only, while Cdh1-bound APC mediates the ubiquitination of proteins with either D- or KEN-box motifs. Progression throughout meiosis in oocytes utilizes the same molecular players although the start-stop nature of the female meiosis invokes additional levels of regulation. Recently, we reported that cyclindependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK) play compensatory roles in regulating the APC by suppressing its activity early in prometaphase I, thereby allowing accumulation of the APC substrates essential for meiosis I. In this study we have investigated which of the APC coactivators is responsible for this APC activity. Remarkably, we find that depletion of either Cdc20 or Cdh1 failed to prevent APC activity and that depletion of both co-activators led to increased destruction of the APC substrates. Similarly, persistent destruction of the APC substrate, securin, in Cdh1 and Cdc20-depleted prophase I-arrested oocytes points to a second cell cycle stage in which APC activity can be detected even when the known co-activators are depleted. Incomplete depletion of the coactivators seems an unlikely explanation for these findings. Western blotting reveals that the proteins are efficiently depleted in both cases and functional studies show that depletion has the predicted effects on meiotic cell cycle progression. Furthermore, the fact that co-depletion leads to an increase in APC activity, points to novel additional means of APC activation in mouse oocytes. The APC activity revealed in this study targets D-box substrates only. This observation provides further support that Cdh1 is not active, as it would also be expected to recruit KEN-box containing substrates to the APC. However, the observations are also not consistent with Cdc20-mediated APC activation given that in both prometaphase and prophase I Cdc20 depletion is without effect, Cdk1 activity is required for Cdc20 function and there is no evidence that Cdc20 can stimulate the APC during the prophase I-arrest. All these findings suggest that the instability of the D-box substrates revealed in prometaphase I and also during the prophase I-arrest might well be due to activation of the APC independently of its two major co-activators.

SUNDAY-POSTER PRESENTATIONS

P184 Board Number: B292

Multiple Pathways for Spindle Assembly in Mouse Oocytes. L. Bury1, P.A. Coelho1, M. Zernicka-Goetz2, D. Glover1; 1 Department of Genetics, University of Cambridge, Cambridge, United Kingdom, 2Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom Mammalian oocytes are naturally acentriolar, and assemble their spindles by self-organization of cytoplasmic microtubule organizing centers (MTOCs) that congress at the center of the oocyte at the time of nuclear envelope breakdown. Although Ran-GTP contributes to the generation of sufficient microtubule density, it appears that bipolar spindles are still able to form in the absence of a stable RanGTP gradient, implying the existence of other factors that might support spindle assembly in the maturing oocyte. A Recent study investigating acentriolar spindle formation in the early mouse embryo has discovered that centriolar proteins, namely Plk4 and Cep152, which are present despite the absence of centrosomes, are crucial for microtubule nucleation and spindle assembly. However, the mechanism of acentrosomal spindle formation in the mouse oocyte is thus far unknown. It has been hypothesized that the long duration of metaphase (6-8 hours) would allow time for multiple spindle assembly factors to form a robust microtubule network, even in the absence of a Ran-GTP gradient. On the other hand, the fact that enucleation of oocytes abrogates bipolar spindle formation, has supported the idea that indeed other nuclear factors might exist that are responsible for regulating microtubule nucleation. Consistent with studies in Xenopus egg extracts that point to an important role of Aurora A in chromosomal MT nucleation, we find using small molecule inhibitors that Aurora A but not Auroras B/C contribute to bipolar spindle assembly in meiosis I. Using a combination of pathway-specific inhibitors and dominant negative constructs, we find that Plk4 also contributes to this process. These two protein kinases appear to act in addition to Ran-GTP to locally stabilize microtubules surrounding chromatin, and synergism between these pathways is required to build the bipolar meiotic spindle. Interestingly, these proteins and several of their partners are concentrated in the nucleus and accumulate around chromatin at the time of the initial MT nucleation following nuclear envelope breakdown. Inhibition of any of these pathways strongly reduces the early stages of MT nucleation, but does not interfere with the actin-dependent migration of chromatin to the oocyte cortex. Eventually, cytoplasmic MTOCs nucleate small aster-like spindles and meiotic exit is able to occur.

P185 Board Number: B293

Spindle Matrix Formation is Required for Cell Cycle Progression. J. Johansen1, C. Yao1, C. Wang1, Y. Li1, J. Girton1, K.M. Johansen1; 1 Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA A paramount issue in mitosis is the synchronization of events required for cell cycle progression, and although many previous studies have identified critical regulatory factors that act to coordinate the

SUNDAY-POSTER PRESENTATIONS myriad activities involved in setting up a spindle and organizing chromosomes upon it, how these diffusible regulatory factors are spatially organized and confined to the spindle region in the absence of a diffusion barrier after nuclear envelope breakdown (NEB) is not well understood. However, in Drosophila we have identified four nuclear proteins, Skeletor, Chromator, Megator, and EAST from two different nuclear compartments that interact with each other and that redistribute during prophase to form a dynamic, gel-like spindle matrix that embeds the microtubule spindle apparatus (MBoC 23:3532, 2012). This matrix exists independently of microtubules and the NE and specific interactions between spindle matrix molecules are necessary for complex formation and cohesion. In order to address how the spindle matrix interacts with cell cycle components we have taken a live imaging approach to determine the relative timing of localization and cross-interactions of these proteins. Previously, we have shown that the spindle matrix protein Megator and its human homolog Tpr have an evolutionarily conserved function as spatial regulators of the spindle assembly checkpoint proteins Mad2 and Mps1. Here we show that a number of key cell cycle proteins such as Cyclin B, Polo, Ran, 14-3-3 and Endos also are co-localized at enriched levels within the spindle matrix during mitosis and that this localization is independent of microtubules. Furthermore, prevention of spindle matrix formation by injection of a function blocking antibody to the spindle matrix protein Chromator results in cell cycle arrest prior to NEB phenocopying the triple RNAi knockdown of Cyclins A, B, and B3 (McCleland and O'Farrell, Curr. Biol. 18:245, 2008) Interestingly, in such embryos the dynamic relocation of Polo and Cyclin B to the nuclear rim and kinetochores is abrogated and Polo is not imported into the nucleus. This is in contrast to colchicine-arrested embryos where the wild-type dynamics of these proteins are maintained. Furthermore, we show that the spindle matrix prevents Pdi-GFP-marked vesicular membranes from entering the nuclear space after NEB although they are permeable to microtubules. These studies promise to provide a mechanistic framework for understanding how cell cycle factors are physically confined and organized in the spindle region in organisms with open or semi-open mitosis, allowing for spatial and temporal integration of signaling events leading to mitotic progression and chromosome segregation.

P186 Board Number: B294

Plk4 regulation of acentriolar MTOC architecture is essential for meiotic division of mouse oocytes. M. Luksza1, J. Kannaan1, V. Marthiens2, R. Basto2, M. Verlhac1; 1 CIRB, UMR-CNRS 7241/INSERM U1050, Coll De France, Paris Cedex, France, 2Institut Curie, Paris, France Spindles of most animal cells are assembled from canonical centrosomes, build up of two centrioles and their surrounding pericentriolar material. However, most oocytes lose their centrioles during their growth in the ovary yet do assemble functional meiotic spindles able to segregate chromosomes. In fully-grown mouse oocytes, acentriolar MTOCs (aMTOCs, MicroTubule Organizing Centers) gather as large pericentriolar material foci around the nuclear envelope in prophase I of meiosis. At meiosis resumption, these foci are rapidly stretched around the nuclear envelope, fragmenting into smaller

SUNDAY-POSTER PRESENTATIONS pieces in a dynein- and MT-dependent manner (Łuksza 2013). We address here the importance of such timely reorganization of aMTOCs. We show that Plk4, a major kinase in centriole duplication, plays an essential role in aMTOCs organization. First we show that endogenous Plk4 protein is present on aMTOCs in the mouse oocyte. Second, transgenic overexpression of Plk4 specifically during oocyte growth impacts the morphology of aMTOCs. Indeed it induces their precocious fragmentation in prophase I. This in turn has major consequences; increasing their MT-nucleating capacity and promoting early meiotic spindle bipolarization as well as assembly of larger spindles. Such robust spindles contain abnormal levels of incorrect bivalent attachments, which delay SAC inactivation impeding on the transition to anaphase I. These defects are not due to increased levels of Plk4 activity during meiotic divisions, since the injection of cRNAs encoding for Plk4 into fully grown oocytes does not perturb meiosis I progression nor affects aMTOCs fragmentation. We describe here a novel function for Plk4, namely in controlling pericentriolar material organization in the absence of centriole, a function that is essential for proper progression into meiosis I of mouse oocytes. Furthermore we show here that overfragmented aMTOCs compromise Kinetochore-MTs attachments and chromosome congression during female meiosis. Ref : Łuksza M., Queguiner I., Verlhac M.-H.* and Brunet S.* 2013. Rebuilding MTOCs upon centriole loss during mouse oogenesis. Dev Biol, 382: 48-56 (* : co-senior authors)

P187 Board Number: B295

The Relationship Between Localization and Function of the Chromosomal Passenger Complex in Drosophila melanogaster Oocytes. R.A. Battaglia1, S.J. Radford1, K.S. McKim1; 1 Waksman Institute, Piscataway, NJ Accurate segregation of chromosomes during meiosis is required to preserve the correct amount and distribution of genetic material in eggs and sperm. Errors in chromosome segregation are the leading cause of miscarriages and also lead to genetic diseases such as Down syndrome. In female meiosis of many animals, spindle assembly occurs without centrosomes, the microtubule organizing centers of the cell. In Drosophila oocytes, the chromosomal passenger complex (CPC), which consists of INCENP, Aurora B kinase, Survivin, and Borealin, is required for chromosome-based spindle formation and biorientation of the centromeres. In addition, the CPC promotes the localization of additional spindle assembly factors, supports spindle maintenance, and participates in kinetochore assembly. How the CPC functions in all of these processes is not known, but we hypothesize that the localization of the CPC is important. At metaphase in oocytes, the CPC localizes with central spindle microtubules in a ring around the chromosomes and may be transiently at centromeres. One possibility is that the CPC starts on centromeres and then moves onto the central spindle. Alternatively, there may be two separate populations of CPC, one that localizes to centromeres and a separate population that localizes to microtubules. By creating RNAi-resistant mutant INCENP transgenes that will manipulate the localization of the CPC, we will test whether centromere-localized and/or central spindle-localized CPC

SUNDAY-POSTER PRESENTATIONS provide the functions of the CPC in oocytes. We will force the CPC to localize to the centromeres, kinetochores, chromatin and microtubules. Conversely, we will prevent CPC localization to the centromeres and microtubules. It is possible that CPC localization to chromatin or centromeres is not required for bipolar spindle formation and that when the CPC is unable to localize to centromeres, it will have only bi-orientation defects. However, if the CPC must first localize to centromeres before it can transition to the central spindle, this mutant may fail to assemble a spindle. Observation of the effect on spindle assembly and bi-orientation by these mutants will elucidate the CPC’s mechanism in meiosis.

P188 Board Number: B300

PP1 antagonises Aurora B in Drosophila oocytes for chromosome structure, cohesion and kinetochore localisation. A. Das1, K.S. McKim2; 1 Cell and Developmental Biology, Rutgers University, Piscataway, NJ, 2Waksman Institute, Piscataway, NJ Reversible protein phosphorylation allows cells to direct progression through cell cycle. The significance of protein kinases in cell cycle regulation and progression has been well established. In Drosophila female germline Aurora B kinase, a member of the chromosomal passenger complex (CPC), is required for meiotic spindle assembly and chromosome segregation. We have also seen that if an Aurora B inhibitor is added after spindle assembly is complete, the spindle completely disintegrates and kinetochore proteins fail to localise. This indicates that continual phosphorylation activity is required due to either the presence of multiple phosphatases or degradation. Hence the protein phosphatases may play equally important reciprocal roles in cell cycle regulation and the processes involved. We have looked at the role of the serine/threonine protein phosphatase 1 (PP1) in female meiotic chromosome segregation and spindle assembly. In PP1 depleted oocytes we observe a gross disorganization of spindle microtubules and the length and shape of these spindles resemble prometaphase spindles suggesting a defect in progression to metaphase. In addition the karyosome, a structure into which all the chromosomes are compacted, is broken into several pieces with a loss of sister-centromere cohesion. This is surprising since PP2A is usually required for maintaining cohesion in mitotic cells. Kinetochore proteins like Spc105R however are unaffected by loss of PP1. We have also found that some but not all of these phenotypes are due to phosphorylation by Aurora B. The karyosome defect and loss of cohesion in oocytes lacking PP1 is rescued by Aurora B inhibition. In addition the loss of kinetochore protein Spc105R at centromeres upon inhibition of Aurora B is also rescued when PP1 is absent. These results suggest that PP1 may antagonize Aurora B for maintaining cohesion, karyosome integrity and kinetochore protein localization. However, the complete loss of the meiotic spindle caused by an Aurora B inhibitor is not restored by loss of PP1. Thus, other phosphatases may negatively regulate spindle assembly and/or Aurora B-dependent phosphorylation is required to maintain constant incorporation of spindle associated proteins throughout meiosis.

SUNDAY-POSTER PRESENTATIONS

P189 Board Number: B301

Three-dimensional reconstruction of female meiosis in C. elegans. I. Lantzsch1, M. Srayko2, S. Prohaska3, T. Müller-Reichert1; 1 Medical Faculty "Carl Gustav Carus", TU Dresden, Dresden, Germany, 2Univ Alberta, Edmonton, AB, 3 Zuse Institute, Berlin, Germany In meiosis, haploid gametes are produced from a diploid progenitor cell and this process of genome haploidization differs between oogenesis and spermatogenesis. Similar to many other species, including Drosophila, Xenopus, mice and humans, the female meiotic spindles in C. elegans are anastral and acentrosomal [1,2]. At early metaphase, the microtubules form an elongated bipolar spindle, which is about 10 μm in length. The metaphase spindles then shorten to roughly 3-4 μm and adopt a barrel-like shape. During late anaphase, microtubules are only observed in the interzone between the separating chromosomes/chromatids. The mechanism of this reorganization of the microtubules during the metaphase-to-anaphase transition is not understood [3]. Here, we show three-dimensional reconstructions of meiotic spindles at different stages. The 3D models were obtained by correlative light and electron tomography. Our anaphase I model shows about 4000 microtubules in between the chromosomes. Currently, we are analyzing the average length and the length distribution of microtubules to test models of chromosome segregation. In addition, we are investigating, whether microtubule assembly in this anaphase ‘inside-out’ spindle nucleation is taking place close to the surface of the chromosomes. Parallel to this wild-type characterization of the female meiotic spindles, we are characterizing candidate genes involved in various aspects of spindle assembly. One candidate protein for 3D reconstruction is the microtubule depolymerising kinesin-13 member, KLP-7/MCAK. 1. 2. 3.

Manandhar et al. (2005). Biol Reprod. 72, 2-13. Müller-Reichert et al. (2010). Cell Mol Life Sci. 67, 2195-2213. Dumont et al. (2010). Nature Cell Biology. 12, 894-901.

P190 Board Number: B302

Feedback control of chromosome separation by a midzone Aurora B gradient. O. Afonso1, I. Matos1,2, A. Pereira1, P. Aguiar1,3, H. Maiato4,5; 1 Chromosome Instability and Dynamics Laboratory, IBMC, Porto, Portugal, 2Laboratory of Mammalian Cell Biology and Development, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, 3Center for Mathematics, Universidade do Porto, Porto, Portugal, 4Chromosome Instability Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal, 5 Cell Division Unit, Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto, Porto, Portugal Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids prior to nuclear envelope reassembly (NER). However, how these two processes are coordinated

SUNDAY-POSTER PRESENTATIONS remains unknown. Using live-cell imaging, RNAi, pharmacological inhibitions and laser microsurgery in Drosophila S2 cells, we identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteract this gradient to trigger chromosome decondensation and NER. Thus, the Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids. This promotes the correction and re-integration of lagging chromosomes in the main nuclei prior to completion of NER.

P191 Board Number: B303

Chromosome segregation during male meiosis in C. elegans. G. Fabig1, J. Brugues2, C. Eckmann2, T. Müller-Reichert1; 1 Medical Faculty "Carl Gustav Carus", TU Dresden, Dresden, Germany, 2MPI-CBG, Dresden, Germany We aim to characterize male meiosis I and II in C. elegans. Germ cells in the male worm mature while slowly moving from the distal to the proximal part of the gonad [1]. During this process undifferentiated germ cells leave the mitotic region at the distal tip of the gonad and undergo a transition to early stages of meiotic prophase I. After getting disconnected from the syncytial core (rachis), the germ cells form primary spermatocytes, while undergoing a series of cytological events during the formation of the bipolar spindles. Segregation of homologous chromosomes in meiosis I and sister chromatids in meiosis II results in four haploid spermatids from one single primary spermatocyte [2]. We apply both light microscopy and electron tomography to gain novel insights into the dynamics and ultrastructure of the male meiotic spindles. Using light microscopy, we image meiotic divisions within immobilized males that are fluorescently labeled with GFP::gamma-tubulin and mCherry::histone H2B. We currently determine the dynamic properties of both meiotic spindles in wild-type specimens. Furthermore, we apply laser microsurgery to manipulate the segregation of the single sex chromosome. For electron tomography, we apply high-pressure freezing followed by freeze-substitution of whole animals [3]. After semi-thick sectioning (300 nm serial sections) through whole worms, we aim to localize the meiotic region inside the male gonad in order to reconstruct and segment different stages of meiotic spindles in ultrastructural detail. Comparing wild-type and mutant situations [4, 5], our long-term goal is to integrate the data on spindle dynamics and ultrastructure to develop a mechanistic model of spindle assembly and sex chromosome segregation in C. elegans male meiosis. [1]

Kimble, J. and Crittenden, S. L.; Germline proliferation and its control; 2005; WormBook.[2] Chu, D. S. and Shakes, D. C.; Spermatogenesis; 2013; Germ Cell Development in C. elegans.[3] Müller-Reichert, T., et al.; Cryoimmobilization and 3D visualization of C. elegans ultrastructure; 2003; J Microsc.[4] O‘Connell, K. F., et al.; A genetic screen for temperature-sensitive cell-division mutants in C. elegans; 1998; Genetics.[5] Dernburg, A. F., et al.; Meiotic recombination in C. elegans initiates by a conserved mechanism and is dispensable for homologous chromosome synapsis; 1998; Cell.

SUNDAY-POSTER PRESENTATIONS

P192 Board Number: B304

Metabolic Imaging and the Role of Cellular Energy in Cell Division. T. Sanchez1, D. Needleman1; 1 Harvard School of Engineering and Applied Sciences, Cambridge, MA Metabolic defects and chromosome segregation errors co-occur in cancer, non-competent embryos resulting in failed pregnancies, and aging. It is unclear to what extent metabolic defects cause chromosome segregation errors, or chromosome segregation errors cause metabolic defects, or both are caused by other factors. To study this issue, we are investigating the relationship between mitochondrial metabolism and chromosome segregation in C. elegans and mouse oocytes and embryos. We are performing quantitative metabolic imaging by measuring endogenous NADH and FAD fluorescence with fluorescence lifetime imaging microscopy (FLIM), allowing us to determine the concentrations, lifetimes, and enzyme interactions of these central metabolites. Our preliminary results show clear distinctions between metabolic states of various cell types. We are quantitatively characterizing how defined biochemical and genetic perturbations produce metabolic changes in these cells, and we will study the extent of corresponding changes in spindle morphology and dynamics, and chromosome segregation rates.

P193 Board Number: B305

Adaptability of Intracellular Structures to Variations in Cell Size. M.C. Good1, M.D. Vahey2, D.A. Fletcher3,4,5, R. Heald1; 1 Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, 2Bioengineering, University of California, Berkeley, Berkeley, CA, 3Department of Bioengineering, University of California, Berkeley, Berkeley, CA, 4Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA, 5Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA Cells exist in a wide variety of shapes and sizes – from round eggs millimeters in diameter to highly elongated neurons with sub-micron extensions. Despite dramatic physical differences, growth and division of these cells is dependent on functional organelles, requiring that these intracellular structures adapt to a wide range of cell geometries. The mitotic spindle, a dynamic microtubule-based structure required for chromosome segregation, provides an important example of this flexibility. During Xenopus early embryo development, cell volume reduces nearly one million-fold due to division in the absence of growth, and spindle size scales with the spatial dimensions of the cell. An interesting question is how intracellular size regulation is achieved. Possible explanations include changes in cytoplasmic composition tied to a developmental program or direct coupling of spindle assembly to physical parameters such as cell volume and diameter. The difficulty of modulating cell size in embryos makes it hard to differentiate between these hypotheses, necessitating the construction of cell-like system with

SUNDAY-POSTER PRESENTATIONS controllable size. By combining droplet microfluidics and cell-free cytoplasmic extracts prepared from various stages of development, I was able to generate functional spindles and nuclei inside compartments whose diameter can be tuned from microns to millimeters, recapitulating the spindle size-scaling trend observed during Xenopus embryogenesis. By modulating droplet diameter and geometry, I discovered that metaphase spindle size is set by compartment volume, not shape, suggesting that limiting amounts of cytoplasmic material can restrict spindle growth. Various experimental results and a mathematical model support the hypothesis that tubulin is at least one component whose levels become limited by the cell volume decrease in early development, thereby constraining spindle length and matching it to cell size. In the future, by extending this encapsulation technology to other organelles and intracellular processes, it should be possible to uncover additional principles of size regulation and identify new pathways that respond to cell size. The significance of this research lies in the intimate connection between cell size and embryo development, and the observation that cell and organelle size are often misregulated in disease.

P194 Board Number: B306

Role of heat shock protein 70 in the assembly of mitotic spindles. C. Fang1, H. Kuo1, L. Yih1; 1 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan The expression of heat shock protein 70 (HSP70) is high in many tumors. The elevated level of HSP70 negatively correlates with prognosis and efficacies of chemotherapeutics, implying a supporting role of HSP70 in tumor cell survival. Accumulated studies also demonstrated that HSP70 interacts with proteins of mitotic structures including spindle poles, microtubule motors, and kinetochores, indicating that HSP70 may involve in the regulation of assembly of mitotic structures and mitosis progression. In this study, we explored the role of HSP70 during mitosis by treating cells with a HSP70 inhibitor or transducing cells with HSP70-specific shRNA. The effects of these treatments on mitosis progression, mitotic spindle assembly, and cell viability were investigated. Our results showed that HSP70 accumulated at the spindle pole and co-localized with γ-tubulin and pericentrin during mitosis. Inhibition of HSP70 or depletion of HSP70 by shRNA transduction led to disruption of microtubule assembly, formation of abnormal mitotic spindles, and interference of mitosis progression. These results indicate that HSP70 may be required for the assembly and/or maintenance of a functional mitotic spindle.

SUNDAY-POSTER PRESENTATIONS

P195 Board Number: B307

Clinically Relevant Concentrations of Taxol Cause Chromosome Missegregation Rather than Mitotic Arrest. L.M. Zasadil1, M. Burkard2, B.A. Weaver1; 1 Cell and Regenerative Biology, Univ Wisconsin-Madison, Madison, WI, 2Department of Medicine, Univ Wisconsin-Madison, Madison, WI Taxol, a microtubule-stabilizing drug, is commonly used to study fundamental mechanisms of mitosis. It is also used in the clinical setting as a chemotherapeutic agent to treat a variety of cancers. For the three decades that taxol has been used in humans, it has been thought to exert its antitumor effects by arresting cells in mitosis, since this is observed at concentrations typically used in cell culture. After treatment with µM or high nM concentrations of taxol, cells with an intact mitotic checkpoint arrest in mitosis, which often leads to cell death. Though not directly shown, it has been widely assumed that taxol also causes mitotic arrest in patient tumors in vivo. In a clinical study, we have measured tumor response and intratumoral drug concentration in breast tumors following taxol treatment. Our data indicate that intratumoral concentrations of taxol are lower than those traditionally attained in cell culture. Moreover, mitotic arrest is not necessary for tumor shrinkage in response to taxol therapy. However, the incidence of multipolar spindles uniformly increased in patient tumors following taxol treatment. These features are mimicked by treatment of cultured cells with clinically relevant concentrations of taxol, which vary by cell type but are in the low nM range. Unlike treatment with higher concentrations, treatment with clinically relevant doses of taxol does not cause a substantial mitotic arrest or an increase in death from interphase without passage through mitosis in the presence of the drug. Instead, cells treated with clinically relevant concentrations of taxol divide with relatively normal kinetics on multipolar spindles to form two or three daughter cells, resulting in chromosome missegregation. Timelapse imaging reveals that initially bipolar spindles evolve into multipolar spindles, which are then often partially focused before anaphase onset, to produce two-or-more-way divisions of DNA. Exposure to clinically relevant doses of taxol is sufficient to induce aneuploidy in chromosomally stable cells and causes cell death in both chromosomally stable and unstable cells. The success of taxol and its presumed mechanism of action have resulted in the development of additional drugs intended to cause mitotic arrest, including those targeting Eg5 and Plk1. These drugs have not yet shown the level of clinical success taxol has, perhaps because the antitumoral effects of taxol are due to chromosome missegregation rather than mitotic arrest. Although taxol is considered highly effective, only approximately half of all patients experience tumor shrinkage after taxol treatment. At present, there is no way to identify and selectively treat patients that will benefit from taxol therapy. These data reveal an alternate mechanism of action for taxol that may be useful in identifying mechanisms of resistance to this type of chemotherapy.

SUNDAY-POSTER PRESENTATIONS

P196 Board Number: B308

An RNAi screen to identify genes required for acentrosomal spindle function in C. elegans. I. Wolff1, T.J. Mullen2, C.M. Heath1, A.C. Davis1, M. Tran1, S.M. Wignall1; 1 Northwestern Univ, Evanston, IL, 2Northwestern Univ, Chicago, IL During cell division in most cell types, duplicated centrosomes nucleate microtubules and ultimately form the poles of the spindle. However, in many species, oocytes lack centrosomes and therefore spindle assembly occurs through a different mechanism. Our aim is to investigate acentrosomal spindle formation by identifying proteins that are required for proper spindle assembly and chromosome congression in C. elegans oocyte meiosis. We have completed a screen of 2,025 genes that were previously classified as embryonic lethal in genome-wide screens. This class includes genes known to be required for acentrosomal meiotic spindle function, such as mei-1 and mei-2 (regulators of microtubule stability), aspm-1 (spindle organization) and bub-1 (kinetochore component). We screened for defects in spindle formation and/or chromosome alignment using worms co-expressing GFP::histone and GFP::tubulin to visualize chromosomes and microtubules simultaneously. Additionally, our screening strain carried a temperature-sensitive mutation affecting the Anaphase Promoting Complex (APC) that prevents entry into anaphase I, allowing us to arrest oocytes at metaphase of meiosis I to better focus on spindle formation and chromosome alignment. Out of 2,025 genes screened, depletion of 291 led to gross defects in the worm, such as aberrant gonad morphology, sterility and defective worm growth; therefore, these could not be screened for meiotic spindle defects. Of the remaining genes, 63% were embryonic lethal under our conditions. 400 of these showed defects in spindle morphology such as monopolar spindles, multipolar spindles, disorganized spindles, as well as defects in chromosome congression. As expected, MEI-1, MEI-2, ASPM-1 and BUB-1 were in this class and resulted in dramatic spindle morphology defects following RNAi. In addition, a number of genes that are uncharacterized in worms were shown to cause defects in spindle morphology, some of which have putative homologs in other organisms. Initial results from our screen and preliminary characterization of our candidate genes will be reported. Future characterization of the genes identified in our screen may help us better understand the mechanisms involved in acentrosomal spindle assembly.

SUNDAY-POSTER PRESENTATIONS

P197 Board Number: B309

Cenexin, a mother centriole appendage protein, is required for spindle orientation during mitosis. H. Hung1, H. Hehnly1,2, S. Doxsey1; 1 Univ Massachusetts Med Sch, Worcester, MA, 2Univ of Washington, Seattle, WA Polarized epithelial cells are required for organ function, but whether this polarization is initially established during late cell cycle has not been clearly resolved. One argument for establishment during late cell cycle is that the epithelial layer can undergo constant regeneration to replace dead and/or damaged cells. During this time, it is known that appropriate orientation of the mitotic spindle relative to cell polarity cues can contribute to cell fate determination, which would thus establish tissue architecture. Another contributor to spindle orientation is the spindle pole. More specifically, one of the two spindle poles contains more mother centriole appendage proteins than the other. Based on this, we propose a model where the centrosome with elevated appendage proteins and polarity cues at the cell cortex establish spindle orientation. One cell cortex polarity complex examined, NuMA/LGN/Gαi, is implicated in spindle orientation through its interaction with astral microtubules. In geneticallyengineered kidney epithelial cells (MDCK) we found that cells depleted of the sub-distal appendage component, Cenexin, lead to asymmetric spindle orientation in both two- and three-dimensional (3D) cell culture systems. In contrast, depletion of the distal appendage protein, Cep164 had no effect on spindle orientation. In 3D culture, Cenexin-depleted cells induced a 2-fold increase in the number of lumina compared to control, which correlated with increased spindle misorientation. Surprisingly, an increase in cortical NuMA localization, and disorganization of a microtubule destabilizer, Kif2a, at the pole was also observed with Cenexin-depletion. Taken together we propose that Cenexin modulates spindle orientation by maintaining spindle pole localized Kif2a, which regulates microtubule organization and contributes to cortical NuMA localization.

P198 Board Number: B310

Mitotic Repertoire of Acetylated Survivin. S.P. Wheatley1, A.M. Ajaberi1; 1 School of Life Sciences, Univ Nottingham, Nottingham, United Kingdom Survivin is a cancer associated protein that was originally cloned as a member of the inhibitor of apoptosis (IAP) family, and later shown to be an essential mitotic protein. The dual pro-survival functions of survivin depend on the localization of the protein within immunochemically distinct pools located in cytoplasm, mitochondria, nucleus and on the chromosomes. During mitosis, survivin operates in a complex with the chromosomal passenger proteins, aurora-B kinase, borealin and INCENP, which localises initially to the centromeres where it regulates chromosomal movements, and later to the equatorial cortex of the cell where it is required for cytokinesis. The mitotic activity of survivin is

SUNDAY-POSTER PRESENTATIONS phospho-regulated by a number of kinases including Cdk1, aurora-B and plk1. Recently, it has been shown that residue K129 in its C-terminal alpha helical coil is acetylated and that this post-translational modification affects the subcellular localisation of survivin during interphase, however, whether this acetylation event contributes to the regulation of mitosis remains unknown. In this study, we ask whether mimicking acetylation by mutating this site to an alanine alters the behaviour of during mitosis. Our data demonstrate that constitutive acetylation of this site inhibits cell proliferation and causes errors in chromosome segregation during mitosis.

P199 Board Number: B311

Microtubule dynamics are modulated by changes in cytoplasmic volume: a possible mechanism for mitotic spindle scaling. A. Milunovic-Jevtic1, A. Matov1, J.W. Hazel1, A.C. Groen2, K. Ishihara2, T.J. Mitchison2, J. Gatlin1,3; 1 Department of Molecular Biology, University of Wyoming, Laramie, WY, 2Department of Systems Biology, Harvard Medical School, Boston, MA, 3Molecular Cellular Life Sciences Program, University of Wyoming, Laramie, WY Spindle size is determined by mechanisms intrinsic to the spindle itself, such as motor-dependent sliding and microtubule polymerization dynamics, and by extrinsic mechanisms like cell size and cortical/cytoplasmic pulling forces. The combined action of these processes ensures constancy of spindle size and shape and a high fidelity of chromosome segregation during cell division. During early development in animals, changes in cell size that accompany rapid reductive divisions result in cells with smaller and smaller spindles, likely due to a progressive reduction in the available amounts of essential spindle components. The mechanistic link between component limitation and spindle size remains poorly understood. Recent studies suggest that changes in microtubule polymerization dynamics, specifically growth rate, could account for spindle scaling during development, with spindle length exhibiting a positive correlation with microtubule growth rates. This led us to predict that microtubule growth rates might be influenced by cell size. To investigate the relationship between microtubule dynamics and cell size, we combined microfluidics and cell-free extracts to encapsulate artificial microtubule organizing centers and sperm nuclei in discrete droplets of X.laevis egg extract. The extract was spiked with fluorescently labeled tubulin and mCherry-EB1, a protein which associates with the growing microtubule ends forming fluorescent comets. Microtubule dynamics we characterized by EB1+TIP tracking using ClusterTrack MATLAB software package. Our measurements indicate that microtubule growth velocities correlate with extract volume. These results suggest that volumedependent modulation of microtubule growth rates is mechanism linking limiting cytoplasmic pools with changes in spindle size.

SUNDAY-POSTER PRESENTATIONS

P200 Board Number: B312

Minus-End Directed Kinesin-14 Motors Align Anti-Parallel Microtubules to Control Metaphase Spindle Length. A. Hepperla1, P. Willey2, C.E. Coombes1, S. Mukherjee1, M. Winey*3, D. Odde4, E. Otoole5, M. Gardner1; 1 Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, 2Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 3MCDB, University of ColoradoBoulder, Boulder, CO, 4Biomedical Engineering, University of Minnesota, Minneapolis, MN, 5MCB, University of Colorado-Boulder, Boulder, CO During cell division, a microtubule-based mitotic spindle mediates the faithful segregation of duplicated chromosomes into daughter cells. Proper length control of the metaphase mitotic spindle is critical to this process, and is thought to be achieved through a mechanism in which spindle pole separation forces from plus-end directed motors are balanced by forces from minus-end directed motors that pull spindle poles together. However, in contrast to this model, metaphase mitotic spindles with inactive Kinesin-14 minus-end directed motors often have shorter spindle lengths, along with poorly aligned spindle microtubules. A mechanistic explanation for this paradox is unknown. Using computational modeling, in vitro reconstitution, live-cell fluorescence microscopy, and electron microscopy, we now find that the budding yeast Kinesin-14 molecular motor Kar3-Cik1 can efficiently align spindle microtubules along the spindle axis. This then allows plus-end directed Kinesin-5 motors to efficiently exert the outward microtubule sliding forces needed for proper spindle bipolarity.

Cytokinesis 1 P201 Board Number: B313

Investigating the role of F-BAR proteins in cytokinesis. M. Marino1, Y. Geng1, O.A. Quintero1, E.R. Griffis2; 1 Department of Biology, University of Richmond, Richmond, VA, 2Centre for Gene Regulation and Expression, University of Dundee, Dundee, Scotland The goal of this research project is to identify the role that F-BAR proteins play during cytokinesis in D. melanogaster S2 cells. It was discovered that F-BAR proteins are essential for fission yeast cell division and act cooperatively as membrane-bending proteins (Martin-Garcia et al. J. Cell Sci. 2014); and recent work has shown that a Drosophila F-BAR protein is also required for coupling the membrane to the contractile ring (Takeda et al. Open Biology 2013). The first component of this research project is to identify if there are other F-BAR proteins involved in cytokinesis in Drosophila cells, and if they behave synergistically. This will be achieved by using dsRNAs to knock down six specific F-BAR containing proteins alone and in combinations. Cells treated with dsRNAs were imaged and the images were

SUNDAY-POSTER PRESENTATIONS analyzed to determine the percentage of multinucleate cells. Preliminarily, each F-BAR knockdown induced a higher percentage of multinucleate cells than untreated control cells. However, a combinatorial knockdown of two F-BARs at once was not shown to induce a stronger multinucleate phenotype. The second component of this research project is to discover the cellular localization of the six F-BAR domain-containing proteins. The localization of these proteins will be made possible by creating GFP tagged F-BAR constructs. These constructs can be transfected into cells for fixed and live-cell imaging. When we observed GFP-F-BAR-CG8176 and mcherry-tubulin, in S2 cells spread on ConA, we discovered that the F-BAR localized in spots on the basal cell membrane radiating from the center of the cell. GFPF-BAR-CG33094 exhibited an identical localization. Further dsRNA knockdown experiments are required to confirm whether certain F-BAR depletions cause cytokinetic defects. Also, the matching localization of two separate F-BAR proteins suggests a shared localization among many F-BAR domain-containing proteins in S2 Cells. Further cloning and imaging is necessary to determine the localization of F-BAR proteins during the process of cytokinesis.

P202 Board Number: B314

Cdc14 Regulates Actin Ring Formation in Budding Yeast Via Dephosphorylation of Iqg1. K.B. Shannon1, D.P. Miller1; 1 Biological Sciences, Missouri ST, Rolla, MO In budding yeast, the IQGAP protein Iqg1 is required to recruit actin filaments to the actomyosin ring, as well as for contraction of the ring during cytokinesis. Iqg1 contains four perfect CDK consensus sites in the N-terminus, and mutation of these sites to alanine (iqg1-4A) leads to premature formation of the actin ring prior to anaphase onset, and prevents proper actomyosin ring contraction. Since Cdc14 dephosphorylates many CDK targets, we looked at the effects of manipulating Cdc14 levels on actomyosin ring formation. Overexpression of Cdc14 induces premature actin ring formation in nocodazole arrested cells. Using a cdc14-1 temperature sensitive mutation to inactivate Cdc14 and overexpression of Sic1 to bypass the mitotic arrest of cdc14-1 cells, we show that Cdc14 is required for actin ring formation. This defect can be rescued by expressing iqg1-4A, demonstrating that Iqg1 is the sole target of Cdc14 that regulates actin ring formation. Iqg1 is the first actomyosin ring target identified for Cdc14.

SUNDAY-POSTER PRESENTATIONS

P203 Board Number: B315

Anillin proteins regulate intercellular bridge stability to maintain syncytial organization of the C. elegans germline. R. Amini1, A. St-Pierre-See 2, N.T. Chartier2, J. Labbe3; 1 Systems Biology, IRIC, Univ Montral, Montreal, QC, 2Pathology and Cell Biology, IRIC, Univ Montral, Montreal, QC, 3Institute for Research in Immunology and Cancer, Department of Pathology and Cell Biology, University of Montreal, Montreal, QC Cells typically complete their division with cytokinesis. However, during development of certain tissues, mitotic division is followed by incomplete cytokinesis, giving rise to interconnected cells in a shared cytoplasm, or syncytium. Syncytia are a conserved feature of female and male germline of most species. Despite this, how syncytium formation is regulated remains elusive. To understand this, we studied the syncytial C. elegans germline wherein germ cells (GCs) remain connected via intercellular bridges. Using live-cell imaging, we found that the contractility regulators ANI-1 (Anillin), NMY-2 (myosin), UNC-59 (septin), ZEN-4 (MKLP1) and CYK-7 localize to GC intercellular bridges. ANI-2, a shorter isoform of Anillin, is enriched between the two primordial GCs from the onset of GC specification during embryogenesis and further accumulates at the intercellular bridges in the gonad. Analysis of ANI-2 localization revealed that syncytial architecture occurs progressively during larval development. Loss of ANI-2 disrupted intercellular bridge integrity and resulted in GC multinucleation, and thus severe gonad disorganization. In contrast, ANI-1 depletion partially restored syncytial organization in ani-2 mutants, suggesting that a balance of activity between the two Anillin proteins at the intercellular bridge is required for the proper organization of the syncytial germline. We are currently investigating the mechanism by which the germline syncytium is formed to understand how controlled incomplete cytokinesis promotes formation of the syncytial C. elegans gonad.

P204 Board Number: B316

The role of cortical dynein during C. elegans female meiotic spindle rotation. J. Flynn1, M. Ellefson1, K. Messina1, P. Kuehnert1, F. McNally1; 1 Univ California-Davis, Davis, CA Meiosis is a specialized cell division required for sexual reproduction characterized by a single round of DNA replication followed by two rounds of chromosome segregation. The molecular machine responsible for accurately segregating chromosomes is the meiotic spindle. In female meiosis, to properly segregate chromosomes the spindle must correctly orient its pole-to-pole axis perpendicular to the cell cortex. In C. elegans, this perpendicular orientation is achieved through a dynein dependent process called spindle rotation. Spindle rotation has been shown to correlate with increased dynein localization on the spindle poles (Ellefson 2011, J. Cell Biol. 193:1229). However, in the female meiotic embryo, dynein has two distinct subcellular localizations: the spindle poles and the cell cortex. This begs

SUNDAY-POSTER PRESENTATIONS the question of whether or not cortical dynein plays a role in spindle rotation during female meiosis. To address this question, we will identify proteins required for cortical localization of dynein during meiosis then test whether these proteins are required for spindle rotation. We found that dynein recruitment to the cortex is not microtubule dependent, but is LIN-5 dependent. Other candidates that may be involved in recruiting dynein to the cortex during meiosis include proteins that are known to recruit dynein to the cortex in the C. elegans one cell mitotic embryo (Galpha and GPR), a protein that has been shown to recruit dynein to the cortex in an anaphase specific pathway in HeLa cells (Band4.1), and a cortical protein shown to suppress lethality induced by a conditional dynein heavy chain mutant (efa-6). We are currently testing whether depletion of these proteins prevents cortical dynein localization and meiotic spindle rotation.

P205 Board Number: B317

The Chromosomal Passenger Complex (CPC) couples the actin and microtubule cytoskeletons during cytokinesis. J.E. Landino1, R. Ohi1, M.E. Dumas1; 1 Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN In animal cells, the microtubule (MT) and actin cytoskeletons are remodeled throughout cell division to facilitate chromosome segregation and cytokinesis. During anaphase, MTs organize into the spindle midzone while actin and myosin assemble into the cleavage furrow. Anaphase events – chromosome segregation, centrosome separation and cleavage furrow contraction – are closely coupled in space and time, implying that mechanisms may exist to link them. Indeed, other cellular processes that rely on both cytoskeletons, such as cell migration, use signaling-based feedback mechanisms to coordinate the activities of the actin and microtubule networks. We identified a novel pathway that stabilizes spindle midzone MTs (mMTs) at the onset of cleavage furrow ingression. Stabilization of mMTs during late anaphase depends on acto-myosin contractility, strongly suggesting that cross-talk between the actin and MT cytoskeletons occurs. We found that cells lacking CPC activity do not stabilize mMTs following cleavage furrow ingression, suggesting a novel role for the CPC in regulating MT stability during cytokinesis. The CPC is transported to mMT plus ends at the onset of anaphase by the kinesin Mklp2, and we have determined that the CPC also localizes to the cell cortex during anaphase via direct actin binding. Additionally, overexpression of CPC components rescues cytokinetic defects caused by Mklp2 depletion indicating there may be multiple pathways for targeting the CPC to the midzone. We propose a model in which actin binding by the CPC provides a second pathway to help recruit the complex to the cleavage furrow, and maintain its localization during anaphase and cytokinesis. We also propose that actin binding allows the CPC to mediate cross-talk between the actin and MT cytoskeletons, ultimately stabilizing mMTs following cleavage furrow ingression.

SUNDAY-POSTER PRESENTATIONS

P206 Board Number: B318

Rac Antagonism of Cytokinesis in the Early Sea Urchin Embryo. A. Ellis1, S.P. Sepulveda1, A. Alvarez2, R. De La Rosa1, J.H. Henson3, C.B. Shuster1; 1 Biology Department, New Mexico State University, Las Cruces, NM, 2New Mexico State University, Las Cruces, NM, 3Dickinson Coll, Carlisle, PA z, John Henson and Charles B. Shuster Rho GTPases are critical modulators of the actin cytoskeleton, and while the role of RhoA during cytokinesis is well established, less is known about how Rac and Cdc42 contribute to cytoskeletal organization during the final stages of cell division. Neither Cdc42 nor Rac is thought to be required for cytokinesis, and several studies indicate that Rac inactivation is necessary for the completion of cytokinesis, possibly by suppressing Rac-mediated cell adhesion at the cell equator. To better understand the regulation of actin in the early embryo as well as the role of Rac during cytokinesis, we performed live cell imaging of actin dynamics during the first cell division of the sea urchin embryo. Studies to date revealed that in addition to microvilli and the contractile ring, there are other distinct populations of actin filaments that underwent dramatic changes during the first mitotic division. A thin layer of cortical actin could be detected that underwent a transient thinning upon anaphase onset, only to recover shortly before cytokinesis. Additionally, there was an explosive elaboration of cytoplasmic actin occurring just prior to the metaphase-anaphase transition, which initiated at the cell surface, and extended inward towards the cell center. Interestingly, RhoA activity was necessary for all actin structures in the egg, whereas neither Rac nor Cdc42 were required. However, expression of constitutively active Rac (Q61L) resulted in early cleavage arrest with aberrant cortical actin projections. Because there are several possible downstream pathways by which Rac may antagonize cytokinesis, well-characterized, effector-binding mutants were employed. Expression of Q61L Rac containing the PAK-binding mutation (Y40C) suppressed the aberrant cortical actin projections, but still resulted in cytokinetic failure during the first two cleavages. In contrast, expression of Q61L Rac carrying the F37A mutation, which affects Arp2/3 mediated actin polymerization, resulted in normal cleavage and actin organization. Together, these findings suggest that in non-adherent, spherical cells, Rac activity plays a direct role in antagonizing the cytokinetic apparatus, possibly through the promotion of Arp2/3-nucleated actin arrays. Current efforts are focused on verifying this notion, as well as examining whether Rac plays an active role in suppressing Rho activation during anaphase and cytokinesis.

SUNDAY-POSTER PRESENTATIONS

P207 Board Number: B319

Regulation of Rho GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis. R. Davidson1, D. Laporte2, J. Wu1,3; 1 Department of Molecular Genetics, The Ohio State University, Columbus, OH, 2Biochemistry and Cellular Genetics, Institute of Biochemistry and Cellular Genetics, Bordeaux, France, 3Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH Rho GTPases, activated by guanine nucleotide exchange factors (GEFs), are essential regulators of polarized cell growth, cytokinesis, and many other cellular processes.However, the regulations of Rho GEFs themselves are not well understood. Rgf3 is an essential GEF for Rho1 GTPase in fission yeast cytokinesis. Here we show that Rgf3 protein levels and localization are regulated by arrestin-related protein Art1. art1∆ cells lyse during cell separation with a thinner and defective septum. Same as Rgf3, Art1 concentrates to the contractile ring starting at early anaphase and spreads to the septum during and after ring constriction. Moreover, Art1 localization depends on its C-terminus and Art1 is important for maintaining Rgf3 protein levels. Biochemical experiments reveal that Rgf3 C-terminus binds to Art1. Using an Rgf3 conditional mutant and with mislocalization experiments, we find that Art1 and Rgf3 are interdependent for localization to the division site. As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells. Taken together, these data reveal a role for the arrestinfamily protein Art1 in regulating the protein levels and localization of the Rho GEF Rgf3, which in turn modulates active Rho1 levels to build a division septum during cytokinesis.

P208 Board Number: B320

The putative Rho-GEF Gef3 interacts with the septin complex and regulates the GTPase Rho4 during fission yeast cytokinesis. N. Wang1, M. Wang1, Y. Zhu1, T. Grosel1, D. Sun1, J. Wu1,2; 1 Department of Molecular Genetics, The Ohio State University, Columbus, OH, 2Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH Rho GTPases, activated by guanine nucleotide exchange factors (GEFs), are highly conserved molecular switches for signal transduction and regulate diverse cellular processes including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1-Rho5) and seven Rho GEFs (Scd1, Rgf1-Rgf3, and Gef1-Gef3). The Rho GEFs for Rho2-Rho5 have not been unequivocally assigned. Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins to a non-constricting double rings at the cell equator during division-septum formation. Gef3 physically interacts with and depends on the septin complex to localize. Purified Gef3 strongly interacts with Rho4 GTPase in vitro. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and cell separation. Although localization of Rho4 is not affected in gef3∆, the localizations of two potential Rho4 effectors, glucanases Eng1 and Agn1, are

SUNDAY-POSTER PRESENTATIONS abnormal, indicating a compromised activation of Rho4. Moreover, overexpression of active Rho4 or Eng1 rescues the cytokinesis defects of exocyst mutants and mutants containing gef3∆. Together, our data suggest that the putative Rho-GEF Gef3 interacts with the septin complex and regulates GTPase Rho4 for septum formation and cell separation during cytokinesis.

P209 Board Number: B321

The RhoGAP domain in centralspindlin directly contributes to activation of RhoGEF ECT-2 during cytokinesis. D. Zhang1, M. Glotzer1; 1 Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL In animal cells, the small GTPase RhoA is essential for the contractile actomyosin network that drives cytokinetic furrowing. Counterintuitively, the evolutionarily conserved protein complex, centralspindlin, which contains a subunit, CYK-4, with a Rho family GAP domain, promotes RhoA activity. The regulatory N-termini of CYK-4 and the RhoA GEF ECT-2 directly interact. Various studies have indicated that the GAP domain is important for CYK-4 function, but the underlying mechanism remains controversial. To determine whether the catalytic activity of CYK-4 GAP domain contributes to cytokinesis, we established single-copy transgenes in C. elegans, and mutated the catalytic residue R459 to specifically perturb the GAP activity of CYK-4. cyk-4R459A mutant embryos fail to complete cytokinesis. RhoA activation involves a parallel, non-essential, nematode-specific activator of ECT-2, NOP-1. cyk-4R459A;nop1 embryos exhibit extremely weak furrow ingression. Mutations in CED-10/Rac1 do not modulate the defect in cyk-4R459A;nop-1 mutant embryos, demonstrating that the defect resulting from loss of catalytic activity of the GAP domain is not due to hyperactivation of CED-10/Rac1. To investigate whether furrow ingression defects result from failure to fully activate or inactivate RhoA, we examined how the defect was modulated by depletion of the primary RhoA GAP, RGA-3/4. Remarkably, depletion of RGA-3/4 allowed cytokinetic completion in both cyk-4R459A and cyk-4R459A;nop-1 mutant embryos. Binding assays revealed that CYK-4R459A binds to RhoA more strongly than CYK-4. To test whether the CYK-4R459A cytokinesis defect results from sequestration of RhoA, we mutated charged surface residues in CYK-4. These variants reduce RhoA binding and cause more severe RhoA activation defects than CYK-4R459A. These results suggest that CYK-4 has to bind to RhoA in order to fully activate ECT-2 and that CYK4R459A•RhoA•GTP complex does not effectively fulfill this role. To obtain independent insight into how the CYK-4 GAP domain promotes cytokinesis, we identified mutations that suppress cyk-4(or749) to viability (or749 has GAP domain mutation, causing cytokinetic failure). One isolate containing a substitution mutation in ECT-2 was proven to be causative. ect2(xs110) mutant embryos exhibit hypercontractility during cytokinesis in a CYK-4-dependent manner. ect-2(xs110) and cyk-4(or749) mutually suppress. These results demonstrate that the primary defect in cyk-4(or749) is a failure to activate RhoA.

SUNDAY-POSTER PRESENTATIONS We show that the GAP domain of CYK-4 promotes ECT-2-dependent RhoA activation, and suggest that this activity is enhanced by RhoA binding to the GAP domain of CYK-4.

P210 Board Number: B322

MgcRacGAP’s GAP activity spatially restricts active RhoA and Rac1 during cytokinesis and maintains proper adherens junction structure in epithelial cells. E.B. Breznau1, A. Semack2, A.L. Miller2; 1 Graduate Program in Cell and Molecular Biology, University of Michigan, Ann Arbor, MI, 2Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI Cytokinesis is the last step of cell division where one cell is physically separated into two. In animal cells, localized activation of the small GTPase RhoA (RhoA-GTP) in a focused equatorial zone is essential for cytokinesis. Activation of RhoA is dependent on the guanine nucleotide exchange factor Ect2, which is spatially confined at the equatorial membrane by Centralspindlin, a complex of the kinesin MKLP1 and the GTPase activating protein (GAP) MgcRacGAP (Mgc). Both the identity of the GTPase targeted by Mgc’s GAP activity and the question of whether phosphorylation of Mgc at serine 386 affects the specificity of Mgc’s GAP activity are controversial. Here, using Xenopus laevis embryos as a model system, we examine for the first time Mgc’s role in the intact vertebrate epithelium. We show that Mgc’s GAP activity regulates the proper spatially restricted accumulation of both RhoA-GTP and Rac1GTP during cytokinesis in epithelial cells. Phosphorylation at serine 386 within Mgc’s GAP domain is not required for proper regulation of GAP activity toward RhoA or successful cytokinesis in the intact epithelium. However, perturbing Mgc’s GAP activity leads to cytokinesis failure, abnormally increased accumulation of RhoA-GTP at the cleavage furrow, and increased local accumulation of both RhoA-GTP and Rac1-GTP at cell-cell junctions. Furthermore, Mgc regulates adherens junction but not tight junction structure, and Mgc’s ability to regulate adherens junctions is dependent on its GAP activity. Cells expressing a Mgc GAP-dead mutant had defects in adherens junctions, which could be rescued by expressing dominant negative RhoA, suggesting that the defects are due to misregulated active RhoA. These results indicate that Mgc’s GAP activity is necessary to down-regulate the active populations of RhoA at the division site and both RhoA and Rac1 at cell-cell junctions in epithelial cells, and failure to do so results in defects in both cytokinesis and cell-cell junctions.

SUNDAY-POSTER PRESENTATIONS

P211 Board Number: B323

The GAP activity of C. elegans CYK-4 is required for incomplete cytokinesis in the syncytial germline but not for contractile ring constriction during embryonic cytokinesis. K. Lee1, R. Green1, A. Desai1, K. Oegema1; Ludwig Institute for Cancer Research, La Jolla, CA

1

Contractility-based remodeling of the actomyosin cell cortex partitions nuclei into distinct cells during cytokinesis. An incomplete cytokinesis-like process also partitions nuclei into membrane-bound compartments that remain cytoplasmically connected in syncytial tissues such as the C. elegans germline and Drosophila embryo. Cortical remodeling during cytokinesis is directed by regulators concentrated on the anaphase spindle, including the Rho family GTPase-activating protein (GAP) CYK-4, a subunit of the centralspindlin complex, that also includes kinesin-6. CYK-4 is also required for nuclear partitioning in the germline and thus for organismal fertility. While the importance of CYK-4 is undisputed, the activities responsible for its specific functions are controversial. CYK-4 has two functional domains: (1) a GAP domain, predicted to target a Rho family GTPase, and (2) a C1 domain, which confers association with the plasma membrane. We have tested the importance of CYK-4 GAP activity as well as of the C1 domain in C. elegans by deleting or mutating each domain and analyzing embryonic cytokinesis and germline nuclear partitioning. As complete inhibition of CYK-4 prevents the generation of embryos, we engineered strains with an additional wild-type CYK-4 copy that is tagged with a degron that results in its elimination at the meiosis-to-mitosis transition to enable selective analysis of the consequences of engineered mutations on embryonic cytokinesis. All mutants in the GAP and C1 domains supported central spindle assembly. Deletions of either domain, or point mutations disrupting the C1 domain, disrupted contractile ring assembly and cytokinesis. However, mutation of GAP activity (by either a single point mutation in the catalytic residue R459A or a triple point mutation targeting the catalytic Arginine plus two other conserved residues important for GTPase binding: R459A/K495A/R499E: AAE) did not disrupt contractile ring assembly and constriction in the early embryo, although it did appear to compromise cytokinesis completion as abscission failed in ~16% of R459A and ~45% of AAE embryos. In contrast to embryonic cytokinesis, all mutants in the GAP and C1 domains, including the R459A and AAE GAP activity mutants, disrupted the incomplete cytokinesis-like process that partitions nuclei in the germline and severely reduced fertility. Thus, the GAP and C1 domains are essential for all CYK-4 functions, but GAP activity is required to promote abscission during embryonic divisions and for incomplete cytokinesis in the germline but not for contractile ring assembly and constriction.

SUNDAY-POSTER PRESENTATIONS

P212 Board Number: B324

Role of GTP in Septin Assembly and Dynamics in Ashbya gossypii. A. Khan1, A. Gladfelter1; 1 Dartmouth College, West Lebanon, NH Septins are GTP binding proteins that can come together to form rod-shaped hetero-oligomers. These rod complexes can give rise to filaments that organize into higher order structures. Septins have been shown to bind and hydrolyze GTP, however the relationship between septin assembly and GTP binding/hydrolysis is unclear. Here we use the filamentous fungus Ashbya gossyppii to study the role of GTP in septin assembly and dynamics. In ashbya, there are six mitotic septins encoded by CDC10, CDC3, CDC12, CDC11A, CDC11B and SHS1. While the position of most septins in the rod complex is fixed, Cdc11 and Shs1 can compete for the terminal position where they bind and interact with Cdc12. Using GTP binding mutants, we show that nucleotide binding can change Cdc12 binding partners in the septin rod complex, possibly by changing the oligomerization interfaces. This preference for Shs1 over Cdc11 changes the rod composition, which translates to behavioral changes of higher order structures in vivo.

P213 Board Number: B325

Protease-dead separase is dominant negative in the C. elegans embryo. X. Bai1, D. Mitchell1, L. Uehlein1, J.N. Bembenek1; 1 Univ Tennessee-Knoxville, Knoxville, TN Separase is a protease that promotes chromosome segregation at anaphase by cleaving cohesin. Several non-proteolytic functions of separase have been identified in other organisms. We found that separase is required for vesicle exocytosis during anaphase, and hypothesize that its protease activity is required. To address this, we expressed protease-dead separase in C. elegans embryos. We find that expression of protease-dead separase is dominant-negative in embryos, not previously reported in other systems. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes and have applied them to characterize expression of protease-dead separase in embryos. Using these methods, we show that protease-dead separase causes embryo lethality, and that protease-dead separase cannot rescue separase mutants. Furthermore, expression of protease dead separase interferes with chromosome segregation and vesicle trafficking during cytokinesis. In addition, ectopic accumulation of protease dead separase is observed at several putative sites of action within the cell during anaphase, including the furrow and midbody. These data suggest that protease-dead separase interferes with endogenous separase function, possibly by binding substrates and protecting them from cleavage. Identification of relevant substrates will be an important goal to understand the mechanism of separase dependent exocytosis.

SUNDAY-POSTER PRESENTATIONS

P214 Board Number: B326

Reconstituted Cytokinesis Signaling. A.C. Groen1, P.A. Nguyen1, M. Loose2, K. Ishihara1, M. Wühr1, T.J. Mitchison1, C.M. Field1; 1 Department of Systems Biology, Harvard Medical School, Boston, MA, 2Harvard Med Sch, Boston, MA Cell division is the process whereby one cell divides into two daughter cells and is essential for many aspects of biology, including growth, immune response, and cancer (when unregulated). Successful symmetric cell division relies on precise positioning of the cleavage furrow at the midplane. During early zygotic cell divisions, astral microtubules grow from the spindle poles out to the cortex after anaphase to anchor the spindle apparatus during chromosome segregation. We developed a novel cell-free system from frog egg extracts that reconstitutes growth and interaction of asters nucleated from artificial centrosomes. Similar to embryos, these zones recruit critical cytokinesis protein complexes, such as Kif4-PRC1, Centralspindlin, and the chromosomal passenger complex (CPC). Most importantly, we observed the recruitment of cleavage furrow markers (anillin, actin, and GTP-bound RhoA) at interaction zones assembled over supported lipid bilayers (which mimic the plasma membrane). This is the first time a spindle midzone-like structure was reconstituted that can signal to the cortex to initiate the cleavage furrow. We are currently exploring the role of microtubule dynamics on recruitment of cleavage furrow signals at the zone.

P215 Board Number: B327

Differential survivin expression at the single-cell level as revealed by flow cytometry and microscopy. A. Miletic1, W. Kang1, B. Scott1, X. Li1, L. Ma1, S.G. Becker-Catania1, S. Lee1, N. Schrantz1, P. Just1; 1 Research and Development, eBioscience, An Affymetrix Company, San Diego, CA Survivin (also known as Birc5) is a member of the inhibitor of apoptosis (IAP) family that also includes XIAP, c-IAP1, c-IAP2, and NAIP. Like other members of the IAP family, one major function of survivin is inhibition of caspase activation, which leads to the negative regulation of apoptosis. Nevertheless, survivin also plays a critical role in mitosis and cytokinesis, during which it is expressed only during the G2/M-phase and can be found localized to the mitotic spindle and cleavage furrow/midplate. Due to its role in cell cycle progression, survivin has been found to be highly expressed in tumors and fetal tissue, but is absent in terminally-differentiated and non-proliferating cells. Moreover, deletion of survivin in developing mouse thymocytes, as well as in mature T and B cells, demonstrated that survivin is critical for cell division in lymphoid cells but not for survival. To date, very few tools are available to detect and visualize survivin expression and localization ex vivo. Here, we describe a novel mouse monoclonal antibody that specifically recognizes human survivin and demonstrate its utility in flow cytometry and immunocytochemistry to examine survivin expression in actively proliferating T cells. With this antibody,

SUNDAY-POSTER PRESENTATIONS researchers will be able to gain a better understanding of the role survivin plays in the balance between cell proliferation and apoptosis during cancer and development.

G1, G1-S, and S Phase Regulation P216 Board Number: B328

Effect of pituitary tumor transforming gene (PTTG1) on the self-renewal activity of placenta-derived mesenchymal stem cells. H. Lee1, J. Choi1, J. Kim2, S. Lee3, G. Kim1; 1 Dept. of Biomedical Science, CHA University, Seoul, Korea, 2Dept. of Pharmacy, CHA University, Seongnam, Korea, 3Dept. of Oral Pathology, College of Dentistry, Gangneung-Wonju National University, Gangneung, Korea In addition to differentiation potential, self-renewal capability is an important characteristic of stem cells. The limited self-renewal activity of mesenchymal stem cells is the biggest obstacle to the application of stem cell therapy in regenerative medicine. The human TERT gene enhances the selfrenewal of MSCs, but the mechanism of self-renewal and the interactions among TERT gene-related molecules remain unknown. The objectives of this study were to generate immortalized MSCs derived from mesenchymal stem cells isolated from placenta (Naïve) by human TERT gene transfection using the AMAXA gene delivery system, compare their characteristics, and investigate whether increased TERT expression affects the pituitary tumor transforming gene (PTTG1) (also known as securin), which is involved in chromosome segregation during mitosis. TERT-immortalized cells (TERT+) with prolonged life span displayed high PTTG1 expression. TERT+ cells also retained the stemness capacity and multipotency of naïve cells and displayed high PTTG1 expression. However, down-regulation of PTTG1 by treatment with siRNA induced cell senescence and decreased telomerase activity. In addition, TERT bound to PTTG1 forms complex with chaperones such as Ku70 and heat shock protein 90 (HSP 90). Taken together, placental MSCs immortalized by TERT gene transfection display differentiation potential and enhance the self-renewal of MSCs through a balanced interaction of PTTG1 and chaperones. The results suggest that the interaction between TERT and PTTG1 by association of Ku70 could be important for the enhancement of the limited self-renewal activity of MSCs as well as for understanding the regulatory mechanisms of self-renewal.

SUNDAY-POSTER PRESENTATIONS

P217 Board Number: B329

Cell Cycle Dependent Transcription is Regulated by Nuclear Positioning in a Syncytium with Asynchronous Nuclear Division. S.E. Roberts1, A.S. Gladfelter1; 1 Biological Sciences, Dartmouth College, Hanover, NH In multinucleate cells of the fungus Ashbya gossypii, nuclei divide independently despite sharing a common cytoplasm. Internuclear distance is tightly regulated, with neighboring nuclei ~5 μm apart. We demonstrated that heterogeneous localization of cyclin transcripts by their association with protein aggregates promotes asynchronous division. To determine whether variability in the production of transcripts also promotes transcript spatial heterogeneity, nuclei producing cyclin transcripts were identified using single molecule RNA FISH. Cyclin transcription correlated with the cell cycle phase of the nucleus indicating that even in a shared cytoplasm nuclei have “knowledge” of their cell cycle phase. It is possible that variable transcription of cyclins contributes to variable local cytosolic concentrations of cyclin protein. If so, the volume of cytosol between neighboring nuclei may be crucial to nuclear cycle autonomy. In fact, previous data showed when nuclei cannot control their spacing they undergo synchronous division. To further investigate the relationship between nuclear spacing and division autonomy we utilized a mutant strain lacking the dynactin complex in which large nuclear clusters form with 10-30 nuclei

P218 Board Number: B330

IRBIT is a novel regulator of Ribonucleotide reductase in high eucaryotes. M. Dasso1, A. Arnaoutov1; 1 National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD Ribonucleotide reductase (RNR) is an enzyme that supplies the balanced pools of deoxynucleotide triphosphates (dNTPs) necessary for DNA replication and maintenance of genomic integrity. RNR is subject to allosteric regulatory mechanisms in all eukaryotes, as well as to control by small protein inhibitors Sml1p and Spd1p in budding and fission yeast, respectively. We have found that the metazoan protein IRBIT controls RNR activity via a novel allosteric mechanism: IRBIT forms of a complex with RNR in dATP-dependent manner, stabilizing dATP in the activity site of RNR, and thus inhibiting the enzyme. Formation of the RNR-IRBIT complex is regulated through phosphorylation of IRBIT, and ablation of IRBIT expression in HeLa cells causes imbalanced dNTP pools and altered cell cycle progression. We have begun to develop model systems for examination of IRBIT function in intact organisms, and have particularly focused on analysis of the Drosophila melanogaster midgut, a wellcharacterized system for tissue generation and homeostasis. Our preliminary findings suggest that these processes are misregulated in the absence of IRBIT.

SUNDAY-POSTER PRESENTATIONS

P219 Board Number: B331

Ran GTPase activates DNA damage response and cell cycle resumption, driving the evasion of cell senescence in normal and cancer cells. P. Cekan1, S. Ryu1,2, K. Hasegawa1,3, J. Lee1, P. Kalab1; 1 NCI/NIH, Bethesda, MD, 2Well-Aging Research Center, Samsung Advanced Institute of Technology, Osan, Korea, 3Biochemistry-Biophysics, Amherst College, Amherst, MA Several lines of evidence indicated that the small GTPase Ran could have a role in activating cell cycle resumption in the interphase. First, Ran is required for the nuclear-cytoplasmic transport of cell cycle regulators, including nuclear export of cyclin mRNAs and nuclear import of DNA replication factors. Second, the activity of Ran-regulated nuclear transport was found to be reduced in non-dividing senescent cells. Finally, previously we showed that rapidly dividing tissue culture cells expressed increased levels of RCC1, the factor required for the formation of the GTP-loaded active form of Ran. Here we show that stable cell cycle exit in normal senescent cells led to the decline of RCC1 expression and reduced RanGTP levels. In contrast, RCC1 expression was required for cell cycle resumption in colorectal carcinoma cells recovering from DNA damage-induced senescence-like state. Remarkably, RCC1 overexpression was sufficient to override the DNA damage-induced cell cycle arrest in normal epithelial cells, leading to the rise of aneuploid proliferative cells. The effects of RCC1 on cell senescence evasion in normal and cancer cells required the role of RanGTP and Exportin 1 in nuclear export and involved the activated expression of many factors promoting DNA damage repair, DNA replication and cell cycle. These results indicate that, through its interphase nuclear functions, RanGTP promotes DNA damage repair and cell cycle initiation, biasing the cellular response to genotoxic insults towards continuous proliferation in the presence of severe genomic alterations.

P220 Board Number: B332

Elm1 regulates Hsl1/Hsl7 interaction in the morphogenesis checkpoint. H. Kang1; 1 Pharmacology, Duke University, Durham, NC The yeast morphogenesis checkpoint prevents nuclear division prior to bud emergence through Swe1mediated inhibition of Cdc28. After bud formation, Swe1 is targeted for degradation via sequential phosphorylation events that require localization of the kinases Elm1 and Hsl1, as well as the Swe1binding protein Hsl7, to septin ring at the mother-bud neck. Elm1 activates Hsl1, which recruits Hsl7 to septin ring only in budded cells. How do these proteins “know” when budding has occurred? By tethering the small Hsl7-binding domain of Hsl1 to a septin, we found that this domain is sufficient to confer Hsl7 recruitment. Although the Hsl1 fragment was present before budding, Hsl7 was only recruited after budding. The Elm1 kinase was also localized to the septins only after budding. Using a Bni4-Elm1 fusion that targets the Elm1 kinase to the septin ring prior to budding, we found that

SUNDAY-POSTER PRESENTATIONS premature Elm1 targeting led to premature Hsl7 recruitment by the Hsl1 fragment. Over expression of Elm1 leads to its early localization before budding. Similarly, early localized Elm1 can also contribute to Hsl7 recruitment by Hsl1 fragment in unbudded cells. These findings suggest that Elm1 localization is the process that coordinates with bud emergence, and that it regulates Hsl1/Hsl7 interaction to promote Hsl7 recruitment and Swe1 degradation only after budding has begun.

P221 Board Number: B333

Yeast metabolic cycle: Entry into oxidative phase is tightly coupled to the cell cycle in diverse strains and growth conditions. A.J. Burnetti1, N. Buchler2, M. Aydin2; 1 University Program in Genetics and Genomics, Duke University, Durham, NC, 2Biology, Duke University, Durham, NC The budding yeast Saccharomyces cerevisiae is capable of synchronous metabolic oscillations with a period between 45 minutes and several hours when grown in chemostat conditions. This oscillation consists of alternating periods of buildup and oxidation of the storage carbohydrates trehalose and glycogen, and changes in expression of a large fraction of the transcriptome. The cell division cycle (CDC) couples strongly to this oscillation, with a single pulse of cells entering S phase once per yeast metabolic cycle (YMC). Early work showed that in some strains DNA replication and oxidative phase never overlapped, presumably to prevent damage to DNA during replication. However, more recent work in other strains has shown that DNA replication can occur both during and outside of oxidative phase. To better understand strain-dependent and strain-independent phenotypes, we examined YMC-CDC coupling in different laboratory strains and wild-isolates across varying chemostat conditions. We show that all strains exhibit a linear relationship between YMC and CDC period, that oxidative phase can overlap DNA replication in many strains, and that the interval between entry into oxidative phase and DNA replication is invariant for a given strain across growth conditions. These data suggest that cell cycle Start is tightly coupled to events surrounding the entry into the high-metabolic-rate oxidative phase. This excludes mechanisms and rationales for cell cycle coupling involving protection of DNA from metabolic damage, and instead suggests a role for coupling in ensuring DNA replication occurs when sufficient cellular energy is available. Moreover, when combined with recent work revealing direct metabolic effects on cyclin gene expression it suggests a positive feedback loop between carbon metabolism and cell cycle Start which synchronizes compatible cellular and metabolic events to ensure they occur together.

SUNDAY-POSTER PRESENTATIONS

P222 Board Number: B334

The anaphase-promoting complex works together with the SCF complex for proteolysis of the S-phase cyclin Clb6 during the transition from G1 to S phase. S. Wu1, V.J. Kuan2, Y. Tseng3, S.C. Schuyler2, Y... Juang4; 1 Microbiology and Immunology, Tzu-Chi University, Hualien, Taiwan, 2Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan, 3Institute of Medical Sciences, Tzu-Chi University, Hualien, Taiwan, 4 Mackay Medical College, New Taipei City, Taiwan In Saccharomyces cerevisiae, the S-phase cyclin Clb6 is expressed shortly at the G1/S border. It has been shown that the SCFCdc4 ubiquitin ligase controls Clb6 proteolysis requiring cyclin-dependent kinases and that Clb6-3A, bearing non-phosphorylatable mutations of S6A, T39A, and S147A, is hyperstabilized but still unstable in mitosis. In this study, we found that the APCCdh1 form of anaphase-promoting complex, an E3 ubiquitin ligase, was required for Clb6 proteolysis in both early and late G1. In vitro ubiquitination assay further confirms that Clb6 is a substrate of the APCCdh1. Clb6 has a KEN box and a destruction box in its N-terminus. Mutations in the KEN box (mkb) and/or the destruction box (mdb) could enhance Clb6 stability in G1. Clb6mkd bearing mutations of mkb and mdb could make cells bypass the late G1 arrest caused by the cdc4-1 temperature-sensitive mutation depending upon CDK phosphorylation at residues S6, T39 and S147. As compared to Clb6, overexpression of Clb6-ST, bearing mutations of S6A, T39A, S147A, mkb, and mdb, had a greater effect on promoting expression of Clb2 and S-phase entry, and caused a greater G2 delay and a greater defect in cytokinesis. Furthermore, Swe1 was required for occurrence of bud emergence when Clb6-ST was overexpressed. Overall, our observations suggest that both APCCdh1- and SCFCdc4-dependent proteolysis of Clb6 at the G1/S border is crucial for proper expression of Clb2, G1/S transition, G2/M transition, and cytokinesis.

P223 Board Number: B335

Cyclin oscillations in living cells: Endogenous reporters for a quantitative view on cyclin expression and proteolysis. M. Lachnit1, I. Gak1, J. Mansfeld1; 1 Cell Cycle, Biotechnology Center, Technische Universität Dresden, Dresden, Germany Cell cycle progression is tightly controlled by oscillations of cyclins that activate distinct cyclindependent kinases (Cdk) at the right time. While the protein level of the Cdk generally remains constant, the levels of cyclins are precisely controlled by ‘tug of war’ between cyclin expression and ubiquitinmediated proteolysis. Decades of research have revealed the key players and pathways regulating cyclin stability. Ljttle is known, however, about the in vivo dynamics of cyclin oscillations and their response to cell cycle challenges due to the lack of reporters that faithfully reflect the regulation of endogenous cyclins. Using non-transformed retinal pigment epithelial cells as a model we have here employed

SUNDAY-POSTER PRESENTATIONS homologous recombination techniques to insert the gene of the fluorescent protein mVenus sitespecific and in frame into the loci of cyclins A2, B1 and D1. Notably, the fluorescent fusion proteins behave comparably to the untagged cyclin indicating that measuring mVenus fluorescence is a valid readout to visualize cyclin oscillations in living cells. To relate the fluorescence data to the exact cell cycle stage we have further tagged endogenous the proliferating cell nuclear antigen (PCNA) and histone H3.1 with mRuby and mTurquoise2, respectively. Therefore, we can automatically segment and classify single cells into the exact cell cycle stage. Furthermore, the same fluorescent tag on different cyclins allows us to precisely determine their absolute and relative expression and proteolysis kinetics by quantitative live cell microscopy. As expected and validating this approach, cyclins A2 and B1 are rapidly degraded in mitosis and accumulate in S- and G2-phase, respectively. Cyclin D1 in contrast to the prevalent view oscillates in G1-phase, strongly decreases before S-phase, and then re-accumulates in G2-phase. Allowing nuclear segmentation (histone), cell cycle stage classification (PCNA) and cyclin expression (mVenus)- all dependent on endogenously-tagged proteins - the cyclin reporters will be valuable tools to get a more quantitative view on cyclin oscillations and to assay the behaviour of single cells when the cell cycle is challenged.

P224 Board Number: B336

Phase separated RNA-protein droplets position cyclin transcripts to organize the cytosol of multinucleate cells. H. Zhang1, A.A. Bridges1, P. Occhipinti1, A.S. Gladfelter1; 1 Biological Sciences, Dartmouth College, Hanover, NH Syncytial cells are found throughout the biosphere from filamentous fungi to early animal development, muscle, placenta and tumor cells. Multinucleate cells face unique organizational problems and in many cases must functionally compartmentalize their cytosol for diverse processes including nuclear division and symmetry breaking. We found that in the filamentous fungus Ashbya, in which nuclei cycle asynchronously despite sharing a cytosol, that cyclin transcripts are clustered and kept near nuclei by binding to the RNA-binding protein, Whi3. Whi3 has a substantial polyQ tract and this aggregationprone region is essential for clustering transcripts and asynchronous nuclear division. We hypothesized that Whi3-cyclin complexes form functional, dynamic aggregates that phase separate from bulk cytosol, potentially to control the location of cyclin protein production. We have reconstituted the cyclin transcript-polyQ protein complex and found that at physiological concentrations Whi3 and cyclin mRNAs phase separate and form liquid droplets both in buffer and in concentrated cell extracts. Biophysical measurements of the droplet properties indicate that they are dynamic, fluid and highly dependent on having mRNA and full length Whi3 protein. Similarly, in cells the ability of Whi3 protein to bind mRNA is essential for its heterogeneous localization into dynamic puncta and tubules, as well as nuclear asynchrony. These results demonstrate a function for RNA-protein granules in regulating variable and asynchronous nuclear progression and show mRNA is a critical component for the assembly process. We

SUNDAY-POSTER PRESENTATIONS propose that phase-separated droplets may be widely employed to pattern cytosol for diverse processes beyond the regulation of mRNA turnover and are likely critical for large, syncytial cell organization.

P225 Board Number: B337

The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells. T. Gotoh1, M. Vila-Caballer2, C.S. Santosa2, J. Liu2, J. Yang2, C.V. Finkielstein1; 1 Biological Sciences, Virginia Tech, Blacksburg, VA, 2Biological SCiences, Virginia Tech, Blacksburg, VA Circadian rhythms are mechanisms that measure time on a scale of about 24 h and that adjusts our bodies to external environmental signals. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Disruption of circadian rhythm has recently emerged as a new potential risk factor in the development of cancer, pointing to the core gene period 2 (per2) as a tumor suppressor. However, it remains unclear how the circadian network regulates tumor suppression, nor which, if any, of its components is either the ultimate effector that influences the fate of the cell. The relevance of hPer2 to human disease is underlined by alterations in its function that impacts numerous biochemical and physiological processes and, when absent, result in the development of various cancers. We found hPer2 binds the C-terminus half of human p53 (hp53) and forms a stable trimeric complex together with hp53’s negative regulator Mdm2. We determined that hPer2 binding to hp53 prevents Mdm2 from ubiquitinating and targeting hp53 by the proteasome. Accordingly, downregulation of hPer2 expression directly impacts hp53 levels whereas its overexpression influences both hp53 protein stability and transcription. Furthermore, we spatially defined the distribution of the trimeric complex within the nucleus. Lastly, we established that hPer2 binding influences hp53-mediated gene transcription (e.g., CDKN1a, SFN, GADD45α) in response to DNAdamage by reconstituting the p53-mediated signaling pathway in a p53-/- cellular model. Overall, our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to rapidly respond to a stress conditions.

P226 Board Number: B338

Functions of PP1-NIPP1 in cell cycle progression. C. Winkler1, C. Kig1, B. Lesage1, M. Beullens1, A. Van Eynde1, M. Bollen1; 1 Department of Cellular and Molecular Medicine, Laboratory of Biosignaling Therapeutics, Leuven, Belgium Protein Ser/Thr phosphatase 1 (PP1) forms heterodimers with dozens of structurally unrelated polypeptides that regulate substrate selection and enzymatic activity. One of the major nuclear interactors of PP1 is NIPP1, which has a ForkHead-Associated (FHA) domain that recruits a subset of

SUNDAY-POSTER PRESENTATIONS Cdk-phosphorylated proteins for dephosphorylation by associated PP1. To delineate the role of PP1NIPP1 in cell-cycle progression we have generated cell lines (Flp-In T-REx HeLa) that inducibly express NIPP1 variants or fusions. The expression of NIPP1 caused a block in early-mid-mitosis associated with lagging chromosomes, spindle defects and a permanently activated spindle-assembly checkpoint, often culminating in mitotic cell death. This heterogeneous phenotype was also observed after expression of a FHA-binding mutant of NIPP1 but was completely absent after expression of a PP1-binding mutant. Additional data suggested that the mitotic defects induced by the overexpression of NIPP1 stem from the competitive disruption and inhibition of PP1 holoenzymes that are essential for mitotic progression. Hence, NIPP1 expressing cell lines can be used to further delineate the mitotic functions of PP1. A completely different phenotype was observed after the expression of a PP1-NIPP1 fusion. This fusion caused replication stress, associated with the accumulation of gH2AX foci and impaired DNA repair via homologous recombination. This effect was dependent on both a functional FHA domain and PP1 activity, and could be explained by the dephosphorylation and destabilization of two FHA ligands of NIPP1 that function in homologous recombination, namely protein kinase Melk and the tumor suppressor protein CtIP. Our data show that PP1-NIPP1 contributes to the reversal of Cdk-mediated cellcycle progression.

P227 Board Number: B339

The deubiquitinase USP37 promotes the efficient S-phase progression and the cellular response to replication stress. M.K. Summers1, A.C. Burrows1; 1 Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH Ubiquitin-mediated proteolysis is a key regulatory process in cell cycle progression. Recently we identified the deubiquitinase USP37 as regulator of S-phase entry. Here we report that depletion of USP37 leads to diminished cellular proliferation and loss of viability. USP37-depleted cells exhibit altered replication kinetics and increased levels of DNA damage markers γH2AX and 53BP1 . Moreover, USP37-depleted cells display significantly increased sensitivity to replication stress. Consistent with this increased sensitivity, activation of the checkpoint kinase Chk1 is attenuated in USP37-depleted cells. We have determined that decreased stability of the replication checkpoint mediator Claspin underlie the defects in Chk1 activation. USP37 interacts with and deubiquitinates Claspin. Our data suggest a model whereby temporally regulated interplay of USP37, APCCdh1, and the replication checkpoint machinery determines the timing of replication checkpoint activity that sharpens the G1/S transition and promotes the efficient completion of replication. These data provide an improved understanding of the replication checkpoint, control of APCCdh1, and maintenance of genome stability.

SUNDAY-POSTER PRESENTATIONS

P228 Board Number: B340

Survival of proliferative, radio-resistant polyploid cells in Drosophila requires FANCD2. H. Bretscher1, D. Fox1; 1 Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC Maintaining a stable genome prevents damaged DNA, altered cellular function, and ultimately diseases such as cancer. Genome instability can result from a wide variety of cellular stresses including DNA damage. In order to prevent propagation of genome instability, diploid mitotic cells employ a DNA damage checkpoint to ensure that they do not divide with damaged DNA. Entering mitosis with damaged DNA can result in mutations and chromosome number imbalance (aneuploidy). While the response to DNA damage is well characterized in diploid mitotic cells, it has been recently observed that passage through the endocycle, a variant cell cycle in which mitosis is truncated, can alter the response to DNA damage. As a result of this alteration, endocycling murine trophoblasts and several Drosophila larval tissues are highly resistant to DNA damage. Unlike diploid cells, these non-proliferative cells continue to endocycle despite high levels of DNA damage. While, most endocycled cells are terminally differentiated and do not re-enter the mitotic cell cycle, we previously established a model to address mechanisms by which the endocycle promotes genome instability during mitosis. In the Drosophila rectum, we found endocycled papillar cells can re-enter mitosis as polyploid cells. We show that, like other endocycled cells, papillar cells acquire radioresistance during the endocycle. Rather than undergoing full repair of DNA breaks, papillar cells re-enter mitosis with broken chromosomes. Despite frequent DNA breaks, lagging DNA during anaphase and organism-lethal amounts of DNA damage, papillar cells develop normally and undergo surprisingly little cell death. To survive, we find that papillar cells enlist a non-canonical DNA damage mechanism to ensure that anaphase is extended to allow for incorporation of broken DNA into daughter nuclei. This survival depends on ATM and ATR, but not chk1 and chk2 kinase activity or the transcription factor p53. In addition to ATM and ATR, we find that the DNA repair protein FANCD2 is also required for cytokinesis delay and survival of damaged polyploid papillar cells. Cells lacking FANCD2 fail to incorporate broken DNA into daughter cell nuclei prior to the onset of cytokinesis, and subsequently die by mitotic catastrophe. We speculate that the mechanism used by polyploid papillar cells to survive mitosis with high levels of DNA damage may contribute to radio-resistance in cancer cells that have undergone an endocycle.

SUNDAY-POSTER PRESENTATIONS

P229 Board Number: B341

Store-operated calcium entry (SOCE) controls G1/S cell cycle transition. Y. Chen1, M. Shen1,2; 1 Department of Pharmacology, National Cheng Kung University Hospital, Tainan, Taiwan, 2Department of Obstetrics and Gynecology, National Cheng Kung University Hospital, Tainan, Taiwan Background: The well-defined store-operated Ca2+ entry (SOCE) pathway is a ubiquitous Ca2+ influx pathway activated in response to depletion of intracellular Ca2+ stores. The major components of SOCE are stromal-interacting molecule (STIM1, STIM2) and Orai (Orai1, Orai2, Orai3). STIM1 serves a dual role as the endoplasmic reticulum Ca2+ senor, and activator of SOCE. The clinical relevance of STIM1 has been highlighted in several cancers but the molecular mechanism by which SOCE involved in G1/S transition of cell cycle progression remains unclear. However, this study explores the regulatory mechanisms by which STIM1-dependent Ca2+ signaling controls cell cycle progression. Results: Here, we showed that SOCE increased in the S phase of cell cycle in different cell lines. Ca2+ microdomain is necessary for G1/S transition. We discovered that SOCE activity is necessary for G1/S transition but not important for S to G2/M transition. SOCE inhibitor, SKF96365 significantly inhibited G1/S transition to a similar extent to that of components of SOCE (STIM1, STIM2, Orai1) silencing. Total internal reflection fluorescence (TIRF) microscopy of living cells indicated that the increase of STIM1 trafficking to the plasma membrane in S phase but not in G0/G1 phase, suggesting STIM1 is important for the regulation of the G1/S transition. Inhibition of SOCE induced cyclin E autophage as well as blockage of G1/S transition induced cell apoptosis. Taken together, our results revealed that the novel role of SOCE activity in controlling cell cycle progression.

P230 Board Number: B342

DRG2 binds to Wee1 for regulating cell cycle progression during G2/M phase. S. Jang1, N. Park2, S. Kim3, J. Park4, I. Han4; 1 Biomedical Research Center, University of Ulsan, Ulsan, Korea, 2Department of Medical Science, University of Ulsan, Ulsan, Korea, 3Department of biological sciences, Univeristy of Ulsan, Ulsan, Korea, 4 Department of Biological Sciences, University of Ulsan, Ulsan, Korea Developmentally regulated GTP-binding protein 2 (DRG2) plays important role in cell growth. Here we explored that the linkage between DRG2 and G2/M phase checkpoint function in the cell cycle progression. We observed that HeLa cell lacking DRG2 has the slower wound-healing rate than that of control cells. Flow cytometry assay and 3H incorporation assay indicated that G2/M phase arrest leads to decreased proliferatio of this cell. Depletion of DRG2 elicited the changed expression of major cell cycle determinant factors, such as cyclin A2, B1, p21Waf1/Cip. In addition, DRG2 interacts with Wee1 kinase, a mitotic gatekeeper. These findings elucidate a noble role for DRG2 as regulator of Wee1, and therefore serve to progress G2/M phase in cell cycle.

SUNDAY-POSTER PRESENTATIONS

P231 Board Number: B343

Phosphorylation of Xenopus p31comet potentiates mitotic checkpoint exit. M. MO1, M. Dasso2, A. Arnaoutov2; National Institute of Health, Bethesda, MD, 2National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD

1

The spindle assembly checkpoint (SAC) ensures faithful segregation of mitotic sister chromatids by monitoring interactions between kinetochores (KTs) and microtubules (MTs). Defective MT-KT interactions trigger SAC activation, causing the formation of a potent inhibitory complex, the mitotic checkpoint complex (MCC), which in turn blocks the metaphase-anaphase transition. Once all KTs have formed correct MT attachments, the SAC is silenced. The MCC is then disassembled through a series of poorly understood processes, allowing anaphase to ensue. It has previously been shown that the p31comet protein helps to inactivate the MCC by binding directly to Mad2, an essential MCC component. However, p31comet‘s regulation and mechanism of action are unclear. Xenopus egg extracts (XEEs) are an excellent in vitro system that recapitulates many aspects of cell cycle control, including the SAC. We confirmed that Xenopus p31comet was important for SAC silencing in XEEs, and observed that it was phosphorylated during the course of mitosis. Unexpectedly, our data suggest that this modification was mediated by IKK-β (Inhibitor of nuclear factor κ-B kinase-beta), but not by a panel of other well-known mitotic kinases. Depletion or chemical inhibition of IKK-β compromised SAC silencing and delayed mitotic exit, suggesting that this modification was important for p31comet regulation. We identified p31comet phosphorylation sites, and found that a p31comet phosphomimetic mutant (p31comet-EEE) shows enhanced binding of Mad2, an event critical for MCC disruption. Moreover, p31comet-EEE exhibited increased ability to disrupt MCC in XEEs, and it accelerated mitotic exit. Notably, p31comet-EEE also disrupted the association of SAC components to KTs, where MCC is formed, in a dosage dependent manner. By contrast, wild type p31comet or a phosphodeficient mutant (p31comet-AAA) had no effect on KT-associated MCC. Taken together, our data indicate that phosphorylation of p31comet by IKK-β potentiates its ability to silence the SAC and may contribute to SAC silencing at the KT.

SUNDAY-POSTER PRESENTATIONS

P232 Board Number: B344

Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion. G. Zheng1, K. Hara1,2, Q. Qu1, H. Liu1, Z. Ouyang1, Z. Chen1, D. Tomchick1, H. Yu3,4; 1 University of Texas Southwestern Medical Center, Dallas, TX, 2School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan, 3Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, 4Howard Hughes Medical Institute, Dallas, TX Sister-chromatid cohesion, mediated by ring-shaped cohesin complex, is a prerequisite for proper chromosome segregation and genomic stability. Errors in chromosome segregation cause chromosome aberrations and aneuloidy, which can promote tumorigenesis. Human cohesin complex consists of four core subunits, Smc1, Smc3, Scc1, SA1/2. The loading and release of cohesin from chromosomes are tightly regulated during the cell cycle. During S phase, cohesin core subunit Smc3 is acetylated by the acetyltransferase Esco1/2, which enables the binding of the cohesin protector Sororin to cohesin through the adaptor protein Pds5. Sororin antagonizes the cohesin inhibitor Wapl to stabilize cohesin on replicated chromatin and establishes sister chromatid cohesion. In prophase, mitotic kinases phosphorylate cohesin and Sororin, triggering Wapl-dependent cohesin removal from chromosomes. A pool of cohesin at centromeres is protected by the Sgo1-PP2A complex, which binds to cohesin, dephosphorylates Sororin, and protects cohesin from Wapl at centromeres. After all sister kinetochores attach properly to the mitotic spindle and are under tension, Separase cleaves centromeric cohesin to initiate sister chromatid separation. The separated chromatids are evenly partitioned into the two daughter cells. We reported the crystal structure of human cohesin subcomplex SA2-Scc1. SA2 has multiple helical repeats, which form a dragon-shaped structure. Scc1 makes extensive contacts with SA2, with one hot spot being particularly critical for binding. SA2-Scc1 complex is the interaction hub for cohesin regulators. Cohesin protector Sgo1 and inhibitor Wapl compete for binding to a conserved site on SA2– Scc1. Mutations of SA2 residues at this site that disrupt Wapl binding bypass the requirement for Sgo1 in cohesion protection. Thus, in addition to recruiting PP2A to dephosphorylate cohesin and sororin, Sgo1 physically shields cohesin from cohesin inhibitor Wapl. These two mechanisms collaborate to protect centromeric cohesion to the fullest extent, which makes centromeric cohesion strong enough to resist the spindle pulling force at sister kinetochores and enables the generation of kinetochore tension necessary for spindle checkpoint inactivation and accurate chromosome segregation. Sgo1 is also known to protect meiotic cohesin at centromeres from separase-mediated cleavage during meiosis I. In preliminary results, I have shown that SA1/2, as the interaction hub, is required for separase cleavage of Scc1 at anaphase onset. It is possible that separase is recruited by SA1/2 to cleave centromeric cohesin. Overexpression of Sgo1 reduces the separase cleavage efficiency. This indicates that Sgo1 might directly compete with separase for binding SA1/2 and shield cohesin from separase. To

SUNDAY-POSTER PRESENTATIONS test this hypothesis, cohesin cleavage by separase will be reconstituted in vitro using purified proteins. Cohesin cleavage efficiency will be compared in the presence and absence of Sgo1 peptide.

P233 Board Number: B345

Actin-capping proteins play essential roles in spindle migration and polar body protrusion in maturing mouse oocytes. S. Namgoong1, Y. Jo1, N. Kim1; 1 Animal Science, Chungbuk National University, CheongJu, Korea Actin polymerization is essential for various stages of mammalian oocyte maturation, including asymmetric spindle migration, cortical actin cap formation, polar body extrusion, and cytokinesis. The heterodimeric actin capping protein (CP) is an essential element of the actin cytoskeleton. It binds to the fast-growing (barbed) ends of actin filaments, thereby blocking their elongation, and plays essential roles in various actin-mediated cellular processes. However, the roles of CP in mammalian oocyte maturation are poorly understood. We investigated the roles of CP in mouse oocytes, and found that CP is essential for correct asymmetric spindle migration and polar body extrusion. Experiments in which CP was knocked down or ectopically overexpressed revealed that this protein is critical for maintenance of cytoplasmic actin mesh. Perturbations in the level of CP impaired spindle migration, cytoplasmic actin mesh and polar body extrusion during oocyte maturation. Expression of capping protein inhibitory region of CARMIL reduce polar body extrusion rate of oocyte and significantly reduce cytoplasmic actin mesh. Taken together, this study shows that CP is an essential component of the actin cytoskeleton machinery, presumably controlling cytoplasmic actin mesh during oocyte maturation.

P234 Board Number: B346

Ptychography: Label free imaging of the cell cycle and mitosis. R. Suman1, T.J. Henser-Brownhill1, K. Langley2, P. O'Toole1; 1 University of York, York, England, 2Phasefocus, Sheffield, United Kingdom The use of fluorescent probes has become commonplace for staining fixed biological specimens; however when used in living cells they can be toxic and ultimately perturb the natural function of the cell. With this in mind, there has been an emerging need for label free microscopy. Existing techniques such as Zernike phase contrast and DIC have limitations and most importantly are not quantitative. Here we report a novel stain-free, high-contrast and quantitative method for imaging live cells using a ptychographic microscope (Phasefocus VL21). The technique reconstructs an image from overlapping diffraction patterns using a ptychographical algorithm (Maiden et al., 2010), to give high contrast, quantitative information of the sample.

SUNDAY-POSTER PRESENTATIONS We have previously demonstrated the power of ptychography to quantitatively analyse cells during mitosis (Marrison et al., 2013). Here we demonstrate further measurement of the mitotic index (MI) of A549 cells in response to the cell cycle inhibitor, Nocodazole. An advantage to this imaging approach is the ability to acquire large fields of view in which data from 500+ cells can be analysed in a manner comparable to flow cytometry. Following the 6hr treatment with 10, 25 and 50ng/ml Nocodazole, the MI value also increased to 0.05, 0.23 and 0.36 respectively when compared to the control (MI= 0.03). Furthermore we were able to use ptychography to analyse the cell cycle yielding phase characteristics related to cell thickness, cell volume and nuclear volume. Following mitosis the volume of the daughter cell increases linearly during the cell cycle and approximately doubles immediately prior to the next mitotic event. We also propose that nuclear volume remains constant through G1, but starts to increase during S phase. There is an increasing need for quantitative label-free imaging methodologies. Here we demonstrate that ptychography generates high contrast and quantitative images that not only enable cell cycle analysis using a label free non-invasive approach, but can now be used in studies where cell volume/mass are underpinning the biological function of the cell. In conjunction with in situ fluorescence imaging, the quantitative phase information can add additional and vital information that goes beyond fluorescence imaging itself. In summary, the work highlighted here demonstrates one potential application of label-free phenotypic analysis of cells. The method is ideally suited for primary and stem cells where labels are undesirable or not possible. Furthermore the technique could also be used in many other assay types, such as anticancer therapeutics, cell motility and migration, cell morphology and neurodegeneration.

P235 Board Number: B347

Identifying candidate molecules through which γ-tubulin acts to inactivate the anaphase promoting complex/cyclosome at G1/S in Aspergillus nidulans. V.K. Paolillo1, H.D. Edgerton1, B.R. Oakley1; 1 Molecular Biosciences, University of Kansas, Lawrence, KS In addition to its well-established role in nucleating microtubules at microtubule organizing centers such as the centrosome and spindle-pole body, γ-tubulin has additional, essential, microtubule-independent functions that are incompletely understood. Experiments with the cold-sensitive γ-tubulin mutant mipAD159 in the filamentous fungus Aspergillus nidulans revealed that γ-tubulin has an important role in inactivating the active anaphase promoting complex/cyclosome (APC/C) bound to the activator CdhA (the A. nidulans Cdh1 homolog) at the G1/S transition (Edgerton-Morgan and Oakley, 2012, J. Cell Biol. 198, 785-791). As a result, cyclin B is continuously destroyed in a subset of nuclei and these nuclei do not progress through the cell cycle (Nayak et al., 2010, J. Cell Biol. 190, 317-330). The goal of this project is to elucidate the mechanism by which γ-tubulin inactivates APC/CCdhA. Based on our data so far, we hypothesize that γ-tubulin acts through regulators of CdhA. In many organisms, initial phosphorylation and inactivation of Cdh1 occurs via cyclin A/Cdk2, which is then thought to trigger Cdh1 ubiquitination

SUNDAY-POSTER PRESENTATIONS by the Skp1-Cullin1-F-box (SCF) complex. We have identified a putative A. nidulans cyclin A gene, which we designate cycA, and a component of the SCF complex, designated culA, and found both to be essential for viability. If CycA is required for inactivation of APC/CCdhA, the deletion of cycA (cycAΔ) is expected to result in lethality due to a continuously active APC/CCdhA. It follows that deletion of cdhA might reverse the lethality of the cycAΔ and we found that this is the case. We have examined the phenotypes of cycAΔ and culAΔ using the heterokaryon rescue technique. cycAΔ inhibited nuclear division as expected, but, more interestingly, caused nuclei to disappear over time (as judged by fluorescently tagged histone H1) raising the possibility that cycAΔ may cause apoptosis. Additionally, we have found that a subset of nuclei fail to accumulate Cdk1 in culAΔ, similar to a phenotype observed in mipAD159, suggesting that APC/CCdhA is constitutively active in these nuclei. Deletion of culA likely prevents degradation of CdhA, resulting in nuclei that accumulate constitutively active APC/CCdhA and fail to progress through the cell cycle. Taken together, our data indicate that CdhA is regulated by CycA and the SCF complex in A. nidulans as in higher eukaryotes. They also indicate that CycA and CulA are valid candidates for proteins through which γ-tubulin acts to control APC/CCdhA activity. Supported by the Irving S. Johnson Fund of the KU endowment.

P236 Board Number: B348

A tale of two phases: How RNA-protein interactions can spatially position transcripts involved in cell cycle control and morphogenesis. E. Langdon1, A.S. Gladfelter2, H. Zhang2; 1 Biology, Dartmouth College, Hanover, NH, 2Biological Sciences, Dartmouth College, Hanover, NH In the multinucleate cells of the fungus, Ashbya gossypii , individual nuclei within this syncytial cell must find a way to functionally compartmentalize their common cytoplasm in order to regulate nuclear division and symmetry breaking events. One way they solve this organizational problem is through the heterogeneous association of low-complexity sequence/polyQ-containing RNA-binding proteins with target RNAs. We have demonstrated that the proteins, Whi3 and Puf2, bind mRNAS to form aggregate structures that create sections of cytoplasm that are differentiated from one another. Further, we showed that assembly of these proteins with key cell cycle and cell polarity transcripts allow the cell to control the diffusivity of mRNA localization through space and time. We hypothesized that the multivalent interactions of these proteins with RNAs are sufficient to drive the assembly of phaseseparated droplets that form non-membranous compartments within the cytoplasm. In support of this, cell-free reconstitution techniques along with structured illumination microscopy has revealed that these proteins form different structures whether in the presence of RNA, and that these structures can physically change over time depending on the local concentration of RNAs. Currently, we are investigating how the specificity and arrangement of the protein binding sites on the mRNAS can catalyze the coalescence of these aggregation-prone proteins through the use of cell-free assays, smRNA FISH, and single molecule pull down (SiMPull) for RNAs. Further investigation will focus on the role of

SUNDAY-POSTER PRESENTATIONS the local environment and cellular stress has on controlling assembly of these physiological RNA-protein complexes.

P237 Board Number: B349

Cyclin mRNA regulation by dynamic Whi3 RNA-protein droplets. T. Gerbich1, A.S. Gladfelter1; 1 Biological Sciences, Dartmouth College, Hanover, NH In the multinucleate filamentous fungus Ashbya gossypii, many aspects of cellular growth and activity are spatially and temporally confined to local zones within a single, large cell., For example, nuclei that are only micrometers apart divide asynchronously despite being bathed in the same continuous cytosol where freely diffusing components would be predicted to be equal in all regions. This raises the problem as to how in Ashbya functional cytoplasmic compartments can be created that give rise to local territories with distinct activity from neighboring territories. One solution employed by the fungus is to create phase-separated droplets of protein and mRNA that are distributed non-homogenously in the cell. Asynchronous nuclear division is dependent upon the non-uniform positioning of cyclin transcripts by Whi3, an RNA-binding protein with a low complexity sequence that allows it to undergo regulated aggregation. This work considers how Whi3 complexes are regulated by environmental conditions and how the complexes function to promote local production of the cyclin,Cln3, protein. Whi3 aggregates are known to respond to cellular stresses by changing their size and distribution in a manner specific to the environmental challenge. Single molecule FISH data will be presented to show how these changes in Whi3 aggregation correspond to changes in Cln3 transcript localization. The degree to which cyclin transcripts are protected or masked by Whi3 protein complexes so as to limit translation during different phases of the cell cycle or in response to environmental stress is also being investigated. This work uses protease digestion assays combined with single molecule FISH. These data provide insight into how multinucleate cells use functional RNA-protein aggregates to create dynamic compartments that organize the cytoplasm.

P238 Board Number: B350

How prolonged prometaphase blocks daughter cell proliferation despite normal completion of mitosis. Y. Uetake1, G. Sluder1; 1 University of Massachusetts Medical School, Worcester, MA We previously reported that when untransformed human (RPE1) cells spend >1.5 hr in prometaphase, their daughters uniformly exhibit a p38 dependent arrest in G1 despite normal division of their mothers. Transient inhibition of p38 activity allows daughters born of prolonged prometaphase to proceed

SUNDAY-POSTER PRESENTATIONS through interphase and mitosis of normal duration, yet all granddaughters arrest in G1 - suggesting a durable change in the properties of the daughter cells that cannot be erased by progression through interphase and mitosis. What happens during prolonged prometaphase and the nature of the change in daughter cell properties were unknown. Here we find that during prometaphase the progressive loss of activity of the anti-apoptotic protein MCL1 and oxidative stress at ambient oxygen levels can act independently or in concert to diminish the proliferative capacity of the daughters. Additional observations provide functional evidence that a slow activation of the apoptotic pathway participates in the initiation of the G1 arrest of daughter cells. Since some spindle defects prolong prometaphase but eventually allow mitotic checkpoint satisfaction, this phenomenon can prevent unresolved defects in mitosis from being propagated to future cell generations.

P239 Board Number: B351

Activation of Plk1 pool in late G2 promotes mitotic commitment. L. Gheghiani1,2, O. Gavet1,2; Sorbonne Universités, UPMC Paris VI, UFR927, F-75005, paris, France, 2 Gustave Roussy, UMR 8200 CNRS, PR2, Villejuif, France 1

Activation of the master driver CyclinB1-Cdk1 in late G2 orchestrates the cell division program. How this trigger event is timely controlled during each cell cycle is still an open question. Here, we investigated upstream regulatory mechanisms focusing on the involvement of Polo-like kinase 1 (Plk1). Its activity is limiting for mitotic commitment during unperturbated cell cycles and we determined that activation of Plk1 pool is taking place in late G2 and reproducibly precedes Cyclin-B1-Cdk1 one by a few minutes. This sudden activation relies on upstream Aurora-A kinase and appears related to CyclinA2-Cdk1&2 activity level. In fact, stimulating CyclinA2-Cdk activity from late S, by inhibiting Wee1 kinase, induced a premature activation of Plk1 preceding unscheduled entry into mitosis. We determined that Plk1 associates with Cdc25C in interphase and that its activation induces concurrent Cdc25C phosphorylation before mitotic entry. Mimicking Plk1-dependent Cdc25C phosphorylation events, we found that this regulatory mechanism is sufficient to promote mitotic entry. Altogether, our results unravel some upstream molecular steps leading to CyclinB1-Cdk1 initial activation and mitotic entry.

SUNDAY-POSTER PRESENTATIONS

P240 Board Number: B352

CATS (FAM64A) ALTERED EXPRESSION REDUCES PROLIFERATION OF HEMATOPOIETIC CELLS. I. Barbutti1, J.M. Xavier1, S.T. Saad1, L.F. Archangelo1,2; 1 Hematology and Hemotherapy Center, University of Campinas, UNICAMP, Campinas, Brazil, 2University of São Paulo (USP) at Ribeirão Preto, Ribeirão Preto, Brazil CATS (FAM64A) is a nuclear protein expressed according to the different cell cycle phases, and its expression peaks during S and G2/M. Moreover, a function in metaphase-anaphase transition has been attributed to FAM64A. The protein can be used as a marker for proliferation since its expression positively correlates with proliferation. The precise role of CATS (FAM64A) in proliferation and cell cycle control remains to be defined. The aim of this study was to investigate the effects of CATS (FAM64A) depletion and overexpression of hematopoietic cells. Lentivirus targeting CATS (FAM64A) silencing were used to transduce the U937 cells. Depletion was confirmed at RNA (qRT-PCR) and protein (Western blotting) levels. FAM64A depleted (shFAM64A) and control cells underwent functional in vitro assays and cell lysates were used for Western blotting analysis. Proliferation was assessed by MTT assay and colony forming unit (CFU), grown in semi-solid media without growth factors, and cell cycle assay was performed by FACS analysis of the DNA content. Overexpression of the murine Cats (Fam64a) was achieved by retrovirus transduction of primary bone marrow cells with pMIG-Fam64a. Successfully transduced cells were sorted for GFP expression, submitted to cell cycle analysis and cultivated in semisolid media supplemented for myeloid differentiation. GM (granulocytes and monocytes), BFU-E (erythrocytes) and GEMM (progenitors) colonies and total number of cells were counted after 8 days. Cells were replated and colonies were counted after 10 days. FAM64A depletion was about 80% in shFAM64A cells. Cell proliferation was reduced in shFAM64A cells by 20% in liquid and by 66% in semisolid media compared to control. Accordingly, a significant reduction of 12% of shFAM64A cells in S phase of the cell cycle was observed. The CYCLIN E and B1 expression were decreased in shFAM64A, whereas CYCLIN A and D1 expression were similar to control cells. Expression of Fam64a in mice cells was 215-fold higher in pMIG-Fam64a than in control cells. A significant reduction of GM (by 55%) and GEMM (by 78%) pMIG-Fam64a colonies was observed when compared to control, whereas GM pMIGFam64a colonies were reduced by 84% after replating. Total number of pMIG-Fam64A cells was also significantly reduced by 58%. Cell cycle analysis of transduced cells showed a significant reduction of 5% of cells in the S phase of the cell cycle in pMIG-Fam64a compared to control cells. Our results demonstrate that abnormal expression of FAM64A on hematopoietic cells leads to a decrease in cell proliferation which correlates with a decrease in percentage of cells in the S phase of the cell cycle. In human cells, this decrease also correlates with reduced CYCLIN E and B1 expression.

SUNDAY-POSTER PRESENTATIONS

P241 Board Number: B353

CDK1-dependent Inhibition of the E3 Ubiquitin Ligase, CRL4CDT2, Ensures Robust Transition from S Phase to Mitosis. L.F. Rizzardi1, D. Varma1, K.E. Coleman1, J.P. Matson1, S. Oh1, J.G. Cook1; 1 Biochemistry and Biophysics, UNC Chapel Hill, Chapel Hill, NC Replication-coupled destruction of a cohort of cell cycle proteins ensures efficient and precise genome duplication. Three proteins destroyed during replication via the CRL4CDT2 ubiquitin E3 ligase, CDT1, p21, and SET8 (PR-SET7), are also essential or important during mitosis making their re-accumulation after S phase a critical cell cycle event. During early and mid-S phase and during DNA repair, PCNA loading onto DNA (PCNADNA) triggers the interaction between CRL4CDT2 and its substrates. We have discovered that beginning in late S phase, PCNADNA is no longer sufficient to trigger CRL4CDT2mediated degradation. A CDK1-dependent mechanism that blocks CRL4CDT2 activity by interfering with CDT2 recruitment to chromatin actively protects CRL4CDT2 substrates. We postulate that deliberate override of replication-coupled destruction allows anticipatory accumulation in late S phase; we further show that (as for CDT1) de novo SET8 re-accumulation is important for normal mitotic progression. In this manner, CDK1-dependent CRL4CDT2 inactivation contributes to efficient transition from S phase to mitosis.

P242 Board Number: B354

Diaphanous formin is required for epigenetic inheritance of CENP-A at centromeres. C. Liu1, Y. Mao1; 1 Department of Pathology and Cell Biology, Columbia University College of Physicians Surgeons, New York, NY Epigenetic landscape has to be well inherited over cellular and organismal generations, largely independent of the underlying DNA sequences. In mammals, as the fundamental unit for chromosome segregation during mitosis, centromeres are defined epigenetically by nucleosomes containing the histone H3 variant CENP-A. In order to maintain centromere identity against CENP-A dilution as DNA duplicates and cell divides, newly synthesized CENP-A proteins are deposited at preexisting centromeres specifically during early G1 of each cell cycle. However, little is known about how accurate CENP-A inheritance is achieved. Recent efforts in the field have elucidated pathways involving different licensing, loading and stabilizing factors required for CENP-A deposition and maintenance, among which a small Rho GTPase molecular switch has being proposed to stabilize newly incorporated CENP-A at the centromere. The FH2 domain of mammalian diaphanous-related formin mDia2, which is a Rho family GTPase effector, has been shown to co-complex with various nucleosome proteins including histone H3

SUNDAY-POSTER PRESENTATIONS in mammalian cell proteomics (Daou et al. MBoC, 2014). Knocking down mDia2 resulted in substantially reduced levels of centromeric CENP-A revealed by quantitative imaging and high-throughput, unbiased measurement with fixed HeLa cells. Notably, this reduction was not caused by the loss of centromeres, as total number of CENP-B foci in knockdown cells was not significantly changed. A siRNA resistant construct of WT mDia2 rescued this phenotype by restoring CENP-A levels at centromeres. High resolution ratiometric live cell imaging (developed in this study) using HeLa cells stably expressing YFPCENP-A, documented defective CENP-A loading over the 10 hour time window after anaphase onset in cells depleted of mDia2. Meanwhile, SNAP-tag pulse chase assay revealed that mDia2 is required specifically for newly synthesized CENP-A at centromeres. Furthermore, in mDia2 depleted cells, expression of actin mutants of mDia2 didn’t restore CENP-A levels at centromeres. Therefore, our findings have uncovered a novel role for diaphanous formin in regulating CENP-A deposition and thus the epigenetic inheritance of centromere identity.

P243 Board Number: B355

Spatial regulation of mitotic timing. B. Strauss1, M. Zernicka-Goetz2, J. . Pines3; 1 The Gurdon Institute, University of Cambridge, Cambridge, England, 2Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom, 3Zoology, The Gurdon Institute, University of Cambridge, Cambridge, UK One of the most prominent characteristics of cyclin B1 is its dynamic localisation throughout the cell cycle. We, and others, have shown that cyclin B1 shuttles between the cytoplasm and the nucleus, but its greater rate of export over import ensures that the majority of cyclin B1 is cytoplasmic throughout interphase. At mitosis, cytoplasmic cyclin B1 is rapidly imported into the nucleus concomitant with the activation of its partner kinase, Cdk1. Although we understand the mechanism by which cyclin B1 is maintained in the cytoplasm, the function of this spatial distribution is still not understood. In order to study the function of cyclin B1 localisation throughout the cell cycle we made use of a cyclin B1 knock out mouse line. We have shown earlier that cyclin B1 null embryos arrest at the four-cell stage and is therefore the only truly essential cyclin in the mouse. This system allowed us to avoid the potential pitfalls of RNAi based approaches by injecting mRNAs encoding mutant forms of cyclin B1 into a genetically null background. Moreover, the large cell size of the early mouse embryo makes this system particularly well suited to study spatial controls using a 3D imaging approach. To increase throughput (as only 25% of embryos are B1 null in each experiment) we have developed a novel imaging chip that allows high resolution imaging of large numbers of mouse embryos as well as their retrieval for genotyping at the end of the experiment. Using this experimental setup we studied the role of nuclear export with respect to cell cycle timing by injecting constitutively nuclear cyclin B1 into B1 null embryos. We found that injected nuclear cyclin B1 can drive up to three division cycles. Furthermore, maintaining cyclin B1 in the nucleus had little effect on the duration of mitosis, nor did it appear to have any measurable effect on DNA replication, because both G1 and S phase durations were the same as in

SUNDAY-POSTER PRESENTATIONS embryos injected with wild type cyclin B1. Nuclear cyclin B1 did, however, significantly accelerate entry to mitosis after the end of DNA replication, as G2 phase was 40% shorter than normal. We have further tested the functionality of membrane tethered Cyclin B1 and found that it cannot drive entry into mitosis in a null background. Thus, the ability to export cyclin B1 into the cytoplasm is important for cells to regulate the correct timing of mitosis.

P244 Board Number: B356

Cdk4/6-dependent activation of FOXM1 controls recovery from DNA damage in G1. I.A. Shaltiel1, M. Aprelia2, A. Llopis2, E.E. Voest1, R.H. Medema3; 1 Cell Biology, Netherlands Cancer Inst, Amsterdam, Netherlands, 2Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, Netherlands, 3Division of Cell Biology, the Netherlands Cancer Institute, Amsterdam, Netherlands Most Cyclin-dependent kinases are redundant for normal cell division. We tested whether these redundancies are maintained in the abnormal situation of cell cycle reentry after a DNA damage-induced arrest in G1. Interestingly, using non-transformed RPE and BJ-Tert cells, we find that while Cdk4 is dispensable both for S-phase entry of normally proliferating cells and for cell cycle re-entry after serum starvation, it becomes essential to drive S-phase entry after irradiation. Overexpression of Cdk6 can overcome this dependence on Cdk6, indicating that the overall level of Cdk4/6 activity becomes ratelimiting during recovery. We show that Cdk4/6 kinase activity is only required once the checkpoint has been turned off, suggesting that Cdk4/6 activity is not required for DNA repair or checkpoint silencing. Indeed, DNA repair and the elimination of Cdk inhibitor proteins p21 and p27 are unaffected after depletion of Cdk4. We find that Cdk4/6 activity is required to phosphorylate pRb after irradiation, yet inactivation of pocket proteins is insufficient to allow S-phase entry after DNA damage when Cdk4 is depleted. Instead, our data demonstrate that Cdk4 drives recovery from a G1 arrest through the activation of FOXM1.

P245 Board Number: B357

Two sequential decisions commit human cells to start the cell cycle. S. Cappell1, S.L. Spencer1, M. Chung1, A. Jaimovich1, T. Meyer1; 1 Department of Chemical and Systems Biology, Stanford University, Stanford, CA One of the most fundamental decision processes in biology is the commitment of cells to enter the cell cycle and generate two daughter cells. Cell-cycle commitment in mammalian cells is thought to be regulated by a single decision point in G1 called the Restriction Point, after which growth factors are no longer required for the cell cycle to proceed. Using live-cell microscopy of cell-cycle sensors and single-

SUNDAY-POSTER PRESENTATIONS cell analysis, we have identified the moment at which cells cross the Restriction Point and find that more than three hours pass between the Restriction Point and the actual start of DNA replication. Furthermore, we find that cell stress after the Restriction Point but before the start of DNA replication results in cell-cycle exit and a return to a pre-Restriction Point state, providing evidence for another later decision to commit to the cell cycle. We present evidence for a second bistable switch regulating Sphase entry. The trigger for this second switch is the CDK2/Cyclin E-mediated partial inactivation of APCCdh1, an E3-ligase that controls the degradation of proteins necessary for S-phase progression. We show that the switch is driven by a double negative feedback between the regulatory protein Emi1 and APCCdh1. Markedly, Emi1 functions as both an inhibitor and a substrate of the APC, which generates a bistable and irreversible switch regulating S-phase entry. Thus, cells commit to the cell cycle by making two sequential decisions. They first monitor mitogen signals to decide whether to cross the Restriction Point, and then monitor stress signals to decide whether to irreversibly inactivate APCCdh1, start DNA replication, and fully commit to the cell cycle.

P246 Board Number: B358

hNek5 interferes negatively in mitochondrial mediated apoptosis and respiration. T.D. Hanchuk1, P.F. Papa1, P.G. La Guardia2, A.E. Vercesi2, J. Kobarg1; 1 NACIONAL LAB OF BIOSCIENCE, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, Brazil, 2 Departamento de Patologia Clínica, Unicamp, Campinas, Brazil Beside to supplying cellular energy, mitochondrias are involved in signaling, cell death andcellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammals protein kinases that plays essential roles in cell-cycle progression, but other functions are recently related. Nek5 is the kinase with the smallest information. To characterize Nek5 functions, the yeast two-hybrid (Y2H) screening was performed. According to GO, mitochondrial proteins were identified as hNek5 partners. The mitochondria location of hNek5 was confirmed by cell fractionation followed by western blot analysis and colocalization between hNek5 and mitochondrial proteins in U2-OS and MCF-7 by confocal microscopy. As the prey are involved on apoptosis, mitochondrial transport and respiratory chain complex assembly, the involvement of hNek5 in the mitochondria context was analyzed by MTS and annexin V-FITC assay, measurement of ROS generation and the oxygen consumption. Apoptosis assay showed protective effects of hNek5 in Hek293-T’s cell death after thapsigargin treatment (2uM). Stables cells expressing hNek5 or the kinase dead (K33A) version were generated using the FLP-In T-REX mammalian expression system and shRNA lentiviral particles were used to silence hNek5. Silenced cells as well as kinase dead displayed an increase on ROS formation after 4 hours of thapsigargin (2uM) treatment, but, the potentiation effects on cells death occur only after 16 hours of thapsigargin treatment. Cells expressing hNek5, showed protective effects on cell death and ROS formation. Mitochondrial respiratory chain activity of stable and depleted hNek5 cells was determined using the Oroboros Oxygraph. Mitochondrial respiratory chain activity is impaired in the presence of hNek5. Cells silenced for hNek5 presented rampant rates of electrons transfer from

SUNDAY-POSTER PRESENTATIONS ascorbate to cytochrome c and in the complex II. The partners identified by Y2H are potential substrates for hNek5 that mediates the defects on respiratory chain. In conclusion our data suggest for the first time the hNek5 mitochondrial localization and its role on cell death and cell respiration defects.

Oncogenes and Tumor Suppressors 1 P1837 Board Number: B360

Involvement of calreticulin in α-integrin mediated survival and chemoresistance in T-cells. C. Liu1,2, S. Yap1,2, P. Leclair1,2, D. He1,2, C. Lim1,2; 1 Pediatrics, University of British Columbia, Vancouver, BC, 2Child and Family Research Institute, Vancouver, BC Cell adhesion mediated drug resistance (CAM-DR) is a major contributor to relapse in hematological malignancies. Previously, we showed that adhesion via α4β1 or α5β1 integrins promotes chemoresistance in Jurkat T-leukemia cells (Mol Cell Biol 33:4334). Likewise, adhesion via other integrins including α2β1 or αLβ2 also promotes chemoresistance, pointing to a common conserved signaling mechanism. The membrane proximal KxGFFKR motif is highly conserved in the cytoplasmic domain of all α-integrins. Expression of a non-integrin chimeric receptor with only KxGFFKR as the cytosolic motif rendered cells highly chemoresistant in an adhesion-independent manner. These cells exhibit high rates of calcium influx coupled with drug efflux, as well as constitutive activation of Akt pro-survival signaling. Furthermore, we found increased interaction of KxGFFKR with the calcium-binding protein, calreticulin (CRT). Recently, high frequency somatic mutations that give rise to a variant CRT protein predicted to be enriched in the cytosol (vs the endoplasmic reticulum) was implicated in the JAK-STAT pathway controlling myelo- and lympho-proliferative disorders. As CRT-integrin association can be stimulated by cell adhesion, we propose that cytosolic CRT may act as an oncogenic protein involved in cell survival and regulation of chemoresistance. To investigate the role of CRT in CAM-DR, we generated CRT null T-cells by CRISPR-Cas mediated gene silencing, and show that CRT is implicated in integrin activation, cell adhesion, and enhanced apoptotic resistance to cytotoxic chemotherapy. Furthermore, interferon-stimulated phosphorylation of STAT3 and STAT5 is disrupted in CRT null T-cells, suggesting CRT has a vital role in coupling cytokine-mediated stimulation of JAK-STAT signaling in lymphoblasts. Ongoing investigation will further delineate the cytosol and endoplasmic reticulum specific roles of CRT in CAM-DR by intracellular targeted CRT expression enriched in the cytosol and/or ER.

SUNDAY-POSTER PRESENTATIONS

P1838 Board Number: B361

iRHOM2 regulation of ADAM17 as a key regulator of epithelial growth factor signalling. M.A. Brooke1, S.L. Etheridge1, B. Fell1, D.P. Kelsell1; 1 Blizard Institute, Barts and The London School of Medicine Dentistry, London, United Kingdom Our group has previously linked point mutations in the proteolytically inactive rhomboid protein iRHOM2 to the dominantly-inherited disease Tylosis with oesophageal Cancer (TOC), a disorder of palmoplantar keratoderma, oral leukokeratosis, and the only known syndrome of inherited oesophageal squamous cell carcinoma susceptibility. In keratinocyte cell lines, these iRHOM2 mutations result in dysregulation of the multi-substrate ectodomain sheddase enzyme ADAM17 (TACE), an enzyme whose maturation and activation is regulated by iRHOM2. TOC-associated iRHOM2 mutations result in increased ADAM17 maturation and translocation to the plasma membrane of keratinocytes (in both monolayer and three-dimensional epidermal-equivalent cultures), and greatly upregulated constitutive shedding of numerous ADAM17 substrates, including the EGF-family growth factors amphiregulin, TGFα and HB-EGF. This is accompanied by a phenotype of significantly increased proliferation and migration of TOC-associated iRHOM2-mutant cells relative to controls, with both the increased levels of growth factor shedding and the pro-migratory phenotype sensitive to pharmacological ADAM17 inhibition or siRNA knockdown. Excessive EGF-family growth factor signalling was also shown to result in the upregulation of Transglutaminase 1 activity in the epidermis of TOC-affected individuals and in three-dimensional TOC keratinocyte cultures relative to controls, whilst the epidermis of TOC-affected individuals is also characterised by the expression of desmosomes lacking electron-dense midlines, suggestive of the presence of a pro-migratory, woundhealing-like state in the skin. The presence of a constitutive wound-healing-like state is also supported by the very strong constitutive upregulation of IL-6 and IL-8 expression and secretion by TOC keratinocytes, secretion which is sensitive to ADAM17 knockdown despite these cytokines not being ADAM17 substrates. In addition to regulation of EGF-family growth factor shedding, iRHOM2 may also play a role in the transactivation of EGFR signalling in response to G-protein coupled receptor (GPCR) activation, a process dependent on ADAM17 activity. Activation of the GPCR FGFR2b on keratinocytes by the growth factor KGF leads to phosphorylation of iRHOM2 and ADAM17, suggesting that activation of iRHOM2, and the interaction between it and ADAM17 may play an important role in regulating EGFR transactivation in response to GPCR signalling. These findings reveal the central role of iRHOM2 in growth factor regulation in the skin. This, and the association of iRHOM2 mutations with oesophageal cancer may offer new insights into mechanisms of epidermal hyperproliferation and oesophageal carcinogenesis.

SUNDAY-POSTER PRESENTATIONS

P1841 Board Number: B362

Pro-apoptotic role of human YPEL5, yeast ortholog MOH1, in DNA damageinduced apoptosis in tumor cells. J. Lee1, D. Jun2, L. Xie1, Y. Kim1; 1 School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu, Korea, 2Institute of Life Science and Biotechnology, Kyungpook National University, Daegu, Korea During comprehensive sequence analysis of human chromosome, a novel gene family consisting of five members (YPEL1 through YPEL5) showed high conserved among eukaryotes from yeast to human. To understand the functional role of YPEL5, which is distinct from the other four members, we employed the budding yeast (Saccharomyces cerevisiae) mutant possessing disrupted yeast ortholog MOH1 gene, and compared its physiological characteristics with those of the wild-type strain. The mutant for the MOH1 gene exhibited an enhanced resistance to UVC (254 nm) irradiation and genotoxic agents as compared with the wild-type. This phenomenon was abrogated when the disrupted MOH1 gene in the mutant was restored by transformation of the human YPEL5 gene. In tumor cells, the level of YPEL5 was up-regulated following treatment with genotoxic agents and the up-regulation of YPEL5 at the transcription level appeared to depend on the tumor suppressor p53. Whereas the overexpression of YPEL5 augmented DNA damage agent-induced caspase cascade activation, the down-regulation of YPEL5 could suppress the apoptotic events. These results demonstrate the functional conservation of YPEL5 protein between lower and higher eukaryotes, which is involved in DNA damage-induced apoptosis, and provide insights into the molecular and cellular function of the YPEL family proteins.

P1842 Board Number: B363

Mechanisms of p53-dependent Cell Fate Choice. K.D. Sullivan1, J.M. Espinosa1; 1 HHMI and MCDB Department, University of Colorado, Boulder, CO The p53 tumor suppressor governs cellular stress responses ranging from cell cycle arrest to apoptosis; however, the molecular mechanisms of these cell fate decisions remain poorly understood. Attempts at small molecule activation of p53 have fallen short clinically as they result in reversible cell cycle arrest in the majority of cancer cell types. We have performed genome-wide shRNA screens with one such small molecule, Nutlin-3, to identify genes that are lethal with non-genotoxic activation of p53 and discovered that the ATM and MET kinases regulate p53-dependent cell fate choice. Genetic or pharmacological interference with ATM or MET converts the cellular response to Nutlin-3 from cell cycle arrest to apoptosis. Nutlin-3 and ATM synthetic lethality owes to functions of ATM outside the DNA damage response. We show that inhibition of ATM results in accumulation of intracellular reactive oxygen species (ROS), and that these ROS are involved in the synthetic lethality as the phenotype is abrogated by addition of the ROS scavenger N-acetyl-cysteine. Furthermore, knockdown of several p53-inducible

SUNDAY-POSTER PRESENTATIONS antioxidant genes including SESN1/2 and TP53INP1 further sensitizes cells to Nutlin-3 and ATM synthetic lethality. We demonstrate that ROS increases associated with a loss in ATM activity correlate with accumulation of mitochondria and provide evidence that this may be linked to defects in autophagy. Finally, to identify genes that are required for Nutlin-3 and ATM induced synthetic lethality; we performed a positive selection screen that identified several promising candidates of diverse biological function. Further studies are also under way to better understand the conservation of synthetic lethality across cancer types and between drugs with different mechanisms of action.

P1845 Board Number: B364

Profiling the Molecular changes during malignant transformation and response to different oxygen levels, using a combined transcriptomics and proteomics approach. F. Danielsson1, E. Lundberg2, M. Uhlén3, A.K. Gad4; 1 Proteomics, Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden, 2 Affinity Proteomics, Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden, 3Affinity Proteomics, Science for Life Laboratory, KTH - Royal Institute of Technology, Solna, Sweden, 4Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, Stockholm, Sweden Transformation of normal cells to cancer cells that are able to form tumors and even metastasize is a multi step process where molecular changes of high complexity are acquired. By using a four-stage isogenic human cell model that mimics the route to malignancy we demonstrate how a transcriptomics approach with subsequent protein analysis can be used to define the molecular changes that accompany the mechanisms related to immortalization, transformation and invasion/metastasis separately and identify potential biomarkers for malignancy. Additionally, we are currently investigating the impacts of hypoxia by studying differential protein expression and spatial distributions of proteins under different oxygen levels. This project is performed in close collaboration with the Human Protein Atlas (HPA) project and with the use of RNA sequencing in combination with the HPA collection of antibodies covering the whole human proteome and high-resolution microscopy, protein expression can be studied on a single cell level. Our results show that about 1300 genes are differentially expressed across the cell model and that the majority of the genes are downregulated, supporting the principle of dedifferentiation en route to malignancy. [1] 1. Danielsson, F., et al., Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model. Proceedings of the National Academy of Sciences of the United States of America, 2013. 110(17): p. 6853-8.

SUNDAY-POSTER PRESENTATIONS

P1846 Board Number: B365

p21-activated kinase 4 promotes breast cancer initiation and progression. T. Costa1, T. Zhuang1, M. Zhao1, Z. Li1, U. Rabenhorst1, H. Olofsson1, P. Hernandez Varas1, J. Lorent2, S. Strömblad1; 1 Department of Biosciences and Nutrition, Karolinska Institute, Stockholm, Sweden, 2Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden p21-activated kinase 4 (PAK4) is overexpressed in diverse human cancers and plays a pivotal role in several cancer-associated cellular events. To elucidate the role of PAK4 in breast cancer initiation and progression in vivo, we have generated transgenic mice that overexpress PAK4 in the mammary gland (MMTV-PAK4). MMTV-PAK4 mice exhibit atypical hyperplasias that eventually progress to cancer, indicating that PAK4 overexpression in the mammary gland is sufficient per se to increase the spontaneous mammary carcinoma incidence. Conversely, we have also established mice with conditional PAK4 depletion in the mammary epithelium (PAK4 floxed;MMTV-Cre). Conditional knockout of PAK4 significantly delays PyMT-driven mammary tumourigenesis. Consistently, abalation of PAK4 inhibited human breast cancer xenograft growth in mice. Through the combination of several global approaches (including exome sequencing to identify genetic events that accompany PAK4-driven tumourigenesis and global expression profiling of cancer cells upon PAK4 inhibition) we aim to gain a comprehensive insight into how PAK4 regulates breast cancer initiation and progression.

P1848 Board Number: B366

Characterization of the SW13 adrenal adenocarcinoma cell line and induction of subtype switching by HDAC1 inhibition. M.R. Davis1, J.J. Daggett1, A.S. Pascual1, K.E. Cooper1, K.J. Leyva2, E.E. Hull1; 1 Biomedical Sciences, Midwestern University, Glendale, AZ, 2Microbiology Immunology, Midwestern University, Glendale, AZ The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses the tumorsuppressor gene Brahma (Brm), SW13+, and one that does not, SW13-. Unlike many tumor suppressor genes, which are often mutated, in SW13- cells loss of Brm expression occurs post-transcriptionally and can be restored via histone deacetylase (HDAC) inhibition. However, many of the previously characterized Brm inducing HDAC inhibitors are toxic, broad-spectrum inhibitors which have limited downstream use, and provide little insight into the specificity of epigenetic Brm suppression. In this work we sought to further characterize the oncogenic potential of the two SW13 subtypes, and to

SUNDAY-POSTER PRESENTATIONS investigate the potential epigenetic regulators that contribute to the subtype switching. Cell growth and colony formation assays revealed that Brm deficient SW13- cells have both a faster cell number doubling time and an increased rate of anchorage-independent growth, while SW13+ cells appear to express higher levels of MMP. Next, we screened multiple HDAC inhibitors for their ability to induce switching between the SW13 subtypes in an effort to identify the specific HDAC enzyme(s) necessary for this conversion. Immunofluorescence and gene expression analyses suggest that inhibition of HDAC1 most efficiently increases the rate of conversion between SW13- and SW13+ cells, and that it does so in a dose dependent manner. Furthermore, after conversion to SW13+ there is increased acetylation on histone H3 but not histone H4 when compared to levels in SW13-, suggesting that the role of acetylation on the H3 tail may play an essential role in the subtype switch. Pathway-specific quantitative RT-PCR profiling arrays uncovered significant differences in the expression of multiple genes involved in epigenetic chromatin remodeling as well as epigenetic chromatin modification, indicating that these two subtypes also have unique gene expression profiles that could contribute to Brm regulation and maintenance of the subtype switch. These studies provide new insight into the functional consequences of Brm loss in the SW13 cell type and reveal potential targets for the reversal of Brm epigenetic silencing.

P1851 Board Number: B367

Proteomic analysis of EWS-Fli-1 associated proteins shows the contribution of lysosomes in EWS-Fli-1 turnover. D.J. Elzi1, M. Song1, K. Hakala2, S.T. Weintraub2, Y. Shiio1,2; 1 Greehey Childrens Cancer Research Institute, Univ Texas Hlth Sci Ctr-San Antonio, San Antonio, TX, 2 Biochemistry, Univ Texas Hlth Sci Ctr-San Antonio, San Antonio, TX Ewing sarcoma is an aggressive pediatric cancer of the bone and soft tissues characterized by a chromosomal translocation involving EWS and an Ets transcription factor, usually Fli-1. EWS-Fli-1 fusions comprise 85% of Ewing sarcoma cases. Ewing sarcoma cells are dependent upon EWS-Fli-1 for growth and survival, yet little is known about EWS-Fli-1’s biochemical characteristics. Using a proximitydependent biotinylation technique, BioID, we identified cation-independent mannose 6-phosphate receptor (CIMPR) as a protein located in the vicinity of EWS-Fli-1. CIMPR is a cargo transporter which facilitates the movement of lysosomal hydrolases between the trans-Golgi network and the endosome, which are then transferred to the lysosome. We verified the interaction of CIMPR with EWS-Fli-1 by coimmunoprecipitation and discovered the role of lysosomes in regulating EWS-Fli-1 stability. Inhibition of lysosome function by chloroquine stabilized EWS-Fli-1 protein levels, while inhibition of the mTORC1 pathway by torin 1, which activates the TFEB-lysosome pathway, degraded EWS-Fli-1. These results suggest that lysosomal activation may provide a therapeutic means to modulate EWS-Fli-1 levels.

SUNDAY-POSTER PRESENTATIONS

P1852 Board Number: B368

Hypersensitivity to DNA double strand breaks in late G2 protects against checkpoint adaptation. L. Krenning1, F.M. Feringa1, R.H. Medema1; 1 Division of Cell Biology, the Netherlands Cancer Institute, Amsterdam, Netherlands The cellular response to DNA damage ranges from transient cell cycle arrest to the induction of apoptosis or senescence. It is generally thought that permanent cell cycle withdrawal is initiated in response to severe, irreparable DNA damage. This way cells can prevent the propagation of a damaged genome. Alternatively, some cells may enter mitosis in the presence of DNA damage, a process known as checkpoint adaptation. This is a potentially dangerous process, as the propagation of damaged DNA may compromise genome integrity. Using live-cell microscopy to study the effects of DNA damage on cell fate decisions we have identified that a fraction of cycling cells are hypersensitive to DNA double strand breaks. This fraction represents cells that are damaged just prior to mitotic entry. These cells respond to DNA damage by activating the anaphase promoting complex / cyclosome in complex with its coactivator Cdh1 (APC/CCdh1) and undergo permanent cell-cycle exit, even in response to low amounts of DSBs. During unperturbed G2 progression the APC/CCdh1 is inhibited by Early mitotic inhibitor (Emi1)-binding to Cdh1, and CDK-dependent phosphorylation of Cdh1. However, as Emi1 levels decline towards the later stages of G2, these cells become critically dependent on CDK activity for APC/CCdh1inhibition. Indeed, inhibiting CDKs in unstressed cells is sufficient to induce APC/CCdh1-activation specifically in late G2 cells. Conversely, preventing DNA damage-induced CDK inhibition prevents APC/CCdh1 activation in late G2 cells. In addition, overexpression of Emi1 prevents the DNA damage induced activation of APC/CCdh1. We continue to show that this cell cycle exit mechanism functions to prevent late G2 cells from entering mitosis in the presence of DNA double strand breaks, and therefore acts to prevent checkpoint adaptation and guard genome stability.

P1855 Board Number: B369

The role of RhoGAPs in basal-like breast cancer. C.D. Lawson1,2, K.L. Rossman2, C. Fan3, C.M. Perou3, K. Burridge1, C.J. Der2; 1 Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 2 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 3Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC The basal-like subtype of human breast cancer accounts for a disproportionately high percentage of overall breast cancer recurrence and death, and the current therapeutic options for this cancer are ineffective. Hence, elucidating the signaling pathways that are responsible for driving the growth of basal-like tumors may identify novel targets for the development of effective therapies. Rho family small GTPases have been implicated previously in promoting tumor cell proliferation, invasion, and metastatic

SUNDAY-POSTER PRESENTATIONS growth in a variety of cancers. These proteins are activated by guanine nucleotide exchange factors (GEFs) and, in the context of cancer, overexpressed RhoGEFs can function as oncogenes which cause hyper-elevated Rho GTPase activity. In contrast, GTPase-activating proteins (GAPs), which return Rho GTPases to an inactive, GDP-bound state, have generally been presumed to act as tumor suppressors. Surprisingly, microarray analysis of the expression of Rho GTPases, GEFs, and GAPs across a panel of human breast tumors revealed that a number of RhoGAP genes were significantly upregulated in basallike breast cancers (BLBCs). These preliminary results suggest that RhoGAPs may play an unexpected role in promoting tumor growth. The aim of our research is therefore to validate and characterize the role of two of these RhoGAPs, RacGAP1 (also known as MgcRacGAP and CYK4) and ArhGAP11A (also known as MP-GAP), in BLBC development. In human BLBC cell lines, shRNA-mediated knockdown of either of these genes resulted in a significant defect in proliferation relative to control cells, in addition to distinct morphological changes. Furthermore, knockdown of RacGAP1 caused an increase in the number of multinucleated cells, consistent with a previously-established role for this Rac-specific GAP in regulating cytokinesis. In contrast, ArhGAP11A, a relatively poorly-characterized Rho-specific GAP, caused BLBC cell lines to accumulate in the G1 phase of the cell cycle upon its knockdown, indicative of a role in regulating cell cycle progression. In summary, we have established that two RhoGAPs, RacGAP1 and ArhGAP11A, are critical to the growth of BLBC cell lines. Experiments to establish the precise mechanisms through which these GAPs elicit each phenotype are ongoing. This work was supported by the U.S. Army Medical Research and Materiel Command under Award No. W81XWH-14-1-0033.

P1856 Board Number: B370

Epigenetic silencing of MARVELD1 gene attenuates nonsense-mediated mRNA decay in lung cancer and represents a potential biomarker for detection of malignancy. M. Shi1; 1 School of Life Science and Technology, Harbin Institute of Technology, Harbin, China Nonsense-mediated mRNA decay (NMD) pathway, a well-known mRNA quality control system, rapidly degrades mRNAs with harboring translation premature termination codons (PTC) in post-transcriptional process. Recent studies suggest that NMD regulation might implicate in tumorigenesis; however, the mechanism is poorly defined. Here, we found that novel nuclear factor MARVELD1 and the epigenetic modification of MARVELD1 promoter are involved in NMD regulation. We demonstrated that the expression of MARVELD1 gene is down-regulated in lung cancer, especially in small cell lung cancer. The reduced MARVELD1 level in lung cancer is due to its promoter hypermethylation. Treatment with DNA methyltransferase inhibitor can restore MARVELD1 expression. In addition, MARVELD1 physically interacts with NMD factor SMG1 and NMD target PTC-mRNA. The decreased MARVELD1 expression in lung cancer reduces NMD efficiency through diminishing the association of NMD complex component UPF1/SMG1 with PTC-mRNA. These results suggest that the epigenetic modification of MARVELD1 promoter links with the regulatory mechanism of NMD pathway in lung cancer cells.

SUNDAY-POSTER PRESENTATIONS

P1858 Board Number: B371

Identifying the c-MET phosphorylation site regulated by CD82 in prostate tumor cells. V. Janardan1, J. Shady1; 1 Biomedical Sciences; Cell and Molecular Biology, Grand Valley State University, Allendale, MI CD82 (KAI), a metastasis tumor suppressor protein is under-expressed in prostate as well as several other types of metastatic cancers. It inhibits cancer metastasis, but the mechanism through which regulation happens remains unclear. Various pathways are being explored in this lab, including regulation of c-MET, a growth factor receptor observed to have increased activation in tumor cells. CD82 and c-MET do not co-localize, suggesting that CD82 indirectly downregulates c-MET. To be expressed, c-MET first needs to bind to its ligand, HGF. This growth factor encourages phosphorylation of c-MET, consequently activating it. C-Met has four tyrosine phosphorylation sites that include p-Tyr 1003, p-Tyr 1234/1235, p-Tyr 1349 and p-Tyr 1365. Knowing how each phosphorylation site of c-Met affects downstream signaling event, our lab is focused in identifying which site is regulated by CD82. Another tetraspanin CD151, which promotes tumor progression and metastasis, has been shown to associate with c-Met and the integrins, which CD82 associates with. We are currently exploring the levels and expression of CD151 and its association with c-Met in the presence and absence of CD82. This we believe will provide additional insight into how CD82 overall regulates c-Met and prevents prostate tumor metastasis.

P1859 Board Number: B372

p53 protein reduces centrosome amplification in human cells exposed to subtoxic levels of 2-chloroethyl ethylsulfide (2-CEES). J.D. Fulkerson1, E. Behrens2, A. Zinn2,3, C. Duncheon2, T.J. Lamkin4, R.A. Bennett5; 1 Biology, University of Southern Indiana, Evansville, IN, 2University of Southern Indiana, Evansville, IN, 3 Biological Sciences, University of Toledo, Toledo, OH, 4Molecular Signatures Section - Molecular Genetics, Air Force Research Laboratory, Wright-Patterson AFB, OH, 5Department of Biology, University of Southern Indiana, Evansville, IN Mustard gas is a simple molecule that was first used as a chemical weapon in World War I. It is a powerful vesicant and alkylating agent that causes painful blisters on epithelial surfaces and increases the incidence of cancer in those exposed. The mechanism of mustard gas toxicity and tumorigenesis is not well understood but is thought to be mediated by its ability to induce oxidative stress and DNA damage. Interestingly, several proteins (including p53) that have been shown to either be targets of mustard gas or mediate mustard gas toxicity have also been shown to regulate centrosome duplication.

SUNDAY-POSTER PRESENTATIONS Centrosomes are small, non-membrane bound organelles that direct the segregation of chromosomes during mitosis through the formation of the bipolar mitotic spindle. Cells with more or less than two centrosomes during mitosis can segregate their chromosomes unequally, resulting in chromosome instability, a common phenotype of cancer cells. In our studies, we show that subtoxic levels of a mustard gas analog, 2-chloroethyl ethylsulfide (2-CEES), induces centrosome amplification and chromosome instability (CIN) in cells lacking p53. Additionally, we show that the reintroduction of p53 protein to cells exposed to subtoxic levels of 2-chloroethyl ethylsulfide (2-CEES) reduces centrosome amplification in, which may retard the mutation rate necessary for tumorigenesis. These data may support previous demonstrations of mustard gas toxicity targeting p53.

P1862 Board Number: B373

BRG1 Promotes Breast Cancer by Reprogramming Lipid Biosynthesis. Q. Wu1, P. Madany1, J. Akech1, J. Dobson2, S. Douthwright1, G. Browne3, J.L. Colby1, J.M. Schnabl1, J. Pratap4, G. Sluder5, R. Bhargava6, S.I. Chiosea7, A.J. Van Wijnen8, J.L. Stein3, G.S. Stein3, J.B. Lian9, J.A. Nickerson1, A.N. Imbalzano1; 1 Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA, 2 Department of Computer Science, Brown University, Providence, RI, 3Cancer Center, Univ Vermont Coll Med, Worcester, MA, 4Department of Anatomy and Cell Biology, Rush University, Chicago, IL, 5University of Massachusetts Medical School, Worcester, MA, 6Department of Pathology, Magee-Womens Hospital, Pittsburgh, PA, 7Anatomic Pathology Department, UPMC Presbyterian Hospital, Pittsburgh, PA, 8Mayo Clinic, Rochester, MN, 9Biochemistry, Univ Vermont Coll Med, Burlington, VT Epigenetic mechanisms can drive the development and progression of cancer. Epigenetic changes in tumor cells hold particular promise as therapeutic targets because, unlike gene mutations, they are reversible. In one well characterized epigenetic mechanism the SWI/SNF enzymes control gene expression through their ATP-dependent remodeling of chromatin structure. We found that the levels of BRG1, one of two mutually exclusive SWI/SNF ATPases, were up-regulated in human and mouse primary mammary tumors. Reduction of BRG1 expression decreased cancer cell proliferation and tumorigenesis in a panel of breast cancer cell lines in vitro, and in MDA-MB-231 xenografts. Depletion of BRG1 reprogrammed cancer cell metabolism, decreasing DNA, RNA and lipid synthesis. BRG1 controlled de novo fatty acid synthesis via transcriptional regulation of key lipogenic enzymes, including Acetyl-CoA Carboxylase, Fatty Acid Synthase, and ATP Citrate Lyase. We propose that targeting BRG1 could reverse the higher proliferation-supporting rates of de novo fatty acid synthesis observed in tumors even when exogenous fatty acids are abundant. In support of this idea, reduction of BRG1 sensitized breast cancer cells to chemotherapeutic drugs used clinically. Therefore, BRG1 may be a novel and promising target for the epigenetic treatment of breast cancer.

SUNDAY-POSTER PRESENTATIONS

P1863 Board Number: B374

Is Hsp90 inhibitor efficacy dependent on Cullin 5 expression?. S. Wicks1, G. Heck1, T. Atuobi1, E. Ehrlich1; 1 Biological Sciences, Towson University, Baltimore, MD Cancer is the second leading cause of death in the United States. Despite considerable progress in development of new cancer therapeutics, over half a million people are expected to die from cancer this year. Hsp90 inhibitors target Hsp90, a chaperone whose clients include oncogenes that are overexpressed in many different cancers. Inhibition of Hsp90 results in proteasomal degradation of clients and inhibition of related signaling pathways in cancer cells. Despite its promise, Hsp90 inhibitors have shown little efficacy in clinical trials. Cullin 5 (Cul5), a ubiquitin ligase involved in regulation of Hsp90 clients, has been shown to be down-regulated in breast cancer cells suggesting that variation in Cul5 gene expression may be negatively impacting Hsp90 inhibitor efficacy. We have previously reported that Cul5 is required for the geldanamycin-induced degradation of two clinically relevant Hsp90 clients, ErbB2 and Hif1a. Through analysis of gene expression data from 62 cases of breast invasive carcinoma obtained from The Cancer Genome Atlas, we observed decreased expression of Cul5 in tumor compared to normal tissue in all but 11 patients (t = -5.6155, df = 61, p-value = 5.119e-07). Here we demonstrate that Cul5 expression correlates with sensitivity to 17-AAG. Downregulation of Cul5 expression in AU565 cells resulted in complete resistance to 17-AAG, where as control cells exhibited almost 80% cytotoxicity. Sensitivity to 17-AAG correlated with Cul5 transcript levels in breast cancer cell lines. MCF-7 cells expressed Cul5 at levels two-fold higher than MDA-MB-468 cells and were more than three times as sensitive to killing by 17-AAG. This data suggests that variation in Cul5 expression may be responsible, in part, for the variation in sensitivity to Hsp90 inhibitors observed in the clinic. By establishing Cul5 as a critical player in the response to Hsp90 inhibitors, we can study Cul5 downregulation in cancer with the goal of increasing Hsp90 inhibitor efficacy, thereby enhancing survival and quality of life for affected individuals.

P1866 Board Number: B375

CDKN3 plays an important role in cellular transformation by Abl oncogenes. J. Chen1, K. Chen1, G. Guo1; 1 Chinese Academy Scis-Inst Microbiol, Beijing, China Previous studies have revealed that deregulation of multiple signaling pathways associated with cell survival and proliferation, including phosphoinositide-3-kinase PI3K)/AKT, RAS, and Janus kinase (JAK)/signal transducer and activator of transcription (STAT), underlies Abl oncogene induced tumorigenesis. However, the precise mechanisms by which Bcr-Abl causes leukemogenesis are not fully clarified. CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell

SUNDAY-POSTER PRESENTATIONS cycle progression. Deregulation or mutations of CDNK3 have been shown to play roles in various cancers. However, the function of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a key inhibitor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Forced expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

P1867 Board Number: B376

Inhibitors of Skp2 E3/Cks1 ligase complex prevent degradation of p27kip1 as a novel therapeutic approach to estrogen-induced type I endometrial cancer. S.C. Pavlides1, B.R. Rueda2, E. Rothenberg1, S.V. Blank1, K.R. Mittal1, T. Cardozo1, L.I. Gold1; 1 New York Univ Sch Med, New York, NY, 2Vincent Center for Reproductive Biology, Harvard medical school, Boston, MA In many human cancers, the tumor suppressor, p27kip1 (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization; lack of nuclear p27 causes aberrant cell cycle progression and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that estrogen (E2) induces Skp2-dependent degradation of p27 and cell proliferation in primary endometrial epithelial cells (EECs) and endometrial carcinoma (ECA) cell lines (e.g., ECC-1) suggesting a pathogenic mechanism for type I endometrial carcinoma (ECA), an estrogen (E2)-linked cancer. The current studies show that treatment of ECC-1 cells with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) increase protein levels of p27, increase p27 half-life by 6 hours and inhibit cell proliferation (IC50 14µM) by G1 phase block and not by apoptosis. Two of five SKP2E3LIs, C2 and C20 specifically increase p27 in the nucleus by 2-fold while decreasing p27 in the cytoplasm by 33% in 18 hours in both ECC-1 cells and primary ECA cells. Similarly, by Super Resolution Microscopy, Skp2E3LIs increased nuclear p27 by 1.8-fold. In addition, C2 and C20 prevent both E2-induced proliferation and degradation of nuclear p27. Importantly, C2, C5, and N1injected into ovariectomized, E2-primed mice increase nuclear p27 in uterine epithelial cells with a concomitant 42-62% decrease in proliferation. Taken together, these data suggest that Skp2E3LIs function in the nucleus to prevent E2-induced

SUNDAY-POSTER PRESENTATIONS degradation of p27 to regain growth control by directly blocking p27 ubiquitylation and subsequent degradation suggesting that these inhibitors have potential to prevent ECA since E2-induced hyperplasia precedes carcinoma. Skp2E3LIs enable a chemical biology approach to understanding the functional significance of the ubiquitin pathway in p27-mediated dysregulation of the cell cycle in cancer, and importantly, are the first specific proteasome inhibitors that pharmacologically target the binding interaction between the E3ligase, SCF-Skp2/Cks1 and p27 to critically stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors represent a major therapeutic advancement over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of p27.

P1870 Board Number: B377

Molecular and cellular mechanisms of Pin1-mediated Grb7 degradation in regulation of cancer progression. Y. Tai1, L. Tung1, Y. Lin1, T. Li2, T. Shen1; 1 Natl Taiwan Univ, Taipei, Taiwan, 2Natl Taiwan Univ Coll Med, Taipei, Taiwan Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein in cooperation with numerous tyrosine kinases to regulate various cellular signaling and functions. Indeed, cooverexpression of Grb7 with EGFR or Her2 is related to cancer progression and malignancy, therefore, Grb7 serves a prognostic marker and therapeutic target in cancers. However, it remains largely unknown the regulatory and signaling mechanisms of Grb7 in regulating these various cellular functions. Here, we observed a novel interaction between Pin1 and Grb7 by a yeast two-hybrid screening. The prolyl cis/trans isomerase Pin1 functions as a regulator to isomerize the pSer/Thr-Pro bond(s), which conferring a conformational change and leading to alterations in protein functionality, stability, and/or intracellular localization. By mutational analyses, we found that Pin1 enables directly interacting with Grb7 through the phospho-Ser194-Pro motif on Grb7 and the WW domain of Pin1. In addition, knockdown of Pin1 expression through lentiviral-mediated gene silencing could prolong Grb7 protein stability. Utilizing cycloheximide pulse-assay and quantitative RT-PCR analysis, we conclude that Pin1 could negatively regulate Grb7 protein stability at the post-translational level. In fact, we found that Pin1 could exert its isomerase activity to modulate Grb7 protein expression in an ubiquitin/proteasomedependent proteolysis pathway. Collectively, our data suggested that the Pin1-Grb7 complex formation enables negatively regulating tumor progression and/or malignant processes due to the influence of Grb7 protein expression.

SUNDAY-POSTER PRESENTATIONS

Tumor Invasion and Metastasis 1 P247 Board Number: B378

NR2F1 regulates epithelial identity and maintains quiescence of a stem cell-like subpopulation of pre-malignant mammary cells. M.S. Sosa1,2, J.A. Aguirre-Ghiso1,2; 1 Division of Hematology and Oncology, Mount Sinai School of Medicine, Department of Medicine, NY, 2 Black Family Stem Cell Institute, Manhattan, NY Invasive tumor cells are known to be able to complete all steps of the metastatic cascade. However, new evidence shows that pre-malignant cells (from lesions histopathologically defined as non-invasive, i.e. DCIS, ADH) can disseminate and, as disseminated tumor cells (DTCs), enter a non-proliferative state for prolonged periods. Early DTCs are proposed to contribute to metastasis, but the mechanisms that would allow cells considered sessile to complete all steps of metastasis are unknown. Here we report that the orphan nuclear receptor NR2F1 is downregulated in human pre-malignant and malignant lesions. Further, in MMTV-ErbB2+ pre-malignant mammary epithelial cells (MECs) downregulation of NR2F1 activates a motile phenotype and this is coincident with the detection of circulating tumor cells (CTCs) and DTCs in lungs and bone marrow. Knock down of basal NR2F1 levels in pre-malignant ErbB2+ MECs induced cell motility, loss of laminin-V deposition, β-catenin delocalization from the membrane and dramatic loss of E-cadherin junctions. Interestingly, TWIST, SNAIL and ROR1 mRNA levels became upregulated upon NR2F1 depletion. These results suggest that NR2F1 expression maintains epithelial identity and suppresses epithelial-mesenchymal transition (EMT), possibly by blocking WNT signaling. Knock down of NR2F1 in ErbB2+ pre-malignant MECs also enhanced mammosphere formation efficiency and this was accompanied by upregulation of the pluripotency transcription factor NANOG. These findings suggest that NR2F1 limits the spreading of ErbB2+ pre-malignant cells and it promotes a differentiated epithelial identity by promoting quiescence and limiting the activation of a stem cell-like program. Our findings provide for the first time evidence that NR2F1 functions in mammary epithelial cells as a suppressor of pluripotency and dissemination during early stages of tumor progression. We propose that therapies that might restore expression of NR2F1 might allow limiting early dissemination and the progression to metastasis of already disseminated tumor cells.

SUNDAY-POSTER PRESENTATIONS

P248 Board Number: B379

Bundling of the actin cortex by Erk phosphorylated Eps8 is essential for high cortex tension and the bleb-based migration of melanoma cells. J. Logue1, A. Cartagena-Rivera2, R. Chadwick2, C. Waterman3; 1 NHLBI/NIDCD, National Institutes of Health, Bethesda, MD, 2NIDCD, National Institutes of Health, Bethesda, MD, 3National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD Melanoma is a cancer that is known for its rapid spreading and low survivability. Within the confines of tissues, a growing body of evidence suggests that cancer cells frequently use blebs to migrate. Melanoma cells are hyper-contractile because of an activating mutation in the Erk signaling pathway (BRaf V600E) that pre-disposes cells to blebbing. Epidermal growth factor pathway substrate 8 (Eps8) is an actin bundling and capping protein whose capping activity is inhibited by Erk phosphorylation. We hypothesized that Eps8 may act as a key effector of Erk to modulate actin cortex mechanics and thereby mediate bleb-based migration of cancer cells. We used live cell imaging of fluorescent probes and atomic force microscopy (AFM) to test this hypothesis in human melanoma A375 cells. We found that Eps8 rapidly localized to newly formed bleb membranes, co-incident with the assembly of cortical Factin, suggesting it regulates actin in blebs. When cells were confined under agarose, they migrated rapidly in the direction of a very large bleb (50% or more of total cell area), which we call a “leader bleb,” and which exhibited co-localization of Eps8 with actin along its cortex. Depletion of Eps8 by siRNA reduced leader bleb size and prohibited bleb-based migration. Re-expression of wild-type Eps8 but not a bundling-defective mutant rescued leader bleb size, indicating a critical role for actin bundling by Eps8 in bleb-based migration. Re-expression of a capping-deficient Eps8 mutant in Eps8 knockdown cells rescued leader bleb size, but re-expression of a mutant with constitutive capping activity induced by substitution of Erk phosphorylation sites (S624A/T628A) did not. This suggests that down-regulation of Eps8 capping activity by Erk is required for leader bleb formation. Total Internal Reflection Fluorescence (TIRF) microscopy showed that WT or capping defective Eps8, but not bundling defective or nonphosphorylatable Eps8 could effectively bundle the actin cortex, suggesting that the capping activity of Eps8 antagonizes its efficiency as an actin bundler. Using a Fluorescence Resonance Energy Transfer (FRET)-based biosensor of Erk activity we found that Erk activity exhibited a gradient from front-to-back in leader blebs. Atomic Force Microscopy (AFM) of blebbing cells showed that cortex tension was reduced in cells over-expressing a dominant negative bundling-defective Eps8 mutant or nonphosphorylatable Eps8, while cells over-expressing a dominant negative capping-deficient Eps8 had elevated cortex tension. Collectively, our results suggest a model in which a gradient of Erk activity in leader blebs locally regulates Eps8 to create a gradient of actin bundling and high cortex tension to drive the bleb-based migration of melanoma cells.

SUNDAY-POSTER PRESENTATIONS

P249 Board Number: B380

Contact inhibition of locomotion in a fibrillar-like microenvironment during breast cancer progression. D.F. Milano1, S.K. Muthuswamy2, A.R. Asthagiri1,3; 1 Department of Chemical Engineering, Northeastern University, Boston, MA, 2Princess Margaret Cancer Center, University of Toronto, Toronto, ON, 3Department of Bioengineering, Northeastern University, Boston, MA Recent work demonstrated that cancer cells invade through a 3-D microenvironment composed of fibers. Tumor-associated fibroblasts help facilitate this invasion by aligning fibers perpendicular to the tumor-stromal interface. Invasion of the tumor boundary is more efficient when metastatic cancer cells migrate along these fibers, which serve as adhesive, directional cues out of the primary tumor. Yet, much of our current understanding about how cancer cells acquire invasive capabilities is based on studies using non-fibrillar, standard 2-D environments. A hallmark of invasive cancer cells is the loss of contact inhibition of locomotion (CIL). To begin to examine the nature of CIL in confined, fibrillar-like microenvironments, we utilized surfaces with micropatterned stripes whose high aspect ratio emulates a protein fiber. Time-lapse microscopy was used to quantitatively compare homotypic interactions between non-transformed mammary epithelial cells (MCF-10A) and metastatic breast cancer cells (MDA-MB-231). Cells were confined on patterned lines with widths from 6 to 33 microns. Pairwise collisions between cells were imaged in real-time and scored as bouncing or sliding collisions using frame-by-frame image analysis. The fraction of sliding collisions was measured as a representative metric to quantify the loss of CIL and reported as a function of patterned line width. Our data reveal that MDA-MB-231 cells do overcome CIL even in confined 1D microenvironments. In fact, our results demonstrate the ability of these metastatic cells to evade CIL is significantly enhanced in more confined 1D-like microenvironments, suggesting the migratory advantage of triple negative breast cancer cells may be particularly well tuned to the spatial constraints of the local tumor microenvironment. Additionally, we examined the role of partitioning defective homolog 3 (PARD3), a known suppressor of metastasis in breast cancer and regulator of CIL in vivo, by itself and in combination with the protooncogene ErbB2, on CIL in confined microenvironments. Our analysis showed PARD3 knockdown alone had no effect on the observed frequency of sliding collisions but did decrease the duration of bouncing collisions to a level indistinguishable from MDA-MB-231 cells. Activation of ErbB2 alone in MCF-10A cells did lead to a slight increase the ability of cells to slide, however, the greatest increase in sliding ability was observed when ErbB2 was activated in PARD3 knockdown cells. The cooperative nature of ErbB2 activation and PARD3 knockdown to facilitate a more invasive phenotype agrees with previous work performed in vivo and validates this platform as a novel tool to quantitatively investigate CIL in a physiological ex vivo microenvironment.

SUNDAY-POSTER PRESENTATIONS

P250 Board Number: B381

Role of aberrant vimentin expression in early and late events of human oral cancer. M. Vaidya1, C.C. Dmello1, S.S. Sawant1, H. Alam2; 1 Vaidya Lab, ACTREC-TMC, Navi Mumbai, India, 2Dept. of Molecular and Cellular Oncology, The University of Texas M D Anderson Cancer Center, Houston, TX Vimentin is an intermediate filament protein predominantly expressed in mesenchymal cells (Lazarides E, 1982). Its association has been shown with progression of many cancers including carcinomas (Satelli A, Li S, 2011). In our earlier study, we had seen the expression of vimentin in oral leukoplakia and submucous fibrosis which are known premalignant lesions for human oral cancer (SS Sawant et al, 2013). Its presence in premalignant lesions suggests the possibility of its role in early oncogenesis. Hence we overexpressed vimentin in human oral premalignant derived cell line to ask if vimentin is one of the causes or just a consequence of the process of neoplastic development. Although no difference was seen between the transformation potential of vimentin overexpressing vs vector control clones using soft agar colony forming assay, critical EMT and stemness related molecular changes were observed amongst them using reverse transcriptase PCR. Therefore further chemical carcinogenic stimuli were given using benzopyrene to both the clones till they showed an anchorage-independent state of growth in soft agar. Remarkably vimentin expressing clones showed more number of soft agar colonies as compared to vector control clones. Vimentin is well reported in late events of cancer progression but the molecular pathway underlying it is unclear hence we downregulated vimentin in oral cancer derived cell line using shRNA technology. We found increased surface levels and decreased biological turnover of β4 integrin in absence of vimentin. It was seen that the normal function of β4 integrin as mechanical adhesive device was enhanced while its signaling function remained unaltered upon vimentin downregulation. Another important proteins that we found altered upon vimentin knockdown, were the pair of K5/K14. Our data suggests that vimentin may regulate the pair of K5/K14 transcriptionally through delta p63 alpha to contribute in transformation potential of the cancer cell. Also similar correlations in expression levels of vimentin/beta4 and vimentin/K14 were seen in human oral tumor samples. Collectively this study provides insights into role of vimentin in the process of human oral oncogenesis.

SUNDAY-POSTER PRESENTATIONS

P251 Board Number: B382

Identification of an ErbB2+ early disseminating pre-malignant cancer cell subpopulation with metastatic potential. K. Harper1, M. Sosa1, H. Hosseini2, A. Avivar-Valderas1, N. Chandandeep1, R.J. Davis3, C. Klein2, D. Entenberg4, J.S. Condeelis5, E. Farias1, J.A. Aguirre-Ghiso6,7; 1 Hematology and Oncology, Icahn School of Medicine at Mount Sinai, New York, NY, 2Experimental Medicine and Therapy Research Faculty, University of Regensburg, Regensburg, Germany, 3Univ Massachusetts Med Sch, Worcester, MA, 4Albert Einstein College of Medicine, Bronx, NY, 5Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 6Black Family Stem Cell Institute, Manhattan, NY, 7Division of Hematology and Oncology, Mount Sinai School of Medicine, Department of Medicine, NY Recent clinical evidence suggests that cells are capable of disseminating from “pre-invasive” breast cancer lesions. It has been proposed that these early disseminating cancer cells (eDCCs) lodge into secondary sites, enter a state of dormancy, escape conventional therapy, and potentially evolve through different pathways from the primary tumor to form metastases. However, the mechanisms that endow pre-malignant cells to disseminate and their behavior in target organs remain unknown. Here we show that ErbB2+ pre-malignant cancer cells, while non-tumorigenic are highly efficient in disseminating. While a majority of these eDCCs remain dormant in target organs they are still endowed with a metastatic capacity. We identified an ErbB2HIGH/p38LOW/E-cadherinLOW population of pre-malignant mammary epithelial cells (MECs), which undergo a Wnt-dependent EMT and disseminate. Using intravital imaging of transgenic ErbB2-T-CFP pre-malignant lesions we imaged this process at high resolution revealing that ErbB2HIGH/p38LOW/E-cadherinLOW cells display activate cancer cell motility, invadopodia formation and intravasation capacity. This correlated with CK8/18+/HER2+ circulating cancer cells as well as eDCCs in the lung and bone marrow of mice, a process that was enhanced following 2 weeks of systemic p38α/β inhibition. We conclude that at pre-malignant stages p38α/β antagonizes ErbB2 signaling restricting early dissemination of cells, and that early dissemination is a source of predominantly dormant eDCC with metastasis initiating capacity that might contribute to relapse.

P252 Board Number: B383

Heterogeneity in cell-matrix adhesion as an indicator of metastatic state. A. Fuhrmann1, T. Tlsty2, A.J. Engler1; 1 Bioengineering, University of California, San Diego, La Jolla, CA, 2University of California, San Francisco, San Francisco, CA Cancer cells have great genetic diversity, which may be reflected in varied metastatic potential. As it has been difficult to determine a comprehensive set of genetic markers to define a metastasizing cell population, we propose a common behavior, i.e. cell-matrix adhesion, that may be differentially

SUNDAY-POSTER PRESENTATIONS regulated in metastasizing versus non-metastasizing cancer cells. To assess cell-matrix adhesion strength in a heterogeneous population of breast cancer cells, we employed a spinning disc device where cells adhering to matrix-coated substrates were exposed to radially-dependent shear. Within a cell population, cells at the center or edge experience low or high matrix detachment forces, respectively. Highly metastatic cancer cell lines, e.g. MDA-MB-231 and MDA-MB-468, exhibit the same detachment behavior as non-malignant MCF10A and non-metastatic MCF7 cells in the presence of tumor-like concentrations of magnesium and calcium, which regulate integrin activation. However at stromal concentrations of these cations, only the adhesion of metastatic cells was drastically lowered and broadly distributed, suggesting that the most metastatic cells may be more sensitive to lower cation concentrations. Average attachment strength–defined as the shear required to detach 50% of cells, i.e. τ50–and restoration of attachment-homogeneity gradually increased with cation concentration with the greatest sensitivity occurring between 0.01 and 0.50 mM, which falls within the physiological range for both stroma and tumor. Moreover, focal adhesion assembly in MDA-MB-231 cells was highly dependent on cations: at stromal concentrations, cells had more labile adhesions compared to large, stable adhesions at tumor-like concentrations. These changes were absent in non-metastatic cells, indicating that variances in attachment strength and adhesion assembly in the absence of cations may be cell state dependent. Indeed after selecting for strongly attaching metastatic cells, cells maintained their strong attachment phenotype for several days, but stochastically return to heterogeneous adhesion strength over several weeks. Migration and invasion of selected MDA-MB-231 cells were markedly impeded versus their non-selected or non-metastatic counterparts, i.e. sensitivity to cation removal indicates higher invasion potential. These results suggest a potential biophysical mechanism to differentially regulate cell metastasis.

P253 Board Number: B384

Mismatch in cell mechanical properties in co-cultures can lead to enhanced cell motility: A Brownian Dynamics Simulation. J. Butcher1, D. Kolbman1, M. Das1; 1 Physics and Astronomy, Rochester Institute of Technology, Rochester, NY Recent experiments suggest that the mechanical stiffness of cells and their interaction with their surroundings undergo remarkable changes during tumor progression [1,2]. An intriguing experimental result in this area suggests that the mismatch in the elasticity and adhesive properties between cancer cells and non-cancerous cells in a co-culture may lead to enhanced cancer cell motility [2]. Motivated by this, we study a mathematical model of a 2D co-culture of cells with different stiffnesses and adhesivity using active Brownian Dynamics simulations. We characterize cell motility by studying particle trajectories, mean square displacements and correlation functions. We find that in a binary system of softer and stiffer particles, the softer particles are more motile, and the difference in motility between the two types of particles is enhanced with increase in difference between their stiffnesses. We also find that introducing particle-particle adhesion to the stiffer particles can further increase the motility of the

SUNDAY-POSTER PRESENTATIONS softer particles. Our study may provide insights into the interplay of mechanical and statistical mechanical properties underlying the enhanced motility of cancer cells during metastasis [2]. References: [1] S. Suresh, Biomechanics and biophysics of cancer cells, Acta Biomaterialia 3, 413 (2007). [2] M. H. Lee, P. H. Wu, J. R. Staunton, R. Ros, G. D. Longmore, and D. Wirtz, Mismatch in Mechanical and Adhesive Properties Induces Pulsating Cancer Cell Migration in Epithelial Monolayer 102, 2731 (2012).

P254 Board Number: B385

Joined forces of cancer cells and fibroblasts against the basement membrane. A. Glentis1, V. Gurchenkov1, M. Schoumacher1, F. Zaccarini2, P. Mariani3, D. Vignjevic1; 1 Institut Curie, Paris, France, 2AP-HP Hopitaux de Paris, Paris, France, 3Hopital Curie, Paris, France In carcinoma in situ, the basement membrane represents a physical barrier that prevents spreading of primary tumor to adjacent tissues. It is believed that cancer cells perforate basement membranes. However, stromal cells such as carcinoma-associated fibroblasts also secrete matrix proteinases. Therefore, the question is who is invading whom – do cancer cells invade stroma or possibly stroma is invading tumor cells? Using human colon cancer cells and primary human fibroblasts isolated from tumors and adjacent normal tissues, we addressed if cancer cells and fibroblasts are invading the basement membrane simultaneously or they work together but have distinct functions. Analyzing human colon cancer samples, we observed that invasive tumors contain higher amount of fibroblasts in general and higher proportion of CAFs (αSMA+) compared to non-invasive tumors or healthy tissues. We isolated fibroblasts from fresh human colon tumors of different stages (CAFs) and from the adjacent normal tissues (NAFs). A combination of markers was used to discriminate fibroblasts from other cell types to validate the purity of isolated cells and to discriminate NAFs from CAFs. In co-culture experiments on Matrigel-coated transfilters, both NAFs and CAFs induced migration and invasion of HT29, intrinsically non-invasive colon cancer cells. On contrary, in an assay containing native, mesenteric basement membrane that separates cancer cells on one side and fibroblasts embedded in collagen I on the other, we found that only CAFs are able to stimulate invasion of cancer cells. CAFs stimulated invasion of cancer cells when physically present in the assay and, in a lesser amount, via paracrine ways. Proteomic study using SILAC, showed that CAFs secrete more proteases, extracellular matrix proteins, and proteins that modify the matrix compared to NAFs, pointing to a matrix-remodeling role in invasion. Live cell imaging showed that fibroblasts and cancer cells are communicating through the basement membrane via cellular protrusions long time before the actual translocation of cancer cells is detected. We are currently testing a role of CAF-derived molecules in basement membrane remodeling in order to dissect the interplay between cancer cells and CAFs in basement membrane invasion.

SUNDAY-POSTER PRESENTATIONS

P255 Board Number: B386

Cortactin, a key regulator of tumor progression, promotes exosome secretion. S. Sinha1, D. Hoshino1, K. Kirkbride2, N.E. Grega Larson3, C. French2, M.J. Tyska3, A.M. Weaver1,4; 1 Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, 2Vanderbilt University Medical Center, Nashville, TN, 3Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, 4Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN Exosome secretion from cancer cells is thought to drive cancer aggressiveness. Presumably, exosome secretion is a regulated event; however little is known about the molecular regulators or how this is altered in cancer. In this study, we show that the tumor-overexpressed cytoskeletal protein cortactin plays an important role in controlling exosome secretion. Inhibition or overexpression of cortactin respectively inhibited or upregulated the number of exosomes secreted from cells. Proteomic analysis indicated that cortactin only affected the number of vesicles but not their cargo content. Live imaging experiments revealed that cortactin controls exosome secretion by controlling trafficking from late endosomes. These data may explain the reported association of cortactin overexpression with tumor aggressiveness and poor patient prognosis.

P256 Board Number: B387

Effect of Vitamin C on the Cytotoxicity of Vemurafenib or Doxorubicin Human Malignant Melanoma Cells. G. Gao1, G. Yang1, Y. Ma1, l. wang1, Y. Yang1; 1 Emporia State University, Emporia, KS Objective: The goal of this study is to identify the effect of vitamin C on the cytotoxicity induced by Vemurafenib or Doxorubicin in human malignant melanoma cells. Methods: The effect of vitamin C on the cytotoxicity induced by Vemurafenib or Doxorubicin were examined by cell viability assay, wounding healing assays, and Transwell migration assay to measure its effects on cell proliferation, migration, and invasion. The apoptotic cell percentage were determined by flow cytometry. Results: Vitamin C alleviated the cytotoxic effects of Doxorubicin or Vemurafenib on proliferation, migration, and invasion of human malignant melanoma cells when the concentration of vitamin C was relatively low (=5mM). Vitamin C significantly reduced the percentage of apoptotic cells induced by Doxorubin or Vemurafenib when the concentration was low (1mM). Conclusion: Vitamin C showed the dual roles in the cytotoxicity induced by Vemurafenib or Doxorubicin in malignant melanoma cells depending upon the concentrations. Vitamin C alleviated the cytotoxic effect of Doxorubicin or Vemurafenib on human melanoma cells when the concentration was low. However, when the concentration of vitamin C become higher, it enhanced the cytotoxic effect of Doxorubicin or Vemurafenib on malignant melanoma cells.

SUNDAY-POSTER PRESENTATIONS

P257 Board Number: B388

Rosehip (Rosa canina) extracts prevent serum-stimulated migration of human glioblastoma cells. F.S. Ogunsemowo1; 1 Department of Biology, North Carolina AT State University, Greensboro, NC Glioblastoma multiforme (GBM) are the most common and most aggressive intracranial tumors. Malignant gliomas are characterized by their diffuse invasion of surrounding brain tissue. Despite modern diagnostics and treatments the median survival time is less than a year; and over the past decades no significant increase in survival of patients suffering from this disease has been achieved. Thus, alternative treatment and prevention options are necessary to combat this disease.Natural products represent a source of potential chemical molecules exhibiting anti-oncogenic properties. An example is Rosehip (Rosa canina); rosehips are blossoms from the wild rose and frequently used as an herbal medicine. Recently our laboratory has shown that rosehip extracts exert anticancer activities in vitro, by inhibiting proliferation of GBM cells. Preliminary data from our laboratory shows that rosehip extracts are able to prevent breast cancer cell migration. Therefore, we investigated the efficacy of rosehip extracts in preventing cell migration of human GBM cell lines. We hypothesize that GBM cell migration will be prevented when exposed to rosehip as a result of interference of cell signaling pathways. Wound healing assays were performed to examine the anti-migratory effects of rosehip extracts (1mg/ml) on human GBM cell lines, U-251 MG and U-87 MG. The data demonstrate that rosehip extracts facilitate inhibition of serum-stimulated cell migration more effectively than MAPK (UO126) and AKT (LY294002) inhibitors. We extended this investigation by examining whether rosehip extracts could prevent EGF-induced cell migration. Rosehip extracts did not prevent EGF-induced migration, suggesting that an alternative mechanism is promoting EGF-associated migration in these cells. These data suggest that rosehip extracts may serve as an alternative therapy for treating certain populations of malignant gliomas.

P258 Board Number: B389

Metastasis Upregulated Genes Have Distinct Function in C. elegans Cell Migrations. S.A. Vetrone1,2, M. Kato2, P. Sternberg2; 1 Biology, Whittier College, Whittier, CA, 2HHMI, Biology and Biological Engineering, California Institute of Technology, Pasadena, CA Cell migration is vital for normal animal development but also contributes to the invasive spreading of early stage metastatic cancer. From two published databases, we complied 107 genes unregulated in either breast cancer or melanoma metastases and investigated their requirement in two Caenorhabditis

SUNDAY-POSTER PRESENTATIONS elegans cell migrations: the male linker cell (LC) and hermaphrodite distal tip cells (DTC) which have similar functions as gonadal leader cells that undergo a complex migration while pulling non-motile followers. We performed an RNAi screen to identify genes implicated in normal LC and DTC migrations. Thirty-two genes from the metastasis list were required for the cell migration of which 13 genes effected the migration of both LC and DTC, 18 genes effected only LC migration, and 4 genes only DTC migration. The genes used by both cell types corresponded to genes involved in cell cycle activity, adhesion, cytoskeleton organization, protein degradation activity, spliceosome activity, ubiquitin-like modification and function, and peptidase inhibition. The genes used by only the LC corresponded to genes involved in adhesion, cytoskeletal organization, methyltransferase activity, metalloproteinase activity, and signaling. Those genes used by only DTC corresponded to genes involved in signaling, peptidase inhibition, and utrophin activity. The significant differences among the developmental cell migrations and the overlap in genes shared between the two metastases underscores the value of characterizing diverse genes and considering cell type in developing treatments.

P259 Board Number: B390

DIBUTYLTIN-INDUCED ALTERATIONS OF INTERLEUKIN 1 beta SECRETION FROM HUMAN IMMUNE CELLS. S. Brown1, S. Tehrani2, M. Whalen2; 1 Biological Sciences, Tennessee State University, Nashville, TN, 2Chemistry, Tennessee State University, Nashville, TN Dibutyltin (DBT) is an organotin compound that is used as a stabilizer in polyvinyl chloride (PVC) plastics (including pipes that distribute drinking water and bottles) and as a de-worming agent in poultry. DBT is found in human blood samples, and DBT exposures alter the secretion of tumor necrosis factor alpha (TNFα) from lymphocytes. Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine that promotes cell growth, tissue repair, and immune response regulation. Produced predominately by both monocytes and macrophages, IL-1β appears to increase the invasiveness of certain tumors. The aim of the current study is to determine whether exposure to DBT alters the secretion of IL-1β from increasingly reconstituted preparations of human immune cells. We examined whether exposure to DBT concentrations of 0.05 to 5 µM after 24h, 48h, or 6 days alters IL-1β secretion in a preparation of highly enriched human NK cells, a monocyte-depleted preparation of human peripheral blood mononuclear cells (PBMCs) (MD-PBMCs), PBMCs, granulocytes, and a preparation combining both PBMCs and granulocytes. The levels of IL-1β were monitored using an enzyme-linked immunosorbent assay (ELISA). The results indicated that DBT alters IL-1β secretion from all of the above cells preparations. The high concentrations of DBT (5 and 2.5 µM) decreased the secretion of IL-1β, while some of the lower concentrations of DBT (0.1 and 0.05 µM) increased the secretion of IL-1β. The concentrations and lengths of exposure to DBT that caused statistically significant increases in IL-1β secretion from human immune cells varied from one donor to the next. Therefore, the data indicates that DBT-induced alterations of IL-1β secretion from immune cells may potentially affect immune function and cancer

SUNDAY-POSTER PRESENTATIONS invasiveness. An additional aim is to examine the signaling pathways involved in the DBT-induced increases in IL-1β secretion from MD-PBMCs, which are a preparation that primarily consists of T and NK lymphoyctes. MD-PBMCs will be treated with inhibitors of pathways that regulate IL-1β secretion (IL-1β cleavage inhibitor, ERK ½ pathway inhibitors (PD98059, U), p38 inhibitor (SB202190), and NFκB inhibitor (BAY 11-7085)) 1h prior to adding DBT concentrations of 0.1 and 0.05 µM for 24h.

P260 Board Number: B391

Oncogene-expressing cells collaborate to evade microenvironmental restraint. A.J. Hughes1, N. Jee2,3, M. Todhunter2,4, Z.J. Gartner2,5; 1 Physiology Course at Marine Biological Laboratory, Woods Hole, MA, 2Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, 3Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, San Francisco, CA, 4Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA, 5Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA Healthy tissue can harbor a high frequency of “occult” cancer; in which isolated transformed cells do not progress into overt tumors. We hypothesized that multiple transformed cells can collaborate to accelerate the earliest stages of tumor progression. To test this hypothesis, we studied the growth trajectories of small (~8 cell) 3D microtissues comprising wild-type MCF10A (WT) mammary epithelial cells intermixed with MCF10AT (Ras) cells that expressed low levels of a constitutively active H-Ras oncogene. H-Ras expression confers cells with increased rates of cell motility and proliferation. During culture in Matrigel over 7 days, we quantified the growth rates of each cell type by tracking fluorescent nuclear reporters of cell identity by confocal microscopy. Mixed tissues containing single Ras cells showed a broad diversity of growth trajectories over 7 days of culture. Ras cells either expanded rapidly through the WT population (an "escape" trajectory), did not expand through the population ("restraint"), or reached a steady-state fraction of the cell population while also triggering rapid expansion of both the WT and Ras cell populations ("collaboration"). In contrast, cell assemblies containing two or more Ras cells were far more likely to show collaboration rather than restraint trajectories. Our data suggests that a tipping point can exist wherein an increase in the frequency of oncogeneexpressing cells amongst wild-type neighbors triggers a transition between restrained and collaborative proliferation of entire cell collectives.

SUNDAY-POSTER PRESENTATIONS

P261 Board Number: B392

Mechanisms of Transendothelial Migration by Invasive Breast Carcinoma Cells from Patients. J. Pignatelli1, S. Goswami1,2, J.G. Jones1,3,4, X. Chen1, J.S. Condeelis1, M.H. Oktay1,4; 1 Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 2Department of Biology, Yeshiva University, New York, NY, 3Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY, 4Department of Pathology, Albert Einstein College of Medicine, Bronx, NY The majority of breast cancer related deaths are not due to the primary tumor, but rather to the spread of distant metastatic tumors. Our lab has previously described the identification of the tumor microenvironment of metastasis (TMEM) in mouse and human mammary tumors, sites where transendothelial migration (TEM) occurs and therefore intravasation. The constituent cells of TMEM are an endothelial cell, a perivascular macrophage and an invasive Mena-over expressing tumor cell in direct contact. TMEM are present in human invasive breast tumors and the density of TMEM is positively associated with the risk of developing metastases. Using invasive ductal carcinoma cells of the breast obtained from patients by fine needle aspiration, we demonstrated that intravasation-directed transendothelial migration of these cancer cells requires macrophages and, depending on clinical subtype, involves either paracrine, or both paracrine and autocrine signaling. Compared to the total population of primary breast cancer cells assayed, cells capable of transendothelial migration expressed relatively high MenaINV and low Mena11a levels, independently of clinical subtype. MenaINV and Mena11a are functionally distinct isoforms of Mena, a key regulator of motility and invasion. Furthermore, relative MenaINV expression correlated with the density of TMEM, which were previously shown to correlate with risk of metastasis in patients. Our data reveal targetable signaling events required for transendothelial migration of human breast cancer cells, and indicate that relative MenaINV levels and TMEM frequency are correlated prognostic markers of metastasis and therapeutic targets for most human breast cancers.

P262 Board Number: B393

Transendothelial migration of uveal melanoma cells. M.D. Onken1, J. Li1, J.A. Cooper1; 1 Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO Uveal melanoma (UM) is cancer arising from the pigmented layers of the eye that is highly metastatic and only spreads through the bloodstream. Almost half of UM patients develop distant metastatic disease, most often in the liver, even after the tumor-bearing eye is completely removed. The key to understanding and targeting UM metastasis is in understanding how circulating tumors cells enter and

SUNDAY-POSTER PRESENTATIONS exit the bloodstream and then invade and colonize distant organs. We use primary human dermal microvascular endothelial (HDMVEC) monolayers grown on polyacrylamide soft substrates that mimic the physiological stiffness of normal tissue as a model of the blood vessel wall. To these monolayers, we add UM cells and follow their transendothelial migration. We found that UM cells transmigrate via a unique route that is distinct from the transendothelial migration common to immune cells. During this multistep process, UM cells identify HDMVEC cell-cell junctions and then intercalate between adjacent HDMVECs, taking on a flattened morphology while maintaining contacts with the adjacent endothelial cells. UM cells remain intercalated for an extended period in a state reminiscent of vasculogenic mimicry. After intercalation, UM cells extend invasive projections beneath adjacent HDMVECs, and use these to migrate beneath the monolayer. Immunofluorescence in fixed monolayers revealed cortical actin networks with strong cortactin staining within these invasive projections. We used UM cells expressing F-tractin, a fluorescent fusion protein that specifically binds filamentous actin without disrupting actin function, to image of actin dynamics in live cells. Time-lapse confocal images revealed complex actin-rich projections invading and sampling the interface between the endothelial monolayer and the substrate. Dynamic actin cytoskeleton reorganization was also apparent as cells migrated under the monolayer. We used blocking antibodies to identify cell adhesion molecules that were involved in UM transmigration. We found that blocking VCAM1 inhibited early intercalation but enhanced overall migration, suggesting VCAM1 might be necessary for initiation and maintenance of intercalation. BAP1 is the major metastasis suppressor in human UM tumors. We knocked down BAP1 in our uveal melanoma cell lines, and found that loss of BAP1 had no significant effect on initiation or intercalation of UM cells. However, overall migration was significantly enhanced by BAP1 knockdown. Our studies show that UM transendothelial migration occurs in two phases: vasculogenic intercalation that depends upon VCAM1 adhesion, and subsequent invasion under the monolayer, which is regulated by BAP1.

P263 Board Number: B394

Dissecting the Mechanism of Tumor Cell and Macrophage Streaming Towards Blood Vessels. E. Leung1, P. Rougerie1, V.P. Sharma1,2, R.J. Eddy1, J.S. Condeelis1; 1 Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 2Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY In vivo, breast tumor cells have been observed to migrate with macrophages on collagen fibers towards blood vessels in a process known as “streaming.” Mechanistically, the interactions of the macrophages and tumor cells have been characterized to involve an EGF and CSF-1 paracrine loop with macrophages secreting EGF and tumor cells releasing CSF-1 to attract each other. However, the signaling molecule that is responsible for the directional migration of both the tumor cells and macrophages towards the blood vessel has not been determined. By plating MTLn3 breast tumor cells and primary bone-marrow derived macrophages (BMMs) on micropatterned substrates that mimic the diameter and composition of the collagen fibers found in vivo, we

SUNDAY-POSTER PRESENTATIONS observed that tumor cells and macrophages align together to form “pairs.” However, the tumor cells and macrophages did not demonstrate directional or biased migration towards one direction within the assay. However, directional migration of tumor cells could be established when beads coated with either NIH3T3 fibroblasts or human umbilical vein endothelial cells (HUVEC), both know sources of HGF, were placed at one end of the micro-patterned “collagen fibers” within the assay. This directional migration was inhibited with an HGF inhibitor, suggesting that HGF is the molecule responsible for the directional migration of tumor cells towards blood vessels. This combination of cells; tumor cell, macrophage and endothelial/fibroblast, when placed on a linear substrate, reconstituted streaming in vitro with speed, directional persistence and cell migration characteristics like those observed in vivo during tumor cell streaming toward blood vessels.

P264 Board Number: B395

A Novel Bone Bioreactor Used to Model Bone Metastasis Ex Vivo. R.J. Romero Moreno1, K. Jackson2, T. Coughlin2, T. Kriepke2, L. Nystrom3, G. Niebur2, L.E. Littlepage1; 1 Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, 2Aerospace and Mechanical Engineering, University of Notre Dame, Notre Dame, IN, 3Orthopaedic Surgery and Rehabilitation, Loyola University Chicago, Chicago, IL Bone metastases are incurable. Understanding what drives cancer to metastasize to bone and identifying treatments that eliminate bone metastasis are essential to improving the survival and quality of life of cancer patients with metastasis to bone. The current methods used to study bone metastasis are restricted to in vitro tissue culture models and to in vivo animal models, both of which have several limitations. The in vitro tissue cultures lack the 3-D environment of heterogeneous cell types of the bone and marrow, and in vivo animal models often are limited by the confounding primary tumor burden. Both options generally are not applicable to rapid screening aimed at targeting bone metastases. In this interdisciplinary project, we use a novel bone bioreactor to culture mouse bone explants, study bone metastases, and develop therapies to help breast cancer patients that have developed bone metastases. The objective of this research is to develop an experimental system that preserves the 3-D environment and heterogeneous culture conditions (bone, marrow, and cancer cells) within the physiological context of an intact bone environment and apply the technology to develop faster screening techniques than the ones available in current animal models. We will use this ex vivo bone culture bioreactor to identify the molecular factors that contribute to develop bone metastases and to aid in the screenings of new drugs aimed at targeting bone metastasis in breast cancer patients. We will validate the bioreactor as a means to understand the stages of metastatic tumor colonization, progression, and response to therapies. After validation in a murine model, our bioreactor will make it possible to study metastatic cancer progression temporally and independently from primary tumor growth. Because this system is amenable for investigating bone colonization by multiple cancer types, this study also has general application beyond breast cancer. Due

SUNDAY-POSTER PRESENTATIONS the usage of bone explants and vibrational technology that is currently available to patients, this study has high translational value. Due the limitations of current methodologies and lack of exploration to improve them, our unique interdisciplinary perspective to attack this problem will result in high impact publications in the fields of cancer and bone behavior.

P265 Board Number: B400

Multiclonal Seeding is a Frequent Route to Metastatic Spread. K. Cheung1, V. Padmanaban1, K. Schipper1, V. Silvestri1, A. Ewald2; 1 Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 2Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD Introduction: A foundational concept in metastasis research is that primary tumors first dissociate into single cancer cells in order to seed distant sites. The requirement for a single-cell seed forms an important tenet for common molecular models of metastasis including the epithelial-mesenchymal transition. However, the experimental evidence for the single-cell seed model of metastasis remains limited. Furthermore, circulating tumor cell clusters are detectable in the bloodstream and therefore multiclonal seeding events are also possible. In this study, we experimentally isolated the relative contributions of single and multiclonal seeds and uncovered that multiclonal seeding is a frequent route to metastatic spread. Methods: We devised a lineage tracing strategy using Rainbow transgenic mice to reveal the clonal origin of lung metastases in MMTV-PyMT mice, a commonly used mouse model of metastatic breast cancer. Primary PyMT tumor organoids that express a genetically encoded Rainbow construct were treated with Cre in order to induce random assortment of color labeling, and then injected orthotopically into the mammary fat pad of host mice. Lung metastases were then scored for the number of unique colors. Results: If lung metastases arise exclusively from single seeds then each lung metastases should express only one color. Surprisingly, we observed frequent multicolor lung metastases (mean 32%, maximum 61%, N=998 metastases). By implanting tumor organoids labeled with distinct colors into separate flanks of host mice, we observed that these multiclonal seeding events occur predominantly in a single step rather than by serial colonization of single cells. To address why multiclonal seeding might be advantageous, we flow sorted trypsinized tumor cells, gating by size. This enabled us to obtain fractions of single cells and cell clusters of increasing size distributions. Interestingly, we observed that cell clusters had 10-fold higher colony formation and superior survival relative to isolated single cells. Work is underway to test the lung colonization potential of single cells versus cell clusters using tail-vein metastasis assays.

SUNDAY-POSTER PRESENTATIONS Conclusions: Our results challenge the requirement for single cell seeding in metastatic colonization and establish the importance of a parallel route for metastatic spread involving collective multiclonal dissemination. Furthermore, our data suggest a fundamental mechanism for cell growth and survival mediated by cell-cell contacts. We suggest that disrupting cell contacts may limit cancer cell survival and metastatic spread.

P266 Board Number: B401

Establishing 3-dimensional Spheroids from the Nasopharyngeal Carcinoma (NPC) cell line HK1 to investigate their Growth and Invasion behavior and test their Sensitivity to Flavopiridol. N. Mohana Kumaran1, K. Muniandy2, P. Siva Sankar1, A. Khoo Soo Beng3, V. Balakrishnan2; 1 School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia, 2Institute for Research in Molecular Medicine (INFORMM), Penang, Malaysia, 3Molecular Pathology Unit, Institute for Medical Research (IMR), Kuala Lumpur, Malaysia Most anti-NPC drug activity studies have come from work performed with two-dimensional (2D) culture assays. 2D culture systems ignore the fact that NPC cells do not live in isolation but are oriented in a 3D space, establishing contact with the stroma, i.e. extracellular matrix and other cell types such as endothelial cells, fibroblasts and immune cells. Three-dimensional (3D) cultures represent a good compromise between the lack of a microenvironment encountered under 2D culture conditions and the great complexity of the in vivo animal models. 3D culture mirrors the tumour architecture and microenvironment more closely than 2D, recreates the oxygen/nutrient gradient with a hypoxic zone and a central necrosis and allows interaction between NPC cells and their stroma, when provided. This close resemblance to the situation encountered in vivo facilitates more realistic study of NPC growth, invasion and drug response. The objective of this study is to establish a 3D model from the HK1 NPC cell line to determine the growth and invasion behavior of the spheroids on the collagen matrix and to test the sensitivity of the spheroids to chemotherapeutic drugs. Approximately 5000 NPC cells were seeded onto concave agar and left to form spheroids for 72 hours. Once the spheroids have formed, they were harvested and embedded onto the collagen matrix. Spheroid growth and invasion were documented by taking phase contrast images every 24 hours using an inverted fluorescence microscope. Spheroid growth and invasion were quantified by image analysis software’s. The HK1 cells formed compact spheroids within 72 hours. Our observation from the 10 days experiments revealed that spheroids gradually grew and invaded into the collagen matrix, showing that the HK1 spheroids are capable of growth and invasion. We also observed formation of holes in the spheroids, which could be explained by cell death under hypoxia. Progressing from these experiments, the HK1 spheroids were employed to perform a drug sensitivity assay using the chemotherapeutic drug, Flavopiridol. The drug had a dosedependent inhibition of spheroid growth and invasion. Our future endeavor will be to employ the spheroids to study whether Flavopiridol will sensitize the NPC cells to small molecule inhibitors which targets the intrinsic apoptosis pathway for NPC therapy and to establish spheroids from the Epstein -

SUNDAY-POSTER PRESENTATIONS Barr virus (EBV) positive NPC cell line, C666-1 to observe if the spheroids exhibit the same growth and invasion behavior as the HK1 or whether the EBV infection dictates for any changes.

P267 Board Number: B402

A novel 3D cell culture system using FP001 for in vitro evaluation of anticancer compounds. T. Kanaki1; 1 Nissan Chemical Industries, Ltd, Shiraoka, Japan Recently, a number of approaches have been developed to generate 3-dimentional (3D) cell culture models to mimic in vivo environments for cancer studies; e.g. scaffolds, microcarriers, and spheroids. However, these approaches remain some problems such as applying them into high throughput screening (HTS) systems and improving the efficiency of anti-cancer drug discovery. In this study, we identified FP001 as the polymer materials, and developed a novel 3D cell culture medium using FP001 which has the ability to form cancer cell spheroids in normally-distributed and appropriate size. 3D cell cultures system using ultra-low attachment multi-well plates in combination with FP001 exhibited >3fold increase in the number of A549 cells after 5-day culture as compared to that without FP001. The positive effect of FP001 was applicable to a wide variety of cancer cell lines and was clearly beneficial for HTS. As for the cell proliferation of HeLa and A549, the 3D culture system was more sensitive to AKT inhibitors and MEK inhibitors, compared with that employs 2D monolayer culture condition. Furthermore, Heparin-binding EGF-like growth factor, an EGF receptor ligand, clearly stimulated the growth of SKOV3 and A431 cells in the 3D culture system, but not in monolayer culture one. In conclusion, we established a novel method for the 3D culture of cancer cells under low attachment condition by using FP001, which was available for HTS and showed high sensitivity to molecularlytargeted drugs, EGF signal inhibitors. Our approach would facilitate the development of novel models for in vitro evaluation of anticancer compounds.

P268 Board Number: B403

Utilizing 3-D Collagen Cultures to Represent In Vivo Cancerous Tumors and to Test Targeted Molecular Imaging Agents (TMIAs). A.M. Butera1, H.D. Ophardt1, I.M. Evans1; GSOLS, Rochester Institute of Technology, Rochester, NY

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The goal of this research is to develop in vitro assay methods for the evaluation of targeting molecular imaging agents (TMIAs) using confocal fluorescent microscopy (CFM). Culturing cells in two dimensions on a plastic surface results in artificial two-dimensional growth patterns of sheets of cells which do not provide a good model of a tumor growing in vivo. Most cells in the human body interact with a three-

SUNDAY-POSTER PRESENTATIONS dimensional environment, composed of other cells, matrices, fibrous layers, and adhesion proteins. In order to create a more accurate TMIA screening environment that closely mimics how tumor cells grow in the body, 3-D cultures were utilized. Collagen is a very widely used matrix for 3-D cultures due to its chemical and physical simplicity and thus was used for initial experiments. A549 cells were suspended in a liquid collagen matrix which was allowed to solidify on a glass bottom dish. The cells were then stained with a Targeted Molecular Imaging Agent (TMIA) Cy5.5-RGDyk conjugate and other stains such as NucBlue, MitoTracker Red, and Tubulin Tracker. Visualization of cultures was done using confocal microscopy. The cells grown in collagen culture formed spheroid-like structures which more closely resembled a tumor growing in vivo. These cultures are a better in vitro tumor model than cells growing on a 2D surface. Observation of the cells stained with the TMIA agent showed that the TMIA penetrated well into the spheroid structure and that the TMIA was taken into the cells by an endocytosis-like mechanism. The use of 3D collagen culture should allow more efficient TMIAs to be identified and hopefully will lead to identification of TMIA agents that will penetrate and stain tumor tissue in the body.

P269 Board Number: B404

Study on the mechanisms of pipoxolan inhibits the migration and invasion in CL1-5 human lung adenocarcinoma cells by reducing MMP-9 and MMP-2 expression. Y. Chen1; 1 pharmacy, Taichung, Taiwan Pipoxolan has been shown to have antitumor effect. However, the effects of pipoxolan on lung cancer cell metastasis remains undecided. This study was to investigate how pipoxolan affect the lung cancer cell migration and invasion. This study investigated the antimigration and antiinvasion effects of pipoxolan on lung adenocarcinoma cancer cells (CL1-5 cells) and its underlying molecular mechanisms. The data demonstrates that pipoxolan does not effectively inhibit the viability of CL1-5 cells. Pipoxolan markedly suppress cell migration of CL1-5 cells by the assessment of wound scratch assay and transwell assays. Treatment of pipoxolan brought about inhibition of migration and invasion of CL1-5 cancer cells by transwell assay. And, these observations were associated with a reduction in the activities and levels of matrix metalloproteinase (MMP)-2 and MMP-9 in CL1-5 lung cancer cells. Lastly, pipoxolan at 5 μg/mL and 10 μg/mL inhibited phosphorylation c-Jun N-terminal kinase (p-JNK) of CL1-5 cells. Moreover, pipoxolan at 10 μg/mL inhibited phosphorylation p38 MAP Kinase (MAPK) of CL1-5 cells. In addition, our results also demonstrate that administration with JNK1/2 inhibitor (SP600125), and p38 inhibitor (SB203580) reduce MMP-2 and MMP-9 expressions in CL1-5 lung adenocarcinoma cancer cells. Based on these results, our conclusions show that treatment of CL1-5 lung cancer cells with pipoxolan downregulates MMP-2 and MMP-9, which are the down-stream targets of JNK and p38 and play a critical role in CL1-5 cells metastasis. These results show a new therapeutic potential for pipoxolan in antimetastatic therapy.

SUNDAY-POSTER PRESENTATIONS

P270 Board Number: B405

Podoplanin-expressing CAFs Lead and Enhance the Local Invasion of Cancer Cells in Lung Adenocarcinoma. S. Neri1, G. Ishii1, A. Ochiai1; Pathology Division, National Cancer Center Hospital East, Kashiwa, Japan

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Cancer-associated fibroblasts (CAFs) communicate with cancer cells and play important roles in cancer invasion. We previously observed that lung adenoca rcinoma patients with podoplanin(PDPN)expressing CAFs frequently exhibited the local invasion of cancer cells. In this study, we established a novel co llagen invasion assay model in which cancer cells and CAFs were co-cultured; we then analyzed the mechanisms governing how cancer cell invasion was promoted by PDPN(+)CAFs. We found that PDPN(+)CAFs invaded the collagen matrix to a greater extent, with more cancer cells invading within tracks created by the CAFs, compared with control CAFs. After intravenous injection in the mouse tail vein, PDPN(+)CAFs invaded and promoted cancer cellinvasion into the lung parenchyma, compared with control CAFs. Among the lung adenocarcinoma patients, we observed some cases with PDPN(+)CAFs at the invasive front of the tumor. These cases predominantly exhibited pleural invasion of cancer cells, known as pathological invasiveness. Our results indicated that PDPN(+)CAFs were tumor-promoting CAFs that lead and enhance the local invasion of cancer cells.

P271 Board Number: B406

Plant Polyphenols Reduce Migration Ability of Human Melanoma Cells. C. Jones1, C.A. Joseph1, V.S. Parmar2, A.L. DePass1; 1 Department of Biology, Long Island University, Brooklyn, NY, 2Department of Chemistry, University of Delhi, New Delhi, India Melanoma is the most serious type of malignant skin cancer due to its ability to metastasize, and appears highly resistant to many forms of cancer treatments. Plant polyphenols have a number of beneficial health effects, and are well noted for their anti-cancer properties. To develop more effective treatments for melanoma, we assessed the chemotherapeutic potential of various plant polyphenol analogs on the migration ability of A375 human melanoma cells as an in vitro model. Treatment of A375 cells with polyphenols MLN-1249, MLN-1337, and MLN-2287 resulted in inhibition of cell migration. Employing cell viability assays, the compounds resulted in dose-dependent cytotoxicity at concentrations ranging from 0 to 100 µM. Using cell invasion assays, we found that the polyphenols inhibited activity on cell migration at concentrations of 1 µM over a 72 h time period. The inhibition of cell migration correlates to the expression of genes involved in the apoptosis, NFκB, and epithelial-tomesenchymal transition pathways. Together, these results show that the polyphenols have the ability to inhibit cell migration, a crucial step of metastasis.

SUNDAY-POSTER PRESENTATIONS

P272 Board Number: B407

An Examination of Obesity-induced Epithelial to Mesenchymal Transition in Melanoma Cells. S.S. Papas1, E. Brandon2; Biology, Mississippi College, Clinton, MS, 2Biology , Mississippi College, Clinton, MS

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Recent studies have shown that obesity worsens the prognosis of several cancers, including melanoma. Evidence suggests that cytokines such as leptin and resistin, promote the growth, epithelial-tomesenchymal transition (EMT), and subsequent metastasis of melanoma tumor cells. Our research focuses on extending this understanding by discerning the connection between adipocyte’s presence and tumor cell proliferation and migration. Additionally, concentrating on the mTOR signaling pathway as a means for regulation of EMT, we seek to add detail to the otherwise vague mechanisms concerning obesity-associated metastasis of solid tumor cells. To test the hypothesis that tumor cell proliferation is increased by the presence of adipocytes, we co-cultured mouse adipocytes and melanoma cells. Control and experimental melanoma cells were counted at three time points during their treatments; comparisons of the tumor cell growth at 0, 24, and 48 hours, revealed enhanced growth as a result of adipocyte presence. Melanoma cells cultured with adipocytes grew faster than the melanoma cells cultured alone. Growth rates were significantly increased in the co-cultured melanoma cells (13,163 ± 3.22 vs. 10,307 ± 1.17 cells/mL/day, Student’s T-test p = 0.01). Additionally, preliminary data suggests that the co-cultured melanoma cells migrate more than melanoma cells cultured alone. Cells going through EMT have increased levels of vimentin and phosphorylated mTOR. Western blots will be used to determine whether the co-cultured melanoma cells follow this pattern. Adipocytes directly promote the growth of melanoma cells and migration also appears to increase as a result of co-culturing. These results support our previous finding of increased melanoma tumor growth in obese mice. This may occur because of mTOR activation, which remains to be determined. Future experiments will examine the expression of the transcription factors SNAIL, Slug, and Twist in co-cultured melanoma cells.

P273 Board Number: B408

Stiffened extracellular matrix and paracrine signaling from stromal fibroblasts synergistically combine to result in both increased tumor cell invasiveness and a change in invasive strategy in a 3-D m. J.S. McLane1, L.A. Ligon1; 1 Biology, Rensselaer Polytechnic Inst, Troy, NY Several changes have been described in the stroma surrounding a tumor, including changes in cellular composition, altered extracellular matrix (ECM) composition and organization, and increases in tissue

SUNDAY-POSTER PRESENTATIONS stiffness. Tumor cells are influenced by the composition, organization, and mechanical properties of the microenvironment, as well as by signals from stromal cells. The tumor-associated microenvironment can induce changes in tumor cells such as EMT and increased invasiveness. Here we sought to test whether the small increase in stiffness observed in the stroma surrounding a tumor is sufficient to induce tumor cell invasiveness. Further, we sought to determine if signaling from stromal fibroblasts can regulate tumor cell invasion in the stiffened matrix. In order to test the role of physiologically relevant stromal stiffness in initiating tumor invasion, we developed an in vitro model of a tumor in situ in which the stromal stiffness can be controlled independently of collagen concentration. We generated tumor spheroids with a basement membrane-like coating and then encapsulated them in a collagen-I based 3D hydrogel with or without fibroblasts in the “stroma”. We varied the stiffness of the hydrogel from that of normal breast (~200 Pa) to that of observed breast tumor adjacent stiffness (~800 Pa), a difference of less than 5-fold. Our results show a distinct change in invasive cellular behavior with increased stiffness, and a differential regulation of invasion by fibroblast signaling indicating both physical and paracrine links between the altered stroma and tumor behavior.

P274 Board Number: B409

Mechanical Confinement Triggers Glioma Linear Migration Dependent On Formins. P. MONZO1, C. Guetta1, A. Krishnasamy1, B. Ladoux1,2, M.P. Sheetz3,4; 1 Mechanobiology Institute, National University of Singapore, Singapore, Singapore, 2Institut Jacques Monod, Université Paris Diderot CNRS, Paris, France, 3Mechanobiology Institute, National University of Singapore, Singapore, NY, 4Biological Sciences, Columbia University, New York, NY Glioblastoma are extremely aggressive brain tumors characterized by their resistance to radio- and chemotherapy and their highly invasive properties. Glioblastoma cells have the remarkable capability to insinuate themselves throughout the brain. Since they migrate in a confined environment, we analyzed their movement on micropatterned lines coated with laminin to mimic the tracks that glioma cells follow when invading the brain. We found that they adopted an efficient linear migration mode upon confinement to lines. During this linear migration, antiparallel streams of cells were observed along the axis of the laminin tracks. Within these streams, individual cells adopted a spindle shape and used a 2 phase saltatory motion that depended on microtubules and contractile actin bundles, and involved paxillin-containing adhesions. Surprisingly, we found that this linear migration mode was dependent on formins but independent of Arp2/3. Finally, we found that in confined conditions glioma cells expressed a specific mDia2 isoform offering a promising target for cancer therapy development.

SUNDAY-POSTER PRESENTATIONS

P275 Board Number: B410

Co-culture with mature adipocytes promotes morphological changes and growth of breast cancer cells. Y. Lee1, J. Koo1; 1 Department of Pathology, Severance Hosp, Seoul, Korea The crosstalk between tumor cells and adjacent stroma plays a significant role in tumor growth and progression. In the breast, the main stroma cell types are fibroblasts and adipocytes. Fibroblasts are widely suggested as stimulating the invasion of breast cancer cells. Although emerging evidences suggest that adipocytes contribute to tumorigenesis in breast cancer, its mechanism remains to be elucidated. In this study, we show that mature adipocytes promote morphological changes and growth of human breast cancer cells. Co-culture of MCF-7 (luminal type) with mature adipocytes exhibited spindle-like structures without other stimuli, compared to MCF-7 cells co-cultivated with preadipocytes. Furthermore, MDA-MB-435S and MDA-MB-231 cells, well-known Triple Negative Breast Cancer (TNBC type), co-cultivated with mature adipocytes induced extensive morphologic change and incomplete EMT. However, co-culture with mature adipocytes has no effect on MDA-MB-453 (Her2 type) and MDAMB 468 (TNBC type) cells. After co-culture of MDA-MB-231 with mature adipocytes, the cancer cell upregulated mRNA and protein expression of vimentin. Five breast cancer cell lines cultured in the presence of mature adipocytes showed an increase in cell proliferation compared to cancer cell alone culture. These data suggest that mature adipocytes can promote tumor growth and metastasis by causing incomplete EMT via phenotypic change of MCF-7, MDA-MB-435S, and MDA-MB-231 cells.

Cancer Therapy 1 P276 Board Number: B411

Identification of natural products with selective activity against triple-negative breast cancer molecular subtypes. A.J. Robles1, L. Du2, S. Cai2, J.B. King2, R.H. Cichewicz2, S.L. Mooberry1,3,4; 1 Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, 2Natural Products Discovery Group, Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, 3Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, TX, 4Canter Therapy and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, TX Triple-negative breast cancers (TNBCs) are tumors lacking estrogen receptor/progesterone receptor expression and amplification of HER2. These tumors respond initially to cytotoxic chemotherapy but have a poor prognosis and drug resistance often develops. TNBCs are heterogeneous cancers without

SUNDAY-POSTER PRESENTATIONS clear molecular targets for the development of new therapies. Genetic subtyping of 587 TNBC patients by Lehman and Bauer (2011 J Clin Invest) identified 6 subtypes of TNBC with distinct genetic alterations, allowing for the first time the ability to search for targeted therapies for TNBC subtypes. High-content screening was used to evaluate 4,336 fungal and 1,392 plant extracts for selective efficacy against 5 TNBC subtypes. We identified 11 extracts with selective cytotoxic and/or antiproliferative activity against cell lines representing the different TNBC subtypes. Bioassay-guided fractionation of a fungal extract yielded a novel compound, maximiscin, which was found to have selective cytotoxic efficacy against the MDA-MB-468 cell line of the basal-like 1 subtype. Additionally, we isolated deguelin, which had selective activity in the MDA-MB-453 cells of the luminal androgen receptor (LAR) subtype. Cellular studies were conducted to investigate the molecular mechanisms of action of each compound and to potentially identify new pharmacological targets for TNBC. Initial studies showed that maximiscin caused an accumulation of cells in G1. Protein microarray studies gave a preliminary indication that maximiscin increased levels of phospho-p53, which was consistent with the observed G1 accumulation. Additionally, maximiscin caused a reduction of phospho-S6K and phospho-rS6 levels relative to vehicletreated cells after 8 h of treatment, suggesting inhibition of the mTOR signaling pathway may be involved in its mechanism of action. A small decrease in cellular levels of phospho-Stat1 and phosphoStat3 was also observed beginning 8 h after maximiscin treatment. Maximiscin induced apoptosis of MDA-MB-468 cells, but DNA double-strand breaks did not occur prior to the accumulation of cleaved PARP, suggesting that DNA damage is not the primary mechanism of action. Based on published studies of deguelin and the knowledge that LAR TNBC cells are particularly sensitive to Hsp90 inhibition, we hypothesized that deguelin selectively inhibits growth of MDA-MB-453 cells by inhibiting Hsp90 and modulating androgen receptor (AR) function. Preliminary results suggest deguelin modulates AR levels but does not directly bind Hsp90. Studies focused on further defining the mechanisms of action of each of these compounds are ongoing. These results demonstrate that novel compounds with potential therapeutic value for the treatment of TNBC subtypes can be identified from nature.

SUNDAY-POSTER PRESENTATIONS

P277 Board Number: B412

Rosehip (Rosa canina) extracts prevent MAPK and AKT-mediated cell proliferation and migration in African American triple negative breast cancer cells. P. Cagle1, T. Coburn2,3, A. Shofoluwe2, P.M. Martin2; 1 Energy and Environmental Systems, North Carolina AT State University, Greensboro, NC, 2Department of Biology, North Carolina AT State University, Greensboro, NC, 3Energy and Environmental Systems, NC AT State University, Greensboro, NC Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer, characterized by its lack of the human epidermal growth factor receptor-2 (HER-2), the estrogen receptor (ER), and the progesterone receptor (PR). The high prevalence of triple negative tumors is more commonly observed in African American (AA) women. Akt and MAPK have been shown to promote cell proliferation and migration in TNBC and MAPK expression may be an underlying mechanism contributing to the generation of chemo-resistance in triple negative breast cancer. Currently, the existing targeted therapy is of minimal benefit in TNBCs. Furthermore, adverse side effects and the emergence of drug-resistant cancer cells are of great concern. Natural products have received growing interest in recent years as an alternative medicine with potential anti-oncogenic properties. Rosehip extracts have been used as dietary supplements to relieve symptoms associated with gastrointestinal disorders and arthritis, and in our laboratory it has been shown to prevent cell proliferation in glioblastomas. This study investigated the efficacy of rosehip extracts in preventing proliferation and migration of an AA triple negative breast cancer cell line (HCC1806). HCC1806 cells treated with rosehip extracts (1mg/mL -25ng/mL) demonstrated a significant decrease in cell proliferation. The observed decrease in cell proliferation was equal to or better than the decrease of cell proliferation observed when inhibitors of the MAPK (U0126, 10 μM) or AKT (LY294002, 20 μM) signaling pathways were utilized. Rosehip extracts also demonstrate anti-migratory potential. Additionally, pretreatment of this cell line with rosehip extracts selectively decreased AKT, MAPK, p70S6K, and S6 phosphorylation suggesting these extracts prevent AA TNBC cell proliferation and migration by blocking both the MAPK and AKT signaling mechanisms. Western blot analysis and apoptosis studies demonstrate that rosehip extracts inhibit cell proliferation without promoting apoptosis. To investigate the potential clinical application of rosehip extracts we examined whether rosehip extracts could enhance the chemotherapeutic properties of Doxorubicin (20µM). Rosehip extracts enhanced the anti-proliferative effect of Doxorubicin by promoting apoptosis. These data suggest that rosehip extracts are capable of decreasing cell proliferation and migration in an AA triple negative breast cancer cell line. Moreover, rosehip extracts promote apoptosis and demonstrate a synergistic inhibition of cell proliferation, when given in combination with Doxorubicin. This investigation demonstrates that rosehip extracts may serve as either an alternative or complimentary treatment to current chemotherapeutic regimens for TNBC, especially in AA women.

SUNDAY-POSTER PRESENTATIONS

P278 Board Number: B413

Electrophilic Nitroalkenes Cause Degradation of NFκB RelA in Triple Negative Breast Cancer Cells. C. Woodcock1, S.R. Woodcock1, N. Davidson2, B.A. Freeman1, Y. Huang2; 1 Pharmacology and Chemical Biology, Univ Pittsburgh, Pittsburgh, PA, 2University of Pittsburgh Cancer Institute, Pittsburgh, PA Triple negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR) and Her2/Neu, is an aggressive and therapy-resistant metastatic cancer that accounts for up to 20% of breast cancer incidence in the US. Women with TNBC are at higher risk for early relapse within 5 years of treatment, and recurrent tumors become more aggressive and invasive. Current chemotherapy for TNBC is limited because of ill-defined targets and poor efficacy; therefore, there is an unmet need for novel therapeutic agents with improved efficacy that can also prevent disease recurrence. Electrophilic fatty acids are endogenously-generated signaling mediators that modulate cell differentiation and proliferation via post-translational modification of functionally-significant nucleophilic amino acids (Cys, His) of transcriptional regulatory proteins such as NFkB, PPARr, and Keap1/Nrf2. Preliminary studies have revealed that an exemplary electrophilic fatty acid nitroalkene, 10-nitro-oleic acid (OA-NO2), displays potential therapeutic value in TNBC cells, by preferentially reducing the growth and viability of two TNBC human breast ductal epithelial cell lines (MDA-MB-231, MDA-MB-468), but not normal MCF10A cells. Essential to proliferation and survival, NFkB plays an important role in TNBC development, and its signaling actions are constitutively activated in ER-negative breast cancer cell lines and primary tumors. Notably, the protein level of NFkB RelA/p65 subunit was diminished in TNBC cells upon OA-NO2 treatment. Therefore, it is hypothesized that electrophilic fatty acids reduce TNBC cell proliferation and survival through down-regulation of NFkB expression and signaling. We have demonstrated that fatty acid nitroalkenes induce caspase-3 activation and down-regulate transcript levels of NFkB –regulated genes, cyclin D1 and the pro-survival genes, bcl-xL and survivin, in TNBC cells. Moreover, nitro-fatty acids promote p65/RelA protein polyubiquitination via nitroalkylation of p65/RelA in TNBC cells. Overall, this study will help to extend our current knowledge of diverse electrophile functions with proteins, and also serve as a prelude to the clinical study of electrophilic nitrated lipids as therapeutic agents that may display a high selectivity for killing TNBC cells/tumors.

SUNDAY-POSTER PRESENTATIONS

P279 Board Number: B414

Differential Sensitivity of Breast Cancer Cell Lines to Diverse Microtubule Targeting Agents. A.L. Risinger1, N. Dybdal-Hargreaves1, S.L. Mooberry2,3,4; 1 Pharmacology, Univ Texas Hlth Sci Ctr-San Antonio, San Antonio, TX, 2Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, 3Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, TX, 4Canter Therapy and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, TX Microtubule targeting agents (MTAs) are some of the most important drugs used in the treatment of a wide range of malignancies. New evidence suggests that the anticancer effects of these agents are not only due to antimitotic actions, but also to disruption of interphase signaling processes that require a functional microtubule network. Microtubules play fundamental roles in cellular trafficking, metabolism and cell signaling and it is not unreasonable that in the context of an oncogene driven cancer cell, disruption of microtubule-dependent processes could inhibit essential transport and signaling, ultimately leading to cytotoxicity. If this is the case, then cell lines representing different molecular phenotypes and oncogenic drivers would be expected to be differentially sensitive to diverse MTAs that have subtly different mechanisms of action. To test this, the effects of 6 MTAs, including both microtubule stabilizers and microtubule depolymerizers, were evaluated in 8 cell lines representing the major classes of breast cancer, including triple negative subtypes. An initial observation is that there was no correlation between the cell doubling rate and sensitivity for either antiproliferative or cytotoxic effects of any of the MTAs. This discounts the notion that cell lines are differentially sensitive only by virtue of their proliferation rate. Second, profound differences among the cell lines were seen. Some are intrinsically resistant to all the drugs tested, for example HCC1937 and T47D cells, while most of the other cell lines have a wide range of sensitivities to the different agents. The MDA-MB-231 cell line is sensitive to the antiproliferative effects of each of the MTAs, yet total growth inhibition was achieved with only 2 of the 6 drugs, vinorelbine and eribulin, at concentrations up to 10 µM. Third, there was no indication of differential sensitivity across this panel to either microtubule stabilizers or destabilizers, but to individual drugs of each of these classes. For example, Hs578T cells are sensitive to eribulin, vinorelbine, paclitaxel and ixabepilone, yet resistant to docetaxel. Fourth, a variety of dose response relationships were observed, including the classic sigmoidal log dose response curves, inverted Ushaped curves, bi and triphasic curves. Together these data show that there are substantial differences among the potency and efficacy of MTAs even at the in vitro level. Our data suggest that in vitro studies can be useful to begin to understand the mechanistic differences among these agents in the context of different oncogenic signatures. These studies can assist in the elucidation of the multiple mechanisms by which disruption of microtubules can impact cell function and ultimately cell viability.

SUNDAY-POSTER PRESENTATIONS

P280 Board Number: B415

Ceramide-induced secretion of exosome-associated breast cancer resistance protein and reduction of doxorubicin resistance in human breast cancer MDAMB-231 cells. J. Kong1, Q. He2, G. Wang2, S. Dasgupta2, M.B. Dinkins2, G. Zhu1, S.D. Spassieva3, E. Bieberich2; Georgia Regents University, Augusta, GA, 2Department of Neuroscience and Regenerative Medicine, Georgia Regents University, Augusta, GA, 3Med Univ South Carolina, Charleston, SC

1

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA-MB-231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP-associated exosomes, while siRNA-mediated knockdown or GW4869-mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor-labeled breast cancer stem-like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than 5-fold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy. Supported by NIH and NSF.

P281 Board Number: B416

The Microtubule Stabilizer Taccalonolide AJ Inhibits EGFR Signaling and Transport in Breast Cancer Cells. C.C. Rohena1, N. Dybdal-Hargreaves1, S.L. Mooberry2,3,4; 1 Pharmacology, Univ Texas Hlth Sci Ctr-San Antonio, San Antonio, TX, 2Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, 3Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, TX, 4Canter Therapy and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, TX Breast cancer is the second most diagnosed cancer in women, with millions of women suffering from the disease worldwide. Microtubule stabilizers are some of the most important drugs used in the

SUNDAY-POSTER PRESENTATIONS treatment of breast cancer. The taxanes, paclitaxel and docetaxel are some of the most valuable agents used in the treatment of breast cancer. However, there remains a need for new microtubule stabilizers that can overcome some of the limitations of the taxanes. The taccalonolides are a class of microtubule stabilizers isolated from plants of the genus Tacca. The taccalonolides bind covalently to microtubules, have the ability to overcome drug resistance due to the overexpression of PgP, and have shown excellent activity in paclitaxel resistant xenograft models. Recent evidence suggests that the ability of microtubule targeting agents (MTAs) to interrupt microtubule-dependent functions in interphase cells contributes to their anticancer actions. The specific signaling events interrupted by MTAs and mechanistic differences among MTAs are only beginning to be investigated. EGFR is overexpressed in 40% of breast cancers and in triple negative breast cancers EGFR overexpression occurs in 75% of cases. Following ligand binding, activated EGFR is internalized and transported to the endosome, events which are dependent on microtubules.The effects of taccalonolide AJ and paclitaxel on microtubule-dependent EGFR trafficking were evaluated in a panel of breast cancer cell lines, including BT549, MDA-MB-468 and T47D. Our results show that taccalonolide AJ and paclitaxel inhibit EGFR internalization causing the receptor to be retained at the cell periphery following ligand stimulation. This impaired trafficking inhibited the phosphorylation of EGFR’s downstream targets, as evidenced by lower levels of ERK1/2, AKT and STAT3 phosphorylation. Surprisingly, taccalonolide AJ was much more effective at inhibiting the phosphorylation of these downstream targets. These data suggest that microtubule stabilizers can modulate EGFR pathways in distinct ways. Identification of the specific signaling pathways and downstream targets interrupted by the different MTAs might identify new rational drug combinations. This has the potential to identify patient populations that would benefit from different MTAs based on the specific signaling defects in their tumors and thus better responses. Our study evaluating differences among microtubule stabilizers begins to shed some light into the complex and subtly different mechanisms of action of these drugs.

P282 Board Number: B417

Reishi sensitizes EGFR-overexpressing inflammatory breast cancer cells to the tyrosine kinase inhibitor erlotinib. I.J. Suarez-Arroyo1, G. Maldonado-Martinez1, L.A. Cubano1, M. Martinez-Montemayor2; 1 Univ Central Del Caribe - Sch Med, Bayamon, PR, 2Univ Central Del Caribe-Sch Med, Bayamon, PR Inflammatory Breast Cancer (IBC) is the most aggressive, and lethal type of breast cancer. Its lethality stems from its unique ability of forming tumor spheroids, which invade the dermal lymphatics causing the inflammatory phenotype. Within these spheroids, in IBC cell lines and human tissues, the Epidermal Growth Factor Receptor (EGFR) is overexpressed and it is associated with increasing IBC tumor growth rates, invasion and metastasis. Moreover, the high incidence of resistance to EGFR Tyrosine Kinase Inhibitors (TKIs) has greatly diminished the effectiveness of this chemotherapeutic drug. Thus, we aimed to investigate Reishi’s therapeutic potential in IBC, focusing on the regulation of the EGFR signaling cascade and its contribution to the IBC cellular response when treated in combination with erlotinib.

SUNDAY-POSTER PRESENTATIONS SUM-149 IBC cells were treated with various concentrations of erlotinib, Reishi, or their combination for 72h. In order to study the molecular mechanism for the improved effects of the combination treatment we successfully developed an IBC erlotinib resistant cell line, rSUM-149 cells, which were also treated with erlotinib, Reishi or their combination. Treatment effects were tested on IBC cell viability, invasion via Transwell Invasion Assays, cell migration using FluoroBlok™ Cell Culture Inserts, tumor spheroid formation via three-dimensional cell culture, and immunoblots. Statistical analyses include a One-way ANOVA with post-hoc test using the Dunnett’s multiple comparisons estimator and p was set at ≤0.05. Our results show that Reishi sensitizes parental and resistant SUM-149 cells to erlotinib treatment after 72h (ANOVA, F= 104.1; p

P283 Board Number: B418

Ganoderma lucidum decreases the phosphorylation of STAT-3 in in vitro and in vivo inflammatory breast cancer models. Y. Loperena1, L.A. Cubano2, M. Martinez-Montemayor3; 1 Univ Central Del Caribe-Sch Med, Bayamón, PR, 2Univ Central Del Caribe - Sch Med, Bayamon, PR, 3Univ Central Del Caribe-Sch Med, Bayamon, PR Inflammatory Breast Cancer (IBC) is an aggressive form of breast cancer (BC) with symptoms that include a diffuse redness and swelling of the breast. Unique pathological findings indicate the presence of tumor emboli invading the dermal lymphatics of the breast; a process that is partly responsible for the inflammatory phenotype. However, these tumors produce inflammatory mediators such interleukin-6 (IL-6), which may act as a growth factor to contribute to cancer progression, invasion and metastasis via the activation of the Janus Kinase-2/Signal Transducer and Activator of Transcription 3 (STAT-3) pathway. Our published data demonstrates that the medicinal mushroom, Ganoderma lucidum (Reishi), disintegrates tumor emboli and modulates the abundance of proteins involved in invasion cascades. In the current study, we assessed the effects of Reishi on inflammatory markers focusing on the IL-6/JAK2/STAT-3 pathway both in vitro and in vivo. SUM-149 IBC and SUM-102 non-IBC BC cells were treated with vehicle, or Reishi at different time points to determine cancer cell viability, invasion via threedimensional culture assays and protein abundance via immunoblots. In vivo studies were carried out in female severe combined immunodeficient (SCID) mice that were treated with vehicle or 7mg/kgBW, 14mg/kgBW and 28mg/kgBW of Reishi for 2wk. SCID mice were then injected with SUM-149 IBC cells in their lower right mammary fat pad and Reishi treatment was continuously administered for 12 wks. Our data from the in vitro study suggests that Reishi reduces cancer cell viability, and Reishi has an immunomodulatory role in SUM-149 IBC cell line, demonstrated by its ability to reduce the secretion of IL-6 and reduced phosphorylation of STAT-3 in SUM-149 cells. Cell invasion studies show that Reishi reduces invasion in IBC cells. Interestingly, Reishi reduces SUM-102 BC cell viability; however it does not affect STAT-3 signaling in the cell line. Our in vivo results show that Reishi does not change tumor volume, when mice where treated with any concentrations of Reishi. However, the phosphorylation of STAT-3 in tumor lysates significantly decreases in a concentration dependent manner. Future efforts are

SUNDAY-POSTER PRESENTATIONS focused on understanding the invasion effects on the SUM-102 non-IBC BC cell line, and on the implications of STAT3 inactivation in IBC. This project was sponsored by Title V PPOHA US Department of Education #P031M105050, NIH/NIMHD # 8G12 MD 007583, NIH/NIGMS #P20 GM103475, NIH/NIMHD U54 MD008149.

P284 Board Number: B419

The Rac Inhibitor EHop-016 Reduces Mammary Tumor Growth, Metastasis, and Angiogenesis. L. Castillo-Pichardo1,2, T. Humphries-Bickley1, C. de la Parra3, E. Hernandez3, L.A. Cubano4, J. RodriguezOrengo3, S.F. Dharmawardhane3; 1 Biochemistry, University of Puerto Rico Medical Sciences Campus, San Juan, PR, 2Pathology and Laboratory Medicine, Universidad Central del Caribe, Bayamon, PR, 3University of Puerto Rico Medical Sciences Campus, San Juan, PR, 4Anatomy and Cell Biology, Universidad Central del Caribe, Bayamon, PR The Rho family GTPases Rac and Cdc42 play pivotal roles in actin cytoskeleton reorganization during migration/invasion and thus, metastasis; as well as epithelial to mesenchymal transition, transcription, cell proliferation, cell cycle progression, apoptosis, vesicle dynamics, angiogenesis, and cell adhesions. In many human cancers, Rac proteins are hyperactive due to the upregulation of their upstream effectors, guanine nucleotide exchange factors (GEFs). Therefore, we have characterized a series of small molecules designed to inhibit the interaction of Rac and Cdc42 with their GEFs. We reported the development of EHop-016, a small molecule compound, which inhibits Rac1 and Rac3 activities of metastatic cancer cells with an IC50 of 1 μM, and blocks the interaction of Racs with the oncogene and Rac and Cdc42 GEF Vav. EHop-016 also inhibits the activity of Cdc42 at higher concentrations, the Rac downstream effector p21-activated kinase (PAK), lamellipodia extension, migration, and viability of metastatic breast cancer cells. In this study, we further investigated the in vitro and in vivo action of EHop-016 in metastatic cancer, and report that EHop-016 inhibits the Rac activities of breast and prostate cancer cells, and human umbilical vein endothelial cells (HUVEC). EHop-016 may inhibit metastatic cancer cell viability by downregulating Akt and Jun kinase activities and c-Myc and Cyclin D expression, as well as increasing caspase 3/7 activities. Next, we tested the efficacy of EHop-016 in a nude mouse model of experimental metastasis, where female athymic nude mice bearing mammary fat pad tumors established from GFP-MDA-MB-435 cells were treated with 0, 5, 10, 25, or 40 mg/kg body weight (BW) EHop-016. Tumor growth and metastasis were quantified using fluorescence in vivo image analysis. We found that at 25 mg/kg BW, EHop-016 significantly reduces mammary fat pad tumor growth, metastasis, and angiogenesis. As quantified by ultra high pressure liquid chromatography (UPLC)/ mass spectrometry (MS/MS), EHop-016 was detectable in the plasma of nude mice at 17-23 ng/mL levels at 12 h following intraperitoneal (i.p.) administration of 10-25 mg/kg BW EHop-016. The EHop-016 mediated inhibition of angiogenesis in vivo was confirmed by in vitro tube formation assays of HUVECs. In conclusion, EHop-016 has potential as an anticancer compound to inhibit cancer progression

SUNDAY-POSTER PRESENTATIONS via multiple Rac-directed mechanisms. Moreover, EHop-016 is a useful tool to study Rac function in human disease in vitro and in vivo.

P285 Board Number: B420

Inhibition of thromboxane pathway, a promising new treatment against lung cancer. G.G. Chen1, Y. Liu1, R. Huang1, M. Li1, M. Underwood1; 1 Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong The efficacy of available treatments for the majority of lung cancer is still far from satisfactory, mainly due to lack of good therapeutic targets or severe side-effects. Recently, thromboxane (TX) pathway has been demonstrated to play a role in lung cancer development. Since the TX pathway is downstream of cyclooxygenase (COX), the inhibition of relevant targets in the TX pathway can avoid affecting the entire COX system, and thus improve the specificity of the treatment and reduce side-effects. In this study, we tested the TX inhibitor, and the TX receptor (TXR) blocker in lung cancer cells to check their effects on tumor cell proliferation, growth and apoptosis. A smoking carcinogen NNK-induced mouse lung tumor model was established to determine the effect of these inhibitors on the growth of lung tumor in vivo, and to examine the possible toxic effects of these treatments on liver and renal functions. Our results showed that TX was able to function as a critical mediator for tumor-promoting effects of COX-2 in lung cancer. Both TX specific inhibitors and TXR blockers could completely block NNK-mediated cell proliferation via inducing apoptosis. TXR agonist U46619 reconstituted a near full proliferative response to NNK when TXS was inhibited, affirming the role of TXR in tumor cell proliferation and growth. However, the dual TX inhibitor BM567 which inhibits both TX and TXR did not offer better suppression, compared with single TX inhibitor or TXR blocker, suggesting that TX and TXR were likely to function in a same channel to promote the tumor growth. The lung tumor model showed that the TX inhibitor led to the significant arrest of the tumor growth. Furthermore, TX inhibitors neither affected the daily activity of mice, nor caused damages to liver and kidney. In conclusion, the inhibition of TX pathway can lead to the significant suppression of lung tumor in vitro and in vivo without producing significant side-effects.

SUNDAY-POSTER PRESENTATIONS

P286 Board Number: B421

Anti-Mullerian Hormone (AMH) and its Receptor AMHRII provide a TGF-βAssociated Pro-Survival Signaling Loop in Lung Cancer. T.N. Beck1, E. Nicolas2, Y. Zhou3, I. Serebriiskii4, E.A. Golemis4; 1 Molecular and Cell Biology and Genetics, Drexel University/Fox Chase Cancer Center, Philadelphia, PA, 2 Genotyping and Real-Time PCR, Fox Chase Cancer Center, Philadelphia, PA, 3Epidemiology and Biostatistics, Fox Chase Cancer Center, Philadelphia, PA, 4Developmental Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA Signaling changes associated with epithelial-mesenchymal transition (EMT) affect prognosis and response to survival in lung cancer. The molecular chaperone heat shock protein 90 (HSP90) supports the activity of many protein clients associated with lung cancer pathogenesis including ERBB2, MET, BRAF and TGFBR1. Because of the dependence of cancer cells on HSP90 function, HSP90 inhibitors such as ganetespib selectively kill cancer cells, and are showing promising activity in clinical trials. We used RNAi screening to identify genes influencing lung cancer cell resistance to ganetespib. Depletion of AntiMullerian Hormone (AMH) and its receptor, AMH receptor type II (AMHR2), strongly sensitized cells to ganetespib in three RAS-mutated and one EML4-ALK expressing lung cancer cell lines. This was surprising, as neither the expression of AMH nor its receptor has ever been reported in lung cancer. Instead, almost all studies of AMH to date have focused on its role in differentiation of gonadal tissue, where it activates a cell surface receptor upstream of the SMAD genes, which are common effectors in the lung cancer-relevant TGF-ß cascade, which controls EMT. Using multiple approaches, we have experimentally confirmed the expression of AMH and surface expression of AMHR2 in lung cancer cell lines, and mining of public databanks suggests that both proteins are expressed in a subset of lung cancer samples and cell lines. Surprisingly, we observed that depletion of AMH and AMHR2 induced EMT-like features, including downregulation of cadherins and assumption of a mesenchymal morphology. Conversely, ganetespib treatment increased epithelial features. These findings suggest that resistance to HSP90 inhibition may differ from resistance to traditional chemotherapeutics. In line with this hypothesis, we found that depletion of AMH and AMHR2 did not sensitize cells to cisplatin, while depletion of E-cadherin sensitized cells to HSP90 inhibition. Mechanistically, AMH and AMHR2 depletion depressed activity of NF-kB and AKT, both critical EMT and survival regulators. These results for the first time indicate the presence of an AMH-AMHR2-NFkB-AKT pro-survival signaling axis of therapeutic relevance in lung cancer. Further, our results also suggest that AMH, AMHR2 and EMTassociated proteins may serve as biomarkers to predict resistance to HSP90 inhibitors, and that it may be more beneficial to prime lung tumors with HSP90 inhibitors, to induce MET and decrease survival signaling, prior to treatment with chemotherapy.

SUNDAY-POSTER PRESENTATIONS

P287 Board Number: B422

Molecular mechanisms underlying lung cancer progression. J. Hooda1, D. Cadinu1, M. Alam2, A. Shah2, T.M. Cao2, R. Brekken3, L. Zhang1; 1 Molecular and Cell Biology, Univ Texas-Dallas, Richardson, TX, 2Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX, 3Division of Surgical Oncology, University of Texas Southwestern Medical Center, Dallas, TX Altered metabolism is a hallmark of the transformed state in cancer progression. Recent studies have uncovered a plethora of metabolic alterations in cancer cells. One important finding is that mitochondrial respiration is amplified in certain human cancer cells. Recently, using a matched pair of cell lines representing normal nonmalignant HBEC30KT and non-small-cell lung cancer (NSCLC) HCC4017 cells developed from the same patient, we identified metabolic changes linked with the transformation of normal to cancer cells. We found that oxygen consumption and heme synthesis were intensified significantly in lung cancer cells, compared to the normal cells. Furthermore, the levels of heme uptake proteins and oxygen-utilizing hemoproteins were dramatically increased in cancer cells and xenograft tumors. Interestingly, we found that inhibition of heme synthesis or mitochondrial function preferentially suppressed cancer cell proliferation, colony formation and cell migration. These results demonstrated that heme availability is significantly increased in cancer cells and tumors, which leads to elevated production of hemoproteins, resulting in intensified oxygen consumption and cellular energy production for fueling cancer cell progression. Further, we also found that lowering heme availability sensitized NSCLC cells to paclitaxel. We measured the oxygen consumption of 6 NSCLC cell lines in the absence of 3 key metabolites individually and found that the levels of oxygen consumption increased in all the cell lines in the absence of glucose, while it decreased in the absence of glycine or glutamine in 4 of the cell lines. Experiments are currently underway to elucidate the roles of heme in cancer cell bioenergetics and function under different metabolic conditions.

P288 Board Number: B423

Antitumor studies of Modified L– Penetratin Peptide as a molecule targeting E2F in Prostate Cancer. T. Shaik1, N. Bansal1, N.J. Farley1, J. Kerrigan2, E.E. Abali3, D. Banerjee1, J.R. Bertino1; 1 Department of Medicine and Pharmacology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, 2Department of Cancer Pharmacology and Preclinical Therapeutics, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, 3Department of Biochemistry and Molecular Biology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ BACKGROUND: Mutation or inactivation of the retinoblastoma protein (pRB) is frequently involved in castrate resistant prostate cancer tumorigenesis resulting in overexpression/deregulation of E2F activity. E2F1-3a overexpression induces genes involved in DNA synthesis and leads to abnormal cellular

SUNDAY-POSTER PRESENTATIONS proliferation, tumor growth and invasion. Therefore, inhibiting the overexpression of one or more activating E2Fs is a recognized target in cancer therapeutics. In our previous studies we showed that a novel penetratin coupled 7-mer peptide (PEP), bound tightly to an immobilized consensus E2F1 promoter sequence, was cytotoxic to many malignant cell lines including Burkitt lymphoma cells, pRB negative small cell lung cancer cells and DU145 prostate cancer cells. Furthermore, treatment of tumor xenografts of DU145 tumors propagated in mice with a PEGylated liposome encapsulated PEP caused tumor regression; but tumor growth resumed when the drug was stopped. OBJECTIVE: As the PEP was unstable in serum, we examined the antitumor activity and stability of two different modified penetratin peptides – 1. D- Arg penetratin peptide (substituting L- Arginine with DArginine in the peptide sequence); 2. N-acetylated, C-methylated penetratin peptide (end capping the peptide). METHODS: DU145 (prostate cancer), H196 and H82 (small cell lung cancer) cells were used. To compare the efficacy of the peptides, we tested the IC50s of peptides at different time points in various cell lines by MTS assay. Stability tests were performed by incubating the peptides in RPMI media containing 10% FBS for 24 hours and then measuring cell viability using MTS assay. Drug combination experiment results were analyzed using the combination index method developed by Chou and Talalay. RESULTS: D- Arg penetratin peptide was more potent compared to L- Arg penetratin peptide and it was found to be more resistant to degradation by serum proteases than the L- form. The other modified form, N-acetylated, C-methylated PEP was marginally more effective than the unmodified PEP. Drug combination studies showed that D- Arg penetratin peptide in combination with docetaxel, caused additive cytotoxicity against DU 145 cells. Our findings validate D- Arg peptide, an inhibitor of E2F1, as a drug for targeted molecular therapy of prostate cancers with elevated levels of activated E2F’s; and encourage future modifications of the peptide that include all D- amino acid substituted peptide. Acknowledgments: Supported by Lung Cancer Research Foundation and H82 cells were given by Dr. Piyali Das Gupta, Marshall University, West Virginia.

P289 Board Number: B424

Study of the Effects of Lysophosphatidic Acid on Lymphatic Metastasis of Prostate Cancer. W. Chen1, Y. Lin1, K. Lin1, C. Lin1, P. Wu1, H. Lee1; 1 Life science, National Taiwan University, Taipei, Taiwan Clinical evidences suggested that lymphangiogenesis and lymphatic metastasis are important processes during the progression of prostate cancer. Vascular endothelial growth factor (VEGF)-C was shown to be a key regulator during these processes. Our previous studies demonstrated that lysophosphatidic acid (LPA) enhances VEGF-C expression in human endothelial cells. However, the effects of LPA on VEGF-C expression in prostate cancer remain elusive. We demonstrated that LPA enhanced VEGF-C expression

SUNDAY-POSTER PRESENTATIONS in three different human prostate cancer cell lines. Moreover, the enhancement effect is mediated through activating LPA1/3, reactive oxygen species (ROS) generation, and action of lens epithelium derived growth factor (LEDGF). Furthermore, PC-3 cells were injected in nude mice followed by LPA1/3 antagonist Ki16425 treatment to evaluate the effects of chemical inhibition on LPA receptor in blocking lymphatic metastasis of prostate cancer. We monitored the endothelial and lymphatic markers expression in the xenografts by immunochemistry stainings. We demonstrated that Ki16425 treatment reduced lymphatic markers expression in tumors. These results may potentially lead to future strategies for preventing lymphatic metastasis of prostate cancer.

P290 Board Number: B425

Systematic interrogation of druggable pathways in pancreatic adenocarcinoma. E. Ferrer-Lorenzo1, D.P. Nussbaum1, P. Winter1, K. Wood1; 1 Pharmacology and Cancer Biology, Duke University, Durham, NC Despite significant advances in the development of cancer treatment in the last 20 years the best available therapies have marginally improved the survival rate of treated pancreatic cancer patients (from 3% to 6% five-year survival), making it still one the deadliest malignancies. Single standard of care cytotoxic and targeted therapies demonstrate little to no effect on patient survival. Even though we now understand some of the main mutated genetic factors present in pancreatic tumors, their role in drug response remains poorly understood. To identify drug-sensitizing genetic targets in cancer cells we have developed a high-throughput screening platform. In this methodology, pooled lentiviral libraries containing hundreds of knockout constructs specific for cancer-related genes are used to infect cancer cells. By treating the infected populations with therapeutic agents we are able to identify genetic targets that when inhibited synergize with existing drugs to cause increased cell death. We piloted this type of screening platform in HCT-116 cells, a KRAS mutant, MEK inhibitor resistant colorectal cancer cell line, using the MEK-inhibitor AZD-2644 and a pooled shRNA library. This study identified 4-7 target genes that when knocked down sensitized the previously resistant HCT-116 cells to AZD-2644. To conduct a more robust and representative sensitization screen we developed a lentiviral CRISPR-Cas9 library. By performing screens in a panel of pancreatic cancer cell lines, we have identified both cell line dependencies and sensitizers to targeted and cytotoxic chemotherapies. The findings from these screens provide potential mechanistic insights into the key survival signaling programs in pancreatic cancer as well as new therapeutic strategies.

SUNDAY-POSTER PRESENTATIONS

P291 Board Number: B426

Treatment of pancreatic cancer cell lines with high-affinity galectin-3 inhibitor further confirms novel intracellular function of galectin-3 and implications in anticancer therapy. J. Stegmayr1,2, G. Sharma1, M. Carlsson1,3, S. Oredsson2, U.J. Nilsson4, H. Leffler1; 1 Department of Laboratory Medicine, Lund University, Lund, Sweden, 2Department of Biology, Lund University, Lund, Sweden, 3Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark, 4Department of Chemistry, Lund University, Lund, Sweden Galectin-3, a protein with the affinity for β-galactosides, has been implicated in a number of cancerrelated functions. In pancreatic cancer cell lines, for example, galectin-3 knockdown results in decreased proliferation and migration, and increased gemcitabine-induced apoptosis. Surprisingly, treatment of the pancreatic cancer cell lines MIA PaCa-2 and PanC-1 with a novel high-affinity (Kd in the low nM range) galectin-3 antagonist, bis-{3,3’-deoxy-3,3’-[4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl]}-1,1’sulfanediyl-di-β-D-galactopyranoside, had no effect on cell proliferation or gemcitabine-induced cell death. The lack of effect was not due to insufficient intracellular accessibility of the galectin-3 antagonist, because it efficiently inhibited accumulation of galectin-3 around glycyl-L-phenylalanine 2naphthylamide (GPN)-damaged lysosomes, a phenomenon known to depend on the carbohydratebinding activity of cytosolic galectin-3. The inhibition of galectin-3 accumulation around GPN-damaged lysosomes by the antagonist was seen to be both time- and dose-dependent, as investigated by quantifying galectin-3 puncta formation using immunocytochemistry and confocal microscopy. This indirectly suggests that the proliferation promoting and apoptosis inhibiting effects of galectin-3 may not require its carbohydrate-binding activity. Additionally, treatment of both of the pancreatic cancer cell lines with GPN in combination with the galectin-3 antagonist led to an increased reduction in cell viability compared to treatment with GPN alone, suggesting a protective role of galectin-3 after lysosomal insult. Taken together these results further endorse a novel intracellular function of cytosolic galectin-3, which is to accumulate around and protect lysosomes upon damage, which in turn may render galectin-3 antagonist interesting agents in anti-cancer therapy.

SUNDAY-POSTER PRESENTATIONS

P292 Board Number: B427

Pharmacological activation of myosin II to correct pancreatic cancer cell mechanics. A. Surcel1, Q. Zhu2, E.S. Schiffhauer1, H. West-Foyle1, R.A. Anders2, D. Robinson1,3; 1 Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, 2Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 3Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD Pancreatic ductal adenocarcinoma (PDAC) annually affects 44,000 people in the U.S. and has an abysmal five-year survival rate of around 6%, which is nearly unchanged over the past 40 years. Pharmacological strategies for treating cancer have primarily focused on inhibiting cell growth through specific genetic pathways, which typically either fail to abolish the disease or lead to compensatory regulatory changes and subsequently, to drug resistance. Importantly, alterations in mechanical properties are a common feature of cancer cells, yet targeting cell mechanics remains an under-utilized approach for drug development. Here we develop a system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators in Dictyostelium discoideum. We characterized 4-hydroxyacetophenone (4-HAP), which increases the cellular cortical tension by enhancing the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation regulation. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke. We further establish that changes in key cytoskeletal protein distributions correlate with the changes in the biomechanical profile of PDAC progression. In addition to actin-crosslinkers, we detect that non-muscle myosin II distributions vary across PDAC states: specifically myosin IIA increases, myosin IIB decreases, and myosin IIC increases in metastatic cells. We further demonstrate that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP. Tests of 4-HAP in mouse models of metastatic pancreatic disease are underway. Overall, 4-HAP modifies non-muscle myosin II-based cell mechanics across phyla and disease states and provides proof-of-concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior.

P293 Board Number: B428

Modulation of the Unfolded Protein Response in Multiple Myeloma. J. Thomas1, y. argon2; 1 children's hospital of philadelphia, Philadelphia, PA, 2children's hospital of philadelphia, philadelphia, PA Multiple Myeloma (MM) is the second most common hematological malignancy in the U.S.A. with over 20,000 new cases reported each year and a life expectancy in the range of 3-6 years. MM is

SUNDAY-POSTER PRESENTATIONS characterized by increased numbers of bone marrow plasma cells that secrete copious quantities of antibodies. The IRE1 kinase/ribonuclease arm of the unfolded protein response (UPR) is activated in MM cells and is necessary for their ability to sustain antibody production. The UPR is initially cytoprotective in that it alleviates ER stress that results from this sustained antibody production. However, IRE1 activity can be modulated to promote apoptosis through both the JNK and regulated IRE1-dependent decay of mRNAs (RIDD) pathways. The clinical proteasome inhibitor Bortozomib (Bz) is the most common drug used to treat MM. Since IRE1 activity is transient, the progression of MM that is characterized by Bz resistant clones under selection pressure may occur due to eventual attenuation of IRE1. Therefore, a single-cell assay method to detect and tract IRE1 activity in Bz resistant clones will be invaluable in understanding how to target therapy resistance. Our lab is developing a fluorescent protein reporter that will be able to identify individual MM cells and determine if a correlation exists between Bz resistance and attenuation of IRE1 activity. Our way IRE1 activity can be attenuated in Bz resistant clones is through a new modulator of IRE1 that was discovered in the Argon lab: the ER enzyme PDIA6, which binds to IRE1 and terminates its XBP1s activity. We are interested in determining if knockdown of PDIA6 in multiple myeloma will increase sensitivity to Bz due to prolonged activation of IRE1. The ability to identify Bz resistant clones, and modulate their IRE1 activity could be a novel therapeutic approach to treat MM.

P294 Board Number: B429

Extracellular matrix mechanics causes systematic variation in cancer cell proliferation and responses to drugs against myeloid leukemias. J. Shin1,2, D.J. Mooney1,2; 1 School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 2The Wyss Institute for Biologically Inspired Engineering, Boston, MA Emerging evidence pinpoints the bone marrow (BM) milieu as a cell extrinsic contributor to the pathophysiology and chemotherapeutic resistance of cancer. Cancer progression is frequently associated with profound alterations in the BM microenvironment by collagen deposition that leads to fibrosis and changes in mechanical properties of the matrix. While the relevance of extracellular matrix mechanics in solid tumors has been investigated, its role in causing the progression and drug resistance of blood cancers or “liquid tumors” remains unclear. Myeloid leukemias consist of a number of disease subtypes defined by distinct genetic mutations and constitutive activation of signaling pathways. Some of these pathways, including Protein Kinase B (AKT), are involved in mechanotransduction, and hence may influence sensitivities of leukemia cells to matrix stiffness. The study is thus based on the hypothesis that matrix stiffness differentially regulates cell proliferation of myeloid leukemia subtypes. By culturing cells in an engineered 3D hydrogel system with tunable stiffness and independent control of ligand density, we demonstrate distinct ex vivo cell proliferation profiles and colony growth patterns in response to matrix mechanics for different leukemia subtypes, including human acute and chronic myeloid leukemias (AML and CML). Importantly, ex vivo growth of leukemia cells in this system reflects

SUNDAY-POSTER PRESENTATIONS in vivo growth observed in a xenograft human myeloid sarcoma model, which is generated by subcutaneously implanting leukemia cells encapsulated in the hydrogel with different stiffness. Varying ligand density further modulates leukemia cell proliferation. Interestingly, CML cells are resistant to an AKT inhibitor in the 3D hydrogel compared to the standard plastic culture, while AML cells are generally responsive to the treatment. Further studies reveal that CML cells in the viscous matrix are more resistant to existing drugs (e.g. Imatinib) than those in the solid matrix. Drug screening studies show that different small molecules can be classified based on pharmacodynamics and cell proliferation kinetics profiles of CML cells in different matrix mechanics. Using this approach, we have identified a subset of drugs that may be useful to inhibit CML cell proliferation regardless of matrix mechanics. The results suggest a potential approach to design a drug regimen to reduce the probability of minimal residual disease that could be present in different microenvironments, which can be tailored to individual blood cancer subtypes prior to administration.

P295 Board Number: B430

Blockage of prion protein-HOP engagement impairs glioblastoma growth and improves overall survival. M.H. Lopes1, T.G. Santos2, B.R. Rodrigues2, V.R. Martins2; 1 Cell and Developmental Biology, University of Sao Paulo, Sao Paulo, Brazil, 2Cellular and Molecular Biology, A C Camargo Cancer Center, Sao Paulo, Brazil Glioblastomas (GBMs) are resistant to current therapy protocols and identification of molecules that target these tumors is crucial. Interaction of secreted heat-shock protein 70 (Hsp70)–Hsp90-organizing protein (HOP) with cellular prion protein (PrPC) triggers a large number of trophic effects in the nervous system. We found that both PrPC and HOP are highly expressed in human GBM samples relative to nontumoral tissue or astrocytoma grades I–III. High levels of PrPC and HOP were associated with greater GBM proliferation and lower patient survival. HOP–PrPC binding increased GBM proliferation in vitro via phosphatidylinositide 3-kinase and extracellular-signal-regulated kinase pathways, and a HOP peptide mimicking the PrPC binding site (HOP230–245) abrogates this effect. PrPC knockdown impaired tumor growth and increased survival of mice with tumors. In mice, intratumor delivery of HOP230–245 peptide impaired proliferation and promoted apoptosis of GBM cells. In addition, treatment with HOP230–245 peptide inhibited tumor growth, maintained cognitive performance and improved survival. Thus, together, the present results indicate that interfering with PrPC–HOP engagement is a promising approach for GBM therapy.

SUNDAY-POSTER PRESENTATIONS

P296 Board Number: B431

Effect of MiR-26a on Human Malignant Melanoma Cells. G. Yang1, G. Gao1, J. Jiang1, Y. Yang1; 1 Emporia State University, Emporia, KS Objective: The goal of this study is to examine the effect of Let-7a on the proliferation and invasiveness of human malignant melanoma cells and identify the gene(s) that Let-7a targets to affect the growth and metastasis of melanoma cells. Methods: The effect of Let-7a on proliferation, migration and invasion was measured by cell viability assay and Wounding healing assays, and Transwell migration assay, respectively. The expressions of MITF, and MAP4K3 genes at protein level were measured by Western blot. Cell cycle progression was determined by flow cytometer. Results: Let-7a displayed a significant (p 30,000 genes. We found reduced activity of the p53 interactome is associated with progressive disease at two years and elevated betatubulin class III was linked to a trend for disease-free survival over the same time period.

P298 Board Number: B433

Characterization of particle-endothelium interaction using particles functionalized with dual antibodies in a complex synthetic microvascular network. Y. Tang1,2, G. Lamberti3, M.F. Kiani4, B. Wang1,2; 1 Temple University, Philadelphia, PA, 2Biomedical Engineering, Widener University, Chester, PA, 3 Department of Mechanical Engineering, Temple University, Philadelphia, PA, 4College of Engineering, Temple University, Philadelphia, PA The interaction between leukocytes and receptors on the endothelium play a key role in the early response to tissue injury. Research has shown that drug carrier particle rolling velocity increases dramatically with the increase of carrier size which affects their final attachment efficiency. The objective of this study is to characterize the rolling and adhesion profile of different size functionalized particles in a synthetic microvascular network (SMN). Functionalized particles were prepared by coating their surfaces with different ratios of antibodies against adhesion molecules ICAM-1 and E-selectin. Our modified Geographic Information System (GIS) approach was used to digitize the microvascular networks and generate the SMN on PDMS. The rolling velocity and adhesion profile of functionalized 2 & 10 µm particles was quantified in the SMN precoated with TNF-α activated human umbilical vein endothelial cells under shear conditions from 0 – 280 sec-1. Single antibody coated 10 µm particles roll at a higher speed than its 2 µm counterparts. Particle adhesion increased significantly with decreasing shear in SMNs. Increasing particle size from 2 µm to 10 µm did not change its binding efficiency significantly in SMNs, which is different from what have been reported in the traditional parallel plate flow channel. Particles functionalized with both a rolling molecule and an adhesion particle can more realistically mimic the leukocyte-endothelium interaction in a SMN. These findings have important implications for better understanding the mechanisms behind inflammatory pathologies and for optimizing the design of carriers for targeted drug delivery.

SUNDAY-POSTER PRESENTATIONS

P299 Board Number: B434

The Effect of Androgen on Cell Proliferation in Prostate Cancer In Vitro Models. S. Turner1, D. Lamb2; 1 Department of Biology, Prairie View AM University, Prairie View, TX, 2Departments of Urology and Molecular Cellular Biology. , Baylor College of Medicine, Houston, TX Prostate Cancer (PCa) is the second most common cancer amongst older men, and one of six men is diagnosed with PCa in their lifetime. Of note, one in 30 men diagnosed with PCa will succumb to their disease. African-American males and men over the age of 50 are at an increased risk. PCa has a multifactorial etiology. Typically, PCa is a slow growing cancer; most men will never know they have the disease. During the early stages of PCa, men experience few to no symptoms. Currently, 85 percent of American men are diagnosed with PCa in its early stages, which are treatable, allowing these men to carry out a moderately normal life. Unlike other cancers, victims of PCa will die due unrelated causes. The growth of PCa is androgen responsive. The effect of androgens, such as testosterone (T) and dihydryotestosterone (DHT) is mediated through its interactions with the androgen receptor (AR), which plays crucial roles in cell proliferation and differentiation,(processes important for prostate development and PCa, respectively). AR activity is associated with disease progression and therefore, the men with advanced disease are treated with androgen ablation therapy. For this study, human PCa cell lines, LNCaP cellsand PC3, which lack androgen receptors, were used. LNCaP are androgen responsive while PC3 are androgen insensitive. R1881 is a synthetic androgen used to study the effects of androgen action. Studies in LNCaP cells show a bimodal effect of androgens on cell proliferation. Low concentrations (10nM) inhibit proliferation. The purpose of this project was to examine the effect of several concentrations of R1881 on cell proliferation in LNCaP and PC3 cells using several cell quantification techniques. These included counting using the vi-cell, a trypan blue cell analyzer, a Hemocytometer, and a WST (tetrazolium salt viability) assay to determine cell proliferation for a range of days. It was hypothesized that the growth of LNCAP cells at higher concentrations of R1881 would be inhibited; however PC3 cells would not be inhibited. Using the aforementioned techniques, it was found that more than 10nM of R1881 inhibits cell proliferation in LNCaP cells, and less than 10nM of R1881 stimulated cellular proliferation in these cells, indicating these are sensitive to androgens. Furthermore, no effects were seen on PC3 cellular proliferation with lower concentrations of R1881. In conclusion, androgens working through the androgen receptor exhibit a dosage sensitive action on LNCaP growth in vitro.

SUNDAY-POSTER PRESENTATIONS

P300 Board Number: B435

A Cure for Prostate Cancer is Possible. A.A. Sinha1; 1 Univ. of Minnesota , V A Med Ctr Genetics, Cell Biology , Minneapolis, MN Androgen ablation/deprivation by castration, chemical castration by diethylstilbestrol (DES), or by a variety of FDA-approved drugs has provided palliative treatment for prostate cancer (PCa). Our objective was to find the reasons for the treatment failures. To do this we studied untreated and DES treated biopsies for localization of androgen (AR) and estrogen (ER) receptors using antibodies and immunogold techniques. We examined biopsies of eight DES treated (ranging from 37 days to 18 years) and 18 untreated PCa patients with Gleason histological score 7 and higher grade tumors. Epon-embedded thick sections prepared with a Reichert-Jung microtome were stained with methylene blue. We used two types of androgen receptor antibodies (AR, N-20, Santa Cruz Biotechnol., CA, and anti-AR-V-7 (Precision antibody, A& G Pharmaceutical, Inc. Columbia, MD) and estrogen receptors (ERbeta, H-150, Santa Cruz). Receptors were localized using immunogold techniques and protein A conjugated to 15nm gold particles enhanced with silver. Androgen and estrogen receptors were localized in acini and invasive cells of untreated and DES treated sections. At the electron microscopic level, estrogen receptors localized on the nuclear membranes and the adjacent heterochromatin, but not in nucleoli. Analysis of data indicated that androgen ablation was incomplete since androgen receptors were present in both treated and untreated samples. This led to review of the steroid biosynthetic pathway. This pathway utilizes one or more specific enzymes to catalyze, for example, cholesterol into pregnenolone followed by enzymes that leads to production of progesterone then to testosterone and finally to estrogen. Androgen ablation, undoubtedly, decreases the supply of testosterone in prostate, but it does not inhibit the production of the enzymes in the steroid pathway. Enzymes involved in catalyzing progesterone to testosterone ought to be inhibited for androgen ablation. Since progesterone, testosterone and estrogen and their receptors are found in malignant prostate, inhibitions of enzymes are essential for suppressing production and supply of gonadal steroid hormones to cancer cells. Presence of estrogen receptors in untreated and DES treated PCa indicates that androgen ablation was bypassed in the prostate. Our study indicates that in addition to androgen ablation, we must also inhibit enzymes that catalyze progesterone for the production of testosterone. In conclusion, we suggest that inhibition of enzymes catalyzing cholesterol to pregnenolone have the potential of not only significantly improving treatment of PCa, but also of breast cancer and other hormone-dependent cancers. The author is grateful to the Research Service of the Minneapolis Veterans Affairs Medical Center for providing laboratory and research facilities. Since the funding expired, this work was completed by the volunteered effort of AAS. There is no conflict of interest in publication of this abstract. We give special thanks to Mr. Francis E. Pomroy, Jr. who is expert in immunogold localization and electron microscopy autoradiography.

SUNDAY-POSTER PRESENTATIONS

P301 Board Number: B436

CuS + Graphene Oxide + Folate nanocompound = X factor in Photothermal Therapy. D.M. Peterson1, D. Gilbert2, S. Henderson-Jones3, A. Novelo2, K. Manns2, L.E. Carson4, A. Oki5, E.C. Regisford2; 1 Biology, Prairie View AM University, Prairie View, TX, 2Department of Biology, Prairie View AM University, Prairie View, TX, 3Prairie View AM University, Prairie View, TX, 4Cooperative Agricultural Research Center, Prairie View AM University, Prairie View, TX, 5Chemistry Department, Prairie View AM University, Praire View, TX Photothermal Therapy (PTT) is a novel approach at cellular irradiation. PTT manipulates electromagnetic radiation of select particles at an excitable wavelength. Inferred, radiofrequency, microwaves, and ultrasonic wavelengths have all been used to elicit excitation. Excitation, due to photons transfer of electromagnetic energy to selected particles, leads to release of heat energy via vibration. Heat builds up in the cell causing cell membrane lysis leading to cellular death. Copper sulfide (CuS) a black colloid, is used as irradiant. To transport CuS into the cell, Graphene Oxide a single layer form of graphite, is used as its vehicle. In order to deliver CuS + Graphene Oxide to a targeted cancer cell, a known cancer biomarker must be identified. Many cancer cell lines increase up-regulation of the folic acid receptors, these receptors bind to Folate as its ligand. Thus, this study takes advantage of folate’s ability to act as ligand to bind the CuS + Graphene Oxide to cell surface and induce its cellular uptake via endocytosis. Once the compound is inside the cell, a practitioner may irradiate the targeted area with an external light source. This targeted approach will only kill cells that have up-taken the CuS + Graphene Oxide+ Folate compound. Finding the optimal concentration of the nanoparticle compounds is paramount. SKOV-3, Hela, A549, and hFOB-1 cell lines were used. Cells were treated with [50ng/ml], [100ng/ml], [200 ng/ml], [400 ng/ml], and [800 ng/ml] concentrations of CuS, Folate, Graphene oxide, Graphene oxide + Folate, CuS + Folate, and Graphene oxide + Folate + CuS, and an untreated control group. An assay , specifically, 3-(4,5-dimethylthiazolyl-2)-2, 5 diphenyltetrazolium bromide (MTT) assay was then used to measure cell viability and proliferation.

P302 Board Number: B437

Dihydrocucurbitacin B potently induces apoptosis in acute leukemic cell lines of B, T and promyelocytic origin. M. Martlock1, T. Laakko Train1; 1 Department of Biology, Elon University, Elon, NC Leukemia is responsible for 25% of cancer diagnoses in patients under age 20. While major advances have been made in treatments, effectiveness varies depending on the type of leukemia and there is still

SUNDAY-POSTER PRESENTATIONS significant mortality. Cucurbitacins are a group of compounds derived from plants that have been shown to have anti-inflammatory and anti-tumor effects. The cucurbitacin dihydrocucurbitacin B (DHB) is a compound found in Cayaponia tayuya, a Brazilian tree thought to have anti-inflammatory properties. Previous work from our lab has shown that treatment of leukemic cells with DHB or tayuya extract causes an increase in apoptosis in an acute T-cell leukemia cell line (Jurkat). To determine whether DHB had similar effects on other types of leukemic cells, additional cell lines were investigated. Cells were cultured with or without DHB at concentrations ranging from 0.25-25 µM from six to 24 hours. Apoptosis was detected by Annexin V and propidium iodide (PI) staining of cells followed by flow cytometric analysis. Significant increases in Annexin V+/PI- (early and late apoptotic) cells were observed within hours of exposure to the lowest (0.25 μM) concentration tested. After 24 hours with 2.5 μM DHB, the mean percent of apoptotic cells significantly increased from 13.2% in controls to 44.6% (a 3.4-fold increase) in treated samples. To determine if this response was similar in different types of leukemic cells, the RAJI cell line (Burkitt B-cell lymphoma) and HL-60 cell line (an acute promyelocytic leukemia) were cultured with varying concentrations of DHB. Results similar to those observed in Jurkat cells were observed with RAJI cells; after 24 hours without or with 2.5 μM DHB, the average percent apoptosis increased nearly 3-fold from 11.5% in controls to 31.2%, respectively. Interestingly, the promyelocytic line, HL-60, showed a more dramatic increase in apoptosis under the same conditions. The average percent apoptosis increased nearly 20-fold, from 4.5% in controls to 89.0% in treated samples. This data indicates that DHB potently induces apoptosis in a variety of different leukemic cell types and cells of myeloid lineage had a much greater response than those of lymphoid origin. Future studies will investigate how primary immune cells respond to DHB and explore potential mechanisms for induction of apoptosis.

P303 Board Number: B438

POTENTIAL THERAPEUTIC STRATEGY FOR NON-MUSCLE INVASIVE BLADDER CANCER (NMIBC) TREATMENT: EFFECTS OF DOXORUBICIN AND CISPLATIN LOADED IN REDUCED GRAPHENE OXIDE ON THE PI3K AND PTEN REGULATION. R. VILLELA1, P.V. Garcia1, M.A. Aguiar e Silva1, N. Duran2, H.J. Ceragioli3, W.J. Fávaro1; 1 Structural and Functional Biology, University of Campinas (UNICAMP), Campinas, Brazil, 2Institute of Chemistry, University of Campinas (UNICAMP), Campinas, Brazil, 3FEEC, University of Campinas (UNICAMP), Campinas, Brazil Background: Bladder cancer is the most common malignancy of the urinary tract. The recommended first-line treatment for non-muscle invasive bladder cancer (NMIBC), following transurethral resection, is an induction course plus maintenance with intravesical Bacillus Calmette-Guerin (BCG). However, BCG immunotherapy causes undesirable effects, which contributes to treatment interruption besides increasing cancer index recurrence after treatment. In this context, graphene oxide (GO) and/or reduced graphene oxide (rGO) has attracted increasing interest in the field of biological detection, drug delivery,

SUNDAY-POSTER PRESENTATIONS and cancer therapies. Thus, the aims of this study were to characterize and to compare the antitumor effects of Doxorubicin (DOX) and Cisplatin (CIS) functionalized in rGO on NMIBC. Methods: The protocol to induce NMIBC was the following: twenty-five female Fischer 344 rats received 1.5 mg/kg dose of Nmethyl-N-nitrosourea (MNU) intravesically (I.V.) and the 5 Control animals (Group 1) had received physiological saline. After MNU treatment, the Cancer group (Group 2) received the same treatment as Group 1; The Cancer+rGO group (Group 3) received 2.0 mg/mL dose of reduced graphene oxide; The Cancer+rGO+CIS group (Group 4) received 1.5 mg/kg dose of cisplatin; The Cancer+rGO+DOXO group (Group 5) received 3.0 mg/mL dose of doxorubicin; The Cancer+rGO+CIS+DOXO group (Group 6) received the same treatment as Groups 3, 4 and 5; all treatments I.V. for 6 weeks. After 16 weeks, all bladders were collected for histopathological and immunohistochemical analysis. Results: The results demonstrated that 100% of animals from Group 2 showed carcinoma with invasion of the lamina propria (pT1). Similarly, the Groups 3 and 4 showed high-grade papillary carcinoma (pTa), carcinoma in situ (pTis) and pT1 in 60%, 20% and 20% of animals, respectively. The Group 5 showed pTis, pTa and low-grade intraurothelial neoplasia in 60%, 20% and 20% of animals, respectively. There was a better histological recovery in the Group 6, which showed 40% of flat hyperplasia, 40% of low-grade intraurothelial neoplasia and 20% of pTis. Intensified PI3K (Phosphatidylinositol-4,5-bisphosphate 3kinase) immunoreactivities was observed in the Groups 2, 3, 4 and 5 when compared to Group 6. In contrast, the Groups 2, 3, 4 and 5 showed weak PTEN (Phosphatase and tensin homolog) immunoreactivities in relation to Groups 1 and 6. Conclusions: The tumor regression grades were correlated with increased PTEN and decreased PI3K reactivities in the Cancer+rGO+CIS+DOXO group. Taking together, the data suggest the great potential of reduced graphene oxide as carrier system of doxorubicin and cisplatin, and as nanoformulation for NMIBC.

P304 Board Number: B439

EVALUATION OF NEW HDACs INHIBITORS OVER TUMORAL AND NORMAL CELL LINES. E.M. Jiménez1, C.T. Muñoz2, T.M. Percino2; 1 ESM-CINVESTAV, Instituto Politécnico Nacional, México, D.F., Mexico, 2Instituto Politécnico Nacional, México, D.F., Mexico HDAC inhibitors (HDACi) have been prove over diverse antitumoral effects such as cell growth inhibition, differentiation induction and apoptosis triggers1. In this work we prove the antitumoral action of new HDACi previously synthetized with HDAC inhibition activity reported 2, over three tumoral cell lines: MCF-7, HepG2 and HeLa, as well as, two normal cells that normally present damage in the chemotherapy process like endothelial cells and kidney cells. HDACs are considered as one promising target in the development of new antitumoral agents, due to their actions on apoptosis, cell proliferation, metastasis and cell growth inhibition. Different HDACi have been reported as antitumoral compounds, between them there are the isoindoline analogues3y 4. In 2014 this group reported a series of insoindolines-2-sustituted as new HDACi, these compounds show a good HDAC inhibitory activity,

SUNDAY-POSTER PRESENTATIONS that is why in this work there was evaluated their antitumoral capacity over different tumoral cell lines from mammary cancer, hepatocarcinome and cervical uterin cancer. Due the importance of the adverse reactions in pacients treated with several antitumoral agents, we considered of relevance to prove this new agents over possible toxic effects on turn over cells like endothelial, and cells usually very exposed to chemical agents due to elimination process as kidney cells. Our findings showed that all isoindolines evaluated produced growth inhibition of tumoral cells, specially compounds 1b, 1f and 2d. Whereas isoindolines 1a, 2a, 2b and 2c showed minor cytotoxicity on endothelial cells and kidney cells. By microscopy we observed the effects that denote a possible apoptotic pathway in tumoral cells. Therefore isoindolines-2-subtituited can be used as possible antitumoral agents with low cytotoxicity in normal cells in the treatment of mammary cancer, hepatocarcinome and cervic uterine cancer. Kirsten GrØnbÆk, Christoffer Hother and Peter A. Jones. Epigenetic changes in cancer, Journal Compilation, 115: 1039–1059, 2007. Cynthia R. Trejo Muñoz, Elvia Mera Jiménez, Rodolfo Pinto Almazan, José Alfredo Díaz Gandarilla, José Correa Basurto, José G. Trujillo Ferrara, Christian Guerra Araiza, Patricia Talamás Rohana y Teresa Mancilla Percino. Effect of two of isoindolines over HDAC8 activity and expression. Medicinal Chemistry Research 23: 3227-3234, 2014. Shoukou L., Chihiro S., Kiyoshi O., Motomu S., Satoko M., Mayumi S., Minoru Y., Yuichi H. y Hiroyuki M., Design, synthesis, and evaluation of isoindolinone-hidroxinamic acid derivatives as histone deacetylase (HDAC) inhibitors, Bioorganic and Medicinal Chemistry Letters, 17: 4895-4900, 2007 Chihiro S., Takamuri N., Satoko M., Minoru Y., Yuichi H., Hiroyuki M., Design and synthesis of phtalidamide-type histone deacetylase inhibitors, Bioorganic and Medicinal Chemistry Letters, 15: 44274431, 2005

P305 Board Number: B440

Using confocal laser scanning microscopy to monitor targeted delivery of chemotherapeutic agents. L.L. Stojanovic1, S. Pokropinski1, B.M. Hoffman2, E.R. Trivedi2, T. Hai1, J.P. DiOrio1; 1 Baxter Healthcare Corporation, Round Lake, IL, 2Northwestern University, Evanston, IL OBJECTIVE: The objective of this study is to use confocal laser scanning microscopy (CLSM) to monitor drug uptake and movement within MDA-MB-231 breast cancer cells. BACKGROUND: A favorable property of chemotherapeutic agents is the preferential delivery of cancer drug to the tumor tissue, lessening damage to healthy surrounding tissue. An experimental drug conjugate was used for this study and consisted of two components. Component A (A) acts as a delivery vector for cancer therapeutics, binding to LDL and entering the cell via the LDL-receptor pathway. Once the drug conjugate is in the cell, it moves into the lysosomes where the low pH environment cleaves off

SUNDAY-POSTER PRESENTATIONS drug component B (B). Drug component B is the cancer therapeutic doxorubicin that travels to the nucleus where it intercalates into the DNA to prevent transcription and eventually leads to cell death. METHODS: Human breast cancer cell line, MDA-MB-231 cells were maintained in DMEM/1% PenicillinStreptomycin/10% Fetal Bovine Serum. The cells were plated in poly-l-lysine coated Mattek dishes and incubated overnight. The drug was added to the cells followed by overnight incubation. After rinsing the cells, wheat germ agglutinin (WGA)-Alexa Fluor 488 was added, followed by fixing and cover slipping using Prolong® Gold containing DAPI. Samples were imaged on a Nikon A1Rsi CLSM at 405nm (DAPI, blue), 488nm (WGA, green), 561nm (B, orange) and 638nm (A, red). Each channel was acquired separately to avoid emission cross over between channels. To demonstrate the movement of the drug to the lysosomes, the cells were incubated with the drug as above, stained with LysoTracker® and imaged live. In addition, cells were blocked with LDL/heparin and imaged live to prevent the uptake of the drug into the cells. In each case, negative controls were imaged in parallel. RESULTS: Confocal laser scanning microscopy was utilized to: 1) image the drug incorporation into MDAMB-231 breast cancer cells, 2) demonstrate the localization of the drug in the lysosomes and 3) show that the conjugate uptake is LDL-mediated. CONCLUSION: We used confocal microscopy to demonstrate the uptake of the drug into the cell, movement to the lysosome, followed by the nucleus. We also verified the mechanism by blocking the uptake of the drug.

P306 Board Number: B441

The effects of vanadyl sulphate on rat glioma cells ultrastructure. H.M. Kutlu1, D. Vejselova1; 1 Biology, Anadolu Univ, Eskisehir, Turkey Vanadium compounds were stated to have different effects on the cells as to be genotoxic, lipid peroxidative, erythrocyte hemolytic and effective in inhibition of neoplastic progression. One of the most popular vanadium salts is vanadyl sulphate that is investigated in different types of cells, also in vivo. In this study vanadyl sulphate was used with an aim to detect the effects of a vanadium compound on the rat glioma cells (C6) ultrastructure. The effects of vanadyl sulphate on glioma cell ultrastructure were investigated using a transmission electron microscopic assay. For this manner, C6 cells treated with IC50 concentration (100 µM) of vanadyl sulphate for 24 hours were fixed in glutaraldehyde (2,5% in 0,1 M phosphate buffer) at pH 7.4 and embedded in agar. Embedded samples were left in phosphate buffer at +4 °C, overnight and post fixed in osmium tetroxide. Post-fixed cells were dehydrated in graded ethanol: 70, 90, 96 and 100% than embedded in EPON 812 epoxy and left 48 hours in oven for polymerization. The samples sectioned on ultramicrotome (Leica, UC6) were stained in uranyl acecate and lead citrate than photographed on transmission electron microscope (FEI, Tecnai BioTWIN). In our

SUNDAY-POSTER PRESENTATIONS results it has been showed major changes on C6 cells ultrastructure indicating apoptosis caused by vanadyl sulphate as membrane blebbings, nuclear fragmentation, horseshoe nucleus and shrinkage on cells. From our findings it can be concluded that vanadyl sulphate possess anticarcinogenic effects on C6 cells showing its effects with clear apoptotic sparks and may be successful agent in drug desingning for cancer treatment but further investigations are required.

P307 Board Number: B442

Antitumoral effect of doxazosin on C6 rat glioma cells. M. Maier Gaelzer1, H. Flores Mello1, B. Paranhos Coelho1, A. Hoffmann de Quadros1, J. Bender Hoppe1, M. Barea Guerra1, C. Saraiva Gonçalves1, V. Usach2, P. Setton-Avruj2, C. Gazzana Salbego1; 1 Biochemistry Department, Federal University of Rio Grande do Sul, Porto Alegre, Brazil, 2Departamento de Química-Biológica, Universidad de Buenos Aires, Buenos Aires, Argentina Glioblastoma (GB) is the most frequent and most malignant human brain tumor. Prognosis for patients with GB remains dismal, as median survival after diagnosis varies from six months to one year. This is due to the inability of current treatment strategies to address the highly invasive nature of this disease. Thus, new therapeutic strategies are required. Doxazosin, a quinazoline compound, is a selective α1adrenoceptor antagonist used for treatment of high blood pressure and benign prostate tumor. In addition, it has also been demonstrated that doxazosin affects prostate cancer cells survival. In this context, the aim of this study was to evaluate the effect of doxazosin on C6 rat glioma cells. C6 cells were cultured with DMEM and treated with doxazosin at different concentrations. Cellular density and viability were determined by Suforhodamine B (SRB) and MTT assays, respectively. Cell death was analyzed by Propidium Iodide (PI) and Annexin V/PI staining. Cell cycle profiles were determined by flow cytometry (FACS Calibur, BD Bioscience). Cytotoxicity assays were performed in non-tumor cells, organotypic hippocampal culture slices and primary astrocyte cultures. Data are expressed as means±SD. All results are representative of at least 4 independent experiments. Differences between mean values were considered significant when p20%) reduced their velocity. A decrease in transport velocity is consistent with application on dynein of an external load by the kinesin-1 motors. Activation of kinesin-2 by pigment dispersion stimuli never led to a decrease in velocity of minus-end runs of melanosomes. Our data show that forced simultaneous activation of

TUESDAY-POSTER PRESENTATIONS cytoplasmic dynein and kinesin-1 leads to tug-of-war between these motors and suggest that cellular mechanisms exist that regulate cargo runs in each direction thus preventing tug-of-war.

P1708 Board Number: B225

Regulation of dynein motility by the antenna structure of dynactin p150. T. Kobayashi1, T. Miyashita1, H. Kajita1, K. Saito1, T. Murayama2, Y.Y. Toyoshima1; 1 Dept of Life Sci, The University of Tokyo, Tokyo, Japan, 2Graduate School of Medicine, Juntendo University, Tokyo, Japan Dynactin is an intricate complex and has been known as a dynein activator. Dynactin consists of 11 species of polypeptides, and among those p150 subunit constitutes a sidearm which extend from Arp1 rod. The coiled coil 1 region (CC1) of p150 is considered to bind directly to the N-terminal region of dynein intermediate chain (IC). Previously, we have shown that CC1 of p150 forms an antenna structure which extrude from the head within a sidearm. To elucidate the mechanism of dynein regulation by dynactin complex, we examined the effect of dynactin complex and the isolated CC1 on thedynein motility in vitro. Single molecules of cytoplasmic dynein diffusively moved on microtubules and exhibited slightly biased displacement to the minus end of microtubules as previously reported. Single molecules of dynactin complex showed diffusive movement on microtubules without unidirectional bias. When we mixed dynein and dynactin together, the fluorescent spot derived from dynein disappeared from microtubules. This was not caused by the competition of binding to microtubules between dynein and dynactin because both molecules are much fewer to occupy the binding sites on microtubules. Next, we prepared the recombinant CC1 and confirmed that the isolated CC1 directly bound to cytoplasmic dynein with a high affinity (Kd, 20 nM), but did not bind to microtubules. Single molecules of dynein associated with the CC1 released easily from microtubules and did not stay on microtubules for enough time to be detected, as is the case for the dynein-dynactin-complex. Intriguingly, when we added the CC1 to the gliding microtubules on the dynein coated surface, the moving microtubules gradually dissociated and finally disappeared from the surface. These observations suggest that the effect of CC1, the antenna structure of p150, on dynein motility is to reduce the affinity of dynein to microtubules, which appears to be inconsistent to the accepted idea that dynactin activates dynein motility by increasing its processivity.

TUESDAY-POSTER PRESENTATIONS

P1709 Board Number: B226

Dynein, Dynactin and Lis1 are required for endosome maturation in Drosophila oocytes. P. Sanghavi1, G. Liu1, N. Townes1, G.B. Gonsalvez1; 1 Cellular Biology and Anatomy, Georgia Regents University, Augusta, GA Several studies have demonstrated a requirement for Dynein in the transport of early and late endosomes. However, the extent to which Dynein contributes to endocytic uptake and maturation is poorly understood. Our initial experiments were focused on examining the role of p50/Dynamitin in oskar mRNA localization. Dynamitin is a component of the Dynactin complex, a core regulator of Dynein. Our findings indicate that Dynamitin is indeed required for efficient oskar mRNA localization in Drosophila oocytes. During the course of these studies, we unexpectedly discovered an additional role for Dynamitin in endocytic uptake and maturation. Large quantities of Yolk proteins are internalized into mid-stage oocytes via Clathrin-mediated endocytosis. Subsequently, maturation of these endocytic vesicles results in formation of condensed yolk granules. A defect in endocytosis is reflected by a reduction or loss of yolk granules. Our ultra-structural analysis indicated that oocytes depleted of Dynamitin contained significantly fewer yolk granules, but instead accumulated numerous endocytic intermediate structures. Particularly prominent were large vesicular structures that reflect an endocytic block downstream of Rab5. Similar endocytic defects were also observed upon depleting Dynein heavy chain or Lis1, another regulator of the Dynein motor. Consistent with a role for Dynein in endocytosis, we demonstrate that Dynein heavy chain is recruited to the oocyte cortex in an endocytosis-dependent manner. Our results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor in turn functions to ensure efficient endocytic uptake and maturation, ultimately resulting in the production of abundant yolk granules.

P1710 Board Number: B227

Molecular Mechanism for Dynactin Regulation of Processive and Diffusive Dynein Transport. S.K. Tripathy1, S.J. Weil2, C. Chen3, S. Gross4, R. Vallee2; 1 UC Irvine, Irvine, CA, 2Department of Pathology and Cell Biology, Columbia University, New York, NY, 3 biological sciences, Columbia university, New York, NY, 4Developmental and Cell Biology, UC Irvine, Irvine, CA The dynactin complex is an important cytoplasmic dynein regulator implicated in both dynein cargo recruitment and motor regulation. Dynactin increases dynein run length along microtubules in in vitro assays, a function long thought to involve the N-terminal CAP-Gly microtubule-binding site within the major dynactin subunit p150Glued . However, recent experiments from several labs argue against such a

TUESDAY-POSTER PRESENTATIONS role for this domain, and the mechanism for dynein regulation by p150Glued remains unknown. We have produced and characterized well-behaved bacterially- and baculovirus-expressed subfragments of the Nterminal half of the major dynactin subunit p150Glued and its neuronal splice variant p135, which lacks the CAP-Gly domain (Tripathy et al., ASCB 2013). We tested the effects of these constructs on dynein motor behavior by single molecule laser trap bead assays. We found that the largest p150Glued and p135 fragments consisting of the N-terminal half of the full polypeptides each induce both diffusive and processive bead movement, as is the case for the complete dynactin complex. An internal fragment, CC1B, which is sufficient for dynein binding, was also sufficient to promote dynein processivity. The immediate upstream fragment, CC1A, increased dynein diffusion, whereas a complete CC1 fragment potently inhibited dynein motility. To address how these activities and that of the N-terminal globular domain of p150Glued and p135 might be interrelated, we tested for interactions between domains. We identified a clear interaction between the individual CC1A and CC1B fragments using both size exclusion FPLC and pull-downs. In single molecule assays we also found clear CC1A dose-dependent neutralization of the ability of CC1B to stimulate dynein processivity. Together our data provide a comprehensive model for p150Glued mediated dynein regulation, involving CC1B as the prime locus for dynactin autoregulation by CC1A and, we speculate, by the N-terminal globular region of p150Glued. Supp. by NIH GM102347 to RV and GM070676 to SG.

P1711 Board Number: B228

Dynactin functions as both a dynamic tether and brake during dynein-driven motility. S. Ayloo1, J. Lazarus1, A. Dodda2, M. Tokito1, E. Ostap1, E.L. Holzbaur3; 1 University of Pennsylvania, Philadelphia, PA, 2University of Massachusetts, Amherst, MA, 3Department of Physiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA Dynactin is a large multi-subunit complex that is an important cofactor for cytoplasmic dynein in cellular processes including organelle trafficking and mitosis. Dynactin increases the processivity of dynein and also targets dynein to specific cellular locations. Although the role of dynactin in dynein-mediated processes is well established, the underlying mechanisms involved are unknown. Here, we use single molecule approaches to investigate the contribution of the dynactin subunit p150Glued to dynein motor function. We first characterized the motility of purified GFP-tagged mammalian dynein isolated from a knock-in mouse brain tissue (Zhang et al., 2013). Consistent with previous reports (Mallik et al., 2005; Ross et al., 2006), single molecules of dynein-GFP switch stochastically from processive to diffusive states of motion. We analyzed this motion by a novel algorithm that we developed to parse trajectories into different states and found that dynein is processive 60% of the time. To examine the activation of dynein by dynactin, we investigated the formation and co-migration of a dynein-p150Glued co-complex using dual-color TIRF microscopy. We provide direct evidence that p150Glued is sufficient to recruit and tether dynein to the microtubule and that this recruitment is concentration-dependent; p150Glued

TUESDAY-POSTER PRESENTATIONS increases the on-rate and decreases the off-rate of dynein from microtubules. Single molecule imaging of motility in cell extracts demonstrates that the CAP-Gly domain of dynactin is essential for the decreased detachment rate of the dynein-dynactin complex from the microtubule and also acts as a brake to slow the dynein motor. Consistent with this important role, two neurodegenerative diseasecausing mutations in the CAP-Gly domain abrogate these functions in our assays. Together, these observations support a model in which dynactin enhances the initial recruitment of dynein onto microtubules and promotes the sustained engagement of dynein with its cytoskeletal track. Thus, our work reveals new insights into the functioning of dynein and dynactin and how this process is perturbed in neurodegenerative disease.

P1712 Board Number: B229

Characterization of a dynactin p62 variant associated with increased risk of chronic Pseudomonas infection in cystic fibrosis. T. Yeh1, T.A. Schroer1; 1 Department of Biology, Johns Hopkins University, Baltimore, MD Dynactin is an essential component of the cytoplasmic dynein motor that contributes to interactions with dynein cargoes and other cellular components. The dynactin subunit p62 (DCTN4), along with Arp11, p27, and p25, comprise a subcomplex that is located distal to dynactin’s dynein-binding site. We have shown that p62 is essential for dynactin complex integrity and targeting to specific cellular cargoes, but its full range of functions have not yet been defined. Exome sequencing has revealed that the presence of a single copy of the p62 variant (Y270C) in individuals with cystic fibrosis (CF) correlates with early onset of chronic Pseudomonas aeruginosa airway infection. We find that exogenously expressed p62-Y270C incorporates into the dynactin complex like p62-WT and does not affect dynactin integrity. p62 Y270, which is known to be phosphorylated in vivo, is a consensus site for kinases linked to cell adhesion and locomotion, so we investigated the impact of p62 Y270C on cell migration and focal adhesion dynamics. Cells expressing p62-Y270C migrate slower than those expressing p62-WT in a scratch wound assay. Centrosome reorientation in cells at the wound edge appears normal, but the morphology, location and number of actin stress fibers and focal adhesions (FAs) are altered. The level of the phosphorylated form of focal adhesion kinase (pFAK) at FAs is also significantly reduced, suggesting that the signaling at FAs may be perturbed. Live cell imaging showed that turnover of stress fibers (labeled with GFP-F-tractin) and FAs (labeled with RFP-zyxin) are also suppressed. Taken together, our results suggest that the p62-Y270C variant may reduce cell migration in the CF airway epithelium by altering stress fiber and FA dynamics, leading to decreased repair and increased susceptibility to infection. Further investigation of dynactin’s contributions to cell migration is underway.

TUESDAY-POSTER PRESENTATIONS

P1713 Board Number: B230

Dynein regulation by its microtubule track: Roles of tubulin carboxy-terminal tails in vivo. C.E. Estrem1, J.K. Moore1; 1 Cell and Development, University of Colorado School of Medicine, Aurora, CO Dynein is a conserved microtubule motor with a multitude of cellular roles such as vesicle transport, mitotic spindle assembly, and spindle positioning. Despite these diverse roles, most eukaryotes express a single cytoplasmic dynein heavy chain; therefore, its activity must be highly regulated to provide specified force for different cellular functions. Here we identify a novel mode of regulation, in which dynein is regulated by tubulin carboxy-terminal tails (CTTs) that decorate its microtubule track. CTTs are negatively charged regions that differ across species and tubulin isotypes, and are major sites of posttranslational modification. We examine CTT mutants in budding yeast, where dynein generates force to move the mitotic spindle by sliding astral microtubules along the cell cortex. Using live cell imaging of fluorescently labeled microtubules, we demonstrate that truncation mutants that lack the βtubulin CTT exhibit increased spindle movement. Microtubule sliding events in β-tubulin CTT mutants are more frequent and significantly longer-lived, producing greater displacement of the nucleus and mitotic spindle. These effects depend on dynein -- conditionally depleting dynein eliminates spindle movement in β-tubulin CTT mutants. Our results indicate that β-tubulin CTT negatively regulates dynein in vivo. We are investigating whether the β-tubulin CTT directly affects dynein’s affinity for the microtubule or alters the activity of a different dynein regulator. This continued research offers new insight into how tubulins regulate microtubule binding proteins such as motor proteins, which in turn affects cellular processes.

P1714 Board Number: B231

The motility of axonemal dynein is regulated by the tubulin modifications. J. Alper1, F. Decker2, B. Agana3, J. Howard1; Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 3Chemistry, Missouri State University, Springfield, MO 1

Microtubule diversity, arising from utilization of different tubulin isotypes and post-translational modifications, regulates many cellular processes including cell division, centriole assembly, and neuronal differentiation. In cilia and flagella, microtubule diversity is important for axonemal assembly and motility. However, it is unknown whether microtubule diversity directly regulated the activity of the axonemal dyneins, the motors that drive the beating of the axoneme, or whether the regulation is indirect, perhaps through pathways upstream of the motors. To test whether microtubule diversity directly regulates the activity of axonemal dyneins, we asked if in vitro acetylation or deacetylation of

TUESDAY-POSTER PRESENTATIONS lysine 40 (K40), a major post-translational modification to tubulin, or proteolytic cleavage of the Cterminal tail (CTT) of tubulin, the location of detyrosination, polyglutamylation, and polyglycylation modifications as well as most of the genetic diversity, influences the activity of outer-arm dynein in motility assays using purified proteins. By quantifying motility, we found that K40 acetylation increases and CTTs decrease axonemal dynein motility. Further mathematical modeling suggests that these modifications regulate the power stroke speed of dynein, likely by altering the unbinding rate of the microtubule. These results show that axonemal dynein directly deciphers the tubulin code, which has important implications for eukaryotic cilial beat regulation.

P1715 Board Number: B232

Emergence of large-scale vortices of microtubules collectively driven by axonemal dyneins. N. Kanatani1, K. Oiwa2; 1 1Graduate School of Life Science, University of Hyogo, Kamigori, Hyogo, Japan, 2Advanced ICT Res Inst, Natl. Inst. Info. Commun. Technol., Kobe, Japan Self-propelled particle models have been often used in trial that the motions often observed in biology (schools of fish, some flocks of birds, cell migrations during development and so on) are understood quantitatively. In addition to computer simulations of the models, substantial experimental systems have been demanded for the study and in vitro motility assays commonly used in protein-motors biophysics have provided some solutions. Using conventional in vitro motility assays, we reported collective behavior of microtubules driven by axomemal dynein molecules which are attached on glass surface [Sumino, Nagai, et al., Nature 483, 448-452, 2012]. Under certain experimental conditions, the collective movement of microtubules generates nematic order, millimeter-scale meandering streams or millimeter-scale vortices. To explore the conditions causing such phase-shift, we have investigated collective behavior using differrent types of axonemal dynein subspecies and microtubules with various length. The motor proteins we used are the inner arm dynein subspecies c and g (dynein c and g) of Chlamydomonas flagella, both of which are single-headed dyneins composed of one heavy chain and two types of light chains, actin and p28 (for dynein c) or centrin (for dynein g). Dynein c and g are capable of moving microtubules on glass surface at velocities of ca. 12 and 6 µm/sec in the presence of 1 mM Mg-ATP at 23 C, respectively. Purified dynein molecules were adsorbed on a glass surface at densities higher than 1000 molecules µm-2. Microtubules with length less than ca. 60 µm did not move in the absence of ATP but bound to dynein g-coated glass surface being slightly aligned by flow. Upon the addition of 1 mM Mg-ATP, microtubules started moving smoothly. Moving microtubules often collide each other and upon these collisions they are aligned through nematic interactions. Within a few minutes, microtubule-alignment gradually increases its size and finally forms streams meandering across a very large distance (longer than 400 µm). Under the experimental conditions with microtubules of less than ca. 60 μm, vortices with 200-500 μm diameter emerge from the stream tens of minutes after the ATP addition. On the other hand, microtubules with mean length of longer than 60 μm do not form

TUESDAY-POSTER PRESENTATIONS vortices. The features of vortices driven by dynein g, i.e. number, diameter and time necessary for their emergence, are different from those driven by dynein c. These results now suggest that the vortex formation is not dynein-c-specific but may reflect the mechanical properties of inner-arm dyneins. One parameters which can shift the phase is the length of microtubules but the mechanism how their length can modify the emergence still requires further investigation.

P1716 Board Number: B233

Regulation of Cytoplasmic dynein activity by GSK-3β. F. Gao1; 1 biology, university of south carolina, Columbia, SC Cytoplasmic dynein is a vitally important microtubule motor in animal cells, contributing to many aspects of cellular and intracellular motility. Genetic lesions that lead to altered dynein function have been associated with disease, but relatively little is known about signaling pathways regulating motor activity. Our studies now indicate that a prominent insulin response pathway culminating in inhibition of GSK-3β (glycogen synthase kinase 3 β) contributes to dynein activation and increases minus end directed transport in both neuronal and non-neuronal cells. Moreover, GSK-3β coprecipitates with dynein in mouse brain extract and phosphorylates dynein in vitro. Dynein phosphorylation by GSK-3β reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Because GSK-3β inhibition occurs downstream of multiple receptor signaling pathways it has the potential to act as a switch, stimulating transport of dynein and local cargo away from peripheral membrane sites upon receptor activation.

P1717 Board Number: B234

Structure of human cytoplasmic dynein-2 primed for its powerstroke. R. Zalyte1, A.P. Carter1, H. Schmidt1, L. Urnavicius1; 1 MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Dyneins are a family of microtubule motor proteins belonging to the AAA+ (ATPases associated with diverse cellular activities) family.The cytoplasmic dynein-2 subfamily play a key role in intraflagella transport and in humans deficiencies are associated with a wide range of skeletal ciliopathies such as Jeune syndrome.Here we present the crystal structure of the human cytoplasmic dynein-2 motor bound to the ATP hydrolysis transition state inhibitor ADP.vanadate. This pre-powerstroke structure explains how the dynein motile element, called the linker, is bent and primed to generate force. It also reveals how the movements within the ring of AAA+ domains are coupled to the relative sliding of two αhelices in the dynein stalk that lead to release of dynein from microtubules.

TUESDAY-POSTER PRESENTATIONS

P1718 Board Number: B235

Direct observation by cryo-EM of cytoplasmic dynein motors stepping along microtubules. H. Imai1, T. Shima2, K. Sutoh3, M.L. Walker4, P.J. Knight1, T. Kon5,6, S.A. Burgess1; School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, Univ Leeds, Leeds, United Kingdom, 2QBiC, RIKEN, Osaka, Japan, 3Waseda University, Tokyo, Japan, 4MLW Consulting, Cornwall, United Kingdom, 5Department of Frontier Bioscience, Hosei University, Tokyo, Japan, 6JST PRESTO, Saitama, Japan 1

Cytoplasmic dynein motor proteins perform critical roles in eukaryotic cells, transporting subcellular cargoes towards the minus ends of microtubule (MT) tracks and maintaining the positions of organelles. Tracking of fluorescently-tagged dimeric dynein suggested an uncoordinated stepping by the two heads, but there is also evidence of communication between them. We have now imaged directly by cryoelectron microscopy individual Dictyostelium discoideum dynein dimers flash-frozen while stepping along MT at near-physiological Mg-ATP concentration. About half of the molecules show diverse configurations of the two motor heads. The other half of the molecules has the two heads closely superposed with both their linker domains in post-powerstroke conformations. This unpredicted superposed configuration reveals more clearly the coiled-coil stalk domains and their terminal stalkheads that bind the dimer to the MT. The stalks are angled at ~42±11° to the MT, and are flexible mainly about the stalkhead-stalk junction, resulting in axial and radial variation of the heads. The superposed configuration is different from any configuration of the two other cytoskeletal motor families, kinesin and myosin, and thus direct motor-motor interactions may be functionally important in stepping by dynein.

P1719 Board Number: B236

The crystal structure of the dynein heavy chain dimerization domain. A.G. Diamant1, M.A. Schlager1, H. Schmidt1, M. Yu1, A.P. Carter1; 1 MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Cytoplasmic dynein is a molecular motor responsible for the vast majority of retrograde traffic along microtubules. The largest subunit of the dynein complex is called the dynein heavy chain (DHC). The DHC includes a C-terminal motor domain, which converts ATP hydrolysis into mechanical force, and an N-terminal tail domain. An intermediate chain (DIC) and light intermediate chain (DLIC) bind directly to the DHC tail, while light chains (DLCs) bind to the DIC. This tail complex is important for both cargo binding as well as homodimerization of the DHC, which is necessary for processive movement. Previous studies suggest that the DLCs play an important role in homodimerization of the dynein complex, but it remains unclear whether the other subunits also contribute to the dimer interface.

TUESDAY-POSTER PRESENTATIONS Using Saccharomyces cerevisiae as a model, we co-expressed all four dynein subunits and purified functional dynein motors that were active in an in vitro motility assay. In this background, we found that truncating the DHC to include only the first ~1000 residues eliminates the motor domain, while preserving binding to the DIC, DLIC and DLC. Truncating another 50 residues off of the C-terminus led to a loss of all accessory subunits, suggesting this may disrupt a domain required for binding both the DIC and DLIC. Furthermore, a construct including only the first ~500 residues of the DHC dimerizes despite not being able to bind any of the other subunits. We solved the crystal structure of this smaller DHC fragment and found that the N-terminal ~200 residues of the DHC constitute an intricate dimerization domain. Not only is this is the first crystal structure of any part of the DHC N-terminus, but it reveals a previously undocumented dimerization domain within the DHC itself. These studies shed new light on the biochemical organization of this important motor complex and contribute to a better understanding of its function.

P1720 Board Number: B237

Dynein light chain 1 locates at the stalk of the outer arm dynein. M. Ichikawa1, K. Saito1, H. Yanagisawa2, T. Yagi3, R. Kamiya4, Y. Kushida5, K. Nakano5, O. Numata5, Y.Y. Toyoshima1; 1 Dept of Life Sci, The University of Tokyo, Tokyo, Japan, 2Dept Cell Biology, The University of Tokyo, Tokyo, Japan, 3Pref. Univ. of Hiroshima, Hiroshima, Japan, 4Dept Life Science, Gakushuin University, Tokyo, Japan, 5Univ. of Tsukuba, Tsukuba, Japan Cilia and flagella are highly conserved microtubule (MT)-based organelles in eukaryotes. The bending motion of cilia and flagella is driven by the motor protein called axonemal dyneins. The outer arm dynein (OAD) complex generates ~80% of propulsive force and thus OAD is an important component of cilia and flagella. OAD complex is composed of three heavy chains (α, β, γHCs), two intermediate chains (ICs), and eleven light chains (LCs). Each of the three HCs comprises the tail and the head domains. Three HCs form a heterotrimer through their tail domains and thus the OAD complex appears as a threeheaded structure. The head domain is composed of the linker, the AAA+ ring, the stalk, and the microtubule binding domain (MTBD). The stalk is a coiled-coil structure, which protrudes from the AAA+ ring, and the MTBD resides at the tip of stalk region. LC1 is a 22-kDa light chain of OAD and widely conserved among many species. While all the other smaller subunits (ICs and LCs) have been shown to associate with the tail domain, LC1 was found to associate with the head domain of the γHC. LC1 was found to be essential for proper beating of cilia and flagella in many species. Mutation of human LC1 also leads to primary ciliary dyskinesia. Despite its importance in vivo, LC1's regulatory mechanism of OAD is not clearly understood because the structural basis for interaction of LC1 and γHC has not been elucidated. Here, by using single particle analysis of electron microscopy (EM), we have found that there is an additional density at the tip of the γHC stalk region. By expressing His-tagged LC1 in Tetrahymena and Chlamydomonas and performing EM observation aided by Ni-NTA-nanogold labeling, we have shown

TUESDAY-POSTER PRESENTATIONS that this additional structure corresponds to LC1. This is in remarkable contrast to the previous assumption that LC1 locates at the AAA+ ring of the γHC (Patel-King and King 2009). Pull-down assay using bacterially expressed fragments has shown that LC1 preferentially binds to the γ-stalk at a 1:1 molar ratio. Further biochemical mapping has revealed that the MTBD region of the γHC is sufficient for the binding of LC1. These results raise the possibility that LC1 regulates OAD activity by changing γHC s affinity to MTs.

P1721 Board Number: B238

Characterizing the Role of Cytoplasmic Dynein in HTLV-1 Intracellular Trafficking. R.E. Alvarado1, T.M. Gibson2; 1 Biology, University of Texas of the Permian Basin, Odesssa, TX, 2Biology, University of Texas of the Permian Basin, Odessa, TX Human T-Cell Leukemia Virus type 1 (HTLV-1) is a human retrovirus that is associated with a variety of human diseases, which include adult T-Cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A hallmark for retroviral infection requires the integration of the viral genome into the host cell genome. One problem the virus faces is trafficking through the cytoplasm in order to reach the nucleus. Some viruses have overcome this obstacle by hijacking molecular motors that move through the cytoplasm on microtubules (MT) in a retrograde fashion. The motor protein, termed cytoplasmic dynein, mediates retrograde movement of intracellular cargo along microtubules. In previous experiments, our laboratory has demonstrated that HTLV-1 particles co-localized with cytoplasmic dynein and dynactin within HEK293T cells. Also immunoprecipitation studies revealed that HTLV-1 directly or indirectly interacts with cytoplasmic dynein in-vitro. Thus, we have hypothesized that HTLV-1 utilizes cytoplasmic dynein to travel to the nucleus in order to insert its DNA into the host genome. To investigate the role of cytoplasmic dynein in HTLV-1 trafficking, we have optimized conditions to perform experiments using short interfering RNAs (siRNA). By inhibiting the expression of this protein, we can better understand the role cytoplasmic dynein has in retrovirus movement. Through these studies, we expect to further elucidate the molecular details of HTLV-1: host cell interactions. Furthermore, these studies should reveal insights to potential therapeutic targets.

TUESDAY-POSTER PRESENTATIONS

P1722 Board Number: B239

A Ras-Like Domain in the Light Intermediate Chain Bridges the Dynein Motor to a Cargo-Binding Region. C.M. Schroeder1,2, J.M. Ostrem1,2, N.T. Hertz1,2, R.D. Vale2,3; 1 University of California, San Francisco, San Francisco, CA, 2The Howard Hughes Medical Institute, San Francisco, CA, 3Department of Cell and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA Cytoplasmic dynein, a microtubule-based motor protein, transports many intracellular cargos by means of its light intermediate chain (LIC). However, it is not understood how the LIC carries out intracellular transport, and structural information about the LIC is lacking. We have determined the crystal structure of the conserved LIC domain, which binds the motor heavy chain, from a thermophilic fungus. We show that the LIC has a Ras-like fold with insertions that distinguish it from Ras and other previously described G proteins. Despite having a G protein fold, the fungal LIC has lost its ability to bind nucleotide, while the human LIC1 binds GDP preferentially over GTP. We show that the LIC G domain binds the dynein heavy chain using a conserved patch of aromatic residues, whereas the less conserved C-terminal domain binds several Rab effectors involved in membrane transport. These studies provide the first structural information and insight into the evolutionary origin of the LIC as well as revealing how this critical subunit connects the dynein motor to cargo.

P1723 Board Number: B240

Dual Role for Cdk1 in Triggering Early and Late G2 Dynein Recruitment to the Nuclear Envelope. A. Baffet1, D.J. Hu1, R. Vallee1; 1 Department of Pathology and Cell Biology, Columbia University, New York, NY Cytoplasmic dynein is recruited to the nuclear envelope (NE) during G2 via two distinct nuclear poreanchored mechanisms involving, respectively, RanBP2-BicD2-dynein/dynactin/Lis1 and Nup133-Cenp-FNudE/L-dynein (Splinter et al, PLoS Biol., 2010; Bolhy et al., JCB, 2011). These pathways also function in neuronal progenitor cells, where they control early vs. late G2 apical nuclear migration (Hu et al., Cell, 2013). How they are activated specifically during G2 is unknown. This question has particular relevance in neuronal progenitors, in which nuclei must reverse their direction of transport from kinesindependent basal movement during G1 to dynein-dependent apical movement to the ventricular surface of the developing brain during G2. Failure to reach the ventricle blocks mitotic entry and neurogenesis. To address this important question we have undertaken experiments to test the role of cell cycledependent protein kinases in NE dynein recruitment and nuclear migration. We previously found that small molecule Cdk1 inhibitors block the recruitment of dynein, as well as dynactin and Lis1 to the HeLa

TUESDAY-POSTER PRESENTATIONS cell NE and inhibit apical nuclear migration in the developing rat brain (Baffet et al., ASCB 2013 abstract). We have now further characterized the underlying molecular mechanism for Cdk1 action. We show that Cdk1 inhibition displaces the most upstream factors in each pathway, BicD2 and Cenp-F, whereas Cdk1 stimulation results in more intense and even earlier BicD2 and Cenp-F NE decoration. We identify novel Cdk1 phosphorylation sites in the BicD2-binding region of RanBP2, phosphorylation of which strongly enhances binding to BicD2-CT in vitro. Constitutive targeting of BicD2-NT to the NE of Cdk1-inhibited cells restores dynein, dynactin and Lis1 recruitment to the HeLa cell NE and rescues apical nuclear migration in neuronal progenitor cells. These results reveal that Cdk1 phosphorylation of factors downstream of BicD2 (dynein, dynactin, Lis1) is not involved in their targeting to the NE. Surprisingly, Cdk1 inhibition affects the late dynein NE recruitment pathway by preventing Cenp-F from exiting the nucleus. In conclusion, Cdk1 independently controls both the early and late NE dynein recruitment pathways. Cdk1 phosphorylates RanBP2 to activate BicD2 recruitment to the NE, which appears to serve as the trigger for apical nuclear migration in neuronal progenitor cells. Cdk1 also activates the late NE dynein recruitment pathway by a novel mechanism, controlling the exit of Cenp-F from its default intranuclear localization. Supported by HD40182 to RV and an AHA PD Fellowship to AB.

P1724 Board Number: B241

Differential Roles for NudE1 and NudEL1 In Rat Brain Development. D. Doobin1, S. Kemal1, R. Vallee1; 1 Department of Pathology and Cell Biology, Columbia University, New York, NY During development of the mammalian neocortex, radial glial progenitor (RGP) cells divide while undergoing cell cycle dependent interkinetic nuclear migration (INM). We have found apical nuclear migration to be dependent on cytoplasmic dynein, recruited to the nuclear envelope (NE) by sequential G2-specific mechanisms (Hu et al. Cell. 2013, 154:1300-13), which also control G2-depedent dynein recruitment to the NE in cultured non-neuronal cells. The specific roles of two of these component proteins – NudE and NudEL – in the NE dynein recruitment process, however, remain unexplored. NudE and NudEL are closely related throughout their length (55% identity; 70% similarity), and each interacts with cytoplasmic dynein and Lis1. However, knockout mice exhibit different brain developmental phenotypes (Feng & Walsh. Neuron. 2004, 44:279-93; Sasaki et al. Mol Cell Biol. 2005, 25:7812-27), consistent either with different functional roles or distinct brain developmental expression patterns. Using in utero electroporation of Nde1 and Ndel1 shRNAs in E16 rat brains, followed by fixed or live imaging in brain slice culture , we tested the effects of NudE and NudEL RNAi on neurogenesis and migration. NudE RNAi resulted in severe inhibition of apical INM, and strongly reduced the number of dividing cells at the ventricular surface. Ectopic divisions away from the ventricular surface were undetectable. In contrast, depletion of NudEL had no effect on INM or the frequency of dividing cells at the ventricular surface. RNAi for each gene led to an accumulation of multipolar cells in the intermediate zone, and severe inhibition of further neuronal migration of postmitotic neurons in the cortical plate. To test for inherent differences in NudE and NudEL function, we have begun series of

TUESDAY-POSTER PRESENTATIONS complementation experiments. We find that the post-mitotic neuronal migration defect seen in NudEL RNAi is largely rescued by NudE overexpression. Thus, NudE and NudEL may exhibit significant functional redundancy in the brain. Of further importance, the specific inhibition caused by NudE RNAi in INM, and the failure of NudE knockdown cells to reach the ventricle to divide, may play a substantial and previously unappreciated role in the clinical microcephaly reported in human patients with mutations in NDE1 (Alkuraya et al., Am J Hum Gen. 2011, 88:536-547). Supp. by NIH HD40182.

P1725 Board Number: B242

NuMA links the mitotic spindle with plasma membrane lipids. S. Kotak1, C. Busso2, P. Gonczy3; 1 ISREC/EPFL Lausanne, 1015, Lausanne, Switzerland, 2ISREC/EPFL, Lausanne, Switzerland, 3EPFL SV ISREC UPGON, Lausanne, Switzerland Correct positioning of the mitotic spindle during metaphase and spindle elongation during anaphase are critical for error-free mitosis. In human cells, the evolutionary conserved ternary complex components NuMA/LGN/Gαi1-3 anchor dynein at the cell cortex, thus ensuring metaphase spindle positioning. Whether the ternary complex also participates in cortical localization of dynein during anaphase was previously not known. Importantly, mechanisms by which mitotic spindle behaviour is coupled with mitotic progression remained elusive. We discovered that cortical levels of dynein markedly increase as cells transit from metaphase to anaphase, and this in a NuMA- and CDK1-dependent manner. We showed also that this increase of cortical dynein is crucial for proper spindle elongation during anaphase. Notably, we uncovered that LGN/Gαi1-3 are dispensable for NuMA-dependent cortical dynein enrichment during anaphase. In addition, we established that NuMA is excluded from the cortical equatorial region by the centralspindlin complex components CYK4 and MKLP1. Interestingly, we discovered that NuMA directly interacts with membrane phosphoinositides in vitro and in vivo, and that cortical localization of NuMA during anaphase is dependent on these lipids. In summary, our study uncovered a novel mechanism by which NuMA links cell cycle progression with proper spindle behavior by directly associating with membrane lipids to ensure faithful execution of mitosis.

P1726 Board Number: B243

The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1mediated inhibition. L.G. Lammers1, S.M. Markus1; 1 Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO Cortically anchored dynein motors orient the mitotic spindle through interactions with spindle pole anchored astral microtubules. In budding yeast, dynein is offloaded to cortical Num1 receptor sites from

TUESDAY-POSTER PRESENTATIONS the plus ends of dynamic microtubules. Rather than walking toward minus ends, dynein remains associated with microtubule plus ends due to its association with Pac1 (LIS1 homolog), a potent inhibitor of dynein motility. The mechanism by which dynein is switched from “off” at the plus ends to “on” at the cell cortex remains unknown. Here we show that overexpression of the dynein-interacting coiled-coil domain of Num1 (Num1CC) – which is insufficient for cortical anchoring – depletes dynein-dynactinPac1 complexes from microtubule plus ends, and reduces the extent of colocalization of dynein and Pac1. Num1CC-mediated depletion of dynein from plus ends requires an interaction between Num1 and dynein, and also requires the dynein microtubule-binding domain. Live cell imaging reveals minus enddirected motility of dynein molecules along astral microtubules upon Num1CC overexpression – an event that is never observed in wild-type cells. An enhanced Pac1-affinity mutant of dynein, or overexpression of Pac1 partly restores Num1CC-mediated depletion of dynein from plus ends, suggesting that Num1CC activates dynein-dynactin motility by inducing the release of Pac1. Our findings indicate that dynein activity can be directly switched “on” by Num1, which, upon binding to dyneindynactin complexes, may induce the removal of the Pac1/LIS1 inhibitor.

P1727 Board Number: B244

Budding Yeast She1 Restricts Loading of Ipl1/Aurora B onto the Spindle to Coordinate Spindle Integrity with Spindle Positioning. Y. Zhu1, K. Kalutkiewicz2, S.M. Markus3, W. Lee2; 1 Molecular Cellular Biology Graduate Program, Univ Massachusetts Amherst, Amherst, MA, 2Biology, Univ Massachusetts Amherst, Amherst, MA, 3Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO In budding yeast, the mitotic spindle protein She1 is a key regulator of dynein pathway activity that functions to dampen productive microtubule-cortex interactions in the mother cell in order to achieve polarized spindle movements toward the bud. While the role for She1 is well established in the cytoplasm, its role in the nucleus is poorly understood. Here, we show that She1 is required for proper timing of Ipl1/Aurora B recruitment onto the mitotic spindle to ensure spindle integrity for dyneindependent pulling from the cell cortex. Ipl1/Aurora B localizes in the nucleus with a diffusive pattern during metaphase and loads onto the mitotic spindle immediately prior to anaphase onset. Loss of She1 leads to premature loading and enhancement of Ipl1 on the spindle, whereas overexpression of She1 delays loading. In she1∆ metaphase spindles, enhanced Ipl1 resulted in a decreased level of microtubule-stabilizing +TIP protein Bim1 and an increased rate of anaphase spindle elongation due to cortical dynein activity. Additionally, she1∆ bim1∆ double mutant exhibited a synthetic broken spindle phenotype. The N-terminal conserved region of She1 is sufficient for restricting Ipl1, whereas the Cterminal domain is required for dampening cortical dynein. Our results establish a new role for She1 in ensuring spindle integrity through restricting Ipl1 in the nucleus, while promoting spindle positioning through cortical dynein.

TUESDAY-POSTER PRESENTATIONS

P1728 Board Number: B245

Development and characterization of a cytoplasmic dynein mutant mouse carrying a human Charcot Marie Tooth mutation. S. Nandini1, T. Sabblah1, N. Patwardhan1, A. Ledray1, V. Nascimento1, J. Pasos1, R. Love1, L. Ehler1, S.J. King1; 1 Burnett Sch. Biomed. Sci., Univ Central Florida, Orlando, FL Charcot Marie Tooth disease (CMT) is the most common inheritable peripheral neuropathy in the United States. Individuals with CMT experience a range of symptoms, ranging from mild loss of motor coordination and some muscle weakness to debilitating pain and muscle weakness, often leading to the need for therapeutic intervention. Dynein is an essential molecular motor that functions to transport cargos in all cells. Multiple human dynein mutations have recently been shown to cause a variety of neurological disorders, including CMT, suggesting that dynein transport along nerve axons may be more sensitive than in other cells to genetic and physical dysfunction. To test this hypothesis we developed and are characterizing a novel mutant mouse model that carries a human dynein mutation found in a hereditary form of CMT. We found that heterozygous dyneinCMT-/+ mice have motor skills behavioral phenotypes that are consistent with a loss of muscle strength and/or muscle coordination. The phenotypes we observed appear to be distinct and less severe than the motor skills phenotypes seen in Cra, Loa, and Swl mutant dynein mice, which do not have corresponding mutations in humans. Most interesting, homozygous dyneinCMT-/ dyneinCMT- mice have survived up to 9 months and counting, whereas Cra/Cra, and Loa/Loa mice die within 24 hours of birth. We have determined that homozygous dyneinCMT-/ dyneinCMT- mice have more severe motor skills behavioral phenotypes. With this mouse model in hand, it is now possible to study the effect of a known human disease causing DHC mutation on development, whole animal neurodegeneration, and axonal transport at the cellular level. In vitro analyses of pure mutant protein can now be performed without the confounding background of wildtype DHC dimers or mixed DHC/+ dimers. By correlating the behavioral phenotypes with the molecular characterization of the mutant dynein and cellular transport assays, we hope to generate a more comprehensive understanding of the key role dynein plays in neurological disease, ultimately leading to better human therapeutics including potential drug intervention strategies.

TUESDAY-POSTER PRESENTATIONS

P1729 Board Number: B246

Role of NDE1 in T cell effector functions. S. Nath1, S.F. Lee2, J. Artz3, S. Wong1, M. Poenie1; 1 Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, 2Neogenomics Laboratories, Fort Myers, FL, 3Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX Helper and cytotoxic T cells exert their effector functions mainly though focused secretion. This process is thought to be accomplished by clustering of secretory vesicles at the centrosome or microtubule organizing center (MTOC) and translocation of MTOC up to the site of contact (the immunological synapse or IS). Our studies and those of other have implicated dynein in the process of MTOC translocation. Here we have examined the role of the dynein-binding protein NDE1 in the process of MTOC translocation. We recently found that NDE1 is recruited to the immunological synapse when Jurkat T cells engage superantigen-coated Raji cells or when cells from an NK cell line (NK92) engage Daudi cells. In both the cases both NDE1 and dynein form a ring-like structure corresponding to the pSMAC of the immunological synapse. Immunoprecipitation of either dynein or NDE1 show that both proteins form a complex that includes Lis1. When expression of NDE1 was reduced using siRNA, MTOC polarization process was reduced nearly to levels of a negative control. Moreover, when NDE1 expression was reduced by siRNA, dynein failed to localize at the synapse. Depletion of NDE1 did not interfere with other aspects of T cell activation such accumulation of actin or phosphorylated LAT at the synapse or calcium signaling in response to stimulation with anti-TcR mAb. Localization of NDE1 was also examined in live cells using GFP-NDE1 chimeras. Interestingly, studies of GFP-NDE1 (GFP at the Nterminus) showed that the expressed construct failed to accumulate at the synapse, that it completely inhibited MTOC translocation, and that it displaced dynein from the synapse. On the other hand, expressed NDE1-EGFP (C-terminal EGFP) did accumulate at the synapse and did not interfere with either MTOC translocation or accumulation of dynein at the synapse. Dynein forms mutually exclusive complexes either with dynactin or with NDE1 and Lis1. These results combined with previous studies of Lis1 and dynein trapping using molecular traps show that the dynein-Lis1-NDE1 axis is required for MTOC translocation.

P1730 Board Number: B247

Characterization of Novel Splicing of Dynein’s Microtubule-Binding Domain. S. Bernard1, J. Wildonger2; Integrated Program in Biochemistry, University of Wisconsin-Madison, Madison, WI, 2Biochemistry, University of Wisconsin-Madison, Madison, WI 1

The distribution of mRNAs, proteins and organelles within cells depends on the activity of molecular motors. While the kinesin family of motor proteins includes many members that transport cargo to the

TUESDAY-POSTER PRESENTATIONS plus-ends of microtubules, dynein is the major cytoskeletal motor responsible for minus-end-directed transport along microtubules. We have identified novel alternative splicing of the microtubule-binding domain in the Drosophila dynein heavy chain gene (Dhc64C) encoding the motor subunit of dynein. This alternative splicing affects helices within the microtubule-binding domain that are predicted to directly interact with microtubules. These isoforms are expressed throughout Drosophila development and in the adult. To determine the biological function of alternatively spliced microtubule-binding domains, we are utilizing the CRISPR-Cas9 system to generate animals that selectively express either isoform. We are also analyzing the microtubule-binding domain of the heavy chain gene in other organisms to determine how this alternative splicing evolved. These studies of novel microtubule-binding domain splicing will serve to define the effect of microtubule-binding domain residues upon dynein function.

P1731 Board Number: B248

Initial Characterization of a Probe to Measure Microtubule Sliding Forces in Living Cells. P. Mooney1,2, M. Tomschik1, J. Gatlin1,2; 1 Department of Molecular Biology, University of Wyoming, Laramie, WY, 2Molecular Cellular Life Sciences Program, University of Wyoming, Laramie, WY Microtubules play critical roles during many cellular processes, often functioning directly in the transport and movement of various intracellular components. Microtubules can affect these movements actively by using energy stored in their lattice to push or pull, or passively by acting as a scaffold upon which motor proteins and their associated cargoes can travel. In either capacity, microtubules must bear loads imparted by forces. Thus, full understanding of microtubule function within the cell will require measuring and characterizing these forces. Toward this end, we have developed a genetically-encoded probe designed to measure the forces that slide microtubules relative to one another in living cells. This probe consists of a FRET-based tension sensor flanked on either side by microtubule binding domains derived from tau. Truncated versions of this probe decorate spindle microtubules homogeneously, in contrast to similar probes derived from the microtubule binding domain of ensconsin. Using in vitro microtubule gliding assays, we demonstrate that the full-length tension sensor can crosslink microtubules and that it is indeed sensitive to the forces that are generated between them. Future work will involve calibration of the tension sensor, development of single molecule imaging strategies, and construction of a spatial map of microtubule sliding forces in the mitotic spindle.

TUESDAY-POSTER PRESENTATIONS

P1732 Board Number: B249

Role of Dynactin in HTLV-1 Infection. T.M. Gibson1, R. Sperl1; 1 Biology, University of Texas of the Permian Basin, Odessa, TX The cytoskeleton is responsible for overall cell shape and structure, as well as movement and organization of cellular organelles. Motor proteins, which associate with components of the cytoskeleton, facilitate many different types of intracellular movement. This includes organization of organelles and movement of chromosomes during cell division. Cytoplasmic dynein is a motor protein complex that mediates some of the retrograde movement in the eukaryotic cell. Dynein is known to interact with an activator protein called dynactin, which has been proposed to be critical for most of cytoplasmic dynein’s activity. Evidence has shown that viruses, such as adenovirus and retroviruses, utilize the cytoskeleton for intracellular movement. One retrovirus termed Human T-cell Leukemia Virus1 (HTLV-1) exhibits microtubule-dependent movement. HTLV-1 is a retrovirus that can cause a rare blood cancer called Adult T-cell Leukemia (ADL). HTLV-1 movement toward the nucleus during the infection process is poorly understood. Our laboratory has evidence that HTLV-1 binds to the dynein motor protein complex. Therefore, we hypothesize that dynactin is required for HTLV-1 intracellular movement. To investigate the role of dynactin in HTLV-1 movement during infection, siRNA will be used to knockdown dynactin expression in HEK 293 T-cells. The dynactin siRNA is not tagged, therefore a GFPtagged GAPDH siRNA knockdown will be performed simultaneously to determine transfection efficiency of the dynactin knockdown. Next, the cells be infected with GFP-tagged HTLV-1, and infection and intracellular movement will be monitored using fluorescent microscopy. It can be predicted that HTLV-1 will not be able to properly attach to the dynein motor protein complex, resulting in little to no retrograde movement based on the fact that dynactin is required for most of dynein’s activity. Western blot analyses of knockdown cell lysis have revealed a decreased GAPDH expression. Additionally, quantitative analysis of these Western blots has shown a 90 – 95% reduction in GAPDH compared to control samples. If a dynactin knockdown results in limited intracellular movement of HTLV-1, these results will reveal new information about the mechanism used by viruses to maneuver throughout the host cell. Additionally, antivirals and therapeutics could be developed to stop this interaction, thus halting the viral cycle.

TUESDAY-POSTER PRESENTATIONS

P1733 Board Number: B250

VezA, a novel factor required for dynein-mediated early endosome transport. X. Yao1, H.N. Arst2, X. Wang3, X. Xiang1; 1 Department of Biochemistry and Molecular Biology, USUHS - F. Edward Hébert School of Medicine, Bethesda, MD, 2Microbiology Section, Department of Medicine, Imperial College London, London, UK, 3 Department of Plant Genetics and Breeding, College of Agronomy and Biotechnology, China Agricultural University, Beijing, China Cytoplasmic dynein transports organelles/vesicles and proteins/mRNA along microtubules. However, how dynein function is regulated in vivo remains unclear. We have been using Aspergillus nidulans as a model organism to dissect the regulation of dynein-mediated transport of early endosomes. Using a classical genetic approach, we have identified genes that are required for dynein-mediated early endosome distribution, and we recently reported the identification of A. nidulans HookA and FhipA proteins that link dynein-dynactin to early endosomes (Zhang et al., 2014; Yao et al., 2014). From the same genetic screen that allowed us to identify HookA and FhipA, we have recently identified VezA (vezatin-like protein in A. nidulans), another novel factor critical for dynein-mediated early endosome transport. The identification of the vezA gene was achieved by using a combination of classical genetic and whole genome sequencing approaches. The VezA protein contains a vezatin domain that shows weak similarity to the mouse vezatin protein and the budding yeast protein Inp2p. Vezatin in mammalian cells binds myosin VIIA to bring it close to the cadherin-catenins complex (KüsselAndermann et al., 2000), while the yeast Inp2p is a peroxisomal receptor for the myosin V protein Myo2p (Fagarasanu et al., 2006). Our current data show that, unlike HookA or FhipA that associate with early endosomes, VezA does not co-localize with early endosomes. In addition, VezA is not required for HookA-early endosome interaction, which is in contrast to the function of FhipA. We are in a process of further studying the mechanism of VezA action in dynein-mediated early endosome transport.

P1734 Board Number: B251

Functional analyses on the p25 and p27 components of dynactin in Aspergillus nidulans. R. Qiu1, J. Zhang1, X. Xiang1; 1 Department of Biochemistry and Molecular Biology, USUHS - F. Edward Hébert School of Medicine, Bethesda, MD Within the dynein-accessary complex dynactin, the two left-handed beta-helical proteins p25 and p27 locate at the pointed-end of the Arp1 mini-filament and are required only for a subset of dynein functions (Eckley et al., 1999; Lee et al., 2001; Schroer 2004; Parisi et al., 2004; Zhang et al., 2011; Yeh et al., 2012; 2013). In Aspergillus nidulans, dynein-mediated early endosome transport requires p25, which is necessary for dynactin-dynein to interact with the early endosome adapter protein, HookA (A.

TUESDAY-POSTER PRESENTATIONS nidulans Hook) (Zhang et al., 2011; 2014). In the vertebrate dynactin complex, p25 and p27 form a heterodimer (Eckley et al., 1999), and RNAi of p25 makes p27 unstable and vice versa (Yeh et al., 2012). Thus, whether it is p25 or p27 that is more directly involved in early endosome transport and HookAdynein-dynactin interaction would need to be addressed. Here we found that in A. nidulans, loss of p27 does not apparently affect p25-dynactin-dynein interaction or p25 protein level, but loss of p25 abolishes p27-dynactin-dynein interaction and reduces the level of p27. However, p25 function in early endosome transport is not mediated by p27 because loss of p27, unlike loss of p25, does not significantly affect HookA-dynein-dynactin interaction and dynein-mediated early endosome movement. We performed homology-based modeling of p25 structure and predicted that A. nidulans p25 contains, besides the core structure of the left-handed beta-helix core, two loop regions and a C-terminal alpha helix. We also identified several amino acid clusters whose side chains face outside of the core structure. Our functional analysis revealed three peripheral regions of p25 that contribute to dynein-mediated early endosome transport. These regions include Loop-1, the C-terminal region and the K36-Y83-H113 cluster. Importantly, these amino acids only play a minor role in p25-dynactin-dynein association but play a significant role in HookA-dynein-dynactin interaction. These data support the notion that within the dynactin complex, p25 is the protein specifically required for mediating HookA-dynein-dynactin interaction.

P1735 Board Number: B252

The manner by which dynactin regulates mammalian cytoplasmic dynein is dependent on the dynein intermediate chain isoforms. W. Smiley1, K. Blasier1, D.J. Mitchell1, K. Pfister1; 1 Cell Biology, Univ Virginia Sch Med, Charlottesville, VA Cytoplasmic dynein is a motor protein responsible for the movement of membrane bounded organelles and other cargo toward microtubule minus ends. The dynactin complex is associated with several of dynein’s functional properties. We previously reported that specific phosphorylation of the dynein intermediate chain isoforms (IC) was important for dynein binding to endosomes and that this phosphorylation did not modulate IC binding to the p150 subunit of dynactin. We therefore sought to directly examine the functional significance of the interaction of dynactin and dynein complexes for dynein motility in axons. We disrupted the interaction of dynactin and cytoplasmic dynein by overexpressing the first coiled-coil region (CC1) of the p150 subunit of dynactin in neurons. This domain competes with endogenous p150 for binding to the dynein IC, but does not dissociate the dynactin complex. Using live cell imaging of axons expressing fluorescent–tagged ICs, we found that there was significantly less movement of dynein containing IC-2C in the presence of CC1 than in the control. The motility of dynein containing IC-2C was significantly reduced in both directions of movement, 40% in the anterograde direction and 70% in the retrograde. These results are consistent with previous observations that disruption of the dynein-dynactin interaction reduces organelle motility. Importantly, we also observed that there was no inhibition of the motility of dynein complexes containing IC-1B in

TUESDAY-POSTER PRESENTATIONS either direction. We then examined the motility of two dynein cargoes. In the presence of CC1 we observed reduced motility of mitochondria, which we previously reported were transported by IC-2C containing dynein, but no inhibition of the motility of Rab7 containing endosomes, a cargo transported by IC-1B containing dynein. In the presence of CC1, we also observed a 33% decrease in run length for IC-2C dynein, but no effect on IC-1B dynein run length. These results demonstrate that when cytoplasmic dynein is detached from the dynactin complex in vivo, there are two distinct dynein motility responses which are correlated with specific dynein intermediate chain isoforms. The disruption of dynactin binding has minimal effects on the motility of dynein containing IC-1B or its cargo, while the disruption significantly inhibits the motility of dynein containing IC-2C and its cargo. Furthermore, the data strongly suggest that the interaction between the IC and p150 is not required for dynein containing IC-1B to bind to cargo, and that dynein containing IC-1B does not require signals transmitted through the intact dynactin complex (via p150) for normal motility. These data have important implications for studies of dynein populations that have mixtures of IC isoforms.

Microtubule Nucleation and Organization P1736 Board Number: B254

Microtubule asters grow to span a millimeter-sized egg by nucleation remote from centrosomes. K. Ishihara1, P. Nguyen1, A.C. Groen1, C.M. Field1, T.J. Mitchison1; 1 Department of Systems Biology, Harvard Medical School, Boston, MA The large cells in early vertebrate development face an extreme physical challenge in organizing their cytoplasm. For example, frog embryos are 1.2 mm in diameter but have to divide every 30 min. In these cells, radial arrays of microtubules called asters grow, interact and move to precisely position the cleavage plane. Although the rapid expansion of asters, spanning the entire cytoplasm, is required for cells to explore its size and shape, it is unknown how microtubules assemble at such large distances from their presumable nucleation centers or centrosomes. Here, we combine quantitative imaging and cell-free reconstitution to show that microtubule asters grow by microtubule nucleation away from the centrosome. Aster growth occurs in the absence of microtubule transport while plus ends undergo both polymerization and depolymerization. Further, we report that the embryonic interphase cytoplasm supports spontaneous microtubule assembly independent of centrosomes, consistent with previous reports in vivo. We propose that aster growth, initiated by the centrosome, is supported by microtubule nucleation and is stimulated by the presence of pre-existing microtubules.

TUESDAY-POSTER PRESENTATIONS

P1737 Board Number: B255

A new microtubule end-binding protein, NOCA-1, coordinates with patronin to control assembly of non-centrosomal microtubule arrays in C. elegans. S. Wang1, K. Oegema1, A. Desai1; 1 Ludwig Institute for Cancer Research, La Jolla, CA In contrast to the centrosomal microtubule arrays in dividing cells, many differentiated cells assemble non-centrosomal microtubule arrays adapted for specific cellular functions. In an RNAi screen in C. elegans, we identified NOCA-1 as a novel protein that does not contribute to centrosome-driven embryonic cell divisions but is required to form microtubule arrays in the germline that are essential for organismal fertility. The noca-1 gene encodes 8 isoforms expressed in a variety of tissues. Two distinct long isoforms control assembly of microtubule arrays in the germline and embryonic epidermis, respectively. In contrast, a short isoform functions in parallel to the microtubule minus end-binding protein Patronin (PTRN-1) to control assembly of microtubule arrays in the post-embryonic epidermis. Evidence for the redundant activity of NOCA-1 and PTRN-1 includes synthetic lethality and early larval stage dye permeability of noca-1Δ;ptrn-1Δ double mutants, both of which are rescued by selectively expressing PTRN-1 in the post-embryonic epidermis. In support of the genetic interaction, NOCA-1 cosediments with taxol-stabilized microtubules from worm lysates and purified recombinant NOCA-1, like PTRN-1, binds to microtubule ends in a microtubule-anchoring assay. Live imaging of the epidermal syncytium where NOCA-1 and PTRN-1 function redundantly revealed an array of evenly spaced microtubule bundles that run circumferentially around the animal. This array, comprised of both stable and dynamic microtubules, is subtly affected in single noca-1∆ and ptrn-1∆ mutants but nearly completely eliminated in noca-1Δ;ptrn-1Δ worms, suggesting that this specialized non-centrosomal array supports epidermal integrity during animal growth. We conclude that NOCA-1 represents a new class of microtubule end-binding proteins with essential functions of its own, as well as parallel functions with Patronin, in the assembly of non-centrosomal microtubule arrays in multiple C. elegans tissues.

P1738 Board Number: B256

A Detailed Centrosome Interactome Reveals a Novel Role for Asterless/CEP152 in Controlling Centriole Length Via Cep97. B.J. Galletta1, K.C. Jacobs2, C.J. Fagerstron2, K.C. Slep3, N.M. Rusan1; 1 National Heart Lung, and Blood Institute, NIH, Bethesda, MD, 2NHLBI-NIH, Bethesda, MD, 3Department of Biology, University of North Carolina, Chapel Hill, NC A critical gap in our understanding of how centrosomes are built and subsequently mature into microtubule organizing centers (MTOC) is the relative lack of information on the direct interactions

TUESDAY-POSTER PRESENTATIONS among the constituent proteins. To address this deficiency in our knowledge, we performed a directed yeast two-hybrid screen to identify the possible, direct interactions among 22 conserved Drosophila centrosome proteins. To provide additional detail about how centrosomal proteins interact, these 22 proteins were subdivided into smaller fragments, keeping known domains and predicted secondary structures intact. In total, 67 full-length proteins and subfragments were tested. We identified more than 100 interactions among these proteins, the majority of which are novel, significantly increasing our understanding of the protein-protein interaction network within the centrosome. With this new interaction information, we attempted to better understand the role of Asterless (Asl)/CEP152. Since Asl is essential for the duplication of the centrosome, the molecular mechanism of its function and if it plays roles in addition to centriole duplication remains somewhat unclear. We have studied the loss of asl in the male germline stem cells (mGSCs) of Drosophila melanogaster larvae, which reproducibly harbor Aslfree centrioles. These remnant centrioles were built during embryogenesis using maternally provided Asl, but by this stage no longer have any associated Asl protein. Amazingly, in the mGSCs of asl mutants, the remnant centrioles are 10 to 30 fold longer than in wild-type mGSCs. Examining the localization of all of the Asl interacting proteins identified in our screen, we found proteins that localize to the proximal end of the giant centrioles in wild type spermatocytes extend the entire length of the centriole in the mGSCs of asl mutants. Interesting, the localization of proteins along the long centrioles in asl mGSC appears unique in comparison to previously described long centrioles, suggesting they may arise by a novel mechanism. To understand the possible mechanism of centriole length control by Asl in mGSCs, we examined flies with a mutation in cep97, which has been suggested to regulate centriole length. These flies have long centrioles in mGSCs, similar to Asterless. This is consistent with a model where in mGSCs, Asl plays a role in controlling the length of centrioles via Cep97. To test this model we have engineered a centriole-targeted version of Cep97 and show that expression of this construct can partially rescue the length of centriole in asl mGSCs. Finally, we are exploring the consequences of loss of Asl and the resulting increased length of centrioles on the interphase positioning of centrioles in mGSCs.

P1739 Board Number: B257

From Yeast to Human: Kinesin Regulators of γ-TuRC Structure and Nucleation. Z.T. Olmsted1, T. Riehlman1, A. Colliver1, J.L. Paluh1; 1 SUNY Colllege of Nanoscale Sci and Eng, Albany, NY Members of two conserved mitotic kinesin-like protein (Klp) families, Kinesin-14 and Kinesin-5, have emerged as direct regulators of the γ-tubulin ring complex (γ-TuRC) microtubule organizing center (MTOC) in fission yeast and humans. While Klp members in these families are most studied for microtubule based organization roles in spindle assembly, a subset of members also localize to γ-TuRC at spindle poles to regulate microtubule nucleation. Genetic analysis, biochemical sedimentation of γTuRC complexes, co-immunoprecipitation assays and in vivo cell studies reveal a competitive mechanism between Kinesin-5 and Kinesin-14 that balances γ-TuRC structure and nucleation capacity.

TUESDAY-POSTER PRESENTATIONS We recently investigated details of the Kinesin-14 Pkl1 mechanism in fission yeast and the contribution of Kinesin-5 Cut7 to perturb the functional relationship between Kinesin-14 Pkl1 and γ-TuRC. We demonstrate that similar but counteracting forces by Kinesin-5 on γ-TuRC interfere with Pkl1 Motor and Tail domain interactions at γ-TuRC. We designed multiple unique Pkl1 Tail peptides as tools for capturing γ-TuRC or disrupting its structure by removal of γ-tubulin subunits (Cell Cycle 2013). This action is reversible and also occurs on γ-TuRC of rat and human centrosomes. New data from our lab further indicates that the normally essential fission yeast Kinesin-5 Cut7 motor protein becomes dispensable in the absence of Pkl1, emphasizing the importance of this antagonistic MTOC regulatory relationship. In yeast the action is asymmetric and additionally influences spindle microtubule numbers and microtubule overlap. We demonstrate by in vivo studies ihuman breast cancer cell lines that similar to as in yeast our Kinesin-14 peptides block microtubule nucleation from γ-TuRC. Previously we demonstrated in vivo that human Kinesin-14 HSET, but not Drosophila Ncd, functionally replaces Kinesin-14 Pkl1 in fission yeast iand more recently that the genes encoding human γ-TuSC proteins GCP2 and GCP3 fully replace fission yeast genes for the analogous Alp4 and Alp6 MTOC proteins (JCS 2013). Our research highlights conserved in vivo regulation of γ-TuRC that informs on its structure and capacity for nucleation and reveals novel underlying mechanisms.

P1740 Board Number: B258

In vitro reconstitution of efficient microtubule formation by the combined action of TPX2 and chTOG. J. Roostalu1, N.I. Cade1, T. Surrey1; 1 Cancer Research UK London Research Institute, London, United Kingdom Spindle assembly relies on a number of microtubule-associated proteins that contribute to microtubule organisation through microtubule crosslinking and transport, regulate microtubule dynamics and/or facilitate microtubule nucleation. TPX2 is a multi-functional microtubule-binding protein required for proper spindle formation in all higher eukaryotes. It is one of the key targets of the Ran pathway that is essential for chromatin-dependent microtubule assembly in Xenopus laevis egg extracts. Total spindle microtubule mass in Xenopus laevis is additionally controlled by the processive microtubule polymerases of the XMAP215/Dis1 protein family. The molecular mechanism of how these two proteins contribute to microtubule formation and whether they directly facilitate the nucleation process is not understood. Here we use a combination of microscopy-based in vitro reconstitution assays to elucidate the respective roles of human TPX2 and chTOG, the human ortholog of XMAP215/Dis1, in microtubule assembly. TPX2 is preferentially recruited to the microtubule lattice at growing microtubule ends by its central domain, where it prevents microtubule catastrophes without affecting microtubule growth rate. These properties are markedly different from the microtubule growth-promoting activity of the chTOG proteins. The microtubule stabilising effect of TPX2 is directly related to its function in microtubule nucleation: TPX2 also stabilises early sheet-like nucleation intermediates at the initial stages of microtubule assembly. Efficient tube formation and sheet closure, however, require the additional

TUESDAY-POSTER PRESENTATIONS presence of the microtubule polymerising activity of chTOG. Together, our results demonstrate that a combination of very distinct activities of two microtubule binders is both necessary and sufficient for efficient microtubule formation in solution. Furthermore, these findings provide important insight into the mechanism of microtubule nucleation stimulated by the Ran pathway during spindle assembly.

P1741 Board Number: B259

Genetic analysis of microtubule array organization in Arabidopsis hypocotyls. A.W. Elliott1, D. Thoms2, M. Kline2, S.L. Shaw2; 1 Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN, 2Department of Biology, Indiana University, Bloomington, IN Microtubules (MTs) organize through time and in space to affect critical processes such as cell division, morphogenetic events, and vesicle trafficking. Flowering plants produce distinct MT array patterns in the absence of centrosomes or defined MT-organizing centers. Highly dynamic cortical microtubule arrays are proposed to influence cell morphology through alignment of cellulose microfibrils in the cell wall. However, the mechanisms controlling the organizational states of these MT arrays remain largely unknown. Cortical MTs in Arabidopsis hypocotyl cells nucleate from γ-tubulin complexes distributed throughout the plasma membrane and remain laterally attached to the membrane. Redistribution of polymer via treadmilling leads to MT-MT interactions and formation of higher order structures such as MT bundles. We have used plant hormones to synchronously induce hypocotyl cells to reorganize the cortical MTs into a transverse co-aligned array on a 2 hr time scale. Reorganization initiates at the cell midzone and progresses bidirectionally to the apical and basal ends of the cell without transiting through an obligate intermediate pattern. To determine the genes required for this MT array patterning event, we initiated a forward genetic screen in Arabidopsis. EMS-mutagenized seedlings were screened for hypocotyl growth defects, including reduced hypocotyl length, radial cell expansion, and twisting cell files, and candidate mutants were rescreened for defects in hormone-induced transverse MT patterning. A systematic quantitative evaluation of cell shape and MT array pattern coupled with time-lapse imaging and supersensitivity tests for MT disrupting drugs is providing data for clustering phenotypes into distinct groups for complementation and epistasis tests. Five phenotypic groups have been initially identified with one group verified as new katanin (MT severing protein) alleles. A temperature-sensitive mutant class displays a novel radial-like MT array (termed pinwheel) with the focal point at the midpoint of the outer periclinal cell face. Pinwheel mutants fail to reorganize MT arrays to the transverse coalignment after hormone treatment and exhibit reduced axial hypocotyl growth following long-term exposure to hormones. Outcomes from the screen and analysis of pinwheel mutants will be presented.

TUESDAY-POSTER PRESENTATIONS

P1742 Board Number: B260

Linker Scanning Mutagenesis of Yeast Microtubule Nucleating Components Spc97 and Spc98. K. Fong1, J. Tien1, C. Payen2, A. Zelter1, B. Graczyk1, M. Dunham2, T. Davis1; 1 Department of Biochemistry, University of Washington, Seattle, WA, 2Department of Genome Sciences, University of Washington, Seattle, WA The mitotic spindle is a large structure responsible for the regulation and organization of chromosome segregation. Misregulation of chromosome segregation can lead to aneuploidy or genomic instability. Microtubules play several key roles during mitosis—mediating chromosomal movements, stabilizing spindle structure, and positioning the spindle within the cell. The γ-tubulin small complex is a highly conserved complex, essential for microtubule nucleation in all organisms. In budding yeast, the γ-tubulin small complex is localized to the spindle pole bodies and is comprised of two γ-tubulin molecules and one copy each of Spc97 and Spc98. Despite our advances in understanding microtubule function, dynamics and nucleation, purified γ-tubulin complexes fail to efficiently recapitulate nucleation in vitro, suggesting that additional regulatory processes activate the nucleating complex. Based on EM structure, we hypothesize that Spc98 undergoes a conformational change that enables the γ-tubulin small complex to serve as a template for microtubule growth. We have performed a saturating linker-scanning mutagenesis to define the regions of Spc97 and Spc98 required for the small complex to function in vivo and promote the formation of a microtubule. In vitro transposition reactions generated a library of random 15 base pair insertions in each gene, with approximately 90% coverage. This extensive transposition library was collected and transformed into yeast to screen for viability and conditional lethality using a plasmid shuffle. Mutants that abolish function identify the essential regions of the protein and mutants that are temperature sensitive can be studied after shifts to restrictive temperatures to examine nucleation activity. Over 50,000 yeast colonies were screened and 4% of the colonies contained lethal mutations and 0.02% contained temperature-sensitive mutations. Sites of lethal insertions were identified and mapped by high-throughput sequencing. Representative lethal mutations have been studied to determine the effects on protein expression, localization, and small complex formation. Temperature sensitive mutants were inspected by fluorescence microscopy and in vitro nucleation assays to reveal several nucleation hyperactive and deficient mutants. When combined with high-throughput sequencing, linker-scanning mutagenesis was a powerful approach to identify essential regions of the γ-tubulin small complex and provide novel temperature-sensitive mutants with specific defects in microtubule nucleation.

TUESDAY-POSTER PRESENTATIONS

P1743 Board Number: B261

MAP65 acts as a molecular yardstick to minimize microtubule overlap in Dictyostelium syncytia. M.P. Koonce1, K. Irizarry1, I. Tikhonenko1; 1 Wadsworth Center, Albany, NY The MAP65/Ase1/PRC1 family is known as bundling proteins important for managing regions of overlapping antiparallel microtubules during animal cell division and for crosslinking parallel and antiparallel microtubule arrangements during interphase in plants and fungi. Homodimers work in combination with kinesins and other factors to link adjacent microtubules in a compliant manner. We postulate here that the MAP65/Ase1/PRC1 family has an additional role in sensing and coordinating the elimination of antiparallel microtubule arrangements during interphase, a process important to maintain intranuclear spacing in syncytial cells. Dictyostelium cells often fail cytokinesis and form multinucleated cells that remain viable. A striking feature of these cells is that nuclei are generally dispersed in the cytosol and their associated microtubule arrays roughly occupy spatially distinct territories. Treatment of cells with nocodazole shrinks distances between centrosomes, indicating that microtubule length plays a role in array spacing, and suggesting that nuclear spacing is actively maintained. Dictyostelium contains two homologs of MAP65 (A,B); knockout of MAP65A results in a significant collapse of inter-centrosome distances and enhances microtubule array co-mingling. The average spacing between centrosomes in wild type binucleate cells is 7.2 μm, but is only 3.7 μm in the MAP65A knockout. Disruption of MAP65B accentuates cellular branching and formation of elongated microtubules. The overall growth rate of cells lacking either MAP65 isoform is similar to wild type. However, for the MAP65A knockout, there is a marked increase in multinucleated cells. We have previously shown that the interphase microtubule array in Dictyostelium is a highly dynamic structure whose position is influenced by dynein, Kinesin-4, and Kinesin-8 activities. Our current work indicates that MAP65 also plays a role in maintaining microtubule organization, in particular with separation of multiple interphase arrays in a common cytosol. Kinesins-4 and -8 are known to interact with MAP65/Ase1/PRC1 proteins and thus potentially could be targeted to microtubule overlaps to foster sliding or depolymerase activity that minimizes antiparallel microtubules. These roles would not only facilitate endomembrane management in complex environments, but also minimize the potential for misintegration of multiple centrosomes into the same nucleus during division. For this project, we are grateful to support from the National Science Foundation (IOS-1051612 and DBI-1062963).

TUESDAY-POSTER PRESENTATIONS

P1744 Board Number: B262

Identifying Pathways Directing Plant Cortical Microtubule Array Organization. D. Thoms1, A.W. Elliott2, R. Chaudhari1, A.J. Hill2, M.A. Kline1, S.L. Shaw1; 1 Department of Biology, Indiana University, Bloomington, IN, 2Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN The interphase microtubules at the plant cell cortex play a defining role in plant cell morphogenesis. Cell shape arises through a combination of forces, but relies heavily upon the mechanical properties of the cell wall. Cortical microtubules aid in patterning the extracellular deposition of cellulose microfibrils, the major structural fiber impacting wall mechanics. Cells undergoing anisotropic growth typically have coaligned microtubule arrays oriented transverse to the dominant growth axis suggesting that the anisotropic growth arises because of strongly oriented cellulose deposition. The molecular pathways that determine how the cell organizes microtubules into a transverse coaligned pattern have not been explicitly described. We discovered a hormone treatment that synchronously induces plant hypocotyl cells to form transverse coaligned microtubule arrays and resume anisotropic growth. To identify molecules involved in the related genetic pathways, a genetic screen is underway. Candidate mutants are classed based on statistical analysis of array pattern distributions, growth morphology, and chemical sensitivity to drugs targeting microtubule and cellulose synthase activity. Approximately 60,000 seedlings from 1350 mutant lines were screened, and 55 initial lines were identified as having a hypocotyl growth defect along with the inability to form transverse arrays. The majority of mutants show subtle hypocotyl growth phenotypes such as cells with slightly decreased cell anisotropy or twisting cell files. The most common phenotypes are found in mutants with disorganized, basket arrays, and normal cell anisotropy, but are hypersensitive to treatment with oryzalin. Other common phenotypes occur in mutants which form co-aligned array patterns other than transverse, but have significantly decreased cell anisotropy. Less common phenotypes are in mutants with dense and disorganized arrays, abnormal cell files and cell morphology, normal or slightly increased anisotropy, and varying sensitivities to oryzalin treatment. Rarer phenotypes are in mutants with a threefold increase in cell anisotropy in comparison to light grown wild type seedlings, and a novel array patterning consisting of multiple swirl-like regions of co-alignment in each cell. Single mutant phenotypes include an inefficient ability to bundle microtubules, a wispy microtubule defect with extreme oryzalin hypersensitivity, or a hypocotyl defect exhibiting projecting cell files when grown in 30°C with extreme oryzalin hypersensitivity. Future work includes full genomic sequencing to identify underlying mutations, epistasis tests, and localization studies of identified proteins during the formation of transverse arrays.

TUESDAY-POSTER PRESENTATIONS

P1745 Board Number: B263

Paternal Poc1 is Essential for the Function of the Zygote Centrosome. A. Khire1, S. Hynek1, E.D. Rodriguez1, T. Avidor-Reiss1; 1 Biological Sciences, University of Toledo, Toledo, OH Centrioles are conserved microtubule-based organelles that, together with pericentriolar material (PCM), form the centrosome. During oogenesis in animals, the centrioles are eliminated, and therefore, the ovum does not contribute centrioles to the zygote. Instead, centrioles are provided to the zygote by the sperm. However, compared to centrioles in somatic cells, in many animals, including Drosophila, the spermatozoa centriole has a different structure and/or protein composition. Recently, we showed that Drosophila spermatozoa provide, in addition to the giant centriole (GC), a centriole precursor, which we named the proximal centriole like (PCL). The PCL resembles a centriole precursor, but lacks the centriole hallmark of microtubules. In the zygote, the GC and PCL recruit maternally contributed PCM, and form a centrosome. This centrosome, which is found near the male pronucleus, forms astral microtubules that reach out to the female pronucleus. Then, the female pronucleus migrates along the astral microtubules until it fuses with the male pronucleus. Later, the GC and PCL duplicate and form the poles of the zygote’s spindles. Here, we report that the centriolar protein, Poc1, plays a role in fertilization in Drosophila. Poc1 is a conserved centriolar protein that is essential for centriole elongation and centrosome function. In an EMS screen, we identified a missense point mutation in poc1 that effects the recruitment of Ana-1 to the PCL, but does not effect Ana-1 recruitment to the GC or the length of the GC in the sperm. Males with this mutation, which preferentially affects the sperm PCL, have reduced fertility. This reduced male fertility associates with two distinct phenotypes in the zygote: failure in female pronucleus migration and formation of monopolar spindles. These findings demonstrate that poc1 is a paternal affect mutation due to dysfunction of the zygotic centrosome. It also suggests that the PCL is essential for zygotic centrosome function.

P1746 Board Number: B264

A Chlamydia effector manipulates host microtubule dynamics to promote bacterial survival. M. Dumoux1, A. Menny1, D. Delacour2, R. Hayward1; 1 Biological sciences, Institute of structural and molecular biology (Birkbeck college-UCL), London, United Kingdom, 2Institut Jacques Monod - CNRS, Paris, France Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes sexually transmitted disease worldwide and is the leading cause of acquired blindness (trachoma) in developing nations. Chlamydiae employ a type III secretion system to deliver virulence effector proteins into host cells. These effectors subvert fundamental cellular processes, allowing bacterial intracellular survival and replication within a specialized membrane-bound compartment termed an inclusion. We discovered

TUESDAY-POSTER PRESENTATIONS that C.trachomatis dramatically reorganizes the microtubule cytoskeleton within infected cells to form a filamentous superstructure. This comprises a microtubule scaffold surrounding the inclusion and an associated nest of microtubules that originate at the inclusion and extend towards the plasma membrane. Using a bioinformatics approach, we identified a chlamydial effector (inclusion protein acting on microtubules; IPAM) that shares limited sequence similarity with eukaryotic centrosomal and microtubule-binding proteins. Using pull-down and co-immunoprecipitation assays we demonstrate that IPAM, a hydrophobic effector known to associate with the inclusion membrane, interacts with host centrosomal protein 170kDa (CEP170), a centrosome and kinesin-interacting protein, via a domain exposed to the cytosol. Ectopic expression of this IPAM domain impairs microtubule dynamics. This defect was restored by simultaneous CEP170 knock-down, demonstrating functional interplay between IPAM and CEP170 in cells. CEP170 is essential for chlamydial control of host microtubule dynamics and organelle repositioning during infection, and is necessary for inclusion morphogenesis and bacterial infectivity. Moreover, as IPAM stimulates CEP170 functions in Chlamydia-infected cells, this reveals potential physiological roles of CEP170 repressed in non-dividing uninfected cells. Together our data show how a chlamydial type III secretion effector stimulates a host target to promote the reorganization of the microtubule cytoskeleton in infected cells, an event required for inclusion biogenesis and bacterial intracellular survival. In turn this provides broader insight into the control of cellular microtubule dynamics.

P1747 Board Number: B265

Role of osmotic pressure in the regulation of the cytoskeleton during mitosis. I. Dasgupta1, C. Koh1,2; School of Biological Sciences, Nanyang Technological University, Singapore, Singapore, 2 Mechanobiology Institute, National University of Singapore, Singapore, Singapore 1

The actin cytoskeleton plays an important role in the regulation of many cellular activities, including cell motility and cell division. The flat, adherent cells during interphase, assume a spherical shape at the onset of mitosis, termed as “mitotic cell rounding”. This change in cell shape is essential for the proper positioning and stabilization of the mitotic spindle, thus ensuring chromosome segregation with high fidelity. The remodeling of the actin cytoskeleton and the ability of the cell to regulate its osmotic pressure are among the key forces that primarily govern this cell rounding. A balance between the outward directed osmotic pressure and the inward actomyosin contraction is instrumental in driving this process. The ERM (Ezrin, Radixin, Moesin) family of proteins, known to cross-link the plasma membrane with the underlying actin cortex, are associated with mitotic cell rounding. Previous reports suggest the ERM proteins, upon phosphorylation and activation during mitosis are redistributed towards the cell cortex, thereby contributing to the cortical rigidity. Our results show an upregulation in the p-ERM levels and increase in the cortical rigidity of the mitotic cells upon exposure to a hypertonic environment. The actin filaments in the cells undergo realignment and distribution to the spherical cortex during mitosis. The interaction of the cortical actin with the astral microtubules that radiate towards the cell cortex

TUESDAY-POSTER PRESENTATIONS from the spindle poles generates tensile forces, thus favoring proper spindle assembly. Exposure to the hypertonic stress resulted in defects in the astral microtubule arrangement and centrosome integrity. The impaired astral microtubule arrangement was restored to normal when the cells were recovered in an isotonic medium, following a hypertonic shock. We further observe that the increase in the astral microtubules is directly proportional to the duration of the hyperosmotic shock. Perturbation of the osmotic gradient also leads to a significant decrease in the length and width of the mitotic spindle, as well as shrinkage of the cortical actin. Thus, our studies suggest the existence of possible functional links between cytoskeletal regulators and proteins modulating the osmotic pressure in cells.

P1748 Board Number: B266

Bayesian analysis of FRET to quantitatively measure microtubule nucleation in vivo and in vitro. B. Kaye1, D.J. Needleman2,3,4; 1 Applied Physics, Harvard University, Cambridge, MA, 2FAS Center for Systems Biology, Harvard University, Cambridge, United States, 3School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 4Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA The mitotic spindle is a self-organizing structure that segregates chromosomes during cell division. The spindle is composed of microtubules, and microtubules are composed of the protein tubulin. Understanding microtubule nucleation is critical to understanding the mitotic spindle. Microtubule nucleation can be inferred from the temporal dependence of the amount of tubulin in polymer. We use FRET, a technique to probe protein-protein interactions, to measure the amount of tubulin in polymer in cell extracts. To detect FRET, we measure the lifetime of the excited state of the donor fluorophore, a technique commonly referred to as FLIM. We developed a Bayesian framework for analyzing FLIM measurements that rigorously accounts for all known experimental complications. This novel technique will allow us to quantitatively measure microtubule nucleation in situ and dissect the biophysics, biochemistry and cell biology of microtubule nucleation.

P1749 Board Number: B267

Mechanisms controlling the microtubule organizing center state of the cell. J.L. Feldman1; 1 Biology, Stanford Univ, Stanford, CA The centrosome is the major microtubule organizing center (MTOC) in dividing animal cells. In many types of differentiated cells, however, MTOC function is reassigned to non-centrosomal sites. We are using C. elegans intestinal cells as a model to study non-centrosomal MTOCs. The apical surfaces of intestinal cells become MTOCs during polarization: microtubule-nucleating proteins that are centrosome

TUESDAY-POSTER PRESENTATIONS components in dividing cells become localized apically and apical membranes nucleate the assembly of microtubules. Concurrently, the centrosome is turned off as an MTOC in polarized cells; centrosomes lose pericentriolar material, microtubule association, and microtubule nucleating capacity. Thus, intestinal cells switch from a centrosome MTOC during mitosis to an apical MTOC during polarization. A subset of intestinal cells divides following polarization. In these cells, the apical MTOC is removed and the centrosomal MTOC is reactivated, indicating that cells can switch in either direction between MTOC states but do not maintain both. To determine how the cell “chooses” its MTOC state, we tested dominance of MTOC states by fusing a polarized cell with a mitotic cell in developing embryos. Cell fusion experiments indicate that mitotic cytoplasm is capable of dictating the MTOC state: fusing a mitotic cell with a polarized cell results in the rapid loss of MTOC components from the apical surface and the growth of MTOCs at normally quiescent centrosomes. We are now pursuing the mechanism that “flips the switch” in cellular MTOC state and will present our results.

P1750 Board Number: B268

A Novel Proximal Centriole-Like (PCL) Structure Becomes the Second Centriole in the Zygote of Drosophila melanogaster . A. Khire1, S. Blachon2, E. Fishman1, T. Avidor-Reiss1; 1 Biological Sciences, University of Toledo, Toledo, OH, 2Hybergenics, Paris, France Centrioles are conserved organelles that form centrosomes, or microtubule organization centers that are essential for cell division and function. As a result, they are essential for fertilization, development, and physiological functions. During oogenesis in animals, centrioles are lost, and thus, the oocyte does not contribute any centrioles to the zygote, the first cell of the embryo. Instead, it has been reported that zygotic centrioles are inherited from the sperm. In animals like sea urchins, frogs, and Caenorhabditis elegans, the sperm provides two centrioles, which is the normal number of centrioles in a cell. In contrast, in humans and in most mammals, the sperm contributes only a single centriole. This reduced contribution is due to a process known as “centrosome reduction,” in which one of the spermatid’s two centrioles degenerates during the last phase of spermatogenesis. Thus, the origin of the second zygotic centriole is uncertain. Like humans, Drosophila melanogaster sperm contains one centriole (Giant Centriole/GC). However, we have recently discovered that Drosophila melanogaster sperm contains a centriolar structure that is composed of centriolar proteins but lacks the structural characteristics of a centriole; we have named it the Proximal Centriole-Like (PCL). Based on the PCL’s presence, we propose the PCL Hypothesis, which states that the PCL is a second zygotic centriolar structure. Here we report that like the GC, the PCL also loses many of its components during the late stage of spermiogenesis. Because of centrosome reduction, centriolar proteins do not label the PCL in the sperm and therefore, we indirectly visualize the PCL and GC by their recruitment of centrosomal markers in the fertilized zygote. Immediately after fertilization, we find two centriolar structures, the GC and the PCL. As expected from the PCL hypothesis, we find that early maternally contributed centriolar proteins like Sas 4 and Sas-6, do not

TUESDAY-POSTER PRESENTATIONS label the first two-centriolar structures of the zygote, but do label their daughter centrioles. Lastly, as the PCL hypothesis predicts, we found that zygotes lacking Asterless, which is essential for centriole duplication, have two centriolar structures. Altogether, we have found that the PCL is a centriole precursor in the sperm, which functions as the second zygotic centriole in Drosophila melanogaster. The PCL hypothesis may be applicable to other animals; it would explain why only three centrioles have been observed in a mammalian zygote during mitosis, despite the requirement of four centrioles for normal mitosis. Further studies aim to identify the range of animals with a PCL, to identify the specific role of the PCL in the zygote, and to identify the molecular mechanism underlying PCL function

Ciliopathies P1751 Board Number: B270

Deficiency of tuba, a cdc42 GEF, disrupts normal zebrafish ciliogenesis and kidney development. J. Baek1, S. Choi1, X. Zuo2, J.H. Lipschutz2; 1 Med Univ South Carolina, Charleston, SC, 2Department of Medicine, Medical University of South Carolina, Charleston, SC Background: Dysfunction of renal primary cilia leads to polycystic kidney disease (PKD). The exocyst, a highly-conserved eight-protein membrane trafficking complex, is essential for ciliogenesis, and is regulated by Rho and Rab family GTPases, such as cdc42. We previously showed that cdc42 deficiency disrupts renal ciliogenesis and causes PKD in zebrafish and mice, and that Tuba, a cdc42-specific GEF, is necessary for ciliogenesis in cultured renal tubular MDCK cells. Methods: To determine how tuba affects ciliogenesis and kidney function in vivo, tuba knock-down was performed in zebrafish by microinjection of a translation blocking morpholino (MO). The phenotypic and histological defects caused by tuba deficiency were analyzed using whole-mount in situ hybridization, H & E staining and immunofluorescence. Results: Tuba was expressed in several tissues containing primary cilia, including the brain, neuromast cells, and renal tubules. We found that tuba morphants phenocopied cdc42 morphants, with ciliary mutant phenotypes including: a curly tail, hydrocephalus, and abdominal fluid accumulation. These defects were rescued by human tuba mRNA, which was resistant to the morpholino due to a difference in primary base pair structure, indicating that the phenotype was due to tuba deficiency, and not off-target effects. In the tuba morphant kidney, pronephric cilia were short and disordered, and the glomeruli were also disorganized. Because tuba is a known GEF of cdc42, and tuba and cdc42 morphants shared the same phenotype, we next performed a genetic synergy experiment in which we knocked down both tuba and cdc42. Co-injection of tuba and cdc42 morpholinos at low concentrations, which individually resulted in no phenotype, caused a severe phenotype, suggesting that tuba and cdc42 act in the same pathway. Conclusion: Our study showed that tuba deficiency causes an abnormal renal ciliary phenotype in zebrafish, demonstrating that tuba plays a critical role in

TUESDAY-POSTER PRESENTATIONS ciliogenesis and kidney development in zebrafish. We are currently generating kidney-specific tuba knockout mice to confirm these results in metanephrogenesis.

P1752 Board Number: B271

The Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) regulates Wnt signaling pathways. A. Caceres1, D.S. Wheeler1, S. Barrick1, Y. Yang1, D.B. Stolz2, M. Tsang3, P.A. Friedman1, G.G. Romero1; 1 Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, 2Cell Biology, Univ Pittsburgh Sch Med, Pittsburgh, PA, 3Developmental Biology, Univ Pittsburgh, Pittsburgh, PA The Na+/H+ Exchanger Regulatory Factor (NHERF1) is a scaffolding protein of the PSD-95/Discs-large/Zo1 (PDZ) family. NHERF1 contains two tandem PDZ domains through which it mediates protein-protein interactions. Mice that do not express NHERF1 present various pathological phenotypes including hydrocephalus associated to disorganized ependymal cilia and reduced number of ciliated cells. Our studies demonstrate that these phenotypes can be explained by deregulated Wnt signaling in the absence of NHERF1. We show that NHERF1 binds a subset of Frizzled receptors (Fzd) and inhibits activation of β-catenin. NHERF1-/- animals have increased levels of nuclear -catenin in the ependyma. We further demonstrate that NHERF1 binds the Planar Cell Polarity proteins Vangl1/2 and promotes their traffic to the plasma membrane, and in particular to the apical surface of ependymal cells. Furthermore, we show that NHERF1 assembles a ternary complex containing Fzd4 and Vangl2. We hypothesize that NHERF1 is a “signaling switch” that regulates the input to the canonical and noncanonical Wnt signaling pathways by promoting the assembly of multimolecular complexes containing Fzd receptors and PCP proteins. Funding: This work was supported by NIH Grants DK079864 (G.R.), DK069998 (P.A.F.), T32GM008424 (Molecular Pharmacology Training Grant: A.C. and D.S.W.), F30DK083211 (D.S.W.), and by internal funds from the University of Pittsburgh School of Medicine.

P1753 Board Number: B272

Functional studies of NPHP5, a novel centrosomal protein deficient in human ciliary diseases. M. Barbelanne1,2, W. Tsang2,3,4, J. Song2, M. Ahmadzai2; 1 Medicine, University of Montreal, Montreal, PQ, 2Institut de recherches cliniques de Montréal (IRCM), Montreal, QC, 3University of Montreal, Montreal, PQ, 4McGill University, Montreal, PQ The purpose of this project is to understand the regulatory mechanisms of centrosomes and cilia function. Centrosomes are small organelles composed of two centrioles surrounded by a pericentriolar

TUESDAY-POSTER PRESENTATIONS matrix. They are the major microtubule-organizing centers in mammalian cells and regulate diverse cellular processes such as cell division. In addition, centrosomes promote the formation of cilia, cellular protuberances on the surface of quiescent cells critical for signal transduction. Centrosome and cilia dysfunction are linked to a wide variety of human diseases such as retinal degeneration or polycystic kidney disease. I am studying the function of one novel centrosomal protein named NPHP5. First, mutations in NPHP5 have been identified in patients suffering from Senior-Løken syndrome or Leber congenital amaurosis; two diseases with renal and/or retinal failure. Second, NPHP5 mRNA is upregulated in gastrointestinal cancer but down-regulated by p53 after DNA damage; and its depletion causes pronephric cysts in zebrafish embryos. Finally, NPHP5 interacts with another centrosomal protein involved in ciliary diseases, Cep290. Despite its importance in health and disease, the precise function of NPHP5 is not fully understood. Our hypothesis is that NPHP5 cooperates with Cep290 to modulate cilia function and that its dysfunction contributes to the development of ciliary diseases. Using indirect immunofluorescence, NPHP5 was shown to localize to the distal region of centrioles in interphase cells. I have also demonstrated by RNA interference, immunofluorescence and immunoblot that NPHP5 depletion inhibits the migration of centrosomes to the cell surface, an early step of cilia formation. Using mapping studies and immunoprecipitation experiments, it was determined that NPHP5 interaction with Cep290 via its C-terminal region is critical for ciliogenesis. During those mapping studies, I also showed that the Cep290- and CaM-binding domains of NPHP5 are separable. Both domains are also distinct from the centrosomal localization domain, which contains the coiled-coil motif. Furthermore, it was observed by immunofluorescence that NPHP5 interaction with CaM prevents its self-aggregation. It was also demonstrated that disease-causing mutations of NPHP5 lack binding to Cep290 and are mis-localized from centrosomes, thus rendering the resulting proteins non-functional. Finally, using a pharmacological approach to modulate downstream events in the ciliogenic pathway, I showed that cilia formation could be rescued in the absence of NPHP5. These studies will improve our understanding of the biology of centrosomes and cilia and can lead to the development of new diagnostic and therapeutic applications.

P1754 Board Number: B273

The coiled coil domain containing proteins CCDC39 and CCDC40 are required for assembly of the nexin-dynein regulatory complex and affect tubulin polyglutamylation. H. Lin 1, Z. Zhang1, S.K. Dutcher1; 1 Genetics, Washington University School of Medicine, Saint Louis, MO The proteins, CCDC39 and CCDC40, were identified first as causative mutations in primary ciliary dyskinesia patients (Antony et al., 2013). In Chlamydomonas, the pf7 and pf8 alleles are causitive mutations in CCDC40 and CCDC39, respectively, based on reversion and rescue experiments. The Chlamydomonas mutations result in short, paralyzed flagella in which a fraction of the outer doublets fall into the center of the axoneme by electron microscopy as was observed in the PCD patients. CCDC39 and CCDC40 were hypothesized to be part of the nexin-dynein regulatory complex (N-DRC) based on the

TUESDAY-POSTER PRESENTATIONS patient phenotypes. To test this hypothesis, we used sliding assays and immunoblots to N-DRC proteins in Chlamydomonas. Isolated wild-type axonemes incubated with 0.1mM ATP slide along their entire length after treatment with protease, but show no sliding with just 0.1mM ATP. Axonemes from pf7 and pf8 with 0.1mM ATP and no protease show splaying of the microtubules, but remain attached at the proximal end as has been observed for other N-DRC mutants (Bower et al., 2013). By immunoblots, the CCDC39 protein is absent in isolated pf7 and pf8 axonemes. Immunoblots with antibodies to N-DRC proteins (DRC1, 2, 3, 4, 5, 7, and 11) show a reduction or absence in isolated single and double mutant axonemes (pf7, pf8 and pf7; pf8), which demonstrate that the mutational loss of CCDC39 and 40 is associated with the loss/reduction of known N-DRC proteins. Several lines of evidence suggest that CCDC39 and CCDC40 play an additional and novel role in tubulin polyglutamylation. The ssh1 allele was identified as a suppressor of mutants lacking dynein arms that have short flagella (LeDizet and Piperno, 1985). Surprisingly, ssh1 did not act as a suppressor of the short flagellar length in pf7 and pf8 strains, but further reduced flagellar length. The ssh1 allele is a mutant in TPG2, which is needed for localization of the TTLL9/TPG1 polyglutamylase (Kubo et al., 2013; 2014). By electron microscopy, a fraction of the B-tubules fail to close properly in pf7 and pf8 axonemes. This phenotype is observed previously in various mutants that reduce polyglutamylation. Supporting these two lines of evidence, mutants in CCDC39 and 40 show a significant reduction in tubulin polyglutamylation in isolated axonemes by immunoblots. These results suggest that CCDC39 and 40 may function or alter the tubulin polyglutamylation pathway. We are using a temperature-sensitive ccdc39 allele identified by whole genome sequencing to investigate the effects of loss of CCDC39 on IFT and the TPG proteins during temperature shifts and in dikaryons. In summary, CCDC39 and CCDC40 play roles in assembly of the NDRC and in interactions with the tubulin polyglutamylation pathway

P1755 Board Number: B274

The human ciliopathy protein Jbts17 is required for basal body docking and intraflagellar transport. M. Toriyama1, P.K. Jackson2, J.B. Wallingford1; 1 Molecular Biosciences, University of Texas at Austin, Austin, TX, 2Department Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA Ciliopathies are a broad class of human disease with a common etiology involving defective cilia structure or function. Jbts17 (C5orf42) is a recently identified gene that has been found to be mutated in human patients with Joubert, Meckel-Gruber, and Oral-Facial-Digital Type VI syndromes, but its cellular and molecular functions have yet to be characterized. Here, we report the expression and functional analysis of Jbts17 in Xenopus embryos and elucidate the pathogenic mechanism of human ciliopathy diseases associated with Jbts17 mutations. We find that Jbts17 is highly expressed in neural tissues and multiciliated cells (MCCs), and the expression of sonic hedgehog target genes, which is mediated through primary cilia function, is attenuated in Jbts17 knockdown embryos. These embryos also exhibit several developmental abnormalities, including defective neural tube closure, eye formation, and

TUESDAY-POSTER PRESENTATIONS embryonic growth. We follow up by demonstrating that Jbts17 is strongly localized at basal bodies in MCCs and that Jbts17 knockdown cells are impaired in the capacity to dock basal bodies to the apical membrane. We also identified the specific domain required for Jbts17 basal body localization, which is truncated in some human ciliopathy patients. Finally, the normal expression and localization of Jbts17 at basal body is required for the basal body localization of both Inturned and Rsg1, proteins that are involved in basal body docking and intraflagellar transport. Our study provides direct evidence for the role of Jbts17 in ciliogenesis and the pathogenic mechanism of its associated human ciliopathy.

P1756 Board Number: B275

Cdc42 and sec10 are required for normal retinal development in zebrafish. S. Choi1, J. Baek1, X. Zuo1, J. Dunaief2, J.H. Lipschutz1; 1 Department of Medicine, Medical University of South Carolina, Charleston, SC, 2Department of Medicine, University of Pennsylvania, Philadelphia, PA Cilia are essential organelles required for a broad spectrum of functions, including left-right patterning, nephrogenesis, and vision. In the eye, development/maintenance/function of the photoreceptor requires transport of vesicles containing ciliary proteins from the Golgi to the outer segment by a modified cilium, called the connecting cilium. We previously demonstrated that the exocyst, a highlyconserved eight-protein complex, is required for renal ciliogenesis, most likely due to its role in targeting and docking vesicles carrying ciliary proteins. We also showed that Cdc42, a small GTPase, localizes with the exocyst at cilia, and biochemically interacts with Sec10, a crucial exocyst component. In this study, we examined the function of cdc42 and sec10 in eye development in zebrafish. Cdc42 and sec10 knockdown in zebrafish resulted in both abnormal eye development and increased retinal cell death. Cdc42 morphants had a relatively normal retinal structure, aside from the absence of most connecting cilia and outer segments, while in sec10 morphants, much of the outer nuclear layer, which is comprised of the cell bodies of photoreceptor cells, was missing and retinal-pigmented epithelial cell thickness was markedly irregular. Consistent with the proposed mechanism, knockdown of cdc42 and sec10 resulted in an intracellular transport defect affecting retrograde melanosome transport. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 act in the same pathway in retinal development. Based on these data, we propose a model whereby sec10 and cdc42 play a central role in trafficking ciliary proteins to the outer segment of photoreceptor cell for ciliogenesis and phototransduction.

TUESDAY-POSTER PRESENTATIONS

P1757 Board Number: B276

HDACs and their role in Polycystic Kidney Disease. P. Paul1; 1 Stowers Institute for Medical Research, Kansas City, MO Autosomal-dominant polycystic kidney disease (ADPKD) is a monogenic renal disease characterized by progressive development of bilateral renal cysts, decline in renal function and many extrarenal effects. ADPKD has a very high worldwide prevalence rate of 1:400 to 1:1000 and it typically results in end-stage renal disease. ADPKD is caused by mutation in one of the two genes PKD1 and PKD2. PKD1 encodes polycystin-1 (PC1), which is a large G-protein coupled receptor-like protein and PKD2 encodes polycystin-2 (PC2), which is a TRP calcium channel. PC1 can interact with PC2 via its C-terminal coiledcoil domain to form a calcium permeable mechanosensor. Fluid flow on renal epithelium cell stimulates calcium influx, which activate kinases like protein kinase C. Our lab has showed that this kinase then directly or indirectly phosphorylates the 14-3-3 binding sites on histone deacetylase 5 (HDAC5) and causes nuclear export of HDAC5. This leads to the derepression of MEF2C target genes, one of which is missing in metastasis. It has been shown that pan-HDAC inhibitors can reduce cyst formation in PKD2-/mouse embryos supporting the important role HDACs play in ADPKD pathogenesis and thus in maintaining the normal organization of epithelial cells in kidney tubules. Further we confirmed that HDAC5 knockout can rescue polycystic kidney phenotype in adult PKD1 mouse model. Preliminary data suggest that even HDAC5 heterozygotes have significantly less percentage of cyst area and improved renal function compared to HDAC5 wild-type in a PKD1 deletion background. This further emphasizes the potential clinical implication of using specific HDAC inhibitors to reduce the progression of renal cyst in ADPKD patients. To better understand the mechanism of epigenetic regulation of cyst formation in ADPKD and to find novel therapeutic targets we wanted to understand the biochemical complex that HDAC5 forms in renal epithelium cells. HDAC5 forms complex with a class I HDAC, HDAC3 for its enzymatic activity. So a good therapeutic approach is to find specific inhibitors for HDAC3. To find drugs that can modulate the nuclear-cytoplasmic shuttling of HDAC5 we performed a screen, using a small pool of FDA approved compounds with well characterized targets involved in diverse signaling pathways using HDAC5 export as a paradigm. These studies together will give us mechanistic insights and also has the potential to identify novel therapeutic targets for ADPKD.

TUESDAY-POSTER PRESENTATIONS

P1758 Board Number: B277

Loss of ciliary gene, Thm1, reduces expression of anorectic POMC, causing hyperphagia-induced obesity, fatty liver disease, diabetes, and hypertension. D.T. Jacobs1, M.P. Schonfeld1, L.M. Silva1, A. Chatterjee1, G.C. Talbott2, D.R. Beier3, P.V. Tran1; 1 Anatomy and Cell Biology, Univ Kansas Med Ctr, Kansas City, KS, 2Genetics Division, Brigham and Women's Hospital, Boston, MA, 3Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, WA Obesity and metabolic syndrome are serious worldwide epidemics with treatments that are costly, invasive and largely ineffective. Primary cilia are antenna-like structures present on most vertebrate cells that mediate signaling pathways. Cilia defects cause ciliopathies, which manifest clinical features that are also common in the general population, such as obesity. The ciliary gene, Thm1, negatively regulates Hedgehog signaling and is most commonly mutated in patients with ciliopathies. We examined a postnatal role for murine Thm1 in obesity using a Thm1 conditional knock-out (cko) allele together with a ubiquitous, tamoxifen-inducible Cre recombinase. Thm1 deletion during adulthood caused a two-fold weight gain relative to control mice over a 13-week period. In adult Thm1 cko mice, adipose depots were significantly larger than in wild-type, and Thm1 cko livers had numerous, large lipid droplets, indicative of fatty liver disease. Results from serum glucose, leptin and insulin levels, as well as glucose tolerance tests indicate a diabetic state in Thm1 cko mice. Thm1 cko mice are also hypertensive relative to control mice. These observations indicate metabolic distress or ‘metabolic syndrome’. To determine if the 19% increase in food intake observed in Thm1 cko mice caused the obese phenotype, Thm1 cko mice were pair-fed for 13 weeks following Thm1 deletion. Indeed, body weights and fat depots of pair-fed Thm1 cko mice were similar to controls, indicating hyperphagia is a primary cause of obesity. In the Thm1 cko hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity, we observed stunted primary cilia and reduced levels of anorectic proopiomelanocortin (POMC) transcripts. These data indicate that THM1 regulates response to satiety signals. To examine this role further, we are performing a genome-wide expression analysis on extracts of Thm1 cko arcuate nucleus using RNA sequencing. We are also examining the role of Thm1 in mouse hypothalamic cell lines using lentiviruses expressing Thm1 shRNA. Our data show that primary cilia are important in regulating energy homeostasis and provide an opportunity to identify therapeutic targets against the onset of obesity.

TUESDAY-POSTER PRESENTATIONS

P1759 Board Number: B278

Loss of Occludin caused mucociliary clearance defect with reduced number of multiciliated cells in mouse. E. Herawati1, H. Tanaka1, K. Tateishi1, A. Tamura1, S. Tsukita1; 1 Graduate School of Frontier Bioscience and Graduate School of Medicine, Osaka University, Osaka, Japan This study aims at characterizing phenotype of mice lacking occludin, a transmembrane protein exclusively localized at tight junction strands, with particular reference to the respiratory system. In vivo data were acquired from previously generated Occludin knock out mice in trachea. On the other hand, in vitro data were collected from either wild-type or knock out derived mouse tracheal epithelial cells (mTEC) in culture. Occludin localizes at tight junction (TJ) in mTEC, the expression level of which was markedly increased in FOXJ1 positive cells until cells generated mature cilia. Occludin knock out mouse displays reduced number of the ciliated cells in trachea and accompanied by excess mucus plug in nasal cavity, suggesting a defect in mucocilary clearance function. Although the differentiation of trachea epithelial cells are normal, the detail analysis using electron microscopic images revealed that basal bodies of the ciliated cells were often incompletely docked. Further investigation is currently underway to examine the regulatory mechanism of how occludin is involved in maintaining normal ciliogenesis.

P1760 Board Number: B279

Repurposing of targeted cancer therapies for autosomal dominant polycystic kidney disease (ADPKD). A.S. Nikonova1, T. Seeger-Nukpezah2, H. . Hensley1, A. Klein-Szanto3, B. Egleston1, E.A. Golemis1; 1 Developmental Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, 2'Department of Internal Medicine, Center of Integrated Oncology, University of Cologne, Cologne, Germany, 3Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA Autosomal dominant polycystic kidney disease (ADPKD) is an inherited progressive genetic disease that affects 1 in 500 people. With typical onset in middle age, with normal kidney tissue is gradually replaced with fluid-filled cysts resulting in end-stage renal disease for most patients. ADPKD is caused by mutational inactivation of polycystins 1 or 2 (PC1 and PC2, encoded by PKD1 and PKD2), which abnormally activates multiple signaling pathways regulating cell proliferation, migration, and response to environmental cues. A number of these signaling pathways are also activated in cancer, and targeted drugs are available to inhibit these pathways. There are currently no approved therapies to slow or cure ADPKD. We have been exploring inhibitors of Aurora-A, EGFR, and HSP90, using mouse models for ADPKD. We have previously reported that HSP90 inhibition with the small molecule STA-2842 in Pkd1-/mice reduces initial renal cyst formation and slows the progression of these phenotypes in mice with

TUESDAY-POSTER PRESENTATIONS pre-existing cysts. We now find similar effects in Pkd2-/- mice. This is accompanied with defects in ciliogenesis and striking changes in signaling. In contrast, treatment of Pkd1-/- mice with the Aurora-A kinase inhibitor MLN8237 (alisertib) increased the rate of cystogenesis in these mice. This was associated with defective ciliary resorption and morphological abnormalities of cilia. Intriguingly, while the EGFR inhibitor erlotinib had little independent effect on cystogenesis in Pkd1-/- mice, combination of this agent with alisertib strikingly reduced alisertib-dependent promotion of cyst growth. The mechanistic basis for these signaling relationships will be discussed.

Ciliary/Flagellar Motility P1761 Board Number: B280

Structural insights into the motility generation and regulation of cilia and flagella by cryo-electron tomography. J. Lin1, D. Nicastro1; Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 1

Cilia and flagella play important roles in normal development and health of many eukaryotes, including humans [1, 2]. Their motility is driven by thousands of dynein motors [3, 4] that are coordinated by regulatory complexes, such as the Nexin-dynein regulatory complex and I1 dynein [5-8]. Our recent cryoelectron tomography study of intact sea urchin sperm flagella that were rapidly frozen while actively beating, revealed the 3D structures of dynein in three distinct post- and pre-powerstroke conformations [9]. These results provided important new insights into the structural basis of dynein’s force generation [9], but how ciliary dyneins are regulated in detail and their different roles in generating ciliary motility remain unclear. Here, we analyzed cryo-tomograms of actively beating sea urchin sperm flagella to determine the 3D structures and conformational changes of all major regulatory complexes and dynein isoforms in relation to different regions of the sinusoidal wave of beating flagella (i.e., curved regions of principal or reverse bend, or straight regions between bends). For some of the dynein isoforms and predicted regulatory complexes we resolved distinct structures and distribution patterns of different conformations that correlate well with specific regions along the flagellar waves. Our comprehensive high-resolution analysis provides a new understanding of the molecular mechanisms underlying ciliary and flagellar motility and suggests a model that shifts previous hypotheses. References: [1] Fliegauf et al. (2007) Nat Rev Mol Cell Biol. 8:880-93; [2] Mitchell (2007) Adv Exp Med Biol. 607:130-40; [3] Summers and Gibbons. (1971) Proc. Nat. Acad. Sci. USA. 68:3092-6; [4] Sale and Satir (1977) Proc. Nat. Acad. Sci. USA. 74:2045-9; [5] Huang et al. (1982) Cell. 28:115-24; [6] Piperno et al. (1994) J Cell Biol. 125:1109-17; [7] Bower et al. (2009) Mol Biol Cell. 20:3055-3063; [8] Toba et al. (2010) Mol Biol Cell. 22:342-53; [9] Lin et al. (2014) Nat Cell Biol. 16:479-85.

TUESDAY-POSTER PRESENTATIONS

P1762 Board Number: B281

Single-molecule three-dimensional mapping of protein trafficking in primary cilia. W. Luo1, A. Ruba1, W. Yang1; 1 Biology, Temple University, Philadelphia, PA Primary cilia are a critical sensory organelle containing microtubule-based transport paths for proteins trafficking in cilium. Yet, the size of cilium (~ 300 nm in diameter) makes imaging three-dimensional (3D) transport routes for either membrane or soluble proteins inside live cilia still a major challenge. To localize protein trafficking in cilia, we employed a newly developed super-resolution microscopy imaging technique, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to real-time track various proteins in cilia with a spatiotemporal resolution of 5 nm and 2 ms in 3D. We found that both a membrane associated protein Arl13b and a transmembrane protein HTR6 possess spatially multiple distinct pathways in the primary cilium of Odora cells, agreeing well with a 9+2 microtubule pattern in Odora cilia identified by electron microcopy. Given the successful applications in mapping protein trafficking inside the cilia and the nuclear pore complexes, SPEED microscopy is proven to be an effective super-resolution high-speed imaging approach to map 3D tomography of macromolecules in sub-micrometer biocavities.

P1763 Board Number: B282

The asymmetric flagellar beat of Chlamydomonas corresponds to a sperm-like beat traveling around a semicircular arc. V.F. Geyer1, P. Sartori2, F. Jülicher2, J. Howard1; 1 Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 2Max Planck Institute for the Physics of Complex Systems, Dresden, Germany Cilia and flagella are motile protrusions of eukaryotic cells that contain an evolutionary conserved internal structure, the axoneme. The axoneme is composed of a cylindrical arrangement of 9 doublet microtubules, transiently interconnected by dynein motors. Force generation by dyneins slides adjacent doublet microtubules. As a result of periodic sliding, the axoneme bends rhythmically, leading to its characteristic waveform. Interestingly, cilia and flagella generate two different types of waveforms, a symmetric, snake-like beat that pushes sperm through the surrounding fluid, and an asymmetric, breaststroke-like beat that pulls biflagellated microorganisms such as Chlamydomonas through the fluid. The latter beat is similar to that of cilia that move fluid across epithelia. We have asked the question: what is the geometric difference between these different beating patterns? To answer this question, we have developed a simple, yet complete, shape characterization of the axonemal beat that can be used to compare waveforms both quantitatively and through theoretical modeling. We applied this approach to wild-type Chlamydomonas as well as to flagella from MBO2 mutants that have a symmetric beat. The

TUESDAY-POSTER PRESENTATIONS characterization uses Fourier analysis to decompose the periodic beat, expressed as tangent angle as a function of arc length and time, into a set of bending modes that include the time-averaged shape (“zeroth mode”), the waveform at the beat frequency (“first mode”) and higher order modes. We find that in both waveforms, the higher order modes make only a minor contribution. While the properties of the first order mode are similar in amplitude and phase, the zeroth modes are very different. While the MBO2 beat has a very small zeroth mode, corresponding to a mean shape that is almost straight, the wildtype beat has a much larger zeroth mode whose amplitude increases approximately linearly with arc length. This corresponds to a time-average angular shape that is a semicircular arc, around which the first mode propagates. We conclude that the dramatic differences between flagellar waveforms can be accounted for by differences in mean shapes.

P1764 Board Number: B283

Primary cilia bend and pivot in response to intracellular and extracellular forces. C.M. Ott1, C. Battle2, J. Lippincott-Schwartz3,4, C.F. Schmidt5; 1 NIH/NICHD, Bethesda, MD, 2Drittes Physikalisches Institut, Georg-August-Universität, Göttingen,, Germany, 3Physiology Course at Marine Biological Laboratory, Woods Hole, MA, 4Cell Biology and Metabolism Program, NICHD, NIH, Bethesda, MD, 5III. Physikalisches Institut-Biophysik, Georg-AugustUniversität, Göttingen, Germany Primary cilia are ubiquitous, microtubule-based organelles which play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing and mechanosensing, using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells (MDCK-II), whose role it is to sense fluid flow in renal ducts. We found that upon manipulation with an optical trap, cilia deflect by bending along their length and by pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack inter-doublet dynein motors. Nevertheless we found that primary cilia display active motility. Three-dimensional tracking demonstrated correlated fluctuations of the cilium and basal body. These angular movements appeared random, but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.

TUESDAY-POSTER PRESENTATIONS

P1765 Board Number: B284

IDA6 encodes a conserved subunit required for assembly of the N-DRC and several inner arm dyneins. R. Bower1, D. Tritschler1, K. VanderWaal1, E. O'Toole2, T. Heuser3, D. Nicastro4, M.E. Porter1; 1 Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN, 2 MCD Biology, University of Colorado, Boulder, CO, 3Biology, Brandeis University, Waltham, MA, 4 Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA The nexin-dynein regulatory complex (N-DRC) is a group of polypeptides thought to coordinate dynein activity and interconnect doublet microtubules (Piperno et al., 1994; Gardner et al., 1994; Heuser et al., 2009; Lin et al., 2012), but its precise role in flagellar motility is poorly understood. We have analyzed several drc mutants in Chlamydomonas to identify novel N-DRC subunits, to study N-DRC assembly, and to probe N-DRC function. To better understand the relationship between the N-DRC and the inner dynein arms, we characterized the IDA6 gene. ida6 is a motility mutant that fails to assemble dynein e and is associated with reduced levels of tektin, similar to the drc mutant, pf3 (Kato et al., 1993; Gardner et al., 1994; Yanagisawa & Kamiya, 2004). Electron microscopy and 2D averaging revealed a major defect in assembly of the N-DRC, and so we tested candidates in the flagellar proteome (Li et al., 2004; Pazour et al., 2005) for rescue of ida6 and identified the DRC2 subunit, FAP250, as the IDA6 gene product (Austin-Tse et al. 2013). Mutations in the zebrafish and human orthologue CCDC65 are linked to defects in ciliary motility and primary ciliary dyskinesia. More recently we identified sup-pf5 as another drc2 mutation. DRC2 co-extracts, co-purifies, and co-IPs with other DRC subunits (Bower et al., 2013). iTRAQ labeling and MS/MS identified >10 axonemal proteins that are missing or reduced in ida6 and sup-pf-5, and are restored in rescued strains. Spectral counting of dynein heavy chains (DHCs) revealed deficiencies in DHC8 and several other DHC isoforms. Cryo-electron tomography and 3D averaging further demonstrated that both ida6 and pf3 display similar defects in the assembly of the N-DRC and inner dynein arms, plus additional irregularities in the positioning of radial spokes. Thus IDA6 encodes FAP250, which corresponds to DRC2, and DRC2 together with DRC1 plays a key role in assembly at the junction between the N-DRC, the radial spokes, and the dynein arms. Current studies focus on identifying the components that specify the binding sites for DRC1 and DRC2 on the outer doublet (Supported by NIH).

TUESDAY-POSTER PRESENTATIONS

P1766 Board Number: B285

The ciliary N-DRC and B-tubule polyglutamylation are required for axoneme integrity and outer doublet alignment. L.M. Alford1, E.L. Hunter1, M.E. Porter2, W.S. Sale1; 1 Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, 2Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN The N-DRC is a large multi-subunit complex that interconnects adjacent doublet microtubules (Heuser et al., 2009). The N-DRC is predicted to limit microtubule sliding and to align outer doublet microtubules in the distal axoneme (Bower et al., 2013). To test this hypothesis, we examined ATP-induced reactivated motility of demembranated drc cells, commonly termed “reactivated cell models.” As described previously (Kamiya and Witman, 1984), ATP-induced reactivation of wild-type cell models resulted in the forward swimming of >90% cell models, similar to the movement of live cells. In contrast, ATP-induced reactivation failed in a subset of drc-cell models, despite obvious motility in live drc- cells. Dark field microscopic observations of drc-cell models revealed various degrees of axoneme splaying in pf2, pf3, ida6, sup-pf3, and sup-pf5. Immunofluorescence microscopy using an anti-tubulin antibody revealed that the distal axoneme was splayed in 60-85% of reactivated drc-mutant cell models, in contrast to wild-type cells in which >85% of axonemes remained intact. sup-pf4, unlike the other drc-mutants, retains most of the interdoublet linker domain of the N-DRC (Heuser et al., 2009). Notably, sup-pf4 reactivated cell models displayed nearly wild-type levels of in vitro motility, and most sup-pf4 and suppf4-rescued axonemes remained intact. These data strongly support the hypothesis that the N-DRC, particularly the interdoublet linker domain, is required for the integrity and interdoublet alignment of the distal axoneme. We also tested the idea that tubulin polyglutamylation plays a role in interdoublet interactions and axoneme integrity (see Kubo et al., 2012). Like the N-DRC mutants defective in the interdoublet linker domain, ATP-induced splaying of the distal axoneme was also observed in tpg mutant cell models. As proposed by Kubo et al., (2012), we postulate that the functional interaction of the N-DRC with the adjacent B-tubule is facilitated upon tubulin polyglutamylation.

P1767 Board Number: B286

Basal foot MTOC organizes pillar MTs required for coordination of beating cilia. D. Clare1, J. Magescas2, T. Piolot3, M. Dumoux1, C. Vesque4, E. Pichard5, T. Dang5, B. Duvauchelle5, F. Poirier5, D. Delacour2; 1 Biological sciences, Institute of structural and molecular biology (Birkbeck college-UCL), London, United Kingdom, 2Institut Jacques Monod - CNRS, Paris, France, 3Curie Institute CNRS, Paris, France, 4INSERM, Laboratoire de Biologie du Développement, Paris, France, 5Institut Jacques Monod CNRS, Paris, France Coordination of ciliary beating is essential to ensure mucus clearance in the airway tract. The orientation and synchronization of ciliary motion are controlled by the polarized organization of cilium bases at the

TUESDAY-POSTER PRESENTATIONS apical domain of epithelial cells, as illustrated by basal feet aligned in the same direction, and respond in part to the organization of the underlying cytoskeletal networks. Using dual-axis electron tomography procedure on mouse trachea, we show that microtubule (MT) and actin networks are independently organized in tracheal cells. On one hand, we observed sinuous actin microfilaments forming a dense meshwork encircling the basal bodies (BBs). On the other hand, the cortical cytoplasmic MTs originate from the BBs, mainly from the basal foot caps, where γ-tubulin is enriched. Most of the MTs are orientated obliquely and perpendicularly, with only a few that run parallel to the apical membrane connecting neighboring BBs. Then, BBs are collectively hooked at the cortex by a regular MT array composed of 4-5 MTs, and these cortical MTs may behave as cilium “pillars”. Removal of Galectin-3, one of basal foot cap components, provokes misrecruitment of γ-tubulin and disorganization of this cortical MT framework. Perturbation of the subciliary MT network at the base of the cilium had direct consequences on BB organization. The BBs were normally spaced but their alignment was impaired in gal3-/- multiciliated cells, clearly showing that rotational cell polarity is disrupted in the absence of Galectin-3. Motile cilia do exist in gal3-/- tracheas, but their structure and organization are affected, and reduced fluid flow is generated in mutants. Our data further emphasize the longstanding hypothesis that the basal foot cap is the main cilium base MicroTubule-Organizing Center (MTOC). In addition, we conclude that Galectin-3 plays a crucial role in the maintenance of the BB MTOC and the “pillar” MT units, and that this network is instrumental for the coordinated orientation and stabilization of motile cilia.

P1768 Board Number: B287

A spinning puzzle of the release of a giant multinucleate multiflagellate zoospore. J. Urzay1, D. Ott2, M. Prakash1; 1 Bioengineering, Stanford Univ, Stanford, CA, 2University of Akron, Akron, OH Asexual reproduction in aquatic algal species of Vaucheria occurs by the formation of large multinucleate multiflagellate single cell. Termed as a zoospore, it is an elongated club-shaped zoosporangia at the tips of young branches. During development, the zoosporangia are separated from the rest of the thallus by membranes, resulting in multiple chambers hosting zoospores which will be released and dispersed in the surrounding aqueous environment. The apical gelatinization of the zoosporangial tip, together with the turgor pressure in the segregated portion of the filament, lead to a narrow aperture through which the zoospore escapes. However ordinary this may seem, Vaucheria zoospores have a unique multiflagellated patterned surface that warrants helicoidal flow entrainment at relatively high speeds, and which enables them to undergo a spinning motion that elastohydrodynamically assists the rather unfavorable escape maneuver. Experimental observations of this phenomenon, together with quantitative interpretations, are provided.

TUESDAY-POSTER PRESENTATIONS

P1769 Board Number: B288

Structural analysis of the intraflagellar transport trains. L. Stepanek1, G. Pigino1; 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Intraflagellar transport (IFT) is a non-vesicular transport pathway, which moves cargo proteins along the outer microtubule doublets of the axoneme. While primarily necessary for flagella/cilia assembly and maintenance, IFT also plays a role in cellular signaling and membrane trafficking. The anterograde IFT trains move from the flagellar base to the tip, where they undergo rearrangement, possible cargo unloading and return in the form of retrograde IFT trains. Many aspects of these processes are poorly described, including the structural differences between the anterograde and retrograde particles. To characterize the differences in movement patterns of the two directions of transport I use total internal reflection fluorescence (TIRF) microscopy and single particle tracking techniques. To verify the results and to study the different morphology of the trains, I use TIRF correlated with transmission electron microscopy (TEM). The model organism of choice is biflagellate alga Chlamydomonas reinhardtii. The results of particle tracking show that the IFT tracks are not evenly distributed throughout the crosssection of the flagellum of the gliding cells. Majority of the particles are moving along the lateral parts of the flagellum, indicating preference of some microtubule doublets as tracks for the IFT. To verify and refine these results, I use time-resolved correlative TIRF and TEM microscopy. Briefly, the IFT movement is stopped by fixative addition during time-lapse TIRF acquisition, so the directionality of the particles is known when the sample is imaged by TEM / TEM tomography. The high resolution of TEM is used to identify the microtubule doublets involved in IFT transport and to characterize the morphological difference between the anterograde and retrograde particles. This is the first time to show the structural properties of IFT trains related to their in vivo motility, opening further possibilities for more detailed studies of the IFT machinery.

P1770 Board Number: B289

Growth Arrest Specific 8 Mutant Mouse Reveals a Conserved Role for Motile Cilia Function. W.R. Lewis1, E.B. Malarkey1, R.C. Pasek2, J. Porath3, S.E. Birket4, S. Saunier5, C. Antignac6, M.R. Knowles7, M. Zariwala8, B. Kesterson9, S.M. Rowe4, K.F. Lechtreck10, I.A. Drummond11, J.M. Parant12, M.E. Porter13, F. Hildebrandt14, B.K. Yoder1, N.F. Berbari15; 1 Cell, Developmental, and Integrative Biology, Univ Alabama-Birmingham, Birmingham, AL, 2Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 3Nephrology, Boston Children's Hospital, Boston, MA, 4Pulmonary, Allergy, and Critical Care Medicine, Univ Alabama-Birmingham, Birmingham, AL, 5Paris Descartes University, Paris, France, 6Institut National de la Santé et de la Recherche Médicale , Paris, France, 7Cystic Fibrosis/Pulmonary Research and Treatment Center, Univ North Carolina-Chapel

TUESDAY-POSTER PRESENTATIONS HIll, Chapel Hill, NC, 8Cystic Fibrosis/Pulmonary Research and Treatment Center, Univ North CarolinaChapel Hill, Chapel Hill, NC, 9Genetics, Univ Alabama-Birmingham, Birmingham, AL, 10Cellular Biology, University of Georgia, Athens, GA, 11Massachusetts Gen Hosp/Harvard Med Sch, Charlestown, MA, 12 Pharmacology/Toxicology, Univ Alabama-Birmingham, Birmingham, AL, 13Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN, 14Nephrology, Boston Children’s Hospital, Boston, MA, 15Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN Ciliopathies are genetic disorders of cilia dysfunction. These cellular appendages possess a microtubule based axoneme with either a doublet arrangement of 9+0 (primary/non-motile) or 9+2 (motile). Primary cilia have diverse sensory and signaling roles and as such, defects in primary cilia are associated with a wide spectrum of clinical features. Abnormalities in motile cilia result in Primary Ciliary Dyskinesia (PCD) which is characterized by chronic respiratory infections, situs inversus, infertility, and occasionally hydrocephalus. Here we demonstrate that mutations in the Growth Arrest Specific 8 (GAS8) gene, a putative homolog of Chlamydomonas Paralyzed Flagella 2 (PF2), cause PCD phenotypes in mice. We show Gas8 is required for proper assembly of the inner dynein arms (IDA) and the Nexin-Dynein Regulatory Complex (NDRC), and for normal cilia motility. Gas8GT mice display abnormal cilia beat pattern and a lower beat frequency that Gas8WT mice. Additionally, a screen for GAS8 mutations in PCD patients with situs defects identified two independent homozygous missense mutations whose pathogenicity is being confirmed utilizing model systems. The two independent mutations at c.595G>A and c.1172C>T interrupt a Gas8 Microtubule Association Domain (GMAD) and a Smoothened binding domain respectively. Currently, the pathogenicity of these human mutations is being tested in zebrafish and chlamydomonas. Utilizing CRISPR technology, we generated several Gas8 mutant zebrafish of which we will attempt rescue experiments with human WT, CT, and GA mRNA. Similarly, we are generating zebrafish with corresponding human mutations to analyze pathogenicity. Chlamydomonas is being used to investigate mechanistic defects associated with these human mutations by creating humanized mutations in PF2 and studying the effects on NDRC component docking. We hypothesize that both of these mutations will result in disruption of NDRC assembly and dyskinetic motile cilia.

Kinetochore Assembly and Functions 2 P1771 Board Number: B291

Measuring Force at the Drosophila Kinetochore. A.A. Ye1, S. Cane1, T.J. Maresca1; 1 Biology Department, Univ Massachusetts-Amherst, Amherst, MA To maintain genomic stability, cells need to properly segregate their replicated chromosomes through cell division. As cells prepare to divide, a multi-protein structure called the kinetochore assembles at the

TUESDAY-POSTER PRESENTATIONS centromeres of each sister chromatid to mediate interactions with spindle microtubules. Proper chromosome segregation requires that microtubules emanating from opposing spindle poles bind sister kinetochores to form bioriented kinetochore-microtubule (kt-MT) attachments. Erroneous kinetochore kt-MT interactions lead to unequal chromosome segregation and aneuploidy. Therefore, kt-MT attachment stability must be carefully and tightly regulated during cell division to ensure the fidelity of the process. The application of opposing forces to kinetochores by dynamic MTs upon chromosome biorientation leads to a structural rearrangement of the kinetochore called intrakinetochore stretch in which the outer plate moves away from the inner plate. Since intrakinetochore stretch is an important regulator of kt-MT attachment stability, characterizing the properties of force-transducing kinetochore components and the magnitude of forces applied to these components are essential prerequisites for understanding how forces impact kinetochore structure and function. Existing measurements of the amount of force that is applied to kinetochores differ by orders of magnitude. To address this major discrepancy, calibrated force sensors were inserted into the Drosophila kinetochore. Since we acquired evidence that CENP-C is a central force-transducing component of the Drosophila kinetochore, two different calibrated force sensors were inserted into the middle of CENP-C to measure the force applied to bioriented kinetochores in living cells. One CENP-C reporter was built using a previously calibrated spider-silk-based FRET sensor and the second force reporter was made by inserting the rod domain of the focal adhesion protein Talin into CENP-C, and simultaneously expressing the head domain of Vinculin (a force-sensitive Talin binding partner) in the same cell line. Quantitative measurements of both reporters indicated that each CENP-C molecule is, on average, under 1 pN of force at bioriented kinetochores. Given that a Drosophila kinetochore has between 12-30 CENP-C molecules per MT that are envisioned as linkages arranged around the MT as a set of parallel springs, we propose that, on average, a kt-MT in Drosophila generates ~12-30 pN of poleward-directed force at metaphase. Furthermore, since there are 11 microtubules bound to Drosophila S2 cell kinetochores, we propose that each bioriented kinetochore, as a whole, experiences poleward-directed pulling forces between ~130 and 330 pN of force. It is noteworthy that this magnitude of force, in the hundreds of piconewtons range, differs significantly from recent measurements but is in close agreement with those initially measured by Bruce Nicklas in insect spermatocytes.

P1772 Board Number: B292

Mechanical Deformations Within Kinetochores During Chromosome Directional Instability. C. Smith1, A.D. McAinsh2, N. Burroughs1; 1 Mechanochemical Cell Biology, University of Warwick, Coventry, United Kingdom, 2Mechanochemical Cell Biology, University of Warwick, Coventry, United States During mitosis, chromosomes move towards and away from spindle poles, and can undergo directional switches (Skibbens et al. 1993). These are driven by forces generated by the kinetochore, whose attached microtubules can switch between either polymerising or depolymerising states. These states

TUESDAY-POSTER PRESENTATIONS are associated with deformations within the kinetochore itself (Uchida et al. 2009, Dumont et al. 2012), and are also thought to respond to tension generated by the inter-sister linkage. To further understand these dynamics, we have recently advanced our kinetochore tracking assay (Jaqaman et al. 2010) in order to track eGFP-labelled CENP-A position at high temporal resolution (every 2s), which allows directional switch events to be carefully interrogated. We have also used Monte Carlo Markov chain methods to compare this data to a model of kinetochore dynamics, yielding information about forces within the system, providing new insight into the force-dependent mechanism that controls sister kinetochore switching. However, this does not incorporate kinetochore deformations, and therefore forces therein. Here I will present the evolution of our assay to simultaneously track mCherry-labelled Ndc80 position every 2s, thus allowing measurements of kinetochore deformations. Our initial analysis of inter- and intra-kinetochore distances yields key dynamic behaviour in the CENP-A-to-Ndc80 linkage both during continued directional motion and at directional switch events. We are also currently integrating these deformations in CENP-A-to-Ndc80 linkage into our modelling framework. References: Skibbens RV et al., Journal of Cell Biology 122, 859 (1993). Uchida KSK et al., Journal of Cell Biology 184, 383 (2009). Dumont S et al., Science 337, 355 (2012). Jaqaman K et al., Journal of Cell Biology 188, 665 (2010).

P1773 Board Number: B293

Investigation of the in vivo mechanical properties of the kinetochore. I. Ross1, P.S. Maddox1, I. Filiatreault2, J. Dorn2; 1 Department of Biology, University of North Carolina, Chapel Hill, NC, 2Univ De Montreal, Laval, QC The kinetochore is a macromolecular structure that is assembled on chromosomes during mitosis. The kinetochore provides the mechanical linkage between chromosomes and microtubules to translate microtubule dynamics into coordinated chromosome movement. As the sole connection of microtubules to chromosomes, kinetochores are essential to maintaining the fidelity of the genome during mitosis and, therefore, it is paramount to understand the mechanical properties of the kinetochore. In order to establish the in vivo mechanical properties of the kinetochore, I am using superresolution live-cell imaging of chromosome movement upon depletion of kinetochore components. This assay is composed of acquiring spatially oversampled data followed by sub-pixel (super) resolution segmentation and tracking. I am using a strain of C. elegans expressing two GFP fusion proteins: GFP-γtubulin and GFP-histone H2B, allowing precise tracking and measurement of chromosome movements relative to the spindle poles (GFP-γ-tubulin). Each dataset is collected in 4D (x, y, z, t) to maximally capture chromosome movements from nuclear envelope breakdown through anaphase. In preliminary

TUESDAY-POSTER PRESENTATIONS experiments, I have achieved diffraction-limited 3D imaging at 100x 1.4NA with 3 second sampling. Each dataset is subjected to analysis using a custom Matlab program (celX) designed to automatically segment and track individual chromosomes in 4D. The output of celX manifests as numerous metrics of chromosome dynamics including, but not limited to, distance from the spindle axis, orientation of chromosomes relative to microtubules, and chromosome velocities. celX will allow me to track dynamic behaviors, such as oscillations or bending of chromosomes, with high spatial and temporal resolution. Chromosome movements are directly proportional to force imbalances, thus the tracking data can be transformed into relative force revealing the roles of individual kinetochore protein complexes. Initial RNAi screening of kinetochore components has focused on outer kinetochore complexes that are directly implicated in microtubule binding. Of particular interest is the KMN network that is proposed as the mechanical linkage between microtubules and kinetochores. The KMN network is composed of the Knl1, Mis12, and Ndc80 complexes and is the major player in end-on kinetochore-microtubule attachment. The measured velocities will provide insight into the mechanical properties of the kinetochore and begin to build a more comprehensive picture of the force landscape of chromosome movement. In sum, this analysis will provide accurate in vivo measurements of the movement and forces of chromosomes within the complex setting of the spindle.

P1774 Board Number: B294

Laser microsurgery reveals conserved viscoelastic behavior of the kinetochore. G. Cojoc1, E. Roscioli2, L. Zhang3, E. Civelekoglu-Scholey4, A. García-Ulloa5, J. Shah6, M. Berns7, D. Cimini2, I.M. Tolic5,8, J. Gregan3,9; 1 Max Planck Instute of Molecular Cell Biology and Genetics, Dresden, Germany, 2Biological Sciences, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, 3Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria, 4Molecular and Cellular Biology, University of California - Davis, Davis, CA, 5Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 6Harvard Medical School, Boston, MA, 7University of California, San Diego, La Jolla, CA, 8Dept. Molecular Biology, Ruder Boškovic Institute, Zagreb, Croatia, 9Department of Genetics, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia Accurate chromosome segregation depends on proper attachment of kinetochores to microtubules. Mounting evidence suggests that the mechanical properties of the kinetochore are fundamentally important for faithful segregation of chromosomes. We developed an assay where we use merotelic kinetochore as a model for studying mechanical properties of the kinetochore in vivo. A merotelic kinetochore is attached to microtubules emanating from both spindle poles and during anaphase, pulling forces exerted by microtubules lead to lateral stretching of such kinetochore. In our assay, we use laser ablations to sever microtubules attached to a stretched merotelic kinetochore, thus releasing the forces acting on this kinetochore. The mechanical properties of the kinetochore can then be inferred from the change of the kinetochore shape after microtubule severing. We used Hec1/Ndc80 and CenpA/Cnp1 fused to GFP to visualize outer and inner kinetochore domains, respectively, in mammalian

TUESDAY-POSTER PRESENTATIONS PtK1 cells and in the fission yeast Schizosaccharomyces pombe. In both model systems, kinetochores shortened after severing microtubules. An initial rapid shortening was followed by a period of slow relaxation. Interestingly, the inner kinetochore relaxed faster than the outer kinetochore. Whereas yeast kinetochores typically regained their unstretched size, many PtK1 kinetochores remained stretched over a longer period of time after microtubule severing. Our analysis of the time dependent kinetochore shortening reveals a viscoelastic behavior of the kinetochore that is evolutionarily conserved between yeast and mammalian cells.

P1775 Board Number: B295

Nanoscale patterning of the Ndc80 complex using a programmable DNA origami scaffold for architecture-function analysis of the kinetochore. V. Verma1, R.F. Hariadi1, S. Sivaramakrishnan1, A.P. Joglekar1,2; 1 Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 2Biophysics, University of Michigan, Ann Arbor, MI The eukaryotic kinetochore is a macromolecular machine that uses multiple copies of many different proteins to mediate bidirectional chromosome movements during cell division. In budding yeast, three different microtubule-binding proteins: Ndc80, Dam1, and Stu2 are necessary for chromosome motility and their accurate segregation. In vivo studies also reveal that the yeast kinetochore incorporates a specific number of copies of each of these proteins: 8 copies of Ndc80, 16-20 copies of the of the Dam1 complex, and 7-8 Stu2 dimers. Moreover, these molecules are situated in a well-defined nanoscale organization at the microtubule plus end. This precise organization may be the key to achieving persistent kinetochore movement coupled to the polymerization and depolymerization of the microtubule. To directly test this hypothesis, we are developing an in vitro assay to pattern kinetochore proteins in pre-defined architectures and numbers. We have engineered a programmable DNA origami scaffold that provides up to six stable anchoring positions specified in an accurately defined geometry. Each position can be individually addressed so that either the number or the geometry or both can be varied to suit experimental design. Additionally, we are creating DNA origami scaffolds of different shapes and sizes to mimic/understand the kinetochore geometry at nanometer scale. We can achieve > 90% position occupancy with hybridizing DNA oligomers to the DNA origami scaffold as revealed by stepwise photobleaching using TIRF microscopy. We can attach recombinant Ndc80 complexes with a SNAP tag fused to the centromeric globular domain of the Ndc80 complex, and obtain homogenous populations of origami scaffolds for the given number of molecules as confirmed by gel-shift assays. Using this technique, we are systematically exploring the influence of the number and geometry of Ndc80 complexes on microtubule-binding and on generating persistent attachment. This technique provides a versatile and effective platform for a systematic study of the role of kinetochore architecture in defining the mechanism of kinetochore motility.

TUESDAY-POSTER PRESENTATIONS

P1776 Board Number: B300

Chromosome segregation depends on a negatively-charged region of the βtubulin carboxy-terminal tail. C.P. Fees1, J.K. Moore2; 1 Univ Colorado Sch Med, Aurora, CO, 2Cell and Development, University of Colorado School of Medicine, Aurora, CO Proper chromosome segregation requires carefully choreographed interactions between kinetochores and dynamic spindle microtubules. Whereas the roles of kinetochore proteins are relatively well understood, how tubulin proteins contribute to the fidelity of chromosome segregation is poorly understood. Here we investigate the negatively-charged carboxy-terminal tail (CTT) domains of the αand β-tubulins, which are thought to promote electrostatic interactions with kinetochore proteins. CTT sequences are highly variable across species and tubulin isotypes, and are major sites of posttranslational modifications. CTTs are, therefore, a possible point of regulating kinetochore-microtubule interactions that determine the fidelity of chromosome segregation. Using a series of mutants that alter or ablate CTTs of α- and β-tubulin in budding yeast, we identify a specific role for β-CTT in chromosome segregation. Mutant strains lacking the β-CTT exhibit delayed progression into anaphase and elevated rates of chromosome loss. In contrast, mutants lacking the α CTT appear similar to wild type. Using live cell imaging to measure the dynamics of kinetochores labeled with Nuf2-GFP, centromeres labeled with CENP-A/Cse4 –GFP, and single centromeres labeled with CENIV-GFP, we show that loss of the β-CTT disrupts the bi-orientation of sister kinetochores. To elucidate the molecular role of the β-CTT, we map the residues that are necessary for function, and identify a short region of negatively-charged residues. Altering the charge of these residues disrupts chromosome segregation and microtubule dynamics. Furthermore, this negatively charged region may play an important role in facilitating interactions between the CTT and microtubule binding proteins. We provide evidence that this region supports the activity of the kinetochore protein, Ndc80. Based on these results, we propose that the β-CTT promotes proper chromosome segregation in two ways; by regulating the dynamics of spindle microtubules and by tuning kinetochore-microtubule interactions.

P1777 Board Number: B301

A Novel Role for the Rod-Zw10-Zwilch (RZZ) Complex in Modulating Microtubule (MT) Attachments at Kinetochores. A.Z. Raja1, J.M. Kasuboski1, J. Kaneshiro1, M. Joyce1, W. Boggess1, P.S. Vaughan1, K.T. Vaughan1; 1 University of Notre Dame, Notre Dame, IN Kinetochores utilize a combination of dynamic and stable MT attachments during the processes of chromosome alignment and chromosome segregation. Although phosphorylation is implicated in

TUESDAY-POSTER PRESENTATIONS regulating the MT-binding activities of some candidates, a mechanism that explains when each protein is dominant remains elusive. In previous work, we reported premature formation of stable MT attachments by HEC1/Ndc80 as a consequence of inhibiting Aurora B (AurB). This phenotype reflected the loss of the RZZ complex and all proteins dependent on RZZ, including cytoplasmic dynein, dynactin, spindly and MAD1/2. Testing a model in which RZZ masks the MT-binding activity of HEC1/Ndc80 until late stages of chromosome alignment, we sought additional kinases that regulate RZZ and identified Monopolar Spindle 1 (MPS1). MPS1 inhibition induced chromosome alignment defects including premature formation of stable MT-attachments. MPS1 inhibition also blocked recruitment of transient kinetochore proteins including RZZ and proteins that depended on RZZ. To define the requirements for MPS1 activity, we tested relevant candidates and identified Zwilch as a novel substrate for MPS1. MS/MS analysis revealed phosphorylation at three sites near the N-terminus of Zwilch and mutant analysis demonstrated that MPS1 phosphorylation is essential for RZZ function. 3A-mutant (T85A/S88A/S91A) Zwilch mimics MPS1 inhibition and blocks RZZ recruitment to kinetochores. 3Emutant (T85E/S88E/S91E) Zwilch mediates recruitment of RZZ and RZZ-dependent proteins to kinetochores and renders cells resistant to MPS1 inhibition. Finally, phospho-mimetic Zwilch binds directly to Zw10, revealing a novel requirement for Zwilch phosphorylation in RZZ assembly. These findings support a model in which RZZ assembly and recruitment are responsible for modulating MTattachments during prometaphase, perhaps through regulation of access to MTs by stable MT-binding proteins such as HEC1/Ndc80.

P1778 Board Number: B302

Ndc80 Tail Phosphorylation Controls Kinetochore-Microtubule Attachment Formation via Modulation of the Adjacent Calponin Homology (CH) Domain. D.K. Cheerambathur1, M. Moyle1, K. Oegema1, A. Desai1; 1 Ludwig Institute for Cancer Research, La Jolla, CA The 4-subunit Ndc80 complex is a central component of the attachments between kinetochores and spindle microtubules that segregate chromosomes during cell division. The Ndc80 subunit of the complex has a conserved architecture with an unstructured N-terminal tail (N-tail) adjacent to a calponin homology (CH) domain. Although the N-tail is divergent in primary sequence between species, it harbors two conserved features: a highly basic nature (pI =10-12) and the presence of Aurora kinase target sites. Both the CH domain and N-tail have been proposed to make essential contributions to microtubule binding. The CH domain docks onto the microtubule lattice, while the basic N-tail is thought to promote binding through electrostatic interactions with the acidic C-terminal tails of α/β-tubulin. Correction of aberrant kinetochore-microtubule attachments requires Aurora B kinase, which is proposed to phosphorylate Ndc80 N-tails to counter their electrostatic interaction with the microtubule surface. Here we use the C. elegans embryo as a model system to test these ideas and examine the interplay between the CH domain, N-tail and Aurora B-mediated phosphorylation in controlling the formation of microtubule attachments. We show that, as expected, mutation of the CH domain prevents

TUESDAY-POSTER PRESENTATIONS the formation of load- bearing kinetochore-microtubule attachments in vivo. In contrast, whereas the Ntail is essential for microtubule binding in vitro, it is dispensable for both attachment formation and embryonic viability in vivo. Despite the fact that the N-tail is not essential for attachment formation in vivo, introducing phosphomimetic mutations into the 4 Aurora target sites in the N-tail perturbs attachment formation, phenocopying mutations in the CH domain. This observation suggests that rather than releasing an electrostatic interaction between the N-tail and the microtubule lattice, N-tail phosphorylation promotes attachment release by allowing the tail to function as an intra-molecular inhibitor of the CH domain. In support of this idea, synthetic genetic analysis in one-cell embryos revealed that the phosphomimetic N-tail resembles mutations in the CH domain instead of the N-tail deletion. These results lead to a new model in which Aurora kinase-mediated Ndc80 N-tail phosphorylation modulates activity of the adjacent CH domain to control kinetochore-microtubule attachment formation and ensure accurate chromosome segregation.

P1779 Board Number: B303

Spindle checkpoint protein Bub3 promotes mitotic cell cycle progression by activating APC/C-Cdc20 in budding yeast. Y. Yang1, D. Tsuchiya1, S. Lacefield1; 1 Indiana University Bloomington, Bloomington, IN The spindle checkpoint is a well-conserved surveillance mechanism, which functions at the metaphaseanaphase transition. In the presence of improperly attached kinetochore, the spindle checkpoint forms an effector complex to delay anaphase onset by binding and inhibiting Cdc20, the co-activator of APC/C (Anaphase Promoting Complex/Cyclosome). This effector complex is comprised of the spindle checkpoint proteins Bub3, Mad2 and Mad3. Here we show that deleting BUB3, but not MAD2, results in a prominent delay of anaphase onset. The delay is not a result of the other cell cycle checkpoints, or a result of aneuploidy. Surprisingly, co-immunoprecipitation studies show that Cdc20 binding to the APC/C complex is impaired in bub3Δ cells. In addition, the delay is also observed when Bub3 is prevented from localizing to the kinetochore. Bub3 and Cdc20 co-localize to the kinetochore in metaphase, indicating their interaction at the kinetochore could be important for timely anaphase onset. This study reveals the multifaceted function of the spindle checkpoint by illuminating a novel role of Bub3 in APC/C-Cdc20 modulation.

TUESDAY-POSTER PRESENTATIONS

P1780 Board Number: B304

Mad1 and Bub1 – more than kinetochore recruitment factors in the spindle assembly checkpoint. K. Sewart1, N. Schmidt2, S. Heinrich2, M. Langegger2, S. Hauf3,4; 1 Department of Biological Sciences, Virginia Tech, Blacksburg, VA, 2Friedrich Miescher Laboratory of the Max Planck Society, Tuebingen, Germany, 3Dept. of Biol. Sciences and VBI, Virginia Tech, Blacksburg, VA, 4 Friedrich Miescher Laboratory, Max Planck Society, Tuebingen, Germany To maintain genome integrity, the genetic material has to be equally distributed during cell division. The spindle assembly checkpoint is a conserved surveillance mechanism that delays anaphase as long as any of the chromosomes is not properly attached to the mitotic spindle. Malfunction of this checkpoint causes erroneous chromosome segregation and has been implicated in tumorigenesis. Despite a wealth of information on the localization and interaction of checkpoint proteins, the in vivo signaling mechanism is still only partially understood. A complex between the proteins Mad1 and Mad2 is crucial for the spindle assembly checkpoint. Kinetochore-bound Mad1:Mad2 binds a second molecule of Mad2 through Mad2:Mad2 dimerization, which is necessary for binding of this second Mad2 to the anaphase activator Cdc20. This ultimately prevents anaphase. Work of several labs, using different organisms, consistently showed that Bub1 is required for kinetochore recruitment of Mad1 and that kinetochore-bound Mad1 serves as binding platform for Mad2. Employing a combination of biochemical methods, yeast molecular genetics and live cell imaging approaches, we recently showed that the roles of Mad1 and Bub1 go beyond this role in bringing Mad2 to the kinetochore. We introduced mutations in the Mad1 C-terminus that preserved Mad1 kinetochore localization, its interaction with Mad2, and the capability for Mad2 dimerization, but nevertheless impaired checkpoint functionality. Similarly, artificial tethering of Mad1 to the kinetochore in cells depleted of Bub1 also did not rescue checkpoint functionality, although Mad2 was co-recruited with Mad1 and was able to dimerize. These results indicate that Mad1 and Bub1 are more than mere recruitment factors for Mad2. We are in the process of determining, which step, downstream of Mad2 dimerization, is defective and use the separation-of-function mutants of Mad1 to investigate the molecular role of the Mad1 C-terminus in this process.

TUESDAY-POSTER PRESENTATIONS

P1781 Board Number: B305

Toward quantitative theoretical description of the kinetochore-microtubule interactions and their roles in ensuring accurate chromosome segregation. A. Zaytsev1, F.I. Ataullakhanov2,3, E.L. Grishchuk1; 1 Physiology Department, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, 2 Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow, Russia, 3Physics Department, Moscow State University, Moscow, Russia Mitotic chromosomes segregate equally between two daughter cells with the help of spindle microtubules (MTs). MTs attach dynamically to chromosomal kinetochores, but little is known about how molecular characteristics of kinetochore proteins contribute to the dynamicity and robustness of MT attachments, and how quantitative characteristics of these interactions affect the accuracy of chromosome segregation. Our long term goal is to use quantitative “bottom-up” modeling to link the molecular parameters of key kinetochore proteins with physiology of mitotic processes, seeking novel insights and predictions. To investigate chromosome segregation at different organizational scales we developed several theoretical models. The molecular-kinetic determinants of kinetochore MTs stability were examined using probabilistic single-molecule model of the MT binding at the kinetochore site with 12-20 molecules. We show that when these molecular interactions are independent and stochastic, the physiological rate of MT turnover is achieved only when the bindings are transient, each lasting fraction of a second (Zaytsev et al., CaMB 2013). MT bindings in vitro by the single molecules of the core kinetochore protein NDC80 last only 20-400 ms, so we next modeled the kinetochore interface using the experimentally determined NDC80 constants. We demonstrate that the stability of kinetochore MTs and its regulation during mitosis can be largely explained by the phosphoregulatable MT-binding affinity of just this one kinetochore component. Interestingly, the interactions between NDC80 complexes and kinetochore MTs are predicted to be unconstrained, meaning that the NDC80 molecules are able to alternate their binding between adjacent kinetochore MTs (Zaytsev et al., JCB 2014). Finally, we developed a probabilistic model for dynamic kinetochore-MT interface to systematically and quantitatively examine the impact of transient MT attachments on the accuracy of chromosome segregation. We show that the rate of kinetochore-MT turnover on its own has little impact on the final accuracy of MT attachments. Strikingly, a model that combines the optimal rate of MT turnover with the plate-like kinetochore geometry predicts the frequency of chromosome loss (2.6 – 47) × 10-2. The lower value is similar to what is seen in tumor-derived cells, but this simplified model cannot explain low chromosome loss frequency in normal human cells. Together, these theoretical approaches provide novel mechanistic insights into the molecular mechanisms of accurate chromosome segregation. This work should assist developing the comprehensive quantitative understanding of chromosome segregation in normal cells, and help to uncover how pathological chromosomal instability can be prevented.

TUESDAY-POSTER PRESENTATIONS

P1782 Board Number: B306

Studying kinetochore-microtubule attachment using fluorescence lifetime imaging microscopy with Bayesian analysis. T. Yoo1, D.J. Needleman2,3,4; 1 Harvard University, Cambridge, MA, 2FAS Center for Systems Biology, Harvard University, Cambridge, United States, 3School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 4 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA Accurate segregation of chromosomes requires the proper attachment of microtubules to chromosomes via the kinetochore. Kinetochore-microtubule attachments are thought to be predominately mediated by the Ndc80 complex, which is modified by Aurora B kinase, but the mechanism by which attachment is maintained and regulated is poorly understood. We are studying these processes with Bayesian analysis of Fluorescence Lifetime Imaging Microscopy (FLIM), a form of Forster Resonance Energy Transfer (FRET) capable of quantitatively measuring the dynamics of protein-protein interaction in cell with millisecond time resolution. We are investigating the dynamics of the binding and clustering of Ndc80 complexes on microtubules at kinetochores in human tissue culture cells. We are also studying phosphoregulation of kinetochore-microtubule attachment in cell by monitoring the activity of Aurora B kinase using a Aurora B biosensor.

P1783 Board Number: B307

A minimal model of kinetochore-microtubule dynamics captures metaphase oscillations, error-correction and effects of Aurora B hyperactivation. K.K. Chiou1, E.R. Ballister2, E.J. Banigan3, A.M. Mayo2, M.A. Lampson2, A.J. Liu1; 1 Physics and Astronomy, University of Pennsylvania, Philadelphia, PA, 2Department of Biology, University of Pennsylvania, Philadelphia, PA, 3Physics and Astronomy, Northwestern University, Evanston, IL Successful eukaryotic cell division requires the formation of correct attachments between kinetochores and spindle microtubules. In the correct (bioriented) configuration, sister kinetochores attach exclusively to microtubules from opposite spindle poles; these microtubules exert opposing poleward forces, resulting in inter-kinetochore tension. Kinetochore-microtubule interactions are dynamic and subject to tension-dependent regulation that destabilizes improper attachments. Tension acts directly via catch-bond-like effects and indirectly through Aurora B phosphorylation of kinetochore proteins. We developed a minimal stochastic model incorporating previous in-vitro biophysical measurements of kinetochore-microtubule interactions and their dependence on both tension and phosphorylation. These previous experiments used kinetochores that bind single microtubules; our model builds on these measurements to describe kinetochores that bind bundles of microtubules and qualitatively captures key phenomena including error correction and metaphase chromosome oscillations. We challenged this

TUESDAY-POSTER PRESENTATIONS model by experimentally manipulating kinetochore phosphorylation using chemically induced dimerization in living cells. Recruiting Aurora B to metaphase kinetochores caused a dramatic disorganization of the metaphase plate. Systematic analysis of our computational model showed that varying the simulated rates of microtubule catastrophe, rescue and detachment, but not other parameters, provided a good fit to experimental results. These are the same parameters which were independently shown to be modulated by Aurora B phosphorylation in vitro. These results establish a miminal physical model for kinetochore-microtubule dynamics that provides a framework for further theoretical development and quantitative interpretation of in vivo experiments.

P1784 Board Number: B308

Bi-stability of Aurora B kinase activity underlies spatial gradients in dividing cell. D. Segura-Pena1, A.V. Zaytsev2,3, E.R. Ballister1, R. Stamatov4, F.I. Ataullakhanov5,6, M.A. Lampson1, E.L. Grishchuk2; 1 Department of Biology, University of Pennsylvania, Philadelphia, PA, 2Physiology Department, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, 3Center for Theoretical Problems of Physico-chemical Pharmacology, RAS, Moscow, Russia, 4Bioinformatics, Duke University, Durham, NC, 5Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow, Russia, 6Physics Department, Moscow State University, Moscow, Russia Aurora B kinase, the enzymatic component of the chromosome passenger complex, is a key regulator of cell division that provides temporal and spatial coordination for chromosome segregation. Gradients of Aurora B activity are implicated in regulating kinetochore-microtubule interactions and cytokinesis, but the mechanisms underlying these spatial patterns are unknown. Spatial phosphorylation gradients extend beyond known locations of Aurora B binding sites, suggesting that these patterns form by a Turing mechanism through dynamic self-organization of a coupled kinase-phosphatase system. Such a mechanism implies that the system contains positive feedback and exhibits bi-stability. To test the hypothesis that self-organization can establish spatial patterns of Aurora B activity, we use a coupled Aurora B kinase and phosphatase system in vitro, which is biochemically and theoretically tractable. We show that Aurora B kinase activation by auto-phosphorylation in trans leads to a bi-stable switch, and we experimentally define a hysteretic region for Aurora B activity that matches the theoretical prediction. In living cells, we find similar bi-stability using a FRET-based phosphorylation sensor: partial kinase inhibition leads to two distinct populations of mitotic cells with either high or low levels of Aurora B substrate phosphorylation. Together these results suggest that spatial patterns of Aurora B activity in cells are established around sites of CPC concentration by a Turing self-organization mechanism.

TUESDAY-POSTER PRESENTATIONS

P1785 Board Number: B309

The spindle checkpoint components Mad1 and Mad2 have an essential, checkpoint-independent function in C. elegans. P. Lara-Gonzalez1, M. Moyle1, T. Kim1, D.K. Cheerambathur1, K. Oegema1, A. Desai1; 1 Ludwig Institute for Cancer Research, La Jolla, CA The spindle assembly checkpoint promotes accurate chromosome segregation by restraining progression through mitosis until all the chromosomes are attached to the microtubules of the mitotic spindle. The most downstream components of this pathway are the “mitotic arrest deficient” (Mad) proteins Mad1, Mad2 and Mad3, which are conserved throughout evolution. While the spindle checkpoint and its components have been studied extensively from a single-cell perspective, we aimed to understand the consequences of disrupting this pathway in a developing organism, and ask whether the resulting phenotypes are explained by lack of checkpoint function. For this, we employed CRISPRCas9 to generate strains of C. elegans in which mad-2 and mad-3 were fully deleted; a full deletion of mad-1 was previously generated. We found that, consistent with previous observations using partially deleted alleles, complete deletion of mad-1 and mad-2 has a profound effect on viability and fertility. Surprisingly, deletion of mad-3 has only a mild effect on viability. Thus, despite all of the MAD proteins being essential for the spindle checkpoint, MAD-1 and MAD-2 have additional, checkpoint-independent roles, which are essential for organismal development. We confirmed this conclusion by analyzing mutations that disrupt the ability of MAD-1 to localize to unattached kinetochores; despite their lack of checkpoint activity, these mutant forms of MAD-1 supported normal viability and fertility. In addition, MAD-1 localizes to the nuclear envelope during interphase, but we engineered a mutant form of MAD-1 that abolishes this localization and found that it still supports normal viability and fertility. In contrast, a MAD-1 mutant that cannot interact with MAD-2 was phenotypically similar to a mad-2 deletion, exhibiting severe loss of viability and fertility. These data indicate that the MAD-1/MAD-2 complex has an essential function during organismal development in C. elegans, which is unrelated to its known role in the spindle checkpoint. We are currently testing if the well-described MAD-1/MAD-2 complexmediated control of the anaphase promoting complex/cyclosome (APC/C) underlies this essential function. In addition we are performing unbiased biochemical, molecular genetic, and developmental analyses to elucidate the non-checkpoint function of MAD-1/MAD-2 that is essential for organismal development.

TUESDAY-POSTER PRESENTATIONS

P1786 Board Number: B310

The nanoscale organization of Spc105 in metaphase and its role in silencing the Spindle Assembly Checkpoint. B. Roy1, J. Sim1, A.P. Joglekar1,2; 1 Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 2Biophysics, University of Michigan, Ann Arbor, MI Faithful chromosome segregation during cell division requires that the kinetochore, a multi-protein machine built on every chromosome, is stably attached to spindle microtubules. Whether or not the kinetochore is attached is monitored by a surveillance mechanism known as the Spindle Assembly Checkpoint (SAC). The SAC delays the cell cycle as long as even a single kinetochore is unattached. It gets silenced rapidly once stable attachments form. The physical platform for the SAC is provided by Spc105, a conserved kinetochore protein that is large protein (~ 900 amino acids) and consists of a large, unstructured amine-terminal (~ 500 amino acids) signaling domain with microtubule-binding activity. Upon phosphorylation by the Mps1 kinase, this domain recruits SAC proteins to activate the SAC. Just as the organization of microtubule-binding kinetochore proteins plays a key role in executing kinetochore movement, the organization of Spc105 in the attached kinetochore may be important for the reliable silencing of the SAC. Therefore, using in vivo Forster Resonance Energy transfer (FRET) and high resolution co-localization measurements, we defined the average localization and distribution of five different positions spanning the length of the Spc105 molecule. We find that the entire Spc105 molecule is compacted within a 30 nm zone bookended by the centromeric end of the Ndc80 complex (C terminus of Spc24) up to the C terminus of Ndc80 (subunit of the Ndc80 complex). Detectable FRET between Stu2, a microtubule plus-end binding protein, indicates that the N-terminus is proximal to the microtubule lattice. FRET between two different positions in the N-terminus of adjacent Spc105 molecules can also be detected, which is consistent with the disordered nature of this domain. This compact Spc105 organization is surprising, given its large size and disordered nature. We hypothesize that the microtubule-binding activity in Spc105 restrains it within the inner kinetochore in metaphase, and that this organization is required for reliable silencing of the SAC. We will test these hypotheses by abrogating the microtubule-binding activity of Spc105, and studying whether its nanoscale organization changes and if timely SAC silencing is impaired.

TUESDAY-POSTER PRESENTATIONS

P1787 Board Number: B311

TRIP13 AAA-ATPase is a novel mitotic checkpoint silencing protein. K. Wang1, B. Sturt-Gillespie1, J. Hittle2, D. Macdonald3, G. Chan3, T.Y. Yen4, S. Liu1; 1 Biol Sci, University of Toledo, Toledo, OH, 2Fox Chase Cancer Center, Philadelphia, PA, 3Cross Cancer Inst, Edmonton, AB, 4Developmental Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA The mitotic checkpoint (or spindle assembly checkpoint) is a fail-safe mechanism to prevent chromosome missegregation by delaying anaphase onset in the presence of defective kinetochoremicrotubule attachment. The target of the checkpoint is the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Once all chromosomes are properly attached and bi-oriented at the metaphase plate, the checkpoint needs to be silenced. Previously we and others have reported that Thyroid hormone receptor interacting protein 13 (TRIP13) AAA-ATPase interacts with the mitotic checkpoint silencing protein p31comet. Here we show that endogenous TRIP13 localizes to kinetochores. TRIP13 knockdown delays metaphase-to-anaphase transition. The delay is caused by prolonged presence of the effector for the checkpoint, the mitotic checkpoint complex (MCC) and its association and inhibition of the APC/C. These results suggest that TRIP13 is a novel mitotic checkpoint silencing protein. The ATPase activity of TRIP13 is essential for its checkpoint function, and interference with TRIP13 abolished p31comet mediated mitotic checkpoint silencing. TRIP13 overexpression is a hallmark of cancer cells with chromosomal instability, particularly in certain breast cancers with poor prognosis. We suggest that premature mitotic checkpoint silencing triggered by TRIP13 overexpression may promote cancer development.

P1788 Board Number: B312

BUB-1 promotes the onset of anaphase independently of its role in the spindle checkpoint and chromosome alignment. T. Kim1, M. Moyle1, C. De Groot1, K. Oegema1, A. Desai1; 1 Ludwig Institute for Cancer Research, La Jolla, CA Bub1 is a conserved kinase implicated in spindle checkpoint signaling and chromosome alignment. Bub1 targets Mad1 to the kinetochore to initiate spindle checkpoint signaling. In addition, Bub1 promotes chromosome alignment by phosphorylating histone H2a to direct recruitment of the chromosomal passenger complex via the adaptor Shugoshin. Unexpectedly, we found that depletion of BUB-1 significantly extended the duration of mitosis in one-cell C. elegans embryos: the NEBD-anaphase interval was extended by 61% (from 188 ± 2.0 sec in control embryos to 303 ± 5.6 sec in BUB-1 depleted embryos). This extension was independent of checkpoint signaling as it was unaffected by co-depletion of MAD-2 and was observed in a checkpoint signaling-defective allele of bub-1. The promotion of anaphase onset by BUB-1 required kinetochore localization—mutants of BUB-3 and of the BUB-1/BUB-3 kinetochore receptor KNL-1 that perturbed kinetochore recruitment of BUB-1 delayed anaphase onset

TUESDAY-POSTER PRESENTATIONS to a similar extent as BUB-1 depletion. In addition, engineered mutant forms of KNL-1 that recruit variable amounts of BUB-1 to the kinetochore resulted in NEBD–anaphase onset durations that correlated with the magnitude of BUB-1 recruitment. To analyze the biochemical events underlying anaphase onset, we engineered a sensor for activation of separase, the protease that cleaves cohesin. Use of this sensor indicated that BUB-1 depletion delayed separase activation. Once activated, the kinetics of separase-mediated cleavage of the sensor was not affected by BUB-1 depletion, suggesting that the delay in separase activation is a consequence of delayed APC/C activation. Mutational analysis revealed that the BUB-1 kinase domain, but not its kinase activity, are important for promoting separase activation and anaphase onset. Notably, a mutant allele in the kinase domain that did not perturb chromosome alignment and rescued a bub-1 null mutant delayed separase activation and anaphase onset. These results reveal an unexpected role for Bub1 in the promotion of anaphase onset, independently of its functions in checkpoint signaling and chromosome alignment, and suggest a new mechanism contributing to the switch-like transition from metaphase to anaphase.

P1789 Board Number: B313

N-terminal modification of Ndc80 can induce a spindle assembly checkpoint arrest in mouse oocytes. Y. Yun1, S. Lane1,2, J. Holt1, K. Jones1,2; 1 University of Newcastle, Callaghan, Australia, 2University of Southampton, Southampton, United Kingdom Correct microtubule-kinetochore (MT-KT) interactions are essential to achieve faithful chromosome segregation and prevent aneuploidy. The spindle assembly checkpoint (SAC) helps ensure the fidelity of this process, by coupling anaphase-onset with MT-KT attachment. A key component of the MT-KT interface is Ndc80 (Hec1 in humans), which is essential for maintaining kinetochore integrity and the stability of MT-KT interactions during mitosis. However, in mammalian oocytes, which are susceptible to aneuploidy, its function remains unclear. In this study, by expressing fluorescently tagged C- or Nterminally coupled Ndc80 protein in mouse oocytes, we examined the effect on chromosome dynamics and SAC activity. Both of the fused proteins showed specific kinetochore localization, however, majority of the N-terminal tagged Ndc80 (Ndc80-N) oocytes failed to complete meiosis I, and instead arrested at metaphase I (MI completion rates: 12.5% of Ndc80-N oocytes, n=80; vs. 67.5% of Ndc80-C oocytes, n=40; pGA], despite retaining the ability to flip PE, is not able to suppress the ergosterol and membrane protein mislocalization phenotypes of drs2Δ cells. In contrast, DNF1 N550S, which gains the ability to flip PS in addition to its normal lysophospholipid substrates, is able to suppress the drs2Δ defects. These results suggest that PS flip by Drs2p plays an important functional role in the generation and maintenance of lateral membrane organization and provides new insight into how lateral domains are established in budding yeast.

P2013 Board Number: B531

Structure and function of the N-terminal domains of Sec7/BIG1/2 regulating Arf1mediated trafficking from the trans-Golgi network. B.C. Richardson1, J.C. Fromme2; 1 Cornell University, Ithaca, NY, 2MBG/WICMB, Cornell University, Ithaca, NY Export of trafficked proteins from the trans-Golgi network (TGN) is an essential function in all eukaryotes, controlled by the small GTPase Arf1. The proximal upstream regulator of Arf1 is the conserved Sec7 (yeast) / BIG1/2 (mammal) / Sec71 (fly) guanine nucleotide exchange factor (GEF), which activates Arf1 specifically at the TGN. This action initiaties the terminal cascade of events at the Golgi leading to cargo sorting and vesicle formation with multiple different cargos and cargo adaptors. Sec7 consists of a single well-characterized catalytic domain responsible for its GEF activity on Arf1, and six similarly sized domains, two N-terminal and four C-terminal to the GEF domain. We recently demonstrated that these latter six domains serve varying and essential functions both to localize Sec7 to its point of action and to regulate its activity negatively when unlocalized and positively when properly localized. Together, the regulatory domains of Sec7 tightly restrict its activity to the trans-Golgi network, ensuring proper maturation and targeting of all transported proteins. However, the mechanistic details of how each domain mediates its autoregulatory functions and its interactions with other proteins remain obscure. Here, we present the 2.7 Å crystal structure of the two N-terminal regulatory domains of Sec7, representing the first structural model available for regions of Sec7 outside the catalytic domain. The structure identifies a pair of conserved surface regions, one in each domain, mutation of which affects

TUESDAY-POSTER PRESENTATIONS the activity of Sec7 on Arf1 both in vitro and in vivo. Paired with small-angle x-ray scattering and multiangle light scattering data of a suite of Sec7 fragments, this structure provides insight into the nature of dimerization mediated by the N-terminal domains and into the relationship between the catalytic domain and the N-terminal domains.

P2014 Board Number: B532

Defining the assembly determinants and architecture of the yeast exocyst complex. M.R. Heider1, C.M. Duffy1, Z. Hakhverdyan2, R. Kalia3, M. Gu3, M. Rout2, A. Frost3, M. Munson1; 1 Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, 2Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, 3 Biochemistry, University of Utah, Salt Lake City, UT The exocyst is a highly conserved, hetero-octameric protein complex, which is proposed to function at the tethering step of exocytosis in all eukaryotes. The classification of the exocyst as a multisubunit tethering complex (MTC) stems from its known interacting partners, polarized localization at the plasma membrane, and structural homology to other putative MTCs. The presence of 8 subunits begs the questions: why are so many subunits required for vesicle tethering and what are the contributions of each of these subunits to the overall structure of the complex? Additionally, are subunit or subcomplex dynamics a required feature of exocyst function? In order to answer these questions, it was critical that we accurately define the subunit connectivity within the endogenous exocyst complex. Using Saccharomyces cerevisiae as our model system, we purified intact exocyst complexes under physiological conditions. Regardless of the exocyst subunit used as purification handle, all 8 subunits are stoichiometric. In contrast to early studies of the exocyst, these complexes remained assembled over a wide range of pH and ionic strength conditions, suggesting greater intra-complex stability than previously expected. However, at higher extremes of pH and salt concentration, biochemically stable subcomplexes including Sec10-Sec15 and Sec3-Sec5-Sec6-Sec8 emerged. To further dissect these connections and because most of the exocyst subunits are encoded by essential genes, we used the auxin-inducible degradation (AID) system to selectively and rapidly degrade individual exocyst subunits and monitor the effects on complex assembly. Using the AID system, we identified critical subunits that are required for the overall assembly and for connecting modular subcomplexes. However, given the highly stable assembly of the complex, we hypothesize that any subunit dynamics or complex disassembly require additional factors. To address this hypothesis, we are using the AID system to identify which components within the post-golgi secretory pathway are required for the assembly and disassembly of the exocyst. By selectively depleting GTPases, myosin, and SNAREs and then purifying endogenous exocyst complexes, we will pinpoint the steps in the pathway when these complex rearrangements may occur. Finally, piecing together these subunit connections into the overall structure of the exocyst will provide clues as to how the subunits are poised to capture secretory vesicles. To this end, we visualized its structure for the first time using negative stain electron microscopy. 2D class

TUESDAY-POSTER PRESENTATIONS averaging reveals an elongated conical structure, and we are currently working to position the individual exocyst subunits within this overall structure.

P2015 Board Number: B533

Trafficking of the Kir2.1 potassium transporter is regulated by the ubiquitin ligase Rsp5 and a select subset of alpha-arrestins in yeast. A.F. O'Donnell1,2, T. Mackie3, A. Kolb3, A. Dempsey4, C. Szent-Gyorgyi4, M.P. Bruchez4, J.L. Brodsky5; 1 Cell Biology, Univ Pittsburgh, Pittsburgh, PA, 2Dept. of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, 3Dept. of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, 4Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, 5Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA To ensure optimal cell growth and survival, protein composition at the plasma membrane is tightly regulated, with both controlled protein internalization and selective protein targeting to the cell surface in response to environmental changes. To identify factors that regulate this selective protein trafficking, we developed a yeast expression system for the Kir2.1 potassium channel, which maintains potassium homeostasis in heart cells and is thereby critical for cardiac function. By monitoring the growth of Kir2.1 yeast on low potassium medium, Kir2.1 activity at the cell surface can be assessed. Using a highthroughput screening methodology we identified the ESCRTs and retromer complex as novel regulators of Kir2.1 trafficking. We now show that three specific alpha-arrestins – Ldb19/Art1, Aly1/Art6, and Aly2/Art3 – control Kir2.1 trafficking. In marked contrast to the well-described role for alpha-arrestins as endocytic adaptors, these alpha-arrestins promote Kir2.1 trafficking to the cell surface, increasing Kir2.1 activity at the plasma membrane and raising intracellular potassium levels. Consistent with the alphaarrestin requirement, we also demonstrate that the ubiquitin ligase, Rsp5, and the protein phosphatase calcineurin regulate Kir2.1 plasma membrane targeting. Furthermore, by fusing a single chain antibody to Kir2.1 and employing live cell fluorescence microscopy, we can selectively monitor Kir2.1 cell surface localization or its intracellular distribution, thus providing a complementary tool to delineate the intracellular sorting pathway for this channel. Current work is focused on using the yeast model to define additional factors and regulatory elements required for alpha-arrestin-mediated trafficking of Kir2.1 to the cell surface. Together, these findings identify Kir2.1 as new alpha-arrestin substrate and lay the foundation to further define the molecular mechanisms governing alpha-arrestin-mediated intracellular sorting.

TUESDAY-POSTER PRESENTATIONS

P2016 Board Number: B534

CLATHRIN ADAPTOR AP-1 CONTROL BASOLATERAL SORTING OF THE CHLORIDE CHANNEL 2 THROUGH A SOLE DILEUCINE MOTIF. E. de la Fuente-Ortega*1, D. Gravotta*2, C. Lagos3, A. Perez-Bay2, G. Lehmann2, I. Benedicto2, E. Rodriguez-Boulan2; 1 Department of Biomedical Sciences, Faculty of Medicine, Universidad Católica del Norte, Coquimbo, Chile, 2Department of Ophthalmology, Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY, 3Department of Pharmacy, Faculty of Chemistry, Pontificia Universidad Catolica de Chile, Santiago, Chile *Authors contributed equally. Voltage-regulated chloride channel 2 (ClC2) is a polytopic protein ubiquitously expressed in the basolateral membrane of many epithelial cells that function in the regulation of cell volume, ion transport and acid-base balance. As for most chloride channels, the molecular signals and cellular machinery that determine the subcellular distribution of ClC2 are poorly understood. Here, we combined in silico modeling, biochemical and cellular approaches to study the mechanisms that control the basolateral localization of ClC2 in polarized MDCK cells. ClC2 exhibit three putative dileucine motifs in its C-terminus, two in cystathionine β-synthase domain 1 (CBS1) and a third motif, previously implicated in basolateral sorting, within the CBS2. Our in silico model of ClC2 however revealed that CBS2-motif, TIFS812LL, was buried within the dimeric structure of the channel and inaccessible, while CBS1-motifs, ESMI623LL and QVVA635LL, displayed a higher exposure toward the solvent. Consistently yeast three hybrid assays and in silico modeling of AP-1 demonstrated that ESMI623LL favorably interacted with a highly conserved pocket in the gamma-sigma1A hemicomplex of AP-1. Single alanine mutagenesis of these motifs and trafficking analysis of these mutants in MDCK cells demonstrated that ESMI623LL acted as a sole basolateral sorting signal. Single and double knockdown experiments indicated that clathrin adaptors AP-1A and AP-1B were individually necessary and sufficient for basolateral localization of ClC2. Our study constitutes the first structure-function analysis of a dileucine-based basolateral sorting mechanism in epithelial cells. Supported by NIH grants EY08538 and EY022165 to ERB, a chilean government fellowship to EdlF and a PEW Latin American postdoctoral fellowship to GL.

P2017 Board Number: B535

2-deoxyglucose induces α-arrestin-dependent trafficking of hexose transporters via signaling through the Snf1/PP1 phosphatase pathway. A.F. O'Donnell1,2, R.R. McCartney3, D.G. Chandrashekarappa3, B.B. Zhang3, J.W. Thorner4, M.C. Schmidt3; 1 Cell Biology, Univ Pittsburgh, Pittsburgh, PA, 2Dept. of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, 3Dept. of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, 4Dept. of Molecular and Cell Biology, University of California, Berkeley,

TUESDAY-POSTER PRESENTATIONS Berkeley, CA 2-deoxyglucose (2DG), a non-metabolizable analogue of glucose thought to block glycolysis, prevents cancerous growth in animal models and in human tumor cell lines. Despite its widespread use and important clinical applications, the mechanism of 2DG action remains unclear. Our study demonstrates that 2DG is a potent inhibitor of yeast cell growth, even when cells are grown in the presence of abundant glucose. Using a comprehensive high-throughput screen, we demonstrate that overexpression of either of the two low-affinity hexose transporters – Hxt1 and Hxt3 – can suppress yeast sensitivity to 2DG. Furthermore, we find that in response to 2DG addition, these transporters are rapidly internalized and trafficked to the vacuole for degradation. 2DG-induced internalization of Hxt1 and Hxt3 requires Rod1/Art4 and Rog3/Art7, members of the alpha-arrestin family of trafficking adaptors. Consistent with a role for Rod1 and Rog3 in mediating cellular response to 2DG, cells lacking these alpha-arrestins are resistant to 2DG and 2DG fails to trigger Hxt1 and Hxt3 internalization and trafficking to the vacuole. Importantly, Hxt1 and Hxt3 internalization in response to 2DG is regulated by the Snf1 protein kinase, the ortholog of which is mammalian AMPK. In the presence of 2DG, Snf1 becomes moderately activated, as indicated by phosphorylation on its activation loop, and selectively phosphorylates Rod1 and Rog3, while other known Snf1 substrates, including the transcriptional repressor Mig1, are not phosphporylated under these conditions. Addition of 2DG not only increases the Rod1 and Rog3 phosphorylation, but dramatically increases the ubiquitination of these alpha-arrestins. We further show that binding to the ubiquitin ligase Rsp5 is required for Rod1- and Rog3-mediated trafficking of the Hxts and induction of 2DG sensitivity. However, while phosphorylation of Rod1 and Rog3 may facilitate their ubiquitination it is not required for ubiquitination as in the absence of Snf1, both alpha-arrestins are ubiquitinated and able to stimulate Hxt internalization. Based on these findings we propose a mechanism for 2DG-induced toxicity in yeast whereby Snf1 becomes activated, triggering phosphorylation and subsequent ubiquitination of the alpha-arrestins Rod1 and Rog3, which in turn induces Hxt1 and Hxt3 endocytosis and trafficking to the vacuole. By reducing glucose transporter levels at the cell surface, 2DG causes a glucose starvation response, even when cells are grown in a glucoserich environment. Our findings may shed light on the long elusive mechanism of 2DG in inducing cancer cell death.

P2018 Board Number: B536

In vivo imaging reveals that restricted exocytic trafficking drives cell shape changes required for intercalation during chondrocyte stacking. D.S. Levic1, E.W. Knapik2; 1 Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, 2Medicine, Vanderbilt University Medical Center, Nashville, TN Mechanisms that control cell shape changes and 3D tissue organization play a crucial role in morphogenesis. A striking example occurs during vertebrate cartilage development, when clusters of

TUESDAY-POSTER PRESENTATIONS small chondrocytes transition to stereotypical rows of elongated cells that build intricate skeletal structures by a process called stacking. Although this process has been observed for nearly a century, the cellular programs that execute chondrocyte shape changes during stacking are largely unknown. Here, using imaging, genetic and cell biology approaches we identify Erc1 as a novel component of the vesicle trafficking pathway that regulates chondrocyte cell elongation in vivo. During stacking, Erc1-deficient cells become hyper-elongated but fail to expand in width, leading to chondrocyte cell death and craniofacial dysmorphology. We show that Erc1 is required to promote orientation of microtubules, and Erc1 depletion results in aberrant microtubule localization at polarized cortical regions of intercalating chondrocytes. These changes in microtubule polarity do not drive cell shape change in an active mode, but instead correlate with vesicle trafficking defects. We observed that this mechanism is engaged exclusively during chondrocyte cell shape changes. Erc1 depletion leads to accumulation of vesicular structures at the chondrocyte cell cortex as well as the intracellular retention of glycosylated ECM components, suggesting impaired exocytosis. We demonstrate by in vivo mosaic analysis and in situ imaging in the developing cartilage that Rab8 and Kinesin-1 contribute to Erc1-dependent chondrocyte cell shape changes. Our study identifies for the first time a cellular mechanism of chondrocyte cell shape regulation that drives stacking and organ morphogenesis while utilizing microtubule polarity and vesicle trafficking machinery.

P2019 Board Number: B537

Lipid Binding by Osh4p, an OSBP homologue, is Required for Polarized Exocytosis. R.J. Smindak1, D.M. Hyatt1, R.C. Deutscher1, K.G. Kozminski2; Biology, University of Virginia, Charlottesville, VA, 2Biology and Cell Biology, University of Virginia, Charlottesville, VA

1

Polarized exocytosis is an essential cellular process required for diverse events such as polarized cell growth and secretion, and cell migration. The seven-member Osh protein family, the S. cerevisiae homologues of the mammalian oxysterol-binding protein (OSBP) family, is required for polarized exocytosis (Alfaro et al., Traffic, 2011). Within the Osh protein family, Osh4p (Kes1p) is known to bind and transfer specific lipid species between membranes in vitro, however whether lipid binding and transfer by Osh4p is an essential activity in vivo has remained elusive (de St. Jean et al., JCB, 2012; Georgiev et al., Traffic, 2011). Two questions regarding Osh4p function are whether Osh4p regulates non-polarized exocytosis in addition to polarized exocytosis and whether lipid binding by Osh4p regulates its role in polarized exocytosis. First, we show data excluding a role for Osh4p in non-polarized exocytosis as marked by invertase secretion. Further we show that the dominant lethality of the osh4Y97F allele, which encodes a sterol binding null Osh4p, is dependent on the ability of Osh4p to bind phosphatidylinositol-4-phosphate (PI4P) and that Osh4p must be able to bind both sterols and PI4P for polarized exocytosis, marked by Bgl2p, to occur. These studies show that Osh4p is dependent on its

TUESDAY-POSTER PRESENTATIONS lipid binding activity to function in polarized exocytosis, with PI4P serving as a positive regulator of Osh4p function. These results suggest that other Osh or OSBP family members will be shown to regulate polarized exocytosis or other membrane-membrane contact sites in a lipid dependent manner.

P2020 Board Number: B538

GGA3 modulates the trafficking of G protein-coupled receptors via direct interaction. M. Zhang1, C. Li2, J. Davis2, G. Wu2; Georgia Regents University, AUGUSTA, GA, 2Georgia Regents University, Augusta, GA

1

The molecular mechanisms underlying the cell surface transport of newly synthesized G-protein-coupled receptors (GPCRs) remain poorly defined. Here, we determined the role of GGA3 [Golgi-localized, gamma-adaptin ear domain homology, ADP ribosylation factor (ARF)-binding protein 3] in the cell surface targeting of alpha2B-adrenergic receptor (AR), a prototypic GPCR. As an adaptor protein for clathrin-coated vesicles, GGA3 modulates the transport between the trans-Golgi network (TGN) and endosomes and its function is tightly controlled by specific interactions of the VHS domain with the acidic LL motifs presented in the C-terminus of cargo molecules. We found that siRNA-mediated depletion of GGA3 significantly attenuated the cell surface expression of alpha2B-AR and arrested the receptor in the Golgi/TGN compartment. Consistent with the reduction in the cell surface expression, the function of alpha2B-AR measured as MAPK activation was markedly inhibited in response to agonist stimulation in cells transfected with GGA3 siRNA. More interestingly, although alpha2B-AR does not possess any acidic LL motifs, it strongly interacted with GGA3 and the interaction domains were subsequently mapped to the VHS domain of GGA3 and the third intracellular loop of the receptor. These data demonstrate an important function for GGA proteins in the Golgi-to-plasma membrane transport of GPCRs which is likely mediated through a non-conventional mechanism.

TUESDAY-POSTER PRESENTATIONS

P2021 Board Number: B539

A phosphorylation-dependent sorting motif restricts presenilin 2 to late endosomes/lysosomes providing a basis for substrate specificity. R. Sannerud1, C. Esselens1, L. Rochin2, A.K. Tharkeshwar1, G. De Baets3, P. Ejsmont1, V. Baert1, W. Vermeire1, S. Munck4, J. Schymkowitz3, F. Rousseau3, G. Van Niel2, W. Annaert5; 1 Center for the Biology of Disease, VIB / KULeuven, Leuven, Belgium, 2Subcellular Structure and cellular Dynamics, Institut Curie, Paris, France, 3Switch Laboratory, VIB / KULeuven, Leuven, Belgium, 4LiMoNe, VIB, Leuven, Belgium, 5KU Leuven and VIB Center for the Biology of Disease, Campus Gasthuisberg, POB 602, OandN4, Room 7.159, Laboratory of Membrane Trafficking, Center for Human Genetics, Leuven, Belgium Regulated intramembrane proteolysis (RIP) abrogates or initiates downstream signalling by cleaving substrate proteins within their transmembrane domains. γ-Secretase governs such mechanism by targeting >100 type I proteins, including amyloid precursor protein (APP) and Notch. Its activity is confined to a complex comprising presenilin (PSEN), nicastrin, APH-1 and PEN-2. The two homologous (PSEN1&2) and several APH-1 isoforms give rise to complex heterogeneity within the cell of which the functional relevance is largely unexplored. Using biochemical and confocal approaches, we now reveal that, as opposed to the broad distribution of PSEN1/γ-secretase, PSEN2/γ-secretase is essentially confined to late endosomes/lysosomes in different cell types including primary hippocampal neurons. Domain swapping and mutational analysis identified a unique N-terminal motif in PSEN2 that directs PSEN2/γ-secretase to late endosomes through a direct phosphorylation-dependent interaction with the AP-1 adaptor complex. This restricted localisation limits substrate availability for PSEN2/γ-secretase, providing the first cell biological basis and relevance of complex heterogeneity.

P2022 Board Number: B540

Palmitoylated Ras-driven MAPK signaling and transformation is spatially regulated by plasma membrane microdomains. J.V. Michael1, J.G. Wurtzel1, L.E. Goldfinger1,2; 1 Department of Anatomy and Cell Biology and the Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, 2Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA This study addresses the spatiotemporal regulation of Ras/MAP kinase (MAPK) signaling and mitogenic and tumorigenic outcomes. Somatic mutations in the Ras-superfamily of small GTPases are highly prevalent in cancers. Activating mutations in Ras (e.g., H-Ras) cause uncontrolled signaling through the MAPK pathway via activation of Raf kinase. After recruitment to the plasma membrane Raf propagates a mitogenic cascade by phosphorylating MEK, leading to ERK-mediated transcription. H-Ras also has been

TUESDAY-POSTER PRESENTATIONS shown to support induction of tumor angiogenesis. Related-Ras (R-Ras) has a nearly identical sequence with H-Ras, but is unable to drive MAPK signaling. R-Ras is anti-angiogenic and weakly mitogenic, but is not tumorigenic, and activating mutations have not been linked with human cancers. R-Ras can bind Raf in vitro, but does not bind in vivo. These paralogues have identical effector-binding regions, and differ primarily in the C-terminal domains, which harbor sequence-specific target sites for lipid modifications that direct targeting to distinct plasma membrane (PM) microdomains: H-Ras to lipid raft/non-raft borders, R-Ras to rafts. We hypothesized that the lateral spatial segregation at the PM regulates effector interactions, downstream signaling, and functional effects. We used targeting domain (tD) swap mutants between H- and R-Ras, and assessed the signaling, cellular and tumorigenic effects. Activated RRas harboring the H-Ras tD (R-Ras-tH), stably expressed in NIH3T3 fibroblasts, interacted with Raf and propagated MAPK signaling through MEK and ERK phosphorylation in the absence of growth factors. Furthermore, R-Ras-tH stimulated cell proliferation under these conditions, and supported anchorageindependent growth in soft agar, similar to activated H-Ras, indicating induction of a transformed phenotype in vitro by the R-Ras-tH. Conversely, H-Ras harboring the R-Ras tD (H-Ras-tR), did not stimulate MEK or ERK phosphorylation, or proliferation, and these cells displayed significantly reduced colony formation in soft agar compared with active H-Ras cells. Thus, access to Raf at the plasma membrane is a critical step for Ras-driven mitogenic signaling and transformation. A tumor xenograft model showed that R-Ras-tH promoted tumor formation whereas R-Ras cells yielded few tumors; however, R-Ras-tH tumors showed no neovascularization, which blunted tumor progression. Both H-Ras and H-Ras-tR xenografts formed very large, highly vascularized tumors. Thus, factors outside of the tDs affect tumor progression distinct from in vitro transformation, as H-Ras-tR appears to drive tumorigenesis through a MAPK-independent pathway in vivo. Together, these results indicate that the angiogenic function of Ras isotypes is independent of PM microdomain localization.

P2023 Board Number: B541

Cytosolic N-terminal proline-rich domains of MAL2 confer specificity to protein trafficking in polarized hepatic WIF-B cells. A. Lopez Coral1, P. Tuma2; 1 Biology, The Catholic University of America, Washington, DC, 2Department of Biology, Catholic Univ America, Washington, DC Epithelial cells rely on the formation of tight junctions at the plasma membrane to create two major membrane domains, the apical (AP) and basolateral (BL), which separates the external from the internal environments. The establishment and maintenance of these membrane domains are driven by a highly regulated protein trafficking pathway that selectively deliver newly-synthesized AP or BL resident proteins to their respective membrane domains. Polarized hepatocytes use the indirect route to deliver newly-synthesized apical resident proteins from the trans-Golgi network (TGN) to the BL from which they are selectively endocytosed into basolateral early endosomes (BLEE), transcytosed to the subapical compartment (SAC) and finally delivered to the AP. Myelin and lymphocyte protein 2 (MAL2) has been

TUESDAY-POSTER PRESENTATIONS identified as an important apical protein trafficking regulator of the indirect route in polarized WIF-B cells in at least two stages: from the TGN to the BLEE and from the BLEE to the SAC. More recently, we have determined that MAL2 also regulates constitutive secretion from the TGN to the BL. Our hypothesis is that MAL2 coordinates with multiple binding partners thereby conferring specificity to cargo selection and vesicle targeting to distinct locations. Once the cargo is packaged into discrete vesicles, we propose that sorting specificity may be conferred by interactions of distinct MAL2 binding partners (e.g. rab17 and Ser/Thr kinase 16) with the divergent MAL2 N-terminal domain. Thus, we predicted that the MAL2 cytoplasmic domains mediate interactions with other proteins or lipids that in turn confer specificity to apical sorting/targeting. Interestingly, the MAL2 N-terminus encodes VPPPP and FPAP sequences that resemble the F/L/W/YPPPP recognition sites for EVH1 (enabled, VASP, homology1) motifs present in Ena/VASP proteins, suggesting a possible role of MAL2 in actin dynamics. The N-terminus also encodes a predicted phosphorylated site (ser 17) that may be important in regulating protein-protein interactions. To test our hypothesis, we will overexpress C-terminal FLAGtagged MAL2 constructs using recombinant adenoviruses in WIF-B cells to allow their detection relative to endogenous MAL2. We will change the VPPPP and FPAP motifs to alanines either alone or together, deleting them individually or together and adding them to the MAL N-terminal sequence. We will also mutate ser 17 to ala or asn (a phospho-mimetic) and analyze their distribution and function in protein trafficking. Finally, we will examine if the C-terminal LRRW in MAL2 mediates lipid association like in MAL. Such careful and rigorous analysis will identify which (if any) of the domains confer specificity of MAL2 binding interactions and subsequent vesicle targeting.

P2024 Board Number: B542

Specific targeting/cellular localization of HCMV tegument protein pUL71 plays a critical role in production of infectious virus particles. R. . Grabski1, W.J. Britt1; 1 Pediatrics-I.D., University of Alabama at Birmingham, Birmingham, AL The human cytomegalovirus (HCMV), also known as human herpesvirus-5, is a common virus that can cause life-threatening infection in immunocompromised patients. The HCMV UL71 gene product, a component of the viral tegument layer, is a palmitoylated, oligomeric protein that plays a critical role in secondary envelopment of viral particles. Although previously described as non-essential, deletion of pUL71, mutation of palmitoylation sites, or deletion of oligomerization sequences severely impairs production of infectious virus. To explore involvement of pUL71 in viral assembly, we generated a series of C-terminal truncation mutants. Co-localization studies in transfected cells showed that pUL71 has strong preferential localization to the trans-Golgi compartment. At the position 23-26AA of pUL71 we identified a YxxΦ sorting signal responsible for interaction with the µ subunit of Adaptor Protein complex. The mutation of YVLL sequence of pUL71 to alanines (mutant 23A) exhibited a strikingly different phenotype as compared to that of the wild type (WT) protein, with characteristic plasma membrane localization and decreased presence at the Golgi apparatus. Considering the close proximity

TUESDAY-POSTER PRESENTATIONS of the mutations to the palmitoylation sites of pUL71 we tested incorporation of modified palmitic acid in transiently expressed WT and 23A mutant. The lack of differences in palmitoylation showed that the phenotype of the 23A mutant was caused by the replacement of the YVLL with AAAA. To elucidate the functional significance of the YxxΦ motif in assembly of the virus, a recombinant mutant virus was created. The 23A mutant virus showed extremely slow growth and infectivity rate. The electron microscopy analysis of cells infected with the 23A mutant virus showed accumulation of empty capsids and partially tegumented sub-viral particles in the cytoplasm. Our results indicate the existence of a new functional motif in pUL71 distinct from previously described palmitoylation and oligomerization signals, and one that is vital for localization of pUL71 and its function in HCMV assembly. It is attractive to speculate that tegument protein pUL71 may cycle between trans-Golgi, TGN, endosomes and plasma membrane, compartments that are dramatically modified during the process of infection, in a similar manner to that of envelope glycoproteins of HCMV.

P2025 Board Number: B543

Molecular basis of the phenotypic heterogeneity in congenital sucrase-isomaltase deficiency. B. Gericke1, M. Amiri1, D. Cooper2, H.Y. Naim1; 1 Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany, 2QOL Medical LLC, Vero Beach, FL The etiology of chronic, severe and common gastrointestinal symptoms (GI) such as osmotic diarrhea and abdominal pain is oftentimes due to carbohydrate malabsorption triggered by reduced levels of disaccharidase activity at the apical membrane of the enterocytes. Congenital sucrase-isomaltase deficiency (CSID) is a prominent form of carbohydrate malabsorption disorders that is characterized by altered trafficking and functioning of sucrase-isomaltase (SI), the most active intestinal disaccharidase. CSID is commonly detected as a recessive inborn defect that is elicited by mutations in the coding region of SI. Biochemical and cellular analyses of several mutations have established the phenotypes concept of CSID according to the cellular location, intracellular processing and function of SI (for a review see Naim et al., JPGN 2012). While the clinical presentation of CSID in most reported cases is severe, it is worthwhile to investigate whether a mild to severe spectrum of GI symptoms exists in heterozygotes or other less severe genotypes that would occur at a higher frequency in the general population. Here, we investigated the molecular pathogenesis of 14 naturally occurring mutations of the SI gene in patients with a prior diagnosis of CSID and varying enzyme activity levels. (Uhrich et al, JPGN 2012). The mutations were introduced into the cDNA of wild type SI by site-directed mutagenesis and expressed in COS-1 cells. Analysis of the structural and biosynthetic features of the mutants, their cellular localization and enzymatic function revealed three major categories of CSID. The first category is comprised of mutants that are trafficked and processed in a fashion similar to wild type SI and with mostly normal, but varying, levels of enzymatic activity. The second class of mutants revealed delayed trafficking and maturation kinetics as well as reduced enzymatic activities. Finally, the third group of mutants is entirely

TUESDAY-POSTER PRESENTATIONS transport-incompetent and persists in the ER as mannose-rich, immature and enzymatically inactive proteins that are ultimately degraded, presumably in the proteasome. Notably, the expression of active, wild-type SI proteins is negatively impacted by malfolded SI mutants in heterozygotes. The current study supports the existence of heterogeneous forms of CSID that vary in their degree of enzyme activity and clinical severity. These forms are primarily generated by the interaction of two mutants of SI or a mutant SI with its wild type counterpart. Furthermore, variations in the degree of severity are dictated by the malfolded structure of SI and its trafficking rate, which may vary in heterozygotes.

P2026 Board Number: B544

Mx1, a large GTPase involved in intracellular transport. F. Hoff1, C. Greb1, E. Hoenig1, K. Ringer1, R. Jacob1; 1 Institute for Cytobiology Cytopatholgy , Philipps-University of Marburg, Marburg, Germany The plasma membrane (PM) of polarized epithelial cells can be subdivided into an apical and basolateral domain, both separated by tight junctions. Targeting cargo molecules to their correct site of delivery is provided by a complex intracellular sorting and transport machinery. The analysis of post-TGN (Trans Golgi Network) exocytic carriers for new components involved in apical trafficking revealed the presence of the large GTPase Mx1 on these vesicles in Madin Darby Canine Kidney (MDCK) cells. Mx1 is known so far as an antiviral factor induced by the Interferon system after viral infections. However Mx1 is constitutively expressed in MDCK cells. Interestingly, the intracellular amount of Mx1 increases during early days of epithelial polarization. We could see that this dynamin-related protein associates with cargo vesicles guided to the apical PM. Furthermore, siRNA mediated knockdown of Mx1 significantly reduces the apical delivery of both soluble and membrane proteins. Our recent data show an interaction of this GTPase with the tubulin and actin cytoskeleton in the subapical region of MDCK cells. Fluorescence microscopy data indicate that Mx1 also colocalizes with different motor proteins.

P2027 Board Number: B545

The Role of Sec3p in Secretory Vesicles Targeting and Exocyst Complex Assembly. G. Luo1, W. Guo1; Department of Biology, University of Pennsylvania, Philadelphia, PA

1

During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before SNARE-mediated membrane fusion. The exocyst complex was thought to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We have developed an ectopic targeting assay in yeast, in which each of the eight exocyst subunits was expressed on the surface of

TUESDAY-POSTER PRESENTATIONS mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mis-targeting of the exocyst led to secretion defects in cells. On the other hand, Sec3p, but not other members of the exocyst, is capable of recruiting secretory vesicles. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.

Endocytic Trafficking 3 P2028 Board Number: B546

A flow cytometry based mutagenesis screen to identify novel endocytic factors. K. Wrasman1, Y. Zhang2, M. Khairy1, B. Wendland1; 1 Department of Biology, Johns Hopkins University, Baltimore, MD, 2Scripps Research Institute, San Diego, CA Clathrin-mediated endocytosis involves the coordination of many proteins to uptake nutrients, and respond to extracellular signals. With the plethora of proteins that need to be internalized from the plasma membrane, and the needs of the endocytic machinery to respond to many types of signals, it is unlikely we have identified all the factors involved in endocytosis. Using Saccharomyces cerevisiae, we performed an EMS mutagenesis screen to identify novel genes that contribute to endocytosis using an endogenous cargo, the methionine permease protein Mup1 to measure endocytic activity. Mup1 localization was visualized and quantified using a pHluorin (pHl) tag, which is a pH-sensitive GFP variant that is quenched upon delivery to the acidic environment of the vacuole lumen. In cells grown in low methionine conditions, Mup1 accumulates at the plasma membrane, whereas Mup1 is internalized and trafficked to the vacuole in high levels of methionine. We used flow cytometry to enrich for mutagenized cells with elevated fluorescence, where Mup1-pHl failed to traffic to the vacuole following addition of methionine. Over 1000 positive colonies were secondarily screened by microscopy. Defects in endocytosis and trafficking of Mup1-pHl were confirmed in 256 mutants, and these were categorized according to Mup1 subcellular localization. Eight recessive mutants with general endocytic defects (defined as having defects for all endocytic cargos thus far tested) were selected for further characterization, and two of these mutants are truncations of the known endocytic genes, SLA1 and SLA2. We are currently identifying the remaining general endocytic mutants using whole genome sequencing. As expected, some mutants with specific defects in the endocytosis of Mup1 are complemented by LDB19/ART1, a protein known to contribute to Mup1 internalization. Our screen allows us to directly identify endocytic mutants using a high-throughput, phenotype-based approach to study endocytic mechanisms of specific cargo proteins, and to find mutants with altered rates of transit from the plasma membrane to early/late endosomes.

TUESDAY-POSTER PRESENTATIONS

P2029 Board Number: B547

Defining the adaptor function of endocytic protein Syp1. K. Hoban1, A. Reider Apel2, S. Chuartzman3, M. Schuldiner4,5, B. Wendland1; Department of Biology, Johns Hopkins University, Baltimore, MD, 2Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 3Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 4Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel, 5Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel

1

Endocytosis is an important cellular process. One class of endocytic proteins is the adaptors, which bind to specific cargos, membranes, and other components of the endocytic machinery. One of the earliestarriving proteins recruited to sites of clathrin-mediated endocytosis (CME) in yeast is Syp1, a member of the muniscin family of adaptors that includes multiple mammalian homologs. Syp1 is known to bind Ede1, another early-arriving CME factor, and cargos such as Mid2, via its homology domain ( HD). However, the molecular mechanisms underlying Syp1's adaptor functions, including the sites on the HD necessary for its physical interactions with Ede1 and cargo proteins, as well as the sorting motifs that allow Syp1 to select specific cargos for internalization, have not been determined. Therefore, we aimed to locate the Ede1 binding site on the HD and ascertain the mechanisms of cargo recognition. Also, few proteins have been classified as Syp1 cargos; consequently, we sought to identify additional cargos of this adaptor. Using a collection of alanine scanning HD mutants, we isolated an evolutionarily conserved region of the domain’s surface that appears important for its physical interaction with Ede1. This construct, in which a seven residue-long sequence of the HD is mutated, shows reduced binding to Ede1 in assays such as affinity pull-downs. However, the mutant does bind Mid2, indicating that the mutant HD is folding properly and the decreased affinity for Ede1 is due to loss of side chains present in the WT domain. We also confirm the identity of Mep3, an ammonium permease isolated in a high throughput microscopy screen, as a novel Syp1 cargo using fluorescence microscopy. Mep3-GFP internalization is inhibited in the absence of Syp1; the protein instead accumulates at the plasma membrane. Its endocytosis is also HD-dependent, as the introduction of truncated Syp1 lacking this domain fails to internalize Mep3. Similarly, truncation of a portion of the Mep3 cytoplasmic, C-terminal tail prevents its endocytosis in WT cells. This indicates that Mep3 is a novel Syp1 cargo recognized specifically by the HD, possibly via a specific sorting motif. We are searching for this potentially novel endocytic motif involved in Syp1 cargo selection. We predict that a motif present in the cytoplasmic tail of Syp1 cargos such as Mid2 is important for cargo binding to the HD. Mutant Mid2 in which a small cytoplasmic segment is mutated to alanines exhibits diminished affinity for the HD in pull-down assays. We plan to use our collection of alanine scanning HD mutants

TUESDAY-POSTER PRESENTATIONS to identify the cargo binding site on this domain, complementing our studies to define the sorting motif within cargo proteins.

P2030 Board Number: B548

CALM is an adaptor for Gyrating-clathrin coated membranes. A. Gorin1, Y. Luo1, L. Ruggiero1, J.H. Keen1; 1 Biochemistry/Mol. Biol., Thomas Jefferson Univ, Philadelphia, PA We have identified highly dynamic clathrin-coated structures in the endosomal region of mammalian cells, and their rapid but highly localized movement, suggestive of clathrin-coated buds on membrane tubules, led to the term “Gyrating-“ or “G-clathrin” (1). Endocytic cargo such as transferrin can be visualized in G-clathrin downstream of the sorting endosome, and the structures have been implicated in rapid recycling of transferrin, b1-integrin and c-met receptor. While the tetrameric TGN and plasma membrane adaptors AP-1 and AP-2 are virtually undetectable in G-clathrin the single chain adaptors GGA1 or GGA3, expressed as fluorescent protein fusions, are almost completely colocalized with Gclathrin. Here we report that another single chain adaptor, GFP-tagged CALM (Clathrin assembly lymphoid myeloid leukemia protein) is also present in G-clathrin. Importantly, endogenous CALM can also be detected in G-clathrin structures followed through fixation. CALM contains an unstructured assembly domain which binds clathrin, and an N-terminal phosphoinositide-binding ANTH domain which has also recently been shown to bind SNARE proteins (2). Consistent with this, we could readily detect GFP-tagged forms of VAMP3, VAMP4 and VAMP8 in G-clathrin. However, SNARE binding is not critical for G-clathrin formation, as G-clathrin persisted in cells over-expressing either CALM mutants lacking SNARE binding or a CALM construct lacking the entire ANTH domain, and all were incorporated into Gclathrin structures. In contrast, expression of the CALM ANTH domain alone was cytosolic (3). G-clathrin also was unaffected by siRNA-mediated knockdown of CALM. However, this treatment abolished appearance of VAMP3 in G-clathrin. As endocytosis of VAMP3 is blocked under these conditions, as previously reported (2), these observations indicate that G-clathrin functions primarily in VAMP3 recycling rather than in biosynthetic transport, and that CALM is not required for G-clathrin formation. Finally, in cells expessing tagged forms of both CALM and GGA1, their presence in G-clathrin appeared mutually exclusive. While structures with GGA1 are primarily Arf6-dependent and are resistant to BFA, those with CALM show substantial sensitivity to BFA. This suggests that G-clathrin, until now defined only by its mobility and clathrin coat, likely exists in structurally and functionally distinct forms in the endosomal regions of cells. Further, our results suggest that G-clathrin is a significant trafficking compartment for other potential CALM cargos. 1- Zhao et al., Traffic 9:2253 (2008) 2- Miller et al., Cell147:1118 (2011) 3- Tebar et al., Mol Biol Cell10:2687 (1999)

TUESDAY-POSTER PRESENTATIONS

P2031 Board Number: B549

The use of phospholipase A2 Inhibitors to investigate the localization and trafficking of amyloid precursor protein. S.K. Boyle1, N. Belko1, E.B. Cluett1; 1 Biology, Ithaca College, Ithaca, NY Evidence from our lab has implicated phospholipase A2 activity in the intracellular transport of LDLderived free cholesterol in the endomembrane system. The effects of phospholipase inhibitors on the localization of proteins associated with endocytic compartments implicated the early endosome and the Endocytic Recycling Compartment (ERC) in the transport of cholesterol. siRNA knockdown of rab5 isoforms revealed differential effects on the distribution of cholesterol in both control and ONO-treated cells. Using isoform-specfic antibodies and immunofluorescence microscopy, treatment with ONO altered the localization of rab5A and rab5B but not rab5C. The ability to alter the distributions of cholesterol and marker proteins associated with specific intracellular compartments suggest that phospholipase A2 inhibitors may be useful in investigating the trafficking and processing of amyloid precursor protein (APP), a normal cellular protein that undergoes complex proteolytic processing. Misprocessing of this protein is associated with Alzheimer’s Disease. However, it is still not clear where the different processing events occur in the cell or what causes the misprocessing. Cholesterol is implicated in the onset of AD, and evidence suggests that the amount of cholesterol in the membrane may play a role in the misprocessing of APP. An emerging model suggests that one set of processing steps occur on the plasma membrane, but alternative processing, which can lead to misprocessing, may occur in an intracellular compartment. In CHO cells that have been stably transfected with APP, the protein is found in several different intracellular compartments, which is consistent with existing evidence. Treatment of these cells with ONO significantly changes the distribution of APP. However, APP does not appear to be appreciably associated with cholesterol in the ERC. Knockdowns of rab5 isoforms or rab11 change the distribution of APP suggesting that the protein traffics through the endocytic pathway. Experiments are now underway using immunoblotting and sandwich ELISAs to investigate the processing of the protein in the presence and absence of ONO.

P2032 Board Number: B550

cholesterol trafficking between the plasma membrane and the endocytic recycling compartment. S. Mao1, D.B. Iaea2,3, F.R. Maxfield2; biochemistry, Cornell University Weill cornell medical college, New York, NY, 2Cornell University Weill Cornell Medical College, New York, NY, 3Tri-Institutional Program in Chemical Biology, Weill Cornell Medical College, New York, NY 1

TUESDAY-POSTER PRESENTATIONS Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane (PM) is the largest cholesterol pool in the cell and contains ~60% of total cellular cholesterol. Cholesterol is also highly enriched in the endocytic recycling compartment (ERC). The abundance and distribution of intracellular cholesterol within membranes are tightly controlled by complex and poorly characterized homeostatic mechanisms. To analyze cholesterol transport between organelles, a fluorescent cholesterol analog dehydroergosterol (DHE) was used. We used fluorescence recovery after photobleaching (FRAP) to measure cholesterol delivery to ERC. Within 30 min after photobleaching, the fluorescence in ERC recovered about 90%. Recovery of DHE fluorescence in the ERC can be described by a single first order rate constant. The recovery t1/2 into the ERC is 14.5±1.5 min in U2OS cells. We developed a novel efflux method, in which a large exogenous cholesterol pool from red blood cells (109 per ml) rapidly exchanges with DHE at PM. This new method allows for accurate measurement of DHE efflux that can be modeled as a single exponential to characterize sterol transport rates from the ERC to the PM. DHE in the ERC effluxed completely out of cells with a t1/2 of 14.9±0.9 min in U2OS cells. Using these experimental improvements in the measurements of DHE trafficking, we accurately measured the effect of intracellular cholesterol level, the role of cholesterol transporters, and the energy-dependent and -independent transport processes in sterol trafficking. High intracellular cholesterol levels increase cholesterol the transport rate constant between the ERC and the PM, while mildly reduced intracellular cholesterol levels do not statistically change the rate constants for cholesterol transport into and out of the ERC. The increased amount of a cholesterol transporter (STARD4) significantly enhances the rate of cholesterol transport between ERC and PM. Energy depletion slightly reduces the rate of cholesterol transport between the ERC and the PM. These results suggest that while there is some contribution from vesicular transport, nonvesicular transport plays the major role in sterol transport between the ERC and the plasma membrane.

P2033 Board Number: B551

CFTR transcytosis in human respiratory epithelia: a constitutive rescue mechanism of basolaterally missorted channel from the biosynthetic and recycling pathways. A. Bidaud (Bidaud-Meynard)1, F. Bossard1, G. Veit1, G.L. Lukacs2,3; 1 Physiology, McGill University, Montreal, QC, 2Physiology, McGill University, Quebec, QC, 3Biochemistry, McGill University, Montreal, QC Cystic Fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), a chloride channel in the apical (Ap) plasma membrane (PM) of secretory epithelia. Considering that CFTR density at the Ap PM correlates with the CF disease severity, understanding the cellular/molecular mechanisms that determine the channel delivery to and removal from the Ap PM has fundamental importance. The mechanism responsible for CFTR polarized expression, however, remains poorly defined. Using morphological and biochemical assays, here we show that CFTR undergoes constitutive transcytosis (basolateral-to-apical) in differentiated, filter grown immortalized (CFBE) and conditionally

TUESDAY-POSTER PRESENTATIONS reprogrammed primary (HBE) human bronchial epithelial cells expressing CFTR. Transcytotic CFTR partly originates from basolaterally delivered channel from a) non-selective Ap and basolateral (Bl) delivery of secretory vesicles (cca. 30%) and b) from reversed or Ap-to-Bl transcytosis (cca. 70%) as a consequence of the limited fidelity of Ap recycling internalized CFTR. A similar trafficking rescue mechanism may be involved in mutations leading to small C-terminal truncations that have been implicated in compromising CFTR polarized expression by disrupting the interaction between the C-terminal PDZ (Postsynaptic density/Disc large/ZO-1)-binding motif of CFTR and NHERF-1, a PDZ-domain containing scaffolding protein. Truncation of the PDZ-binding motif increased internalization and decreased endocytic recycling of the CFTR-Δ6 mutant without discernible effect on Ap expression and stability in CFBE cells contrary to previous reports. The preserved Ap expression of CFTR-Δ6 could be explained by its increased transcytosis rate, similarly to that of the wild-type CFTR upon NHERF-1 knockdown and accounts for the lack of CF clinical phenotype in individuals carrying C-terminal truncations. Using morphological, genetic and biochemical tools some unique characteristics of CFTR transcytosis were revealed. Internalized CFTR delivery to contralateral PMs avoids both Ap and Bl recycling endocytic compartments contrary to Transferrin and IgA receptors, but likely involves EEA1 positive early endosomes. On the other hand, microtubule disruption interferes with the transcytosis of multiple cargoes, including CFTR, without interfering with its internalization and recycling. In summary, we have uncovered some of the cellular-molecular mechanisms that contribute to CFTR transcytosis and plays a role in the channel polarized expression in airway epithelial cells by redirecting basolaterally missorted molecules to the apical PM.

P2034 Board Number: B552

Shear Stress Dependent Regulation of Apical Endocytosis in Renal Proximal Tubule Epithelia. V. Raghavan1, Y. Rbaibi1, F. Carattino1, M.D. Carattino1, O.A. Weisz1; 1 Univ Pittsburgh Sch Med, Pittsburgh, PA The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorption over a wide range of glomerular filtration rates (GFRs). Internalization of filtered low molecular weight proteins, vitamins, hormones, and other small molecules is mediated by the proximal tubule (PT) multiligand receptors megalin and cubilin. Changes in GFR and the accompanying fluid shear stress (FSS) modulate acute changes in PT ion transport thought to be mediated by microvillar bending. We recently discovered that FSS also modulates apical endocytosis in PT cells via a mechanosensitive pathway regulated by primary cilia. FSS caused an acute and transient increase in intracellular calcium in cells that preceded the endocytic response. Neither the FSS-stimulated increase in intracellular calcium or apical endocytosis was observed in deciliated cells. Addition of ATP to the medium rescued both responses, suggesting the involvement of purinergic receptors in the signaling cascade. Consistent with this, inclusion of the ATP hydrolyzing enzyme apyrase or the pan-purinergic receptor inhibitor suramin in the perfusion medium blocked the FSS-stimulated endocytic response. Both basal and FSS-stimulated

TUESDAY-POSTER PRESENTATIONS endocytosis were inhibited by perturbants of clathrin and dynamin function. Additionally, treatment of PT cells with small molecule inhibitors of cdc42 or siRNA mediated knockdown of cdc42 ablated the FSSstimulated increase in endocytosis. Similarly knockdown of N-WASP or inhibition of Arp2/3 complexmediated nucleation of branched actin filaments prevented the FSS stimulated endocytic response. Our data suggest that exposure of PT cells to FSS enhances apical clathrin-mediated endocytosis via an actindependent pathway that is modulated by activation of Cdc42, N-WASP, and Arp2/3. Studies are underway to determine how the FSS-stimulated increases in intracellular calcium are transduced to increases in endocytosis.

P2035 Board Number: B553

Apical and Basolateral Recycling in Epithelial Cells. A.E. Perez Bay1, R. Schreiner2, I. Benedicto1, E. Rodriguez-Boulan3; 1 Ophthalmolgy department, Weill Cornell Medical College, New York, NY, 2Weill Cornell Medical College, New York, NY, 3Department of Ophthalmology, Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY Epithelial cells mediate vectorial transport of solutes and nutrients, by accurately sorting transporters and receptors to the apical and basolateral plasma membrane (PM) domains. Whereas some sorting occurs in the biosynthetic route, the major sorting of fast recycling receptors, such as Megalin and Transferrin receptor (TfR), occurs in the recycling route. Megalin is an apical recycling receptor responsible for the absorption of ultrafiltrate proteins in the kidney proximal tubule and its mutation in several human genetic syndromes causes proteinuria. TfR is the best-studied basolateral recycling receptor, which mediates iron uptake from the blood. Whereas the basolateral recycling pathway has been extensively studied using several endocytic receptors (i.e. TfR, LDLR, EGFR, etc), the apical recycling pathway is poorly understood. Here we show that apically internalized Megalin recycles successively through WGA-positive apical sorting endosomes, TfR-positive common recycling endosomes (CRE) and Rab11a-positive apical recycling endosomes (ARE) with half-times of 5, 12 and 21 minutes, respectively. Apically internalized Megalin mixes with basolaterally internalized TfR at CRE, suggesting that this is the compartment where sorting of these recycling receptors occurs. Very little is known on the mechanisms that mediate sorting and recycling of Megalin from CRE to the apical PM. Previous work has identified NPXY-like apical sorting signals (Takeda et al., AJP, 2003; Marzolo etal., Traffic, 2003) and our preliminary results suggest that clathrin is involved. In contrast, basolaterally internalized TfR is known to be sorted at CRE, by the clathrin adaptor AP-1B and clathrin, into a recycling route to the basolateral membrane. In certain epithelia that constitutively lack AP-1B, such as Retinal Pigment Epithelium, and in AP-1B knocked-down MDCK cells, TfR is relocated to the apical surface through a novel transcytotic pathway that involves its apical sorting at CRE by an N-glycan sorting signal and the sorting lectin galectin-4 (Perez Bay et al., JCS, 2014) and its transport from CRE to ARE and the apical surface by the kinesin KIF16B, non-centrosomal microtubules and the small GTPase rab11a (Perez Bay et al, EMBO J, 2013). In summary, our results describe the complete endosomal itinerary of apically

TUESDAY-POSTER PRESENTATIONS recycling megalin, provide the first evidence for a role of clathrin in apical sorting in mammalian cells and identify a novel transcytotic pathway for basolateral recycling receptors in AP-1B deficient epithelia. Supported by a NIH KO1 award 1K01DK102836-01 to APB and NIH grants GM34107 and EY08538 to ERB.

P2036 Board Number: B554

The Interaction of the Human Papillomavirus type 18 E6 Oncoprotein with Sorting Nexin 27 Modulates Retromer Dependent Cargo Recycling Function. K. Ganti1, J. Manzo-Merino2, L.M. Banks1; 1 Tumour Virology, ICGEB, Trieste, Italy, 2Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, Mexico City, Mexico Human Papilloma viruses are the causal agents of a large number of human malignancies, chief among which is cervical cancer. The continued over expression of the viral oncoproteins E6 and E7 is required for the initiation and maintenance of the neoplasm. Cancer causing HPV types are termed as high risk and a defining feature of the E6 oncoprotein from these types is the presence of a C-terminal PDZ binding motif. This allows the E6 protein to bind to and target various cellular PDZ domain containing proteins for degradation. Recent proteomic studies by Rozenblatt-Rozen et al (2012) and Belotti et al (2013) show that E6 may potentially bind to SNX27, which is a component of the endosomal transport system. Sorting Nexin 27 is unusual in that it is the only sorting nexin known to have a PDZ domain. This PDZ domain is known to facilitate the interaction of SNX27 with various PBM containing cargoes, thus directing their transport through the endosomal system. In this study, we show that SNX27 is a bonafide binding partner of HPV18E6 and this interaction is PDZ dependent. Furthermore, this interaction is conserved across various high risk cancer causing HPV types. We also show that E6 alters the subcellular distribution of SNX27 in HeLa cells and disrupts the SNX27-retromer link in a PDZ dependent manner. Analysis of known SNX27 cargoes such as the glucose transporter GLUT1 shows a perturbation in cargo retrieval and transport in the presence of E6. We show that the loss of E6 reduces surface GLUT1 levels in HeLa cells and this loss is reflected in increased lysosomal accumulation as seen in endosomal fractionation studies. We also demonstrate that the loss of E6 re-establishes the interaction between the retromer and GLUT1 in HeLa cells; further supporting the hypothesis that E6 is involved in the perturbation of the SNX27-retromer recycling pathway. Thus, these studies demonstrate a novel function for high risk HPV oncoproteins in the regulation of endosomal sorting pathways.

TUESDAY-POSTER PRESENTATIONS

P2037 Board Number: B555

Defining Cytosolic Components That Regulate Endosomal Membrane Protein Sorting. M.B. Gireud1,2, S. Reyes1,2, N. Sirisaengtaksin1,2, S. Tsunoda3, A.J. Bean1,2,4; 1 Neurobiology and Anatomy, University of Texas Health Science Center at Houston, Houston, TX, 2 University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, 3Biomedical Sciences, Colorado State University, Fort Collins, CO, 4Pediatrics, M.D. Anderson Cancer Center, Houston, TX Endocytosis is required for the uptake of essential nutrients from the extracellular environment and to limit the signaling by molecules (e.g. ion channels, receptors) present on the plasma membrane. The endocytic pathway is separated into stages based on cargo movement and identification of morphologically defined compartments. After internalization and transport from early to late endosomes, proteins destined for degradation in the lysosome are internalized into the lumen of late endosomes via membrane invagination and vesicle fission. The resulting organelle, the multivesicular body (MVB), is characterized by a limiting membrane and the presence of internal vesicles. Proteins that are sorted into internal vesicles are subject to degradation while those that remain on the limiting membrane are recycled. To facilitate sorting of proteins into the internal vesicles of the MVB, ubiquitin is added to the membrane protein. Ubiquitin allows for interaction with components of the sorting machinery, a group of cytosolic multi-protein complexes, the endosomal sorting complexes required for transport (ESCRTs), that play a role in protein sorting and internal vesicle formation. To examine the mechanisms underlying endosomal sorting, we have established a cell-free based assay that measures both MVB formation (ultrastructure) and the sorting of a cargo protein into the endosomal lumen. The sorting event is dependent on cytosolic components, ATP, time and temperature. Since this sorting event is dependent on soluble proteins, we examined the requirements and regulation of endosomal sorting by proteins present in the cytosol. We have undertaken a biochemical examination of cytosolic components by taking advantage of yeast and fly genetics, to understand the regulation of endosomal sorting and MVB biogenesis. By following the movement of a prototypical membrane protein, the epidermal growth factor receptor (EGFR), we have determined that cytosol obtained from Drosophila Melanogaster fly strainsand Saccharomyces cerevisiae yeast strains are sufficient to support the endosomal sorting of the EGFR with reasonable efficiency (20-40%). Further, cytosol derived from yeast strains deleted of ESCRT-0-III components decreased EGFR sorting into endosomes. The addition of recombinant mammalian ESCRT-0 homologs rescues the sorting defect caused by the absence of these yeast proteins. We have also examined other non-ESCRT cytosolic proteins that regulate the MVB sorting process. Understanding the molecular details of this critical endosomal sorting step will allow a better appreciation of the molecular pathways that ultimately may affect membrane protein signaling.

TUESDAY-POSTER PRESENTATIONS

P2038 Board Number: B556

Exploring retromer de-regulation in Parkinson’s disease. K.J. McMillan1, I.J. McGough2, F. Steinberg3, D. Jia4, P.A. Barbuti5, K.J. Heesom1, A.L. Whone6, M.A. Caldwell5, D.D. Billadeau7,8, M.K. Rosen9,10, P.J. Cullen1; 1 School of Biochemistry, University of Bristol, Bristol, United Kingdom, 2Developmental Biology, MRC National Institute for Medical Research, London, United Kingdom, 3Centre for Biological Systems Analysis, University of Freiburg, Freiburg, Germany, 4University of Texas Southwestern Medical Center, Dallas, TX, 5School of Clinical Sciences, University of Bristol, Bristol, United Kingdom, 6Institute of Clinical Neurosciences, University of Bristol, Bristol, United Kingdom, 7Department of Immunology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN, 8Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 9Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, 10Howard Hughes Medical Institute, Dallas, TX Retromer is a protein assembly that plays a central role in orchestrating export of trans-membrane cargo proteins from endosomes into recycling pathways destined for the Golgi apparatus and the plasma membrane [1]. Recently, a specific mutation in the retromer component VPS35, VPS35 (p.D620N), has linked retromer dysfunction to familial autosomal dominant and sporadic Parkinson's disease [2, 3]. Here we established that in cells expressing VPS35 (D620N) there is a perturbation in endosome-to-Golgi transport but not endosome-to-plasma membrane recycling, which we confirm and functionally rescue in Parkinson's disease patient cells harboring the VPS35 (p.D620N) mutation [4]. Through comparative stable isotope labeling by amino acids in cell culture (SILAC)-based analysis of wild-type VPS35 versus the VPS35 (p.D620N) mutant interactomes, we establish that the major defect of the D620N mutation lies in the association to the actin nucleating Wiskott-Aldrich syndrome and SCAR homologue (WASH) complex [4,5]. Consistent with this we quantify, using isothermal calorimetry that the affinity of VPS35 (p.D620N) for the WASH complex component FAM21 is decreased by 2.2 +/- 0.5 fold compared to wildtype [4]. In addition, we confirm the interaction between retromer and DNAJC13 [6], a mutation in which, DNAJC13 (N855S), is also linked with Parkinson's disease [7]. Together with evidence that retromer associates with LRRK2 [8] these data highlight retromer-mediated endosome sorting as a new pathway for studying the pathophysiology of Parkinson’s disease. Ongoing studies seek to utilize iPSCs and mouse models to further elucidate the role of retromer in dopaminergic neuron function and viability. [1] Cullen PJ, Korswagen HC. Nat Cell Biol 14, 29-37 (2012). [2] Vilarino-Guell et al. Am J Hum Genet 89, 162-167 (2011) [3] Zimprich et al. Am J Hum Genet 89,168-175 (2011). [4] McGough et al. Curr Biol 24, 1670-1676 (2014). [5] Zavodszky et al. Nat Commun 5, 3828 (2014). [6] Freeman et al. J Cell Sci 127, 2053-2070 (2014). [7] Vilarino-Guell et al. Hum Mol Genet 23, 1794-1801 (2014). [8] MacLeod et al. Neuron 77, 425-439 (2013).

TUESDAY-POSTER PRESENTATIONS

P2039 Board Number: B557

Numb Diverts Notch to Late Endosomes to Antagonize Notch Membrane Recycling in Drosophila Sensory Organs. S. Johnson1, F.J. Roegiers2; Cell and Molecular Biology, University of Pennsylvania, Philadelphia, PA, 2Fox Chase Cancer Center, Philadelphia, PA

1

Notch signaling is a well conserved signaling pathway that controls a wide variety of developmental functions including cell fate determination and patterning. Errors in the regulation Notch signaling have been implicated in forms of colon cancer and leukemia, as well as other leukodystrophies including CADASIL syndrome. To elucidate Notch signaling regulation, we took advantage of sensory organ differentiation in Drosophila, a process that requires regulation of both Notch activation and inhibition. Sensory organs of Drosophila arise from a pair of cells, one of which must activate Notch signaling, while the other must inhibit signaling. Blockage of Notch signaling in one of the cells requires the presence of Numb, a membrane-associated protein whose mechanism of Notch inhibition is currently unknown. We are working to show that Numb functions through preventing the recycling of the Notch receptor back to the plasma membrane. We show that Numb promotes targeting of full-length Notch to late endosomes. Conversely, Notch levels in early endosomes are equivalent between the Numb positive and negative cells, supporting a model where Numb functions in Notch trafficking at a postinternalization step. Furthermore, we find that Numb itself may localize to recycling endosomes. To evaluate Notch recycling directly, we employed a novel assay that allows for marking of recycled vs. internalized Notch. Using this technique we found that Numb suppresses Notch receptor recycling back to the plasma membrane. Our work will provide new insight into the inhibition of Notch via Numb, which may allow for the creation of new drugs which can function similarly to Numb to regulate Notch activity.

P2040 Board Number: B558

Clathrin Independent Endosomal Trafficking is required for T Cell Conjugate Formation and Activation. D. Johnson1, J.M. Wilson2, J.G. Donaldson3; 1 Univ Arizona, Tucson, AZ, 2Univ Arizona Coll Med, Tucson, AZ, 3Cell Biology and Physiology Center, NHLBI/NIH, Bethesda, MD The clathrin independent endosomal system is important for cellular homeostasis and specialized modifications of the plasma membrane. We characterized clathrin independent endocytosis (CIE) in the human Jurkat T cell line and compared it other cells. We found that major histocompatibility complex I

TUESDAY-POSTER PRESENTATIONS (MHCI) was endocytosed separately from the clathrin-mediated endocytic marker the transferrin receptor. Subsequently, MHCI passed through EEA1-positive endosomes, prior to trafficking to LAMP-1 positive vesicles for degradation or to endosomes for recycling back to the plasma membrane. In contrast, another CIE cargo protein, CD98, avoided residence in EEA1 positive endosomes after internalization and was rapidly recycled back to the cell surface. Thus CIE in Jurkat cells is similar to what is observed in other human cell lines. Two G proteins, Arf6 and Rab22, have been shown to regulate endosomal trafficking and recycling of CIE cargo proteins. They are also required for rearrangement of the cell surface during events like cell spreading. We found that wild type Arf6 co-localized with CIE cargo and Rab22 co-localized with internalized MHCI in resting T cells. To test whether Arf6 or Rab22 activities were required for T cell activation we incubated Jurkat cells with activated antigen presenting cells (APC). Expression of the dominant negative mutants of Arf6 or Rab22 decreased T cell engagement with the APC and reduced actin polarization at the site of the immunological synapse. In addition, there is reduced T cell signaling as indicated by decreased phosphorylated tyrosine at the synapse of cells expressing mutant Arf6. Arf6 and Rab22 dominant negative mutants were also inhibited in their ability to spread when plated on antibody-coated coverslips that cause activation. Our results confirm that CIE and subsequent endosomal trafficking is a highly conserved process. The clathrin independent endosomal system is used by T cells to redistribute and polarize membranes to the site of activation. Our findings suggest a requirement for Arf6 and Rab22 in mediating membrane trafficking and signaling events at the immunological synapse. Our future goal is to understand how CIE and endosomal trafficking affects T cell signaling and function.

P2041 Board Number: B559

Macronutrient stress modulates antigen trafficking and MHC class II presentation by altering HSC70. S.N. Deffit1, J.S. Blum1; 1 Indiana University School of Medicine, Indianapolis, IN B lymphocytes exploit macroautophagy to capture cytoplasmic and nuclear proteins within autophagosomes. Autophagosome fusion with lysosomes and endosomes facilitates content proteolysis, with the resulting peptides selectively binding MHC class II molecules which are displayed for recognition by T lymphocytes. Nutrient stress amplified this pathway, favoring increased class II presentation of cytoplasmic antigens targeted to autophagosomes. By contrast, nutrient stress diminished class II presentation of membrane antigens including the B cell receptor (BCR) and cytoplasmic proteins that utilize the chaperone-mediated autophagy (CMA) pathway. While intracellular protease activity increased with nutrient stress, endocytic trafficking and proteolytic turnover of the BCR was impaired. Addition of high molecular mass proteins restored endocytosis and antigen presentation, evidence of tightly regulated membrane trafficking dependent on macronutrient status. Altering the

TUESDAY-POSTER PRESENTATIONS abundance of the cytosolic chaperone HSC70 was sufficient to overcome the inhibitory effects of nutritional stress on BCR trafficking and antigen presentation. Together, these results reveal a key role for macronutrient sensing in regulating immune recognition and the importance of HSC70 in modulating distinct membrane trafficking pathways during cellular stress.

P2042 Board Number: B560

Alterations in Trafficking and Degradation of FGFR4, a commonly mutated protein in High-Risk Neuroblastoma Cases. S. Reyes1,2, M.B. Gireud3,4, N. Sirisaengtaksin3,4, P.E. Zage5,6, A.J. Bean3,4,7; 1 Department of Neurobiology and Anatomy, University of Texas Health Science Center at Houston, Houston, TX, 2University of Texas Graduate School of Biomedical Sciences, Houston, TX, 3Neurobiology and Anatomy, University of Texas Health Science Center at Houston, Houston, TX, 4University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, 5Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine, Houston, TX, 6Cancer and Hematology Centers, Texas Children’s Hospital, Houston, TX, 7Pediatrics, M.D. Anderson Cancer Center, Houston, TX Overall survival rates for children diagnosed with high-risk neuroblastoma remain low despite current treatment regimens. Resistance of these tumors to conventional therapies is a major problem and the molecular mechanisms that underlie resistance remain unclear. Growth factor receptors and their signaling cascades have been suggested to cause tumorigenesis and/or metastasis. Therapies targeting these receptors are often effective anti-cancer agents. The fibroblast growth factor receptor 4 (FGFR4) signaling cascade promotes cell division and differentiation. We have observed that expression of a polymorphic FGFR4 correlates with increased mortality in neuroblastoma patients. This polymorphism generates a glycine-to-arginine change in codon 388. It has been linked to aggressive phenotypes in various other cancers and is associated with increased signaling. FGFR4 signaling can be regulated by the endocytic pathway such that the duration and amplitude of receptor signal can be altered by receptor trafficking. Upon ligand binding, FGFR4 is internalized and moves through a series of endosomal compartments prior to being degraded in the lysosome. We hypothesize that the polymorphism (Arg388) interferes with trafficking of this receptor through the endocytic pathway and that sustained signaling from non-degraded receptors promotes neuroblastoma growth. Given the apparent importance of this FGFR4 mutation in neuroblastoma tumorigenesis, and the role that endocytic trafficking can play in receptor signaling kinetics, we explored whether this polymorphism displayed altered trafficking that might underlie enhanced signaling. We examined (wt) Gly388 and (mutant) Arg388 FGFR4 degradation in SY5Y cells following ligand (bFGF) stimulation. We observed that FGFR4 Arg388 receptors are degraded at a slower rate than wild-type FGFR4 receptors. To understand where in the endocytic pathway the mutant FGFR4 is delayed, we compared the movement of FGFR4s through various steps in the endocytic pathway. For example, we used a cell-free assay that measures movement of membrane proteins into internal vesicles of late endosomes to examine whether wt or mutant FGFR4 are differentially sorted at the late endosome.

TUESDAY-POSTER PRESENTATIONS Proteins sorted into internal vesicles within late endosome/MVBs are degraded upon fusion with the lysosome and decreased MVB sorting could therefore account for decreased degradation. However, no significant differences in MVB sorting were observed between wild-type FGFR4 and FGFR4 Arg388. Analysis of other endocytic steps whose inhibition could account for decreased FGFR4 Arg388 degradation (e.g. internalization from the plasma membrane, recycling to the plasma membrane, and movement from early endosomes) as well as effects on downstream signaling are ongoing.

P2043 Board Number: B561

Divide and Polarize: The Role of Cytokinesis and Endocytic Transport during Epithelial Cell Polarization. A. Mangan1, D. Li1,2, R. Prekeris1; 1 Cell and Developmental Biology, Univ Colorado Anschutz Medical Campus, Aurora, CO, 2University of Colorado Anschutz Medical Campus, Aurora, CO Epithelial cells are structurally and functionally polarized to transport specific molecules while maintaining a trans-epithelial barrier. This cellular asymmetry is essential for the proper functioning of epithelial tissues and depends on polarized endocytic transport routes. Additionally, epithelial cells coordinate their polarization with neighboring cells to form an apical lumen, a key step in the establishment of renal and gut architecture, and thereby function. Recent work from several laboratories including ours identified Rab11 and its binding protein FIP5 as major components that regulate apical endosome transport during apical lumen formation. We demonstrated that Rab11/FIP5containing endosomes (FIP5-endosomes) mediate the formation of apical lumen by targeted delivery of apical lumen proteins, and that FIP5 functions by interacting with sorting nexin 18 and Kinesin-2. It was also shown that targeting of FIP5-endosomes to the apical membrane initiation site (AMIS) is a key step during the initiation and expansion of the apical lumen during formation of MDCK epithelial cysts in 3D tissue culture system. Despite recent advances in our understanding of the mechanisms mediating lumen formation, many questions remains unanswered. How are FIP5-endosomes targeted during apical lumen formation? How do cells establish the site of single apical lumen? In this study we focused on the identification of the machinery that mediates AMIS formation and FIP5-endsosmes targeting during apical lumen formation. We identified a new FIP5-interacting protein, cingulin, which is a tight junction protein that localizes to the AMIS. Thus, cingulin is perfectly suited to serve as a tether to mediate FIP5endosome targeting during apical lumen formation. Importantly, our data demonstrate that AMIS forms around the midbody of the mitotic epithelial cells during late telophase. Finally, we demonstrate that AMIS formation is initiated by Rac1-dependent burst of actin polymerization at the midbody. Based on all these studies we propose a novel hypothesis that AMIS formation around the midbody and FIP5/cingulin-dependent endosome targeting to the midbody during late telophase are the first “symmetry breaking” events leading to the initiation of apical lumen during epithelial morphogenesis.

TUESDAY-POSTER PRESENTATIONS

P2044 Board Number: B562

Novel mechanistic insights into anthrax toxin endocytosis. S. Friebe1, L. Abrami1, F. Van Der Goot1; 1 EPFL, Lausanne, Switzerland Anthrax is a disease largely caused by the exotoxins of Bacillus anthracis. Entry into cells is mediated by one of the two known anthrax toxin receptors. The main receptor is Capillary Morphogenesis Gene 2 (CMG2) also known as ANTXR2, a single pass type I transmembrane protein of unknown physiological function. How these toxins act on cells is well studied, yet the exact initial steps of toxin entry are still poorly understood. We therefore aimed to better understand this complex and fascinating process and to shed light on the physiological role of the main anthrax toxin receptor. For this we identified new potential partner proteins of CMG2 by using several Yeast Two Hybrid screens: Currently our work is focusing on a kinase and an E3 ubiquitin ligase as well as a protein identified from a genome wide screen for anthrax susceptibility, ARAP3. ARAP3 is a PI3K effector protein and a RhoGAP implicated in integrin regulation and growth factor receptor trafficking. Lastly, the downstream effector of ARAP3, RhoA is a protein known to mediate actin reorganization upon extracellular stimuli. We could show that all proteins interact with CMG2 by co-immunoprecipitation and have started to pinpoint the interacting domains on the proteins by biochemical and microscopical methods. We also show that interaction between the proteins is constant and not ligand-triggered for all four proteins. To understand the functional implications of these interactions we studied the different steps of anthrax toxin endocytosis. Knockdown of all four proteins did not have an effect on binding of the toxin, yet the effect on later steps of the process was striking. In the absence of the kinase and the E3 ligase, endocytosis of the receptor-toxin complex was almost completely inhibited. This might be due to a lack of phosphorylation and ubiquitination of the receptor after toxin binding in the absence of these enzymes. Knockdown of ARAP3 strongly delayed endocytosis and knockdown of RhoA led to a defect of heptamer formation, a prerequisite for efficient endocytosis. To ensure that these proteins act specifically on CMG2, we looked at entry of Diphteria Toxin, which also enters cells via clathrin-mediated endocytosis. So far it seems that the proteins are specific for endocytosis of anthrax and are not general regulators of clathrin-mediated endocytosis. By gaining important insights into the endocytosis of anthrax toxin with the help of this small-scale protein network, we also hope to broaden our knowledge about the physiological behavior of its receptor.

TUESDAY-POSTER PRESENTATIONS

P2045 Board Number: B563

Ack1 Overexpression Results in the Interruption of Several Endocytic Pathways and Vesicular Trafficking. C. Ye1, L. Guan1, T. Lin1, V.E. Prieto-Echagüe2, D.A. Brown3, W.T. Miller4, A.L. Gucwa1; 1 Biomedical Sciences, Long Island University At Post, Brookville, NY, 2Molecular Human Genetics, Institut Pasteur de Montevideo, Montevideo, Uruguay, 3Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, 4Physiology and Biophysics, Stony Brook University, Stony Brook, NY Ack1 is a direct effector of the Rho GTPase member Cdc42, and is amplified and overexpressed in several types of cancers. Its strong implications in tumor progression and metastasis make it of great interest. However, until recently, little was known about this non-receptor tyrosine kinase (NRTK) and its regulation. The NRTK is known to play an integral role in clathrin-dependent internalization and the down-regulation of activated EGFR. We were interested to see whether Ack1 also plays a role in the internalization and trafficking of ErbB2, a member of the ErbB family of growth factor receptors. Treatment of cells in culture with the ansamycin antibiotic, geldanamycin (GA), results in the internalization and degradation of ErbB2 in a clathrin-independent manner. The overexpression of Ack1 disrupted the down-regulation of ErbB2 in GA-treated cells. Cholera toxin uptake, which occurs via lipid raft-dependent mechanisms, was also blocked by its overexpression. We did not find any effect of Ack1 on bulk fluid uptake, as the internalization of high-molecular weight dextran was unchanged. Additionally, we found this effect was not limited to endocytosis; Ack1 overexpression induced the disruption and tubularization of the Golgi apparatus, similar to that seen in Brefeldin A-treated cells. To further investigate the role of Ack1 on the trans-Golgi network, we studied its effect on mannose 6phosphate receptor (MPR). This receptor binds newly synthesized lysosomal hydrolases in the transGolgi network (TGN) and delivers them to pre-lysosomal compartments. A GFP-tagged form of cationindependent MPR (CI-MPR) was sequestered at the plasma membrane when co-expressed with Ack1, suggesting Ack1 may also alter retrograde transport. Taken together, our data suggests the overexpression of Ack1 plays a global and more significant role in endocytosis and intracellular trafficking than previously thought.

P2046 Board Number: B564

FAM21 regulates recycling of SNX27-retromer cargoes to the plasma membrane. S. Lee1, J. Chang1, C.D. Blackstone1; 1 NINDS, National Institutes of Health, Bethesda, MD The endosomal network controls numerous cellular functions such as signaling, nutrient uptake, and development through balanced trafficking of diverse plasma membrane proteins. After receiving membrane protein cargoes, endosomes sort them into lysosomes for degradation, or else to the transGolgi network or plasma membrane for recycling. These pathways are elaborately controlled by the

TUESDAY-POSTER PRESENTATIONS retromer complex, a multi-subunit complex associated with the cytosolic face of endosomes, along with its interacting proteins. Recently, the retromer complex in association with SNX27, a PDZ-domaincontaining SNX protein, has been identified as a major endosomal hub to regulate endosome-to-plasma membrane recycling. Even though the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex, an activator of Arp2/3-mediated actin nucleation in endosomes, has been identified to interact with the SNX27-retromer complex, a specific role for the WASH complex in the recycling pathway has not been elucidated. Here, we have identified the FAM21 subunit of the WASH complex as a direct interacting partner of SNX27. A FERM domain of SNX27 binds to -helical domains at the N-terminus of FAM21, which are distinct from the binding sites for the VPS35 subunit of the retromer complex. We have also demonstrated that the interaction is required for proper recycling of SNX27-retromer cargoes by distributing SNX27 within an endosomal subdomain to aid export of cargoes to their destination. Thus, we suggest that the WASH complex regulates endosome-to-plasma membrane recycling in collaboration with the SNX27-retromer complex.

P2047 Board Number: B565

A CLATHRIN AND DYNAMIN-INDEPENDENT PATHWAY OF SYNAPTIC VESICLE RECYCLING MEDIATED BY BULK ENDOCYTOSIS. Y. Wu1,2,3, E. O'Toole4, M. Girard5, B. Ritter6, P.S. McPherson7, S.M. Ferguson1, P. De Camilli8; 1 Department of Cell Biology, Yale University, New Haven, CT, 2department of cell biology, 1Howard Hughes Medical Institute, New Haven, CT, 3CNNR program, Yale University, New Haven, CT, 4MCD Biology, University of Colorado, Boulder, CO, 5Neurology and Neurosurgery, Montreal Neurological Institute, Mcgill university, Montreal , Canada, 6Boston Univ Sch Med, Boston, MA, 7McGill University, Montreal, QC, 8Department of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT The massive exocytosis of synaptic vesicles (SVs) elicited by a potent nerve terminal stimulation is rapidly compensated by bulk endocytosis of SV membranes leading to large endocytic vacuoles. Subsequently, these vacuoles (“bulk” endosomes) disappear in parallel with the reappearance of new SVs. We have capitalized on synapses of dynamin 1 and 3 double knock-out (DKO) neurons, where clathrin-mediated endocytosis is dramatically impaired, to gain insight into the poorly understood mechanisms underlying this process. Massive formation of bulk endosomes was not impaired in these neurons. Conversion of bulk endosomes into SVs during recovery was delayed, but not blocked, even after further clathrin knock-down, showing that this process is independent of clathrin-mediated budding, consistent with the absence of clathrin coats on bulk endosomes. These findings support the existence of a pathway for SV reformation that bypasses the requirement for clathrin and dynamin and demonstrates the plasticity of the SV recycling process.

TUESDAY-POSTER PRESENTATIONS

Establishment and Maintenance of Polarity 2 P2048 Board Number: B567

Molecular insights into zipcode activated cytoplasmic mRNA transport. H. Shi1; 1 Dept. of Cell biology, Rockefeller University, New York, NY Myo4p is one of five distinct myosins of yeast, which is dedicated to cytoplasmic transport of two types of cargos, zipcoded mRNPs and tubular endoplasmic reticulum (tER). Neither cargo binds directly to Myo4p. Instead, She3p serves as ‘adaptor’ that contains three binding modules, one for Myo4p and one each for zipcoded mRNP and tER. The assembly of a transport-competent motor complex is poorly understood. Here, we report that [Myo4p•She3p] forms a stable 1:2 heterotrimer in solution. In the [Myo4p•She3p] crystal structure, Myo4p’s C-terminal domain (CTD) assumes a lobster claw-shaped form, the minor prong of which adheres to a pseudo-coiled coil region of She3p. Because the [Myo4p•She3p] heterotrimer contains only one myosin molecule, it is not transport-competent. By stepwise reconstitution, we found a single molecule of synthetic oligonucleotide (representing mRNA zipcode element) bound to a single tetramer of zipcode binding protein She2p to be sufficient for [Myo4p•She3p] dimerization. Hence, cargo initiates cross-linking of two [Myo4p•She3p] heterotrimers to an ensemble that contains two myosin molecules obligatory for movement. An additional crystal structure comprising an overlapping upstream portion of She3p showed continuation of the pseudocoiled coil structure and revealed another highly conserved surface region. We suggest this region as candidate binding site for a yet unidentified tER ligand. We propose a model, whereby zipcoded mRNP and/or tER ligands couple two [Myo4p•She3p] heterotrimers and thereby generate a transportcompetent motor complex either for separate or co-transport of these two cargos.

P2049 Board Number: B568

Proinflammatory cytokine-induced Tight Junction remodeling through dynamic self-assembly of claudins. C.T. Capaldo1, A.E. Farkas1, C.A. Parkos1, A. Nusrat1; 1 Pathology, Emory University, Atlanta, GA Tight Junctions (TJs) are dynamic, mutiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiologic development and pathologic mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a bio-sensor of TJ remodeling, we observed increased Claudin 4 Fluorescence Recovery after Photobleaching (FRAP) dynamics in response to inflammatory cytokines.

TUESDAY-POSTER PRESENTATIONS Interferon gamma (IFN-gamma) and Tumor Necrosis Factor alpha (TNF–alpha) increased the proportion of mobile claudin 4 in the TJ. The claudin 4 PDZ (PSD95/Dlg/ZO-1) motif was required for cytokineinduced changes in dynamics. Upregulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin binding incompatibility.

P2050 Board Number: B569

From Microdishes to microniches.3D microenvironmental control around single cells. Application to single cell apico basal polarization and lumenogenesis control. V. Viasnoff1,2, Q. Li1, J. Thiery3,4, H. Yu1,5,6, Z. Weng7; 1 MBI/DBS, Mechanobiology Institute, Singapore, Singapore, 2UMI3639, CNRS, Singapore, Singapore, 3 Biochemistry, NUS, Singapore, Singapore, 4IMCB, Astar, Singapore, Singapore, 5School of Medicine, NUS, Singapore, Singapore, 6IMB, Astar, Singapore, Singapore, 7Mechanobiology Institute, Singapore, Singapore The influence of the microenvironment on cell behavior and fate is increasingly recognized as of key importance. It follows that new techniques to control the 3D environment around cells are key to understand the processes by which cells probe and respond to their microniches. We present here an approach that allow to transform microwells into artificial microniches where the chemical coating, rheological properties and topographical properties can be differentially controlled on the top, sides and bottom of the wells and assembled in a combinatorial way. This technique is also compatible with high and super resolution imaging that allows to probe the dynamics of cell cytoskeleton and regulatory proteins with unprecedented resolution down to the single molecule level in 3D. We exemplify how theses bone fide artificial microniches can be used to induce full apico-basal polarization of single epithelial cells and as well as to control intercellular stresses driving the anisotropic growth of intercellular lumens. Other on going applications will also be briefly discussed. Ref: Li et al, Nature Cell Biology, in revision

P2051 Board Number: B570

Apical lumen formation in single pluripotent human and mouse embryonic stem cells initiates via establishment of Rho-GTPase dependent intracellular apical membrane initiation site. K. Taniguchi1, R. Townshend1, D. Chue1, Y. Tsai1,2, C. Harris3, S. Lopez1, A. Freddo1, S. Kalantry3, J. Spence1,2, B. Margolis2,4, S. O'Shea1, D. Gumucio1;

TUESDAY-POSTER PRESENTATIONS 1

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, 3Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 4Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 2

Establishment of an apical surface and formation of a lumen is an essential process employed during morphogenesis of epithelial tissues and organs. We have found that single dispersed human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) organize apical and baso-lateral proteins at a peri-nuclear site prior to their first cell division. As the cell divides, this collection of proteins becomes the lumen between the two daughter cells; thus, it functions as an intracellular apical membrane initiation site (intra-AMIS). With further growth, these hESCs and hiPSCs form hollow cysts while maintaining the expression of NANOG and POU5F1. At the one-cell stage, the intra-AMIS structure contains highly distinct apical and baso-lateral compartments. A central apical domain is enriched for EZRIN and aPKC proteins (apical markers), surrounded by ring-shaped ZO1 protein (tight junction), a zone of RAB11 (early endosome) and GM130 (Golgi apparatus), as well as by junctional markers such as E-CADHERIN, β-CATENIN and β1-INTEGRIN. This clearly shows the presence of intracellular baso-lateral domain in addition to apical. We show that the activation of Rho-mDia and Rac1-Arp2/3 signaling is essential for intra-AMIS formation and consequent patterning of the apical lumen, whereas inhibition of Rho-ROCK-myosin-II signaling promotes lumen formation. Other cell types, such as cultured Caco-2 (human colonic adenocarcinoma) and MDCK.2 (adult canine kidney) cells form intra-AMIS structures that evolve to lumens as well, suggesting that intra-AMIS structures are an important aspect of lumen formation in multiple settings and in multiple species. Like hESCs and hiPSCs, mouse embryonic stem cells (mESCs) also form intra-AMIS structures. Seeking an in vivo correlate to this phenomenon, we show that 4-day-old mouse blastocysts display intra-AMIS structures in primitive endoderm, trophectoderm and epiblast precursor cells. Together, these data reveal that multiple cell types, including pluripotent human and mouse cells, have propensity to form lumina, and that the organization of the lumenforming machinery begins with the formation of an intra-AMIS at the one-cell stage.

P2052 Board Number: B571

KLF4 controls epithelial stratification via non-canonical WNT5A signaling. M. Tetreault1, D. Weinblatt1, J.P. Katz1; 1 GI/Medicine, University of Pennsylvania, Philadelphia, PA Background: Krüppel-like factor 4 (Klf4) is a DNA-binding transcriptional regulator highly expressed in gastrointestinal epithelia, specifically in regions of cellular differentiation. In the esophagus, Klf4 is expressed in differentiating cells of the suprabasal and superficial layers. Loss of Klf4 perturbs normal squamous differentiation pathways and alters squamous epithelial stratification. Wnt5a is a noncanonical Wnt ligand down-regulated by gene expression analyses in Klf4 conditional knockout mice. Wnt5a is important for the regulation of a variety of cellular functions, such as proliferation,

TUESDAY-POSTER PRESENTATIONS differentiation, migration, and polarity. We hypothesized that KLF4 controls esophageal epithelial stratification by direct transcriptional regulation of WNT5A. Methods: We used tissue specific gene ablation to delete Klf4 in esophagus and sacrificed mice at 3 months of age for analyses. Primary esophageal keratinocytes were isolated for in vitro studies of gene regulation, cell migration, and stratification. Results: Tissue-specific Klf4 knockout mice (ED-L2/Cre;Klf4loxp/loxp) had altered morphology of cells in the suprabasal and superficial layers. Wnt5a, a non-canonical Wnt ligand, was decreased by 3.0-fold on microarray, and we confirmed a 2.9-fold decrease by quantitative real-time PCR. These decreases in WNT5A were seen predominantly in the suprabasal layers of EDL2/Cre;Klf4loxp/loxp mice. Klf4 inducible knockdown with shRNAs in primary esophageal keratinocytes also produced a 30% decrease in Wnt5a expression levels. We found marked induction of Wnt5a promoter activity by KLF4 in primary esophageal keratinocytes using the luciferase reporter pGL4Wnt5a. Moreover, we also demonstrated KLF4 binding to WNT5A by ChIP in human primary esophageal keratinocytes. Primary esophageal keratinocytes with Klf4 knockdown had decreased polarity during cell migration, which was restored by recombinant WNT5A. Furthermore, we demonstrated that human primary esophageal keratinocytes with KLF4 knockdown had evidence of delayed cellular maturation, and defects in stratification in an organotypic 3D cell culture system. Treatment with recombinant WNT5A restored proper stratification in 3D cultures with decreased KLF4 expression. Conclusion: KLF4 is a key regulator of esophageal epithelial cell migration and stratification via WNT5A.

P2053 Board Number: B572

CRB3 restores epithelial cell shape and actin organization in cancer cells. E. Loie1, L. Charrier1, K. Sollier1, P. Laprise2; BMBMP, Université Laval, QUEBEC, QC, 2BMBMP, Centre de Recherche sur le Cancer de l'Université Laval - CRCHU de Québec, Québec, QC

1

BACKGROUND AND OBJECTIVES: Apico-basal polarity is a common feature of epithelial cells. Loss of the polarized architecture of epithelial cells is associated with many pathologies, including cancer. The transmembrane protein CRB3 is an important determinant of the apical domain in epithelial cells. Recently, CRB3 has emerged as a potential tumor suppressor, as it represses tumor growth and metastasis in animal models. However, the molecular mechanisms used by CRB3 to prevent tumor development and progression are unknown. Our goal is to decipher these mechanisms.METHOD: HeLa cells, which are devoid of endogenous CRB3, were transfected with a tagged form of CRB3, or truncations of CRB3. The phenotype associated with expression of these proteins was analyzed by biochemistry and cell biology techniques.RESULTS: Expression of CRB3 in HeLa cells results in a drastic change in cell morphology and a strong reorganization of the actin cytosqueleton. Cells also show tighter contacts with each other, suggesting an improvement of cell-cell junctions. These changes depend on CRB3 cytoplasmic tail, but do not require the extracellular domain. This indicates that CRB3 probably controls intracellular signaling pathways to trigger actin rearrangements. CONCLUSION: Expression of CRB3 in HeLa cells drives important structural changes similar to a mesenchyme-to-epithelial transition

TUESDAY-POSTER PRESENTATIONS in cancer cells. Expression of CRB3 in aggressive carcinomas could then prevent cancer progression and metastasis. Identification of the mechanisms controlled by CRB3 to limit tumor progression will likely highlights new targets for cancer treatment.

P2054 Board Number: B573

Alternative transcription of Amot80 and Amot130 in response to inflammation in breast cancer. L.R. Bringman1, C. Wells2; 1 Indiana University School of Medicine, Indianapolis, IN, 2Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN The HIPPO signaling pathway is of central importance in controlling the growth of cells in tissues. Conversely, inactivation of HIPPO signaling underlies the progression and promotion of a multitude of cancer types, including breast cancers. HIPPO signaling inhibits the transcriptional co-activator YAP in response to cell-cell contacts and cell polarity. Reduced HIPPO signaling prompts the accumulation of YAP in the nucleus where transcription factors such as TEADs switch on pro-growth, -survival and invasion programs. YAP is known to be inhibited by members of the Amot family of adaptor proteins. Because Amot proteins also bind the key polarity proteins that drive a differentiated morphology, this directly links YAP inhibition to formation of apical-basal polarity in epithelial cells. Because the Amot gene produces a 130 kDa form (Amot 130) that inhibits YAP and promotes cell polarity and an 80 kDa form (Amot 80), which acts as a dominant negative to promote YAP activation and cell growth, it is important to understand their differential regulation. The balance between expression of Amot80 versus Amot130 is shown to dictate whether breast cells will remain differentiated or undergo dysregulated growth. My analysis of breast tissues finds that total Amot is expressed weakly and localizes to the apical membrane in normal mammary cells. Conversely, Amot expression is significantly higher and localized to intracellular puncta (a hallmark of high Amot80 expression) in human breast cancers, especially in triplenegative forms of this disease. In hormone negative DCIS, Amot expression is high and peri-nuclear, specifically in the more disorganized regions adjacent to stromal areas flooded with inflammatory cells. This led us to examine how Amot transcription is modulated by inflammatory cytokines such as interleukin-6 (IL-6), a canonical activator of STAT3 that is commonly secreted in aggressive breast cancers. Phosphorylation of STAT3 (P-STAT3) by IL-6 activated Janus Kinase (JAK) receptors drives its nuclear accumulation and pro-growth transcriptional effects. Consistently I have found that IL-6 treatment of breast cancer cells increases Amot mRNA levels and immunoblot analysis indicates the differential induction of the Amot isoforms. The overall impact of these studies will be both the enhancement of our understanding of inflammation and growth control in breast cancer as well as a bioassay to measure Amot80 and Amot130 mRNA levels in human tumors. The lack of knowledge regarding the cross regulation between inflammatory signaling, HIPPO signaling, and cell polarity, allows this study to drive novel insights to the undisputed pro-tumorigenic impact of inflammation.

TUESDAY-POSTER PRESENTATIONS

P2055 Board Number: B574

Farnesylated LKB1 regulates cell polarization and invasion through a 3D collagen microenvironment in a kinase-independent manner. S. Wilkinson1, A. Marcus2; 1 Graduate Program in Cancer Biology, Emory University, Atlanta, GA, 2Hematology and Medical Oncology, Emory University, Atlanta, GA The tumor suppressor LKB1 is a serine/threonine kinase that serves as a master regulator of cell polarity in epithelial tissue, and is mutated in 30% of non-small cell lung cancer tumors. LKB1 contains a Cterminal farnesylation motif, which allows for post-translational addition of a hydrophobic farnesyl group and insertion into the plasma membrane. Although LKB1 contains a farnesylation motif, the functional significance of this motif is largely unknown. We generated lung cancer cell lines stably expressing GFP-tagged: wildtype LKB1, a farnesylation motif mutant LKB1, and the various domains of LKB1, including the C-terminal domain (CTD, lacking kinase activity) and the CTD containing the farnesylation motif mutant, and then embedded spheroids of these cells in a 3D collagen matrix. Using confocal microscopy and live-cell imaging, we show that lung cancer cells re-expressing wildtype LKB1 acquire a unidirectional polarity, while cells expressing the farnesylation-mutant LKB1 are unable to polarize. Importantly, we show that 3D polarization is regulated through the LKB1 CTD alone, also in a farnesylation-dependent manner. These data indicate that farnesylated LKB1 promotes polarization during 3D motility through its C-terminal domain in a kinase-independent manner. We next sought to identify the role of LKB1 farnesylation in regulating invasion through the 3D collagen microenvironment. After acquiring a unidirectional polarity, cells re-expressing either wildtype LKB1 or the LKB1 CTD invade into the collagen microenvironment. Strikingly, we show that cells re-expressing LKB1 with the farnesylation motif mutation exhibit significantly reduced invasion into the collagen microenvironment, with the few cells invading exhibiting no polarization. Taken together, these data implicate LKB1 farnesylation as critical for promoting cell polarization and potentially providing a mechanism of cell invasion into the collagen microenvironment. This information has the potential to lead to novel information regarding the mechanisms of LKB1 farnesylation in regulating polarization and invasion, ultimately providing new insight into the tumor suppressive function of LKB1.

TUESDAY-POSTER PRESENTATIONS

P2056 Board Number: B575

Adenomatous Polyposis Coli Regulates Epithelial Polarity and 3D Morphogenesis by Mediating FAK/Src Signaling. A. Lesko1, J. Prosperi1,2; 1 Biological Sciences, Harper Cancer Research Institute, University of Notre Dame, South Bend, IN, 2 Indiana University School of Medicine, South Bend, IN Adenomatous Polyposis Coli (APC) is a multi-functional protein that is lost or mutated in many epithelial cancers including breast, colorectal, and pancreatic cancer. Although APC is well known as a negative regulator of the Wnt/β-catenin signaling pathway, it also binds to microtubules and polarity proteins, such as Dlg and Scribble, suggesting functions in regulation of epithelial polarity and cell migration. Our lab has previously determined that the mammary glands of ApcMin/+ mice demonstrate mis-regulation of epithelial polarity, exhibit early neoplastic changes, and develop more aggressive mammary tumors when crossed to the MMTV-PyMT model of breast cancer. Cells isolated from these tumors activated FAK/Src signaling. Our lab has also shown that APC knockdown in the Madin-Darby Canine Kidney (MDCK) model altered epithelial morphogenesis, resulted in inverted polarity in 3D culture, and upregulated gene expression of epithelial membrane protein 2 (EMP2). While restoration of the b-catenin binding domain was unable to rescue the phenotype, introduction of either full-length or a c-terminal fragment of APC partially restored these phenotypes. The current studies investigate the Wntindependent mechanisms by which APC regulates these processes using the MDCK model and primary mammary epithelial cells (MECs) isolated from Apc mutant mice. We hypothesize that the c-terminal fragment mediates FAK/Src signaling to regulate 3D morphogenesis and polarity. Treatment of APC knockdown MDCK cells with PP2, a Src kinase inhibitor, or AIIB2, an integrin inhibitor, eliminated the drastic cyst size changes produced by APC knockdown. Furthermore, inhibition of Src partially restored the polarity phenotype in cysts with APC loss. In addition, shAPC-MDCK cells exhibited increased cell migration indicating a role for APC in cell motility. Preliminary data demonstrates that treatment of shAPC-MDCK cells with PP2 and AIIB2 inhibits migration suggesting FAK/Src signaling as a possible mechanism by which APC mediates cell migration. Interestingly, EMP2 has been shown to bind integrin to activate FAK signaling suggesting an interaction in these signaling pathways. Future studies will aim to dissect the role of the c-terminal fragment and further devise the mechanism by which FAK/Src signaling and EMP2 play a role in APC regulating gene expression, cell migration, and polarity and 3D morphogenesis in MDCK cells. Investigating the interactions of APC with several targets such as those in the FAK/Src signaling pathway will help identify key players in the role of APC in Wnt-independent tumor development.

TUESDAY-POSTER PRESENTATIONS

P2057 Board Number: B576

A cytoskeleton-based memory in chemotactic leukocytes. H. Prentice-Mott1, Y. Meroz2, A. Carlson2, M. Levine3, G. Charras4, L. Mahadevan2, J. Shah1; 1 Harvard Medical School, Boston, MA, 2Harvard School of Engineering and Applied Sciences, Cambridge, MA, 3Biophysics, Harvard University, Cambridge, United States, 4London Centre for Nanotechnology, University College London, London, United Kingdom The directional response of cells to asymmetric chemical environments involves a complex sequence of steps including: gradient sensing, internal polarization and migration. To understand how these modules act to direct cell motion requires quantitative measurements of the cellular response and quantitative control of the external chemical environment. We used microfluidics to generate confining microchannels in which the front and back of the cell can be independently exposed to separate chemoattractant concentrations, which are stable over time. Cells exposed to high, static differences with low background (C0=0 nM, ∆C 1 nM) exhibited persistent polari ation, as assessed by H-Akt asymmetry. Smaller differences (C0=0 nM, ∆C=3 nM) or increased background (C0=50 nM, ∆C 5 nM) resulted in fluctuations between polarized and unpolarized states. The direction of polarization was biased toward the higher concentration. When exposed to uniform concentrations of chemokine (C0=0, 3, 10, 100 nM, ∆C nM), cells exhibited similar polarization fluctuations. Unexpectedly, the direction of polarization for all concentrations except C0=0 was biased towards the initial direction of polarization, indicating a directional “memory”. To further test this, cells were exposed to a controlled dynamic environment, in which a uniform concentration (C0=10 nM, ∆C=0 nM) was first introduced, then changed to no chemoattractant (C0=0 nM, ∆C nM) hich caused most cells to depolari e. Subse uent reintroduction of the chemoattractant (C0=10 nM, ∆C=0 nM) after 2 minutes of no chemoattractant caused ~90% of the cells to re-polarize in the same direction as initially polarized. If the chemokine was removed for 10 minutes, this number decreased to ~70%. We measured the decay of internal cellular asymmetries during the no chemoattractant phase and observed dramatic differences in timescales for molecules such as PIP3 and Myosin light chain which were fast (10 seconds), versus the ERM protein moesin (5 minutes). Chemical disruption of the long-lived moesin (NSC668394) or microtubule (colcemid) structures during the no chemoattractant phase, resulted in decreased memory of polarization after reintroduction of the chemoattractant and washout of the drug (60% or 55%, respectively). We developed a phenomenological model incorporating two layers of polarization, a short-lived membrane layer (pm) and a long-lived cytoskeletal layer (pc). The cytoskeletal polarization is driven by the time-integrated pm, while the direction of the membrane polarization is driven by both pc and external concentration differences. This simple model reproduced the observed directional memory. Cells may use this directional memory to navigate the complex chemical environment found in tissues.

TUESDAY-POSTER PRESENTATIONS

P2058 Board Number: B577

TorsinA operates as a membrane-regulated ring-shaped hexameric molecular chaperone within the nuclear envelope during centrosome orientation in migrating fibroblasts. C.A. Saunders1, E.M. Smith2, Y. Chen2, J. Mueller2, G.G. Luxton1; 1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 2Department of Physics and Astronomy, University of Minnesota, Minneapolis, MN The centrosome is oriented between the nucleus and the leading edge in many migrating cells and contributes to directional cell migration. Recent research shows that torsinA, a AAA+ ATPase found within the Nuclear Envelope (NE), is required for centrosome orientation during directional cell migration. AAA+ ATPases typically function as hexameric ring-shaped molecular chaperones to structurally remodel protein complexes by mechanically unfolding and translocating target proteins through a central pore. TorsinA is an unusual AAA+ ATPase in four ways: 1) it is found within the NE; 2) it is a monotopic membrane-associated protein; 3) it does not posses a recognizable target protein interaction domain; and 4) it lacks arginine fingers that are known to be important for ATP-hydrolysis and oligomerization. Therefore, the molecular mechanism of torsinA-mediated centrosome orientation is currently unknown. To begin to identify this mechanism, we tested the ability of different GFP-tagged torsinA constructs to rescue centrosome orientation in torsinA-null fibroblasts. We find that torsinAmediated centrosome orientation requires ATPase activity and the ability of torsinA to form a hexameric oligomer with at least three active subunits. We identify two conserved hydrophobic residues (Y128 and Y147) in torsinA that are required for centrosome orientation as potential pore loops, which interact with and translocate target proteins in the central pore of AAA+ ATPases. To determine if torisnA can form hexamers, we developed z-scan Fluorescence Fluctuation Spectroscopy (FFS) in order to measure the oligomeric state of GFP-tagged proteins within the NE of living cells. Using this technique, we show that torsinA is on average a dimer within the NE while the deletion of the N-terminal membraneassociation domain results in higher-order oligomeric states consistent with hexamerization. Finally, we show that torsinA lacking the membrane-association domain can still rescue centrosome orientation in torsinA-null fibroblasts. Taken together, our results suggest that torsinA operates as a hexameric ringshaped molecular chaperone during centrosome orientation and that membrane-association negatively regulates torsinA oligomerization.

TUESDAY-POSTER PRESENTATIONS

P2059 Board Number: B578

TorsinA is required for retrograde actin flow and TAN line assembly during rearward nuclear movement in migrating fibroblasts. N.J. Harris1, P. Willey1, C.A. Saunders1, G.G. Gundersen2, G.G. Luxton1; 1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 2Department of Pathology and Cell Biology, Columbia University, New York, NY Actin-dependent positioning of the nucleus at the cell rear is critical for the directional migration of several cell types. We have demonstrated that rearward nuclear positioning is mediated by linear arrays of nuclear envelope membrane proteins composed of nesprin-2G and SUN2, called Transmembrane Actin-associated Nuclear (TAN) lines, which couple the nucleus to actin cables undergoing retrograde flow. Currently, the molecular mechanism of TAN line assembly is unknown. Potential regulators of TAN line assembly are the torsin AAA+ ATPases that reside within the nuclear envelope. Recently, it was shown that torsinA interacts with the KASH domain of nesprin proteins within the nuclear envelope and is important for the localization of nesprins to the nuclear envelope. Since AAA+ ATPases typically function as molecular chaperones that structurally remodel protein complexes, we hypothesized that torsins may regulate the interaction of nesprin-2G and SUN2 during TAN line assembly. Here, we report that of the four mammalian torsins (torsinA, torsinB, tor2A, and tor3A) only torsinA is required for rearward nuclear positioning in migrating fibroblasts. Live imaging of fibroblasts where torsinA function has been inhibited reveals that the nucleus does not move directionally towards the cell rear. We find that torsinA localizes to TAN lines and is required for their assembly, supporting a role for torsinA in the regulation of nesprin-2G-SUN2 interactions. While torsinA inhibition does not negatively affect formation or organization of perinuclear actin cables, it does block retrograde actin flow. Taken together, our results suggest that torsinA controls both TAN line assembly and retrograde actin flow in migrating fibroblasts. Therefore, torsinA is a key regulator of cell polarity in during directional cell migration.

P2060 Board Number: B579

Armcx1, a potential gene of planar cell polarity regulation. L. Knapp1, P. Chen1; 1 Department of Cell Biology, Emory University, Atlanta, GA Planar cell polarity (PCP) is essential in development, tissue morphogenesis, and cell migration. PCP refers to the coordination of cell polarity across a sheet of cells or tissues that utilizes communication between cells leading to a cytoskeletal response. The inner ear, specifically the cochlea’s organ of Corti, is a model of PCP. Core PCP proteins have been implicated in coordinating sensory hair cell polarization, leading to uniform orientation of microvilli-derived stereocilia bundles in the organ of Corti. Specifically, membrane proteins Vangl2 along with Frizzled3 and Frizzled6 have been identified as core PCP proteins

TUESDAY-POSTER PRESENTATIONS required for PCP in the cochlea. How these core PCP proteins regulate morphological polarization is currently unknown. To explore and identify novel components of PCP that may bridge the core PCP proteins and cytoskeletal polarization, we performed a yeast two-hybrid screen to find proteins interacting with the cytoplasmic tail of Vangl2. Armadillo repeat-containing X-linked protein 1 (Armcx1) was identified as a Vangl2-interacting protein. Armcx1 contains armadillo repeats known to mediate protein-protein interactions in cell signaling and cytoskeletal morphogenesis. Down-regulation of Armcx1 mRNA has been linked to carcinomas in which PCP is perturbed. These findings support the hypothesis that Armcx1 may link Vangl2 to cytoskeletal polarization during PCP processes. To determine a possible role for Armcx1 in the PCP of the cochlea, we used in situ hybridization to determine whether its temporal and spatial expression pattern is consistent with a role in establishing PCP in the cochlea. We found that Armcx1 was expressed in the cochlear sensory epithelium, the organ of Corti, during establishment of PCP from embryonic (E) 14.5 to E 18.5 in mice. In addition, Armcx1 is also expressed in the vestibular sensory epithelia during PCP establishment. Together, its expression pattern, its interaction with known PCP proteins, and its associated cellular roles implicate Armcx1 as a potential regulator of PCP in vertebrates. We will further test whether Armcx1 interacts with Vangl2 to regulate cytoskeletal change and test its potential roles in PCP regulation.

P2061 Board Number: B580

Mitotic control of planar cell polarity by polo-like kinase 1. R. Shrestha1, K.A. Little1, D. Devenport1; 1 Dept. of Molecular Biology, Princeton University, Princeton, NJ During cell division, polarized epithelial cells require mechanisms to ensure polarity and tissue integrity are preserved as they undergo dramatic structural rearrangements. Planar cell polarity (PCP), which relies on cell communication to align cell polarity across epithelial sheets, is particularly vulnerable during mitosis, as polarity defects can be propagated to neighboring cells. We previously discovered a novel mechanism in the skin epithelium that preserves PCP during mitosis whereby asymmetrically localized, cortical PCP proteins are internalized into endosomes, distributed equally to daughter cells, and recycled to the membrane where polarity is regained. The disassembly and restoration of PCP asymmetry must coincide precisely with cell cycle progression, but the mechanisms controlling PCP dynamics during mitosis are not known. Here we identify Plk1 as a master regulator of PCP dynamics during mitosis. Plk1 interacts with the core PCP component, Celsr1, via a conserved polo-box domain binding motif, localizes to mitotic endosomes and directly phosphorylates Celsr1’s endocytic sorting signal. Phosphorylation activates the endocytic motif, allowing AP2 recognition and bulk recruitment of Celsr1 into clathrin-coated endosomes. Inhibition of Plk1 activity blocks PCP internalization, leading to perturbations in PCP asymmetry. Thus, Plk1-mediated phosphorylation of Celsr1 ensures PCP redistribution is precisely coordinated with mitotic entry.

TUESDAY-POSTER PRESENTATIONS

P2062 Board Number: B581

Mitotic membrane turnover coordinates differential induction of the heart progenitor lineage. C.D. Cota1, B. Davidson1; 1 Biology, Swarthmore College, Swarthmore, PA Matrix adhesion can polarize cells by stabilizing signaling enriched membrane domains While it is known that dividing cells detach from the surrounding matrix and initiate extensive membrane remodeling, the in vivo impact of mitosis on adhesion-dependent signal polarization remains poorly characterized. Here we show that mitotic membrane turnover orchestrates adhesive polarization of heart progenitor induction. We investigate in vivo signaling dynamics in the invertebrate chordate, Ciona intestinalis. In Ciona, adhesion polarizes Fibroblast Growth Factor (FGF)-dependent induction of the heart progenitor lineage. Through targeted disruption and selective rescue of integrin based matrix adhesion in precardiac founder cells, we show that adhesion promotes localized enrichment of FGF receptors by inhibiting mitotic internalization and degradation. Through mutational analysis of ß-integrin subunits, we have experimentally defined the motif required for membrane stabilization and mitotic FGFR retention. Furthermore, targeted manipulations of Caveolin in the heart progenitor lineage indicate that Caveolin-rich membrane domains function downstream of polarized cell-matrix adhesion to retain FGFRs during mitotic rounding. These results support a revised model of adhesion-dependent asymmetric fate induction in which mitotic membrane turnover resets historic, pre-mitotic receptor distribution according to contemporaneous adhesive cues.

P2063 Board Number: B582

SONIC HEDGEHOG SIGNALING CONTROLS GRANULE NEURON MIGRATION IN A RAS-RAF DEPENDENT PATHWAY. T. Ong1, D. Solecki2; 1 Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, 2developmental neurobiology, St. Jude Children's Res Hosp, Memphis, TN Background: Neuronal progenitors in the brain are born in sites known as germinal zones (GZ) where they undergo proliferation and differentiation before migrating long distances to home to their final positions. Developmental cues that control this transition are tightly regulated to ensure proper brain morphogenesis as dysregulation leads to brain malformations, mental retardation and cancer. A major challenge in the field of developmental neurobiology is to delineate the molecular underpinnings that regulate this developmental program. In the cerebellum, cerebella granule neurons (CGN) that fail to exit the external germinal layer (EGL) due to aberrant sonic hedgehog (Shh) signaling continue to proliferate and give rise to Shh driven medulloblastoma (MB). Results: Our laboratory have previously

TUESDAY-POSTER PRESENTATIONS reported that the E3 ubiquitin ligase, Siah2, blocks CGN migration by negatively regulating Par3, a component of the Par complex that is required to recruit the junctional adhesion molecule, JamC to the membrane to initiate cell-cell contacts for GZ exit. Preliminary data show that Shh signaling maintains Siah2 protein expression in a Ras-Raf dependent pathway. We also show a direct correlation between protein levels of active Ras (H and N isoforms) and Siah2, where their levels decrease from post-natal days (P), P4 to P15 respectively. Interestingly, these protein levels remain high in Ptc+/-P18-/- MB cells. In line with these findings are functional studies that show that activation of Shh signaling or gain of function of the Ras-Raf pathway in granule neuron progenitors (GNP) impairs their GZ exit which can be rescued by exogeneous expression of Par3 or inhibition of Siah2 function, respectively. Conclusion: We propose that Shh signaling negatively regulates Par3 in a Ras-Raf-Siah2 dependent manner, hence blocking CGN GZ exit.

P2064 Board Number: B583

Rab35-dependent axon elongation is negatively regulated by p53-related protein kinase (PRPK). D. Villarroel-Campos1, D.R. Henriquez1, M. Fukuda2, C. Gonzalez-Billault3; 1 Biology, Universidad de Chile, Santiago, Chile, 2Tohoku Univ, Miyagi, Japan, 3Faculty of Sciences, Universidad de Chile, Santiago, Chile The appropriate function of the nervous system relies on the neuronal polarized morphology. Axon elongation is a crucial process through neuronal final shape is achieved, and a mechanism sustained mainly by cytoskeletal dynamics and intracellular trafficking. We previously described that microtubuleassociated protein 1B (MAP1B) is required for proper axon outgrowth. The aim of this work is to describe novel MAP1B-interacting proteins which could be involved in axon elongation. We carried out a yeast two-hybrid experiment using MAP1B light chain (LC1) as bait, and detected PRPK as one of the LC1-interacting partners. PRPK overexpression in hippocampal embryonic neurons in culture significantly reduces their axonal length. PRPK-induced phenotype can be rescue by Rab35 over expression, suggesting that both proteins are functionally related. Noteworthy, axonal defects found in MAP1B knock-out neurons can be recovered by over expression of either PRPK kinase dead mutant or Rab35 constitutively active. These evidences show that the MAP1B-interacting partner PRPK negatively regulates axon elongation in vitro, in a Rab35-dependent manner. This work was supported by Fondecyt-1140325 (to CG-B)

TUESDAY-POSTER PRESENTATIONS

P2065 Board Number: B584

Oligodendrocyte-innate programs dictate cell shape changes required to form myelin sheaths and account for variation in lengths of CNS myelin sheaths. M.E. Bechler1, L. Byrne1, C. ffrench-Constant1; 1 MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh, Scotland Myelin internodes are specialized extensions of glial cell plasma membrane spirally wrapped around axons to create a multilamellar sheath. The length of these internodes regulates the speed of axonal signal propagation. This length usually correlates with underlying axon diameters, suggesting the hypothesis that axonal cues regulate the internode properties and instruct the remarkable changes in cell shape required to form a sheath. However, despite decades of work to identify instructive cues, no axonal molecule has been shown to be required for central nervous system (CNS) myelination. Using primary oligodendrocytes cultured with inert microfibers, we tested the alternative hypothesis that myelin sheath formation is an innate property of the oligodendrocyte refined by, but not requiring, axonal signals. Supporting this hypothesis, oligodendrocytes produce internodes of physiological lengths on these microfibers, with compact multi-layered myelin membranes. This oligodendrocyte-innate program can be adapted by specific axonal molecules, with independent regulation of the number and length of internodes. This is distinct from Schwann cells that require axonal cues to differentiate and form compact myelin sheaths. Oligodendrocytes also inherently respond to the physical cue of microfiber diameter, increasing internode length with increased diameters. Surprisingly, we find that oligodendrocytes isolated from distinct CNS regions have differing innate internode lengths. This mirrors the variation in internode lengths found in vivo: spinal cord oligodendrocytes produce longer sheaths than those from cortex. These results indicate the cell shape changes during myelin internode formation are inherently controlled by oligodendrocytes and reveal previously unsuspected differences between oligodendrocytes from different regions of the CNS.

P2066 Board Number: B585

ROCK1 and 2 differentially regulate actomyosin organization to determine cell polarity in fibroblasts and neurons. K.A. Newell-Litwa1, M. Badoual2, H. Asmussen1, H. Patel1, L. Whitmore1, A.R. Horwitz1; 1 Cell Biology, University of Virginia, Charlottesville, VA, 2Laboratoire IMNC, Univ. Paris-Sud, Orsay, PA RhoGTPases give shape to the cell by organizing the underlying actin cytoskeleton. As such, they are implicated in polarity underlying normal and cancer cell migration, epithelial polarization, and even the subcellular organization of the polarized dendritic spine, which supports learning and memory. In particular, RhoA-mediated signaling activates non-muscle myosin IIB (MIIB) by di-phosphorylating its regulatory light chain to form actomyosin filament bundles that define the sides and rear of migrating

TUESDAY-POSTER PRESENTATIONS cells. RhoA also inactivates Rac-driven actin polymerization, via an unclear mechanism, thus segregating the initiation of actin polymerization to the front of the cell to create the leading edge. However, how RhoA coordinates these two processes to drive polarity is unknown. ROCK is the major RhoA effector kinase that activates MIIB to create the cell rear. Yet, it is unclear which ROCK isoform, ROCK1 or ROCK2, is responsible for MIIB activation and Rac inactivation. We examined whether ROCK1 and 2 similarly or separately regulate MIIB activation and Rac inactivation in both front-back migratory cell polarity and dendritic spine polarity. For both systems, we observed a specific role for ROCK1, but not ROCK2, in the di-phosphorylation of myosin regulatory light chain on Thr18 and Ser19. This ROCK1driven MIIB activation was also necessary for proper microtubule organization. In contrast, ROCK2, but not ROCK1, specifically attenuated Rac activity underlying the formation of the leading edge of the migratory cell and the spine head of the synapse. We hypothesized that ROCK2 regulates Rac activity through a negative feedback mechanism with the actin remodeling protein, cofilin. While Rac activation normally inactivates cofilin by Ser3 phosphorylation; knockdown of ROCK2 decreased levels of Ser3phosphorylated cofilin. The loss of phosphorylated cofilin resulted in smaller nascent adhesions, which signal through Rac to drive membrane protrusion. However, cofilin inactivation by expression of phosphomimetic cofilin S3D restored adhesion maturation and rescued the morphology of ROCK2deficient cells. Similarly, cofilin inactivation regulated the size of post-synaptic densities within the spine head. Thus, ROCK1 and ROCK2 regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. These results also suggest that the ROCK inhibitor, Y-27632, which indiscriminately targets both isoforms, is inhibiting two different pathways.

P2067 Board Number: B586

Contribution of Reactive Oxygen Species to the Establishment of Hippocampal Neuronal Polarity. C. Wilson1, T. Nuñez1, C. Gonzalez-Billault1; 1 Faculty of Sciences, Universidad de Chile, Santiago, Chile Basal production of Reactive Oxygen Species (ROS) by the NADPH (NOX) complex contribute to LTP, memory consolidation and neuronal actin cytoskeleton dynamics. The Rho GTPases Rac1 and Cdc-42 and the actin cytoskeleton are essential for the development of the axo-dendritic compartments. Acquisition of polarity is intimately related with neuronal functions. However, the contribution of ROS during neuronal polarity has not been explored. In this work, we propose that physiological ROS produced by NOX complex contributes to the acquisition of neuronal polarity and axonal growth. Hippocampal neurons from rat brain embryos (E18.5) were cultured for different times to evaluate whether ROS are involved in the establishment of neuronal polarity. Epifluorescence microscopy was used to assess development of neuronal polarity and axonal length. ROS content and actin dynamics were visualized using the genetically encoded biosensors Hyper and Lifeact, respectively. In addition, FRET biosensors were used to measure local activity of Rac1 and Cdc-42 after NOX inhibition.

TUESDAY-POSTER PRESENTATIONS The NOX subunits gp91phox, p22phox, p47phox and p67phox were detected in stage 2 and stage 3 hippocampal neurons. NOX complex inhibition (apocynin, gp91ds-tat and transfection of p22phox P156Q (DNp22phox) delayed polarization of hippocampal neurons and decreased axonal length. In addition, DNp22phox-transfected neurons showed a decrease in the number, length and life-time of axonal filopodia. Moreover, the activities of Rac1 and Cdc-42 were also decreased after DNp22phox expression. Our results suggest that ROS produced by NOX complex contributes to the acquisition of neuronal polarity by regulating actin cytoskeleton. We propose that physiological levels of ROS may be necessary for polarization and maturation of hippocampal neurons. Supported by ACT-1140 to CGB, CONICYT doctoral fellowship 21120221 to CW.

P2068 Board Number: B587

In vitro interaction of drugs with canalicular lipid transporters. Z. Mahdi1, B. Stieger1; 1 Department of Clinical Pharmacology and Toxicology, University Hospital, Zurich, Switzerland Background and aims: Canalicular bile formation is maintained by the simultaneous activities of three ABC-transporters: BSEP (Bile Salt Export Pump or ABCB11), ABCB4 (or MDR3) and ABCG5/G8. Dysfunctions in the biliary cholesterol–bile salt–phospholipid secretion (e.g. an increased ratio of cholesterol to bile salts or phospholipids) lead to cholestasis or result in crystallization of cholesterol followed by cholelithiasis. The aim of this project is the development of a cell-based and polarized model system to test the hypothesis that some drugs may induce cholestatic liver disease by specifically interfering with biliary lipid secretion and to identify such inhibitors/stimulators. Methods: Stably transfected LLC-PK1 cell lines (overexpressing human NTCP, BSEP, ABCB4 and ABCG5/G8) were cultured in the Transwell® system. Detection and polar localisation of the transport proteins was accomplished by immunohistochemistry and Western blots. Functionality and direction of transport were verified using radioactively (e.g. 3H-taurocholate and 14C-cholesterol) or fluorescently (NBD-phosphatidylcholine) labelled substrates. The substrates were added to the basolateral or apical compartments and the amounts transported in the opposite compartment were measured. Albumin was used as an acceptor to stimulate efflux. Lipid secretion into the apical compartment was also chemically analysed by thin-layer-chromatography. Results: Western blots and immunohistochemistry indicate that the transfected LLC-PK1 cells overexpress all three ABC transporters and that NTCP is overexpressed in the basolateral membrane while BSEP, ABCB4 and ABCG5/G8 are overexpressed in the apical membrane, as is the case in human hepatocytes. Functional transport assays demonstrate that the model cell line displays a vectorial transport of bile salts and phosphatidylcholine from the basolateral to the apical compartment. First experiments with the antimycotic agent itraconazole show slight changes in phosphatidylcholine secretion but a strong induction of ABCB4 expression.

TUESDAY-POSTER PRESENTATIONS Conclusions: The antimycotic agent itraconazole does not significantly change the amount of phosphatidylcholine secreted but strongly induces ABCB4 expression indicating that itraconazole indeed inhibits phosphatidylcholine secretion in our assay system if the protein amount is considered. This finding is in line with previously published data. Taking this result together with the vectorial transport bile salts and phosphatidylcholine, we have successfully established an in vitro model for canalicular bile salts and lipid secretion. Consequently, we plan to use this model to characterize the effect of additional drugs suspected to interfere with canalicular bile salts and lipid transporters.

P2069 Board Number: B588

The apical polarity of Na+,K+-ATPase in ARPE-19 cells is regulated by the expression of the β2 subunit. L. Shoshani1, J.A. Lobato1, J. Bonilla-Delgado2, R. Gonzalez-Ramirez3; 1 Physiology, CINVESTAV, Mexico City, Mexico, 2Research Unit, Hospital Juerez de Mexico, Mexico City, Mexico, 3Department of Molecular Biology and Histocompatibility, Hospital General Dr. Manuel Gea González, Mexico City, Mexico The Na+, K+ ATPase or sodium pump plays a very important role in the maintenance of epithelial phenotype. This protein is always polarized in epithelia and is crucial for creating and maintaining the electrochemical gradient of Na+ and K+ as well as the cell volume and osmolarity. In most epithelia, the pump is located in the lateral domain. We demonstrated that the β1-subunit of the Na+, K+ ATPase plays an important role in the polarization mechanism as it interacts directly, in the extracellular space with another β1 subunit localized on a neighboring cell and thus stabilizes the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE) the sodium pump is located at the apical domain. The mechanism of polarization in this epithelium is still unknown. Our working hypothesis is that the apical polarity of the pump in RPE cells is regulated by its β2 subunit. We got evidences that relate the apical localization of the pump in RPE with the expression of the β2 subunit, and we are looking for the mechanism by which β2 subunit regulates the pump polarity. Results: ARPE-19 cells cultured for up to 6 weeks in the presence of ITS acquire the RPE phenotype and express the sodium pump in the apical domain (process termed re-morphogenesis). WB and IF analyses show that the β2 isoform detected at the apical domain is co-expressed with the α2 subunit. In order to find out if the β2 expression is regulated on a transcriptional or translational level, we analyzed as function of time, the mRNAs level of the β subunit isoforms by RTqPCR and the protein expression level by WB. We observed that in the presence of ITS, β2 mRNAs are increased during re-morphogenesis, as well as the protein level. Silencing the β2 expression by siRNAs, reduce the apical expression of the pump as gauged by the apical localization of the α2 subunit. Our results suggest that the apical localization of the pump in ARPE19 cells depends on the expression of the β2 isoform and that it is regulated on a transcriptional level. Accordingly, we suggest that the transcription factor Sp1 is involved as it is also expressed in ARPE-19 cells and its amount increases during re-morphogenesis. We therefore expect that silencing Sp1 by siRNA will inhibit the apical localization of the pump (α2β2 complex).

TUESDAY-POSTER PRESENTATIONS

P2070 Board Number: B589

A spatial switch in H,K-ATPase trafficking by Helicobacter pylori VacA-elicited phosphorylation of ACAP4. X. Ding1, X. Liu2, W. Yao2, L. Chen3, Y. Zhang1, X. Yao4; Medicine, Beijing University of Chinese Medicine, Beijing, China, 2Physiology, Morehouse School of Medicine, Atlanta, GA, 3Beijing University of Chinese Medicine, Beijing, China, 4Cellular Dynamics, University of Science Technology of China, Hefei, China

1

Many cellular processes are orchestrated by dynamic changes in the plasma membrane to form membrane projections and endocytic vesicles. Small GTPase ARF6 is involved in coupling actin dynamics to trafficking of vesicular membranes. Our study demonstrated that ACAP4, ARF6 GTPase-activating protein, governs polarized H,K-ATPase trafficking during parietal cell secretion (J. Biol. Chem. 2010. 285, 18769-80). Helicobacter pylori persistently colonize the human stomach and have been linked to atrophic gastritis and gastric carcinoma. Our early study revealed that VacA disrupts apical membranecytoskeletal interactions and perturbs parietal cell secretion (J. Biol. Chem. 2008. 283, 26714-25). VacA binds to tyrosine phosphatase PTP-ζ which leads to gastric ulcer (Nature Genetics. 2003. 33, 375-381). However, it is unclear how PTP-ζ operates its downstream signaling events in parietal cells. Here we show that PTP-ζ interacts with ACAP4 and VacA treatment enhances ACAP4 phosphorylation at Tyr733 which rewires cellular signaling for aberrant proton secretion. Our biochemical characterization and cellular imaging analyses show that VacA-elicited phosphorylation of Tyr733 results in re-distribution of ACAP4 to the basolateral membranes. Surprisingly, persistent expression of phospo-mimicking ACAP4 results in recruitment of H,K-ATPase and v-SNAREs to the basolateral domains. The large basolateral expansion elicited by VacA is predicted to recruit membranes from sources not normally targeted to that surface. Significantly, the phosphorylation of Tyr733 was correlated to hypochorhydria phenotype of clinical VacA-positive patients. In addition, expression of non-phosphorylatable Y733F mutant in ACAP4 knockout mouse parietal cells resumes apical secretion of proton, suggesting that VacA rewires ACAP4 signaling axis by repolarizing parietal cell secretion at basolateral membrane. The results present a previously undefined mechanism by which microbe-host interaction orchestrates a spatial switch of membrane trafficking in gastric parietal cell secretion.

TUESDAY-POSTER PRESENTATIONS

P2071 Board Number: B590

Large, low-complexity MLT1 protein is associated with unique, eyespotassociated microtubules in the green alga Chlamydomonas reinhardtii. T.M. Mittelmeier1, M.D. Thompson1, M. Lamb2, C.L. Dieckmann1; 1 Molecular and Cellular Biology, University of Arizona, Tucson, AZ, 2Biology, University of Puget Sound, Tacoma, WA The photosensory eyespot of the unicellular green alga Chlamydomonas is found at the equator of the cell in a unique position relative to the two anterior basal bodies and flagella. In response to light, eyespot-initiated signals lead to changes in flagellar stroke patterns and swimming direction, such that the incident light intensity is optimal for photosynthesis. The eyespot comprises components in the plasma membrane, cytoplasm, and chloroplast envelope membranes, and in carotenoid-rich lipid granules associated with thylakoid membranes. Following cell division, the eyespot is assembled de novo in each daughter cell adjacent to the D4 microtubule rootlet, one of four rootlets that extend from the basal bodies. During assembly, localization of one or more eyespot components is directed by the D4 microtubules. mlt1 (multi-eyed) mutant cells contain additional eyespots that are mis-localized to more anterior positions, and localization of both major eyespot photoreceptor proteins is delayed. We used polyclonal antibodies to identify wild-type MLT1 on Western blots and in fixed cells by immunofluorescence. The >300 kDa protein was localized along the D4 rootlet, but not the other three rootlets. MLT1 is absent in mlt1-1 cells, which have a nonsense mutation near the middle of the mlt1 coding sequence. Similar to the Arabidopsis microtubule-binding protein BPP1 (basic proline-rich protein 1), MLT1 is of low complexity and proline-rich, and has three exclusively basic regions. We are testing the hypothesis that binding of MLT1 to rootlet microtubules via its basic region(s) stabilizes the microtubules and/or promotes microtubule-guided trafficking of eyespot components. MLT1 is also absent in mlt2-1 mutant cells, which have a phenotype similar to but distinct from mlt1 cells. Sequencing of the mlt2-1 genome identified the MLT2 gene, predicted to encode a large, lowcomplexity, leucine-rich protein. While eyespot localization is dependent on MLT1 and MLT2, the integrity of the ordered membrane layers of the assembled eyespot is dependent on the MIN1 protein which has an N-terminal C2 domain and a C-terminal LysM domain. Extracellular LysM domains are known to bind bacterial or fungal cell wall components, but the function(s) of intracellular LysM domains is unknown. We observe that cells with changes in highly conserved residues, modeled to be on the surface of the folded MIN1 LysM domain, have “fragmented” eyespots. The data are suggestive that the MIN1 LysM domain is involved in specific interactions that maintain the close apposition of eyespot membrane layers. Further analyses of eyespot proteins will aid our understanding of the functional breadth of a number of widely distributed protein motifs. Support: NSF MCB-1157795 to CD.

TUESDAY-POSTER PRESENTATIONS

P2072 Board Number: B591

Congenital Tufting Enteropathy, a new model for studying intestinal villous morphogenesis. J. Salomon1, J. Schmitz2, F. Campeotto2, N. Brousse3, J. Magescas4, F. Poirier4, B. Duvauchelle4, B. Ladoux5,6, O. Goulet2, L. Veyrier4, d. Canioni3, D. Delacour4; 1 Institut Jacques Monod- CNRS, Paris, France, 2Université Paris Descartes, Paris Cité, Hôpital Necker Enfants Malades , Paediatric Gastroenterology, Paris, France, 3Université Paris Descartes, Paris Cité, Hôpital Necker Enfants Malades , Pathology Department, Paris, France, 4Institut Jacques Monod - CNRS, Paris, France, 5Institut Jacques Monod, Université Paris Diderot CNRS, Paris, France, 6Mechanobiology Institute, National University of Singapore, Singapore, Singapore Molecular mechanisms underlying the maintenance of the intestinal villous epithelial layer homeostasis are largely unknown. Congenital Tufting Enteropathy (CTE), (MIM #613217) a rare human cause of intestinal failure, constitutes an interesting working model. It is characterized by aberrant focal stacks of multiple layers of enterocytes, named « tufts » and is due to mutations in the EPCAM or SPINT2 genes. Our study aimed at understanding the cellular functions of EPCAM and SPINT2 in enterocytes and in villous morphogenesis. EPCAM and SPINT2 are located at the lateral membranes and the terminal web in human enterocytes. We analyzed either duodenal biopsies of CTE patients, or stable Caco2 clones depleted for EPCAM or SPINT2. Ultrastructural analyses were performed using transmission electron microscopy. Cell organization, cell differentiation and polarity markers were tested using immunohistology and confocal microscopy analyses. Our results clearly show specific defects in intestinal cell polarity and adhesion in the mutant enterocytes: microvillus atrophy, mislocalization of brush border components, and weakened cell-cell junctions. Although Caco2-depleted cells display a loss of apico-basal polarity, they still formed epithelial monolayers in classical 2D cultures. We thus generated 3D culture supports that recapitulate villus topology and constraints. Placing mutant human enterocytes on synthetic villus cultures revealed strong perturbations in the maintenance and dynamics of the epitelial monolayer, mimicking the tufts observed in CTE patient mucosa. We demonstrated that CTE disorder is characterized by the loss of enterocyte polarity and differentiation. As a consequence, correct cell dynamics along the villous architecture is impaired in mutant enterocytes.

TUESDAY-POSTER PRESENTATIONS

P2073 Board Number: B592

Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking. F.O. Bendezu1, V. Vincenzetti1, D. Vavylonis2, R. Wyss3, H. Vogel3, S.G. Martin1; 1 Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland, 2Department of Physics, Lehigh University, Bethlehem, PA, 3Institut des Sciences et Ingénierie Chimiques (ISIC), Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland How a cell polarizes growth is a fundamental biological question. The conserved Rho-type GTPase Cdc42 is a central player in this process. Activated GTP-bound Cdc42 determines the zone of growth by interacting with effectors necessary for the targeting of secretory vesicles. It is not clear, however, how cells spontaneously polarize to establish a zone of active Cdc42. This symmetry-breaking event is thought to require dynamic Cdc42 recycling off and on the plasma membrane by GDI (Guanine nucleotide Dissociation Inhibitor)-mediated membrane extraction and actin-based vesicle trafficking. Here, we describe the construction of a functional, internally tagged Cdc42-mCherry sandwich (SW) fusion expressed from the endogenous cdc42 locus in fission yeast. Using pharmacological inhibitors and mutant backgrounds, we find Cdc42 dynamics and spontaneous polarization is maintained largely independent of these proposed recycling pathways. Further strengthening these findings, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix supports viability and polarizes spontaneously to multiple sites in both fission and budding yeasts. Using Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS), we find that Cdc42 is highly mobile at the plasma membrane. However, mobility is slower at sites of active Cdc42-GTP, which correspond with areas of total Cdc42 accumulation. By contrast, an engineered near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedbacks, leads to its local accumulation by capture of fast-diffusing inactive molecules at the plasma membrane.

P2074 Board Number: B593

A minimal system to establish microtubule-based cell polarity in vitro. N. Taberner1, P. Recouvreux2, S. Roth1, M. Dogterom1; 1 BionanoScience, TU Delft, Delft, Netherlands, 2IBDM - Aix Marseille University - CNRS, Marseille, France From yeast to fibroblasts, many cell types have a defined polarity that allows them to directionally move, grow or divide. This polarity is typically defined by a polarized distribution of proteins at the cell cortex. We are interested in the emergence of polarity in systems where microtubules are directly involved in its establishment and maintenance by delivering polarity markers to the plasma membrane. We developed two in vitro systems that allow for microtubule-based delivery of proteins to bio-mimetic

TUESDAY-POSTER PRESENTATIONS cortices. These systems, consisting of elongated micro-fabricated chambers or emulsion droplets, allow for dynamic microtubules to self-assemble and organize in response to interactions with the chamber or droplet boundaries. In micro-chambers, single events of microtubule-based delivery to a wall are imaged and quantified. Our experiments show that clustering of proteins at microtubule tips (fission yeast's mal3, kinesin tea2, and tip1) enhances prolonged docking of proteins to the wall receptors as opposed to non-clustering proteins (EB analog protein mal3). Moreover, pre-docked clusters at the wall can capture growing microtubule tips enhancing repeated deliveries at the same spot. These observations are very similar to the observation of clustering of polarity markers in living fission yeast cells. With elongated emulsion droplets the global emergence of polarity in a closed system can be assessed under conditions where proteins can additionally diffuse within the lipid boundaries. With this minimal system we aim to establish the minimal mechanism by which microtubules can establish and maintain cell polarity in living cells. In parallel, we performed experiments in living yeast cells, which suggest that a simple artificial protein that combines membrane affinity with microtubule tip affinity is in principle enough to establish (but not maintain) polarity.

P2075 Board Number: B594

Role of a clutch molecule shootin1 in laminin-induced axon formation. K. Abe1, Y. Sakumura1,2, N. Inagaki1; 1 Grad. Sch. Bio. Sci., Nara Institute of Science and Technology, Ikoma, Japan, 2Sch. Inform. Sci. and Technol., Aichi Prefectural Univ., Nagakute, Japan Neuron requires correctly oriented axon formation to transmit their signals to appropriate target cells. Axon is directed by asymmetric presentation of soluble and adhesion axon guidance molecules. For example, an extracellular matrix protein laminin contact directs neuronal polarization of retinal ganglion cells to basal lamina in vivo [Owen et al, Neuron, 2011]. The growth cone plays a key role in neurite extension to form axon. However, how does laminin affect growth cone and induce directional axon formation remains elusive. Shootin1 mediates a linkage between F-actin retrograde flow and cell adhesion molecule L1-CAM as a clutch molecule at the growth cone and induces traction force for axonal outgrowth [Shimada et al, J. Cell Biol., 2008., Toriyama et al, Curr. Biol., 2013]. As L1-CAM extracellularly interacts with laminin [Hall et al, J. Neurochem., 1997], we hypothesized that axon is induced on laminin by shootin1-mediated linkage between F-actin and L1-CAM. To examine this possibility, we analyzed effect of shootin1 RNAi on directional axon formation of hippocampal neurons cultured on substrates patterned with stripes of laminin and poly-D-lysine. The rate of neurons that formed axon on laminin was significantly decreased by shootin1 RNAi as compared with control. Next, we measured retrograde flow rates of mRFP-actin and EGFP-shootin1 speckles. As L1-CAM binds with laminin, retrograde flow of F-actin and shootin1 would be negatively regulated by laminin on the extracellular substrate. Retrograde flow rates of mRFP-actin and EGFP-shootin1 speckles in the axonal

TUESDAY-POSTER PRESENTATIONS growth cone on laminin were markedly slower than those on PDL. Because retrograde flow rates of mRFP-actin and EGFP-shootin1 speckles were decreased in the axonal growth cone on laminin, traction force that the axonal growth cone generates would be positively regulated on laminin. To examine this possibility, we performed traction force microscopy. Average stress under the growth cone on laminin was larger than those on PDL. Together, our results suggest that the clutch system involving shootin1 and L1-CAM senses an adhesion cue from laminin and produces traction force for axon formation.

Neuronal Cytoskeleton 2 P2076 Board Number: B596

Somal translocation of Retinal Ganglion Cells: a new mode of microtubule involvement in neuronal migration. J. Icha1, C. Norden1; 1 MPI-CBG, Dresden, Germany Neuronal migration is a key step during brain development and defective neuronal migration can lead to cognitive impairments. Importantly, neuronal migration modes differ depending on tissue context and migratory distances covered. Despite this broad variety however, most mechanistic studies have so far focused on radial glial-guided migration To get insights into additional neuronal migration modes we investigate the kinetics and molecular mechanisms responsible for retinal ganglion cell (RGC) migration in zebrafish. RGCs are the earliest born neurons in the retina and migration from their apical birthplace to the most basal neuronal layer spans about 50µm. It was proposed that RGCs move via somal translocation but the exact molecular mechanisms of migration are still elusive. We image migratory processes in the intact embryos using light sheet as well as confocal microscopy at high temporal-spatial resolution. This approach revealed that RGC migration occurs unidirectionally and migratory speeds reach 20 µm/h. To exclude the possibility that somal translocation is a passive process we inhibited nuclear movements of remaining progenitor nuclei. This treatment did not significantly impair RGC migration arguing that it is an active process depending on force generation within the cells. Interestingly, we observe that the centrosomes as well as the Golgi follow the migrating soma during RGC translocation instead of leading it as seen in glia-guided migration. Nevertheless, like in glial-guided migration, both, microtubules and dynein play a role in RGC displacement. Microtubules and dynein are apically enriched during migration, indicating that the force to move cells is generated BEHIND nucleus and soma. This finding revealed the possibility that microtubules push the nucleus, the bulkiest organelle of the cell, in the direction of basal migration and/or prevent re-displacements by acting as an apical barrier. The observation that basal process attachment is crucial for efficient migration underlines this exciting possibility. We currently test this hypothesis further using laser ablation approaches to

TUESDAY-POSTER PRESENTATIONS explore whether apical and basal processes feature different mechanical properties. We also test the possibility that dynein is important to give rigidity to apical microtubules and thereby stabilizes the apical process. Overall our study aims to gain a basic understanding of the molecular mechanism involved in somal translocation that will most likely also apply in brain regions beyond the retina. It furthermore shows that neurons can achieve successful migration differently in different scenarios and underlines the fact that neuronal migration has many different flavours several of which still await further exploration.

P2077 Board Number: B597

Synaptotagmin-1’s Promotion of the Formation of Axonal Filopodia in developing Neurons is associated with Calcium Flux. K.F. Greif1, A. Brandtjen1, C. Weichelsbaum1, M. Currie1, C. Schoonover1, H. Lehman1, N. Ashok1, S. Gandarez1; 1 Bryn Mawr College, Bryn Mawr, PA Synaptotagmin-1 (syt1), a Ca2+-binding protein that functions in regulation of vesicle exocytosis at the synapse, is expressed in many types of neurons well before synaptogenesis begins both in vivo and in vitro. We recently demonstrated (Greif et al, 2013) that syt1 plays a role in regulating axon branching and filopodial dynamics in developing embryonic chick forebrain neurons. Overexpression of syt1 increased the formation of axonal filopodia and branches. Conversely, knockdown of syt1 decreased the numbers of axonal filopodia and branches. The mechanism by which syt1 exerts its influence on axon development is unknown. In order to investigate whether syt1 accomplishes its effects on filopodia using a mechanism similar to its role at the synapse, we manipulated Ca2+ levels using calcium blockers and calcium ionophores. A 15-minute exposure to the calcium ionophore, A23187, significantly increased axonal filopodia; blockade of calcium channels using La3+ or Cd2+ decreased filopodia number. These findings are consistent with the well-defined role of Ca2+ in filopodial dynamics. Overexpression of syt1 using adenovirus-mediated transfection was coupled with La3+ treatment to examine whether blocking Ca2+ influx would reverse Syt1-mediated increases in axonal filopodia. Embryonic chick forebrain neurons were transfected with a syt1-YFP construct or GFP. After 48 hrs, cells were treated with La3+ or vehicle for 15 minutes. Treatment with La3+ reversed the increase in filopodia stimulated by syt1 overexpression. Taken together these data suggest that syt1 regulates the formation of axonal filopodia using its Ca2+-binding functions, prior to engaging in its conventional functions at the synapse. Future experiments involve site-directed mutagenesis of syt1’s C2 domains, to directly study our hypothesis. This research was supported by grants from Bryn Mawr College. Reference: Greif, K.F., N. Asabere, G. J. Lutz and G. Gallo. Synaptotagmin-1 promotes the formation of axonal filopodia and branches along the developing axons of forebrain neurons, Dev Neurobiol. 2013 Jan; 73(1):27-44.

TUESDAY-POSTER PRESENTATIONS

P2078 Board Number: B600

Fidgetin is a novel microtubule-severing protein that controls neuronal morphology by balancing the stable and labile components of the axonal microtubule array. L. Leo1, A. Matamoros2, W. Yu3, D. Marenda4, D. Sharp5, P.W. Baas3; 1 Neuroscience, Drexel University College of Medicine, Philadelphia, PA, 2Neuroscience/Molecular and Cell Biology and Genetics, Drexel University College of Medicine, Philadelphia, PA, 3Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, 4Biology, Drexel University, Philadelphia, PA, 5Albert Einstein College of Medicine, Bronx, NY The dynamicity of the axonal microtubule (MT) array is defined by stable and labile components. All of the MTs in the axon have a stable domain, and most have a labile domain. MT-severing proteins are important for regulating the axonal MT array by transforming one MT into two, for example to increase the number of MTs at sites of impending branch formation. In order to accomplish this, katanin and spastin (the best studied of the MT-severing proteins) preferentially break MTs in their stable domains, so that each of the two daughter MTs has its own stable domain from which a new labile domain can grow. Theoretically, a MT-severing protein that preferentially breaks MTs in their labile domains would have a very different effect. Specifically, such a severing protein would result in one MT with a stable domain and one without, the latter of which would rapidly and completely depolymerize. In the present study, we have focused on a novel MT-severing protein called fidgetin, which we posit acts in this fashion. The function of such severing would be to pare back the labile domains of the MTs so that they achieve a proportion relative to the stable domains appropriate for meeting the specific challenges neurons face in various stages in their lives. A reduction of such severing activity would result in an expansion of the labile domains, which we posit would be conducive to phenomena such as neuronal polarization. An increase in such severing activity, on the other hand, would reduce the labile domains, which we posit would be conducive to phenomena such as cessation of axonal growth or pruning back of supernumerary axons. In support of these ideas, we have found that reduction in fidgetin levels by siRNA causes cultured rodent neurons to polarize faster, and produce more processes per cell body and longer axons, together with a notable increase in labile MT mass. In developing Drosophila embryos, fidgetin reduction in targeted neurons causes axons to grow past their normal targets rather than retract. Previous studies have shown that katanin and spastin target MT regions that have posttranslationally modified tubulins characteristic of stable MTs. Our present studies indicate that fidgetin knockdown results in a dramatic decrease in the ratio of acetylated to total tubulin in the neuron, suggesting that fidgetin targets MT regions that are deficient in acetylated tubulin. In support of this, two different experimental approaches for experimentally increasing the acetylation status of the MTs rendered them less sensitive to the effects of fidgetin reduction. Collectively, these results accentuate the importance of the labile MT component in the axon, which has generally received less attention than the stable component.

TUESDAY-POSTER PRESENTATIONS

P2079 Board Number: B601

Fidgetin inhibition is a novel strategy for altering the balance of stable and labile components of the axonal microtubule array to treat nervous system disease and injury. A. Matamoros1, L. Leo2, D. Jean1, T. Austin1, D. Sharp3, W. Yu4, P.W. Baas4; 1 Neuroscience/Molecular and Cell Biology and Genetics, Drexel University College of Medicine, Philadelphia, PA, 2Neuroscience, Drexel University College of Medicine, Philadelphia, PA, 3Albert Einstein College of Medicine, Bronx, NY, 4Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA Effective strategies for treating diseases and injuries of the CNS remain elusive. Recent studies have converged on microtubules (MTs) as a powerful therapeutic target. Neurodegenerative diseases called tauopathies involve the dissociation of tau from MTs in the axon. As a result, MT mass is diminished, and this compromises the axon’s ability to maintain proper morphology and organelle transport. Treatments able to correct the MT loss would in theory have profound benefits to patients. Drugs that stabilize MTs have produced encouraging results in preclinical studies on tauopathy, with the same drugs also reported to support regeneration after injury to adult CNS axons. Despite these encouraging results, there are persistent questions as to whether this approach appropriately corrects the MT deficiency. MTs in the axon consist of two domains: a stable domain toward the minus end of the MT and a labile domain toward the plus end. These domains are different from one another in their composition, properties and functions. The labile domains comprise a greater component of the total MT array during development, while the stable domains predominate in adult axons. We have suggested that a better strategy for encouraging an injured adult axon to grow would not be to make the MTs more stable, but rather to promote an increase in the levels of the labile component of the MT mass. In the case of tauopathy, our data suggest that it is the labile component that is most vulnerable to degradation, not the stable component. Therefore, we would question whether notable benefit can be achieved by further stabilizing the remaining MT mass. Here we propose a novel approach aimed at increasing the labile component of the MT mass by suppressing a MT-severing protein called fidgetin. Our data suggest that the normal function of this enzyme is to pare back the labile domains of the MTs, in order to regulate the ratio of the stable and labile MT components of the axon at various stages in the life of the neuron. Our hypothesis is that partially suppressing fidgetin will enable the labile component of the MT mass to swell up, which will restore to the tauopathic axon the MT component that had been lost, and which will make the MT array of the injured adult axon more similar to that of a developing axon, and hence more amenable to growth and regeneration. We will present two sets of experiments on primary cultures of rodent neurons. In the first, we show that fidgetin depletion causes an increase in labile MT mass that is conducive to robust axonal regeneration, even on inhibitory molecules that normally suppress CNS regeneration. In the second, we show that tau depletion results in a loss of labile MT mass that is correctable by fidgetin depletion.

TUESDAY-POSTER PRESENTATIONS

P2080 Board Number: B602

Extracellular nicotinamide protects axons from Wallerian degeneration via multiple pathways in cultured DRG neurons. S. Tokunaga1, T. Araki1; PNS, National Ctr Neurology and Psychiatry, Tokyo, Japan

1

Accumulating evidence suggests delaying axonal degeneration as a possible therapeutic strategy for traumatic and neurodegenerative disorders, including stroke, Parkinson’s disease and peripheral neuropathy. Recent studies demonstrated that exogenous application of excess nicotinamide adenine dinucleotide (NAD) or its precursor nicotinamide (Nam), the amide form of vitamin B3, have a therapeutic impact on Wallerian degeneration models, but previous reports demonstrated apparently contradictory results: Wang et. al., showed that application of NAD or Nam at the time of neurite transection in vitro can delay neurite degeneration concurrently with increased intracellular NAD and ATP level, while others demonstrated that FK866, a potent inhibitor of nicotinamide phosphoribosyltransferase (Nampt) which synthesizes the substrate for NAD biosynthetic enzyme from Nam, can also delay neurite degeneration concurrently with decreased intracellular NAD. Here we show that excess Nam ( 3 mM) applica on-mediated delay of injury-induced neurite degeneration was unaffected by co-application of FK866 or down-regulation of endogenous Nampt. In contrast, FK866mediated delay of neurite degeneration was counteracted by Nam ( 1 mM) dose-dependently but not nicotinic acid (Na), the acid form of vitamin B3, while Na served as NAD precursor in primary DRG neurons. Our results suggest that the Nam may protect axons independently from Nampt activity, presumably by affecting another enzymatic activity sensitive to FK866.

P2081 Board Number: B603

Nerve Growth Factor Induces Mitochondrial Fission Which is Required for Axon Branching. L. Armijo Weingart1, R. Sainath1, G. Gallo1; 1 Shriners Hospitals Pediatric Research Center, Temple University, Philadelphia, PA During nervous system development, the formation of branches from axons is essential to the formation of complex neuronal circuitry. In sensory neurons, Nerve Growth Factor (NGF) rapidly (30 min) induces branching through activation of phosphoinositide 3-kinase (PI3K). Recently, mitochondria have emerged as major determinants of the sites of axon branching. However, the mechanism of NGF-induced axon branching remains to be fully elucidated. An understanding of how NGF signaling impacts mitochondria behavior and function during axonal branching is the main goal of our current work. To this end, mitochondria were detected in living axons through labeling with Mitotracker dyes. NGF treatment

TUESDAY-POSTER PRESENTATIONS decreased the length and increased the number of axonal mitochondria after 15 min of acute treatment, indicative of fission. Consistently, live time-lapse imaging of mitochondria following 5 minutes of NGF treatment revealed mitochondria fission. Direct activation of PI3K using a cell permeable peptide in the absence of NGF copied the effects of NGF. Conversely, inhibition of PI3K using LY294002 blocked the effects of NGF on mitochondria. Inhibition of dynamin related protein 1 (DRP1), a required component of mitochondria fission, using mDivi-1 blocked NGF induced axon branching. Live imaging of axons in the presence of NGF revealed that GFP-DRP1 accumulated at sites of mitochondria fission and endogenous DRP1 was detected in axons through immunocytochemistry. Collectively, these observations indicate that NGF induces mitochondrial fission through a DRP1-dependent mechanism and activation of the PI3K pathway, and that fission is required for NGF-induced branching. The NGF induced fission temporally precedes the formation of branches, consistent with the notion that the fission is required to reorganize the axonal mitochondrial array. In future studies we will further address the mechanistic role of mitochondria fission in the generation of axon branches and the role of PI3K signaling in the induction of fission.

P2082 Board Number: B604

Understanding the Axon Initial Segment Diffusion Barrier at the Nanoscopic Level. D. Albrecht1,2, H. Ewers1; 1 Randall Division of Cell and Molecular Biophysics, King's College of London, London, United Kingdom, 2 Institute of Biochemistry, ETH Zürich, Zurich, Switzerland Polarization is a decisive step in neuronal development and once axonal and somatodendritic domains are generated, they persist over the lifetime of an organism. The axon initial segment (AIS), a stretch of 50 – 100 μm in length, bears a specialized cytoskeleton and maintains polarization by restricting the exchange of axonal membrane molecules with the soma. The adaptor protein ankyrinG is essential for the assembly of this diffusion barrier and tethers ion channels and adhesion molecules to the cytoskeleton. However, the structural organization of the AIS and the molecular mechanism, by which protein diffusion is impeded remain unclear. Here, we use novel single molecule localization based superresolution microscopy in primary hippocampal neurons to investigate the molecular architecture of the AIS and to understand the mechanism by which membrane protein diffusion is locally restricted. To do so, we quantify membrane protein diffusion during neuronal polarization in single cells over time by single molecule tracking and correlate these data to the nanoscopic organization of the AIS. We find that the AIS cytoskeleton is organized in a regularly spaced pattern and that coincident with the appearance of this organization during development, the lateral mobility of membrane molecules is locally reduced. Our results clarify the interdependence between cytoskeletal organization and membrane mobility in the axonal plasma membrane.

TUESDAY-POSTER PRESENTATIONS

P2083 Board Number: B605

Mutations in Tau have Differential Effects on the Aggregation and Microtubule Stabilization Properties of its Isoforms. Y. Mutreja1, B. Combs2, T.C. Gamblin1; 1 Department of Molecular Biosciences, University of Kansas, Lawrence, KS, 2Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University, East Lansing, MI Tau is a microtubule associated protein found in six different isoforms in neurons. In the diseased state, tau loses its ability to bind and stabilize microtubules and polymerizes to form large aggregates. Various mutations occur in the tau gene leading to a class of neurodegenerative diseases named FTDP-17. These mutations can alter tau’s ability to stabilize microtubules and also enhance its ability to aggregate. It has been observed that with many of these mutations, only a subset of isoforms of tau is deposited as aggregates in the patient’s brain. Therefore, we hypothesize that these mutations have differential effects on aggregation and microtubule stabilization characteristics of tau in different isoforms. We also sought to determine whether the position of the mutation would differentially affect the underlying biochemical mechanisms of aggregation and microtubule stabilization. We chose to study three different FTDP-17 mutations R5L, P301L and R406W residing respectively in N-terminal, microtubulebinding repeats (MTBR) and C-terminal regions of various isoforms of tau. The extent and kinetics of aggregation induced by arachidonic acid in vitro was monitored using laser light scattering, thioflavin S fluorescence and electron microscopy. The aggregation properties of some isoforms were effected more by the mutations than others. Certain isoforms, such as 2N3R and 1N4R were disproportionately affected by mutations in the N-terminal region and the MTBR. The aggregation kinetics of 0N4R were affected more by mutation than the other isoforms. Mutations in the MTBR have a larger effect than Nterminal and C-terminal mutations on the stabilization of microtubules, but also show differences between isoforms for a given mutation. Our results from this study indicates that aggregation and microtubule stabilization of all the isoforms of tau is not affected in same manner and these effects further are dependent on the location of mutation and could provide a biochemical rationale for their different incorporation into aggregates in various diseases in the FTDP-17 class.

TUESDAY-POSTER PRESENTATIONS

P2084 Board Number: B606

Neurite-specific activation of NADPH oxidase in hippocampal neurons overexpressing cellular prion protein or exposed to Aβ or TNFα. K.P. Walsh1, T.B. Kuhn1,2, L.S. Minamide1, A.E. Shaw1, S.J. Kane3,4, L.G. Whittington1, M.D. Zabel3, G.G. Rodney5, J.R. Bamburg1,4; 1 Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, 2Chemistry and Biochemistry, Univ of Alaska, Fairbanks, AK, 3Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, 4Cell and Molecular Biology Program, Colorado State University, Fort Collins, CO, 5Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX Persistent exposure of neurons to soluble β-amyloid peptide dimer/trimers (Aβd/t) or proinflammatory cytokines (TNFα, IL-1β, IL-6) induce the formation of cofilin-actin rods in a subset of neurons (~23%) and in a subset of neurites (~30%) of those neurons. Rods disrupt synaptic function by blocking transport and/or sequestering cofilin from dendritic spines. Rod formation induced by these agents is dependent on the expression of the cellular prion protein (PrPC) and the subsequent activation of NADPH oxidase (NOX) and production of reactive oxygen species (ROS) (Walsh et al., PLoS One, 9, e95995, 2014). Overexpression of PrPC-EGFP in the absence of other treatments induces rods in up to 40% of neurons, but the percentage of neurites forming rods does not change significantly. Here we utilized p47PHOXroGFP (Pal et al., PLoS One 8, e63989, 2013) as a redox probe for measuring NOX-2 activity in living neurons allowing us to localize ROS to the neurites in which it is produced. We generated adenovirus for expressing the probe in neurons and in some experiments also co-infected neurons with adenovirus for expressing cofilin-R21Q-mRFP, a probe for visualizing cofilin-actin rods but one that does not induce rods through its overexpression (Mi et al., PLoS One 8, e83609, 2013). Our results show that NOX-2 activation and ROS production only occur in a subset of neurites from neurons expressing PrPCEGFP, even though the PrPC-EGFP is relatively uniformly distributed among the neurites. Similarly, neurons treated globally with Aβd/t or TNFα only show NOX-2 activity, ROS production and rod formation in a subset of neurites. These findings suggest that although PrPC levels may limit the overall neuronal rod response to Aβd/t or TNFα, some factor downstream of PrPC is the limiting factor for rod formation and this factor is differentially distributed among neurites. The factor is neither cofilin nor actin, since cofilin-actin rods can be induced in almost all neurons and neurites in a PrPC- and NOXindependent manner by treatment with excitotoxic levels of glutamate. (Supported in part by NIH AG044812 to JRB).

TUESDAY-POSTER PRESENTATIONS

P2085 Board Number: B607

An unbiased genetic screen using the Drosophila wing as a model identifies the essential role of a novel gene, SAD, in maintaining axonal integrity during aging. X. Cao1, Q. Li1, X. Teng2, Y. Zhu2, Z. Ma1, N.M. Bonini2, Y. Fang1; 1 SIOC, IRCBC, Chinese Academy of Sciences, Shanghai, China, 2Department of Biology, University of Pennsylvania, Philadelphia, PA Axon degeneration is a prominent feature of spinal cord injury and many human degenerative diseases. Studies of the Wallerian degeneration slow (WldS) mouse indicate that axon degeneration is an active process, however, the underlying mechanisms remain elusive. To identify novel molecular components controlling axonal integrity, it is desirable to perform unbiased, large-scale screening. We have developed a novel model of axon injury using the Drosophila wing1,2,3. The fly wing is translucent, allowing us to highlight the axons using fluorescent proteins and to monitor axonal changes in response to traumatic injury and aging in live flies. Using this model, we have performed forward genetic screens to discover novel genes that can protect the injured nerve or revert the WldS-mediated axonal protection. We found a mutant line of a functionally uncharacterized gene, which not only diminishes the WldS protection, but also displays striking age-dependent spontaneous axon degeneration on its own. For this phenotype, we named the mutant: Spontaneous Axon Degeneration (SAD). Further examination reveals massive vacuoles in the brain of aged SAD flies, indicating progressive neurodegeneration in the CNS as well. Moreover, the lifespan of the SAD mutant is significantly shortened. In addition, aged SAD flies have elevated sensitivity to stress such as heat and physical disturbance. Feature analysis of the SAD protein suggests that it may be involved in chromatin remodeling and epigenetic modifications. Ongoing experiments include generating UAS-SAD and RNAi-SAD transgenic flies to determine the role and molecular mechanisms of SAD in maintaining neural integrity during aging. And, our most recent behavior assay results point to a non-cell autonomous mechanism of the SAD function in the nervous system. All together, by this study, we hope to provide important foundation for new therapeutic targets of axonal/neural degeneration in acute injury as well as chronic neurological disorders. References: 1.Fang, Y., Soares, L., and Bonini, N.M. (2013) “Design and Implementation of In Vivo Imaging of Neural Injury Responses in the Adult Drosophila Wing.” Nature Protocols, 8(4): 810-819. 2.Fang, Y., and Boinini, N.M. (2012) “Axon Degeneration and Regeneration: Insights from Drosophila Models of Nerve Injury.” Annual Review of Cell and Developmental Biology, 28: 575-597. 3.Fang, Y., Soares, L., Teng, X., Geary, M., and Bonini, N.M. (2012) “A Novel Drosophila Model of Nerve Injury Reveals an Essential Role of Nmnat in Maintaining Axonal Integrity.” Current Biology, 22(7): 590595.

TUESDAY-POSTER PRESENTATIONS

P2086 Board Number: B608

Disruption of Intraneuronal Tau by Extracellular Tau Oligomers Containing Individual or Mixed Tau Isoforms. E.M. Swanson1, L. McMahon 1, S. Som2, G.S. Bloom2; 1 Department of Biology, University of Virginia, Charlottesville, VA, 2Departments of Biology and Cell Biology, University of Virginia, Charlottesville, VA Tau is microtubule-associated protein enriched in the axons of neurons, where its functions include direct binding and stabilization of microtubules and regulation of axonal transport. Tau is found in the central nervous system (CNS) in six isoforms produced by the alternative splicing of a single tau gene, MAPT, with these isoforms characterized by the presence of zero, one or two N terminal inserts, and three or four C-terminal microtubule binding repeats. Neuronal inclusions composed of hyperphosphorylated tau are a major histopathological feature of a series of neurodegenerative disorders known collectively as tauopathies, the most common and well-known of which is Alzheimer’s disease (AD). While the clinical and histological presentation of these disorders is heterogeneous, a majority share the following hallmarks: loss of the normal axonal distribution of tau; accumulation of insoluble, fibrillar tau aggregates in neurites and perikarya; synaptic dysfunction; and eventual neuron death. There is also evidence that the majority of these disorders spread through the brain by a prionlike mechanism, in which pathologically misfolded tau is taken up by neurons, confers this pathological phenotype to the endogenous "normal" protein through direct protein-protein contact, and is eventually released by the cell, perpetuating the cycle. Some evidence points to short tau fibrils as being prion-like, but scant attention has been paid to small tau oligomers. Using a new morphometric method for rigorously quantifiying tau distributions in cultured neurons, we now demonstrate that externally applied tau oligomers are much more effective than tau monomers or fibrils at disrupting the normal tau distribution in primary cortical neurons, that oligomer potency varies according to tau isoform, and that oligomers made from mixtures of all six CNS tau isoforms are much more potent than oligomers made from individual isoforms. These results raise the possibility that soluble, pre-fibrillar oligomers of mixed tau isoforms account for the prion-like spread of tau pathology in vivo in AD and non-Alzheimer's tauopathies.

TUESDAY-POSTER PRESENTATIONS

P2087 Board Number: B609

Tau hyperphosphorylation in human Alzheimer’s Disease (AD) cases: A quantitative histological comparison. D. Havas1, B. Kerschbaumer1, J. Attems2, H. Hutter3, B. Hutter-Paier1; 1 QPS Austria GmbH, Grambach, Austria, 2Institute of Ageing and Health, Newcastle University, Newcastle, UK, 3Institute of Cell Biology, Histology and Embryology, Med Univ Graz, Graz, Austria Alzheimer’s Disease (AD) is characterized by neurofibrillary tangle (NFT) pathology. Yet, the tangle reflects an end stage of paired helical filament aggregations that are built much earlier and are typical found in neutrophil threads. Furthermore, they consist of a large array of differently hyperphosphorylated Tau (pTau), whereas several phospho-sites were found to be especially related to AD. However, today’s histological knowledge about appearance, quantity and location of PHFs with different phosphorylation and in different brain regions during disease progression is rather poor. To elucidate the quantitative distribution of different pTau sites in the AD brain, we analyzed total human Tau, pThr231 as well as pTyr18 Tau in four different cortical brain regions and the hippocampus of each of five AD subjects from three different Braak stages by immunohistochemistry and compared them to the brains of age-matched non-AD controls. In addition ThioflavinS positive NFTs were counted as well. Further markers, especially pSer262 and pSer202/Thr205 will be included. In spite that Tyr18 Tau phosphorylation was published to be an early AD marker, the quantification in the brain rather revealed the drastic load in cortical regions at end stage. The same late stage increase was observed for ThioflavinS positive NFTs and pThr231 Tau positive somata and neurites. However and most importantly both Tyr18 and Thr231 hyperphosphorylation increase much earlier in the hippocampus and are already significant at Braak stages I/II, while NFT formation lags behind. Our findings indicate a special and early vulnerability of the hippocampal formation to Tau hyperphosphorylation at residues Tyr18 and Thr231, which might contribute to the early break-down of memory function in AD subjects. To note, the downstream tangle formation is a sign of the progressive neurodegeneration but turns out to be rather insensitive for the detection of Tau hyperphosphorylation in early AD.

TUESDAY-POSTER PRESENTATIONS

P2088 Board Number: B610

Induced patient neurons as a potential future model system for Alzheimer's disease. H. Toresson1, L. Minthon1, O. Hansson1, N. Schultz1, C. Orbjörn2, M. Parmar3, U. Pfisterer3; 1 Clinical Memory Research Unit, Dept. of Clinical Sciences, Lund University and Skåne University Hospital, Malmö, Sweden, 2Clinical Memory Research Unit, Dept. of Clinical Sciences, Lund University, Malmö, Sweden, 3Wallenberg Neuroscience Center and Lund Stem Cell Center, Department of Experimental Medical Science, Lund University, Lund, Sweden The underlying mechanisms of Alzheimer's disease (AD) remain unknown. According to the main hypothesis in the field, the amyloid cascade hypothesis, β-amyloid (Aβ) is the causing agent and therefore has been the target for massive drug development efforts. Several drug candidates have been tested in phase III clinical trials but so far none has had any clinical effect. These disappointing results highlight the need for intensified research on the disease in humans. We have launched a cell biological initiative within the Swedish BioFinder Study, a large clinical research programme where dementia patients and cognitively healthy elderly are longitudinally phenotyped by e.g. cognitive tests, cerebrospinal fluid (CSF) biochemistry, amyloid-positron emission tomography and advanced magnetic resonance imaging. Additionally, a skin biopsy is taken to obtain fibroblasts. We have thus far banked fibroblasts from 34 individuals. Preliminary data show that the most efficient way (time, number of cells and induced neuron (iN) conversion efficiency) was to obtain cells from skin explant cultures in 20% serum followed by expansion in defined serum free medium. 12 out of 12 lines tested could be successfully converted to iN cells by growth factor treatment and transduction of the three transcription factors Ascl1, Brn2 and Myt1l (under TET-ON transcriptional control) along with the TET-ON transactivator. Several transduced cells adopted neuronal morphology and after 13 days cells expressed the neuronal markers βIII-tubulin and MAP-2. Importantly, iN cells also expressed the AD pathology-associated proteins Aβ and the microtubule-associated protein tau. Isoforms of these proteins used clinically as AD CSF biomarkers (Aβ1-40, Aβ1-42, and tau (including phosphorylated tau)) were secreted into the medium in measurable amounts. The value of iN cells as tools to investigate the underlying disease mechanisms in sporadic AD (SAD) will be assessed by two means. The first is comparing iN cell phenotypes from SAD patients with cells from familial AD (FAD) cases with APP, PSEN1 or PSEN2 mutations. The second, and potentially most powerful method for finding unknown disease mechanisms is to perform an unbiased search for correlations between cellular and clinical phenotypes. Our clinical cell biology approach makes it possible to compare cells from cognitively healthy individuals without clinical signs of AD pathology to healthy or AD cases with a long clinical history of AD pathology. Importantly, this human cell biology approach to AD takes the clinical heterogeneity of dementia diseases into account and thus paves the way for future personalised treatment.

TUESDAY-POSTER PRESENTATIONS

P2089 Board Number: B611

α-Synuclein Modulates Amyloid-β Ιnduced Ectopic Cell Cycle Re-entry of Neurons. S.S. Thomas1, G.S. Bloom2; Department of Biology and Neuroscience Graduate Program, University of Virginia, Charlottesville, VA, 2 Departments of Biology and Cell Biology, University of Virginia, Charlottesville, VA 1

The defining histopathological features of Alzheimer’s disease (AD) are extracellular plaques that comprise amyloid-β (Aβ) peptides and intraneuronal neurofibrillary tangles made from the neuronspecific, microtubule-associated protein, tau. Normal neurons remain post-mitotic through tight regulation of their cell cycle machinery, but in AD and Parkinson’s disease (PD), cell cycle proteins are aberrantly activated, causing the normally quiescent neurons to re-enter the cell cycle. Neurons that reenter the cell cycle progress at least into S-phase, yet instead of dividing they eventually die. In typical cases of AD, approximately 30% of frontal lobe neurons are lost, and aberrant cell cycle re-entry (CCR) may account for as much as 90% of this neuronal death. Similarly, abnormal activation of cell cycle markers, DNA synthesis, and cell cycle related neuron death have been reported to occur in PD. Despite a large body of evidence demonstrating CCR in AD and PD, many questions regarding the underlying mechanism remain unanswered. Previous work from our lab established that amyloid-β oligomers ( βOs) induce ectopic neuronal CCR through a mechanism dependent on tau phosphorylation at Y18, S409 and S416 (Seward, et al. J Cell Sci 126: 1278-1276). Using primary mouse cortical neuron cultures as experimental models, we now report that βOs also promote a tau-dependent increase in endogenous α-synuclein, which accumulates in Lewy bodies in PD and in many cases of AD as well. Furthermore, overexpression of the pathogenic A53T, E46K, or A30P mutations of human α-synuclein induces ectopic neuronal CCR independently of βOs, with the A30P mutation generating the most robust response. In contrast, reduction of endogenous α-synuclein using shRNA ameliorates βOinduced tau phosphorylation at S409 and S416, and consequently prevents neuronal CCR. Taken together, these data implicate α-synuclein as a novel modulator of ectopic neuronal CCR in AD and PD.

P2090 Board Number: B612

Triple miRNA Shank knockdown shows that Shank-cortactin interactions control actin dynamics in neuronal spines and synapses. H.D. MacGillavry1, J.M. Kerr2, N.A. Frost3, T.A. Blanpied4,5; 1 Cell Biology, Utrecht University, Utrecht, Netherlands, 2NINDS, NIH, Bethesda, MD, 3Psychiatry, UCSF School of Medicine, San Francisco, CA, 4Department of Physiology, Univ of Maryland Sch of Med, Baltimore, MD, 5Program in Neuroscience, Univ of Maryland Sch of Med, Baltimore, MD

TUESDAY-POSTER PRESENTATIONS Proper functioning of the brain relies on the rapid and efficient transfer of signals across neuronal synapses. However, we have little understanding of the molecular mechanisms that establish and modulate the molecular architecture at the postsynaptic density (PSD) and thereby shape the efficiency of synaptic signal transmission. The family of Shank scaffolding molecules (comprising Shank1, 2 and 3) are core components of the PSD that via multiple protein interaction domains link surface glutamate receptors to other scaffolding molecules within the PSD, as well as to the actin cytoskeleton via diverse actin-binding proteins. However, determining the function of Shank proteins in neurons has been complicated because the different Shank isoforms share a very high degree of sequence and domain homology. Therefore, to control Shank content while preventing potential compensatory effects, we developed a miRNA-based knockdown strategy to reduce the expression simultaneously of all synaptically targeted Shank isoforms in hippocampal neurons. Using this approach, we achieved a strong (>75%) reduction in total Shank protein levels at individual dendritic spines, prompting a ~40% decrease in mushroom spine density. Furthermore, miRNA-based Shank knockdown reduced spine actin levels and increased sensitivity to the actin depolymerizing agent Latrunculin A, an effect restored by reexpression of full-length miRNA-resistant human SHANK2. Interestingly, a SHANK2 mutant lacking the proline-rich cortactin binding motif (SHANK2-dPRO) was unable to rescue these defects, suggesting that Shank-cortactin interactions are required for the maintenance and stability of the spine actin cytoskeleton. Furthermore, Shank knockdown reduced cortactin levels in spines and increased the mobility of spine cortactin as measured by single-molecule tracking PALM, suggesting that Shank proteins recruit and stabilize cortactin at the synapse. Consistent with this model, we found that Shank knockdown significantly reduced spontaneous remodeling of synapse morphology, a phenomenon dependent on actin dynamics, that could not be rescued by the SHANK2-dPRO mutant. We conclude that beyond their role as stable scaffolding molecules within the PSD, Shank proteins are key intermediates between the synapse and the spine interior that, via cortactin, permit the actin cytoskeleton to dynamically regulate synapse morphology and function.

P2091 Board Number: B613

Molecular Mechanisms of Neuroprotection in a Drosophila Model of SCA5. L.C. Keller1, S. Alabsi2; 1 Biological Sciences, Quinnipiac University, Hamden, CT, 2Biomedical Sciences, Quinnipiac University, Hamden, CT Spinocerebellar ataxias (SCAs) encompass a large heterogeneous group of late-onset progressive neurodegenerative disorders that result in uncoordinated motor function. Currently, no effective treatments or therapeutics exist to cure these devastating diseases. To facilitate the understanding and eventual treatment of SCAs, it is imperative to gain molecular insight into the processes that drive neurodegeneration. Our strategy is to use Drosophila as a model system to explore SCA5. We have identified a pro-degenerative signaling system that responds to mutations in the neuronal spectrin/ankyrin skeleton and ultimately results in the activation of a cysteine-aspartic protease

TUESDAY-POSTER PRESENTATIONS (caspase) that is both necessary and sufficient for motoneuron and synaptic degeneration (Keller et al., 2011). When any one of seven independent genes in this pro-degenerative signaling system is deleted the progression of neurodegeneration in Drosophila is slowed. The identification of genetic perturbations that can protect against degeneration highlight the importance of these signaling molecules as potential therapeutic targets for treatment of neurodegenerative diseases such as SCA. A preponderance of experimental data suggests that protein quality control mechanisms are intimately involved in disease progression of many SCAs, including SCA5. Preliminary data from my lab indicate that components of the conserved autophagic protein degradation machinery are involved in prodegenerative signaling and removal of these components can protect against degeneration. This research is focused on elucidating new approaches to suppress neurodegeneration in our Drosophila model of SCA5 with the goal of discovering novel targets for future therapeutics.

P2092 Board Number: B614

The adhesion-GPCR CELSR3 promotes axonal facisulation in the embryonic zebrafish. A. Ciprian1, F. Sherine1, A.L. Dell1,2; 1 Department of Biology, St. Francis College, Brooklyn, NY, 2Department of Neuroscience, University of Pennsylvania, Philadelphia, PA Using the zebrafish retinotectal projection as a model system, we previously showed that GPCR signaling is required for axon guidance during normal development. Retinal ganglion cell (RGC) axons that express a dominant negative G-protein subunit GαS fail to cross the midline and misproject to the ipsilateral tectum. GαS promotes expression of the axon guidance receptors Nrp1a and Nrp1b, helping retinal axons cross the midline, likely via cAMP-dependent signaling. This suggests a mechanism by which advancing growth cones can respond to local signals that upregulate cAMP and thereby ready themselves for the next guidance cue they encounter. However which GPCRs and ligands contribute to this process is still unknown. We identified CELSR3 as a candidate by analyzing GEO datasets to find GPCRs expressed in the right time and place to direct growing axons in the visual system. CELSR3 is a member of the adhesion-GPCR with a characteristic a long n-terminal tail decorated with adhesion motifs including EGF-, leucine-rich, and cadherin repeats as well as Ig domains. In the zebrafish retina, CELSR3 is expressed in amacrine cells as well as RGCs. CELSR3 mutants fail to carry out a optokinetic response (OKR) but have not been analyzed for guidance defects. We assessed CELSR3-/- embryos response to the dark flash behavioral paradigm and examined the trajectory of RGC axons by lipophilic dye labeling. We find that CELSR3-/- retinal axons can cross the midline but are defasiculated (4/30 projections p=0.03). This result suggests that CELSR3 may primarily function as an adhesion molecule to maintain axonal bundling during neuronal tract formation.

TUESDAY-POSTER PRESENTATIONS

Establishing and Maintaining Organelle Structure P2093 Board Number: B616

Role of the endoplasmic reticulum during Legionella pneumophila infection. V. Ramabhadran1, K. Kotewicz1, E. Haenssler1, R. Isberg1,2; 1 Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, 2Howard Hughes Medical Institute, Boston, MA Legionella pneumophila is an aerobic bacterium that is the causative agent of Legionnaire’s disease. Upon inhalation, the bacteria are internalized by alveolar macrophages, where they replicate. The bacterium’s type IV secretion system (T4SS), Dot/Icm, releases over 300 proteins into the host cytosol. These proteins are required for the establishment of the Legionella containing vacuole (LCV) and replication. Although it is thought that the LCV acquires membrane by hijacking endoplasmic reticulum (ER) derived vesicles, the exact role of various ER domains during infection is still unknown. Here, we show that Legionella pneumophila closely interacts with ER tubules during the early stages of infection. We find that reticulon 4 (Rtn4), a mammalian protein that localizes specifically to the tubular domains of the ER, is mono-ubiquitinated by a family of secreted bacterial effectors, known as SdeA-C. The monoubiquitination of Rtn4 leads to the formation of detergent-resistant, immobile Rtn4 aggregates around the LCV. Our live-cell imaging studies suggest that ER tubules form a cage-like structure around the internalized bacterium. Formation of this cage occurs within minutes of infection and may play a role in the establishment of the LCV. Depletion of multiple reticulon families negatively affects Legionella intracellular growth in Cos7 cells suggesting that ER tubules might play an important role in the establishment of the LCV. Based on these data, our hypothesis is that the LCV also acquires membrane by interacting with ER tubules rather than solely hijacking ER-derived vesicles. We use genetic manipulation of the Legionella genome, gene knockdown, enzymatic profiling of SdeC, and live and high resolution microscopy to test our hypothesis. Our studies will fundamentally change our understanding of how Legionella pneumophila establishes a niche to replicate within host cells and will open up new avenues for examining other pathogen-containing vacuoles.

TUESDAY-POSTER PRESENTATIONS

P2094 Board Number: B617

Investigating the genetic pathway driving ER dynamics in Drosophila melanogaster. S.A. Beyeler1, B.E. Riggs2, A.Q. Sims1, H. Hoffman1; 1 Biology, San Francisco State Univ, San Francisco, CA, 2biology, San Francisco State Univ, San Francisco, CA The endoplasmic reticulum (ER) is the largest organelle in the cell and recently has been shown to change its shape and localization throughout the cell cycle, experiencing the most dramatic changes during mitosis. It has been suggested that these dynamic changes are the result of the homotypic fusion and fission events of the ER. A prior study has shown that homotypic fusion is mediated by integral membrane GTPase Atlastin (Atl), however the pathway driving these dramatic rearrangements is poorly understood. Here we are investigating the interaction between Atl and the integral membrane ER shaping proteins Reticulon-1 (Rtnl1) and Lunapark (Lnp) in Drosophila melanogaster. Our hypothesis is that Atl is regulating the interactions between Rtnl1 and Lnp in a GTP dependent manner mediating homotypic fusions events. To investigate our hypothesis we created 3 different transgenic Drosophila lines containing Gal4-UAS mediated RNAi inhibition of Atl, Rtnl1, and Lnp. Each line was driven in the Drosophila compound eye and examined for a phenotype. Expression of Atl-RNAi displayed a rough eye phenotype in 70% of the progeny, while expression of Rtnl1 and Lnp did not exhibit a phenotype in the eye. Based on our preliminary findings, we plan to examine Rtnl1-RNAi and Lnp-RNAi in combination with Atl-RNAi and examine the compound eye for either an enhancement or suppression of the rough eye phenotype. These results would indicate that Rtln1 and Lnp are in the same pathway as Atl, and provide the bases for a modifier screen of the Drosophila genome.

P2095 Board Number: B618

Novel ER shaping proteins Pex30 and Pex31 are involved in pre-peroxisome vesicle biogenesis. A.S. Joshi1, W. Prinz2; 1 NIDDK, NIH, Bethesda, MD, 2NIDDK, National Institutes of Health, Bethesda, MD The peripheral endoplasmic reticulum (ER) forms a network of flat sheet-like cisternae and highly dynamic interconnected tubules that extend through out the cell. Reticulons and reticulon-like proteins are required to maintain this structure. S. cerevisiae has three of these proteins: Rtn1p, Rtn2p, and Yop1p. Interestingly, in cells missing these proteins (rtn1rtn2yop1delta) the peripheral ER is mostly converted to sheets. However, a few ER tubules are still found in these cells, suggesting that there may be additional ER tubulating proteins. A novel genetic screen was designed to identify the proteins that tubulate ER in rtn1rtn2yop1delta. We found that overexpression of Pex30p and Pex31p restores ER

TUESDAY-POSTER PRESENTATIONS tubules in rtn1rtn2yop1delta suggesting that Pex30 and Pex31 are ER tubulating proteins. Like reticulons and Yop1p, Pex30p and Pex31p are localized mainly in the peripheral ER and absent from nuclear membranes. Unlike reticulons and Yop1p, both Pex30p and Pex31p contain dysferlin domain of unknown function. We show that this domain is required for ER membrane tubulation. Our studies also establish that dysferlin domain is functionally conserved from humans to yeast. In S. cerevisiae, Pex30p and Pex31p maintain the number and size of peroxisomes respectively. Mature peroxisomes are derived from pre-existing vesicles known as pre-peroxisomal vesicles (PPV). While the origin of PPV is not known, a recent study show that Pex14p containing PPV are present even in pex3atg1delta cells. We investigated whether ER tubulating proteins play a role in PPV biogenesis. We found a significant increase in Pex14p containing vesicles in rtn1rtn2yop1pex30pex3atg1delta cells indicating that ER tubulating proteins negatively regulate PPV formation. Taken together, our results suggest that ER shaping proteins are essential for tubulating a subdomain of ER involved in PPV biogenesis. The mechanism of PPV formation remains to be investigated.

P2096 Board Number: B619

Investigating the Role of Myosin 18A in Golgi Morphology. K. Bruun1, J.R. Beach2, S. Murugesan3, K. Remmert4, J.A. Hammer4; 1 Department of Biology, St. Joseph's University, Philadelphia, PA, 2CBPC, NIH/NHLBI, Bethesda, MD, 3 NIH\NHLBI, Bethesda, MD, 4LCB, NIH/NHLBI, Bethesda, MD Class 18 myosins are closely related to conventional class 2 nonmuscle myosins (NM2) but surprisingly lack intrinsic motor activity. Previous work has shown that M18A binds the Golgi protein GOLPH3 to stretch and shape the Golgi apparatus (Dippold et al., Cell, 2009), although it remains unclear how “motorless” M18A could provide the force necessary to accomplish this task. Recent work from our and Jim Seller’s labs has shown both in vitro and in live cells that M18A co-assembles with NM2 into mixed bipolar filaments. Therefore, M18A may serve to link these hybrid NM2 filaments to the Golgi apparatus, allowing NM2 motor activity to then drive changes in Golgi morphology. The goals of the current study were (i) to use high resolution and super-resolution microscopy to image M18A, NM2, the actin cytoskeleton, and the Golgi apparatus during steady-state and dynamic processes, and (ii) to reexamine the functional role played M18A in determining Golgi shape and size. Using standard Golgi reporters and fluorescently-tagged M18A, we showed that M18A localizes to some extent to the Golgi at steady-state. Additionally, we commonly observed enrichment of actin filaments near the Golgi apparatus. To suppress or eliminate M18A expression, we utilized lentiviral-mediated shRNA knockdown in HeLa cells (HeLa-M18A-KD) and CRISPR-mediated knockout in Rat2 fibroblasts (Rat2-M18A-KO). Surprisingly, we observed no obvious differences in Golgi localization or distribution in KD and KO cells at steady-state relative to control cells. To investigate further, we utilized Brefeldin A (BFA), a small molecule that induces Golgi dispersal, to determine if M18A plays a role during a more dynamic Golgi process. Specifically, we localized M18A and actin during Golgi recovery following removal of BFA. Preliminary results suggest that there is a delay in the recovery of normal Golgi architecture in HeLa-

TUESDAY-POSTER PRESENTATIONS M18A-KD cells. Moreover, live-cell imaging showed that the localization of M18A and actin on the Golgi apparatus may be enhanced during the recovery process. Overall, therefore, our data suggest that M18A is not important for Golgi morphology at steady-state, but may be play a significant role during dynamic Golgi movements, such as those that occur during cell migration or following cell division.

P2097 Board Number: B620

Identification of Rab41 effectors implicated in Golgi organization and trafficking. S. Liu1, B. Storrie2; 1 Univ Arkansas Med Scis (UAMS), Little Rock, AR, 2Physiology and Biophysics, Univ Arkansas Med Scis (UAMS), Little Rock, AR By homology, Rab6a, Rab6a’, Rab6b, Rab6c and Rab41 can be grouped together in Rab VI subfamily with Rab6a/a’ and Rab41 sharing 80% over much of the protein. Rab6a/a’, collectively referred to as Rab6, is the most abundant Golgi Rab protein and localizes to the trans-Golgi cisternae and TGN membranes. Neither depletion nor overexpression of GDP-locked Rab6 results in disruption of the Golgi ribbon and in epistasis experiments, co-depletion of Rab6 suppresses ZW10/RINT1- or COG-dependent Golgi disruption. By electron tomography, Rab6 depletion induces an accumulation of COPI- and clathrincoated vesicles that is accompanied by an increase in Golgi cisternal number and cisternal continuity. In sum, Rab6 may be more important to Golgi associated membrane trafficking than to Golgi organization. In striking contrast, both Rab41 depletion and overexpression of GDP-locked Rab41 cause Golgi fragmentation into a cluster of punctate elements, suggesting that Rab41 has an active role in maintenance of Golgi ribbon organization. Here, our goal has been to identify the Rab41 effectors producing this phenotypic contrast. To identify how Golgi organization is regulated by Rab41, we screened for Rab41 effectors using the yeast two-hybrid assay. Approximately 5.6×106 yeast transformants were screened from which 265 colonies were isolated under stringent conditions. Following PCR reactions and restriction digestion, 155 non-repetitive hits were isolated and sequenced. 8 putative Rab41 membrane trafficking or vesicle transport effectors were identified. Significantly, none of the 155 hits overlapped with any published Rab6 effector. Syntaxin-8, dynactin subunit 6 and kif18A were selected for further study as candidate effectors of Golgi organization and trafficking. Comparative yeast two-hybrid experiments indicate a preferential interaction of syntaxin-8, dynactin subunit 6 and kif18A with GTP- versus GDP-locked Rab41. Co-immunoprecipitation experiments are in progress to test the interactions of these putative Rab41 effectors with GTP- versus GDP-locked Rab41. More importantly, our studies show that, if dynactin subunit 6 or syntaxin-8, but not kif18A, were depleted using siRNA, the Golgi apparatus displayed a Rab41 knockdown phenotype, i.e., the Golgi apparatus was disrupted into a cluster of punctate Golgi elements. This strongly suggests that, in contrast to Rab6, Rab41 may act directly to regulate Golgi organization through dynactin/dynein recruitment and and indirectly through syntaxin-8 dependent membrane trafficking. Supported in part by NIGMS grant R01 GM092960

TUESDAY-POSTER PRESENTATIONS

P2098 Board Number: B621

A Trans-Golgi Specific Delay in Cargo Transport Is Sufficient to Explain Increased Golgi Cisternal Number in Rab6-Depleted Cells: A Test of a Delayed Cisternal Progression Model. L. Dickson1, S. Liu1, B. Storrie2; 1 Univ Arkansas Med Scis (UAMS), Little Rock, AR, 2Physiology and Biophysics, Univ Arkansas Med Scis (UAMS), Little Rock, AR Rab6 is the most abundant and studied Golgi-associated Rab protein, and is located predominantly at the exit or trans side of the Golgi apparatus. Knockdown of Rab6 results in a selective dilation of the trans-Golgi network (TGN), an accumulation of trans/TGN proximal COPI- and clathrin-coated vesicles, and an increase in Golgi cisternal number1. These changes in Golgi organization are accompanied by a delay in cargo transport between the Golgi apparatus and plasma membrane1. We hypothesized that this phenotype could be the outcome of a trans-Golgi/TGN specific delay in COPI-dependent Golgi recycling and hence a delay in cisternal progression. Here, we tested a key prediction of this model, namely, there should be a trans-Golgi specific delay in cargo transport through the organelle. Taking VSV-G-GFP as a model cargo protein, we determined the kinetics of cargo transport relative to cis, medial, trans and TGN46 markers using confocal fluorescence line scanning. Our results showed that cargo in Control and Rab6-depleted cells moved at a similar rate through the initial Golgi compartments (cis and medial). Cargo exit from both the medial Golgi and TGN occurred with similar rapidity, ~3 min. However and importantly, VSV-G transport between the medial Golgi and TGN was selectively delayed, 30 min versus 10 min for Control. Co-localization experiments with trans Golgi markers GalT and SialylT show that the delay was trans Golgi-specific. Taking the delay in cargo transport as an indicator of delayed cisternal progression, we propose that the observed increase in cisternal number with Rab6 knockdown is the predictable consequence. Experiments that test whether the “inserted” Golgi cisternae are trans-specific by Immunogold labeling are in progress. Results are being further validated by biochemical assays of protein glycosylation. Supported in part by NIH grant R01 GM 092960 and a minority student supplement. 1. Brian Storrie, Massimo Micaronii, et al. ”Electron tomography reveals Rab6 is essential to the trafficking of trans-Golgi clathrin and COPI-coated vesicles and the maintenance of Golgi cisternal number”. Traffic 13(5): 727-44.

TUESDAY-POSTER PRESENTATIONS

P2099 Board Number: B622

Quantitative analysis of Golgi size changes during interphase in mammalian cells. A. Sin1, R.E. Harrison1; 1 Department of Biological Sciences, University of Toronto Scarborough, Toronto, ON During the cell cycle, organelles and genetic materials need to be duplicated to ensure there is sufficient cellular content for daughter cells. As the Golgi complex plays an essential role in the biosynthetic pathway for all living cells, a fundamental question in cell biology is whether Golgi duplication occurs in mammalian cells. Although this process has been studied in parasitic protozoa, yeast, plants and insects, findings from such research may not be directly applicable to mammalian cells due to the substantial differences in Golgi morphology amongst the species. As careful, extensive research has already been performed on Golgi dispersal and inheritance during mitosis in mammalian cells, the aim of our study is to understand the growth of the mammalian Golgi complex prior to mitosis and elucidate the underlying mechanisms that control this process. Through the use of flow cytometry, we have shown that the mammalian Golgi complex has increased structural protein and enzyme contents as the cell cycle progresses, doubling its levels at late G2. Through 3D reconstruction and volumetric analyses after confocal microscopy, it is observed that the cis-, medial- and trans- cisternae concomitantly double in volume at late G2. To investigate the possibility of cell cycle control on Golgi growth, pharmacological agents were used to prolong specific cell cycle phases for 24 and 48 hours. Our results show that synchronized cells had significantly higher Golgi protein levels than those unsynchronized suggesting a lack of checkpoints that prevent indefinite Golgi growth. Interestingly, cell cycle synchronized cells were significantly larger in cell size than those unsynchronized establishing a correlation between Golgi size and cell size. We aim to further investigate the connections between these parameters in our future studies.

P2100 Board Number: B623

Monoubiquitination regulates Golgi membrane dynamics in the cell cycle. X. Zhang1, S. Huang1, Y. Wang1; 1 Dept. of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process. Several converging lines of evidence have suggested that monoubiquitination plays an essential role in the regulation of post-mitotic Golgi membrane fusion. Monoubiquitination, as a regulatory signal, occurs during mitotic Golgi disassembly and is required for subsequent Golgi reassembly. The AAA ATPase p97 and its adapter protein p47 are involved in membrane fusion during post-mitotic Golgi reassembly. The p97/p47 complex binds to monoubiquitin through the UBA domain of p47, and this interaction is required for p97-mediated Golgi membrane

TUESDAY-POSTER PRESENTATIONS fusion. Proteasome activity is not involved in either Golgi disassembly or reassembly. We have identified a Golgi-localized ubiquitin E3 ligase HACE1 which is involved in mitotic Golgi disassembly; reduced HACE1 activity is human cancer such as Wilms’ tumor results in fragmented Golgi. We have also discovered the p97/p47 binding protein VCIP135 as a deubiquitinating enzyme whose activity is required for post-mitotic Golgi reassembly. VCIP135 activity is regulated by phosphorylation in the cell cycle; it is inactivated by phosphorylation in metaphase and activated upon dephosphorylation in telophase. Recently, we have identified the substrates on the Golgi membranes whose ubiquitination and deubiquitination are regulated by HACE1 and VCIP135 in the cell cycle. These data suggest that cycles of addition and removal of ubiquitin to and from substrates regulate Golgi membrane dynamics during cell division.

P2101 Board Number: B624

Essential role of Golgi-derived microtubules in Golgi positioning and function at the mitotic entry. M. Fomicheva1,2, D. Yampolsky 1, K. Frye1, V. Magidson3, E. Kolobova1, E. Smirnova2, A. Khodjakov3, X. Zhu1, I. Kaverina4; 1 Vanderbilt University medical center, Nashville, TN, 2Lomonosov Moscow State University, Moscow, Russia, 3Wadsworth Center, Albany, NY, 4Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN In interphase vertebrate cells, the microtubule (MT) minus end-directed motor dynein localizes to Golgi membranes and organizes them into a single complex in the proximity of the centrosome. This mechanism is thought to facilitate proper partitioning of the Golgi membranes in mitosis, and Golgi complex reassembly in the daughter cells. Yet, the exact mechanism(s) responsible for segregation of the Golgi during cell division remain unclear. It has been proposed that the Golgi membranes split into two groups and move apart with the separating centrosomes during transition from G2 to mitosis. This view has been recently challenged by the finding that dynein dissociates from the Golgi in late G2, breaking the connection to the centrosome. To discriminate these conflicting ideas, we followed the movements of the centrosome and Golgi in human cells via high-resolution multi-dimensional microscopy, and assessed the distribution of the Golgi membranes by a newly-developed computational approach. We found that late in G2, Golgi stacks redistributed independently of the centrosome separation. However, Golgi stacks did not scatter randomly in the cytoplasm, but slid alongside the nuclear envelope approaching even distribution around the nucleus. Furthermore, we detected extensive nucleation of MTs at the Golgi at this cell cycle stage. Specific elimination of Golgi-associated MT nucleation by a number of alternative approaches prevented Golgi redistribution around the nucleus, indicating that Golgi-derived MTs were essential for this process. We also found that Golgi redistribution required recruitment of dynein to the nuclear envelope, which normally occurs in late G2/prophase. Together, our data indicate that redistribution of the Golgi stacks at the G2-mitosis transition is driven by nuclear envelope-localized dynein that acts along the Golgi-derived MTs. A

TUESDAY-POSTER PRESENTATIONS corollary of this mechanism is that Golgi-derived MTs are responsible for equal segregation of Golgi membranes between the daughter cells. Indeed, we found that Golgi distribution in proliferating cell populations became irregular upon depletion of Golgi-derived MTs. Additionally, we propose that the described mechanism of Golgi redistribution in prophase serves to properly position Golgi-associated membrane coat protein complexes (COPI), which are essential for nuclear envelope breakdown.

P2102 Board Number: B625

Differential Expression, Sorting and Segregation of Golgi Localized Proteins during Germ Cell Differentiation in the Testis. L. Hermo1, C.E. Au1, E. Byrne1, J. Smirle1, A. Fazel1, P. Simon1, R. Kearney1, P. Cameron1, C. Smith1, H. Vali1, J. Fernandez-Rodriguez1, K. Ma1, T. Nilsson1, J.J. Bergeron1; 1 McGill University, Montreal, QC Germ cell differentiation in the testis is accompanied by dramatic changes in Golgi apparatus structure and function. These coincide with mitosis of spermatogonia, meiosis of spermatocytes, acrosome formation by early spermatids, and Golgi migration in late spermatids undergoing extensive changes in plasma membrane and cell shape remodeling. Protein based mechanisms are lacking since the protein complement of germ cell Golgi apparatus during these modifications is unknown. To address this, abundant proteins were characterized quantitatively by tandem mass spectrometry of germ cell Golgi fractions isolated from adult rat whole testis homogenates. From 1318 proteins assigned to 22 functional categories, antibodies were generated or obtained to 20 of these proteins. The most abundant was GL54D, a protein of unknown function not previously uncovered but shown here to be a Golgi localized type II integral membrane glycoprotein and germ cell specific. A further protein of unknown function, TM9SF3, was a new Golgi marker for both somatic and germ cells, with 2 other TM9 protein family members predicted as Golgi markers based on extensive organelle sub-fractionation. Besides GL54D and TM9 family members, there were 240 characterized proteins of unknown function enriched in germ cell Golgi fractions. During acrosome formation, different Golgi localized proteins were segregated to Golgi apparatus or were shared with the forming acrosome. The former retained Golgi identity as the Golgi apparatus migrated away from the acrosome after its formation with the latter Golgi proteins remaining trapped in the acrosome. Based on the maturation model for acrosome formation, such segregation is unexpected. During acrosome formation, a subset of Golgi localized molecular chaperone and protein-folding enzymes were uncovered to handle the enormous biosynthetic load. Spermatocytes and spermatids during and after acrosome formation were associated with specific Golgi proteins along with others expressed throughout differentiation. Also unexpected was the finding of a burst and selectivity of expression of Golgi localized proteins to the terminal step 19 spermatid enriched in unstacked flattened cisternae of previously unknown function. When the selective expression and localization of the 20 Golgi localized proteins were compared with 30 proteins characteristic of PM, endosome, ER, coat and cytosol, organelle based molecular signatures for all 63 morphologically distinct germ cells were uncovered. In this way, a marker-based resource with insight

TUESDAY-POSTER PRESENTATIONS from the cognate protein functions has been established to uncover new mechanisms and proteins in Golgi membrane trafficking and fate during differentiation.

P2103 Board Number: B626

Golgi defects in Alzheimer’s disease. G. Joshi1, Y. Chi1, Z. Huang1, Y. Wang1; 1 Dept. of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI Golgi fragmentation occurs in neurons of patients with Alzheimer’s disease (AD), but the underlying molecular mechanism causing the defects and the subsequent effects on disease development remain unknown. In this study, we examined the Golgi structure in APPswe/PS1∆E9 transgenic mouse and tissue culture models. Our results suggest that Aβ accumulation leads to Golgi fragmentation by activating cdk5, which in turn phosphorylates GRASP65 and perhaps other key proteins critical for maintaining Golgi morphology. Significantly, rescue of Golgi structural defects by inhibiting cdk5 or by expressing nonphosphorylatable mutants of GRASP65 reduces Aβ secretion by elevating nonamyloidogenic APP cleavage. These results reveal Golgi fragmentation as an important mechanism through which Aβ may exert its toxic effects. A major potential unrecognized source of Aβ toxicity may be that it compromises Golgi integrity and perturbs the proper trafficking and processing of many proteins essential for neuronal function. We hypothesize that in AD, Aβ accumulation promotes Golgi defects, which in turn accelerate APP trafficking and Aβ production; this deleterious feedback circuit would impair the integrity of the secretory pathway and thereby compromise neuronal cell function. Our study provides a molecular mechanism for Golgi fragmentation and its effects on APP trafficking and processing in AD, suggesting Golgi as a potential drug target for AD treatment.

P2104 Board Number: B627

Blocking iron release alters the behavior of transferrin-containing endosomes in epithelial cells. A. Das1, M.M. Barroso1, A.B. Mason2, M.J. Gastinger3; 1 Cardiovascular Sciences, Albany Med Coll, Albany, NY, 2Biochemistry, University of Vermont School of Medicine, Burlington, VT, 3Bitplane Inc., South Windsor, CT Intracellular iron transport occurs by the binding of two iron molecules to transferrin, which is then internalized upon binding to the transferrin receptor and subsequent clathrin-mediated endocytosis. Upon endosomal acidification, iron is released from the transferrin-transferrin receptor complex and is finally taken up by the mitochondria, which is the principal site of iron-sulfur clusters synthesis and iron incorporation into haeme group. However, the mechanism at the basis of the endosomal iron transfer to mitochondria remains poorly understood. Recently, it has been established that iron can be

TUESDAY-POSTER PRESENTATIONS transported via a direct interaction (“kiss and run”) between transferrin-containing endosomes and mitochondria. To characterize these “kiss and run” events we have employed time-lapse live-cell TIRF microscopy to simultaneously track fluorescently labeled transferrin-containing endosomes and mitochondria in epithelial cells. Spatio-temporal endosomal parameters such as endosomal speed and distance from mitochondria have been derived using the image analysis software Imaris. We found that a transferrin-containing endosome generally shows significantly reduced speed during its interaction with the mitochondrion (the “kiss” phase) and increased speed during the “run” step of the “kiss and run” event. The role of iron release from transferrin on the behavior of transferrin-containing endosomes and on their ability to interact with mitochondria was investigated using a single point transferrin mutant, which cannot release iron during endocytic acidification. Blocking iron release increased the half-life of the “kiss” phase of transferrin-containing endosome-mitochondria interactions to 416ms compared to 216ms when using wild-type transferrin. Interestingly, we also found that blocking iron release leads to significantly higher endosomal speeds of mutant transferrin containing endosomes (mean speed = 0.58µm/s) than that of wild-type transferrin endosomes (mean speed = 0.46µm/s). These results suggest that blocking iron release affects endosome-mitochondria interactions resulting in longer interactions with mitochondria as well as increased endosomal speed of transferrincontaining endosomes. In order to address the mechanism underlying the effect of iron release on endosomal speed, we will investigate the role of intra-endosomal milieu such as pH, or extra-endosomal cytoskeletal elements such as actin and tubulin. In the future, we also plan to study the functional relevance of transferrin-containing endosomes and mitochondrial “kiss and run” interactions in cancer cell lines owing to their naturally enhanced iron requirements, altered endocytic pathways, and mitochondrial metabolisms.

P2105 Board Number: B628

The unconventional Myosin Vc is crucial for the transport of cargoes and Rab32/38 from early/recycling endosomes to maturing platelet dense granules in megakaryocytes. F. Martin1, J. Bultema2, S.M. Di Pietro3; 1 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, 2 University of Colorado, Colorado Springs, CO, 3Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO Mutations of Rab38 in mice and rats result in phenotypes consistent with the mouse models of Hermansky Pudlak Syndrome (HPS), characterized by pigmentation deficiencies and bleeding disorders. HPS is caused by defects in the biogenesis of melanosomes and platelet dense granules, two lysosome related organelles (LROs). Platelet dense granule biogenesis takes place in megakaryocytes and depends upon Rab38 and its close homolog Rab32 for sorting and transport of cargoes to the maturing organelle. We initially identified Myosin Vc (Myo5c) as a protein that directly interacts with Rab32 and Rab38, and characterized its involvement in melanosome biogenesis. Here we show that disruption of the

TUESDAY-POSTER PRESENTATIONS Rab32/Rab38/Myo5c interaction results in sequestering of Rab32, Rab38 and platelet dense granule cargoes into enlarged early endosomal structures. These structures are marked by Rab5; participate in recycling pathways as demonstrated by co-localization with the transferrin receptor; and appear to be “locked” into a dead-end state as evidenced by fluorescent live-cell microscopy. Characterization of Rab32/38 constitutively active and inactive mutants revealed that the interaction with Myo5c is GTPdependent. Knockdown of Myo5c function using a dominant negative (DN) Myo5c construct consisting of the coiled-coil and globular tail region showed that transport of Rab32 and Rab38 to maturing dense granules is dependent upon wild type Myo5c. Further, Myo5c is absolutely required for transport of cargoes to dense granules as revealed by the failure of known dense granule components (VMAT2, SLC35D3) to co-localize to dense granules in cells transfected with DN Myo5c. Last, we investigated the relationship with another Rab32/38 binding partner involved in LRO biogenesis, Varp. Our experiments show that wild type Myo5c is anti-correlated with the Rab32/38 effector and endocytic recycling protein Varp; however, knockdown of Myo5c function using DN Myo5c results in sequestering of Rab32/Rab38, Varp, dense granule cargos and DN Myo5c at early/recycling endosomes. We propose a handoff mechanism for Rab32/38 marked vesicles from Varp at early endosomes to Myo5c for subsequent transport and delivery to maturing platelet dense granules.

P2106 Board Number: B629

VAMP7 trafficking in melanosome biogenesis: tubular transport into and out of melanosomes. M.K. Dennis1, C. Delevoye2, A. Acosta-Ruiz1, G.G. Hesketh3, P. Luzio4, D.J. Owen4, G. Raposo2, M.S. Marks1; 1 Pathology and Laboratory Medicine, University of Pennsylvania Children's Hospital of Philadelphia, Philadelphia, PA, 2Institut Curie, Paris, France, 3Lunenfeld-Tanenbaum Research Institure, Mt Sinai Hospital, ON, 4Cambridge Inst Med Res, Cambridge, England Melanosomes are lysosome related organelles (LROs) that coexist in pigmented cells alongside classical endolysosomes. Melanosomal membrane proteins are directed towards maturing melanosomes via membranous transport carriers derived from early endosomal domains, but neither the machinery that mediates fusion of the transport carriers with melanosomes nor a defined pathway for recycling of machinery components to the site of origin for additional rounds of transport are known. To address these unknowns, we focused on VAMP7 as a candidate vSNARE for melanosome transport. VAMP7 has been implicated in melanosome biogenesis because it localizes in wild-type melanocytes primarily to melanosomal membranes at steady state (unlike other endosomal VAMPs), and because depletion of VAMP7 results in mistrafficking of melanosomal cargoes and hypopigmentation. To determine the pathway through which VAMP7 is targeted to melanosomes, we first assessed the subcellular localization of GFP-VAMP7 in melanocytes from mouse models of Hermansky-Pudlak syndrome, in which melanosomal maturation is impaired. Whereas GFP-VAMP7 localized nearly exclusively to pigmented melanosomes in wild type melanocytes, it overlapped nearly completely with syntaxin 13

TUESDAY-POSTER PRESENTATIONS (STX13) in early endosomes of BLOC-1-deficient cells, analogous to melanosome cargoes like TYRP1. Upon transient rescue of BLOC-1-deficiency, GFP-VAMP7 exited endosomes in mCherry (mCh)-STX13labeled tubules, and accumulated in nascent STX13-negative structures with features of early stage melanosomes. This suggests that VAMP7 is targeted to melanosomes in STX13-containing endosomal tubules towards maturing melanosomes and is likely the vSNARE on the BLOC-1-dependent cargo transport pathway. To assess VAMP7 recycling from melanosomes, we exploited live cell imaging of tagged VAMP7 in wild-type melanocytes. A second population of tubules labeled by GFP-VAMP7 but not by mCh-STX13 were frequently detected, often emerging from GFP-VAMP7-containing melanosomes. VARP, a scaffolding protein that maintains VAMP7 in an inactive conformation, was largely localized at steady state to puncta that were associated with melanosomes and that were enriched in the tissuerestricted RAB protein, RAB38. By live imaging, tubules labeled by VARP-GFP overlapped with those labeled by mCh-VAMP7 and were frequently detected departing from melanosomes. As expected for recycling structures, these tubules lacked melanosomal cargoes such as TYRP1 and were discrete from the anterograde STX13-positive tubules. These data support a role for VARP in recycling VAMP7 from melanosomes following cargo delivery. Because VARP is a RAB38 effector and overlaps with RAB38 on melanosomal domains, we speculate that RAB38 also functions on this recycling pathway.

P2107 Board Number: B630

A cluster of isolation membrane-associated tubules represents a part of omegasome during final steps of autophagosome formation. S. Waguri1, T. Uemura1, M. Yamamoto1, A. Kametaka1, Y. Sou2, A. Yabashi1, A. Yamada1, H. Annoh1, S. Kametaka1, M. Komatsu2; 1 Dept of Anatomy and Histology, Fukushima Med Univ Sch of Med, Fukushima, Japan, 2Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Recent findings have suggested that autophagic isolation membrane (IM) originates from a domain of endoplasmic reticulum (ER) called “omegasome”. However, its fine structure and detailed positional relationships to the ER and IM during autophagosome formation remain unclear. In the present study, we used Atg3-deficient mouse embryonic fibroblasts (MEFs) expressing a marker of omegasome, GFPtagged double FYVE domain-containing protein 1 (GFP-DFCP1), and found that GFP-DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims by correlative light and electron microscopy and immuno-electron microscopy. Moreover, we developed a fixation protocol for electron microscopy (EM) using a mixture of paraformaldehyde, glutaraldehyde, and osmium tetroxide as a primary fixative, for clear-cut detection of IM and associated vesicular or tubular structures. By EM analyses including serial ultra-thin sections and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. These IM-associated tubular structures (IMATs) were observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1, but not in FIP200-deficient cells, suggesting that they are relevant to earlier events in

TUESDAY-POSTER PRESENTATIONS autophagosome formation. Taken together, our findings indicate that IMATs represent a part of omegasome during completion steps of autophagosome formation.

P2108 Board Number: B631

Phylogenetic conservation and subcellular localization of apicomplexan SNAREs. D. Ouologuem1, D.S. Roos1; 1 University of Pennsylvania, Philadelphia, PA Apicomplexan parasites are highly polarized eukaryotic cells, with an endomembrane system that includes a minimal ER and Golgi apparatus, and a multitude of distinctive membranous organelles. Dense granules, micronemes and rhoptries are secretory organelles associated with parasite-specific functions, including motility, host cell attachment and invasion, and establishment of a specialized niche for survival within the infected host cell (the parasitophorous vacuole). The Inner Membrane Complex (IMC) consists of a patchwork of Golgi-derived flattened vesicles underlying the parasite plasma membrane and associated with a cytoskeletal scaffold; this assemblage is required for partitioning parasite organelles during cell division. In order to further understand the organellar biogenesis and trafficking, we have identified and partially characterized Toxoplasma and Plasmodium SNARE proteins (soluble N-ethylmaleimide-sensitive factor adaptor protein receptors). 25 SNAREs are predicted from the T. gondii genome, and 24 from the P. falciparum genome, and these proteins can be grouped into conventional R/Qabc-SNARE subclasses. Phylogenetic reconstruction, comparison with wellcharacterized human and fungal SNAREs, and subcellular localization permits identification of clear orthologs for most SNAREs involved in the early secretory pathway (ER/Golgi/TGN trafficking), but not those associated with trafficking to the plasma membrane, lysosomes, etc. Several unusual SNAREs were also identified in the Apicomplexa, including some that localize to the parasite plasma membrane, rhoptries, or uncharacterized cytoplasmic vesicles. This study highlights the divergence of the late secretory pathway in apicomplexan parasites, and provides a map of SNAREs likely to participate in trafficking to both conserved and distinctive organelles.

P2109 Board Number: B632

New Automated Methods to Quantitate Lipid Droplet Number, Fluorescence and Total Volume from Fluorescence Images. S. Dejgaard Yilmaz1, J.F. Presley2; 1 Department of Anatomy and Cell Biology, McGill University, Montreal, QC, 2McGill University, Montreal, QC Lipid droplets are the major organelle for intracellular storage of triglycerides and cholesterol esters. Generally, automated segmentation and quantitation of lipid droplets in fluorescence images of cells has

TUESDAY-POSTER PRESENTATIONS relied on thresholding-based or watershed methods. We describe here a novel three-stage hybrid method in which candidate lipid droplets are initially identified by thresholding and then tested for circularity. Circular structures are accepted immediately as lipid droplets, while non-circular structures are considered candidate clusters, which are segmented using a watershed algorithm. A unique feature of this method is that the circularity measure can be used as a quality control for the overall segmentation of either stage. The same method can, in principle, be applied with modifications to the estimation of the total lipid droplet volume within a cell. However, as the volume of smaller lipid droplets are difficult to determine accurately by light microscopy, we apply an alternate approach of automatically identifying and fitting the volume of large lipid droplets in calibration images taken parallel to experimental images and identically stained with volume dyes (e.g., Nile Red or Bodipy) and imaged. We describe quantitatively the accuracy of determination of volume of lipid droplets using this method, with respect to factors that must be taken into account, including the degree of reproducibility of staining, the effects of variation in lipid droplet composition and effects of lipid droplet size with respect to several common dyes.

P2110 Board Number: B633

Rapid color changes in the chameleon sand tilefish, Hoplolatilus chlupatyi. M. Goda1, Y. Fujiyoshi1; 1 Nagoya Univ, CeSPI, Nagoya, Japan The chameleon sand tilefish, Hoplolatilus chlupatyi, inhabits areas off the coast of Philippines and Okinawa islands in Japan at the depth of 30 to 70m below the surface of the sea. It is well known to hobbyists, scuba divers and zoologists that color of the trunk of the sand tilefish changes from blue to red within less than one second. For this reason, this species also called “flashing tilefish”. In this study, our optical and electron microscopic observations revealed that the coloration and color changes of the chameleon sand tilefish were generated by interference phenomenon of incident light occurred in dermal iridophores. The cytoplasm of the iridophores contained stacked, very thin reflecting platelets, resembling damselfish-type iridophores, but the platelets were arranged and distributed in the limited area nearby the cell membrane. The color changes of the sand tilefish are inordinately fast for fish iridophores, which have been reported to take several seconds or minutes. To investigate the motile activities of the iridophores, we performed pharmacological examinations with scales from trunk of the chameleon sand tilefish. An elevation of K+ ions in the perfusing medium gave rise to rapid color changes of the iridophores from blue to red. While acetylcholine did not influence the state of the iridophores, norepinephrine aroused color changes toward reddish color very effectively. The action of norepinephrine on the iridophores was effectively blocked by alpha-adrenergic blocking agent, phentolamine. These results suggest that adrenergic stimulation is a cue to control the motile activities of the iridophores of the chameleon sand tilefish.

TUESDAY-POSTER PRESENTATIONS

P2111 Board Number: B634

The Keap1-Nrf2 redox pathway alters mitochondrial morphology through induced proteasomal degradation of the mitochondrial fission protein Drp1. T. Shutt1, M. Geoffrion2, R. Screaton3, R. Milne2, H.M. McBride4; 1 Medical Genetics, University of Calgary, Calgary, AB, 2University of Ottawa Heart Institute, Ottawa, ON, 3 University of Ottawa, Ottawa, ON, 4McGill University, Montreal, QC Mitochondria, best known for their ability to generate both energy and reactive oxygen species also play important roles in many cellular functions. Various signals can affect mitochondrial morphology by altering the balance between fission and fusion events, ultimately influencing metabolic output and sensitivity to cell death. For example, mitochondrial hyperfusion occurs rapidly following several types of cellular stress, in a process described as Stress Induced Mitochondrial Hyperfusion (SIHM). Mitochondria with a hyperfused morphology are refractive to mitochondrial autophagy and provide protection against apoptotic triggers, suggesting that SIHM may play an important protective role during the early stages of cellular stress. This study examines mechanisms regulating mitochondrial shape in response to oxidative stress. We recently demonstrated a role for oxidized glutathione in the direct activation of the mitochondrial fusion machinery, providing a mechanistic link between oxidative stress and fusion. However, questions remain about the persistence of the fused mitochondrial state and whether fission may also be negatively regulated. As part of a genome-wide screen to find novel factors modulating mitochondrial morphology, we identified Keap1. Under normal conditions, Keap1 promotes the ubiquitination and degradation of several target proteins, including the transcription factor Nrf2 (NFE2L2) a key regulator of the cell's antioxidant response. Given that Keap1 is a redox-regulated protein controlling the cellular response to stress and its knockdown results in a hyperfused mitochondrial network, we characterized this novel pathway using a combination of approaches. Although mitochondrial fusion was not activated with Keap1 siRNA, accumulation and nuclear translocation of Nrf2 resulted in a significant decrease in the levels of the mitochondrial fission protein Drp1, explaining the hyperfused mitochondrial network. We then showed that the transcription factor activity of Nrf2 influences mitochondrial morphology by promoting the expression of proteasome proteins and ultimately increasing the degradation of Drp1. Rather than the acute increase in mitochondrial fusion previously described in response to oxidative stress, this novel pathway occurs on a timescale of hours to days to prolong the hyperfused phenotype, thereby protecting the cell from apoptosis until the stress has been neutralized. In summary, we have identified a novel role for the Keap1/Nrf2 redox pathway in maintaining a fused mitochondrial network following cellular stress.

TUESDAY-POSTER PRESENTATIONS

P2112 Board Number: B635

A direct interaction between actin filaments and the dynamin-like GTPase Drp1 supports dual roles for the actin cytoskeleton during mitochondrial fission. A.L. Hatch1, H.N. Higgs2; 1 Dartmouth College, Hanover, NH, 2Dartmouth-Geisel Sch Med, Hanover, NH As a dynamic network, mitochondria routinely change morphology in response to different cellular demands and stresses. In this regard, mitochondrial fission and fusion serve several roles: to distribute mitochondria appropriately in dividing or polarized cells; working as a quality control mechanism to help eliminate damaged mitochondrial segments; and even as a component of apoptotic pathways. Defective mitochondrial fission/fusion is found in patients with neurological disorders such as CharcotMarie-Tooth disease (CMTD), Alzheimer’s, Huntington’s, and Parkinson’s disease. The dynamin-like GTPase Drp1 is a key component of the fission machinery that provides contractile force needed for mitochondrial fission, but a key unanswered question is how Drp1 gets recruited to the mitochondrial outer membrane (OMM). While a number of Drp1 “receptors” have been identified on the OMM, there is also evidence that a mitochondrial pre-constriction step is required prior to Drp1 action. We have shown that actin filaments contribute to this pre-constriction step through the formin INF2, additionally requiring myosin II. INF2 is an endoplasmic reticulum (ER)-bound actin polymerization factor, and mutations in INF2 lead to CMTD. Still, how do actin filaments and pre-constriction lead to Drp1 recruitment? Here, we show that Drp1 binds directly to actin filaments with high affinity. Drp1 binds evenly along the length of the actin filament (shown by electron microscopy), but the interaction is highly dynamic (shown by TIRF microscopy). Actin filament binding increases Drp1’s GTP hydrolysis activity, suggesting that actin binding promotes productive Drp1 oligomer assembly. In mammalian cells, Drp1 mutants defective in actin binding do not rescue mitochondrial fission defects induced by endogenous Drp1 suppression. From these results, we propose that actin filaments serve two roles during mitochondrial fission: 1) to provide the force for OMM pre-constriction; and 2) to recruit Drp1 at fission sites through direct binding. In the first role, we propose that actin filaments generate force for pre-constriction with myosin II in a manner similar to cytokinesis. In the second role, actin filaments act as a “co-incidence detector”, along with Drp1 receptors such as Mff, Fis1, MiD49, and MiD51. Coincidence detection of two signals (in this case actin filaments + a Drp1 receptor) is widely used in cells to tightly control activation of important processes. We further propose that other molecules, such as cardiolipin, may serve as independent co-incidence detectors for Drp1 in a similar manner to actin.

TUESDAY-POSTER PRESENTATIONS

P2113 Board Number: B636

Mitochondrial network topology in the absence of fission and fusion dynamics. M.P. Viana1, I.A. Mueller1, C. Goul2, S. Rafelski2; 1 Developmental and Cell Biology, University of California Irvine, Irvine, CA, 2Developmental and Cell Biology, University California-Irvine, Irvine, CA Mitochondria form interconnected networks that are constantly reshaped by fission and fusion dynamics. The underlying topological properties of these networks and how fission and fusion together quantitatively contribute to these properties are unclear. We applied an updated version of our MitoGraph method to quantify the topology of mitochondrial networks in budding yeast. We grew cells in microfluidic chambers with constant fresh media in such a way that the cells stopped dividing but continued to grow. This experimental setup permits mitochondrial networks to be at a steady state; because cells are no longer dividing, we avoid effects of the cell being polarized during cell division. We grew cells both in respiring and non-respiring conditions. We found that the number of networks edges and the total mitochondrial content, expressed in terms of the network length, are strongly correlated in both respiring and non-respiring media. This suggests that there is a typical length for edges in mitochondrial networks, similar to what is observed in other types of planar networks. We also investigated the mitochondrial network topology of Δfzo1Δdnm1 yeast cells, which lack the main proteins responsible for mitochondrial fission and fusion. Surprisingly, although networks in these fission/fusion double mutant cells look different by eye, we found no clear topological differences in non-respiring conditions. The only difference we found was that the surface density of mitochondrial tubules was greater in the fission/fusion double mutant cells in respiring conditions and so was their average interconnectivity. To identify the key rules required to generate the topology of mitochondrial networks we simulated many artificial networks generated by different sets of rules and then compared them to the experimentally measured ones. We found two features critical for generating realistic mitochondrial networks both for wild-type and fission/fusion double mutant cells. First, the networks must consist of nodes with only one or three neighbors in a given proportion and second, nodes that are closer to each other in space must be preferentially connected. We conclude that cells in the absence of fission and fusion still generate mitochondrial networks with very similar basic topological properties to wild-type cells, suggesting that there may be processes beyond fission and fusion that determine how mitochondrial networks are generated in yeast cells. We are now using more sophisticated measurements to determine how the visual differences between wild-type and double mutant cells arise.

TUESDAY-POSTER PRESENTATIONS

P2114 Board Number: B637

High-Throughput drug discovery of novel inhibitors of secretion. A. Satoh1, H. Suzuki1, Y. Honjo2, Y. Nishina1; 1 Okayama Univ, Okayama, Japan, 2Hiroshima University, Hiroshima, Japan Finding new candidate molecules with medicinal properties normally depends on serendipity or investment of time and resources. For example, massive chemical library screenings have successfully identified several new drug candidates. To increase the chances of finding such candidates, we used pooled substrates as starting materials. The pools of substrates, respectively consisting of 11 chemicals and 1 catalyst, underwent multiple chemical reactions, resulting in pools of reactants. These pools were tested in cell-based biological assays for cytotoxicity and secretion. One of the pools was not toxic but exhibited secretion inhibition; therefore, we isolated a compound from the pool showing inhibitory activity. As expected, the compound, termed bis-indole 45, disrupted the Golgi without affecting microtubules. Bis-indole 45 was then compared with known secretion inhibitors, Brefeldin A (BFA) and Golgicide A (GCA), in the following tests. BFA and GCA inhibit secretion by inhibiting the function of an Arf-GEF, GBF1, upon freezing Arf and GBF1 on membranes. Bis-indole 45, however, did not exhibit such an effect. GBF1 recruitment to the Golgi membrane is thought to be mediated by the golgin tether p115 and the small GTPase Rab1. Exogenous expression of a dominant negative mutant of Rab1 but not p115 disrupted GBF1 localization to the Golgi in a manner similar to bis-indole 45. These results suggest that bis-indole 45 inhibits secretion by inhibiting GBF1 recruitment to the Golgi through Rab1. As exemplified in this study, drug screening using pooled substrates can be a powerful and useful method for high-throughput drug discovery.

P2115 Board Number: B638

Quantifying Cell Membrane Morphology Changes with a Biophysical Membrane Model. R. Tourdot1, R. Bradley1, R. Natesan2, R. Radhakrishnan2; Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, 2Bioengineering, University of Pennsylvania, Philadelphia, PA 1

Organelle shapes are defining characteristics of different types of cells and can help regulate their function, with endosomes and the Golgi apparatus being examples of biological topologies with tightly controlled shape and curvature. A special subset of membrane-recruited proteins has recently been shown to sense and/or induce curvature in the bilayer: such proteins include BAR domain containing proteins and the ENTH domain containing proteins. Emerging research has revealed that these curvature-inducing proteins play a role in several critical cell processes ranging from their incorporation into endocytic pits, and localization to sites of invadopodia formation in vivo. Mathematical models relating membrane-protein interaction to membrane morphology can provide physical insight into the

TUESDAY-POSTER PRESENTATIONS driving forces governing curvature induction and morphological changes in the cell membrane. These models of cell membrane elasticity have been used previously to describe the biconcave shape of red blood cells, and large-scale morphological changes of the cell membrane such as cell division. Our work focuses on applying a sub-cellular (mesoscale) model based on the Helfrich formalism for cell membranes to a curvature inducing protein system, and utilizing it in concert with a suite of computational free energy methods to define the free energy landscape of cell-membrane morphology changes. In particular, our model provides novel insight into the critical density of proteins needed for membrane tubulation in invadopodia formation, and the interplay between local tension and curvature generation in vesicle nucleation in endocytosis. Our research finds that protrusions are considerably longer and more stable under low tensions, and these results corroborate recent experimental results comparing cells cultured in 2D and 3D media, where cells in 3D matrices are found to have lower tension and more prominent cell protrusions.

P2116 Board Number: B639

Understanding the factors regulating size and shape of dynamic organelle: an holistic approach. D. Bhattacharyya1,2, M. Bhave1; 1 Tata Memorial Centre, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Kharghar, Navi Mumbai, India, 2Neurobiology, University of Chicago, Chicago, IL Size and shape control mechanisms of dynamic organelles are of fundamental importance in basic cell biology. More over several such organelles are known to alter their size and shapes during normal physiological processes as well in diseases especially in cancerous cells and this underlies the importance of the subject. We are presently studying the Golgi apparatus and Nucleus size and shape control mechanism using a combination of cell biology and mathematical modeling. Recently we have shown that the size control mechanism of Golgi apparatus possibly being regulated by altering the cisternal maturation kinetics by ARF1 in budding yeast. Using a genome wide screening approach we have identified several other factors affecting Golgi size. Combining advanced imaging techniques with mathematical simulation based methods we have identified few potential mechanisms through which such regulatory mechanisms may operate. Our results indicate that although size and shape control mechanism may differ for different organelles however there are definitive possibilities of mechanistic similarities in such processes.

TUESDAY-POSTER PRESENTATIONS

P2117 Board Number: B640

Nucleolar size scaling through C. elegans growth and development. S. Uppaluri1, S.C. Weber1, C.P. Brangwynne1; 1 Chemical and Biological Engineering, Princeton University, Princeton, NJ The nucleolus is a non-membrane bound organelle that is the site of ribosome biogenesis and is therefore strongly linked to protein translation and cell growth. Requirements for protein translation vary greatly during C. elegans development - from a single-celled embryo through four larval stages to adulthood. How nucleolar size and function varies in the context of multicellular growth remains poorly understood. We have shown that in the early reductive embryonic cell divisions, nucleoli scale with cell size. Upon hatching and through subsequent larval development we find again that nucleolar size scales with cell size. Altering growth rate, as well as ultimate cell size, through environmental and genetic perturbations leads to the same ‘universal’ scaling. To elucidate the biophysical mechanism governing nucleolar size, we utilize a custom microfluidic platform to instantaneously change cell volume and nucleolar component concentration. Consistent with a simple phase transition model we find that increasing organelle component concentration leads to larger nucleoli. These results point to a cell-size sensing mechanism through which organelle size is regulated.

P2118 Board Number: B641

A dynamin-like ER-shaping protein and FIT proteins regulate lipid droplet biogenesis in yeast. N. Ojha1, V. Choudhary1, W. Prinz1; 1 NIDDK, National Institutes of Health, Bethesda, MD Lipid droplets (LDs) are ubiquitous neutral lipid storage organelles. LD biogenesis occurs in the ER but is not well understood. FITs are a conserved family of proteins that have been implicated in LD biogenesis. Yeast has two FIT proteins, called Scs3p and Yft2p. We found that a mutant lacking Scs3p and the dynamin-like GTPase Sey1p have a dramatic growth defect. Sey1p is an orthologue of atlastins and facilitates ER-ER fusion. Cells missing Scs3p and Sey1p were found to have a significant increase in the number of lipid droplets and at least a two-fold increase in neutral lipids, suggesting that these cells may have altered LD biogenesis. Remarkably, we found that LDs in these cells were wrapped by a membrane. We ruled out that these membranes are derived from autophagy but instead are part of the ER. LDs in cells missing Scs3p and Sey1p appear to bud towards the lumen of the ER instead of into the cytosol, which may account for the aberrant membrane wrapping. Together, our findings suggest that Sey1p and Scs3p proteins regulate LD number and LD biogenesis, perhaps by facilitating LD budding from the ER.

TUESDAY-POSTER PRESENTATIONS

P2119 Board Number: B642

Analysis of mitochondrial proteins and their effect on mitochondrial dynamics in Dictyostelium discoideum. L. Woods1, B.G. Schimmel1, G.W. Berbusse1, K. Naylor1; Biology, University of Central Arkansas, Conway, AR

1

Mitochondrial morphology is maintained by two distinct membrane events -fission and fusion. Altering these conserved processes can disrupt mitochondrial morphology and distribution, thereby disrupting the organelle’s functionality and impeding cellular function. In higher eukaryotes, these processes are mediated by a family of dynamin-related proteins (DRP’s). In the lower eukaryotes, for instance Dictyostelium discoideum, mitochondrial fission and fusion have been implicated but not yet established. To understand the overall mechanism of these dynamics across organisms, we developed an assay to identify fission and fusion events and measure organelle velocity in Dictyostelium and to assess the involvement of the mitochondrial proteins, CluA, and two DRPs, DymA and DymB. Using laser scanning confocal microscopy to observe real time movement of fluorescently labeled Dictyostelium mitochondria, we show that lower eukaryotes mediate mitochondrial fission and fusion. In Dictyostelium, these processes are balanced, occurring approximately 1 event/minute. Quantification of the rates of fission and fusion in cluA-, dymA-, or dymB- strains established that the DRP’s are not essential for these processes. Rates of fission and fusion were significantly reduced in cluA- cells, indicating that CluA is necessary for maintaining both fission and fusion. Interestingly, loss of DymA significantly increases the velocity of organelle movement within the cell though it does not alter the number of mitochondria moving; while loss of CluA has no effect on velocity, it instead decreases the number of organelles that are motile. We have successfully demonstrated that Dictyostelium mitochondria undergo the dynamic processes of fission and fusion. The classical mediators of membrane dynamics, the DRP’s, are not necessary for these dynamics though DymA does affect motility, whereas CluA is necessary for both processes and decreases the percentage of motile organelles. This work contributes to our overall understanding of mitochondrial dynamics and ultimately will provide additional insight into the evolution of these processes.

TUESDAY-POSTER PRESENTATIONS

P2120 Board Number: B643

VAMP7 is a biogenesis of lysosome related organelles complex-1 (BLOC-1) cargo required for pigmentation in melanocytes. A. Acosta-Ruiz1, M.K. Dennis1, R.A. Linares1, C. Delevoye2, G. Raposo2, M.S. Marks1; 1 Pathology and Laboratory Medicine, University of Pennsylvania Children's Hospital of Philadelphia, Philadelphia, PA, 2Institut Curie, Paris, France Melanosomes are lysosome related organelles (LROs) within which melanins are synthesized and stored in pigmented cells. Resident proteins are delivered to nascent melanosomes from early endosomes via membranous transport intermediates in a process that requires Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1) - a protein complex comprised of subunits that are absent in a subset of patients with Hermansky-Pudlak syndrome. The machinery that mediates fusion of these intermediates with maturing melanosomes is not known. As potential fusion machinery components, we focused on VAMP7 and syntaxin13 (STX13), because depletion of either of these SNARE proteins results in hypopigmentation. In normal pigmented melanocytes at steady state, STX13 localizes to early endosomes and tubules that contact maturing pigmented melanosomes, and VAMP7 localizes primarily to the melanosomes themselves. We thus initially proposed a model wherein BLOC-1-dependent, STX13-positive, endosomal carriers fuse with VAMP7-positive early stage melanosomes. We first found that in BLOC-1-deficient pallid melanocytes, STX13-positive endosomal tubules fail to form, consistent with a critical role for STX13-positive endosomal carriers in protein delivery to melanosomes. However, in these cells a GFP-VAMP7 fusion protein was completely retained within mCherry-STX13-positive endosomes rather than in early stage melanosomes as predicted by our initial model; following "rescue" with myc-tagged Pallidin, GFP-VAMP7 localization was restored to newly pigmented melanosomes that segregated from mCherry-STX13-labeled endosomes. These data suggest that VAMP7 is in fact a cargo of the BLOC-1 pathway and of the STX13-containing transport intermediates. To further test this model, we utilized a novel assay to visualize early events in melanosome biogenesis without the confounding effects of predominant steady state localization to mature melanosomes. We transiently rescued BLOC1 expression in pallid melanocytes by transfection with myc-tagged Pallidin and monitored changes in the localization of coexpressed GFP-VAMP7 and mCherry-STX13 at various time points following transfection. As predicted by our model, the number of GFP-VAMP7-containing structures that segregated from mCherry-STX13-labeled structures increased over time. Moreover, at intermediate time points following transfection, GFP-VAMP7 could be observed by live cell imaging to traffic within mCherry-labeled tubules towards nascent VAMP7+ puncta. These data support a model wherein BLOC1-dependent endosomal carriers that contain STX13 and VAMP7 fuse with early stage melanosomes in a process required for pigmentation. We propose that VAMP7 functions as the vSNARE for this fusion event.

TUESDAY-POSTER PRESENTATIONS

P2121 Board Number: B644

ENDOPHILIN RAPIDLY BENDS MEMBRANES TO PROMOTE ENDOCYTOSIS. K.R. Poudel1, J. Bai1; 1 Fred Hutchinson Cancer Research Center, Seattle, WA Synaptic Vesicle (SV) endocytosis is responsible for generating synaptic vesicles at the presynaptic terminals. To sustain high-frequency brain activities, endocytosis must act rapidly to regenerate SVs, which prevents vesicles from depletion. Endophilin is a synaptic protein that plays critical role in promoting SV endocytosis. Endophilin contains a Bin–Amphiphysin–Rvs (BAR) domain that has been shown to bend flat membranes into highly curved tubules in vitro. Our recent findings demonstrate that the membrane-bending activity is required for endophilin’s function in vivo. Here, we perform biophysical analyses to investigate molecular mechanisms for endophilin to generate membrane curvature. Our preliminary results show that the endophilin BAR domain is an active membrane bender instead of a passive sensor. These results will help us to understand how endophilin acts to support SV endocytosis in neurons.

P2122 Board Number: B645

Three dimensional architecture of Extended-Synaptotagmin-mediated ER-plasma membrane contact sites. R. Fernandez-Busnadiego1, Y. Saheki1, P. De Camilli1; 1 Department of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT Growing evidence suggests that the close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca2+ homeostasis, signaling and lipid metabolism. The Extended Synaptotagmins (E-Syts/Tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM. E-Syt cytoplasmic domains comprise an SMP domain followed by five C2 domains in the case of E-Syt1 and three C2 domains for E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syts-mediated ER-PM contacts at molecular resolution. We show that, in vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, ER-PM distance (19-22 nm) correlates with the amino acid length of the cytosolic region of E-Syts (i.e. the number of C2 domains). We further show that the levels of cytosolic Ca2+ regulate ER-PM distance at ESyt1-dependent contacts sites. E-Syt-mediated contacts displayed a characteristic electron-dense layer between the ER and plasma membranes. In contrast, at STIM1-Orai1-mediated contacts the gap between ER and PM was spanned by filamentous structures perpendicular to the membranes. These two types of ER-PM contacts were also observed in the native context of untransfected neurons. Thus, our results define specific ultrastructural features of E-Syts-dependent ER-PM contacts and reveal their

TUESDAY-POSTER PRESENTATIONS structural plasticity, which may impact on specific E-Syt functions and the cross-talk between the ER and the PM.

Mitochondria 2 P2123 Board Number: B646

Regulated by mitochondrial fusion dynamics, lipid droplets serve as central fatty acid conduit to supply mitochondria with fatty acids for oxidation during nutrient stress. A. Rambold1, S. Cohen2, J. Lippincott-Schwartz2; 1 Max-Planck-Institute-IE, Freiburg, Germany, 2Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD Cells must adapt their metabolism to changing nutrient and environmental conditions in order to survive. One key adaptation occurs during nutrient deprivation when cells reprogram their metabolism from glucose to mitochondrial fatty acid oxidation for maintaining cellular energy levels. Although it is well known that endogenous fatty acids act as major energy substrate during nutrient stress, from where and how such fatty acids are mobilized and selectively trafficked to mitochondria without causing damage to membranes and acting as toxic bioactive lipids is a major question. Using a novel imaging based approach to visualize fatty acid trafficking in live cells in combination with metabolic profiling experiments, we revealed that lipid droplets, specialized lipid storage organelles, serve as selective conduit to direct fatty acids into mitochondria for oxidation. We found that fatty acid recycling by bulk autophagy of cellular membranes helped replenish lipid droplets with fatty acids, increasing lipid droplet number over time. From lipid droplets, fatty acids were subsequently liberated by cytoplasmic lipases, and efficiently transferred into mitochondria. Surprisingly, we found that this transfer step required mitochondria to both be localized near lipid droplets and highly fused. Mitochondrial fusion increased the connectivity to lipid droplets and promoted mitochondrial fatty acid distribution and oxidation. When mitochondrial fusion was prevented in cells lacking the major fusion proteins mitofusin 1 or optic atrophy protein 1, fatty acids neither homogeneously distributed within mitochondria nor became efficiently metabolized. Instead, these morphodynamic defects caused massive alterations in cellular fatty acid routing. Not only were non-metabolized fatty acids redirected to and stored in lipid droplets, they became excessively expulsed from cells. Together this study highlights the importance of coordinated organelle dynamics to direct cellular fatty acid flow and oxidation during nutrient stress. Given that fatty acids and their derivatives play important roles in regulating cellular signaling cascades in metabolism and inflammatory responses, future work studying the multi-organelle controlled fatty acid flux system described here might be of high potential for contributing to our understanding of the pathophysiology of lipid-associated diseases, such as obesity, diabetes, cancer and inflammatory disorders.

TUESDAY-POSTER PRESENTATIONS

P2124 Board Number: B647

Mastoparan causes mitochondrial permeability transition not by interacting with specific proteins, but by interacting with the phospholipid phase. T. Yamamoto1, M. Ito1,2, K. Kuwahara1,2, K. Kageyama1,2, K. Yamashita3, Y. Takiguchi2, H. Terada4, Y. Shinohara5,6; 1 Institute for Genome Research, University of Tokushima, Tokushima City, Japan, 2Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima City, Japan, 3School of Dentistry, University of Tokushima, Tokushima City, Japan, 4Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City, Japan, 5Faculty of Pharmaceutical Science, University of Tokushima, Tokushima, Japan, 6Institute for Genome Research, University of Tokushima, Tokushima, Japan Mitochondrial permeability transition causes the release of various proteins from mitochondria, and the released proteins trigger subsequent steps of apoptosis and necrosis. Permeability transition is believed to result from the formation of a proteinaceous pore, but the molecular mechanisms have not yet been revealed. The mastoparan peptide is a tetradeca-peptide isolated from the venom of wasp. Mastoparan is known as an inducer of the mitochondrial permeability transition. Although mastoparan was suggested to interact with a proteinaceous target in mitochondria to induce this transition, the action sites of mastoparan have not yet been investigated. To clarify whether specific interactions of mastoparan with receptors or enzymes are associated with the induction of this permeability transition, we here examined the effects of D-isomeric peptides, which were synthesized by using D-amino acids assembled in endogenous (inverso mastoparan) and reverse (retro-inverso mastoparan) orientations. When we added inverso mastoparan to isolated mitochondria, the peptide caused the permeability transition in a partially cyclosporin A-sensitive manner at lower doses and in a cyclosporin A-insensitive manner at higher ones. The action manners and the potencies of inverso mastoparan were close to those of parent mastoparan, indicating that the targets of mastoparan for induction of the permeability transition were neither receptors nor enzymes in the mitochondria. Retro-inverso mastoparan also had the same effect on the mitochondria as mastoparan, but the potencies of the effect were weaker. Not only on mitochondria but also on phospholipid vesicles, mastoparan and inverso mastoparan showed massive permeabilization effects at the same potencies, but retro-inverso mastoparan showed weaker ones. These results indicate that mastoparan interacted with the phospholipid phase of the mitochondrial membrane, not with specific proteins, to induce the permeabilization in cyclosporin Asensitive manner and -insensitive manners.

TUESDAY-POSTER PRESENTATIONS

P2125 Board Number: B648

Lipid-mediated interactions of the mitochondrial presequence translocase. K. Malhotra1, N. Alder1; 1 Molecular and Cell Biology, University of Connecticut, Storrs, CT Following the original endosymbiotic event, nearly all of the genes that encode mitochondria-resident proteins have been transferred to nuclear DNA. As a result, the biogenesis of mitochondria is dependent on the efficient import of nuclear-encoded proteins to its different sub-organellar compartments, carried out by the protein translocases present in its two membranes. The inner membrane of mitochondria is a highly protein-rich biomembrane, where the lipid molecules are known to modulate protein structure and function, hence affecting mitochondrial processes. This is probably best exemplified by cardiolipin, the signature phospholipid of mitochondria, which regulates numerous mitochondrial functions, from ATP production to cell death. The Translocase of the Inner Mitochondrial Membrane 23 (TIM23) mediates both the transport of nuclear-encoded precursor proteins across the inner membrane and the lateral integration of membrane proteins. The initial steps during precursor protein transport are largely mediated by the interaction between the central component of the TIM23 Complex, Tim23, and its receptor partner, Tim50. The lipid dependence of the functional interactions of subunits within this translocase have largely remained unknown. Using the Tim23 channel reconstituted into soluble nanoscale bilayers (nanodiscs) and site-specific chemical crosslinking, we show for the first time that the interaction between the co-receptor Tim50 and the channel region of Tim23 protein, is mediated by cardiolipin. To confirm the cardiolipin-dependent interactions of the mitochondrial preprotein translocase seen in this model-membrane system, we employ a host of cardiolipin variant yeast strains, to study interactions between Tim23 and Tim50 within active mitochondria. Further, by performing liposome binding studies combined with Molecular Dynamics simulations, we show that the soluble domain of Tim50 interacts specifically with cardiolipin containing membranes, and specific residues in the β-hairpin loop are required for mediating the interaction. Moreover, our study suggests that the interaction between Tim50IMS and cardiolipin is accompanied by secondary structural changes in the soluble domain of the co-receptor. By allowing the study of membrane proteins using solutionbased biophysical techniques combined with in organello and in silico work, this system offers unprecedented insights into the early stages of Tim23-mediated protein import.

TUESDAY-POSTER PRESENTATIONS

P2126 Board Number: B649

Proper inheritance of mitochondrial content is maintained in budding yeast cells with mitochondrial dysfunctions. I.A. Mueller1, M.P. Viana1, S. Rafelski2; 1 Developmental and Cell Biology, University of California Irvine, Irvine, CA, 2Developmental and Cell Biology, University California-Irvine, Irvine, CA The inheritance of mitochondria from mother to bud is asymmetric and highly regulated in budding yeast, Saccharomyces cerevisiae. The mitochondrial-volume-to-cell-volume ratio (mitochondrial volume ratio) is higher in buds compared to mothers at the time of division and is normally maintained independent of the mother's mitochondrial content. Mitochondrial inheritance depends not only on the redistribution of mitochondria between mother and bud but also on mitochondrial biogenesis. Mitochondrial biogenesis requires mitochondrial membrane potential, an indicator of mitochondrial functionality, for protein import into the organelle. Consequently, mitochondrial function should play a pivotal part in proper mitochondrial inheritance, but the exact role of mitochondrial function in mitochondrial inheritance has yet to be investigated. We sought to determine whether mitochondrial dysfunctions of different severities impact mitochondrial inheritance in budding yeast. Mitochondrial dysfunctions were introduced by deletion of cytochrome c oxidase subunit 6 (∆COX6), inducing loss of respiratory function, or by deletion of mitochondrial DNA polymerase MIP1 (∆MIP), resulting in loss of respiratory function and ultrastructural alterations. Live-cell, 3D light microscopy images of fluorescently labeled mitochondria of wild type, ∆COX6 and ∆MIP cells were analyzed using our quantitative imaging analysis (MitoGraph) approach to determine mitochondrial tubular width and content at the time of division. Visually, the mitochondrial network morphology in ∆COX6 cells is wild type-like”, while the mitochondrial network of ∆MIP cells is characterized by regionally collapsed and clustered tubules. Mitochondrial tubules are 3% narrower in ∆COX6 cells and 7% narrower in ∆MIP cells compared to wild types ones, suggesting ultrastructural alterations occur in both deletion mutants but are more pronounced in ∆MIP cells. Mitochondrial volume ratio is higher in ∆COX6 and ∆MIP buds compared to their mothers, and more importantly the mitochondrial volume ratio of mutant buds is maintained compared to wild type buds at the time of division. This suggests that asymmetric, actively regulated mitochondrial inheritance is not affected in cells with loss of mitochondrial respiratory function and ultrastructural alterations. Interestingly, the total mitochondrial volume ratio of ∆COX6 cells (mother and bud) is lower compared to wild type cells despite the mitochondrial volume ratio of the bud being maintained, implying problems in mitochondrial biogenesis in ∆COX6 cells. Further studies will determine why total mitochondrial volume ratio is reduced in ∆COX6 but not in ∆MIP cells, even though ∆MIP cells should have more severe alterations in organelle functionality and thus biogenesis.

TUESDAY-POSTER PRESENTATIONS

P2127 Board Number: B650

Extracts from Alpinia galanga mediate mitochondria dynamics. C. Lo1, C. Chiu2, C. Chang2; 1 Department of Food Science, National Chiayi University, Chiayi, Taiwan, 2The Institute of Biotechnology, National Tsing Hua University, HsinChu, Taiwan Mitochondria are highly dynamics organelle with continuous fission and fusion. The dynamics of mitochondria is linked to mitochondrial function and cell metabolism. More and more evidences indicated that mitochondria dynamics responses to different environmental stress. Alpinia galanga has been used as food spice as well as folk medicine. Curcuminoids purified form A. galanga were known to have cytotoxic effects on human leukemic cells and may induce apoptosis through a combination of mitochondria and endoplasmic reticulum stress pathways. Thus, we aim to clarify whether the extracts from A. galanga affects mitochondrial dynamics. Our results demonstrated that treating cervical cancer cells with curcuminoids from A. galanga changed mitochondrial morphology and function. These results suggest that curcuminoids from A. galanga can be used to further dissect the regulatory pathways of mitochondria dynamics.

P2128 Board Number: B651

Ordered partitioning and constant replication ensure mitochondrial DNA maintenance. R. Jajoo1, M.P. Viana2, S. Rafelski3, M. Springer1, J. Paulsson1; 1 Department of Systems Biology, Harvard Medical School, Boston, MA, 2Developmental and Cell Biology, University of California Irvine, Irvine, CA, 3Developmental and Cell Biology, University California-Irvine, Irvine, CA Mitochondrial DNA (mtDNA) is located in the mitochondrial matrix and is essential for normal eukaryotic cell function. Like nuclear DNA, mtDNA cannot be created de novo and must be accurately replicated and partitioned at each cell division. Unlike nuclear DNA, mtDNA has an endosymbiotic origin, varies in copy number, and does not encode its own replication machinery, complicating its maintenance. Here we uncover the partitioning and replication strategies of mtDNA in the fission yeast Schizosaccharomyces pombe by counting mtDNA clusters (nucleoids) in single dividing cells. We find that nucleoids are segregated with half the error expected by “random” binomial partitioning, ensuring that most cells have similar numbers of mitochondrial nucleoids and that mtDNA is rarely lost by any cell. By measuring nucleoid partitioning in mutants that divide asymmetrically, we find that this accuracy is achieved by evenly distributing mitochondria throughout the cytoplasmic volume. Finally, by counting nucleoids at the beginning and end of the cell cycle, we show that the nucleoid replication rate is independent of the number of nucleoids present. Therefore, it appears that mitochondrial nucleoids

TUESDAY-POSTER PRESENTATIONS avoid the instability inherent in self-replication by ceding replication control to the host cell. In total, mtDNA avoids being lost through a combination of ordered partitioning and constant replication.

P2129 Board Number: B652

Novel mitochondria-microtubule crosstalk regulates mitochondrial biogenesis and morphology. V. Jayashankar1, J. Aiken2, S. Rafelski3, J.K. Moore4; 1 University California-Irvine, Irvine, CA, 2Univ Colorado Sch Med, Aurora, CO, 3Developmental and Cell Biology, University California-Irvine, Irvine, CA, 4Cell and Development, University of Colorado School of Medicine, Aurora, CO Mitochondrial homeostasis requires a complex coordination of biogenesis, organization, function, dynamics, and inheritance. Here we identify novel regulation of mitochondrial homeostasis mediated by cross talk with tubulin proteins. We previously conducted a genome wide screen to identify genetic interactions for mutants of yeast α- and β-tubulins that lack negatively charged carboxy-terminal tail regions (CTTs), which project from the microtubule surface. We uncovered a number of mitochondrial genes, including genes involved in biogenesis, morphology, and inheritance. This suggested a role for tubulin CTTs in regulating mitochondrial homeostasis; especially surprising because the actin network in yeast drives mitochondrial transport. To test the hypothesis that tubulin CTTs are important for mitochondrial homeostasis, we imaged live mutant yeast cells using spinning-disk confocal microscopy and quantified the mitochondrial networks in 3D using our MitoGraph image processing method. We found that mutants lacking α-CTT contain fewer, less branched mitochondria resulting in a 20% decreased mitochondrial to cell volume ratio compared to WT cells. This suggests that α-CTTs are required to maintain mitochondrial content, most likely by regulating mitochondrial biogenesis pathways. α-CTT mutant cells also display wider, swollen mitochondrial tubules in respiring cells. This suggests that α-CTT mutant cells may have altered internal ultrastructure organization, which we are verifying by electron microscopy. These phenotypes are unique to α-CTT – mutants lacking β-CTT contain normal mitochondrial content, but have misshapen and occasionally aggregated tubules. Furthermore, cell growth rate and mitochondrial membrane potential are not affected in respiring α-CTT mutant cells, indicating that mitochondria remain functional, despite defects in content and morphology. To define the role of α-CTT in mitochondrial biogenesis and morphology, we examined the sequence features of α-CTT that are necessary for function. We find that α-CTT function can be rescued by mutants that lack negatively charged residues in α-CTT or by replacing α-CTT with the longer, and more negatively charged, β-CTT. These data suggest that α-CTT function depends on the length of the tail, but not its charge. We are currently investigating the molecular mechanisms behind tubulin-CTT regulation of mitochondrial biogenesis and morphology.

TUESDAY-POSTER PRESENTATIONS

P2130 Board Number: B653

Regulation of Mitochondrial Release from Microtubules during Cell Division. J. Chung1,2,3, J.A. Steen2, T.L. Schwarz2,3; 1 Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, 2The F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, 3Department of Neurobiology, Harvard Medical School, Boston, MA During mitosis, daughter cells depend on proper inheritance of both chromosomes and organelles. Cytoskeletal rearrangement during mitosis is therefore accompanied by changes in organelle behavior and organelle interactions with the cytoskeleton. The mechanism behind ER, Golgi apparatus, and nuclear envelope regulation have been examined, but to date, the mechanism behind mitochondrial redistribution and inheritance remains unknown. During interphase, mitochondria move along microtubules via a motor-adaptor complex, consisting of Miro (also called RhoT1/2), which localizes to the mitochondrial outer membrane; the adaptor protein Milton (also called Trak1/2); and the conventional Kinesin-1 Heavy Chain and Dynein motors. During interphase, most of a cell’s mitochondria are therefore closely associated with microtubules. However, as the cell enters mitosis, the mitochondria detach from microtubules and do not appear to associate with either the spindle apparatus or astral microtubules. In HeLa and COS cells, mitotic mitochondria reside primarily in an ring towards the periphery of the cells, do not overlap the spindle, and remain unattached to microtubules until late in anaphase We have found that mitochondrial disassociation is a consequence of the detachment of the kinesin and dynein motor proteins from their adaptors Miro and Milton. Attaching motors onto mitochondria independent of Miro and Milton mislocalizes the mitochondria onto the mitotic spindle, subsequently causing mitochondrial inheritance and spindle orientation defects. Furthermore, in vitro kinase and phosphatase assays determined that motor release is caused by CDK1 phosphorylation of mitochondrial proteins. An attractive target may be the motor adaptor Milton. We have found that Milton is highly phosphorylated and consequently exhibits a cell-cycle dependent band shift on western blots. Milton phosphorylation is a likely trigger for the shedding of the motor proteins from the mitochondrial surface and consequent dissociation of mitochondria from mitotic microtubules. The dissociation of mitochondria from mitotic microtubules is likely to be important for proper inheritance of chromosomes and mitochondria.

TUESDAY-POSTER PRESENTATIONS

P2131 Board Number: B654

Inner membrane fusion mediates spatial distribution of axonal mitochondria. Y. Yu1, H. Lee1, J. Suhan1, G. Yang1; 1 Carnegie Mellon University, Pittsburgh, PA The morphological and motility dynamics of mitochondria are striking. It is thought that mitochondrial motility is important for mediating the spatial distribution of the organelle in neurons. However, whether and, if so, how morphological dynamics play a role in mediating axonal mitochondrial distribution is unclear. To address this question, we analyzed the relations between mitochondrial inner membrane fusion and the spatial distribution of axonal mitochondria. We used Drosophila larval motor neurons axons as the model system. We collected time lapse images of mitochondria in proximal and distal axon regions. For each mitochondrion, we characterized its movement and shape, which allowed us to perform correlation analysis between the two dynamic processes. We found that knockdown of mitochondrial inner membrane protein dOpa1 led to fragmentation of both stationary and moving mitochondria, with stationary mitochondria affected more substantially. We characterized mitochondrial morphological dynamics in larval motor neuron axons, confirmed that mitochondria underwent frequent fusion and fission in axons. Loss of dOpa1 blocked axonal mitochondrial fusion and reduced fission. Furthermore, we analyzed mitochondrial shape, movement and localization in different regions along the axon. We found that dOpa1 deficiency disrupted spatial distribution of axonal mitochondria. Mitochondria failed to localize to the distal axon regions, but accumulated in the proximal axon regions in dOpa1 knockdown axons. Together, our data revealed a strong connection between mitochondrial inner membrane fusion and spatial distribution.

P2132 Board Number: B655

TRPM2 channels regulate organelle zinc homeostasis and mitochondrial fragmentation. N. Abuarab1,2, J. Li1, L. Jiang1, A. Sivaprasadarao1; 1 Multidisciplinary Cardiovascular Research Centre, School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom, 2College of Medicine, King Saud Bin Abdulaziz University for Health ScienceNational Guard Hospital, Jeddah, Saudi Arabia Mitochondria play a central role in oxidative stress induced cell death. By increasing the production of reactive oxygen species, such as H2O2, oxidative stress causes mitochondrial fragmentation and apoptosis. Here we hypothesised that Transient Receptor Potential Melastatin 2 (TRPM2) channels play a role in mitochondrial fragmentation. The rationale behind this is the previous evidence that TRPM2 channels are activated by H2O2 and conduct ions (Ca2+ and Zn2+) that affect mitochondrial health and cell survival. To test our hypothesis we have used human umbilical vein endothelial cells (HUVECs). Using FluoZin-3, we demonstrate the presence of free Zn2+ in lysosomes, but no detectable free Zn2+ in

TUESDAY-POSTER PRESENTATIONS other compartments of the cell. Exposure of HUVECs to H2O2 (1 mM) or high glucose (33 mM) conditions led to lysosomal membrane permeability (LMP), resulting in a marked decrease in lysosomal Zn2+ and, interestingly, redistribution of Zn2+ to mitochondria. The resultant rise in mitochondrial Zn2+ led to extensive mitochondrial fragmentation, mitochondrial outer membrane permeabilisation and cell death. Importantly, release of lysosomal Zn2+ was dependent on Ca2+ entry through the plasma membrane TRPM2 channels. Silencing of TRPM2 channels with siRNA prevented intracellular Zn2+ redistribution, mitochondrial fragmentation and cell death. We found that H2O2 causes mitochondrial fragmentation by promoting the recruitment of dynamin-related protein 1 (Drp-1, a mitochondrial fission protein) to mitochondria. TRPM2 siRNA and the Zn2+ chelator, TPEN (N,N,N',N'-tetrakis (2pyridinylmethyl)-1,2-ethanediamine), inhibited Drp-1 recruitment as well as mitochondrial fragmentation. Taken together, our data imply a novel mechanism for how oxidative stress leads to excessive mitochondrial fragmentation and cell death: The mechanism involves activation of TRPM2 channels leading to increased Ca2+ entry, translocation of lysosomal Zn2+ to mitochondria, Drp-1 recruitment to mitochondria, mitochondrial fragmentation and finally cell death. The physiological relevance of TRPM2 role was established by the demonstration that TRPM2 siRNA inhibits mitochondrial fragmentation in human endothelial cells induced by hyperglycaemic (diabetes) conditions. Since mitochondrial fragmentation is associated with several age-related chronic illnesses including neuronal (Alzheimer’s, Parkinson’s), cardiovascular (atherosclerosis, myocardial infarction) and metabolic/inflammatory (diabetes) disorders, our results reveal TRPM2 channel as a novel target that could be explored for therapeutic intervention of age-related illnesses. The work is funded by King Saud Bin Abdulaziz University for Health Science-National Guard Hospital and the British Heart Foundation.

P2133 Board Number: B656

Cardiolipin phase transition induced by Dynamin-related protein 1 promotes mitochondrial membrane fission. N. Stepanyants1, P. Macdonald1, R. Ramachandran1; 1 Physiology and Biophysics, Case Western Reserve University, Cleveland, OH Cardiolipin is a unique, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells. Here, using model membranes and a variety of novel fluorescence spectroscopic and microscopic techniques, we show that the mitochondrial fission GTPase, dynamin-related protein 1 (Drp1) associates with sequestered but uncomplexed cardiolipin molecules at the mitochondrial surface and reorganizes them in a nucleotide-dependent manner upon helical self-assembly. We demonstrate further that Drp1mediated cardiolipin reorganization results in the lipid’s rapid phase transition from a lamellar phase to the non-lamellar, inverted hexagonal phase. GTP-dependent cardiolipin phase transition effected by Drp1 results in the formation of localized membrane constrictions that become predisposed for fission. We propose that Drp1thus catalyzes mitochondrial division.

TUESDAY-POSTER PRESENTATIONS

P2134 Board Number: B657

Spire1C promotes actin- and ER-mediated mitochondria constriction and fission. U. Manor1, S. Bartholomew2, H. Higgs3, J.A. Spudich2, J. Lippincott-Schwartz4,5; 1 Cell Biology and Metabolism, NIH/NICHD, Bethesda, MD, 2Stanford University, Stanford, CA, 3 Department of Biochemistry, Geisel School of Medicine at Dartmouth College, Hanover, NH, 4 Physiology Course at Marine Biological Laboratory, Woods Hole, MA, 5Cell Biology and Metabolism Program, NICHD, NIH, Bethesda, MD Mitochondria must regularly undergo fission in order to maintain proper cellular health; impaired mitochondria fission can lead to a plethora of diseases ranging from cancer to neurodegeneration. The molecular mechanism by which mitochondria fission is coordinated with the rest of the cell remains unclear, although a role for actin polymerization has been previously implicated. Recent work suggests that the endoplasmic reticulum (ER) induces mitochondria constriction as a necessary step for mitochondria fission; it is thought that this constriction enables Drp1, a dynamin-related GTPase protein, to oligomerize into a helical ring around mitochondria and induce membrane scission at these constriction sites. This model was corroborated by later work, which showed that the actinpolymerization activity of the ER-anchored formin protein INF2 enhances mitochondria constriction and fission. However, the specific mechanism by which INF2 and actin is recruited to and activated at mitochondria fission sites is unknown. Here we show that a previously undiscovered alternate splice isoform of the actin-nucleating, formin-binding Spire protein localizes to mitochondria. Using structured illumination microscopy and the fluorescence protease protection assay (FPP), we show that this novel isoform, Spire1C, localizes to the mitochondrial outer membrane. Using the FPP assay, we also show that the exon unique to this isoform inserts through the mitochondrial outer membrane with the Nterminal side facing the cytoplasm, and the C-terminal side facing the mitochondrial lumen, and that it binds to cardiolipin. We also found that Spire1C N-terminal KIND domain binds to the C-terminal half of INF2 in vitro, and that removing the KIND domain from Spire1C strongly inhibits mitochondria constriction and fission, whereas overexpression of Spire1C enhances constriction and fission. Disrupting Spire1C actin-nucleating ability similarly disrupts mitochondria fission. While overexpression of a Spire1C KIND domain deletion mutant results in decreased association between mitochondria and the ER, overexpression of full length Spire1C increases association between ER-anchored INF2 and mitochondria. Interestingly, overexpression of the KIND domain deletion mutant did not disrupt Drp1 localization to mitochondria, suggesting that the prior step of constriction is required for proper Drp1mediated mitochondria fission. These results strongly suggest that Spire1C facilitates coordination of the ER and actin to enhance mitochondria fission. These results show, for the first time, that two halves of an actin-regulatory complex can reside at the interface between the membranes of two different organelles, thereby presenting a novel mechanism by which cellular organelles can communicate with and shape one another via the actin cytoskeleton.

TUESDAY-POSTER PRESENTATIONS

P2135 Board Number: B658

Dynamin-related protein 1 (Drp1), inhibited by its A-insert, utilizes mitochondrial fission factor (Mff) as a GTPase-activating protein (GAP). P. Macdonald1, N. Stepanyants1, L. Lehman1, A. Baglio1,2, R. Ramachandran1; Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 2Case Western Reserve University, Cleveland, OH

1

Eight different isoforms or splice variants of the mitochondrial fission GTPase, dynamin-related protein 1 (Drp1) arise from alternative splicing of its single gene-encoded pre-mRNA transcript. Of these, the longest isoform of Drp1 bears a unique stretch of amino acid residues within its GTPase domain termed the “A-insert”, whose function is yet not known. By comparing the properties of this neuronally enriched isoform with that of the ubiquitously expressed Drp1 isoform in which this sequence is absent, the role of the A-insert in Drp1 function is revealed. Utilizing a variety of biochemical and biophysical measures, we find that the A-insert auto-inhibits Drp1’s helical self-assembly as well as cooperative GTPase activity over mitochondrial membranes, without impairing GTP binding, thus necessitating the requirement for an external GTPase-activating protein (GAP) to relieve this auto-inhibition. The MOM-integrated Drp1 adaptor protein, mitochondrial fission factor (Mff), fulfills such a role by selectively, and potently, stimulating the longest Drp1 isoform’s GTPase activity independent of its interaction with the consensus mitochondrial target lipid, cardiolipin (CL). Thus, Mff functions selectively as a GAP for A-insert-autoinhibited Drp1.

P2136 Board Number: B659

Mitochondrial remodeling protein Opa1-like influences Drosophila epithelial tissue homeostasis and stem cell maintenance. S. Banerjee1; 1 Department of Biological Sciences, Tata Inst Fundamental Res, Mumbai, India Mitochondrial remodeling proteins with conserved functions in regulating fusion and fission, have a role in implicating cellular/tissue homeostasis and are thus of immense interest. Mitochondrial remodeling proteins like Dynamin related protein-1 (Drp1), Mitofusin-2 (Mfn-2) and Optic atrophy1 (OPA1) have been shown to be involved in cellular homeostasis and stem cell differentiation. I’m interested in understanding the implication of changes in mitochondrial morphology has on tissue homeostasis. Here, using proteomics approach, we identified bonafide and unknown interactors of Drosophila Optic atrophy1-like (Opa1-like) such as Drosophila Mitofilin (dMitofilin), Mitochondrial assembly regulatory factor (Marf), Rhomboid-7 (Rho-7) along with novel interactors like Mortalin ortholog (Hsc70-5). Further, we showed how Opa1-like influences mitochondrial morphology through interaction with

TUESDAY-POSTER PRESENTATIONS ‘mitochondria-shaping’ proteins like Marf (Mitofusin ortholog in Drosophila) and dMitofilin. Depletion of hsc70-5 in peripodial cells, yields fragmented, dysfunctional mitochondria (with Opa1-like being proteolysed) that were susceptible to degradation leading to induced lysosome-mediated cell death. Similar phenotype was observed in hsc70-5 knockdown precursor stem cells (AMPs-Adult Midgut Progenitors) of larval gut which were reversed by changing the mitochondrial morphology via manipulation of mitochondrial remodeling proteins. To understand the significance of death of AMPs that bring about altered tissue behavior and homeostasis, I investigated the role for mitochondrial remodeling proteins in maintaining adult midgut stem cell niche. Drosophila adult midgut homeostasis is maintained by Intestinal Stem Cells (ISCs) that divide to form diploid progenitors known as enteroblasts (EBs) which further differentiate into enteroendocrinal (ee) cells and enterocytes (ECs). Depletion of those proteins that regulate mitochondrial morphology and metabolism in ISC/EB cells resulted in fragmented mitochondria with increased number of ee cells and clustered EBs, a hallmark of Notch activity deficient gut. Moreover, these depletions led to increased ROS-induced hyper-proliferation of ISCs along with hyper-activation of EGFR signaling and early-onset of intestinal hyperplasia. Both hyerproliferation and hyper-activation of EGFR requires Yorkie function. Finally, using genetic epistasis and clonal analysis, I demonstrated that Opa1-like gain of function reverses the cytological dysregulation through change in mitochondrial morphology. Thus, mitochondrial role in the maintenance of adult fly gut stem cell niche cross-talks with that of Notch and Hippo-EGFR pathways. These findings uncover a role for mitochondrial remodeling leading to cell growth/death in tissues.

P2137 Board Number: B660

Structure and dynamics of Miro, a target for PINK1/Parkin signaling of mitochondrial autophagy. S.E. Rice1, J. Klosowiak1, S. Park1, K.P. Smith1, M.C. Gonzalez1, P.J. Focia2, D.M. Freymann2; 1 Department of Cell and Molecular Biology, Northwestern University, Chicago, IL, 2Biochemistry, Northwestern University, Chicago, IL The outer mitochondrial membrane protein Miro is a highly conserved calcium-binding GTPase that is a key player in regulating mitochondrial shape, movement, organelle interactions, and degradation. We solved and published an X-ray crystal structure of a Drosophila Miro fragment containing the EF hand and C-terminal GTPase domains (termed EF-cGTPase). This is the first protein structure containing interacting EF hand and GTPase domains. The structure reveals putative PINK1 kinase phosphorylation sites and Parkin-ubiquitinated sites in and near the interface between Miro’s EF hand region and the Cterminal GTPase domain. We have new data suggesting that this interface is highly dynamic. While fulllength constructs of both Drosophila Miro and human Miro-1 are monomeric, an isolated fragment of the human Miro-1 C-terminal GTPase (cGTPase) forms a homo-dimer. We observed this homo-dimer formation using analytical SEC-MALS, SAXS, and in new X-ray crystal structures that our lab has solved. Interestingly, the interface between the dimerized cGTPase proteins is in the same region as the EF hand-GTPase interface in our previous EF-cGTPase structure. It is possible that the EF hand/C-terminal

TUESDAY-POSTER PRESENTATIONS GTPase interaction and/or dimerization of the C-terminal GTPase may protect Miro from ubiquitination until it becomes phosphorylated by PINK1 kinase. These data are beginning to provide a structural basis for Miro-dependent PINK1/Parkin signaling of mitochondrial autophagy.

P2138 Board Number: B661

The mitochondrial peptidase subunit alpha defines PINK1 alternative mitochondrial import route. K. Grenier1, N. Shahrzad1, J. Xiong1, S. Nobile1, M. Kontogiannea1, E.A. Fon1; 1 Neurology and Neurosurgery, McGill University, Montreal, QC PINK1, a Parkinson’s disease associated gene encoding a mitochondrial kinase, in concert with Parkin, another PD gene that encodes an E3 ubiquitin ligase, mediate the autophagy of damaged mitochondria. Loss of the mitochondrial membrane potential, defects in mitochondrial import and the activation of the mitochondrial unfolded protein response are stimuli that lead to PINK1 stabilization on the outer mitochondrial membrane, which in turn recruits Parkin from the cytosol to the mitochondria in order to initiate mitophagy. In healthy cells, PINK1 is processed by multiple proteases in the mitochondria followed by proteasomal degradation in the cytosol by the N-end rule pathway. However, the specific manner and route by which PINK1 is imported and degraded remains to be clarified. We have recently shown that PINK1 also accumulates at the mitochondrial outer membrane following the partial depletion of the catalytic, β subunit of the mitochondrial processing peptidase (MPP). Indeed, MPPβ knockdown recapitulates the main features of PINK1/Parkin-dependent mitophagy induced by chemical uncouplers. Here, we report that PINK1 contains an N-terminal MTS that is processed directly by MPP both in vitro and in vivo. Moreover, we show that the regulatory subunit MPPα, is required for PINK1 insertion and accumulation at the outer mitochondrial membrane both upon MPPβ knockdown and mitochondrial depolarization but is not necessary for canonical PINK1 import to the inner mitochondrial membrane. Taken together, our works positions MPPα as a key regulator of PINK1 import, required to divert PINK1 to the outer mitochondrial membrane and to initiate parkin-dependent mitophagy in response to insults that compromise mitochondrial health.

TUESDAY-POSTER PRESENTATIONS

P2139 Board Number: B662

Parkin regulates the activity of MiTF/TFE transcription factors independent of mTORC1 during mitophagy. C. Nezich1,2, A.I. Fogel1, C. Wang1, R.J. Youle1; 1 NINDS, NIH, Bethesda, MD, 2MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom Loss-of-function mutations in the mitochondrial kinase PINK1 and the cytosolic E3 ligase Parkin cause the majority of early-onset familial Parkinson’s disease cases. Together, PINK1 and Parkin regulate a quality-control pathway, termed mitophagy, which selectively eliminates damaged mitochondria by autophagosome-dependent lysosomal degradation. Coordinated expression of autophagy and lysosomal genes is regulated by the transcription factor TFEB; however, TFEB has yet to be linked to mitophagy. Because mitophagy requires robust lysosomal function, we sought to understand the molecular link between mitophagy and lysosomal biogenesis. Here, we show that during mitophagy TFEB translocates from the cytosol to the nucleus, where it is active, in a PINK1 and Parkin-dependent manner. Unlike during starvation-induced autophagy, this activation of TFEB was independent of mammalian target of rapamycin complex I (mTORC1) inhibition. To examine whether TFEB was required for mitophagy we generated TFEB null cells using CRISPR-mediated genomic editing. Unexpectedly, these cells exhibited no defect in mitophagy and, contrary to previous studies that used RNAi to decrease TFEB levels, genomic elimination of endogenous TFEB also had no apparent effect on lysosomal morphology. Exploring the potential for compensation for TFEB loss by other members of the MiTF/TFE transcription factor family (MITF, TFEC, and TFE3), we observed Parkin-dependent nuclear translocation of MITF and TFE3 during mitophagy, similar to TFEB. We generated single knockout cell lines for the remaining MiTF/TFE family members and found that each exhibited a partial mitophagy defect that was further enhanced when two or more MiTF/TFE family members were knocked out in the same cell line. This indicates that members of the MiTF/TFE family coordinately participate in mitophagy. Our identification of PINK1 and Parkin as novel regulators of MiTF/TFE family member activation, via a unique pathway that works independently of mTORC1, illuminates a much more dynamic regulation of their activity than previously appreciated and provides new insight into the molecular mechanisms that link mitophagy and lysosomal biogenesis.

P2140 Board Number: B663

PINK1 is a ubiquitin kinase. L.A. Kane1, M. Lazarou1, A.I. Fogel1, Y. Li1, K. Yamano1, S.A. Sarraf1, S. Banerjee1, R.J. Youle1; 1 NINDS, NIH, Bethesda, MD PINK1 is a gene mutated in certain autosomal recessive early onset forms of Parkinson’s disease (PD). During the decade since its identification, it has been shown to be a mitochondrial kinase whose

TUESDAY-POSTER PRESENTATIONS abundance is controlled by mitochondrial membrane potential. Low mitochondrial membrane potential, signaling an unhealthy organelle, causes the accumulation of PINK1 on the outer mitochondrial membrane. This results in the recruitment of Parkin, a cytosolic E3 ubiquitin ligase that is also mutated in early onset PD. Parkin then induces the recruitment of both the proteasome and machinery responsible for autophagy, thus facilitating turnover of damaged mitochondria. The substrates of PINK1 have been a source of debate and many substrates have been suggested in the literature, including Parkin itself. Using an unbiased proteomic approach, we have now identified that PINK1 directly phosphorylates ubiquitin (Kane, et al. J. Cell Biol. 205, 143-153 (2014)). This phosphorylation occurs specifically on Ser65 of ubiquitin and mirrors PINK1’s known phosphorylation of the ubiquitin-like domain of Parkin. Phospho-Ser65 ubiquitin binds to Parkin and activates its E3 ligase activity in vitro. Overexpression of a non-phosphorylatable mutant ubiquitin blocks Parkin translocation to damaged mitochondria in vivo. This is the first identification of a ubiquitin kinase and the first reported function for phospho-ubiquitin. Phosphorylation of ubiquitin in this clinically-relevant mitochondrial pathway opens the potential for a broader cellular signaling role for ubiquitin posttranslational modification.

P2141 Board Number: B664

Deoxynucleoside salvage and compartmentalization in regulating mitochondrial deoxynucleoside triphosphates levels. C. Hsiung1, V. Kamath1, E.E. McKee1; 1 Foundational Sciences, College of Medicine, Central Michigan University, Mount Pleasant, MI Mitochondrial DNA depletion diseases have been identified, in which there is a failure in maintaining mitochondrial biogenesis due to deficiencies and/or mutations of deoxynucleotide salvage pathway enzymes. However, little is known concerning the role of the salvage pathways in regulating deoxynucleosides (dNs) metabolism and deoxynucleoside triphosphate (dNTP) pool sizes, particularly in tissues that are not replicating nuclear DNA and are much more dependent on the salvage pathway. Mitochondrial dysfunction has been associated with a variety of anti-cancer and anti-viral nucleoside analogs that may adversely affect the salvage pathway in non-replicating tissues. The role of protein transporters in moving precursors, intermediates and products of dNTP synthesis is still largely unknown. The purpose of this study is to determine the role of exogenous deoxynucleoside concentration on the size of dNTP pools and the compartmentation of these pools in isolated intact rat heart mitochondria. To accomplish this goal, freshly isolated rat heart mitochondria were incubated at 30°C for 60 minutes with increasing concentrations of tritium-labeled or unlabeled deoxynucleosides. After incubation, mitochondria were separated from the medium by centrifugation. In some cases, mitochondria were further fractionated into membrane and soluble fractions through repeated freezing and thawing. The synthesis of the labeled deoxynucleotides was measured by separation using UPLC and an in-line liquid scintillation counter. The synthesis of dNTPs from unlabeled deoxynucleosides was measured in a modified template driven dNTP assay. Results obtained from current investigation

TUESDAY-POSTER PRESENTATIONS indicated that the mitochondrial level of TTP and dGTP present were highly sensitive to the exogenous thymidine and deoxyguanosine concentration respectively while the level of dCTP and dATP were insensitive to the exogenous deoxycytidine and deoxyadenosine concentration respectively. Thymidine was the preferred substrate over deoxycytidine for thymidine kinase (TK2) and deoxyguanosine was the preferred substrate over deoxyadenosine for deoxyguanosine kinase (dGK). Further, thymidine was much preferred over TMP as substrate for the synthesis of TTP. The TTP synthesized appeared to equilibrate readily with the medium, while surprisingly, dGTP, being 90% of the total dNTP pools, was highly concentrated inside the mitochondria and remained with membrane fraction on mitochondrial breaking. In conclusion, regulation of the salvage pathway in supporting mitochondrial dNTP pools appears to be compartmentalized and different to each deoxynucleosides.

P2142 Board Number: B665

Calorie restriction: Effects of oxidative and nitrosative stress at the mitochondrial level. A. Saavedra-Molina1, M. Clemente-Guerrero1; 1 Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Mich., Mexico Calorie restriction (CR) is an experimental manipulation without malnutrition that is known to extend the lifespan of a number of organisms including yeast, worms, flies, rodents and non-human primates. In addition, CR has been shown to reduce the incidence of aging-related disorders (for example, diabetes, cancer and cardiovascular disorders) in mammals. The mechanisms through which this occurs have been unclear. The aim of this project is to determine the effects of a prenatal and perinatal calorie restriction in rats on oxidative and nitrosative stress in liver mitochondria. Female Wistar rats of 200-300 g of body weight were used and were caged individually and maintained in a 12:12 (light:dark) cycle at a 22 +/- 2 °C. The control groups were fed ad libitum and restricted animals received daily 50% of control animal’s food intake (50% calorie restriction). After two weeks of treatment the female rats were caged with males for one week and mating was confirmed by the presence of spermatozoa in a vaginal smear. Pregnant rats were submitted to calorie restriction during the course of gestation and during lactation period, pups were calorie restricted too. At the age of one, three and five months the animals restricted were measured and weighed before the sacrifice by decapitation. The liver was immediately processed to isolate mitochondria. Results obtained showed variation depending the age-development of the rats. An increase on mitochondrial nitric oxide and lipoperoxidation in the perinatal and postnatal stages were observed, as well as peroxynitrite synthesis, respect control. Presence of antioxidants as glutathione and glutathione reductase activity were measured. Both antioxidants measurements were decreased both in pre and postnatal stages. These results suggest that CR has negative control over oxidative and nitrosative stress in the perinatal stage. Acknowledgements: The authors appreciate the partial economic support of the grant of CIC-UMSNH (2.16).

TUESDAY-POSTER PRESENTATIONS

P2143 Board Number: B666

Mitochondrial dynamics in astrocytic processes during secondary pathology in an in vitro model of transient ischemic stroke. J.C. O'Donnell1,2, J.G. Jackson1, M.B. Robinson1,2; 1 Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, 2Pharmacology, University of Pennsylvania, Philadelphia, PA Astrocytes are the most abundant cell type in brain. They are responsible for clearing extracellular glutamate, the predominant excitatory neurotransmitter, from the synapse to maintain crisp signaling and prevent excitotoxicity. In forebrain, the astrocytic glutamate transporter, GLT-1, is responsible for the vast majority of glutamate uptake. Mitochondria are invested throughout fine astrocytic processes where they colocalize with GLT-1. We recently discovered physical and functional interactions between GLT-1, multiple glycolytic enzymes, and mitochondria. We also found that mitochondria in astrocytic processes are retained near glutamate transporters and synapses. Our data suggest that this distribution is regulated by neuronal glutamate release, astrocytic glutamate uptake, and reversal of the Na+/Ca2+ exchanger. Mitochondria can support glutamate uptake by providing ATP and buffering ions, and a portion of transported glutamate is oxidized in mitochondria to generate energy in these compartments. Mitochondrial dysfunction and excitotoxicity from failure of astrocytic glutamate uptake are at the core of delayed cell death that occurs after a transient ischemic insult. Aside from inducing hypothermia, this secondary pathology is currently untreatable. The goal of this project is to quantify changes in astrocytic mitochondrial distribution during the secondary pathology period in an in vitro model of stroke. To do so we have employed transient oxygen glucose deprivation (OGD) in organotypic hippocampal slice cultures biolistically transfected to fluorescently label astrocytic mitochondria. In ongoing experiments we have observed a loss of mitochondria in astrocytic processes in response to OGD that precedes the peak of delayed neuronal cell death common to this model and to stroke in vivo. In preliminary experiments, cyclosporin A, which increases mitochondrial capacity for calcium buffering, reduced cell death and attenuated the loss of mitochondria in astrocytic processes after OGD. Preliminary observations also provide evidence for increased mitophagy (a mechanism for degradation of dysfunctional mitochondria) in astrocytic processes after OGD. Further investigation of the mechanisms underlying the loss of astrocytic mitochondria after transient OGD could provide new therapeutic targets for the currently untreatable secondary pathology following transient ischemic stroke.

P2144 Board Number: B667

In vivo tissue-wide synchronization of mitochondrial metabolic oscillations. N. Porat-Shliom1, Y. Chen2, A. Shitara1, M. Tora1, O. Harding1, A. Masedunskas1, R. Weigert1; 1 NIDCR, NIH, Bethesda, MD, 2NCI, NIH, Bethesda, MD

TUESDAY-POSTER PRESENTATIONS Little is known about the spatio-temporal coordination of mitochondrial metabolism in multicellular organisms in situ, and how oxidative phosphorylation adapts to rapid changes in energy demand during processes such as membrane trafficking. To address this issue we investigated how mitochondrial metabolism is modulated during GPCR-stimulated exocytosis in the acinar cells of salivary glands (SGs) in live rats. Specifically, we developed a method to follow the dynamics of the mitochondrial metabolic activity in live rodents that is based on the use of intravital two-photon microscopy to determine NADH levels (the main substrate of the electron transport chain, ETC) and mitochondrial potential. Unexpectedly, we discovered that mitochondrial metabolism undergoes rapid and spontaneous oscillations in SGs under basal conditions (period: 10-15 sec), in contrast with what reported in ex vivo or in vitro model systems where transient metabolic oscillations are observed only after agonist stimulation. These oscillations are regulated by reactive oxygen species but are insensitive to modulations in the levels of intracellular Ca2+. Most notably, we found that mitochondria in vivo behaves as a network of functionally coupled oscillators, which maintain a high level of coordination throughout the tissue. Indeed, mitochondria in different acini oscillates with the same period and phase, and their coordination is disrupted by blocking the activity of gap junctions. Finally, we observed that βadrenergic stimulation, which stimulates regulated exocytosis, transiently halts NADH oscillations and increases mitochondrial potential, thus generating the ATP necessary to complete secretion. The mechanism regulating this process will be discussed in here. In conclusion, the ability to measure and characterize metabolic oscillations through NADH emission in vivo, has revealed novel aspects of cellular metabolism that have not been appreciated in reductionist model systems, and will open new avenues to study mitochondrial metabolism and tissue energetics at a cellular, subcellular, and tissue scale both under physiological and pathological conditions.

P2145 Board Number: B668

Characterization of the zebrafish family of Trak proteins. L.B. Stone1, K.A. Oonk1, C. Reddig2, E. Nickoloff3, D.C. Weiser4, S. Walsh2; 1 Rollins College, Winter Park, FL, 2Biology, Rollins College, Winter Park, FL, 3Center for Translational Medicine, Temple University, Philadelphia, PA, 4Univ of the Pacific, Stockton, CA Mitochondria serve as the site of oxidative phosphorylation and can move to areas that possess high energetic needs within a cell. This movement is necessary to maintain basic metabolic functions, and disruption of this motility often results in cell death. In fruit flies and mammals, Miro, an outer mitochondrial membrane protein, and Trak, a kinesin adaptor protein, comprise one protein complex that regulates mitochondrial trafficking along microtubules. Our research aims to determine the role of the zebrafish Trak family of proteins. Through sequence homology, we have identified three zebrafish Trak proteins: zTrak1, zTrak2, and zTrak1-like. When expressed in COS7 cells, both human Trak1 and Trak2 are mitochondrial. However, in COS7 cells, zTrak1 does not localize to the mitochondria, yet zTrak2 does. A chimeric protein containing the N-terminus of zTrak1 and the C-terminus of human Trak1 relocated to the mitochondria, indicating that the C-terminus of hTrak1 is sufficient to direct

TUESDAY-POSTER PRESENTATIONS mitochondrial localization, and the C-terminus of zTrak1 is likely responsible for its cytosolic localization. Given the low amino acid conservation among these paralogs, these zebrafish proteins might have different cellular functions, and we are currently exploring the role of these proteins in vivo using antisense morpholino oligonucleotides and RNA in situ hybridization in developing zebrafish embryos. Taken altogether, this research will lay the foundation for studies in the role of mitochondrial motility during vertebrate development.

Kinases and Phosphatases P2146 Board Number: B670

Reconstitution of Apoptotic Cell Engulfment to Decipher an Ancient Signaling System. A. Williamson1, R. Vale1; 1 University of California, San Francisco, San Francisco, CA Apoptosis is crucial during development and for maintaining healthy adult tissues. However, the appearance of apoptotic cells is normally rare: dying cells are efficiently recognized, internalized, and digested by phagocytes (a process termed corpse clearance). Since inefficient corpse clearance leads to inflammation and further tissue damage, a mechanistic understanding of how engulfment works and how the process can be activated may spur development of rational therapies for inflammatory diseases. Elegant genetic work has identified the components of an ancient signaling system conserved across flies, worms and mammals that drives engulfment. However how these factors work together to drive the process remains unclear. We have reconstituted apoptotic cell engulfment by a Drosophila pathway driven by the receptor Draper and kinase Shark. Expression of Draper in cultured cells converts inept phagocytes into efficient engulfing cells. Recognition and engulfment of apoptotic cells or physiological mimics is rapid and specific. A clue to the mechanism of Draper and Shark may lie in the signaling pathway that triggers T cells. In the latter case, the T cell receptor complex (TCR) works with a kinase ZAP-70 at the membrane. Both apoptotic cell engulfment and T cell triggering occur at sites of membrane-membrane contact two cells and require actin. Furthermore the molecules that drive each process bear remarkable similarity. We have shown that Shark, like ZAP-70, is recruited to the plasma membrane during signaling. Similar to TCR and ZAP-70, Draper and Shark form clusters at the plasma membrane that undergo retrograde flow, indicating that functional properties of the signaling systems are conserved between these divergent pathways. We have established a live cell imaging platform to interrogate the contribution of specific molecules towards efficient engulfment; in particular we hope to understand how the Draper pathway interfaces

TUESDAY-POSTER PRESENTATIONS with downstream actin machinery. As a similar system has been proposed to function in mammalian cells, we look forward to extending our findings from Drosophila to other organisms.

P2147 Board Number: B671

Amyloid β-Induced Activation of mTOR at the Plasma Membrane Leads to Neuronal Cell Cycle Re-Entry: a Seminal Step in Alzheimer's Disease Pathogenesis. A. Norambuena1, L. McMahon 1, E.J. Kodis1, E.M. Swanson1, H.K. Wallrabe1, S.S. Thomas2, D. Baerthlein1, X. Zhou3, J. Zhang3, S. Oddo4, G.S. Bloom5; 1 Department of Biology, University of Virginia, Charlottesville, VA, 2Department of Biology and Neuroscience Graduate Program, University of Virginia, Charlottesville, VA, 3Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, MD, 4Department of Basic Medical Sciences, University of Arizona College of Medicine, Phoenix, AZ, 5Departments of Biology and Cell Biology, University of Virginia, Charlottesville, VA Mechanistic Target of Rapamycin (mTOR) plays a major role in cell metabolism by sensing availability of resources, such as growth factors and amino acids, and integrating the resulting signals to control cell growth and proliferation. These functions involve finely tuned regulation of mTOR activity at specific membrane locations, and mTOR dysregulation has been implicated in cancer and Alzheimer's disease (AD). AD is characterized by the accumulation in brain of two types of insoluble deposits, extracellular plaques composed of amyloid-β (Aβ) peptides and intraneuronal neurofibrillary tangles comprising the microtubule-associated protein, tau, and by massive synaptic dysfunction and neuron death. Ironically, a large fraction of neuron death in AD occurs after the normally post-mitotic neurons ectopically re-enter the cell cycle. We recently reported that neuronal cell cycle re-entry (CCR) results from a process by which soluble Aβ oligomers (AβOs) stimulate PKA, fyn and CAMKII to catalyze site-specific tau phosphorylation (Seward, et al. 2013. J Cell Sci 126:1278). We now describe further details of the CCR pathway, including a novel and essential role for mTOR. Ki-67 and Cyclin D1 expression, or BrdU uptake were used to identify cultured WT mouse neurons that respectively had exited G0, were in G1, or had entered or completed S-phase after exposure to AβOs. CCR was prevented by knockdown of NCAM, Gαs, RalA, Rac1 or eIF4E, or reducing the activity of mTORC1 or mTORC2 by respective knockdown of raptor or rictor. Pharmacological inhibition of mTOR or Rac1, or exposure of neurons to a cell permeable peptide that blocks Rac1 binding to mTOR also prevented CCR. By using a genetically encoded mTORC1 activity reporter (Zhou, et al. Cell Metab in revision) we detected AβO-induced mTOR activation at neuronal cell membranes, but not in lysosomes. Reducing Rac1-dependent translocation of mTOR to plasma membrane (PM) domains and forcing mTORC1 association to lysosomes also prevented CCR. In vivo studies showed that CCR marker expression in cortical neurons of Tg2576 AD model mice was strongly suppressed in animals with

TUESDAY-POSTER PRESENTATIONS genetically-reduced mTor activity. Finally, we found that this Rac1-modulated mTOR signaling controls in vitro and in vivo tau phosphorylation at S262, which is also necessary for neuronal CCR. AβOs thus initiate two pathways that together lead to mTOR dysregulation and are obligatory for ectopic neuronal CCR. One pathway mistargets Rac1-mTOR complexes to the PM, and the other leads from Gαs and NCAM to activation of fyn, PKA and CaMKII, which then respectively phosphorylate tau at Y18, S409 and S416. The CCR requirement for tau phosphorylation at S262 establishes that regulation of tau and mTor by each other integrates the two pathways.

P2148 Board Number: B672

BAG-1 differentially regulates intermediate filament-based Hsp70 chaperoning of aPKC in intestinal cells under pro-inflammatory signaling. A. Mashukova1, R. Forteza2, P.J. Salas2; College of Medical Sciences, Nova Southeastern University, Fort Lauderdale, FL, 2Department of Cell Biology, University of Miami Miller School of Medicine, Miami, FL

1

Atypical protein kinase C (aPKC) plays an essential role in the establishment of epithelial polarity and tight junction assembly. aPKC acquires inactive conformation after normal activity and can be rescued from ubiquitinylation and degradation by Hsp-70- and intermediate filament-dependent mechanism. We have shown previously that aPKC was strongly downregulated by TNF-alpha-mediated signaling in intestinal epithelial cells and also in vivo during intestinal inflammation. Furthermore we have demonstrated that decrease of aPKC levels under pro-inflammatory conditions was mediated through inhibition of Hsp70 chaperoning activity, resulting in failure of the aPKC rescue machinery. The goal of this work was to identify the molecule (or molecules) responsible for Hsp70 inhibition under TNF-alpha stimulation. First, we conducted a transcriptome PCR screen detecting chaperones and co-chaperones and compared mRNAs from control and TNF-alpha treated Caco-2 cells (human colon carcinoma). Subsequent validation experiments allowed us to focus on BAG-1, a multi-functional protein that assists Hsp70 in nucleotide exchange but also blocks its activity at higher concentrations. We found that BAG-1 isoform BAG-1M was upregulated up to 3 fold in human Caco-2 cells following stimulation with TNFalpha. In addition, BAG-1M levels increased up to 6 fold in mouse enterocytes following treatment with dextran sodium sulfate (DSS) to induce colitis. BAG-1M, but no other isoform, was found to co-purify with intermediate filaments. To test the effect of BAG-1M on Hsp70 chaperoning activity we employed a well-established chemically denatured luciferase refolding assay. Addition of BAG-1M to the assay reaction within the range of concentrations found in epithelial cells resulted in significant inhibition of Hsp70 chaperoning activity in the intermediate filament fraction. To determine if the findings observed in vitro also apply to the living cells, we have performed both overexpression and knockdown of Bag-1M in Caco-2 cells using lentiviral transduction. Overexpression of BAG-1M decreased levels of phosphorylated aPKCs, similar to TNF-alpha stimulation. In contrast, knockdown of BAG-1 abolished the TNF-alpha-induced decrease of phosphorylated aPKC. Overall, we conclude that BAG-1M mediates inhibition of Hsp70 chaperoning activity during epithelial inflammatory response.

TUESDAY-POSTER PRESENTATIONS

P2149 Board Number: B673

Optogenetic modulation of Raf/ERK and PI3K/AKT pathways in PC-12 cells understanding the temporal dimensions of antiapoptotic pathways against oxidative stress. Q. Ong1, K. Zhang1,2, S. Guo1, L. Duan1, E.A. Collier1, B. Cui1; 1 Chemistry, Stanford University, Stanford, CA, 2Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL Intracellular optogenetics offers chemical biologists an unprecedented new set of tools in manipulating and probing signaling pathways with spatiotemporal precision. We have utilised the light proteininteraction pair, CRY2PHR and CIBN, to elicit Raf and AKT recruitment to the plasma membrane via blue light controllably, with reversible dissociation under dark conditions. This optogenetic toolkit is being applied to study how Raf/ERK and PI3K/AKT pathways act as protective mechanisms to the cells under oxidative stress (hydrogen peroxide and singlet oxygen generation), with greater understanding towards their temporal dimensions.

P2150 Board Number: B674

The MLK3-mediated positive feedback loop is a crucial factor in cellular responses to reactive oxygen species. C. Hwang1, H. Lee1, K. Cho1; 1 Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea Reactive oxygen species (ROS) play a central role in the regulation of numerous cellular processes. It is known that opposite cellular response such as proliferation or cell cycle arrest/apoptosis can be made depending on ROS concentration. However, it remains unclear how such different cellular response can be determined through a complex signaling network. In this study, we found that extracellular signalregulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) coordinate ROS-dependent cellular response determination. ERKs mediate proliferation at low ROS concentration, whereas JNKs mediate cell cycle arrest/apoptosis at high ROS concentration. From mathematical simulations combined with biochemical experiments, we found that mixed lineage kinase 3 (MLK3)-mediated positive feedback loop balances the signal flow for proliferation and cell cycle arrest/apoptosis, which results in contrasting cellular response determination. Cells treated with an MLK3 inhibitor or MLK3 shRNA showed increased viability at high ROS concentrations through higher ERKs activation and lower JNKs activation than control cells. Taken together, our systems biology analysis and biochemical studies provide new insight into the mechanism underlying how ROS-dependent cellular response is determined.

TUESDAY-POSTER PRESENTATIONS

P2151 Board Number: B675

Anti-inflammatory Effects of Heparin on Vascular Endothelial Cells are Dependent on Dual-Specificity Mitogen-Activated Protein Kinase Phosphatase-1. S.N. Farwell1, J.B. Slee1,2, L.J. Lowe-Krentz3; Lehigh University, Bethlehem, PA, 2Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, 3 Biological Sciences, Lehigh University, Bethlehem, PA 1

Heparin is widely-used as an anti-coagulant as part of treatment for increasingly prevalent cardiovascular and pulmonary diseases. Anti-proliferative effects of heparin on vascular smooth muscle cells (VSMCs) have been well documented and involve both the down-regulation of the mitogenactivated protein kinase (MAPK) intermediate Erk and the up-regulation of dual-specificity MAPK Phosphatase-1 (MKP-1). Heparin has been also shown to exhibit potent anti-inflammatory properties on vascular endothelial cells (ECs) in vitro, but the molecular mechanism(s) by which heparin accomplishes this remain unclear. We examined heparin’s effects on primary macrovessel ECs. We tested the hypothesis that heparin’s effects are also at least partially dependent on increased MKP-1 expression in ECs. We show that heparin decreases tumor necrosis factor (TNF)α-induced activation of stressactivated protein kinases (SAPKs) c-Jun N-terminal kinase (JNK) and p38 through western blotting and immunofluorescence assays. Using knockdown strategies, we show these effects are partly dependent on MKP-1. Heparin-induced decreases in TNFα-induced actin stress fiber production also require an MKP-1 induction, as shown through quantitative fluorescence microscopy. An advanced understanding of heparin’s anti-inflammatory effects is crucial to identify novel heparin responsive targets and to develop and improve therapeutic strategies. Exploiting heparin as a drug with anti-inflammatory actions is an important direction to pursue in both cardiovascular research and clinical settings. Support from NIH award HL54269 to LLK.

P2152 Board Number: B676

Metabolic control of Ca2+/Calmodulin-dependent protein kinase II (CaMKII)mediated Caspase 2 suppression by the B55β/protein phosphatase 2A (PP2A). B. Huang1, C. Yang1, J. Wojton1, N. Huang1, C. Chen1, E.J. Soderblom1, S. Kornbluth1; 1 Duke University Medical Center, Durham, NC High levels of metabolic activity confer resistance to apoptosis. Caspase 2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway (PPP). This metabolic control is exerted via inhibitory phosphorylation of the caspase 2 pro-domain by activated Ca2+/Calmodulin-dependent protein kinase II (CaMKII)(1). We show here that metabolic activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (T393/S395) and that this is

TUESDAY-POSTER PRESENTATIONS mediated by metabolic activation of protein phosphatase 2A (PP2A) in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and PP2A-regulated physiological processes, as both enzymes appear to be responsive to alterations in glucose metabolized via the PPP. 1. Nutt, L. K., Margolis, S. S., Jensen, M., Herman, C. E., Dunphy, W. G., Rathmell, J. C., and Kornbluth, S. (2005) Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2. Cell 123, 89-103

P2153 Board Number: B677

Functional interaction network of the conserved NDR kinase Orb6. C. Chen1, I. Suarez2, D.J. Wiley2, G. D'Urso2, F. Verde1; 1 Department of Cellular and Molecular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, 2University of Miami Miller School of Medicine, Miami, FL Cell polarization is the common feature of a variety of different cell types and is fundamental to many cellular processes, including cell differentiation, cell motility and cell fate determination. Defects in cell morphogenesis are linked to diseases such as cancer, developmental defects and neuronal disorders. The conserved NDR kinase Orb6 controls cell morphogenesis in different organisms, ranging from yeast to neuronal cells. In mammals, NDR kinase is also thought to function as a tumor suppressor. Our lab has previously shown that the conserved NDR kinase homologue in fission yeast, Orb6 kinase, plays a key role in maintaining polarized cell growth at the cell tips by spatially restricting the localization and activity of Cdc42 GTPase. To identify novel functional targets of NDR kinase Orb6, we used an ordered fission yeast gene deletion library to perform a genome-wide screen for gene functions that display synthetic lethality or suppression interactions with an inhibitor-sensitive Orb6 kinase mutant. This innovative screen further refined the screen results using computational, mass spectrometry, and 2-hybrid analysis approaches. Refined gene candidates fall into four major functional groups, including morphogenesis, nutritional response, transcriptional and translational control. Of particular interest are novel genetic, functional and physical interactions of Orb6 kinase with conserved protein complexes involved in the control of transcription and heterochromatin gene silencing.

TUESDAY-POSTER PRESENTATIONS

P2154 Board Number: B678

An endoplasmic reticulum lipid phosphatase regulates plasma membrane lipid metabolism. E.J. Dickson1, J.B. Jensen1, O. Vivas1, B. Hille1; 1 Physiology and Biophysics, University of Washington, Seattle, WA Phosphoinositides are a family of low-abundance phospholipids located on the cytoplasmic leaflet of cellular membranes that maintain cell structure, cell motility, membrane trafficking, ion channel functions and also play key roles in signal transduction. Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is the signature phosphoinositide of the plasma membrane. How is PM PI(4,5)P2 sourced? We have reported that plasma membrane PI(4,5)P2 levels are supported by at least two, continuously supplying, precursor pools of PI(4)P, one in the plasma membrane itself, and the other in the Golgi. How is plasma membrane PI(4,5)P2 regulated? Many lipid phosphatases and kinases are needed for the maintenance of each cellular phosphoinositide pool; we focused our attention on an integral membrane lipid 4-phosphatase enzyme localized to the endoplasmic reticulum (ER), Sac1. It has the ability to reduce the precursor source of PM PI(4,5)P2, namely PI(4)P. Using a variety of optical (confocal, TIRF, super-resolution microscopies), protein dimerization, and electrical (ion channel recordings) techniques, we have discovered that Sac1 aggregates in puncta, intimately apposed to the plasma membrane in endoplasmic reticulum–plasma membrane (ER-PM) contact sites in tsA-201 cells. Based on FRET measurements between Sac1 and a plasma membrane marker, Sac1 is within 10 nm of the plasma membrane. What causes Sac1 to aggregate in ER-PM contact sites? Multiple lines of evidence suggest a role for the cortical ER protein, extended synptotagmin-2 (E-Syt2). Quantitative super-resolution imaging analysis reveals strong correlation between Sac1 and E-Syt2 distributions. Overexpressing or knocking down E-Syt2 increased or decreased the abundance of Sac1 in close proximity to the PM, respectively, and lead to alterations in plasma membrane PI(4)P and PI(4,5)P2 levels. Activation of Gprotein coupled receptors to deplete plasma membrane PI(4,5)P2 causes a dynamic redistribution of Sac1 puncta away from the plasma membrane. The reaggregation of Sac1 back into ER-PM contacts is dependent on the rate of PI(4,5)P2 resynthesis. Similar results were observed in superior cervical ganglia neurons. Thus, the intimate organization of ER Sac1 next to the plasma membrane gives it the unique ability to act as a cellular ‘thermostat’ controlling plasma membrane PI(4)P and PI(4,5)P2 levels and with that many plasma membrane proteins. Supported by a grant from the NIH National Institute of Neurological Disorders and Stroke R37NS008174 and the Wayne E. Crill Endowed Professorship.

TUESDAY-POSTER PRESENTATIONS

P2155 Board Number: B679

Distinct patterns of phosphatase activation and subcellular localization impact the kinetics of interferon-gamma signaling in CNS neurons. C. Brown1, A. Stahl1, C. Stotesbury1, K. Sweeney1, G. Rall2, W. Rose1; 1 Department of Biology, Arcadia University, Glenside, PA, 2Immune Cell Development and Host Defense Program, Fox Chase Cancer Center, Philadelphia, PA The immune cytokine interferon-gamma (IFN-g) plays an essential role in the elimination of numerous central nervous system (CNS) viral infections. The cellular response to IFN-g, mediated by JAK/STAT signal transduction, involves the upregulation of genes that result in the establishment of the antiviral state, and is controlled by multiple negative feedback mechanisms. However, whether these control mechanisms, previously characterized in non-neuronal cells, operate similarly in CNS neurons has not been well-characterized. Our lab previously demonstrated that the control of the neuronal IFN-g pathway is distinct from that in non-neuronal cells, as the duration of STAT1 phosphorylation and IFN-g responsive gene expression in IFN-g treated neurons was markedly extended as compared to mouse embryonic fibroblasts (MEF). We also showed that compared to MEF, STAT1 dephosphorylation was delayed in IFN-g treated neurons, providing a mechanism for the extended kinetics of neuronal STAT1 phosphorylation. The current study investigated the expression and subcellular localization of two STAT1 phosphatases, TC45 and SHP-2, to address the hypothesis that mislocalization of these phosphatases could result in the observed delay in neuronal STAT1 dephosphorylation. Although no differences in overall expression levels of TC45 were observed between neurons and MEF, neurons demonstrated an approximately 3-fold lower level of SHP-2 activation as compared to MEF, regardless of IFN-g exposure. To examine the subcellular localization of these phosphatases, neurons and MEF were exposed to IFN-g, and nuclear and cytoplasmic fractions were purified at 1, 24, and 48h post-IFN-g treatment. Our results showed that while SHP-2 was found strictly in the cytoplasmic fraction of both cell types, TC45 was found only in the cytoplasmic fraction of the neurons, whereas it was equally distributed between nuclear and cytoplasmic fractions in MEF. These results indicate that neurons (but not MEF) lack TC45 in the nucleus, which provides a likely explanation for the observation that neuronal STAT1 is not dephosphorylated as rapidly as it is in MEF. Understanding the control of the IFN-g response in CNS neurons will ultimately aid in the characterization of antiviral immune mechanisms of the CNS.

TUESDAY-POSTER PRESENTATIONS

P2156 Board Number: B680

A-Kinase Anchoring Proteins, Phosphorylated-Akt/PKB and Phosphodiesterase4A: Players in Schwann Cell Proliferation. A.L. Asirvatham1, K. Spears1, R. Stahl2, D. Carey2; 1 Biology, Misericordia Univ, Dallas, PA, 2Geisinger Health Center, Weis Center for Research, Danville, PA Proliferation of Schwann cells in the peripheral nervous system is dependent upon the heregulin/neuregulin family of growth factors secreted by neurons. The stimulation of Schwann cell division by heregulin occurs in a synergistic fashion only if pathways involving cAMP are also triggered. The molecular mechanisms involved in the synergistic modulation of Schwann cell proliferation by heregulin and cAMP pathways are unknown. Previous studies have shown that A-Kinase anchoring proteins (AKAPs) of the cAMP/PKA signaling pathway play a key role in Schwann cell division. Treatment of neonatal Schwann cell cultures with SiRNA oligos synthesized against AKAP150 and AKAP95 exhibited a significant reduction in cell growth, expression of AKAPs and the cell survival signal, AKt/Protein kinase B. Based on these studies it was hypothesized that the expression of AKAP95 and AKAP150 along with the expression of phosho-Akt/PKB and the cAMP-terminating enzyme, PDE4A would increase when Schwann cells were treated with heregulin, forskolin or heregulin + forskolin. Immunoblot analysis revealed that heregulin treatment of Schwann cells increased the protein expression of AKAP95 and phosphorylated-Akt while forskolin treatment caused an upregulation in the levels of AKAP 150, PDE4A and Akt. To corroborate the results from immunoblot analysis, immunofluorescence experiments in Schwann cells were conducted, which revealed that in comparison to control, cells treated with heregulin and heregulin + forskolin exhibit distinct colocalization patterns for AKAPs and Akt while cells treated with forskolin show colocalization for AKAPs and phospho-Akt. These preliminary observations suggest that a relationship between expression of AKAP proteins and phosphorylation of Akt along with PDE4A may be necessary to mediate mitogen-stimulated Schwann cell growth.

P2157 Board Number: B681

Novel substrates of Rho kinase in the heart. Y. Yura1, T. Bando2, M. Amano1, K. Kaibuchi3; 1 Cell Pharmacology, Nagoya University School of Medicine, Nagoya City, Japan, 2Cell Pharmacology, Nagoya University School of Medicine, Nagoya, Japan, 3Nagoya University School of Medicine, Nagoya, Japan Rho kinase/ROCK/ROK is highly expressed in the cardiac muscle and is activated in failing heart. However, the substrate and function of Rho kinase in the heart have not been reported so far. We previously developed a novel proteomic approach for screening of protein kinase substrates by combining mass spectrometry and affinity column chromatography of catalytic domain of protein kinase. Using this method, we searched for novel substrates of Rho kinase in the rat heart tissue, and

TUESDAY-POSTER PRESENTATIONS more than 200 candidate substrates were obtained. We then confirmed that some of these candidate substrates including cardiomyopathy-related gene products were really phosphorylated by Rho kinase in vitro. Among them, we focused on ANKRD1/CARP. ANKRD1 has been reported to play a critical role in the maintenance of sarcomere integrity and transcriptional regulation of cardiac genes dependent on its subcellular localization. We further searched for the binding partners whose interactions are affected by phosphorylation state of ANKRD1. Among the known partners, transcriptional factor YB-1 preferentially bound to non phosphorylated ANKRD1 rather than phosphorylated one. In addition, we also found that 14-3-3 proteins specifically associated with phosphorylated ANKRD1. These results suggest that Rho kinase modifies the ANKRD1 function by regulating its transcriptional activity and subcellular localization through the phosphorylation.

P2158 Board Number: B682

Exploring signaling through TAK1 during SAN development. C. Awgulewitsch1,2, S. Legros2, M. Doss2, Y. Dai2, A. Foley2; Biology, College of Charleston (Honors College), Charleston, SC, 2Bioengineering, Clemson-MUSC, Charleston, SC

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Following a myocardial infarction, cardiomyocytes in the damaged heart tissue are replaced by collagen and fibroblasts. This structural change leads to a decrease in cardiac function and also arrhythmia. One developing treatment option for this damaged tissue is the cellular transplantation of cardiac cells derived from pluripotent cells such as embryonic stem (ES) cells. However, this therapy will require a deeper understanding of the mechanisms that determine cardiac cell fates. Previous work in the Foley lab has shown that early cardiac formation is dependent upon intracellular signaling between the endoderm and mesoderm, mediated by antagonists of the canonical Wnt/β-Catenin signaling pathway and activators of the Nodal signaling pathway. TGFβ-activated kinase (TAK1), which is located directly upstream of the Wnt antagonist NLK, is thought to be critical for cardiac differentiation toward the sinoatrial node (SAN) fate. We hypothesize that SMAD–independent TGFβ signaling through TAK1/p38/JNK or TAK/NLK pathways influences formation of the heart inducing endoderm and indirectly to direct SAN differentiation within the myocardium. This study focused on the role of TAK1 in mouse ES cells. Mouse ES cells were transduced with the TAK1 gene and the TAK1 ES cells were then differentiated as embryoid bodies (EBs). Western blots compared protein content of different signaling markers of TAK1 cells to unmodified ES cells Continuous TAK1 mRNA overexpression and the effects on patterning were assessed by qRT-PCR. Markers for the endoderm and the sinoatrial node were significantly upregulated while cardiac contractile proteins were downregulated. In addition we found TAK1 cells alter the expression of Jnk and NLK in ES cells. Therefore, we have determined that TAK1 overexpression could be directing pacemaker differentiation by acting through one or both of these downstream targets.

TUESDAY-POSTER PRESENTATIONS

P2159 Board Number: B683

Activin-Beta/TGF-Beta signaling in skeletal muscle controls insulin signaling and metabolism to influence final body size. L. Moss-Taylor1, M. O'Connor1; 1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN Body size is tightly regulated during development to maximize adult fitness. In Drosophila, final body size is mainly determined by the growth rate and the duration of growth during juvenile stages; once maturation occurs, body size is set. The rate of growth is determined by insulin-like peptide (dilp) signaling and metabolism, while the duration of growth is regulated by prothoraciotropic hormone (PTTH) and ecdysone. Manipulation of these pathways alters final body size by either accelerating or delaying the onset of metamorphosis. Skeletal muscle is known to coordinate tissue and body growth during development, influencing final size. Levels of insulin signaling in skeletal muscle proportionately affect body and organ size, but what influences muscle-specific insulin signaling and how the muscle coordinates tissue growth are unknown. We are investigating the newly identified role that the TGF-Beta ligand Activin-Beta plays in regulating Drosophila body size and timing of metamorphosis. We have found that mutations in dActivin-Beta (dAct-Beta) cause accelerated pupariation and reduced final body and organ size. To determine how Act-Beta affects size and timing, we first looked at which cells express Act-Beta and found expression in the Insulin Producing Cells (IPCs), neuroendocrine cells and motor neurons. Overexpression of ActivinBeta in either neuroendocrine cells or motor neurons increases body size. Muscle-specific knockdown of the TGF-Beta signaling transducer/transcription factor dSmad2 reduces body size, indicating muscle is a target tissue of the Activin-Beta signal. Additionally, levels of phospho-dSmad2 are reduced in skeletal muscle samples of Activin-Beta mutants and increased in animals overexpressing Activin-Beta from motor neurons. Levels of phospho-S6K are correlate with phospho-dSmad2 levels, suggesting TGF-Beta signaling in muscle regulates muscle-specific insulin signaling. Because insulin signaling controls metabolism, we used GC/MS analysis to identify and quantify levels of metabolites in whole-larval samples of Activin-Beta mutants. We found intermediates of the energy-producing steps of glycolysis and lactic acid are reduced, indicated reduced flux through glycolysis. Overall, this indicates neuronallyderived Activin-Beta signals to the skeletal muscle to regulate levels of insulin signaling and subsequent glycolysis. We will identify downstream targets of dSmad2 using skeletal-muscle RNAseq and test potential target genes using tissue-specific knock-downs to determine how TGF-Beta signaling influences insulin signaling and how the muscle coordinates body growth and final organ size.

TUESDAY-POSTER PRESENTATIONS

P2160 Board Number: B684

Understanding the mechanisms of cellular growth control with: (1) the Rose Rosette Virus in plants and (2) Angiomotin controlled Hippo signaling in mammals. J. Adler1, C. Wells2; Brescia University, Owensboro, KY, 2Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN

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Rose Rosette Virus: Various cultivars of Rose are currently under attack by the Rose Rosette Virus. This emerging virus is threatening the existence of many Rose cultivars, by inducing rapid proliferation leading to the death of the plant. The Adler laboratory is currently interested in growth control of these plants and any differences between the unaffected and those infected with the virus. We hypothesize that the virus is working through a mechanism that promotes aggressive uncontrolled growth, and we are exploring the role that this virus may be playing to hijack growth control pathways, similar to those seen in mammalian cancer cells. Angiomotin: Mammalian cellular growth is controlled by microenvironmental signals in serum under both normal physiological settings and during cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). In the Wells laboratory, we defined an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and epithelial cell growth.

TUESDAY-POSTER PRESENTATIONS

P2161 Board Number: B685

Tight control of PP2A holoenzyme biogenesis and catalytic subunit recycling. Y. Xing1; 1 Oncology, University of Wisconsin-Madison, Madison, WI The function of metalloenzyme protein phosphatase 2A (PP2A) relies on proper formation of diverse heterotrimeric PP2A holoenzymes, comprising common scaffold and catalytic (PP2Ac) subunits and a variable regulatory subunit. Holoenzyme assembly is highly regulated by carboxylmethylation of PP2Ac. Our previous study demonstrated that the enzymatic reaction of PP2A methylation is highly responsive to PP2A activation via a direct binding of PP2A methyltransferase to the dynamic PP2A active site (Mol. Cell. 2011, 41(3):331-42). Our recent studies showed that PP2A holoenzyme biogenesis requires PP2Ac stable latency and activation. We showed that the entire protein fold of PP2Ac is highly dynamic and tends to partially unfold in the absence of catalytic metal ions, which renders α4-binding that involves the inner structure of PP2Ac near the active site. α4-binding leads to an allosteric relay of conformational changes that perturbs the scaffold subunit binding site at the opposite surface. This unique mode of α4-binding underlies important mechanisms for stable latency of PP2Ac as well as dissociation of PP2A holoenzymes and recycling of PP2Ac in response to cell signaling. Due to the flexible nature of the apo-PP2Ac, the protein fold of apo-PP2Ac needs to be stabilized by PP2A phosphatase activator (PTPA) via broad contacts with the structural elements surrounding the PP2A active site, which stabilizes the PP2Ac active site in a conformation that could bind catalytic metal ions. PTPA-binding also defines a combined ATP-binding pocket that orients ATP phosphoryl groups to bind directly to the PP2Ac active site. This allows ATP to modulate the metal-binding preferences of the PP2Ac active site and drastically enhances binding of Mg2+ by 10,000-fold, which is crucial for acquisition of pSer/Thr-specific phosphatase activity. Consistent with the activation chaperone function of PTPA and activation-dependent PP2A methylation, PTPA stimulates both PP2A phosphatase activity and methylation. Collectively, our studies showed that PP2A stable latency, activation, methylation, and holoenzyme assembly are elegantly coordinated by conformational switches of PP2Ac, underlying novel mechanisms for tight control of PP2A function and regulation in context of cellular signaling.

TUESDAY-POSTER PRESENTATIONS

P2162 Board Number: B686

Real-time Dynamics of the Purinosome Governed by AMPK-associated Signaling Network. D.L. Schmitt1, S. An2; 1 Chemistry and Biochemistry, Univ Maryland- Baltimore County, Baltimore, MD, 2Chemistry and Biochemistry, Univ Maryland Baltimore County, Baltimore, MD

The objective of this study is to determine if signaling networks associated with AMP-activated protein kinase (AMPK) implicate purinosome dynamics in cancer cells. Human de novo purine biosynthesis is a ten-step process catalyzed by six enzymes, using phosphoribosyl pyrophosphate to produce inosine monophosphate. These enzymes have previously been found to compartmentalize together in the cytoplasm upon purine depletion; forming a transient metabolic complex termed the “purinosome.” Recently, there has been growing interest in elucidating signaling networks associated with purinosome dynamics to understand the biological significance of purinosome assembly in cells. Although a metabolic intermediate of purine biosynthesis, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), is a well-characterized allosteric activator of AMPK, human de novo purine biosynthesis has not yet been explored as part of AMPK-mediated signaling circuits. In this study, we have investigated the effect of AMPK activation on purinosome dynamics by monitoring green fluorescent protein-tagged formylglycinamidine ribonucleotide synthase (FGAMS-GFP) as a purinosome marker under fluorescence live-cell microscopy. Dynamic clustering of purinosomes is observed in ~70% of HeLa cells by the addition of AICAR. Subsequently, we reveal that the biguanides, metformin and phenformin, also induce clustering of FGAMS-GFP in ~79% and ~75% of HeLa cells, respectively. These data support our hypothesis that AMPK controls purinosome assembly as a mean to maintain cellular energy homeostasis. We will continue to delineate the coupled network between the purinosome and AMPK in various cancer cell lines by fluorescence live-cell microscopy in combination with established biochemical assays. As de novo purine biosynthesis has been a validated target of cancer therapeutics, we envision that a greater understanding of purinosome dynamics, especially in response to cellular energy needs, possesses high potential to be translated into better chemotherapeutic intervention.

TUESDAY-POSTER PRESENTATIONS

P2163 Board Number: B687

Regulation of IP6K2 activity by TRAF2. H. Rahman1,2, G.J. Miller2; 1 Department of Biology, The Catholic University of America, Washington, DC, 2Laboratory of Biochemistry and Structural Biology, Department of Chemistry, The Catholic University of America, Washington, DC Overexpression of inositol hexakisphosphate kinase-2 (IP6K2) sensitizes multiple cancer cell lines to the apoptotic effect of IFN-α2, IFN-β, TNFα, γ-irradiation and other apoptotic inducing agents, while its knockdown confers resistance to these stressors. IP6K2 binds to tumor necrosis factor receptor associated factor-2 (TRAF2), which serves as an adapter protein for the TNF-receptor superfamily and interleukin-1 receptor and is a major mediator for the regulation of cell survival and cell death for these receptors. Two short amino acid motifs (SXXE) in IP6K2 have previously been shown to be responsible for TRAF2 binding, which inhibits phosphorylation of TAK1 and AKT, thereby inhibiting NF-kB activation and promoting apoptosis. Our objectives are (1) to characterize the interaction of each IP6K2 motif with TRAF2, (2) determine the impact of this interaction on IP6K2 catalytic activity and (3) determine the structural basis of this interaction.

P2164 Board Number: B688

Analysis of secreted protein kinase substrates. A. Sreelatha1, K. Orth1, J. Dixon2, V. Tagliabracci2; 1 Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, 2department of pharmacology, UC San Diego, San Diego, CA Kinases mediate protein phosphorylation of intracellular and secreted proteins to regulate various cellular processes. Until recently, the kinases that phosphorylate secreted proteins were unidentified. One member of the atypical secreted protein kinase family, Fam20C phosphorylates the S-x-E/pS motif of over 100 secreted proteins. Genetic diseases such as Raine syndrome that result from Fam20C mutations highlight the importance of Fam20C phosphorylation of secreted protein. Further analysis of Fam20C substrates reveal that it also regulates wound healing and lipid homeostasis. Using bioinformatics, biochemistry and genetics we study the substrates of Fam20C kinases and the role of phosphorylation on substrate function.

TUESDAY-POSTER PRESENTATIONS

P2165 Board Number: B689

Mechanisms and functional consequences of T cell microclustersMechanisms and functional consequences of T cell microclusters. X. Su1, J. Ditlev2, E. Hui3, J. Rumpf3, S. Banjade2, J. Taunton3, M.K. Rosen4,5, R. Vale3; 1 Department of Cell and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, 2University of Texas Southwestern Medical Center, Dallas, TX, 3University of California, San Francisco, San Francisco, CA, 4Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, 5Howard Hughes Medical Institute, Dallas, TX The assembly of higher-order protein structures has been suggested to promote signaling whereas a clear cause-and-effect relationship has been difficult to establish, largely due to limited knowledge about how these structures are formed. To investigate the mechanisms and functional properties of membrane-bound signaling clusters, we developed an in vitro reconstitution system in which signaling clusters are assembled from individual purified components on synthetic supported lipid bilayers. We found multivalent protein-protein interactions can drive the cluster formation of LAT, a signaling hub protein that recruits multiple downstream effectors for TCR signaling. LAT clusters enrich Sos1(RasGEF), PLCg1(Phospholipase), and Nck1(connecting to actin polymerization). Clustering renders resistance to dephosphorylation of LAT by protein tyrosine phosphatase, which suggests a mechanism of how clustering might maintain TCR signaling. In addition, we found two adaptor proteins Grb2 and Gads are redundant and competitive for clustering LAT. Finally, through reconstituting a signaling pathway from TCR triggering to LAT clustering, and to actin polymerization, we showed LAT cluster nucleates actin filaments and promotes membrane-proximal actin network formation. Together, we propose TCR triggering induces a phase transition of LAT complex that promotes downstream signaling.

P2166 Board Number: B690

Identification of Novel Oncogenic Drivers in Cancer Cell Lines Through a Systems Pharmacology Approach. N. Sakur1, N. Salmen1, M.Y. Fink1; 1 LIU Post, Brookville, NY Oncogenes drive cancer cell growth and identification of novel candidate oncogenes is a necessary step for developing new drugs. Integration of data from genomic, proteomic, and literature findings, in cancer cell lines, can generate models of altered signaling networks in different cancer types and subtypes. Several large-scale systems biology studies have screened cancer cell lines for drug sensitivity. In this study, systems-level network models of cancer cell lines have been generated and, based on these models, drug sensitivity predictions have been made. These predictions were then compared to known sensitivities from published studies. The results identified unexpected interactions between cell

TUESDAY-POSTER PRESENTATIONS line networks and drugs. The ZR-75-30 breast cancer cell line displayed sensitivity to sorafenib, a multikinase inhibitor, that targets several receptor tyrosine kinases and RAF. This reported sensitivity has been confirmed and the specific target and pathway affected in ZR-75-30 cells is currently being explored.

P2167 Board Number: B691

The role of Sp1 in p21CIP1 regulation in B-RafV600E cancer. M. Karkhanis1, J. Park2; 1 Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 2Biochemistry, Med Coll Wisconsin, Milwaukee, WI We have previously reported that mortalin, an Hsp70 family chaperone, can suppress p21CIP1 expression in certain B-RafV600E cancer cells. We observed that mortalin depletion in B-RafV600E cancer cells results in p21CIP1-mediated cell cycle arrest in a MEK-ERK dependent manner. We thus proposed that mortalin upregulation is a potential mechanism for certain B-RafV600E cancer cells to bypass growth arrest caused by aberrant MEK-ERK activation. Interestingly, mortalin depletion could induce p21CIP1 expression independently of p53 in certain B-RafV600E cancer cells. In this study, we investigated the mechanism by which mortalin regulates p21CIP1 expression. Using luciferase reporter truncation analysis, we have identified a proximal p21CIP1 promoter region responsive to mortalin depletion in p53 mutated cancer cells. Interestingly, when Sp1 transcription factor-responsive elements in this region were mutagenized, the p21CIP1 promoter reporter was no longer responsive to mortalin depletion. Consistent with this, our ChIP analysis revealed that mortalin knockdown could induce Sp1 binding to p21CIP1 promoter in a MEKERK dependent manner. Furthermore, RNA interference of Sp1 substantially attenuated p21CIP1 expression induced by mortalin depletion in these cells. In summary, our data demonstrate that, in BRafV600E cancer cells, Sp1 can regulate p21CIP1 transcription even in the absence of wild type p53. This study suggests that Sp1 may have an important role in p21CIP1 regulation in certain cancer types.

P2168 Board Number: B692

Pseudophosphatase MK-STYX regulates RhoA signaling, and enhances branching in PC12 cells. D.A. Banks1, B.M. Flowers1, S.D. Hinton1; 1 Biology, College of William Mary, Williamsburg, VA The pseudophosphatase MK-STYX (mitogen activated kinase phosphoserine/threonine/tyrosine binding protein) is a member of the dual specificity MAPK phosphatases (MKP) that is catalytically inactive due to mutations in its active site (HCX5R to FSX5R). Previously, we reported that MK-STYX induces neuritelike outgrowths in PC12 cells and enhances the effects of NGF stimulation by dramatically increasing the

TUESDAY-POSTER PRESENTATIONS number of neurites. Furthermore, MK-STYX induced neurite formation in the presence of a MEK inhibitor, suggesting that MK-STYX exerts its effects independently of the extracellular signaling related kinase ERK pathway. RhoA activity assays showed that MK-STYX decreases RhoA activation, whereas knockdown of MK-STYX increased RhoA activation- suggesting that MK-STYX is a regulator of RhoA signaling. Here, we further investigate the role of MK-STYX as a regulator of RhoA signaling by examining MK-STYX’s effects on cofilin, a RhoA downstream effector. Cofilin is an actin binding protein that depolymerizes actin, resulting in cytoskeletal reorganization. MK-STYX did not alter cofilin expression but did alter its phosphorylation. Phosphorylated cofilin (p-cofilin) stabilizes actin filaments, causing persistence of neurites and continued growth. MK-STYX caused an increase in phosphorylated cofilin over time after NGF stimulation, whereas, levels of p-cofilin in control cells stayed consistent over time. Furthermore, p-cofilin decreased when MK-STYX was knocked down with shRNA. Throughout the course of these experiments, we noticed that cells overexpressing MK-STYX developed more neurites, which appeared to branch. To address whether MK-STYX induced a distinct branching pattern in PC12 cells, we scored cells to categorize the neurites branching pattern. Initially, cells were scored for the presence of neurites greater than 20 μm. Cells with at least one extension meeting this criterion were scored for primary (extension from cell body) and secondary neurites (extensions from primary neurite). Cells not stimulated by NGF but expressing MK-STYX showed a slight shift in overall distribution of primary and secondary neurites compared to the control. NGF stimulated cells overexpressing MK-STYX showed a greater shift in distribution of both primary and secondary neurites compared to the control. These data demonstrate that MK-STYX causes branching, which is regulated by the Rho GTPases such as RhoA, in PC12 cells. Taken together these data provide more substantial evidence for MK-STYX as a regulator of RhoA signaling and neuronal differentiation.

P2169 Board Number: B693

Characterization of a novel yeast gene, XRR1, which confers resistance to the antifungal rapamycin and whose protein may be localized to stress granules. M.J. Swede1, V. Rivera1, R. Suwal2; 1 Health Sciences, Long Island University, Post, Brookville, NY, 2Biomedical Sciences, Long Island University, Post, Brookville, NY Cell cycle arrest in response to the macrolide antifungal agent rapamycin (sirolimus) in the yeast S. cerevisiae is mediated through the binding of FKBP12 protein (FPR1p) with rapamycin, forming a complex with the TOR1 and TOR2 proteins. Deletion mutations in the FPR1 gene and specific point mutations in TOR1 and TOR2 genes, which prevent interaction with the FKBP12-rapamycin complex, result in a resistance to rapamycin phenotype. We have characterized a novel gene, XRR1 (eXhibits Rapamycin Resistance), whose null mutant exhibits temperature sensitive resistance to rapamycin. The XRR1 gene product was shown via a systematic yeast two-hybrid analysis (Uetz et al, 2000) to physically interact with FKBP12 (FPR1p). The XRR1 gene exhibits sequence homology to the A1pp (Macro) domain

TUESDAY-POSTER PRESENTATIONS specific for binding ADP-ribose. This domain is found in the C-terminus of the mammalian macroH2A histone variant. Characterization of the XRR1 gene suggests a role in the rapamycin resistance pathway. We have observed that the XRR1 null mutation results in an eight-fold increase in growth in the presence of 100 ng/ml rapamycin at 37 C after six hours of growth compared to five-fold increase at 30 C. Wildtype isogenic strains exhibit only a 2.8 fold increase of growth under the same growth conditions. The XRR1 gene product may be localized to stress granules under conditions of nutritional stress. Microscopic imaging of Xrr1p-GFP indicates three subcellular distribution patterns under normal growth conditions: we have observed uniform cytoplasmic staining and staining in which the vacuolar staining is excluded in the majority of the cells observed, and punctate cytoplasmic staining in a small percentage of cells. However, under conditions of glucose starvation, we observed an increase in the punctate staining pattern and a concomitant reduction in cytoplasmic staining. This phenomenon does not appear to occur under conditions of physical stress, as we do not see an increase in punctate staining after a of 42 C heat shock. This work is supported, in part, by a grant from the Dextra McGongle Baldwin Foundation to MJS

P2170 Board Number: B694

Calcineurin Interacting Protein works as a Modulator of tax-6 Signaling Pathway. H. Jung1, S. Lee1, J. Ahnn2; 1 BK21Plus BDR Team, Hanyang Univ, Seoul, Korea, 2Hanyang Univ, Seoul, Korea Calcineurin is a serine/threonine phosphatase regulated by Ca2+-binging calmodulin. Calcineurin consists of two subunits, catalytic subunit A (TAX-6) and regulatory subunit B (CNB-1). Calcineurin substrate candidates of C.elegans have been screened by yeast two-hybrid assays. We found that CNP-2 interacted with the catalytic domain of TAX-6. The CNP-2 is expressed in head neurons, posterior intestine, spermathecal valve, spermatheca-uterus valve and cell junction/ membrane in spermatheca. In males, the CNP-2 is expressed in spicule retractor muscles, rays and at the end of fan, which are mating behavior-mediated organs. Also, CNP-2 is required in the ovulation, arrangement of eggs, ray structure and mating behaviors. CNP-2 seems to be critical in the mating efficiency and sperm activation. We are currently investigating the roles of cnp-2 in tax-6 pathway, regarding IP3 signaling.

TUESDAY-POSTER PRESENTATIONS

P2171 Board Number: B695

Regulation of body size in C.elegans via counteracting between phophatase and kinase. T. Choi1, S. Lee1, J. Ahnn2; 1 BK21Plus BDR Team, Hanyang Univ, Seoul, Korea, 2Hanyang Univ, Seoul, Korea Calcineurin, Ca2+/calmodulin-dependent sertine/threonine phosphateas, has been reported to function in body size regulation in C. elegans. We confirmed neuronal function of calcineurin is important cue in the regulation of body size. RNAi screening to identify kinases which counteract with calcineurin activity revealed 33 kinases that rescued the phenotype of small body size in tax-6(lf) background. Among the positive candidates, we found mbk-2 which encodes one of two C. elegans members of the Dualspecificity Yak1-Related Kinase (DYRK) family proteins partially rescued the body size of tax-6(lf). One of the candidates target gene, oma-1 RNAi also showed partial rescue of tax-6(lf). We are doing experiments to elucidate the underlying working mechanisms.

P2172 Board Number: B696

High Glucose-Induces Oxidative Stress in HEK-293 Cells. E. Bradshaw1, G. Beggs1, M. Cartafalsa1; 1 Biology, Florida Southern College, Lakeland, FL Approximately 366 million people worldwide are affected by diabetes. A long-term complication of diabetes mellitus is kidney disease, also known as diabetic nephropathy. To date, the underlying causes of diabetes-induced kidney failure are not well understood but recent evidence points to mitochondrial dysfunction. In models of diabetic nephropathy, there is increased mitochondrial stress, which is associated with kidney cell death. In this study, HEK-293 cells were treated with 25mM glucose and ATP production and oxidative stress was assessed with an ATP assay and TBARS assay, respectively. HEK-293 cells treated with 25mM glucose for 24 hours increased ATP production as well as lipid peroxidation compared to control. These data indicate high glucose conditions influence the mitochondria in this cell type. To determine the molecular mechanism for high sugar environment changes in ATP production and oxidative stress, we examined the mTOR pathway. Recent clinical data implicates the Akt/mTOR pathway in diabetic patients with kidney disease. Preliminary western blot evidence shows that dextrose increases phosphorylation of p70S6 kinase in HEK-293 cells. This effect is inhibited by rapamycin, an inhibitor of the mTOR pathway. Thus, these data suggest mTOR may play a role in the increased ATP production and oxidative stress induced by a high sugar environment.

TUESDAY-POSTER PRESENTATIONS

P2173 Board Number: B697

Regulation of DAG Lipase Activity – Implications for ‘On-Demand’ Endocannabinoid Signallling. R.L. Markwick1, E. Williams1, M. Maresca2, P. Singh1, F. Howell1, G. Williams1, P. Doherty1; 1 Wolfson CARD, King's College London, London, England, 2Astra Zeneca, Molndal, Sweden Diacylglycerol lipases (DAGL α and β) are key enzymes in the biosynthesis of 2-AG, the major endocannabinoid (eCB) in the brain. 2-AG acts on CB1 and/or CB2 receptors and DAGL-dependent eCB signalling regulates a large number of responses including axonal growth during development, as well as neurogenesis and retrograde synaptic plasticity in the adult (Oudin et al., 2011). The enzymes also play a major role in driving pathogenic inflammatory responses via a DAGL/MAGL pathway that generates AA as a precursor to prostaglandin synthesis. DAGL antagonists are being developed as novel therapeutics based on their ability to regulate eCB-mediated signalling and/or inflammatory responses. The DAGLs appears to display ‘on-demand’ synthesis, generating increasing amounts of 2-AG in response to cellular messengers. Based on homology modelling and analysis of phosphoproteomic databases we have postulated that a phosphorylation dependent displacement of an intramolecular regulatory loop activates DAGLα/β activity by allowing access of the natural substrate DAG to the active site of the enzyme (Reisenberg et al., 2012). We will report a cellular assay to measure DAGLα function. This assay measures DAGL-dependent activation of the CB1 receptor, reporting on physiologically relevant DAGLα activity in a cell. Evidence for phosphorylation-dependent activation of DAGLα, required for eCB signalling, and use of CRISPR/Cas9 System to systematically switch off’ endogenous DAGLα, will be presented. Oudin, M.J., Hobbs, C. & Doherty, P. (2011) DAGL-dependent endocannabinoid signalling: roles in axonal pathfinding, synaptic plasticity and adult neurogenesis. Eur J Neurosci, 34, 1634-1646. Reisenberg, M., Singh, P.K., Williams, G. & Doherty, P. (2012) The diacylglycerol lipases: structure, regulation and roles in and beyond endocannabinoid signalling. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 367, 3264-3275.

P2174 Board Number: B800

Analysis of the Regulatory Mechanism by which Shear Stress and Stretch Force Induce Nitric Oxide Production in Endothelial Cells. J. Wang1,2, K. Kobayashi-Kato1, M. Hagiwara1, N. Ishida1, K. Matsushita1,2; 1 Oral Disease Research, National Center for Geriatrics and Gerontology, Nagoya, Japan, 2National Center for Geriatrics and Gerontrogy, Obu, Aichi, Japan Blood vessels are subjected to constant mechanical forces from blood pressure and flow, which cause internal stresses (endothelial shear stress and circumferential wall stress, respectively). The mechanical

TUESDAY-POSTER PRESENTATIONS forces not only cause morphological changes of the endothelium and blood vessel wall but also trigger biochemical and biological events. Physiologic shear stress and stretch force exert vasoprotective effects s via nitric oxide (NO) and provide a homeostatic oxidative balance. Perturbation of these mechanical forces can disturb biochemical homeostasis and lead to vascular remodelling and possible dysfunction. It is known that these distinct biological endpoints are caused by some common biochemical pathways. However, little is known about the difference of biological pathways evoked by shear stress and by stretch force. In this study, we focused on NO production induced by shear stress and by stretch force and compared the regulatory mechanism of these stimuli with the NO production in endothelial cells. We examined NO production induced by short (30 sec -10 min) exposure of endothelial cells (HUVECs) to laminar shear stress and sustained stretch force. A high level of NO was produced and peaked at 1 min and 4 min after exposure to shear stress in HUVECs. On the other hand, a low level of NO was produced at 30 sec after exposure to stretch force. Chelation of intracellular Ca2+ with BAPTA-AM completely abolished the phosphorylation of Akt and eNOS and the production of NO induced by shear stress, whereas BAPTA-AM suppressed only half of the phosphorylation of Akt and eNOS and NO production induced by stretch force and, conversely, complete depletion of Ca2+ in the culture strongly enhanced NO production by HUVECs. These results suggest that the regulatory mechanism by which endothelial cells produce NO depends on the type of stress and that the role of Ca2+ signal in NO production may differ with stimulation from shear stress and stimulation from stretch force. The results may be useful for understanding the responses of blood vessels to internal vessel stresses induced by blood pressure and flow.

Rho-Family GTPases P2175 Board Number: B801

A Novel Role for Wiskott - Aldrich syndrome Protein (WASP) in the Regulation of Macrophage Nanotubes. K. McCoy-Simandle1, D. Cox1,2; 1 Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 2Albert Einstein College of Medicine, Bronx, NY Macrophages are known to interact with tumor cells, pathogens, lymphocytes endothelial cells, and many other cell types via small molecule intermediaries. However, traditional soluble means of communication do not account for all macrophage interactions suggesting other means of communication. Recently discovered tunneling nanotubes (TNTs) are membranous channels that connect different cells together. These connections are thin and may be up to several cell diameters long. These structures occur in many immune cells in vitro and in vivo allowing for transfer of signals, vesicles, and organelles. Even pathogens can use these TNTs to transfer from cell to cell. Due to their length and the ability to form networks of TNTs, cell signals can be propagated over vast distances quickly and without interaction with extracellular fluid. Because these structures have no known

TUESDAY-POSTER PRESENTATIONS markers and are sensitive to light exposure, shearing force, and chemical fixation, little is known about the formation of these structures in macrophages. In this study, TNTs are characterized in macrophages by in vitro live cell imaging which minimizes TNT breakage. Live visualization of the membrane was achieved by either staining with the fluorescent membrane label, FM1-43FX, or by expression of a fluorescent protein targeted to the plasma membrane using a CAAX motif. Interestingly, macrophages polarized to either the pro-inflammatory or pro-tumorgenic states, using LPS or IL-4, show increased number of TNTs. Formation of TNTs was also investigated when actin-polymerization, PI3 kinase or Src kinase pathways were blocked. Moreover, it was determined that Wiskott-Aldrich syndrome protein (WASP) regulates formation and stabilization of TNTs. Depletion of WASP by shRNA in a macrophage cell line greatly reduces formation of these structures. Furthermore, while phosphorylation of WASP was minimally required for the formation of these structures, it was important for TNT stability. These results reveal a novel role of WASP in macrophage TNT formation. Interestingly, they also suggest that some of the immune deficiencies observed in patients with Wiskott-Aldrich syndrome may be due to the decreased number of TNTs. More research will need to be done to find potential ways to manipulate TNTs therapeutically.

P2176 Board Number: B802

Minding the GAPs: A novel RhoGAP family of RPEL proteins as target for regulation by G-actin. J. Diring1, S. Mouilleron2, R. Treisman1; 1 Signal Transduction and Transcription Laboratory, CRUK London Research Institute, London, UK, 2 Protein Structure Unit, CRUK London Research Institute, London, UK Small GTPases of the Rho family control the dynamics of the cytoskeleton through multiple effector proteins that regulate the assembly, activity and turnover of actin-based structures, thereby controlling cell adhesion, morphology and motility in response to extracellular signals. Activation of these GTPases lead to the accumulation of filamentous actin (F-actin) through both filament stabilisation and de novo polymerisation, with concomitant depletion of cellular levels of monomeric actin (G-actin). RPEL proteins are a novel family of G-actin-binding proteins, which are able to sense fluctuations in cellular Gactin concentration, and respond to maintain cytoskeleton homeostasis and remodelling. Two families of RPEL proteins have been described: the MRTF family, which couples gene transcription to F-actin availability, and the Phactr family, which coordinates actomyosin crosslinking with F-actin level. We identified two novel RhoGAP subfamilies of RPEL proteins, among which Arhgap12 has been the focus of our study. Our data show that Arhgap12 is a GTPase activating protein towards the small GTPase Rac1. It regulates the remodelling of the actin cytoskeleton, cell motility and invasion. Moreover, direct binding of G-actin regulates its subcellular localisation and inhibits its activity by competing with Rac1 binding. Structural studies and point mutation analyses indicate that actin interacts both with the RPEL motif and with a conserved sequence embedded in the GAP domain. We

TUESDAY-POSTER PRESENTATIONS propose that Arhgap12 orchestrates a homeostatic feedback loop to coordinate G-actin level to the activation of Rac signalling.

P2177 Board Number: B803

Development of a proof-of-concept small molecule inhibitor of MgcRacGAP identifies new potential functions of MgcRacGAP in cell division and signaling. A. van Adrichem1, A. Fagerholm2, L. Turunen1, A. Lehto1, J. Saarela1, A. Koskinen2, G. Repasky1, K. Wennerberg1; 1 Institute for Molecular Medicine Finland FIMM, Helsinki, Finland, 2Chemistry, Aalto University, Espoo, Finland The MKLP1/MgcRacGAP/Ect2 protein complex is well-established as a core regulator for initiation and progression of cytokinesis. Interestingly, it also appears to be involved in oncogenesis through a cytokinesis-independent mechanism where overexpression drives invasiveness, epithelial-tomesenchymal transition and chromosomal segregation aberrations. Furthermore, MgcRacGAP is reported to be essential for nuclear translocation of STAT transcription factors, including STAT3, which is a well-known promoter of oncogenesis and cancer drug resistance. Small molecule modulators of MgcRacGAP would be very valuable research tools for the detailed study of the many apparent roles of MgcRacGAP. Therefore, we developed a biochemical high throughput screen for identification of inhibitors of MgcRacGAP and screened 370,000 compounds. From this campaign, we identified the first publicly described small molecule RhoGAP inhibitor. This compound (MINC1) showed selective biochemical inhibition of MgcRacGAP at low micromolar concentrations. Cellular treatment with MINC1 resulted in inhibition of proliferation and increased frequencies of multinucleation, in agreement with it being a cytokinetic inhibitor. Surprisingly, we also observed collapsed mitotic spindles that resembled a failure at prophase/pro-metaphase similar to a PLK1-inhibited phenotype. Since MgcRacGAP is a substrate of PLK1, this indicates that MgcRacGAP may also play a role in PLK1-regulated mitotic processes. In agreement with MgcRacGAP regulating cellular polarity and invasion, treatment with MINC1 or siRNA-mediated knockdown of MgcRacGAP caused a strong inhibition of MDA-MB-231 breast cancer cell line matrigel invasion. Finally, based on previous published reports proposing that the MgcRacGAP-Rac1 complex plays a vital role in STAT family transcription factor nuclear translocation, we examined the capability of MINC1 to disrupt STAT signaling. Surprisingly, we instead found that both MINC1 treatment and siRNA-mediated knockdown of MgcRacGAP caused an activation of STAT3 and STAT5B signaling in several cell lines, suggesting that the proposed role of MgcRacGAP in STAT family transcription factor function may not be generally applicable. In conclusion, we have identified MINC1, a small molecule inhibitor of MgcRacGAP. This compound represents the first described selective small molecule inhibitor of a small G-protein GTPase activating protein. We utilized this inhibitor to study the function of MgcRacGAP in cell division and in other biological contexts with both expected and unexpected results. Ongoing work is focused on optimizing the compound to allow further detailed studies of the role of MgcRacGAP.

TUESDAY-POSTER PRESENTATIONS

P2178 Board Number: B804

Investigating Rho GTPase Pattern Formation During Wound Repair: The Role of RhoGAP1/8. N. Davenport1, W. Bement1,2,3; 1 Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI, 2 Department of Zoology, University of Wisconsin-Madison, Madison, WI, 3Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI The ability to respond to physical damage is an essential and evolutionarily conserved feature of single cells and tissues. The Rho family of GTPases has been shown to coordinate the cytoskeletal rearrangements needed to reestablish the integrity of the plasma membrane and underlying cortex in response to cell damage. The precision with which Rho and Cdc42 are activated and maintained in distinct activity zones throughout the healing process suggests the involvement of multiple Rho GTPase regulators at the wound. To date, a candidate screen for guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) involved in regulating Rho GTPase activity at the wound has identified a single wound regulator. By continuing the candidate screen, we have identified two additional GAPs, RhoGAP1 and RhoGAP8, as potential wound regulators. While initially labeling early and recycling endosomes in resting cells, eGFP-tagged RhoGAP1 is recruited between the zones of active Rho and Cdc42 in Xenopus laevis oocytes upon wounding. These results have been confirmed by the discovery of endogenous RhoGAP1 at wounds via immunofluorescence microscopy. Targeting of RhoGAP1 to wounds is mediated by its C-terminal region which contains both a GAP domain and a proline-rich region. Reduction of active Rho due to overexpression of untagged RhoGAP1 and alteration of GTPase activity zone position by dominant negative RhoGAP1 supports a hypothesis that RhoGAP1 acts as an enzymatic barrier to promote proper GTPase zone segregation throughout wound repair. We have shown that RhoGAP1 and RhoGAP8 colocalize at single-cell wounds which, in addition to previously reported evidence of their interaction, suggests they may form a complex to regulate Rho GTPase activity. We have also used both RNAi and morpholinos to effectively knockdown proteins in Xenopus embryos 24 hours post-fertilization. Knockdown of RhoGAP1 in embryos appears to increase the amount of cytoplasmic F-actin in cells. Further, wounding of Xenopus embryos causes 3XeGFP-RhoGAP1 to be displaced from its punctate, junctional localization to become diffuse and cytoplasmic. This displacement appears to follow an intracellular Ca2+ increase and precedes increases in both active Rho and F-actin at cell-cell junctions, suggesting RhoGAP1 may regulate a key step in reorganizing the actin cytoskeleton during the multicellular wound response.

TUESDAY-POSTER PRESENTATIONS

P2179 Board Number: B805

A Novel GEF Independent Role of P-Rex1 in GPCR Trafficking. M.J. Baker1, E. Maunders1, H.C. Welch1; 1 The Babraham Institute, Cambridge, United Kingdom P-Rex proteins are a family of guanine nucleotide exchange factors (GEFs) for the Rho GTPase Rac. They use their GEF function to catalyse the release of GDP from Rac, thus allowing GTP to bind transforming Rac into its active GTP bound conformation. It has been well established that the GEF function of P-Rex1 mediates membrane ruffling and cell migration, as well as reactive oxygen species production. All known cellular responses to P-Rex1 activity are as a result of Rac signalling, often involving actin filament reorganisation. Therefore, there are no known GEF independent roles of P-Rex1. Furthermore, P-Rex proteins are known to be important for tumour growth and metastasis in several types of cancer including prostate, breast and melanoma. The role of P-Rex family members in trafficking has not been studied in great detail to date, however, there has been work published which suggests that P-Rex1 has roles in vesicle trafficking. This includes evidence to suggest roles for P-Rex1 in trafficking of the GLUT4 transporter1. We therefore decided to investigate a potential role of P-Rex1 in stimulation dependent GPCR internalisation, in an effort to identify the function of P-Rex1 in trafficking. During this study we have identified a GEF independent function of P-Rex1 in membrane trafficking. This novel function of P-Rex1 is the first GEF independent function to be identified. Using fixed cell immunofluorescence imaging we show that overexpressed P-Rex1 has an inhibitory effect on the internalisation of a GFP tagged GPCR and that this function of P-Rex1 does not require its GEF activity. We also identify the domains of P-Rex1 that are required for this inhibitory role as part of a first step to deciphering the mechanism behind this exciting and new role of P-Rex1. 1. Balamatsias, D., et al (2011) Journal of Biological Chemistry286, 43229-43240.

P2180 Board Number: B806

Cdc42 Activation is Necessary for Sustained Oscillations of Ca2+ and PIP2 in RBL Mast Cells Stimulated by Antigen. D.A. Holowka1, M.M. Wilkes1, B. Baird1; 1 Chemistry and Chemical Biology, Cornell University, Ithaca, NY Antigen stimulation of mast cells via FcεRI, the high-affinity receptor for IgE, triggers a signaling cascade that requires Ca2+ mobilization for exocytosis of secretory granules during the allergic response. To characterize the role of Rho GTPases in FcεRI signaling, we utilized a mutant RBL cell line, B6A4C1, that is deficient in antigen-stimulated Cdc42 activation important for these processes. B6A4C1 cells exhibit

TUESDAY-POSTER PRESENTATIONS severely attenuated Ca2+ oscillations in response to antigen, and these are restored to wild type RBL2H3 levels by expression of constitutively active Cdc42 G12V or by a GEF for Cdc42, DOCK7. Ca2+ oscillations are not restored when the C-terminal di-arginine motif of active Cdc42 is mutated to diglutamine, demonstrating a role for these basic residues in this Rho family function. Antigen-stimulated FcεRI endocytosis, which occurs independently of stimulated Ca2+ influx, is also defective in B6A4C1 cells, and Cdc42 G12V reconstitutes this response as well. Thus, activation of Cdc42 occurs prior to and is critical for antigen-stimulated pathways leading separately to both Ca2+ mobilization and receptor endocytosis. Accounting for these downstream functional consequences, we show that Cdc42 G12V reconstitutes antigen-stimulated oscillations of phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane in B6A4C1 cells, pointing to Cdc42 participation in the regulation of stimulated PIP2 synthesis as a key functional role for this Rho family GTPase in mast cells.

P2181 Board Number: B807

Rac1-dependent activation of Cdc42 during CSF-1 response by macrophages. V. Miskolci1, H. Park1, L. Hodgson1, D. Cox1,2; 1 Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 2Albert Einstein College of Medicine, Bronx, NY Rac1 and Cdc42, members of the Rho family of small GTPases, are central regulatory hubs of actin cytoskeleton rearrangement required during directed migration. It was previously reported that genetic deletion of Rac1 had no significant effect on macrophage chemotaxis, although a range of abnormalities were observed both in cell morphology and behavior. These results suggest that some compensatory action may occur, possibly by other Rho family members via increased or re-localized activity. To address these potential scenarios we used FRET-based Rho GTPase biosensors, as they can detect dynamic changes both in the levels and localization of Rho GTPase activity. We found that Cdc42 activity was elevated in response to CSF-1 but not significantly localized at F-actin-rich protrusions in a murine macrophage RAW 267.4 cell line, whereas Rac1 activity was highly localized at these same protrusions. However, there was no detectable alteration in Cdc42 activity or localization following reduction of Rac1 levels using shRNA. Surprisingly, shRNA-depletion of Rac1 in these cells produced inhibitory effects on CSF-1 – mediated Cdc42 activation, suggesting a previously unknown potentiation effect by the Rac1 signaling axis. This new regulatory mechanism of Rac1 activating Cdc42 is not due to defects in CSF-1 receptor expression or signaling, since CSF-1 stimulation resulted in similar levels of receptor tyrosine phosphorylation in control versus Rac1-depleted macrophages. We report here a novel signaling mechanism in which Cdc42 activation is dependent on Rac1, and we hypothesize that a Rac1 – activated GEF targeting Cdc42 is involved in the CSF-1 stimulation pathway in macrophages.

TUESDAY-POSTER PRESENTATIONS

P2182 Board Number: B808

AMPylation of Rho GTPases disrupts multiple host signaling processes. A.R. Woolery1, X. Yu2, J. LaBaer2, K. Orth1; 1 Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, 2Biodesign Institure, Arizona State University, Tempe, AZ Rho GTPases are frequent targets of virulence factors as they are keystone signaling molecules in many processes. Modifications of their switch-1 loop are known to be a potent method of disabling the actin cytoskeletal machinery, but Rho GTPases are also important for many other signaling pathways. We attempted to broaden the understanding of the consequences of switch-1 loop modification in downstream signaling using AMPylation by the Vibrio parahaemolyticus type three secreted effector VopS. Using infection models, biochemistry and versatile self-assembled microarrays, several novel effects of Rho GTPase AMPylation were identified. We found that multiple signaling interactions of GTPases were inhibited, including NFkB, MAP kinase, the Inhibitor of Apoptosis proteins (IAP) and the phagocytic NADPH oxidase system (NOX2.) Phosphorylation of IkB**a **and JNK kinase was inhibited in a VopS-dependent manner during infection with Vibrio parahaemolyticus. VopS also sequestered p65 in the cytosol during infection, while removal of VopS allowed robust translocation. AMPylation also prevented the generation of superoxide by the phagocytic NADPH oxidase complex. Furthermore, the interaction of GTPases with the E3 ubiquitin ligases cIAP1 and XIAP was hindered, leading to decreased degradation of Rac and RhoA during infection. Finally, we screened for novel Rac1 interactions using a nucleic acid programmable protein array (NAPPA) and discovered that Rac1 binds to the protein C1QA, a protein known to promote immune signaling in the cytosol. Interestingly, this interaction was disrupted by AMPylation. In addition to these findings, we also adapted NAPPA to use fluorescent probes to screen for novel substrates of protein transferases, including AMPylation. We conclude that modification of the switch-1 loop by VopS and other toxic proteins is a multifaceted virulence mechanism that counters several host immunity strategies.

P2183 Board Number: B809

Rac1 orchestrates transcriptional and post-transcriptional regulation of matrix remodelling during fibroblast-mediated matrix contraction. H. LI1, V. Tovell1, D. Paull1, D. Simpson2, P.T. Khaw1, M. Bailly1; 1 Department of Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom, 2Centre for Experimental Medicine, Queen's University, Belfast, United Kingdom Fibrosis is a major cause of morbidity in humans, with over 40% of all death in the developed world attributable to some form of fibrotic disease. In the eye alone, tissue contraction and scarring are involved in the pathogenesis or failure of treatment of virtually all major blinding diseases. However, the molecular mechanisms underlying fibrosis and scarring are still poorly understood. We previously

TUESDAY-POSTER PRESENTATIONS identified Rac1 as one of the major player underlying fibroblast-mediated tissue contraction, and showed that an early transient treatment with Rac1 inhibitor NSC23766 is sufficient to block long-term matrix remodelling and tissue contraction in vitro and ex-vivo. We have now performed a genome wide microarray analysis in primary human conjunctival fibroblasts during contraction in vitro with/without treatment with NSC23766. We have found that the cells undergo dramatic shift in gene expression during the early stages of contraction, akin to a wound activation profile, with over 3000 genes changing expression from day 0 to day3, in which a specific gene profile including matrix metalloproteinases (MMP) 1, 3 and 10, inflammatory molecules and cytokines are significantly up-regulated. This “activation” profile recedes when the contraction slows down at day5, with most of the genes changing back in the opposite direction from day3 to day 5, and the gene profile of later contraction is linked to long-term fibrosis with gene expressions found in common with a few human ocular fibrotic diseases. Transient treatment with the Rac1 inhibitor resulted in an almost complete abrogation of the activation phase, with the cells assuming a day5 resting cell profile. In addition, we showed that whilst Rac1 inhibition did not affect the mRNA expression of most MMPs upregulated during contraction, it lead to a significant reduction in overall MMP activity, in accordance with the previously observed reduction in matrix degradation. This suggests that blocking Rac1 activation during the early stages of contraction is sufficient to fully prevent the cells from entering the contractile pathway as a whole, as Rac1 appears to mastermind a complete wound healing response by acting both at the transcriptional and posttranscriptional level.

P2184 Board Number: B810

Rac3 promotes invadopodia maturation and matrix degradation. S.K. Donnelly1, J. Bravo-Cordero1, J.S. Condeelis2, L. Hodgson2; 1 Albert Einstein College of Medicine, Bronx, NY, 2Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY Metastasis is responsible for ninety percent of all cancer related deaths. One of the first steps in the metastatic cascade is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actinbased protrusions that mediate the extracellular matrix degradation necessary for the invasion of tumor cells. Therefore deciphering the mechanisms of invadopodia regulation is critical to furthering our understanding of cancer cell dissemination. A number of studies have implicated the small GTPase Rac3 in tumor cell invasion but its mechanism of action has not yet been elucidated. Despite the similarity of Rac3 and the better-studied Rac1, there is also evidence to suggest that these isoforms have different functional roles. Rac1 has recently been shown to regulate the disassembly of invadopodia. We now show that, in direct contrast to Rac1, Rac3 regulates the maturation of invadopodia in two breast cancer cell lines, Mtln3 and MDA-MB-231. As Rac1 and Rac3 share 93% sequence homology, traditional approaches are not optimally suited to precisely define the spatial and temporal dynamics of Rac3 activation at invadopodia. Thus we have generated and validated a novel single-chain FRET-based Rac3 biosensor. Preliminary results suggest that there is a transient activation of Rac3 at invadopodia. Taken

TUESDAY-POSTER PRESENTATIONS together our data suggests that Rac3 functions as a molecular switch that regulates the transition of invadopodia from precursor to mature structure.

P2185 Board Number: B811

Cell invasion through basement membrane requires multiple GTPases and is initiated by CDC-42 directed invadopodia formation. M.R. Clay1, L.R. Lilley Lohmer1, J.W. Ziel1, Q. Chi1, D.R. Sherwood1; 1 Biology Department, Duke University, Durham, NC Basement membrane (BM) is a thin, dense, sheet-like extracellular matrix that underlies epithelia, endothelia, and enwraps muscle and adipose tissues. Cell invasion through BM occurs during development and physiological processes, and inappropriately in cancer metastasis. Extrinsic signaling cues play key roles in inducing invasion, but how these signals regulate the cellular machinery and behaviors that promote invasion remains poorly understood. A key barrier in understanding how extrinsic cues direct invasion is the challenge of experimentally examining the dynamic interactions between invasive cells, BM, and the tissue being invaded in vivo. Anchor cell (AC) invasion in C. elegans is an experimentally tractable model to uncover mechanisms controlling cell invasion in vivo. To identify new genes required for AC invasion, we performed a sensitized genome-wide RNAi screen and found that the Rho GTPase cdc-42 promotes AC invasion. In response to extracellular signals, CDC-42 can be activated to regulate processes related to cell invasion, such as cell polarity and cytoskeletal organization. Here we show that animals null for cdc-42 have defects in AC invasion and display a highly penetrant delay in initial breach of the BM. An unknown cue secreted from the vulval precursor cells controls the timing and initiation of invasion, but the molecular pathways downstream of this cue have not been identified. Using site-of-action and genetic analyses, we show that CDC-42 functions in the AC downstream the vulval cue. While cdc-42 has been identified as a regulator of cancer invasion in vitro how CDC-42 is localized, activated, and functions in BM breaching is unknown. A genetically encoded biosensor for active CDC-42 shows that CDC-42 is strongly activated at the site of BM breach, and that active CDC-42 colocalizes with highly dynamic F-actin structures called invadopodia, which are responsible BM breaching in AC invasion. RNAi knockdown of cdc-42 resulted in a significant reduction in invadopodia number. These results suggest cdc-42 is downstream of the vulval cue and initiates BM breaching by promoting invadopodia. Interestingly, an additional extrinsic signaling pathway functions in the AC involving the netrin receptor (unc-40) and its effectors, the Rac GTPases ced-10 and mig-2. UNC40 and the Rac GTPases direct formation of a large invasive protrusion after the vulval cue initiates BM breach via CDC-42 and invadopodia. Altogether, our data suggest that multiple, independent cues are required to direct cell invasion through BM (a coincidence system). We speculate that such systems exist to precisely control the site and timing of cell invasion–an essential but potentially destructive biological process.

TUESDAY-POSTER PRESENTATIONS

Signaling Networks Governing Cell Migration P2186 Board Number: B813

A Cell Life under Confinement: how immune cells manage to move, squeeze and survive. H. Thiam1, M. Raab1, M. Le Berre1, Y. Liu1, M. Piel1; 1 UMR 144, Institut Curie, Paris, France The quest to understand how the mechanical and geometrical environment of cells impacts their behavior and fate has been a major force driving the recent development of new technologies in cell biology research. Despite rapid advances in this field, many challenges remain in order to bridge the gap between the classical and simple cell culture plate and the biological reality of actual tissues. In tissues, cells have their physical space constrained by neighboring cells and extracellular matrix. In the recent years, we have developed simple and versatile devices to precisely and dynamically control this confinement parameter in cultured cells. I will present results we obtained on the effect of confinement on cell migration focusing on two questions: how modulating confinement and adhesion of slow mesenchymal migrating cells can make them switch to a fast amoeboid like migration behavior and how cells can squeeze their nucleus when migrating through small gaps. I will conclude presenting intruiguing results showing that large deformations of migrating cells induce strong damages to their nucleus raising the question of how immune cells such as dendritic cells can combine high motility and long term survival.

P2187 Board Number: B814

TRIM9 regulates DCC trafficking and FAK activity during developmental axonal plasma membrane expansion. C. Winkle1, S. Gupton2; 1 Neurobiology, University of North Carolina, Chapel Hill, NC, 2Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC During neuronal development, growth cones at the tips of axons branch and extend toward postsynaptic targets. This involves dramatic expansion of the plasma membrane and requires regulated delivery of new plasma membrane material. The axon guidance cue Netrin-1 and its receptor DCC promote exocytosis, attractive growth cone turning and axon branching at least partially through the activation of the non-receptor tyrosine kinase FAK. We identified the E3 ubiquitin ligase TRIM9 as a novel regulator of exocytosis, axon branching and axon guidance. We show that TRIM9 directly binds the cytoplasmic tail of DCC within the same domain bound by FAK. Using neurons isolated from

TUESDAY-POSTER PRESENTATIONS embryonic mouse cortex, we demonstrate that genetic loss of TRIM9 disrupts DCC trafficking, activation of FAK, the frequency of exocytic vesicle fusion, and the density of axon branching. TIRF microscopy and surface biotinylation assays indicate that following Netrin-1 stimulation, DCC clusters and accumulates in the plasma membrane and is subsequently endocytosed and degraded. In TRIM9-/- cortical neurons, DCC fails to cluster in the plasma membrane and does not get degraded following Netrin-1 stimulation. Whereas clustering is rescued by expression of GFP-TRIM9, structure function studies indicate that the ubiquitin ligase domain of TRIM9 is not required for clustering, but is necessary for Netrin response. Although total DCC protein is reduced in TRIM9-/- neurons, a higher proportion of DCC localizes at the cell surface. These defects in DCC trafficking are associated with hyperphosphorylation of FAK at Y397, an increased exocytic frequency and exuberant axon branching in TRIM9-/- neurons. Pharmacological inhibition of FAK rescues the aberrant exocytosis and axon branching in TRIM9-/- neurons and blocks Netrin-dependent increases in exocytosis and branching in wildtype neurons. Interestingly, the exuberant branching of TRIM9-/- axons in vitro was also observed in vivo within the corpus callosum and correlated with increased callosal thickness. We have crossed TRIM9-/- and FRNKloxp mice to determine if inhibition of FAK blocks the exuberant axon branching and corpus callosum thickening defects found in vivo. Results from cortical explant assays indicate TRIM9 also functions during axon outgrowth toward an asymmetric Netrin source. We are using a membrane-targeted GFP driven by the Tau promoter to define the axonal projection defects in TRIM9-/- mice in vivo. We conclude that TRIM9 regulates DCC trafficking and FAK activation to control vesicle fusion and plasma membrane expansion during Netrindependent axon responses.

P2188 Board Number: B815

Characterizing new genes regulating cell-substrate adhesion to discover novel regulatory mechanisms of cell motility. T.J. Lampert1, P. Devreotes2; 1 Johns Hopkins University, Buffalo, NY, 2Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD The model organism Dictyostelium has greatly facilitated our understanding of the signal transduction and cytoskeletal pathways that govern cell motility. Cell-substrate adhesion is a target of many chemotaxis signaling events and it can be used to screen for cells that have defects in cell migration. In fact, cells lacking PTEN, a negative regulator of cellular extensions, adhere strongly to the surface. This leads to reasoning that other regulators of migration would also effect adhesion, a screening method was devised and isolated overly adherent mutants from a pool of mutagenized cells. Restriction enzyme mediated insertion (REMI) mutagenized cells, comprising more than 50000 insertions, yielded about 100 mutated cell lines with the desired phenotypes. The mutation sites in 20 of the strains have been mapped and many of the phenotypes are similar to those of PTEN knockout cells. The extent of increased adhesion, cell motility, directed migration, cell shape, and new filamentous actin at the periphery are all parameters that have been examined in these new overly adhesive cell lines. The

TUESDAY-POSTER PRESENTATIONS degree in which these parameters have been effected and the correlations between these changes is providing novel insights into the networks controlling cell motility. Many of these genes have human homologs with unknown functions. Therefore, the future study of this new group of regulators of adhesion and motility genes in Dictyostelium will not only advance the knowledge of cell migration in amoeboid cells but elucidate the functions of novel human genes with potential disease relevance.

P2189 Board Number: B816

Synthetic manipulation of PIP2 levels and PIP2-associated chemotactic signaling dissection in Dictyostelium. Y. Miao1, T. Inoue2, P. Devreotes3; 1 Biological Chemistry, Johns Hopkins University, Baltimore, MD, 2Cell Biology, Johns Hopkins University, Baltimore, MD, 3Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD PIP2 is linked to many key chemotactic players, but our understanding of its roles in chemotactic signaling is very limited due to technical difficulties. The idea that the localized decrease of PIP2 level initiates chemotactic signaling events has been around for years, but has never been tested in a direct manner with traditional methods. In order to illuminate the roles of PIP2 and further explore the features of the chemotactic signaling network, I used the chemically-induced dimerization (CID) system, which allows the rapid inducible translocation of cellular proteins, to synthetically manipulate PIP2 level and dissect the signaling events. I have successfully accommodated CID in Dictyostelium, an excellent model system to study eukaryotic chemotaxis. In my current data, after I synthetically recruited the PIP2-specific 5-phosphotase Inp54p to the plasma membrane, the PIP2 biosensor PHplcdelta fell off the membrane and the cells robustly oscillated between a spreading and a crunching morphology. Cells with the spreading morphology, but not the other one, may have further lowered PIP2 levels indicated by PTEN localization, clearly activated chemotactic signaling events as shown by Ras and PIP3 biosensors, and highly elevated F-actin along the periphery. Further, cells still carried out this response when PIP3 production was diminished by the PI3K inhibitor LY. Based on these results, we propose that PIP2 serves as an inhibitory role on Ras; when PIP2 levels are decreased, the chemotactic signaling network containing PKBs is activated following Ras activation. Also based on previous evidence, we propose that PI5K activation is downstream of PKBs activation, which brings up PIP2 levels to a certain degree and leads to the crunching morphology after cell spreading following the synthetic Inp54p recruitment. I will further use this system to dissect the PIP2-initiated signaling and feedback loops, as well as their roles in cell polarity and chemotaxis. Together the results of these experiments should shed light on the unsolved roles of PIP2 in chemotaxis and contribute to our further understanding of the chemotactic signaling network.

TUESDAY-POSTER PRESENTATIONS

P2190 Board Number: B817

The adaptor proteins Crk and CrkL regulate T cell adhesion and selectively promote migration to sites of inflammation. Y. Huang1, M. Karimi2, T. Park1, N. Roy1, T. Curran1, T. Kambayashi2, J.K. Burkhardt1; 1 Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia Research Institute, Philadelphia, PA, 2Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA Effector T cell migration into inflamed sites greatly contributes to local inflammation and disease severity in inflammatory diseases, including GVHD. T cell migration into such sites depends heavily on regulated adhesion and migration, but the signaling pathways that coordinate these functions downstream of chemokine receptors are largely unknown. Using conditional knockout mice, we have found that T cells lacking the adapter proteins Crk and CrkL exhibit reduced integrin-dependent adhesion, chemotaxis, and diapedesis across an endothelium. Our studies reveal significant functional redundancy of the two closely related proteins. We show that Crk proteins coordinate with C3G and CasL to activate the integrin regulatory GTPase Rap1. Importantly, Crk proteins are required for effector T cell trafficking into sites of inflammation, but not for migration to lymphoid organs. This unique differential migration of Crk/CrkL-deficient T cells has important therapeutic implications, since T cells lacking Crk proteins can carry out graft-versus-leukemia responses with minimal graft-versus-host disease. Our studies show that Crk family adaptors proteins selectively regulate T cell adhesion and migration at effector sites, and identify these proteins as important novel therapeutic targets.

P2191 Board Number: B818

Coagulation factor IX regulates cell migration and adhesion in vitro. A. Mamiya1, H. Kitano1, T. Ishikawa1, Y. Fujiwara1, M. Yoshida 1, . Kokubun1, C. Hidai1; 1 Nihon Univ Sch Med, Tokyo, Japan

Introduction Coagulation factor IX (F9) is thought to circulate in the blood as an inactive zymogen before being activated in the coagulation process. The effect of F9 on cells is poorly understood. This study aimed to evaluate the effects of intact F9 and its cleavage fragments on cell behavior. Materials & Methods A431 cells (derived from human squamous cell carcinoma), Pro5 cells (derived from mouse embryonic endothelial cells), Cos7 cells, and human umbilical vein endothelial cells (HUVEC) were utilized in this study. The effects of F9 and its cleavage fragments on cell behavior were investigated in several types of experiments, including wound-healing assays and modified Boyden chamber assays. To investigate intercellular or cell-matrix adhesion, immunohistochemistry was employed. Results The effect of F9 depended on its processing; full-length F9 suppressed cell migration, increased adhesion to matrix, and enhanced intercellular adhesion. In contrast, activated F9 enhanced cell migration,

TUESDAY-POSTER PRESENTATIONS suppressed adhesion to matrix, and inhibited intercellular adhesion. the effect of F9 appears to be nonspecific with regards to cell type, as the effect was demonstrated in cells of diverse origin (i.e., epithelial [A431], endothelial [pro5], and mesenchymal [cos7]). An activation peptide that is removed during the coagulation process was found to be responsible for the activity of full-length F9, and the activity of activated F9 was localized to an EGF domain of the F9 light chain. Our data indicated that the activation peptide inhibited the activity of the EGF domain on cells. Conclusion: We found that in addition to its role in the coagulation process, F9 has several novel effects on cells. Full-length F9 has a sedative effect on cells, which is counteracted by activated F9 in vitro. Thus, F9 may play roles before, during, and after the coagulation process.

P2192 Board Number: B819

Dose-dependent histomorphometric and ultrastructural changes in the testis of adult male Japanese quails (Coturnix coturnix japonica) exposed to di-n-butyl phthalate (DBP), in vivo. U. M. Bello1,2, M. Madekurozwa1, H. B. Groenewald1, A. Arukwe3, T. A. Aire4; 1 Department of Anatomy and Physiology, University of Pretoria, Pretoria, South Africa, 2Department of Vet Anatomy, Faculty of Vet. Medicine, Ahmadu Bello University, Zaria, Nigeria, 3Department of Biology, Norwegian University of Science and Technology (NTNU), Hogskoleringen 5, 7491, Trondheim,, Norway, 4 Department of Anatomy, Physiology and Pharmacology, School of Vet. Medicine, St George's University, True Blue, Grenada, Grenada We have investigated the dose-response effects DBP on histo-morphometric parameters and ultrastructure of the seminiferous tubules of adult male Japanase quails (C. japonica). A total of ninety (90) pre-sexed, 10-week old male quail birds were randomly divided into six experimental groups: The control group (n=15) received a dose of 1 mL/kg of corn-oil (control), or intragastric daily dose of 1, 10, 50, 200, 400 mg DBP/kg/bw (dissolved in corn oil), for a period of 30-day. At study termination, the testes excised from both control and treatment groups and were further evaluated using a computeraided histometric and ultra-structural (TEM) techniques. Our results showed a generalized significant decrease in the diameter and epithelium thickness (height) of the seminiferous tubules occurring predominantly in the high dose (200mg- and 400mg) groups as compared to median (50mg) or low dose (1mg- and 10mg) groups. Ultra-structurally, a dose-dependent degenerative changes in earlier germ cell series, and being significant at the high dose level, were evident. Necrotic round spermatids were prominent in sub luminar part of the seminiferous tubules amidst apparently normal elongated spermatids. A few spermatogonia were necrotic, and a few empty spaces, probably vacated by necrotic early spermatocyctes or spermatogonia, were encountered. Few spermatogonia showed degenerative changes with morphological characteristics of apoptosis-- including shrinkage of cytoplasm and increased nuclear blebbing, occurring mainly in high doses (i.e. 200- and 400mg DBP) groups. Sertoli cells appeared foamy in consistency because they are laden with lipid droplets. Dense bodies were also conspicuous and very importantly, the mitochondria were swollen and showed a disrupted matrix. In

TUESDAY-POSTER PRESENTATIONS addition, Leydig cells were few and showed numerous lipid droplets (scattered throughout the cytoplasm), multivesicular bodies and giant whorl-like smooth endoplasmic reticulum. In conclusion, the present data indicate that DBP(pubertal) exposure produced dose- and parameter-dependent, histometric and sub-cellular alterations in the seminiferous tubules, thereby affecting the adult spermatogenesis. It is evident that DBP pronounced male reproductive toxicities in testes might likely be due to its anti-androgenic effect on reproduction, resulting from increased damaged to earlier spermatogenic series, Sertoli and Leydig cells. The potential negative implication on the reproductive biology, particularly in those avian species, that are vulnerable to EDCs are further discussed.

P2193 Board Number: B820

High glucose reduces migration and viability of human periodontal ligament cells. M.M. Monteiro1, A.R. Tempestini-Horliana2, M.F. Santos3; 1 Cell and development biology, University of São Paulo, São Paulo, Brazil, 2Department of Stomatology, University of São Paulo, São Paulo, Brazil, 3Cell and development Biology, Univ Sao Paulo, Sao Paulo, Brazil Background: Impaired oral wound healing and increased incidence of periodontal diseases are complications of Diabetes Mellitus (DM). DM impairs the migration of dermal fibroblasts, contributing to deficient healing, and some microRNAs (miRs), such as miR-221 and miR-222, are affected by DM and might be involved in cellular functions. Aim: to evaluate the effects of the in vitro exposure of human periodontal ligament (PLC) cells to high glucose on growth, death, migration, actin cytoskeleton and the expression of miR-221 and miR-222. Methods: cells were obtained from premolar teeth of 3 young humans under orthodontic treatment, and cultured under low (LG, 5mM) or high (HG, 25mM) glucose for 7 days before the assays. Results: Cell number was reduced by 33% in HG cells, compared to LG, mostly due to a 3x increased in cell death (TUNEL assay). Cell adhesion on fibronectin was reduced by 40% after 20 minutes of plating, and by 30% after 1 hour. Cell spreading after 1h was reduced by 35%. HG cell migration (transwell assay) was reduced by 54%. HG cells showed increased circularity when compared to LG (> 0.5 vs. 0.2, respectively). The expression of miR-221 and miR-222 was increased by 2x in HG cells. Conclusion: High glucose negatively affects PLC, reducing migration and viability, thus potentially affecting the progression of periodontal disease and healing after periodontitis treatment, as well as the response to orthodontic treatment. miR-221 and miR-222 were upregulated by HG in those cells and might be involved in some of these events. Support: FAPESP, CAPES, PRP-USP (NAPmIR).

TUESDAY-POSTER PRESENTATIONS

P2194 Board Number: B821

High Glucose concentration reduces cellular contractility and tension forces in NIH3T3 fibroblasts, contributing to the impaired migration in 2D and 3D substrates. M.E. Almeida1, M. Badoual2, M. Lamers3, A.R. Horwitz4, M.F. Santos5; Cell and Development Biology, University of Sao Paulo, Sao Paulo, PA, 2Laboratoire IMNC, Univ. ParisSud, Orsay, PA, 3Morphological Sciences, Universidade Federal Do Rio Grande Do Sul, Porto Alegre- RS, PA, 4Cell Biology, University of Virginia, Charlottesville, VA, 5Cell and development Biology, Univ Sao Paulo, Sao Paulo, Brazil 1

Background: Long-term complications of Diabetes Mellitus include impaired wound healing and fibroblast migration and extracellular matrix (ECM) remodeling are essential for this process. We have previously shown that fibroblasts migration is reduced under high glucose conditions as well as the maturation of cellular adhesions to the ECM. Another feature of those cells is the long rear and less stress fibers, which suggest impaired myosin II activity. During migration, the contraction of the cell body generates tension on the substrate through cell adhesions, which is important for adhesion maturation. Aim: To study the effects of a high glucose concentration on fibroblast contractility and its potential contribution to the impairment of migration. Methods: NIH3T3 fibroblasts were exposed to physiological (5mM, LG) or high (25mM, HG) glucose concentrations and submitted to different analyses on two-dimensional (2D) fibronectin and three-dimensional (3D) collagen/fibronectin substrates. F-actin arrangement and the expression and distribution of the myosin regulatory light chain (MRLC), myosin isoforms IIA (MIIA) and IIB (MIIB) were studied by staining with phalloidin, western blotting and immunocytochemistry, respectively. Traction force microscopy (TFM) assays were employed to study tension exerted on the substrate. Results: Cells exposed to HG showed reduced migration velocity (about 40-60%) and larger lamellipodia on 2D assays. In 3D substrates, HG cells showed extensive branching projections at the front. TFM experiments showed lower tension exerted by HG cells on the adhesions to the substrate. HG did not affect the total amount of MRLC, MIIA and MIIB proteins. MIIB distribution, however, was changed. In cells cultured under low glucose MIIB was mostly present in the cell body and rear, whereas in HG cells it was spread throughout the cell, including the lamellipodia. MIIA distribution, on the other hand, was not changed. Conclusion: fibroblasts under high glucose show impaired contractility, which leads to a reduced tension on the substrate through cellular adhesions. This impairment may be involved in the deficient migration of fibroblasts in diabetic patients. Support: FAPESP, CAPES.

TUESDAY-POSTER PRESENTATIONS

P2195 Board Number: B822

Free β-subunit of human chorionic gonadotropin stimulates choriocarcinoma cell invasion independent of LH/hCG receptor. C. Lee1, P.C. Chiu1, L. Hautala2, W.S. Yeung1, K.M. Koli3, U. Stenman2, H. Koistinen2; 1 Department of Obstetrics and Gynecology, The University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong, 2Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland, 3Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland

Human chorionic gonadotropin (hCG) is a heterodimer consisting of an α- and a β-subunit (hCGα and hCGβ) that are non-covalently linked. Intact hCG, but not free subunits, bind to the LH/hCG receptor in the corpus luteum stimulating its progesterone production, which is essential for successful pregnancy. hCG has been found to stimulate trophoblast invasion in an autocrine fashion. In pregnancy, trophoblast invasion is a tightly controlled process, disturbances in which may lead to pregnancy complications. In addition to placenta, several cancers have been shown to express hCG and hCGβ. Most trophoblastic tumors of placental and germ cell origin produce hCG, while many non-trophoblastic tumors, including bladder, renal and gastrointestinal cancers, often produce hCGβ. The stimulatory activity on trophoblast invasion has been ascribed especially to a so-called hyperglycosylated form of hCG (hCG-h) expressed in early pregnancy. hCG-h is also a major form of hCG produced by choriocarcinoma and testicular cancers, although tumor-derived hCG-h is often glycosylated differently than that produced by the placenta. We compared different forms of hCG in their stimulatory activities on the invasion of JEG-3 choriocarcinoma cells. Various forms of hCG and hCGβ were purified from urine of a patient with nonseminomatous testicular cancer, one with invasive molar disease, and a pregnant women. Both hCG and hCGβ, and their hyperglycosylated forms stimulated the invasion of JEG-3 cells to a similar extent. Down-regulation of the LH/hCG receptor by RNA-interference did not significantly reduce the effect of hCGβ and hCG on cell invasion. Increased invasion was associated with increased activity of MMP-2, MMP-9 and uPA in conditioned culture medium from JEG-3 cells. Unlike suggested by some previous studies, highly purified hCG or hCGβ, or their hyperglycosylated forms, did not activate TGFβ-receptor or induce phosphorylation of ERK1/2. Our findings suggest that hCG and hCGβ stimulate the invasion of choriocarcinoma cells by an unknown mechanism, that is independent of the classical LH/hCG-receptor. These results may explain the association of increased serum hCG and hCGβ with adverse prognosis both in trophoblastic and non-trohoblastic tumors.

TUESDAY-POSTER PRESENTATIONS

P2196 Board Number: B823

The role of an R-Ras/ARF6 signaling module in adhesion-induced epithelial spreading and migration. J.C. Salem1, L.C. Santy2; 1 Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, 2Department of Biochemistry , Microbiology and Biochemistry, Pennsylvania State University, University Park, PA Overexpression of ARF-GEF cytohesin-2/ARNO causes immobile epithelial cells to become highly migratory and scatter away from neighboring cells. Through robust activation of ARF6 and assembly of a multi-protein complex, cytohesin-2/ARNO behaves as both an ARF-GEF and a scaffold to promote activation of small GTPase Rac. R-Ras is a small GTPase that specializes in enhancing cell spreading and promoting integrin activation. It was recently shown that small GTPase R-Ras signals upstream of cytohesin-2/ARNO to promote adhesion-induced migration. Given that both R-Ras and cytohesin2/ARNO function in a similar pathway, we wondered if ARF-activation via cytohesin-2/ARNO could direct R-Ras traffic, and if so, through which intracellular compartment. Therefore, transfected HeLa cells were plated on fibronectin, and imaged using fixed and live immunofluorescence. We demonstrate that overexpressed R-Ras traffics through a tubular EHD1 recycling endosome compartment. Destroying the GEF activity of cytohesin-2/ARNO (E156K) traps R-Ras in EHD1 recycling endosomes. In contrast, phorbol 12-myristate 13-acetate (PMA), a cytohesin-2/ARNO stimulator, promotes dissolution of EHD1 tubular endosomes containing R-Ras. Both R-Ras and ARNO each promote cell spreading, as constitutively active (38V) R-Ras can only partially rescue cell spreading in cells co-expressing E156K ARNO. Finally, we provide evidence suggesting α5-integrin is also trapped in an EHD1 compartment upon E156K-ARNO expression. Given that cytohesin-2/ARNO is required for β1-integrin recycling and migration on fibronectin, we propose that R-Ras effects on integrin activation, ruffling, and spreading are highly governed by cytohesin-2/ARNO-induced ARF6 activation.

P2197 Board Number: B824

A novel mechanism of cell locomotion regulation by LOSK/SLK kinase. A.I. Fokin1, T.S. Klementyeva2, E.S. Nadezhdina1,2, A.V. Burakov1; 1 A.N.Belozersky Institute for Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia, 2Institute of Protein Research of Russian Academy of Sciences, Moscow, Russia Cell locomotion is a complex process which requires both actin and microtubule cytoskeleton involvement. Actomyosin component of the cytoskeleton generates force for movement. Microtubules are assumed to induce cell polarization and maintain direct movement. SLK is a serine/threonine-protein kinase associated with microtubules and centrosome, originally described as LOSK (Long Ste20-Like). Inhibition of its activity results in disorganization of microtubule system and reduction of Golgi polarization in moving cells (Burakov et al., 2008, Zhapparova et al., 2013). Hence they demonstrate

TUESDAY-POSTER PRESENTATIONS decreased ability to directional movement. It is known that LOSK phosphorylates a series of cytoskeletal proteins such as RhoA (Guilluy et al., 2008) and p150Glued subunit of dynactin (Zhapparova et al., 2013) which are essential for cell locomotion. To test the role of RhoA and p150Glued phosphorylation by LOSK we performed transfection of Vero fibroblast-like cells with dominant negative LOSK[K63R-ΔT], dominant negative and constutively active RhoA, phospho-mimic and non-phosphorylatable mutants of RhoA and p150Glued at LOSK phosphorylation site. Moreover we co-transfected cells with RhoA and p150Glued constructions described above against LOSK inhibition by LOSK[K63R-ΔT]. Also we used inhibitor of downstream RhoA effector ROCK kinase. Standart wound scratching assay and ImageJ plugin Manual tracking were applied to specify parameters of cell movement. Besides we examined in details the structure of microtubule system and polarization of Golgi in these cells. We estimated that the regulation of cell motility and the microtubule ordering by LOSK occurs independently in two different signaling pathways. It is turned out that cell motility impaired by LOSK inhibition could be restored, using phospho-mimic (inactivated) RhoA as well as ROCK inhibitor; also it partially rescued Golgi polarization despite the fact that the microtubules remains disordered. On the other hand, the p150Glued construct which could not effectively rescue cell motility, successfully restores the microtubule system and Golgi polarization. Thus, it was first shown in this study that LOSK-dependent microtubule disordering does not affect the cell motility and regulation of cell migration is accomplished through another LOSK-dependent signaling pathway. This work is financially supported by the Russian Foundation for Basic Research (grants 12-0433178 mol-a-ved and 14-04-01729a).

P2198 Board Number: B825

Analysis of Diatom Motility in Multi-species Assemblages. S.A. Cohn1, D. Halpin1, A. Ismail1, W. Macke1, S. Olszewski1, A. Wolske1; 1 Biological Sciences, DePaul Univ, Chicago, IL Diatoms are unicellular algae, present in almost all freshwater and marine aquatic environments, known for their characteristic hardened silica cell walls. Diatoms live within complex multi-species algal communities, and provide a large percentage of earth's primary production and oxygen formation. Investigating the environmental effects on cell behavior, such as the light-regulated gliding motility of pennate diatoms that is crucial for their ecological success, is therefore critically important to understanding their overall ecological sustainability. Previous studies in our lab suggested that the presence of multiple diatom species alters the cells' adhesive ability relative to cells from unialgal cultures (Diatom Res. 18 :225). In order to determine if the presence of other species also affects cell motility, we analyzed the motile responses of three diatom species (Craticula cuspidata, Pinnularia viridis, and Stauroneis phoenicenteron) to high intensity irradiations of blue (470nm), or red (650nm) light at their leading or trailing ends, in the presence or absence of one of the other diatom species. Our analysis of the response times of light-stimulated direction changes in these cells suggests that under some conditions there are significant inter-species effects on diatom motility, which may relate to their

TUESDAY-POSTER PRESENTATIONS niche partitioning. For example, while Craticula cells show little change in their response times in the presence of other species, Stauroneis cells show a strong repression in response times for blue light exposures of their leading ends in the presence of Craticula cells (36±3 sec for Stauroneis alone; 88±9 sec for Stauroneis in the presence of Craticula). Stauroneis cells show similar repression to red light exposures of the leading end (96±10 sec for Stauroneis alone; 148±13 sec for Stauroneis in the presence of Craticula). In contrast, Pinnularia cells show decreased response times for red-light leading end exposures in the presence of Craticula (163±20 sec for Pinnularia alone; 92±21 sec for Pinnularia in the presence of Craticula). We have also analyzed the effect of multiple sequential trailing end cell irradiations to determine the degree to which these exposures repress subsequent direction changes at the leading end. With blue irradiations of the trailing end, Craticula and Stauroneis show a strong dosedependent effect that indicates the repression lasts 30-90 sec, while Pinnularia shows a longer lasting repression that remains for more than 2 min. Red irradiations of the trailing end of Stauroneis cells show an effect that lasts 90-120 sec. This work was supported by grants through the DePaul College of Science and Health, the DePaul University Research Council, and equipment purchased previously through NSF Grant IBN-9982897.

P2199 Board Number: B826

Syndecan-4 regulates Eph receptor-mediated fibroblast clustering during wound repair. R. Brooks1, J.A. Roper1, C.D. Nobes1,2, M.D. Bass1; 1 Department of Biochemistry, University of Bristol, Bristol, United Kingdom, 2Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom Fibroblast migration into a wound bed is essential for wound contraction and healing. Migration relies on cell-matrix and cell-cell contacts, both of which are regulated by transmembrane receptors that are differentially regulated during wound healing. The expression of the fibronectin receptor syndecan-4 is increased upon wounding, which in fibroblasts promotes migration through activation of small GTPases and the recycling of integrin α5β1. Members of the Eph family of receptor tyrosine kinases, which regulate cell-cell contacts, are also differentially regulated in fibroblasts upon wounding. However, little is known about the relationship between these receptors and how this might regulate the balance between cell-cell and cell-matrix contacts. We demonstrate an inverse relationship between syndecan-4 and EphA2, which mediates cell-cell repulsion. EphA2 expression is elevated in syndecan-4 knockout mouse embryonic fibroblasts through a Src family kinase-dependent mechanism. Elevated EphA2 expression increases sensitivity to the EphA2 ligand ephrin-A1 and increases the rate and frequency of cell-cell repulsion when compared to wild type. Importantly, our findings are translated in vivo; EphA2 expression is increased in syndecan-4 knockout mouse wounds compared to wild type. This may contribute to the healing defect previously observed in syndecan-4-null mice1.

TUESDAY-POSTER PRESENTATIONS 1

Echtermeyer, F. et al. Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4. J. Clin. Invest. 107, R9-R14 (2001).

P2200 Board Number: B827

Na+/Ca2+ exchanger 1 regulates Ca2+ dependent ERK signaling and migration. S. Balasubramaniam1,2, A. Gopalakrishnapillai2, R.L. Duncan1, S. Barwe1,2; 1 Biological Sciences, University of Delaware, Newark, DE, 2Biomedical research, Nemours, Wilmington, DE The sodium pump, Na+/K+-ATPase, contains a catalytic α-subunit (Na,K-α) and a regulatory β-subunit (Na,K-β). These proteins regulate ion homeostasis, in addition to maintaining epithelial polarity and structural integrity. The activity of Na+/K+-ATPase is closely coupled with another transporter, Na+/Ca2+ exchanger 1 (NCX1). The efflux of 3 Na+ by Na,K-ATPase provides the gradient for NCX1, which enables the influx of 3 Na+ and efflux of 1 Ca2+. Earlier studies from our lab have shown that mice with heartspecific ablation (KO) of Na,K-β were insensitive to ouabain-induced cardiac contractility with reduced NCX1 expression, and no change in other Ca2+ handling proteins (Barwe et al., J. Mol Cell Cardiol, 2009). In addition we show that Na,K-β co-localizes with NCX1 and confirm their association by immunoprecipitation. These data support the hypothesis that Na,K-β acts as a chaperone in regulating the membrane expression and localization of not only Na,K-α, but also NCX1. Other studies have shown a similar role for sodium transporters such as Na,K,2Cl transporter (Carmosino et al., J. Hypertension, 2014). To understand the cause and consequences of this reduced NCX1 expression, we utilized Madin Darby Canine Kidney (MDCK) cells with Na,K-β knockdown (β-KD). β-KD cells express reduced levels of NCX1, accompanied by a 2-fold increase in free intracellular calcium concentration, consistent with the fact that NCX1 is the major calcium extrusion mechanism in MDCK cells. A corresponding increase in the rate of migration was observed using wound healing assay. Replenishment of Na,K-β in β-KD (β-KD/WTR) cells restored NCX1 protein at the cell surface, reduced the intracellular calcium levels and migration rate similar to that in parental MDCK cells. The Ca2+ chelator BAPTA suppressed migration in β-KD cells, indicating that the increased migration was calcium-dependent, thus implying a role for NCX1 in the regulation of migration. MDCK cells treated with NCX1 inhibitor, KB-R7943, showed an increase in cell migration similar to β-KD cells, confirming the role of NCX1 activity in regulating cell migration. To delineate the signaling pathways in the regulation of cell migration by NCX1, we used specific inhibitors of PI3-kinase, Akt and MEK1/2, since β-KD cells display PI3-kinase dependent ERK1/2 and Akt activation (Barwe et al., J Cell Sci, 2012). Our data revealed that migration induced by reduction of NCX1 in β-KD cells or NCX1 inhibition is dependent on PI3-kinase and ERK1/2 (direct downstream target of MEK1/2), but is Akt-independent.

TUESDAY-POSTER PRESENTATIONS

P2201 Board Number: B828

Activation of Rac by Asef2 Promotes Myosin II-Dependent Contractility to Inhibit Cell Migration in both 2D and 3D Environments. L. Jean1, D. Majumdar1, M. Shi1, L. Yang2, Y. Gao2, J. Broussard1, L. Hinkle1, B. Brewer2, D. Li2,3, D. Webb1,3,4; 1 Department of Biological Sciences, Vanderbilt University, Nashville, TN, 2Department of Mechanical Engineering, Vanderbilt University, Nashville, TN, 3Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN, 4Department of Cancer Biology, Vanderbilt University, Nashville, TN Non-muscle myosin II (MyoII) contractility is important to the regulation of many cellular processes, including migration. The small GTPase Rho has been shown to regulate MyoII contractility, but the role of other GTPases, such as Rac, in modulating contractility is not well understood. In this study, we show that activation of Rac by the guanine nucleotide exchange factor (GEF) Asef2 increases MyoII contractility to impair cell migration on two-dimensional (2D) type I collagen. Knockdown of endogenous Rac using short hairpin RNAs (shRNAs) or treatment of cells with the Rac-specific inhibitor NSC23766 results in a significant decrease in the amount of active MyoII, as determined by serine 19 (S19) phosphorylation, and negates the Asef2-promoted increase in contractility. Furthermore, treatment of cells with blebbistatin, which inhibits MyoII activity, abolishes the Asef2-mediated effect on migration. Because adhesion growth and maturation have been linked to actomyosin contractility, we also examined the effect of Asef2 on adhesions. Interestingly, Asef2 increases active β1 integrin levels in adhesions and induces large, mature adhesions that assemble and disassemble (turnover) more slowly compared with controls. In addition, the Asef2-promoted increase in the level of active β1 integrin in adhesions was diminished by treatment with the Rac inhibitor NSC23766. Because threedimensional (3D) matrices more closely mimic the physiologic environment of cells, we investigated the role of Asef2 in regulating migration and MyoII activity in 3D environments. For these studies, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that Asef2 inhibits cell migration in 3D type I collagen matrices, and treatment of cells with blebbistatin abolishes the Asef2-mediated decrease in migration. Moreover, Asef2 enhances MyoII activity as shown by increased S19 phosphorylation, and treatment with NSC23766 abolishes the Asef2-promoted increase in active MyoII. Collectively, these results indicate that Rac activation, promoted by Asef2, is critical for modulating MyoII activity and cell migration in both 2D and 3D environments.

TUESDAY-POSTER PRESENTATIONS

P2202 Board Number: B829

Glycodelin abolishes PMA-induced migration of MCF-7 breast cancer cells. L. Hautala1, R. Koistinen1, H. Koistinen1; 1 Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland Glycodelin is a secreted glycoprotein mainly expressed in well-differentiated epithelial cells in reproductive tissues. Previously, glycodelin has been shown to induce cell differentiation in endometrial and breast cancer cells. We have transfected glycodelin into MCF-7 breast cancer cells to study its effect on cell differentiation. When the cells were grown in Matrigel basement membrane preparation, glycodelin-expressing cells formed glandular structures, while the control cells were less cohesive and had less luminar structures. In addition, glycodelin-producing cells formed significantly smaller tumors in xenograft mice. The effects of glycodelin are, at least partly, mediated by repressed PKCδ activity, rendering the cells resistant to PKCδ activation by phorbol 12-myristate 13-acetate (PMA). Here we continued these previous studies by addressing the effect of glycodelin on the invasion and migration of MCF-7 breast cancer cells, and studying the affected signaling route in more detail. Activation of signaling proteins was detected with an antibody array of 43 phospho kinases. Cell migration was studied using wound healing test. The cells were grown on chamber slides until they formed a monolayer and then a scratch was made using a pipet tip. After addition of PMA or DMSO control, the migration of the cells was monitored under microscope. For control, PKCδ was down-regulated with RNAi prior to the migration assay. Boyden chamber assay was used to study the invasion through Matrigel. The cells were grown with and without PMA and the amount of invasive cells was quantified. Phospho-kinase array did not reveal any widespread changes in the amount of phosphorylated signaling molecules between the glycodelin-producing and control cells irrespective of the PMA treatment. However, in addition to previously detected decrease in PKCδ levels, increased p27 and Chk-2, and decreased p38α were observed in glycodelin-producing cells. In wound healing test PMA caused a twofold increase in the migration of the control cells, but had no effect on the glycodelin-producing cells. When the expression of PKCδ was down-regulated in the control cells using siRNA, the addition of PMA did not increase the migration of the cells. PMA also increased the invasiveness of the cells, but no difference was observed between glycodelin-expressing and control cells. Without PMA treatment both glycodelin-expressing and control cells behaved similarly in wound healing and invasion tests. In conclusion, glycodelin abolished PMA-induced migration in MCF-7 breast cancer cells, while it did not have any effect on the invasion of the cells. Our results suggest that the effect of glycodelin in migration is mediated by reduced activation of PKCδ.

TUESDAY-POSTER PRESENTATIONS

P2203 Board Number: B830

Regulation of cancer cell motility by secreted exosomes. B. Sung1, T. Ketova2, D. Hoshino1, A. Zijlstra1,2, A.M. Weaver1,2; 1 Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, 2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN Directional movement of cells through tissues is critical for embryonic development and cancer metastasis, yet how cells maintain polarity in the face of complex environmental cues is poorly understood. Here, we use intravital live imaging in the chick embryo chorioallantoic membrane (CAM) to demonstrate that secretion of exosomes from late endosomes is required for directionally persistent in vivo movement of cancer cells. Knockdown (KD) of three distinct exosome secretion regulators leads to defects in tumor cell migration associated with excessive formation of unstable protrusions and directional switching. Rescue experiments with purified exosomes and matrix coating identify adhesion assembly as a key function of exosomes that promotes cell motility. We propose that autocrine secretion of exosomes is a powerful way to promote directionally persistent cell motility by reinforcing otherwise transient polarization states.

P2204 Board Number: B831

Loss of Dlg5 expression promotes the migration and invasion of prostate cancer cells via Girdin phosphorylation. L. Tomiyama1, T. Sezaki1, M. Matsuo1, K. Ueda1,2, N. Kioka1; 1 Agriculture, Kyoto University, Kyoto, Japan, 2iCeMS, Kyoto University, Kyoto, Japan Dlg5 has been reported to participate in cancer progression; however, its role in prostate cancer still remains poorly understood. In this study, we demonstrate that Dlg5 is frequently downregulated in prostate cancer. We show here that Dlg5 is involved in the regulation of cell migration and cancer cell invasion. Knockdown of endogenous Dlg5 markedly increased prostate cancer cell migration and invasion. Our studies, for the first time, demonstrate the interaction between Dlg5 and Girdin, an actinbinding Akt substrate. Importantly, we found that levels of Akt-mediated Girdin phosphorylation (pGirdin-Ser1416) are increased in Dlg5 -depleted cells. Small interfering RNA directed against Girdin and wortmannin treatment, which was found to reduce Girdin phosphorylation, impaired the effect of Dlg5 depletion on cell migration. Taken together, our findings demonstrate that Dlg5 interacts with and inhibits the activity of Girdin, thereby suppressing the migration of prostate cancer cells.

TUESDAY-POSTER PRESENTATIONS

P2205 Board Number: B832

Novel biosensors for Dbl family RhoGEFs elucidate control of Rac1 by multiple GEFs during breast cancer cell migration. D.J. Marston1, M. Vilela2, G. Danuser2, J. Sondek1, K.M. Hahn1; 1 Department of Pharmacology, University of North Carolina, Chapel Hill, NC, 2Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX Dbl-family RhoGEFs are broadly relevant signaling molecules that regulate cytoskeletal dynamics by integrating the effects of growth factors and adhesion molecules. The timing and localization of GEF activity is precisely controlled to generate different Rho family GTPase responses, thereby producing different actin dynamics and specific types of cellular protrusions. We describe a novel design approach, capable of generating biosensors for multiple GEFs, applied here to visualize activation of Vav2, ASEF and Tiam1 in living cells. A pair of fluorescent proteins was inserted in a region of the GEF with no known binding activity, such that relief of GEF autoinhibition altered FRET between the fluorescent pair. High content screening assays were used to optimize donor/acceptor pairs, insertion site, linkers and circular permutants, increasing the dynamic range of the biosensors to produce FRET ratio changes of up to 300%. The Vav2 biosensor reported phosphorylation of Vav2 by Src kinase and reversible Vav2 activation upon EGF stimulation. This biosensor was used to examine activation dynamics during constitutive migration of MDA-MB-231 breast cancer cells, revealing periodic activation of Vav2 during protrusion retraction cycles at the leading edge. Current studies using computational multiplexing are examining the coordination of Vav2 and Tiam1 to regulate Rac-mediated protrusion and cell polarization.

P2206 Board Number: B833

“A Novel Approach for Identifying Cytohesin Variants and their Isoforms in HGF Stimulated Cell Migration using High Resolution Melting Analysis.”. A. Taylor1, L.C. Santy1; Department of Biochemistry , Microbiology and Biochemistry, Pennsylvania State University, University Park, PA 1

Cytohesins are guanine nucleotide exchange factors that play major roles in cell migration, adhesion, and cytoskeleton recycling. The cytohesin family members include: cytohesin1, cytohesin2/ARNO, cytohesin3/Grp1, and cytohesin4. They function by controlling the activation of small GTPases ADP riobsylation factors (ARF1 through 6). These members are closely related and share a similar domain structure. The NH2 terminal domain is comprised of a ~60 residue coil-coil domain that mediates homodimerization scaffolding and localization properties. The central ~200 amino acid catalytic domain is immediately followed by a PH domain. The pleckstrin homology (PH) domain of each of these proteins

TUESDAY-POSTER PRESENTATIONS is the key determinant of the phosphoinositide binding affinity of these PH domain. Cytohesins undergo alternative splicing of a single three- nucleotide exon, resulting in isoforms containing either a diglycine or triglycine motif in their PH domain. We have previously demonstrated that the number of glycine residues in the PH domain of cytohesin2/ARNO and cytohesin3/Grp1 determines their effects on cell adhesion, spreading and localization. It is important to be able to distinguish between the diglycine or triglycine variants in order to help elucidate their roles in cells. To date, an effective assay to differentiate the diglycine or triglycine splice variants has not been developed. High Resolution Melting (HRM) analysis has recently become an efficient, quick, and robust method for genotyping, mutation scanning, and sequencing matching. HRM has proven to be very efficient in even identifying single nucleotide polymorphisms. This study investigated whether HRM analysis can be used to identify and distinguish between cytohesin2/ARNO and cytohesin3/Grp1 and their respective “2G” and “3G” isoforms in madin darbin canine kidney epithelial cells. Specifically, we employed HRM amplicon melting, HRM “snapback” genotyping (asymmetric and symmetric), and unlabeled probe genotyping analyses. The cytohesin amplicons were cloned and sequenced to assist with the interpretation of HRM data. The cDNA HRM curves correlate well with the DNA plasmid controls, which either contained Cytohesin2/ARNO “2G” or “3G” and Cytohesin3/Grp1 “2G” or “3G”. We show that HRM coupled with LC-Green-PCR is a robust, closed-tube, inexpensive analysis for the rapid detection of canine cytohesin2/ARNO and cytohesin3/Grp1 and their respective isoforms. By combining HRM with a simple PCR design, this novel approach is the first assay capable of distinguishing between cytohesin splice variants. Cytohesins are important regulators of proliferation, migration and invasiveness in cancer. This study provides an assay to identify the specific isoforms that are being upregulated, and thus aid in the understanding of cytohesin function in cancer. In addition, identifying cytohesin isoforms as prospective therapy targets for different cancer types and other diseases that affect cell motility could be possible.

P2207 Board Number: B834

Haptotaxis requires a Rac1/WAVE/Arp2/3-based pathway that regulates differential lamellipodial dynamics. S.J. King1, S.B. Asokan1, C. Wu1,2, J.D. Rotty1, K.T. Chan1,3, I.P. Lebedeva1,2, J.E. Bear1; 1 UNC Lineberger Comprehensive Cancer Center, Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC, 2Howard Hughes Medical Institute, Chevy Chase, MD, 3Howard Hughes Medical Institute, Chapel Hill, NC Cells in vivo migrate in response to directional cues such as differing concentrations of extracellular matrix (haptotaxis); however, a mechanistic understanding of how cells sense and respond to these haptotactic cues is lacking. In order to generate the forces required to drive cell migration, actin filaments must be nucleated and organized in a precise manner. One primary mechanism by which new actin filaments are nucleated is the Arp2/3 complex. Using a small molecule inhibitor of the Arp2/3 complex, CK666, and Arp2/3-depleted cells we have previously shown that the Arp2/3 complex is essential for haptotaxis in fibroblasts on a variety of extracellular matrices (Wu, C., S. B. Asokan et al,

TUESDAY-POSTER PRESENTATIONS Cell, 2012). In order to dissect the mechanism of haptotaxis on fibronectin, an agnostic approach using microfluidic chambers that allow direct imaging of fibroblast haptotaxis on gradients of fibronectin has been utilized. The requirement for the Arp2/3 complex for haptotaxis on fibronectin was confirmed using CK666 on vascular smooth muscle cells, indicating that this result is not cell type specific. It has been proposed that the alignment and maturation of focal adhesions could regulate haptotaxis. However, our experiments suggest that fibronectin based haptotaxis of fibroblasts does not appear to be reliant upon a specific integrin pairing or upon the presence of ‘mature’ focal adhesions. One important nucleation promoting factor (NPF) for Arp2/3 is the WAVE complex. Our data suggests that the WAVE complex is the primary NPF required for fibronectin based haptotaxis, with N-WASP and WASH being dispensable. Rac1 is required for WAVE activation, lamellipodial formation and is a component of focal complexes. Inhibition or depletion of Rac1 abrogates fibronectin haptotaxis. Additionally, inhibition of other focal complex components, Src or FAK, also blocks fibroblast haptotaxis on fibronectin, implicating focal complexes in the regulation of fibronectin haptotaxis. Kymography has revealed differential protrusion dynamics across haptotaxing cells, which have the potential to provide the force necessary for differential migration. Furthermore, FRAP studies investigating actin dynamics during haptotaxis have revealed that there are differential actin dynamics across a cell on a gradient on fibronectin that is not present in a cell on uniform fibronectin or upon Arp2/3 inhibition. Our current hypothesis is that differential engagement of integrins triggers a Src/Rac/WAVE pathway that leads to Arp2/3 activation. Resulting in differential actin dynamics and protrusion dynamics, ultimately leading to directed migration towards higher concentrations of extracellular matrices.

P2208 Board Number: B835

A simple assay that distinguishes cell migration from proliferation. H.L. Glenn1, D.R. Meldrum1; 1 Center for Biosignatures Discovery Automation, The Biodesign Institute at Arizona State University, Tempe, AZ Cell motility is a fundamental characteristic of most mammalian cell types and plays a crucial role in normal and pathological processes such as development, wound healing, immune response, and cancer metastasis. Migration is therefore one of the most commonly assayed cellular behaviors. There are a variety of techniques used to evaluate this phenomenon, which range in cost, reliability, and necessary technical expertise. One of the most popular methods is the barrier, or void-filling, assay in which cells are seeded on a surface within a restricted area then allowed to migrate into an adjacent space. This approach is inexpensive, relatively high throughput, can be accomplished with widely available equipment, and does not require advanced expertise or training. The readout is the number of cells that are located in the initially cell-free zone at the end of the migration period. One significant drawback of this approach is that the readout necessarily represents the combined effects of both cell migration and proliferation. We have developed a simple alteration to this approach that employs a commercially available, fluorescent, live-cell stain that is retained through cellular generations. Cell motility is

TUESDAY-POSTER PRESENTATIONS measured as the ratio of the fluorescent signal in the migration area relative to the seeding and migration areas combined. This method allows the contribution of cell migration to be directly measured without the confounding effects of proliferation. We demonstrate the efficacy of this method with cells that have both high and low proliferation or migration rates. We also show that this method can be used to directly compare migration rates between different cell types.

P2209 Board Number: B836

Arpin-mediated idling and reductions in cell speed are associated with turns. R. Gorelik1, A. Gautreau1; 1 CNRS, Gif-sur-Yvette, France We recently reported a novel protein, Arpin, which inhibits the Arp2/3 complex, a molecular machine that nucleates actin filaments at the cellular front in the lamellipodium. There, new actin filaments polymerize and exert force to protrude the edge forward. Arp2/3-based positive feedback signaling is the basis for lamellipodial persistence, which drives effective directional migration. Indeed, we observed that Arpin is associated with decreases in two major parameters of cell migration: speed and directional persistence. To understand how Arpin controls these parameters, in this work we re-examined migration trajectories of three different cell types with Arpin perturbations. In all three systems, we found that speed and turn angle are negatively correlated, suggesting a universal linkage between these parameters. Arpin-mediated decreases in instantaneous speed were associated with increases in turn angle. Importantly, in all three systems Arpin induced episodes of idling, defined as moving below a speed threshold. This threshold is a percentage of the average speed, calculated using the first derivative of log mean squared displacements. Observed idling episodes interrupted and effectively shortened duration of the active phase of migration, consistent with a model in which Arpin counteracts a positive-feedback circuit that sustains the lamellipodium over time. Finally, Arpin-associated idling episodes were followed by turns, implicating idling as a means for enacting a turn.

P2210 Board Number: B837

The Role of Cytohesins in Cell Migration After Acute Kidney Injury. M.M. Reviriego-Mendoza1, L.C. Santy2; Pennsylvania State Univ, State College, PA, 2Department of Biochemistry , Microbiology and Biochemistry, Pennsylvania State University, University Park, PA

1

Acute kidney injury (AKI) is a common and potentially fatal complication that affects 5-9% of hospitalized patients, and with a greater incidence (> 10%) in intensive care unit patients every year. The molecular aspects of kidney injury and repair are still uncertain. Studies have shown that hepatocyte growth factor (HGF) promotes recovery of the injured kidney by inducing survival and migration of

TUESDAY-POSTER PRESENTATIONS tubular epithelial cells to repopulate the bare tubule areas. We have demonstrated in-vitro that HGFstimulated kidney epithelial cell migration requires the activation of Arf6 via the cytohesin family of Arf guanine nucleotide exchange factors (GEFs), and the subsequent activation of Rac1. We hypothesize that this signaling module is required to promote the HGF-stimulated recovery of damaged kidney. We have established an ischemia and reperfusion injury (IRI) mouse model to analyze the effects of modulating this signaling pathway on kidney recovery. We have observed that treatment of injured mice with HGF results in lower creatinine (Cre) and blood urea nitrogen (BUN) when compared to untreated IRI mice, indicative of a faster kidney recovery. In addition, histological analysis of injured kidney tissue samples show that HGF treatment promotes recovery by enhancing kidney tubule repair. We have also treated IRI mice with the cytohesin inhibitor SecinH3. Cre and Bun levels as well as histological analyses show that, unlike HGF, SecinH3 treatment halts injured kidney recovery and delays repair when compared to untreated IRI mice. Analyses to detect active Rac1 and active Arf6 are being carried out to further determine the role of cytohesins and Arf6 in HGF-stimulated kidney recovery. Nevertheless, our results suggest a role of cytohesins in renal ischemia and reperfusion injury repair. We believe that HGFstimulated cell migration in the injured kidney requires the activation of cytohesin. By testing our hypothesis, we expect to broaden the understanding of cell migration in AKI recovery.

P2211 Board Number: B838

The Role of Active ARF6 in Cytohesin-Induced Rac Activation. E. Diekema1, L.C. Santy2; 1 Biochemistry and Molecular Biology, Pennsylvania State Univ, State College, PA, 2Department of Biochemistry , Microbiology and Biochemistry, Pennsylvania State University, University Park, PA Hepatocyte growth factor (HGF) is a potent signaling molecule that causes epithelial cells to scatter and migrate. Small GTPases play an important role regulating the changes downstream of HGF that lead to the initiation of migration. For example, crosstalk between the small GTPases ARF6 and Rac stimulates epithelial cell migration. The ARF-GEF Cytohesin-2 and the Rac-GEF Dock180 have also been implicated in HGF signaling as treatment with the cytohesin inhibitor SecinH3 or expression of the dominant negative form of Dock180 impairs HGF-stimulated wound healing. We have previously shown that the GEF activity of Cytohesin-2 and its promotion of a complex containing the scaffolding protein GRASP and Dock180 is required for cytohesin-induced Rac activation. However, the role of active ARF6 in this process is unclear. Using live-cell imaging, we found that GRASP and Dock180 co-localize with the recycling endosome markers EHD1 and Rab11 at internal locations. Here, we show that upon stimulation, exogenously expressed GRASP and Dock180 translocate from their internal location to the cell periphery when Cytohesin-2 is also expressed. In addition, we are able to detect a FRET signal in live cells using GRASP and Dock180 as FRET pairs, indicating that we can now use FRET to study the spatial and temporal dynamics of the complex. We hypothesize that activation of ARF6 by Cytohesin-2 regulates trafficking of GRASP and Dock180 from recycling endosomes to the plasma membrane. As either overexpression or up-regulation of HGF has been implicated in both cancer progression and

TUESDAY-POSTER PRESENTATIONS wound healing, respectively, increased understanding of HGF signaling would be beneficial to lead to the development of novel therapeutics to either fight metastasis or promote injury recovery.

P2212 Board Number: B839

A NOVEL MICROFLUIDIC ASSAY THAT MIMICS THE IN VIVO LEUKOCYTEENDOTHELIUM INTERACTIONS. G. Lamberti1, F. Soroush2, M.F. Kiani3, B. Prabhakarpandian4, A. Smith4, B. Wang5, C. Garson 4, K. Pant4; 1 Department of Mechanical Engineering, Temple University, Philadelphia, PA, 2Temple University, Philadelphia, PA, 3College of Engineering, Temple University, Philadelphia, PA, 4Biomedical Technology, CFD Research Corporation, Huntsville, AL, 5Department of Biomedical Engineering, Widener University, Chester, United States Due to its significance, several in vitro models have been developed to study different aspects of the leukocyte adhesion cascade. However, currently none of these in vitro models can be used for real-time studies of the entire leukocyte adhesion cascade including rolling, adhesion and migration in a single assay. In this study, we have developed and validated a novel bioinspired microfluidic assay (bMFA) and used it to test the hypothesis that blocking of specific steps in the adhesion/migration cascade significantly affects other steps of the cascade. The bMFA consists of an endothelialized microvascular network in communication with a tissue compartment via a 3 μm porous barrier. Human neutrophils in bMFA preferentially adhered to activated human endothelial cells near bifurcations with rolling and adhesion patterns in close agreement with in vivo observations. When the tissue compartment of bMFA was filled with fMLP, 50% of adhering neutrophils migrated into the tissue compartment after 120 min. Treating endothelial cells with monoclonal antibodies to E-selectin or ICAM-1 or treating neutrophils with wortmannin reduced rolling of neutrophils to ~60%, adhesion to ~20% and migration to ~18% of their respective control values. Furthermore, antibody blocking of specific steps in the adhesion/migration cascade (e.g. mAb to E-selectin) significantly downregulated other steps of the cascade (e.g. migration). This novel in vitro system provides a realistic human cell based model for basic science studies, identification of new treatment targets, selection of pathways to target validation, and low-cost test bed for rapid screening of candidate agents.

TUESDAY-POSTER PRESENTATIONS

Dynamics of Focal Adhesions and Invadosomes P2213 Board Number: B840

Linking sub-cellular mechanical forces to integrins mobility using Image Correlation Spectroscopy. R. De Mets1,2, M. Balland3, O. Destaing4, A. Delon 1, I. Wang5; 1 UMR 5588, Laboratoire Interdisciplinaire de Physique, Grenoble, France, 2Inserm U823, Institute Albert Bonniot, Grenoble, France, 3Materials, Optics and Instrumental Techniques for the Life Sciences (MOTIV), Laboratory of Interdisciplinary Physics (LiPhy), Grenoble, France, 4Differentiation and Cell Transformation, Institute Albert Bonniot (IAB) Inserm U823, Grenoble, France, 5UJF/UMR 5588, Laboratoire Interdisciplinaire de Physique, Grenoble, France The ability of cells to sense physical properties of the extracellular matrix (ECM) is essential to regulate their proliferation, migration and differentiation. Cells feel the different properties of ECM and respond to it by exerting forces on their microenvironment. This process is mainly based on the activity of integrin receptors. Integrin activities depend on their ability to bind-unbind cyclically ECM and are then characterized by their diffusion regime at the plasma membrane (Rossier & al, 2012) which might be modulated by active forces. The present project aims to understand the link between cellular forces and integrin activity in the regulation of mechanotransduction processes. Our work focuses on 2 types of integrins: b1 that plays a role as force generator and b3 that rather regulates cellular forces (Roca-Cusachs & al, 2009). The strategy adopted consists in modulating the spatial distribution of cellular forces by using micropatterns of various geometries and coupling forces modulation to temporal Image Correlation Spectroscopy (tICS). Practically, tICS allows us to determine in any point of the cells the mobility and concentration of b1 and b3 integrins tagged with GFP. The principle of tICS is based on the temporal autocorrelation of a series of images. From the amplitude and the shape of the autocorrelation functions inside a given ROI, we can extract information about the molecule number, their mean diffusion constant and the immobile molecule fraction. Each chosen ROI corresponds to a particular force arrangement, previously measured using patterned traction cytometry (Tseng & al, 2011). Based on our set-up, we were able to modulate the spatial distribution of actin generated forces at the single cell level . We then measured the relation between those sub-cellular forces and the local concentration and mobility of those integrins. Interestingly, we found a strong correlation between the amplitude of traction forces and the mobility of b3, confirming its role of regulator in cell mechanical activity. Strikingly, we did not find the same behaviour in the case of b1 integrins. Overall, this work brings new insights in the field of cell mechanics and provides information at the cell level about integrin dynamics and their correlation to cell mechanical activity.

TUESDAY-POSTER PRESENTATIONS

P2214 Board Number: B841

Structured illumination microscopy reveals that focal adhesions are composed of linear subunits. S. Hu1, Y. Tee1, A. Kabla2, R. Zaidel-Bar1, A.D. Bershadsky3,4, P. Hersen1,5; 1 Mechanobiology Institute, Singapore, Singapore, 2Department of Engineering, University of Cambridge, Cambridge, United Kingdom, 3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 4Mechanobiology Institute, National University of Singapore, Singapore, Singapore, 5 Université Paris Diderot, Laboratoire Matière et Systèmes Complexes, UMR 7057 CNRS, Paris, France The cells ability to mechanically interact with the extracellular matrix (ECM) is a fundamental feature of adherent eukaryotic cells, and is required for cell migration, proliferation, and differentiation. Adhesion is mediated by protein complexes, called focal adhesions. Recent progress in super resolution microscopy revealed that they have an internal organization, yet such methods do not allow observing the formation and dynamics of focal adhesions’ internal structure. Here, we combine structured illumination microscopy (SIM) with total internal reflection fluorescence microscopy (TIRF) to be able to observe focal adhesions at high spatial resolution in live cells. We show that focal adhesion proteins inside focal patches are distributed along elongated subunits, typically 300 nm wide, separated by 300 nm and individually connected to actin cables. These focal adhesions subunits growth speed is in average of 0.3 µm/min. We further show that these structures are intimately linked to actin radial fiber formation. Taken together, our study reveals that, when observed with an appropriate resolution in both space and time, the minimal units of adhesion appear to be elongated, dynamic structures. We anticipate this ultrastructure to be relevant when studying the mechano-biological attributes of focal adhesions and their behavior in response to mechanical stress.

P2215 Board Number: B842

Development of a novel cellular gel substrate system and imaging platform for super-resolution fluorescence microscopy. V.K. Gupta1,2, M.J. Colville3,4, A. Singh5, W.R. Zipfel3,5,6, M.J. Paszek2,3,7; 1 Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY, 2School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 3Field of Biophysics, Cornell University, Ithaca, NY, 4Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, 5 School of Applied and Engineering Physics, Cornell University, Ithaca, NY, 6Department of Biomedical Engineering, Cornell University, Ithaca, NY, 7Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY Cells sense the stiffness of their external environment and use this information to influence cell differentiation, tissue development, and pathological states, including cancer. Large protein assemblies

TUESDAY-POSTER PRESENTATIONS called focal adhesions are primarily responsible for linking the cell to the extracellular matrix (ECM) and converting external mechanical forces into internal biochemical signals. Although recent developments in super-resolution fluorescence microscopy allow one to study the nanoscale architecture of focal adhesions, these studies have only been conducted on glass, due to the challenge of nanometer imaging through standard gel substrates. Our aim was to develop a novel imaging platform and high refractive index gel substrate system of tunable stiffness for super-resolution imaging in both the lateral and vertical dimensions. The gel system that we developed consists of a commercially available silicon elastomer whose stiffness can be systematically varied by altering the ratio of its two components. The fabrication of the gels was optimized to achieve uniform thickness, an even surface, high optical quality and transparency, and minimal background fluorescence, which are critical for super-resolution imaging. The elastic moduli of the gels were measured using Dynamic Mechanical Analysis. By varying the ratio of the two gel parts, we were able to systematically adjust the elastic moduli of our gels from 1.7 kPa to 21 kPa. The gel system was highly compatible with cell culture. We observed that epithelial cells demonstrated similar spreading and growth characteristics on stiff (~21 kPa), fibronectin-conjugated gels compared to glass substrates conjugated similarly. We next developed a custom imaging platform for Photo-activated localization Microscopy (PALM) imaging of adhesion structures in cells on our gel substrates. Our system employed a custom circular scanning Total Internal Reflection Fluorescence Microscopy (TIRFM) system, in which the focus of the laser beam is scanned around the periphery of the focal plane, to compensate for irregularities of the gel surface and unevenness of the TIRF field. By combining our imaging platform and elastomer system, we achieved high signal-to-noise imaging of individual fluorophores in PALM. As a proof of principle, super-resolution images of paxillin-mEos2 in epithelial cells were acquired on our elastomeric substrates. We now are evaluating quantitatively how substrate stiffness impacts the molecular organization of major focal adhesion proteins including FAK, vinculin, paxillin, and talin. This work will lead to a greater understanding of how adhesion complexes sense and respond to the stiffness of their environment.

P2216 Board Number: B843

Kinesin Motor Kid Plays a Role in Cell Adhesion and Migration by Affecting Focal Adhesion Localization and Dynamics. L. Xu1, K. Bartoli2, S. Watkins3, W. Saunders1; 1 Biological Sciences, Univ Pittsburgh, Pittsburgh, PA, 2Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 3Center for Biologic Imaging, Univ Pittsburgh, Pittsburgh, PA The kinesin-10 chromokinesin motor family is traditionally considered to play a role mainly during mitosis influencing chromosome alignment. We believe the Kid/KIF22 member of this group has additional functions during interphase of the cell cycle.

TUESDAY-POSTER PRESENTATIONS We observed that Kid localized to the sites of focal adhesions (FAs) in multiple cell lines and FAs became redistributed when Kid’s function was impaired. In wild-type HeLa cells, FAs are mostly distributed near the peripheral edges of the cell. After knockdown of Kid, we found that the number of FAs significantly increased, and they became more scattered throughout the cytoplasm. FAs are integrin-based multiprotein structures that mediate the attachment of cells to the extracellular matrix (ECM). They play essential roles in various cellular processes including cell morphology, migration, proliferation, and differentiation. Hence, we checked if such changes in the number and distribution of FAs have any effect on cell adhesion and/or migration. Indeed, cells after knockdown of Kid showed a much slower rate of spreading compared to control cells when seeded onto fibronectin, which is a component of ECM. Additional results from our wound healing assay suggested that loss of Kid caused an about two-thirds reduction in the rate of cell migration. Based on these observations, and previous studies showing the role of microtubules in FA stability, we hypothesized that Kid might be involved in regulating assembly and/or disassembly of FAs, thus affecting cell adhesion and migration. Confocal microscopy showed a higher proportion of focal complexes (FCs), which are smaller precursors of FAs, after knockdown of Kid. TIRF microscopy indicated that loss of Kid led to an increased life duration time of FAs. Since the small GTPase Rho is required for the maturation of FCs to FAs, we tested if the regulation of FA dynamics by Kid is Rho-dependent. When wild-type HeLa cells are treated with Rho inhibitor, there were few FAs due to the inhibition of FA maturation. Moreover, there were few FCs as the Rho inhibitor also interrupts actin stress fibers which are required for the assembly of FCs. Interestingly, when we treated cells with Rho inhibitor after knockdown of Kid, there were more FAs and FCs remaining, supporting the idea that Kid functions to destabilize FAs in the absence of Rho activity. Taken together, these data suggested that Kid plays a role in the localization and dynamics of adhesion structures. This study identified a non-conventional role of Kid during interphase and expanded on our knowledge of how a motor protein might be involved in the focal adhesion network affecting cell adhesion and migration.

P2217 Board Number: B844

Novel function of kinesin KIF1C as a component of podosome adhesive rings in vascular smooth muscle cells. K. Frye1, A. Bachmann2, N. Efimova3, A. Straube2, I. Kaverina1; 1 Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, 2Center for Mechanochemical Cell Biology, Warwick Medical School, The University of Warwick, Coventry, United Kingdom, 3Department of Biology, University of Pennsylvania, Philadelphia, PA Extracellular matrix remodeling during physiological processes is mediated by invasive protrusions called podosomes. Podosomes consist of a polymerizing actin core surrounded by an adhesive ring. In vascular smooth muscle cells (VSMCs), podosomes can be rapidly initiated by PKC activation. We have previously

TUESDAY-POSTER PRESENTATIONS shown that kinesin KIF1C is important for efficient podosome formation in VSMCs but the mechanism whereby KIF1C performs this function is largely unknown. Now, our live-cell imaging data show that deposition of KIF1C at distinct sites at the ventral cell membrane correlates with new podosome formation at those sites. In particular, KIF1C accumulates around emerging podosome cores, which are marked by cortactin and TKS5, and acquires a ring-like shape. Our data indicate that KIF1C is delivered and relocated along MTs, but resides at adhesive rings in a MT-independent manner. Because without KIF1C, both the number and size of podosomes are decreased, we hypothesize that this kinesin provides a base for the adhesive ring formation. In addition, KIF1C likely supports relocation of adhesive ring components during movement of existing podosomes to new cellular positions, as seen in relocation of podosomes from the periphery toward the cell center via KIF1C-rich membrane tubes. These findings indicate that microtubule-dependent KIF1C transport and deposition is critical for podosome positioning in VSMCs. Through this mechanism, our study implicates KIF1C as a regulator in the distribution of podosomes over the ventral cell surface that is thought to be important for a more uniform ECM remodeling by VSMCs rather than site specific degradation typical for cancer cells.

P2218 Board Number: B845

Microtubules Regulate Focal Adhesion Dynamics through MAP4K4. J. Yue1, X. Wu1; 1 The Ben May Department, The University of Chicago, Chicago, IL Focal adhesions are dynamic organelles that establish a connection between the ECM (extracellular matrix) and cytoskeletal networks and serve as points of traction for cells. The disassembly of focal adhesions allows cell retraction and integrin detachment from the ECM, processes critical for cell movement. A long-standing and intriguing observation is that growth of MT filaments in migratory cells can be guided toward focal adhesions, and targeting of MT growing ends (plus ends) to focal adhesion often precedes focal adhesion turnover. It has been speculated that MTs can serve as tracks to deliver proteins essential for focal adhesion disassembly. However, the molecular nature of the focal adhesion “disassembly factor” remains elusive. By quantitative proteomics, we identified mammalian MAP4K4 (mitogen-activated protein kinase kinase kinase kinase 4) as a focal adhesion regulator that associates with MTs. Conditional knockout (cKO) of MAP4K4 in skin epidermal cells stabilizes focal adhesions and impairs epidermal migration. Exploring underlying mechanisms, we further show that MAP4K4 associates with MT plus end tracking protein, EB2 (end binding 2) and an Arf6-specific guanine nucleotide exchange factor, ARF-GEP100 (ADP-Ribosylation Factor-Guanine Nucleotide Exchange Protein-100 kDa), which is also known as IQSEC1 (IQ motif and SEC7 domain-containing protein 1). Together, our findings provide new insights for this critical cellular process, suggesting that EB2containing MT plus ends can deliver MAP4K4 toward focal adhesions, where MAP4K4 can in turn activate Arf6 via IQSEC1 and enhance focal adhesion dissolution.

TUESDAY-POSTER PRESENTATIONS

P2219 Board Number: B846

Phosphorylation of Serine 106 Regulates Asef2 GEF Activity and Asef2-Mediated Cell Migration and Adhesion Turnover. B. Erdogan1, J. Evans1, K. Hines2,3,4, J. Forsythe2,3,4, M. Shi1, S. Hill5, K. Rose5,6, J. McLean2,3,4, D. Webb1,4,7; Department of Biological Sciences, Vanderbilt University, Nashville, TN, 2Department of Chemistry, Vanderbilt University, Nashville, TN, 3Vanderbilt Institute for Chemical Biology, Vanderbilt University, Nashville, TN, 4Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN, 5Mass Spectrometry Research Center, Vanderbilt University, Nashville, TN, 6 Department of Biochemistry, Vanderbilt University, Nashville, TN, 7Department of Cancer Biology, Vanderbilt University, Nashville, TN 1

Asef2 is a recently identified guanine nucleotide exchange factor (GEF) that regulates cell migration and adhesion turnover via activation of Rho family GTPases, including Rac. Asef2 is activated by binding of the tumor suppressor adenomatous polyposis coli (APC) to its N-terminal region; however, much less is known about other modes of Asef2 regulation, such as post-translational modifications. In this study, we identified six phosphorylation sites in Asef2 using liquid chromatography coupled with high-resolution mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). One of these phosphorylation sites, serine 106 (S106), is located within the APC binding region, suggesting a potential role in Asef2 regulation. To probe the effects of S106 phosphorylation on Asef2 activity, Asef2 mutants were generated in which S106 was changed to either alanine (S106A) or aspartic acid (S106D), which represent non-phosphorylatable and phosphomimetic analogues, respectively. GFP-tagged Asef2 S106 mutants were transfected into HT1080 cells and activation of Rac was assessed using a pulldown assay. S106A mutation greatly reduced Rac activation by Asef2, while the phosphomimetic mutation (S106D) led to an increase in active Rac levels. Next, we tested the effects of S106 mutation on cell migration using live-cell time-lapse microscopy. Wild-type GFP-Asef2 promoted migration of HT1080 cells on fibronectin, as we have previously shown. Expression of GFP-Asef2-S106A abrogated Asef2-promoted migration, whereas GFP-Asef2-S106D expression promoted migration similarly to GFP-Asef2. Because our previous study showed that Asef2 promotes cell migration by increasing adhesion assembly and disassembly (adhesion turnover), we next determined if S106 phosphorylation affects cell migration by modulating adhesion turnover. Cells expressing wild-type GFP-Asef2 showed an approximately 50% decrease in the t1/2 values for adhesion assembly and disassembly compared to control cells expressing GFP, indicating that adhesions turn over more rapidly in Asef2-expressing cells. Conversely, expression of GFP-Asef2-S106A led to an increase in the t1/2 values for adhesion assembly and disassembly compared to GFP-Asef2 expression, indicating that S106A expression negates the Asef2-mediated effect on adhesion turnover. Moreover, expression of GFP-Asef2-S106D caused more rapid adhesion turnover comparable to that observed with GFP-Asef2 expression. These results demonstrate that Asef2 S106 phosphorylation promotes faster adhesion turnover, which is critical for efficient cell migration. Taken together, we have identified a new regulatory mechanism for Asef2, S106 phosphorylation, which is crucial for Asef2-mediated Rac activation, cell migration, and adhesion turnover.

TUESDAY-POSTER PRESENTATIONS

P2220 Board Number: B847

Septins enhance the mesenchymal-like motility of renal epithelia by promoting stress fiber connectivity and focal adhesion maturation. L. Dolat1, J.L. Hunyara1, J.R. Bowen1, E.P. Karasmanis1, V. Galkin2, E.T. Spiliotis1; 1 Biology, Drexel University, Philadelphia, PA, 2Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA Epithelial-mesenchymal transition (EMT) underlies renal fibrosis and the metastasis of renal cell carcinomas (RCCs). EMT is characterized by disruption of cell-cell adhesions, cell scattering and the enhancement of cell motility with a front-rear polarity. While the disruption of cell-cell adhesion during EMT is extensively studied, little is known about the molecules and mechanisms that enhance the mesenchymal-like motility of epithelial cells. Septins are filamentous G proteins that are over-expressed in metastatic RCCs, but their functions in the mesenchymal-like motility of epithelial cells is unknown. Using the Madin-Darby canine kidney (MDCK) model of partial EMT that allows the study of epithelial motility in 2D and 3D matrices after stimulation with hepatocyte growth factor (HGF), we discovered a novel network of septin filaments that underlies the organization of the transverse arc and radial (dorsal) stress fibers in the leading lamella of migrating epithelia. We show that this lamellar network of septin filaments is required for the maturation of nascent focal adhesions as septin depletion results in smaller and more transient and peripheral focal adhesions with increased levels of phosphorylated paxillin-Y118. In addition, septin-depleted cells consisted of a disorganized transverse arc and shorter and fewer radial stress fibers. These phenotypes were independently rescued by alpha-actinin and the actin-binding motor-dead myosin II N93K, but not by alpha-actinin-deltaABD, which cannot bind or bundle actin, or the constitutively active myosin regulatory light chain RLC-DD. These data suggested that septins function as actin-binding and cross-linking proteins. Using low speed sedimentation assays and negative stain EM, we show that pre-assembled actin filaments are bundled by septin 9 (SEPT9), whose expression is increased after induction of renal epithelial motility with the hepatocyte growth factor. We show that SEPT9 over-expression enhances renal cell migration in 2D and 3D matrices, while SEPT9 knock-down decreases the velocity and alters the mesenchymal-like shape of epithelial cells migrating in 3D matrices. Taken together, these results suggest that septins promote epithelial motility by reinforcing the connectivity of lamellar stress fibers and thereby, stabilizing nascent focal adhesions in the leading edge of motile epithelia.

TUESDAY-POSTER PRESENTATIONS

P2221 Board Number: B848

Evidence of multiple roles for Diaphanous-Related Formins (DRFs) in Invasion. E.W. Miller1, S.D. Blystone1; 1 Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY In cancer cells, the actin cytoskeleton performs multiple functions in invasion and metastasis, partially through F-actin dense, matrix-degradation structures known as invadopodia. Assembly of monomeric actin into filaments is regulated by several different proteins, including a relatively new group of proteins known as formins. A subfamily of this group of proteins, the diaphanous-related formins (DRFs), share a common domain architecture and interact directly with Rho-family GTPases. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), we determined expression levels for all ten DRFs in the highly-invasive breast cancer cell line MDA-MB-231. We then questioned whether depletion of endogenous DRF expression would result in impaired MDA-MB-231 cell invasion. Using DRF-targeting siRNA, we silenced expression of endogenous Dia1, Dia2, and FMNL1 by 75.2%, 65.0%, and 83.5% respectively. Cells treated with DRF-targeting siRNA were then subjected to an in vitro Matrigel invasion assay. Interestingly, knockdown of Dia1 and FMNL1 significantly inhibited invasion while knockdown of Dia2 exhibited only moderate inhibition. This led us to speculate that DRFs may have different roles in the invasion process. Using a combination of two DRF-targeting siRNA oligonucleotides, we simultaneously knocked down three combinations of DRFs, all of which had a significant, but neither all additive nor all compensative, effect on the invasive capacity of MDA-MB-231 cells. In order to determine DRF function within the cell, we used immunofluorescence-based microscopy to understand sites of activity. Interestingly, we found both endogenous FMNL1 and Dia2 to be dispersed throughout the cytoplasm with some localization to the perinuclear region of the cell. We then proceeded to investigate one of these DRFs, FMNL1, more closely. Since knockdown of FMNL1 resulted in a significant inhibition of 3D invasion, we questioned whether knockdown would have the same effect on 2D migration. Cells were treated with DRF-targeting siRNA and subjected to a scratch-wound assay. While cells treated with control siRNA closed the wound within 18 hours, cells treated with FMNL1-targeting siRNA required >24 hours to complete wound closure. FMNL1 is of particular interest as three alternative splice isoforms of this protein have been identified, all of which we detected in MDA-MB-231 cells using PCR. Future examination of all ten DRFs, especially FMNL1, and subsequent meta-analysis will allow us to determine whether there are unique events specific to individual formins which may be targeted by novel therapeutics to treat metastasis.

TUESDAY-POSTER PRESENTATIONS

P2222 Board Number: B849

A plastic relationship between vinculin-transmitted tension and adhesion area defines adhesion complex size and lifetime. U. Berge1, P. Hernandez Varas2, J.G. Lock1, S. Strömblad2; 1 Dept. of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden, 2Department of Biosciences and Nutrition, Karolinska Institute, Stockholm, Sweden Cell-matrix adhesions mediate critical mechanical force transduction, but the interplay between force and adhesion regulation remains unclear. Here, live cell imaging enabled the mapping of timedependent cross-correlations between mechanical tension and adhesion area, revealing a high degree of plasticity in this relationship. Interestingly, while an expected positive cross-correlation dominated in mid-sized adhesions, small and large adhesions displayed negative cross-correlation. Furthermore, while large changes in adhesion area followed tension alterations, small increases in area preceded tension dynamics. Modelling based on this mapping of the tension-adhesion area relationship confirmed its biological validity, and indicated that the tension-adhesion area relationship can explain adhesion size and lifetime limits, keeping adhesions focal and transient. We also identified a subpopulation of adhesions that remain stable in size and tension, whose disassembly may instead be microtubulemediated. In conclusion, we define a plastic relationship between tension and adhesion area that controls cell-matrix adhesion focality.

Integrins and Cell–ECM Interactions 2 P2404 Board Number: B851

Nitric oxide plays a role in transmitting biochemical signal from the extracellular matrix for mammary epithelial morphogenesis. S. Furuta1, M.J. Bissell1; 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA

Nitric oxide (NO) is a short-lived, gaseous signaling molecule produced in response to biochemical or mechanical stress in diverse tissues and organisms. Despite its evolutionary conservation and ubiquitous expression, the bioactivities of NO have been primarily studied in specialized tissues, including mammalian neurons, muscles, endothelial or immune cells, where NO triggers cell membrane hyperpolarization and relaxation or exerts cytotoxic action. In the past decade, however, different functions of NO in other tissues and organisms have been gradually unraveled, including embryogenesis and morphogenesis of Xenopus, pond snail and Drosophila. Nevertheless, whether NO is involved in the establishment of a mammalian tissue is yet to be determined. We found that non-malignant mammary epithelial cells (MECs) produced NO in response to the extracellular matrix (ECM) proteins laminin-5 and -1 and that NO production was essential for their ability to form growth-arrested polarized acini (the functional unit of mammary gland) in three-dimensional (3D) laminin-rich (lr) ECM cultures. While this mechanism was abrogated in breast cancer cells that failed to form acini in 3D cultures, induced NO production by the use of an NO donor allowed them to restore the formation of normal-like polarized

TUESDAY-POSTER PRESENTATIONS colonies. We further found that such an essential role of NO in mammary acinar formation was attributed to its ability to trigger cell membrane hyperpolarization and hence enhance tight junctions through co-localization of E-cadherin and cortical actin. These observations, for the first time, uncover that NO is produced by MECs in response to a biochemical stimulus of the ECM and plays a critical role in mammary acinar morphogenesis, whereas the lack of NO production is relevant to the malignant phenotype of breast cancer cells.

P2223 Board Number: B851

Dynamic and hierarchical reorganization of integrin-associated molecular complexes in nascent adhesions. A. Bachir1, J. Zareno1, K. Moissoglu2, E. Plow3, E. Gratton4, A.R. Horwitz5; 1 Department of Cell Biology, Univ of Virginia, Charlottesville, VA, 2Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, 3Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, 4Laboratory of Fluorescence Dynamics, University California-Irvine, Irvine, CA, 5Cell Biology, University of Virginia, Charlottesville, VA An intricate network of putative molecular interactions comprises cell-matrix adhesions. Most of these interactions are inferred from co-immunoprecipitation, often using over expressed components. However, few have been demonstrated or characterized functionally in living cells, due to challenges in capturing highly regulated and transient associations that may form and evolve in adhesions. Our study uses high-resolution and image-based fluorescence fluctuation microscopy to map the formation and stoichiometry of integrin-associated molecular complexes in the nascent adhesions that populate the leading edge of migrating cells. We focus on putative integrin activating (kindlin and talin) and actinlinking (talin, vinculin and -actinin) molecules and show that all molecules are present in nascent adhesions as soon as they are visible; however, they form integrin containing complexes hierarchically, at different times and with variable stoichiometry within the adhesion itself. These observations suggest a working model for nascent adhesion assembly, whereby transient complexes containing ~ 3 a-actinin and integrin molecules help nucleate nascent adhesions. Subsequently, integrin complexes form that contain kindlin in a 1:1 ratio, but not talin. Talin localizes to adhesions independently of the integrinkindlin complex, and associates with vinculin in molecular complexes. However, once the nascent adhesion has formed, myosin II activity promotes talin association with the integrin-kindlin complex in a 2:1 stoichiometry, suggesting that each talin molecule cross-links two integrin-kindlin complexes. Finally, as adhesions mature into larger structures, talin increases to a 1:1 ratio with integrin and kindlin; whereas the ratio of vinculin to the other molecules continues to increase. In summary, using novel fluorescence fluctuation microscopy, we show that hierarchical adhesion assembly and reorganization accompanies adhesion formation.

TUESDAY-POSTER PRESENTATIONS

P2224 Board Number: B852

Cytoskeletal integrity and dynamics regulate force mediated cell detachment. A. Fuhrmann1, A.J. Engler1; 1 Bioengineering, University of California, San Diego, La Jolla, CA Integrin mediated cell adhesion is regulated by extracellular factors including divalent cations, which vary with niche. Since adhesion is ubiquitous to all adherent cells and involved in many processes, e.g. migration, quantitative information about adhesion strength is fundamental for understanding cell-ECM interactions. While it is commonly assumed that adhesion assays measure integrin-ECM bond strength, reports now show that cell material remains behind after exposure to acute shear and subsequent detachment. Here we show that many focal adhesion (FA) proteins but not actin remain behind after shear induced detachment of HT1080 fibrosarcoma cells suggesting that the cytoskeleton-FA connection disconnects during detachment more so than integrins when bound to planar substrates in the presence of divalent cations. Cytoskeletal stabilization with Phalloidin oleate (PO) did indeed increase attachment strength by 8-fold while crosslinking integrins to the substrate with DTSSP only caused a 50% increase. However, drug treatments can significantly affect cell function well before shear application. Conversely reducing the temperature only during shear application also increased attachment strength 8-fold. Even at lower temperatures, e.g. 8oC, cells leave FA proteins behind that co-localize with actin suggesting that the maximal integrin-ECM bond strength was not exceeded. The importance of temperature in stabilizing adherent cells’ cytoskeleton is not well appreciated in adhesion assays with many of them operating at not further specified ‘room temperature’, and so we determined the dependence of attachment strength on temperature. We found that the rate of attachment strength change is most significant around room temperature (16oC – 28oC), highlighting the importance of carefully monitoring measurement temperature. Finally by observing this fracturing behavior in mouse and human fibroblasts as well as determining that it is ligand-independent, we demonstrate the ubiquity of this cytoskeleton-FA mode of detachment despite small variation in the strength of the response. Together these data demonstrate that the cytoskeleton and its dynamic coupling to focal adhesions are critically important for cell adhesion in niche with divalent cations.

P2225 Board Number: B853

Nanoscale architecture of tension generation within focal adhesions. A.H. Mekhdjian1, M. Morimatsu1, A.C. Chang1, S. Tan1, A.R. Dunn1; 1 Chemical Engineering, Stanford University, Stanford, CA Living cells are exquisitely responsive to mechanical cues from their surroundings. A primary means by which cells sense and transmit mechanical force is through integrins, a class of heterodimeric, transmembrane proteins that physically link the cell cytoskeleton to the extracellular matrix (ECM). The cytoplasmic domains of integrins recruit numerous proteins that collectively comprise focal adhesions

TUESDAY-POSTER PRESENTATIONS (FAs), micron-sized assemblies responsible for mechanosensing and traction force generation. However, the underlying mechanisms responsible for the spatial and temporal organization of force generation within FAs remain poorly understood. We used Förster resonance energy transfer (FRET)-based molecular tension sensors (MTSs) to directly visualize mechanical forces exerted by individual integrins. Simultaneous super-resolution imaging of MTSs and GFP-tagged cellular proteins results in maps of the force-producing structures within FAs with better than 100 nm spatial resolution, allowing us to correlate local force generation with the presence of specific integrin heterodimers and cytoskeletal proteins. We find that αvβ3 integrin localizes to high force regions, whereas α5β1 integrin localization is more diffuse. The canonical FA proteins paxillin, vinculin, talin, and α-actinin colocalize with force production to varying degrees. Surprisingly, paxillin, which is not generally considered to play a direct role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or α-actinin, proteins with hypothesized roles in mechanotransduction. In addition, simultaneous timelapse imaging of either GFP-paxillin or GFP-α-actinin with MTS-measured local traction forces reveals that paxillin and tension are closely related in both space and time in assembling and disassembling adhesions, while α-actinin exhibits a more complex relationship with tension. The high degree of spatial correlation of both paxillin and αvβ3 integrin with mechanical tension suggests that these proteins may work in concert to regulate both FA dynamics and intracellular, mechanically induced signal transduction.

P2226 Board Number: B854

Focal adhesion kinase links lamellipodial actin and nascent adhesions to the ECM through the Arp2/3 complex to regulate leading edge dynamics. V. Swaminathan1, C. Waterman2; 1 Cell Biology and Physiology Center, NHLBI/NIH, Bethesda, MD, 2National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD Efficient cell migration requires proper dynamic interaction between the extracellular matrix(ECM) and the cytoskeletal network through the focal adhesion(FA) system. While FA molecules like vinculin are known to couple stress fibers through mature adhesions to the ECM, what couples dendritic actin at the leading edge through nascent adhesions (NA) to the ECM is unknown. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to nascent and mature adhesions and plays key roles as a scaffold and a signaling molecule. FAK is thought to regulate the disassembly of mature FA, although recent evidence points to FAK playing critical role in early adhesion events. FAK has been shown to recruit the integrin-actin linker talin to NA and also interacts with the Arp2/3 complex that nucleates the actin network in lamellipodia. Based on these results, we hypothesized a novel role for FAK in linking lamellipodial actin dynamics and NA via its interaction with Arp2/3. Using fibroblast cells derived from a FAK knockout (KO) mouse combined with high resolution DIC and TIRF microscopy, we found that FAK KO cells showed faster lamellipodial protrusion and retraction velocities as well as increased frequency in these cycles. Fluorescent speckle microscopy of actin revealed increased actin retrograde flow and

TUESDAY-POSTER PRESENTATIONS weaker engagement of the NA to the ECM marked by their sliding in the lamellipodia and a wider lamellipodia compared to controls. These defects were rescued by expression of FAK. These results implicate FAK in stabilizing NA, attenuating actin polymerization and retrograde flow at the leading edge, and delineating a lamella-lamellipodia border. To test whether FAK’s role at the leading edge was dependent on its interaction with the Arp2/3 complex, we expressed a FAK mutant defective in Arp2/3 binding in FAK knockout cells. The FAK mutant was effectively recruited to NA and FA and resulted in attenuation of leading edge protrusion and actin flow velocities compared to FAK KO, indicating that FAK reduces actin polymerization and retrograde flow independent of its interaction with Arp2/3. However, compared to knockout cells expressing full length FAK, lamellipodial retraction was faster, suggesting an adhesion engagement defect. Indeed, compared to control, in cells with the FAK-Arp2/3 interaction disrupted, NAs often failed to engage to the ECM, had shorter lifetimes, and showed sliding behavior at the same rate as actin retrograde flow, similar to what was observed in the FAK KO cells. Together, these results implicate the specific interaction of the Arp2/3 complex and FAK in stabilizing the NA-ECM linkage at the leading edge of a cell which is required to attenuate edge retraction and result in overall net advance of the leading edge.

P2227 Board Number: B855

Semaphorin3A Shifts the Biphasic Relationship Between Cell Motility and Substratum Concentration Through Increased Focal Adhesion Formation. A. Miller1, S. Gehler2; 1 Augustana College, Bloomington, IL, 2Biology, Augustana College, Rock Island, IL Cell migration plays an essential role in normal and pathological processes such as embryonic development, wound healing, and tumor metastasis. Interactions between integrin-mediated adhesions and the extracellular matrix (ECM) are important regulators of cell migration. Studies have shown that cells exhibit a biphasic relationship between cell migration speed and substratum concentration, suggesting cells experience an optimal level of cell-substratum adhesiveness to facilitate maximal cell migration (DiMilla et, al. 1993). Although these observations are well documented, mechanisms by which extracellular ligands regulate cell migration speed in response to changes in substratum concentration are not clearly understood. Semaphorin3A (Sema3A) has been found to increase integrin receptor expression in breast cancer cells (Staton et, al. 2011) as well as inhibit breast cancer cell motility (Pan et, al. 2009). Because maximal cell motility occurs when there is a balance between substratum concentration and integrin expression, we propose Sema3A alters cell adhesion dynamics to influence breast tumor cell motility on different concentrations of various ECM molecules. First, using a range of concentrations for both collagen type I and fibronectin, MDA-MB-231 cells exhibited maximal migration at intermediate concentrations. However, cells treated with Sema3A exhibited maximal cell migration at lower concentrations of both collagen and fibronectin, while Sema3A inhibited migration at intermediate and high concentrations. Second, integrin-mediated adhesion to different concentrations of either collagen or fibronectin was assessed using a spreading assay. Treatment with Sema3A resulted

TUESDAY-POSTER PRESENTATIONS in maximal cell area at lower concentrations of both collagen and fibronectin, but reduced cell area at intermediate and high concentrations of both substrata. Third, Sema3A increased focal adhesion formation at all concentrations of both collagen and fibronectin. These results suggest Sema3A shifts the optimal level of cell-substratum adhesiveness to lower concentrations of collagen and fibronectin to yield maximal cell migration. Furthermore, the data indicate Sema3A alters cell adhesion dynamics at intermediate and high concentrations of collagen and fibronectin, resulting in reduced cell migration speed.

P2228 Board Number: B856

PAK6 interacts with β-integrin tails and regulates integrin-mediated cell adhesion. E.M. Morse1, B. Ha2, J. Gao2, J.R. Olberding1, K.M. Draheim2, T.J. Boggon2, D.A. Calderwood1,2; 1 Department of Cell Biology, Yale University, New Haven, CT, 2Pharmacology, Yale University, New Haven, CT PAK6 is a member of the p21-activated kinase (PAK) family, a group of six serine/threonine kinases known for their roles in modulating cell growth, survival, adhesion and motility. PAK6 was first identified as a protein that interacts with androgen receptor, and its expression has been correlated to the progression of prostate cancer. However, until recently, little more was known about the cellular roles of PAK6. Here we demonstrate that PAK6 makes a unique contribution to cell growth given that knockdown of PAK6 inhibits cell growth and this growth defect cannot be rescued by its most closely related kinases, PAK4 and PAK5, while it is rescued by re-expression of PAK6. Further, we find that PAK6 regulates cell-matrix adhesion, as knockdown of PAK6 results in increased focal adhesion size and cell spreading. We also find that PAK6 is able to directly interact with the cytoplasmic tails of β-integrins, a family of transmembrane cell adhesion receptors that link the extracellular matrix to the actin cytoskeleton. Taken together, these data support a regulatory role for PAK6 in both cell growth and cellmatrix adhesion, two basic cellular processes crucial to cancer progression.

P2229 Board Number: B857

Super-resolution microscopy reveals a role for the tetraspanin CD82 in regulating integrin molecular clustering. C.M. Termini1, M.L. Cotter1, K.D. Marjon1, K.A. Lidke2, J.M. Gillette1; 1 Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM, 2 Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM Hematopoietic stem and progenitor cell (HSPC) adhesion to the surrounding microenvironment is critical for maintaining HSPC proliferation, differentiation, and survival. However, our present understanding of

TUESDAY-POSTER PRESENTATIONS the mechanisms that regulate this adhesive interaction remains incomplete. Previously, our lab identified the tetraspanin, CD82, as a critical regulator of HSPC adhesion and homing to the bone marrow microenvironment. More specifically, we found that that CD34+ cells treated with monoclonal CD82 blocking antibodies display reduced homing and engraftment in vivo. Tetraspanins, like CD82, are membrane scaffold proteins that promote the organization of membrane proteins, such as integrins, which contribute to cellular adhesion and migration. Therefore, we hypothesize that CD82 regulates HSPC adhesion by modulating integrin organization and density at the nanoscale. In order to test this hypothesis, we generated stable KG1a cells that overexpress wild-type CD82 (CD82OE). We found that the CD82OE cells display increased adhesion to fibronectin when compared to control cells. Pre-treating CD82OE cells with LDV, an α4β1 specific blocking peptide, inhibited the CD82-mediated increase in adhesion, suggesting the involvement of the α4β1 integrin in HSPC adhesion. In order to determine how molecular clustering contributes to HSPC adhesion, we used the super-resolution microscopy technique, dSTORM. We quantified CD82 clustering by pair-autocorrelation analysis and determined that there is an increase in CD82 cluster size in CD82OE cells when compared to the control cells. The palmitoylation of tetraspanins has been shown to regulate lateral membrane protein packing, which is critical for cell adhesion and migration. In order to test the role of CD82 palmitoylation in modulating membrane complex organization and cell adhesion, we also generated KG1a cells that overexpress a mutant form of CD82 that cannot be palmitoylated (Palm-CD82OE). As predicted, we found that the Palm-CD82OE cells display reduced cell adhesion and significantly smaller CD82 clusters than CD82OE cells. Additionally, we used the DBSCAN clustering algorithm to quantify the α4 cluster area from our superresolution data, finding that CD82OE cells display decreased α4 cluster area when compared to control and Palm-CD82OE cells. Interestingly, we found an increase in the number of α4 molecular locations per 0.01 um2 in CD82OE cells when compared to control and Palm-CD82OE cells. Taken together, these data suggest that CD82 regulates α4 molecular density and palmitoylation of CD82 is critical for its regulation of tightly packed α4 clusters within the plasma membrane. Additionally, we will discuss how CD82 expression and mutation control β1 integrin activation and signaling.

P2230 Board Number: B858

Discoidin Domain Receptor 1 Impacts Myosin-Dependent Collagen Remodelling. N.M. Coelho1, P.D. Arora1, C.A. McCulloch1; 1 Matrix Dynamics Group, University of Toronto, Toronto, ON The discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor that is activated by type I collagen, is associated with MMP1-dependent remodelling of collagen. Our objective was to examine the contribution of DDR1 to collagen remodelling by phagocytosis, pericellular proteolysis and traction. Using mouse fibroblasts, human breast cancer cells (MCF7) and cells that do not express β1 integrin (GD25 cells), we analysed the conditioned medium and cell lysates of cells cultured on biotinylated type I collagen. With this approach we tracked the fate of exogenous collagen in different collagen remodelling processes. DDR1 expression levels were positively associated with increased collagen

TUESDAY-POSTER PRESENTATIONS phagocytosis as detected by collagen-coated bead internalization and by immunodetection using streptavidin. With the use of FITC-labeled fibrillar collagen we found that DDR1 expression was associated with higher levels of collagen compaction and fiber reorganization. In cells expressing higher levels of DDR1, there was increased pericellular collagen clearance as detected with FITC-collagen and with collagen ¾ neoepitope antibodies. Inhibition of DDR1 tyrosine kinase activity with nilotinib blocked collagen compaction, fiber alignment and phagocytosis in DDR1 expressing cells but not in DDR1 knockdown cells. Blebbistatin, an inhibitor of non-muscle myosin, did not affect the association of DDR1 and non-muscle myosin IIA in cells plated on collagen but blebbistatin strongly inhibited collagen compaction, alignment and phagocytosis. Collectively these data indicate that DDR1 associates with non-muscle myosin II to mediate collagen remodelling by mechanical reorganization and phagocytosis.

P2231 Board Number: B859

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity. A. Cerchiari1, J. Garbe2, N. Jee3, M. Todhunter3,4, T. Desai5, M.A. LaBarge2, M. Thomson6, Z.J. Gartner3,6; 1 Bioengineering, UC Berkeley - UCSF, San Francisco, CA, 2Lawrence Berkeley National Laboratory, Berkeley, CA, 3Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, 4 Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA, 5Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, 6Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA A combination of cell-intrinsic and extrinsic cues dictates how cell-cell and cell-ECM adhesions orchestrate the self-organization of cells into tissues. The balance between these interactions is central to the outcome of morphogenesis, tissue homeostasis, wound healing, disease progression, and the reconstructions of human tissues in vitro. Characteristics such as differential expression of cadherins and regulation of cortical tension are important cell-intrinsic parameters that influence this process. However, how extrinsic parameters, such as the physicochemical properties of the ECM, modify and relate to intrinsic interactions in the context of tissue self-organization is poorly understood. To clarify how cell-intrinsic and extrinsic cues combine to define tissue architecture in complex epithelia, we FACSsorted human mammary epithelial cells derived from breast reduction mammoplasties into their luminal (LEP) and myoepithelial (MEP) lineages, quantified the differential mRNA and protein expression of key cell-cell and cell-ECM adhesion molecules via qPCR and flow cytometry, quantified their adhesion to each other as well as to the ECM via contact-angle measurements, translated these measurements into works of adhesion, and computationally modeled the self-organization process as a function of variable cell intrinsic and extrinsic parameters using a Metropolis-Hasting algorithm. The mathematical model predicted that MEP-ECM interactions are the dominant factor directing tissue self-organization, and that final tissue architecture is robust to perturbations to other parameters contributing to self-organization. The model also predicted that specific alterations to the balance of parameters directing selforganization could drive catastrophic breakdown of tissue architecture. To test the predictions of our model, we reconstituted compositionally and geometrically defined aggregates of luminal and basal cells

TUESDAY-POSTER PRESENTATIONS fully embedded in different ECMs using 3D cell-patterning technologies we have previously developed. This allowed us to test the consequences of specific perturbations generated with siRNA knockdowns of proteins affecting the primary cell-cell and cell-ECM interactions. We found that talin1 knockdown decreased the work of adhesion between MEPs and the ECM and promoted tissue inversion. In contrast, perturbations such as p120 knockdown in MEPs and/or LEPs decreased work of cell-cell adhesion but had no consequences on cell positioning through self-organization. Contrary to studies of early metazoan development, our results suggest that a single interaction between the basal cells and the ECM overrules cell-cell interactions and directs the self-organization of dynamic and heterogeneous epithelial tissues such as the human mammary gland.

P2232 Board Number: B860

TC21/RRas2 regulates Glycoprotein VI-FcR[gamma]-chain complex-mediated platelet activation and thrombus stability via control of Rap1 and integrin activity. L.E. Goldfinger1,2,3, S. Janapati2, C. Dangelmaier2,4, B. Manne2,4, D. Bhavanasi2,4, J.C. Kostyak2,4, S. Kim2,4, M. Holinstadt5, S.P. Kunapuli2,4; 1 Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA, 2Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, 3Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, 4Pharmacology, Temple University School of Medicine, Philadelphia, PA, 5Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA Many Ras family small GTPases are expressed in human platelets, including Rac, RhoA, Rap, and H-/N/R-Ras1. However, the signaling and cellular functions of most Ras family GTPases in platelets remain poorly understood. Like R-Ras1, TC21/R-Ras2 reverses suppression of integrin activation by oncogenic HRas; hence, TC21 may be important for integrin activation. However, a role for TC21 in platelets has not been explored. In this study, we demonstrate that TC21 is expressed in human and murine platelets and is activated in human platelets in response to agonists for the glycoprotein VI (GPVI)/FcR[gamma] ITAMcontaining collagen receptor (collagen-related peptide (CRP) and convulxin). TC21 activation by GPVI required Src family kinase activity. Platelet aggregation, integrin [alpha]IIb[beta]3 activation, and [alpha]- and dense-granule secretion were significantly inhibited in TC21-deficient platelets in response to GPVI stimulation with CRP ex vivo. In contrast, aggregation and secretion in TC21-deficient platelets were normal in response to stimulation with 2-MeSADP (P2Y receptor agonist), AYPGKF (PAR4 receptor agonist) and fucoidan (CLEC-2 receptor agonist), indicating that the function of TC21 in platelets is GPVI/FcR[gamma]-ITAM-specific. CRP-induced phosphorylation of Syk, PLC[gamma]2, Akt, and ERK were substantially inhibited in TC21-deficient murine platelets. Stimulation with CRP, but not with other agonists, induced enrichment of TC21 at the plasma membrane in human platelets, and TC21 was required for GPVI-induced activation of Rap1b, indicating Ras small GTPase crosstalk. Using a thrombosis model with FeCl3-induced injury to the carotid artery, we observed a delay in initial thrombus formation in TC21-deficient mice compared to WT controls; furthermore, thrombi in TC21-deficient mice were

TUESDAY-POSTER PRESENTATIONS unstable, leading to prolonged time to thrombotic occlusion. Tail clip bleeding times showed similar effects in these mice. Thus, TC21 plays an essential role in GPVI/FcR[gamma]-mediated platelet activation and is important for thrombus stability in vivo via control of Rap1b and integrins.

P2233 Board Number: B861

Defining Phosphatidylinositol Phosphate 5-Kinase Igamma Function in Cell Migration and Anoikis Evasion. N. Thapa1, R.A. Anderson1; Molecular Pharmacology, University of Wisconsin-Madison, Madison, WI

1

Type I phosphatidylinositol-4 phosphate 5-kinases (classified into PIPKIalpha, PIPKIbeta and PIPKIgamma) generate phosphatidylinositol 4,5-biphosphate (PIP2), a lipid signaling molecule that regulate diverse cellular functions, including cell cycle progression, cell survival, endosomal trafficking and cell migration. PIPKIgamma is overexpressed in triple-negative breast cancer tissues and inversely correlates with breast cancer patient survival. In order to define the PIPKIgamma function in tumor progression, we interrogated the PIPKIgamma function in the context of epithelial cells transitioning into the mesenchymal cells, as EMT provides an important platform for understanding how normal cells acquire different traits crucial for tumor progression. Mammary epithelial cells transitioning into mesenchymal cells by TGFbeta1 treatment or by culturing in extracellular matrix proteins displayed significantly increased expression of PIPKIgamma and its focal adhesion targeting variant, PIPKIgammai2. In these cells and other breast cancer cells, PIPKIgamma/PIPKIgammai2 were required for activation of phosphoinositide signaling in response to activation of cells with growth factors and extracellular matrix proteins. During epithelial mesenchymal transition, PIPKIgammai2 lose its association with epithelial protein (E-cadherin) and gain increased association with different pro-migratory molecules, such as talin, moesin, N-cadherin, Src and IQGAP1. Consistently, the loss of PIPKIgammai2 and its associating partners dramatically impaired cell migration. Furthermore, PIPKIgammai2 and talin interaction and their collaborative function were required for providing the signaling inputs from the extracellular matrix proteins and growth factors to maintain the migratory and mesenchymal phenotypes of epithelial cells transformed into mesenchymal cells. Anoikis evasion and the ability to grow in anchorage-independent condition define one of the key properties of metastasizing tumor cells. PIPKIgamma and PIPKIgammai2 shows increased expression in anoikis resistant cancer cells during suspension culture and is crucial for sustaining phosphoinositide signaling. The loss of PIPKIgamma and PIPKIgammai2 dramatically impaired the ability of different tumor cells to grow in anchorage-independent condition. These indicate the diverse pro-tumorigenic function of PIPKIgamma and PIPKIgammai2 in cancer.

TUESDAY-POSTER PRESENTATIONS

P2234 Board Number: B862

Phorbol ester alters cell migration of lung adenocarcinoma on laminin-511. K. Ikari1, S. Fujii2, F. Katagiri2, K. Hozumi2, M. Nomizu2, Y. Kikkawa2; 1 School of Pharmacy, Tokyo Univ Pharm/Life Sci, Tokyo, Japan, 2School of Pharmacy, Tokyo Univ Pharm/Life Sci, Hachioji, Japan Progression of tumor cells results from a multi-step process of carcinogenesis that involves not only sequential genetic alterations but also frequent chemical exposures. Epithelial cells of several organs such as skin and lung are subject to frequent carcinogens and pathogens. Tumor-initiated cells seem to be also transited to malignant tumors by exposure to tumor-promoting agents. Although precancerous cells of epithelium stably anchor to basement membrane, malignant tumors pass through it to achieve metastasis. Of basement membrane components, laminin-511 (α5, β1, γ1; LM-511) has been found to be a major isoform in many adult basement membranes. Several studies have also shown that LM-511 promotes not only cell adhesion but also tumor cell migration. Thus, LM-511 can be viewed as a doubleedged molecule in normal and tumor cells. This is because tumor cells that arise from chemical exposures could alter cell adhesion to LM-511. In this study we examined the effects of potent tumorpromoting agent on lung adenocarcinoma adhering to LM-511. 12-O-tetradecanoylphorbol-13-acetate (TPA) is a phorbol ester that links to the modulation of protein kinase C in epithelial cells. The cells readily attached to LM-511 and Fibronectin (Fn) and spread on the substrata. After adhering to the substrata, the cells were treated with TPA. TPA-treated cells exhibited cuboidal shape on LM-511. On the other hand, the morphology of the cells on FN was flatter or spindle. Flow cytometric analysis showed that the treatment of TPA on A549 cells did not influence the expression of laminin receptors such as integrins and lutheran. Cell attachment assay showed the attachment of TPA-treated A549 cells to LM-511 was weaker than that of control cells, suggesting that the treatment of TPA modulated the binding of laminin receptors. We also examined the cell migration of TPA-treated A549 cells on LM-511. Cell migration was monitored using time-lapse video microscopy. The treatment of TPA promoted cell migration on LM-511 but not FN. We conclude that tumor cell migration on LM-511 requires that the treatment of TPA modulates cell-attachment through the laminin receptors. We suggest that tumor promotion is involved in a transition from static to migratory cell behaviors.

TUESDAY-POSTER PRESENTATIONS

P2235 Board Number: B863

An anti-human Lutheran phage antibody isolated from hepatocellular carcinoma phage library inhibits cell migration on laminin-511. Y. Kikkawa1, Y. Enomoto2, K. Ikari3, F. Katagiri1, K. Hozumi1, M. Nomizu1, Y. Ito2; 1 School of Pharmacy, Tokyo Univ Pharm/Life Sci, Hachioji, Japan, 2Graduate School of Science and Engineering, Kagoshima University, Kagoshima, Japan, 3School of Pharmacy, Tokyo Univ Pharm/Life Sci, Tokyo, Japan The Lutheran glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM) is an Ig superfamily (IgSF) transmembrane receptor for laminin α5, a subunit of laminin-511 that is a major component of basement membranes in various tissues. So far the molecule has been studied primarily in the contexts of blood group antigens and sickle cell disease. B-CAM, a splice variant of Lu, has the same N-terminal extracellular domain as Lu, but it lacks the C-terminal 40 amino acids of the cytoplasmic tail which carries an SH3 binding motif and potential phosphorylation sites that could be involved in intracellular signaling pathways. B-CAM was also identified as an up-regulated antigen in carcinoma, suggesting its involvement in tumor progression. Our previous study showed that although Lu/B-CAM is not present in normal hepatocytes, the expression of Lu/B-CAM is significantly increased in hepatocellular carcinoma cells (HCCs). In this study we isolated thirteen phage antibodies against human Lu/B-CAM from phage library of peripheral blood of HCC patients, suggesting that HCC patients produced autoantibody against endogenous Lu/B-CAM. The RNA of peripheral blood cells was provided from Dr. Hui Kam Man, Natl Cancer Center, Singapore. To characterize the phage antibodies, we determined the epitopes of phage antibodies on Lu. The extracellular domain of Lu contains five IgSF domains, D1-D2-D3-D4-D5. The epitope of A7 phage antibody was localized in D5 domain. The other phage antibodies recognized to D2 domain that is recognized with function blocking mouse monoclonal antibody. Not only C7 phage antibody recognizing to D2 domain inhibited the binding of Lu to ligand, it also prevented tumor migration on laminin-511 (LM-511). However the C7 scFv purified from the periplasm fraction of bacteria could not exhibit the inhibitory effects, indicating that scFv form was not enough to inhibit spatially the binding of Lu to LM-511. Furthermore we identified the amino acid residues to form the epitope recognized by C7 phage antibody. The chimeric mutant study using human and mouse Lu showed that it was formed with amino acid sequences from Ala238 through His257. The mutagenesis study showed that Arg247 is necessary for forming the epitope. C7 phage antibody and its epitope may be useful for developing drugs to prevent HCC progression.

TUESDAY-POSTER PRESENTATIONS

P2236 Board Number: B864

Integrin-integrin Crosstalk Suppresses Fibroblasts Adhesion via PI3K signaling. K. Hozumi1, C. Fujimori2, F. Katagiri1, Y. Kikkawa1, M. Nomizu1; 1 School of Pharmacy, Tokyo Univ Pharm/Life Sci, Hachioji, Japan, 2Tokyo Univ Pharmacy/Life Scis, Hachioji, Japan Various cell surface receptors such as growth factor receptors, GPI anchored receptors, syndecans, and different subtypes of integrin are synergistically related to integrin mediating cell attachment. Several studies has been reported about the crosstalks between different subtypes of integrin and revealed that combination specific integrin-integrin crosstalk is critical on the various biological functions. Here, we focused on various crosstalks of two different subtypes of integrins using the unique integrin binding peptides conjugated polysaccharide (chitosan or alginate) matrix. Six different integrin binding peptides, FIB1 (integrin αvβ3), EF1zz (integrin α2β1), 531 (integrin α3β1), CS1D (integrin α4β1), and A2G10 (integrin α6β1), were mixed and conjugated on the polysaccharide matrix with five different mixture ratios (9:1, 4:1, 1:1, 1:4, 1:9). Human dermal fibroblast (HDF) attachment activities to the 20 different peptide-chitosan matrices were different depending on the peptides and mixture ratios. Ten different pair of mixed peptides-chitosan matrices did not show any special variation by mixture of peptides, and the mixture of FIB1/A2G10, FIB1/CS1D, and EF1zz/CS1D relatively promoted HDF attachment by mixing of two peptides. Interestingly, although EF1zz:531=1:4 showed significant decreasing of cell attachment, but other mixture ratios between EF1zz and 531 did not indicated any differences on both chitosan and alginate matrices, respectively. When we focused on the signal transduction of the EF1zz/531 at 1:4 mixtures, Y397 phosphorylation of FAK was relatively decreased, however Y514 phosphorylation of Src was not. Next we observed the effect of various activator and inhibitor on HDF adhesion to the EF1zz/531-alginate matrices. Integrin activation by TPA or function antibody TS2/16 and PI3K inhibitor of Wortmannin rescue the suppression of HDF attachment at EF1zz:531=1:4, whereas PKC inhibitor of Go 6976 did not affect on that suppression. Further, PKA inhibitors accelerate the suppression of HDF attachment, these suggesting that this suppression was mediated by PI3K signaling through the activation of integrin. From these results, specific integrin-integrin crosstalk promotes or suppresses the cell attachment, and mixed peptide-polysaccharide matrix is useful tool to understand the integrin functions.

TUESDAY-POSTER PRESENTATIONS

P2237 Board Number: B865

Hypoxia enhances differentiation into invasive extravillous trophoblast following BMP4 treatment of human embryonic stem cells. A.K. Wakeland1, M. Moretto Zita2, F. Soncin2, Y. Li2, M. Horii2, M. Parast2; 1 Biomedical Sciences, University of California San Diego, San Diego, CA, 2Pathology, University of California San Diego, San Diego, CA Trophectoderm lineage specification and the bulk of placental growth occur under low oxygen tension. Cytotrophoblast (CTB) are epithelial stem cells in the placenta, which differentiate further into two lineages: hCG-secreting villous syncytiotrophoblast (STB), or invasive extravillous trophoblast (EVT). Proper differentiation into these two lineages is important for appropriate placental function and fetal growth in utero. We have recently shown that human trophoblast lineage specification and terminal differentiation can be modeled in vitro using human embryonic stem cells (hESC). Following treatment with bone morphogenetic protein 4 (BMP4), hESC first differentiate into proliferative CTB, and subsequently into STB and EVT. Differentiation into EVT is characterized by changes in integrin expression as well as expression of matrix metalloproteinases (MMPs), confirming the importance of extracellular matrix (ECM) remodeling in this process. We tested the effects of different ECM and oxygen tension, first on differentiation of primary CTB isolated from first trimester placental tissues. We found that decreasing oxygen levels resulted in impaired differentiation into villous STB based on hCG secretion. Conversely, we found that low oxygen tension directed trophoblast differentiation into invasive EVT lineage, based on increased surface expression of HLAG and integrin alpha-1, as well as secretion of MMP2, which are specific to invasive EVT. EVT differentiation was most optimal when cells were plated on fibronectin, and least optimal on laminin. Similar results were obtained when hESCderived CTB were induced to terminally differentiate, with cells optimally differentiating into EVT when plated on fibronectin and cultured under low oxygen (2%). In addition, hypoxia appeared to accelerate trophoblast lineage specification, with a 20% increase in percentage of EGFR+ CTB after only 2 days of BMP4 treatment under hypoxia. These results show the importance of both ECM and oxygen tension in EVT differentiation, but more importantly, confirm the utility of BMP4-treated hESCs as a model that correctly recapitulates primary EVT differentiation. This is particularly critical as hESC can thus provide a ready alternative to first trimester CTB for the study of EVT differentiation, a process that is highly important to pregnancy success. We are currently testing the role of an intact hypoxia-inducible factor (HIF) complex in both establishment and terminal differentiation of the trophoblast lineage, and hypothesize, based on mouse models of placentation, that it plays a pivotal role in this process, but particularly in differentiation of invasive EVT.

TUESDAY-POSTER PRESENTATIONS

P2238 Board Number: B866

The integrin beta3 tail controls switching between Rac-driven collective migration and Rho-stimulated cell scattering. A. Jimenez Orgaz*1, E. Anthony*2, H. Truong1, I. van den Bout1, I. Kuikman1, P. Secades1, A. Sonnenberg*1, C. Margadant*2; 1 Department of Cell Biology, Netherlands Cancer Institute, Amsterdam, Netherlands, 2Department of Molecular Cell Biology, Sanquin Research, Amsterdam, Netherlands Expression of the fibronectin-binding integrin alpha5beta1 in epithelial cells induces cell scattering, RhoA activity, random single-cell migration, and a switch to a fibroblast-like phenotype. In contrast, the alternative fibronectin-binding integrin alphavbeta3 supports Rac activation, and stimulates collective, directional migration with intact cell-cell contacts. We tested the importance of cytoplasmic domains in the beta3-subunit on cell behaviour. Disruption of the talin-binding NPxY and/or the kindlin-binding NITY motif reduced initial cell adhesion and cell spreading, which was recapitulated by depletion of talin-1 or kindlin-2, respectively. Downstream of cell adhesion, deletion of the NITY motif or Y>A mutation induced an ‘alpha5beta1’-like phenotype, consisting of cell scattering, Rho activity, and random, single-cell migration. These effects are independent of kindlin-2 or Src, but entirely dependent on Rac, and are recapitulated by either the expression of dominant-negative Rac mutants, or constitutively-active Rho. Similarly, the phenotype of cells that express beta3-NITY>A mutants is reversed by overexpression of constitutively-active Rac, or by the inhibition of Rho. Thus, the NITY motif of beta3 controls the balance between Rac-driven collective migration and Rho-stimulated cell scattering, in a kindlin- and Srcindependent manner.

Bioengineering of Cell–Matrix Interactions P2239 Board Number: B867

Minimal matrix model with scar-like fractal heterogeneity reveals a mechanosensitive repressor in stem cell plasticity. P.P. Dingal1, A. Bradshaw1, S. Cho1, J. Swift2, M. Raab3, A. Buxboim4, D.E. Discher1,5; 1 Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, 2University of Pennsylvania, Philadelphia, PA, 3UMR 144, Institut Curie, Paris, France, 4Hebrew University of Jerusalem, Jerusalem, Israel, 5Molecular and Cell Biophysics Lab, University of Pennsylvania, Philadelphia, PA Scarring in higher animals has an adverse and sustained effect on tissue, but the diversity of cell types in a scar complicates understanding of the factors affecting cell phenotype. Here, co-polymerization of collagen-I with polyacrylamide was used to produce a minimal matrix model of a scar in which fractal fiber bundles segregated heterogeneously to the subsurface of an inert hydrogel. Matrix stiffens locally

TUESDAY-POSTER PRESENTATIONS – as in scars – while allowing separate control over adhesive ligand density. Mesenchymal stem cells (MSCs) that are now being used in various tissue repair contexts respond to the model matrices with phenotypes often seen in scars: they polarize (more so than on rigid plastic) and slowly increase expression of ‘scar marker’ smooth muscle actin (SMA). MSCs also traffic to injured sites in vivo, and here they polarize and ‘durotax’ towards scar-like regions of matrices where a subset of stiffnesssensitive proteins respond within hours. Over days, a repressor of SMA transcription, NKX2.5, translocates from nucleus to cytoplasm in response to matrix rigidity, suggesting a mechanism for SMA de-repression. Expression of nuclear-localized mutants of NKX2.5 indeed overrides matrix-rigidity sensing with inhibition of cell spreading and SMA. MSCs cultured for weeks or longer on rigid substrate reinforce a ‘mechanical memory’ by down-regulating NKX2.5, thereby stabilizing an SMA-high, scar-like phenotype.

P2240 Board Number: B868

The Interplay of Duro- and Hapto-taxis in Regulating Stem Cell State. J.H. Wen1, O. Choi2, A.J. Engler1; 1 Bioengineering, University of California, San Diego, La Jolla, CA, 2Biomedical Engineering, National Yang Ming University, Taipei, Taiwan Anchorage dependent cells can sense and respond to extracellular matrix (ECM) properties, e.g. stiffness and ligand density, but gradients of these cues are often found in vivo via normal tissue variation or pathological conditions, such as the post-infarct myocardial scar which is several fold stiffer as well as compositionally different from healthy tissue. Scar tissue and fibrotic tissue are often characterized by the presence of excess matrix proteins such as collagen and fibronectin due to increased deposition and decreased degradation, forming gradients adjacent to healthy tissue. We have previously shown that cell migration can be regulated by substrate stiffness in vitro, but stiffness-directed migration, i.e. durotaxis, may be additionally accompanied in vivo by haptotaxis, i.e. migration up a ligand concentration gradient. To better understand how corresponding and opposing gradients regulate cell migratory behaviors, tunable gradients of stiffness and collagen concentration in a polyacrylamide hydrogel system were constructed using density gradient multilayer polymerization, a technique that utilizes phase separation between liquids of varying density to create layers of distinct composition. Stiffness gradients were obtained by varying the monomer to crosslinker ratio, or by applying a UV photomask in order to activate a photoinitiator gradient. Ligand gradients were obtained by varying the concentration of acrylic acid, thus sequentially varying the density of EDC/NHS linker, in each hydrogel layer; this creates a polyacrylamide substrate with a surface density gradient of covalently bound collagen. Stiffness and ligand gradients were overlaid to investigate the effect of corresponding and opposing haptotactic and durotactic gradients on fibroblast and mesenchymal stem cell (MSC) migratory behavior. Fibroblasts tended to haptotax towards regions of increased collagen concentration and durotax towards stiffer regions of the substrate. While MSCs also underwent durotaxis, surprisingly they

TUESDAY-POSTER PRESENTATIONS did not haptotax. These data suggest that fibroblasts residing in a tissue with such gradients may be more responsive than MSCs, which must home to the tissue from marrow to aide in its repair.

P2241 Board Number: B869

Malignant melanoma cells dynamically construct a tumor biofilm that promotes survival in response to drug treatment and hypoxia. K. Tanner1, A. Afasizheva1; 1 NCI/NIH, Bethesda, MD Cutaneous melanoma cells secrete and assemble a myriad of extracellular matrix (ECM) molecules into dense fibrillar and globular networks. This process is dynamic, mimicking biofilm formation by bacteria in response to nutrient deficiencies, antibiotic treatment and oxidative stress. We hypothesize that the melanoma ‘tumor biofilm’ is similarly adaptive and protective in response to external insults, including pharmaceutical intervention and hypoxic stress. Using quantitative proteomics, we identified the components and determined the temporal evolution of the tumor biofilm. Isogenic melanoma lines were cultured in 3D to recapitulate tumor morphology and interrogated for the establishment of a de novo tumor microenvironment as a function of dimension, oxygen tension and drug treatment. We recreated an established tumor and drug regimen using a modified 3D soft agarose assay to evaluate anchorage independent proliferation. Modulation of the observed tumor biofilm in culture was monitored using confocal microscopy, protein expression analysis and immunofluorescence, and its physiological impact on primary tumor establishment was examined in a mouse subcutaneous injection model. Fibronectin was found to be one of the key architectural components, regulating drug efficacy for a broad spectrum of drug therapies. Stable cell lines engineered to secrete minimal levels of fibronectin became sensitive to cisplatin, BRAF, and ERK inhibition as clonally derived 3D tumor aggregates, emphasizing the importance of 3D architecture in tumor proliferation. Reduced levels of tumor-generated fibronectin also impeded successful tumor formation in mice. We reason that this architectural complexity provides the degree of plasticity and flexibility that allows for better colonial adaptability and survival in response to external perturbations, such as hypoxic stress and drug treatments

TUESDAY-POSTER PRESENTATIONS

P2242 Board Number: B870

Cell-bioactive glass scaffold interactions: The role of substrate nano-structure. T.J. Kowal1, T. Chokshi1, H. Jain2, M.M. Falk1; 1 Biological Sciences, Lehigh University, Bethlehem, PA, 2Material Science and Engineering, Lehigh University, Bethlehem, PA It is not well understood how cells of the human body can interact with biologically active scaffolds used to guide tissue regeneration, especially on the nano-scale level. In order to design the ideal material for the regeneration of different tissues, we must first acquire a comprehensive understanding of the fundamental mechanisms that allow cells to not only attach to these bioscaffold materials, but to also take cues from them as to what tissue should be regenerated. Here we present evidence of the ability of MC3T3-E1 pre-osteoblasts to detect differences in nanostructure on two different substrates. The first substrate, a bioactive glass with a simple composition of 30 mol%CaO – 70 mol%SiO2 contains both nano- and macro-pores. The only variable between samples of this type of substrate is the nano-pore size (3.7 nm vs. 17.7 nm), which is measured using BET (nitrogen adsorption analysis), while total surface area is kept constant. The second substrate, a bioactive glass with a more complex composition of 24.4 mol%Na2O–26.9 mol%CaO–2.6 mol%P2O5–46.1 mol%SiO2 called 45S5 Bioglass® (Hench et al., 2006; J Mater Sci: Mater Med), varies in the nanostructure due to phase separation, resulting in either a spinodal interconnected morphology or a droplet nucleation morphology as determined by SEM analysis (Golovchak et al., 2014; Acta Biomaterialia). Cellular response to either substrate was measured through quantification of initial adhesion to the surfaces using immunofluorescence analyses. On the glass samples varying in nano-pore size, we observed that cells preferred the smaller nano-pores (Wang et al., 2013; Tissue Engineering, PartA). Additionally, a clear preference was observed using the glass that differed in nano-structure in that more adherence was observed on the spinodal interconnected morphology than the droplet nucleation morphology. In the case of both substrates, cells showed a significant preference to one of the nanostructures, indicating that remarkably cells are able to sense morphological details of the substrates that are app. 1000 times smaller than the cells themselves. Research performed in our laboratories aims to uncover the underlying principles that allow cells to respond to such nano-scaled substrate details, allowing to engineer superior tissue regenerative materials.

TUESDAY-POSTER PRESENTATIONS

P2243 Board Number: B871

Cellular response to substrate curvature is cell-type dependent. A. Yip1, K. Chiam1,2; 1 Bioinformatics Institute, A*STAR, Singapore, Singapore, 2Mechanobiology Institute, National University of Singapore, Singapore, Singapore Experiments have shown that cells experience and respond to a variety of mechanical forces in physiological conditions, such as those caused by neighbouring cells as well as those due to geometrical constraints of the extracellular matrix. For example, it has been shown that cells can sense and respond to the rigidity of the extracellular matrix, topographical features of the extracellular matrix, and imposed shear stresses. However, little is known about the effects of substrate curvature on cell behavior. In this study, we fabricated substrates of a range of rigidities with cylindrical grooves of varying radii of curvature. We then characterized the morphology of both mesenchymal NIH 3T3 fibroblasts and A549 human lung adenocarcinoma epithelial cells on these substrates. The radius of curvature of the cylindrical grooves were varied from dimensions comparable to a single cell diameter (20 µm) to dimensions of approximately ten cell diameters wide (250 µm). We found that as the radius of curvature decreased, mesenchymal cells became increasingly elongated with a larger proportion of the cells orienting along the long axis of the cylindrical grooves. In contrast, epithelial cells do not show any variation in cell elongation or orientation when the radius of curvature is changed. We then investigated the role of cadherins in the epithelial monolayers in contributing to this effect by the addition of EDTA. Finally, we developed a quantitative model to explain these results. In summary, our results suggested that mesenchymal and epithelial cells respond differently to substrate curvature, and could provide new insights into the process of epithelial to mesenchymal transition.

P2244 Board Number: B872

Low adhesive scaffold collagen, inducing spheroid formation, promotes the osteogenic differentiation. S. Kunii1, E. Yamamoto1, Y. Horiuchi2, H. Ito1, K. Morimoto1; 1 Biology-Oriented Science and Technology, Kindai University, Kinokawa, Japan, 2Kindai University Faculty of Medicine, Kindai University, Osakasayama, Japan [Background] Collagen has biocompatibility and biodegradability with tissue or organs, therefore, collagen is the most promising material for tissue engineering. In particular, the binding of collagen to specific cells is considered an essential function to develop scaffolds. However, in some cases the binding inhibits the cell motility. We succeeded in developing low adhesive scaffold type I collagen (LASCol) by enzyme treatment (patent pending). In this study, we report that LASCol markedly facilitates osteogenic differentiation of rat marrow mesenchymal stem cells (rMSCs). Furthermore, we investigated the effects of bone wound healing by implanting LASCol in a defect of rat tibia. [Methods] We obtained

TUESDAY-POSTER PRESENTATIONS LASCol by the enzyme treatment of pig type I collagen. The culture dish was coated with LASCol or pepsin-treated collagen (PepCol). Subsequently, rMSCs were cultured on each coated-dish with osteogenic basal medium. We observed the morphology of rMSCs by using a phase-contrast microscope. To evaluate osteogenic differentiation, we quantified the mineralization by SEM-EDX. Furthermore, we transplanted each collagen material into the defect of rat tibiae (φ2.5 mm critical-sized defect). After 15 days, bone regeneration efficiency of each implant treatment was evaluated by histological observation of tibia horizontal section with HE stain. Osteocalcin in each serum was measured by ELISA kit. [Results] Rat MSCs formed spheroid bodies by culturing on the LASCol coated dish. We observed that spheroids adhered to LASCol scaffold. Each cell of spheroid highly expressed alkaline phosphatase activity. The mineralization of rMSCs was significantly promoted by culturing on LASCol. Only spheroid cultured on LASCol showed the activity of mineralization by staining with Alizarin red S reagent. Similarly, the P and Ca contents markedly increased. Interestingly, the results in vivo demonstrated that LASCol graft induces bone regeneration of rat tibia and is more bioabsorbable material than PepCol. Osteocalcin in serum of LASCol implant rats (194 ng/mL) was two-fold higher than that of PepCol (105 ng/mL). [Funding] This work was supported by Adaptable and Seamless Technology Transfer Program through target-driven R&D, Japan Science and Technology Agency (AS2414037P to K.M.).

P2245 Board Number: B873

Quantitative Investigation of Differences in Differentiation of Reactive and Nonreactive Astrocytes in Response to Nanophysical Cues. V. Tiryaki1, V.M. Ayres1, I. Ahmed2, D.I. Shreiber2; 1 Michigan State University, East Lansing, MI, 2Rutgers, The State University of New Jersey, Piscataway, NJ Recent studies of cell responses to elasticity, surface roughness, surface polarity and nano-patterning suggest that controlling aspects of the nanophysical environment holds potential for inducing preferential differentiation of reactive astrocytes into non-inhibitory pathways. In the present studies [1,2], these cues were presented by an implantable electrospun polyamide nanofibrillar scaffold that has demonstrated promising wound healing properties including in vivo mitigation of astrocytic scarring. Only external cues were used to trigger cell responses. Four physical properties of the nanofibrillar polyamide versus poly-L-lysine (PLL) glass, PLL Aclar and Aclar culture surfaces were considered potentially directive: nanoscopic elasticity, work of adhesion, surface roughness, and macroscopic surface polarity. These were quantified using AFM and contact angle measurements. Differentiation of untreated versus reactive-like (dBcAMP-treated) astrocytes was investigated using AFM, superresolution microscopy and confocal laser scanning microscopy z-series, with 50-cell minimum protein quantification for major neuro-reactivity markers: glial fibrillary acidic protein (GFAP), Beta tubulin and neuro-inhibitory chondroitin sulfate proteoglycans (CSPGs) and also Rho GTPase cytoskeletal upstream regulators Cdc42 (filopodia), Rac1 (lamellipodia), and RhoA (stress fiber/stellation).

TUESDAY-POSTER PRESENTATIONS The physical property differences of the culture environments triggered statistically significant (ANOVA with pairwise comparisons by Tukey’s test, P

P2246 Board Number: B874

Label-free quantitative analysis of cancer cell dynamics in topologically and mechanically defined 3D matrices. J. Sapudom1, S. Rubner1, S. Martin1, T. Pompe1; 1 Biophysical Chemistry, Universität Leipzig, Institute of Biochemistry, Leipzig, Germany Interactions between cells and their extracellular microenvironment influence multiple aspects of cellular functions and fate decision in physiological and pathological processes. Cancer cells have a highly distinctive ability to adapt and communicate to their microenvironments. The properties of extracellular microenvironments, namely topology, mechanics as well as composition, regulate the development and progression of cancer. Moreover, migration and invasion of cancer cells through extracellular microenvironments are crucial steps for metastasis. A deep understanding on underlying mechanisms is still hampered by the complexity of the mechanical regulation of cell behavior as well as the heterogeneity of relevant cell populations. Understanding how extracellular matrix properties, regulate cell behavior at single cell level would provide potential strategies in approaches to fight cancer. In this work, we reveal cellular characteristics of melanoma cells, including migration pattern, proliferation and differentiation features, under exogenous influence of ECM topology and mechanics. In order mimic three-dimensional (3D) in vivo like microenvironments, we reconstruct 3D collagen matrices with well-defined pore size, fibril diameter and stiffness. The topological and mechanical parameters are varied by adjusting polymerization conditions and were quantified by image analysis tools and force spectroscopy, respectively. The impact of biophysical parameters of 3D matrices on single cell behavior was analyzed using automated quantitative label-free 3D single cell tracking. First cell experiments reveal distinct migration characteristics of different melanoma cell types in correlation with 3D matrix properties. The matrix-specific migration characteristics of melanoma cells allowed a mechanistic understanding of invasive migratory behavior and prediction of cell fate. Furthermore, we observed that fibril orientation of collagen matrices contribute in guiding migration of melanoma cells in a size-dependent manner. Summarizing, our work emphasize the complex relationship between structural-mechanical properties of the extracellular matrix and invasive migratory behavior of cancer cells and provide new approaches for studying heterogeneous populations of primary cells in a biomimetic 3D microenvironment.

TUESDAY-POSTER PRESENTATIONS

P2247 Board Number: B875

Oral administration of Nano-sized avian eggshell membrane is digested, absorbed, distributed to various organs, and contribute to support good health. E. Ohto-Fujita1, M. Shimizu1, N. Nogawa2, H. Eguchi2, K. Ijiri2, Y. Hasebe3, Y. Atomi1; 1 Div. of Material Health Sci., Dept. of Technol., Tokyo University of Agriculture and Technology, Tokyo, Japan, 2Radioisotope Center, The University of Tokyo, Tokyo, Japan, 3Almado Inc., Tokyo, Japan Eggshell membrane (ESM) is a double-layered insoluble sheet locates between eggshell and egg white and works as a scaffold for biomineralization to fabricate egg shell (Rose and Hincke, 2009). ESM mainly consists of various extracellular matrix (ECM) materials necessary for maintaining homeostasis against stimuli from environment during embryogenesis and development until chick birth. The membrane is formed in the isthmus of the hen’s oviduct before shell mineralization and egg laying. Recently ESM containing cosmetics and supplements are on the market worldwide based on the evidence from traditional folk medicine in Asian countries. Until last year we have reported that topical application of alkaline-solubilized avian eggshell membrane (ASESM) improved gene expression pattern to younger skin after 10 days, like increases of gene trio like type III collagen, decorin, and MMP2 on the skin of hairless mice, and increased elasticity of human skin after 3 months. In the present study digestion, absorption and disposition after single oral administration of tritium-labeled ESM were examined using C57BL/6J mice was examined. Tritium derived from peroral labeled ESM showed peak in blood after 6 hour and later disposed into various tissues including liver. Similar to topical effect to improve dermis ECM condition non-labeled nano-powdered ESM peroral administration showed to improve liver ECM environment, such as significant decreased gene expression of type I and III collagen, which increases with liver fibrosis, compared with control. Chicken ESM may work as materials digested during embryogenesis to contribute the development as well as filter, shelter and as a scaffold for biomineralization. ESM has an ability to keep ECM environment of various differentiated tissue to be young through unsolved mechanism.

P2248 Board Number: B876

Avian eggshell membrane as an effective ingredient for health supplement preventing liver fibrosis by suppressed collagen gene expression. M. Shimizu1, E. Ito1, A. Atomi1, E. Ohto-Fujita1, Y. Hasebe2, Y. Atomi1; 1 Div. of Material Health Sci., Dept. of Technol., Tokyo University of Agriculture and Technology, Tokyo, Japan, 2Almado Inc., Tokyo, Japan Avian eggshell membrane (ESM) has long been utilized as a Chinese medicine for recoveries from burns, injuries, and wound. Even nowadays, Japanese Sumo wrestlers use it for wound healing. Extra-fine powdered ESM is on market and used as a supplement that improve various health problems. ESM has a

TUESDAY-POSTER PRESENTATIONS fibrous network mainly comprised of extracellular matrix (ECM) proteins such as type I, V, and X collagens. We have found that water-soluble alkaline-digested form of ESM can stimulate type III collagen, decorin, and MMP2 expression in human dermal fibroblast cells (Ohto-Fujita et al, 2011). As a part of understanding the whole-body health benefit of ESM, here we report the molecular basis of ESM supplement on liver. Either 6 weeks old male Hairless mouse (Hos HR-1) or Wister rat were used after one-week acclimation. Mice were fed ad labium and additionally let them take either low or high-dose nano-sized powdered ESM supplement daily for 7 days. Rats were fed ad labium and ESM was administrated by gastrointestinal intubation for 14 days. Total RNA from liver was prepared and quantitative real time PCR analysis was performed. Results showed that down-regulated collagen expression (both type I and III collagen for mouse significantly and type I collagen for rat) and upregulated HSP70 (mouse at high dose). These results were comparable to the report on gene expression in rat live (Jia, et al., 2013). Since 45% of deaths in the United States can be attributed to fibrotic disorders (Nature Rev Immunol, 2004), suppressed expression of type I collagen is useful evidence for health. Previous and current data shows ESM is an ECM conditioner and mediate tissue rejuvenation with unsolved mechanism.

P2249 Board Number: B877

Anisotropic effects of three-dimensional confined geometries on cell growth and cell-derived extracellular matrix. J.V. Serbo1, S.C. Kuo2, S. Lewis3, M. Lehmann4, D.H. Gracias5, L.H. Romer6; 1 Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 2Biomedical Engineering, Cell Biology, Johns Hopkins Univ Sch Med, Baltimore, MD, 3Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 4Chemical and Biomolecular Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 5Chemical and Biomolecular Engineering, Chemistry, Johns Hopkins University, Baltimore, MD, 6Biomedical Engineering, Anesthesiology and Critical Care Medicine, Cell Biology, Pediatrics, the Center for Cell Dynamic, Johns Hopkins Univ Sch Med, Baltimore, MD Extracellular matrix is a complex, 3D mesh of proteins, proteoglycans, and bound growth factors that provides mechanical and biochemical environmental cues for cellular growth. Previous work in our lab showed that matrix produced and assembled by human fetal lung fibroblasts (WI38) provided a morphogenic stimulus to endothelial cells, inducing both matrix remodeling and tubulogenesis. Microvascular development in a natural complex scaffold material is an attractive platform for regenerative therapies. The current study tests the hypothesis that confined 3D geometries induce early anisotropy in WI38 growth, actin organization, and matrix deposition. Using microfabrication and photolithography, we created arrays of PDMS wells that included three rectangular shapes with increasing aspect ratios (1:2, 1:4, and 1:8), and corresponding paired squares with the same depth, total surface area, and total volume. Microcontact printing of bovine serum albumin (BSA) was used to block fibroblast adhesion to

TUESDAY-POSTER PRESENTATIONS the top surface of the PDMS block. The orientation of WI38 stress fibers and fibronectin fibrils were examined 1 and 2 days after seeding using laser scanning microscopy and a custom routine that was developed for quantitative autocorrelation analysis (MATLAB (MathWorks)). Anisotropic actin filament organization and fibronectin fibril deposition were observed in the rectangular shapes. In these shapes, cells showed increasing alignment with the long axis as the aspect ratio of the rectangles increased. Actin filaments of cells cultivated in rectangular wells with aspect ratios of 1:4 and 1:8 exhibited anisotropy scores that were significantly higher than those grown in the corresponding squares or in lower aspect ratio rectangular wells. Similar trends were seen with fibronectin. These data suggest that shape-specific effects of 3D confinement on fibroblasts influence actin stress fiber alignment and matrix deposition. Latrunculin A (0.05uM) decreased cytoskeletal and matrix anisotropy at all but the highest (1:8) aspect ratio. Cells treated with nocodazole (0.02uM) did not exhibit alignment of the microfilament or microtubule cytoskeletons or of cell-assembled matrix with anisotropic spatial confinement. In contrast, treatment with Y27632 (4uM) did not inhibit the development of cytoskeletal or matrix anisotropy. These data implicate both actin and microtubule polymerization, but not ROCK-dependent acto-myosin interactions, in anisotropic responses of cellular growth and matrix assembly to spatial confinement. Next phases of this project include definition of the effects of 3D, confined geometries on blood vessel growth and orientation in natural matrix.

Regulation of Aging P2250 Board Number: B879

Defective development of Natural Killer cells during aging. S. Nair1, M. Fang2, L.J. Sigal1; Immune Cell Development and Host Defense Program, Fox Chase Cancer Center, Philadelphia, PA, 2CAS Key laboratory of Pathogenic Microbiology and Immunology, Chinese Academy of Sciences, Beijing, China

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Natural Killer (NK) cells are important in combating both infections and cancer. Using Ectromelia virus (ECTV), the agent of mousepox, as a model of viral infection, our group has shown that mice increasingly succumb to infection as they age since they lack fully mature NK cells that are vital for protection. We have previously shown that under homeostatic conditions, aged B6 mice have a deficit in the maturation of NK cells in the blood, spleen, lymph nodes and bone marrow and that this results in impaired migration of NK cells to the draining lymph node and susceptibility to mousepox. Using an adoptive transfer model, we demonstrate that the age of the host and not the donor bone marrow cells affects the recovery of mature NK cells in the bone marrow suggesting that the radio resistant mesenchymal/stromal cells in aged cannot support the full maturation of NK cells whereas the hematopoietic precursor cells of aged are capable of generating mature NK cells. Aged mice also display

TUESDAY-POSTER PRESENTATIONS reduced proliferation of NK cells, multiple signs of immaturity and altered expression of activating and inhibitory receptors on NK cells. Gene array and q-PCR analysis of bone marrow stromal cells reveal that the message for Type I collagens, Col1a1 and Col1a2 is dramatically reduced in the stromal subset of bone marrow of aged mice. At the protein level, we found that receptors for Type I collagen, CD49b integrin which is a late stage marker of NK cell differentiation, is also significantly down-regulated on total NK cells and mature NK cell subsets in the bone marrow of aged mice when compared with young mice. Moreover, we show that treatment with complexes of the cytokine IL-15 and IL-15Rα induced massive expansion of immature but not of mature NK cells in aged mice. As a result, IL-15/IL-15α treatment did not restore resistance to mousepox in aged mice. Because many infectious diseases in humans spread systemically via lymph nodes and NK cell-deficient patients have significantly increased incidence of viral infections, it is possible that a defective NK cell maturation and migration also contributes to the increased susceptibility of older people to at least some viruses. Our mouse model provides a unique avenue to investigate NK cell dysfunctions with strong potential to advance our knowledge on causes of increased susceptibility to viral disease that we believe should be translatable to humans.

P2251 Board Number: B880

Functional and Phenotypic Alterations in NK Cells in Amnestic Mild Cognitive Impairment (aMCI) and Mild Alzheimer Patients. A. Le Page1, K. Bourgade-Navarro1, J. Lamoureux2, G. Dupuis1, T. Fulop3; 1 Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, 2Faculty of Medicine and Health Sciences, Université de Sherbroooke, Sherbrooke, QC, 3Faculty of Medicine and Health Sciences, IUGS, Argyll Pavilion, Sherbrooke, QC Alzheimer’s disease (AD) is a progressive irreversible neurological brain disorder characterized by accumulation of β-amyloidogenic peptides, amyloid plaques and neurofibrillar tangles. Inflammation and immune alterations have been linked to AD, suggesting that the peripheral immune system plays a role during the asymptomatic period of AD. NK cells are part of innate immunosurveillance against intracellular pathogens and malignancy, their role in AD remains controversial. We have investigated changes in NK cell phenotypes and functions in amnestic mild cognitive impairment (aMCI) (n = 10) and mild Alzheimer (mAD) patients (n = 11) and, healthy elderly controls (n = 10). Patients selected according to NINCDS-ADRDA criteria were classified using neuropsychological assessment tests. Phenotype analysis revealed differences in expression of CD16 (increased in mAD), NKG2A (decreased in aMCI,), TLR2 and TLR9 (both decreased in mAD). Percentage of IL-18 receptor β was lower in aMCI without differences in IL-12 receptors β1- and β2 for the three groups. Functional assays revealed that NK cell killing activity and degranulation (CD107 expression) were unchanged in the three groups. In contrast, expression of CD95 receptor was increased in aMCI and mAD. Granzyme B expressionand cytokine production (TNFα, IFNγ) were increased in aMCI not in mAD. CCL19- but not CCL21-dependent chemotaxis was decreased in aMCI and mAD, despite the fact that CCR7 expression was increased in

TUESDAY-POSTER PRESENTATIONS aMCI. Our data suggest that a limited number of alterations that are predominantly observed in peripheral NK cells of mAD patients may affect more severely their capacity to respond to pathological aggression than aMCI subjects in progressive development of AD.

P2252 Board Number: B881

NK-mediated immune surveillance of senescent cells. A. Sagiv1, D. Burton1, Z. Moshayev1, V. Krizhanovsky1; Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

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NK-cell-mediated immune surveillance of senescent cells is one component of the coordinated process whereby cellular senescence limits the extent of liver fibrosis and facilitates wound repair. Recent studies also suggest that senescent cell recognition and clearance by immune cells promotes tumor regression in established tumors. Our results demonstrate that senescent cells are preferentially recognized and killed by NK cells. We show that DNA damage-induced senescent fibroblasts specifically upregulate the NKG2D immune ligands MICA, ULBP1, ULBP2 and ULBP5. Furthermore, we demonstrate that this immune ligand upregulation is mediated by ERK signaling and Rb, independent of p53 and NFκB. Importantly, the upregulation of MICA and ULBP2 on the cell surface of senescent cells is pivotal for the NK-mediated recognition, since interference with the receptor-ligands interactions inhibits the NK-mediated clearance both in vitro and in vivo to limit liver fibrosis and facilitate tissue repair. Lastly, we demonstrate that the granule exocytosis pathway, but not the death receptor pathway, is necessary for the specific killing of senescent fibroblasts and stellate cells by NK cells and participates in the clearance of senescent activated HSCs to limit liver fibrosis. We suggest that this pathway bias is mediated by the upregulation of Dcr2, a decoy receptor for the death ligand TRAIL, by senescent cells. Therefore, NK-cell-mediated recognition of senescent cells via NKG2D ligands and killing through NKG2D signaling and the granule exocytosis pathway contributes to immune surveillance of senescent cells in vitro and in vivo.

P2253 Board Number: B882

Comparing Nutrient Restrictions and its Gender Specific Effects on Metabolic Output in Drosophila melanogaster. R. Vaders1, A.A. Nagengast2; 1 Chemistry, Widener University, Chester, PA, 2Biochemistry, Widener University, Chester, PA Aging is a complex process that has a variety of underlying causes. Reactive oxygen species (ROS) cause oxidative damage to proteins, lipids and carbohydrates, and contribute to aging. Although calorie restriction has been linked to an increased lifespan, previous results showed that Drosophila melanogaster raised on a high nutrient diet lived longer than those on a nutrient restricted diet. As

TUESDAY-POSTER PRESENTATIONS expected, adult males raised on the high nutrient food had higher triglyceride levels but females raised on the high nutrient food had lower triglyceride levels compared to those raised on the nutrient restricted diet. ROS levels were detected by measuring SOD (superoxide dismutase) expression and were found not to vary between the two nutrient conditions in males. However in females, no expression of cytosolic SOD (SOD1) was detected but mitochondrial SOD (SOD2) expression was robust. Both SOD1 and SOD2 enzyme activity require divalent cations and the nutrient restricted food contains higher levels of iron. Studying iron and its direct or indirect effect on other major and trace minerals as the cause of these unfamiliar results opens windows of opportunity to fully understand iron-response elements (IREs) along the 5’ UTR. These minerals, which contribute to everyday movement, could be the answer to metabolic mutations that occur under specific nutrient conditions, and contribute to slow aging.

P2254 Board Number: B883

D-galactose decreases mitoNEET (CISD1) levels in HepG2 cells. S. Paudel1, L.E. Welker2, B.P. Nathan2, M.E. Konkle3, M.A. Menze4; 1 Biology, Eastern Illinois University, Charleston, IL, 2Eastern Illinois University, Charleston, IL, 3Chemistry, Eastern Illinois University, Charleston, IL, 4Biological Sciences, Eastern Illinois University, Charleston, IL The monosaccharide D-galactose has been used as a tool to study accelerated aging, oxidative stress, and tissue damage in animal models. Supraphysiological levels of D-galactose accelerate aging in rodents by decreasing motor activity and superoxide dismutase activity, and by increasing production of reactive oxygen species (ROS). However, the underlying mechanism of galactose action is still poorly resolved. MitoNEET was discovered as target protein of the drug pioglitazone, a compound widely used to increase peripheral insulin sensitivity. MitoNEET is a small iron-sulfur cluster [2Fe 2S] containing protein localized to mitochondrial membranes. MitoNEET deficiency decreases iron content in the mitochondrial matrix and increases production of ROS in both animal and plant cells. Pioglitazone ameliorates galactose mediated memory deficit in a mouse model of aging. To investigate the role of mitoNEET in galactose-induced aging-related changes, we exposed human hepatocellular carcinoma cells (HepG2) to 10 mM D-galactose and measured endogenous mitoNEET levels by immunoblotting. Galactose treatment for 2 weeks significantly reduced mitoNEET levels in HepG2 cells. This effect was observed both in presence or absence of glucose in the culture medium. D-galactose also decreased levels of mitoNEET-GFP (green fluorescent protein) under the CMV promoter in stably transfected HepG2 cells. Since transcription of the endogenous mitoNEET and mitoNEET- GFP is regulated by different promoters, D-galactose likely increases degradation of mitoNEET. Furthermore, addition of 15 µM pioglitazone in presence of galactose significantly increased the levels of mitoNEET while concentrations above 30 µM, significantly reduced mitoNEET levels as compared to galactose treatment alone. This is the first report that links mitoNEET levels to the D-galactose induced model of accelerated aging processes. [This work was funded by an Eastern Illinois University Council on Faculty Research (CFR) grant to M.A.M and a Research and Creativity Award to S.P.]

TUESDAY-POSTER PRESENTATIONS

P2255 Board Number: B884

Mitochrondria fusion and fission mutants and their roles in cellular degeneration. V.M. Khare1, P. Tran1,2; 1 Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, 2CNRS UMR144, Institut Curie, Paris, France Mitochondria are highly dynamic organelles that undergo fission and fusion. Defects in their dynamics have been linked to diverse diseases. However, at the single-cell level, it is less clear how defects in fission and fusion modify cellular physiology and influence cell health. To gain new insights into the connection between mitochondria dynamics and cell health, we examined four S. pombe genes which are conserved and regulate mitochondria dynamics: dnm1+ and fis1+ (fission), and msp1+ and ugo1+ (fusion). Wild type fission yeast cells have long mitochondria that align with the cell long-axis. Deletion of either dnm1 (dnm1Δ) or fis1 (fis1Δ) resulted in hyper-fused mitochondria. Conversely, deletion of msp1 (msp1Δ) or ugo1 (ugo1Δ) resulted in fragmented mitochondria. All individual mutants showed reduced cell doubling time compared to wild type, especially msp1Δ which had a cell cycle time 3-fold longer than wild type. Interestingly, in a population under good nutrient condition, these mutants exhibited very low cell death not different than wild type. We propose that, at least in one cell cycle, the mitochondria fragmented mutant msp1Δ extends the chronological lifespan of the cell. We are currently testing this hypothesis by examining cellular processes such as cell polarity and growth, and cell division.

P2256 Board Number: B885

Involvement of hexosamine biosynthetic metabolites in endoplasmic reticulum stress and aging. P. Kim1, A. Estrada2, M. Hudson2, J. Kirkwood3, J. Prenni3, D. Wang2, Y. Wei2, M. Pagliassotti2; 1 Biological Sciences, Grambling State University, Grambling, LA, 2Food Science and Human Nutrition, Colorado State University, Fort Collins, CO, 3Proteomics and Metabolomics Facility, Colorado State University, Fort Collins, CO Endoplasmic reticulum (ER) stress and the collapse of proteostasis are recognized as important events in both disease and aging. Glycosylation is one mechanism used by the ER to promote proteostasis. The hexosamine biosynthetic pathway (HBP) provides substrate, in the form of UDP-N-acetylglucosamine, for both N- and O-linked glycosylation. Recent evidence has demonstrated that enhanced HBP activity not only confers resistance to tunicamycin (a drug that inhibits protein glycosylation), but can also promote longevity in C. elegans. The aim of this study was to examine the HBP in the aging liver of mice and tunicamycin-induced ER stress in liver cells. Here, we report that HBP end products UDP-Nacetylhexosamines were reduced in the liver of old (27-29 mo) compared to young (4-6 mo) mice. The liver of old mice was characterized by ER stress, activation of the inflammasome, and injury. We also

TUESDAY-POSTER PRESENTATIONS report that glucosamine, an intermediate metabolite in the HBP, reduced ER stress induced by tunicamycin in H4IIE rat hepatoma cells. Using real time RT-PCR, we found that treatment with 5 µg/ml tunicamycin for 16 hours upregulated splicing of XBP1 and expression of CHOP and GADD34, whereas providing 5 mM glucosamine during the final 10 hours of tunicamycin insult significantly decreased these ER stress markers. These preliminary data suggest that the ability to maintain HBP end products is impaired in aging and that the HBP may be linked to aging-induced ER stress in the liver.

P2257 Board Number: B886

The role of Hsf1 in the maintenance of the endoplasmic reticulum homeostasis during chronological aging in S. cerevisiae. E. Fazio1, M. Duennwald2, P. Lajoie1; 1 Anatomy and Cell Biology, University of Western Ontario, London, ON, 2Pathology, University of Western Ontario, London, ON Cellular aging is a ubiquitous process whose molecular mechanisms remain largely unclear. A deeper understanding of cellular aging is important not only in the context of general lifespan, but also to understand the mechanisms behind the toxicity of aging-related diseases, like Alzheimer’s and Huntington’s disease. The Heat Shock Response (HSR) is a conserved cellular response to various stresses, including exposure to elevated temperatures and the presence of misfolded proteins in the cytosol. In both yeast and humans, the HSR is primarily regulated by the highly conserved heat shock transcription factor, Hsf1. Several genetic studies indicate that the HSR plays a major role in healthy cellular aging, and age-dependent impairment of the heat shock response is well documented in different organisms, including yeast, worms, and flies. Accumulation of misfolded proteins is associated with aging in various model organisms, and to resolve misfolded secretory protein burden, cells have evolved the Unfolded Protein Response (UPR) to help re-establish protein folding homeostasis in the endoplasmic reticulum. The goal of our study is to characterize the interplay between the HSR and UPR during chronological aging in yeast. Chronological aging in yeast is achieved through maintenance of cells in the stationary growth phase, where nutrients have been depleted and cells remain in a quiescent state. We found that chronologically aged yeast cells display increased UPR activation when compared to young cells, suggesting that changes in the ER misfolded protein burden occur during chronological aging. Furthermore, cells with no functional UPR (ire1Δ), or that are unable to upregulate the ER chaperone Kar2 (UPREd), have shorter chronological lifespan (the amount of time that stationary phase yeast cells remain viable) than their wild-type counterparts demonstrating that up-regulation of the UPR is essential for healthy chronological aging. Our work also illustrates that constitutive activity of the HSR through overexpression of HSF1 alleviates ER stress and promotes growth in ire1Δ cells, and that cells expressing a mutated Hsf1 exhibit decreased chronological lifespan. Ongoing studies aim to determine whether Hsf1 expression in ire1Δ and UPREd cells rescues their stunted aging phenotype, and to elucidate the underlying mechanisms. Taken together, our data reveal a role for the HSR in alleviating secretory protein stress and extending chronological lifespan.

TUESDAY-POSTER PRESENTATIONS

P2258 Board Number: B887

Functional Analysis of Vacuolar Membrane Protein Ssg1 Which May Be Involved in the Regulation of Lifespan in Saccharomyces cerevisiae. T. Koyama1, R. Tsubakiyama1, K. Kume1, M. Mizunuma1; Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan

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Aging is an unavoidable phenomenon and common to all living organisms. However, the molecular mechanism associated with aging and longevity is little understood. We previously found that a mutation of SAH1 gene, which encodes S-adenosylhomocysteine (SAH) hydrolase, affected cell growth and chronological lifespan. In fact, the sah1-1 mutant has a growth defect and a short lifespan. Moreover, we have isolated the long-lived mutant by screening for the suppression of the growth defect of the sah1-1 mutant. This dominant-mutation was named SSG1 (Spontaneous Suppressor of Growthdelay of the sah1-1). Interestingly, the SSG1 single mutant was also shown to have a longer lifespan than a wild-type yeast. Therefore, the Ssg1 protein is predicted to be a key factor in longevity. To investigate the function of the Ssg1 protein, we constructed EGFP-Ssg1 and observed its localization by usig a fluorescence microscope. We found that the Ssg1 protein was strongly localized in the vacuolar membrane during exponential growth phase and disappeared in stationary phase. Next, to examine whether it was functionally significant for the Ssg1 protein to localize in the vacuolar membrane, we constructed a double mutant between SSG1 and vps33 which has a defect in vacuole formation. It was revealed that the Ssg1 protein was spread entire in the cell and the longevity effect of the SSG1 mutation was canceled by the deletion of VPS33, even though the Ssg1 protein was produced at roughly the same level as the SSG1 single mutant. This suggests the importance of functions in the vacuolar membrane of the Ssg1 protein. Interestingly, we found that the SSG1 mutation highly accumulated Sadenosylmethione (SAM) and SAH. Considering these results, it was expected that the SSG1 mutation promoted SAM and SAH accumulations in the vacuole and we measured the amounts of SAM and SAH both in the cytoplasmic compartment and in the vacuolar compartment. Accordingly, it turned out that SAM and SAH accumulated in the vacuole as a result of the SSG1 mutation. In conclusion, it is suggested that the Ssg1 protein localized in the vacuolar membrane, and may work as a transporter to carry SAM and/or SAH into the vacuole. We are investigating the role of the Ssg1 in the regulation of longevity in detail.

TUESDAY-POSTER PRESENTATIONS

P2259 Board Number: B888

SCZ genes, which suppress Ca2+-sensitivity of zds1∆, are implicated in the regulation of life span in the budding yeast Saccharomyces cerevisiae. I. Yamana1, R. Tsubakiyama1, K. Kume1, M. Mizunuma1; 1 Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan In any organism, aging is characterized as the gradual decline of multitude of physiological functions which present major risk factors for most diseases and finally leads to death. Much of our understanding of aging derives from studies in model organisms, such as yeasts, worms and mice. However, to date we have not understood of what actually causes aging. In eukaryotic cells, the calcium ion (Ca2+) is involved in the regulation of various cellular functions as a signal molecule. The zds1∆ strain of Saccharomyces cerevisiae shows sensitivity to Ca2+ and the Ca2+induced phenotypes which are G2 cell cycle arrest, and polarized bud growth, due to the activation of cellular Ca2+-signaling pathways mediated by calcineurin (CN) and the Mpk1 MAP kinase pathway. To uncover the growth control regulated by the the Ca2+-signaling pathways in more detail, we screened for suppressor mutants of the Ca2+-sensitive phenotypes of zds1∆ cells (scz mutants). We have clarified into 14 complementation groups and SCZ14 was found to be allelic to SIR3, which is involved in the regulation of gene expression and lifespan. Therefore, it is expected that other mutants would also regulate lifespan. So we have conducted a lifespan analysis using all identified scz mutants. In budding yeast, two different paradigms of aging have been developed: replicative lifespan (RLS) and chronological lifespan (CLS). Previously, we measured the RLS (the number of daughter cells produced by a mother cell) of all scz single mutants. All mutants turned out to have a reduction of their RLS, suggesting that SCZ genes are implicated in the regulation of aging. In particular, the increase in CN activity by the zds1 deletion reduced RLS. Further, increasing the extracellular Ca2+ level causes a shortened RLS in WT. Therefore, it was suggested that Ca2+ homeostasis influences RLS. To investigate whether the SCZ genes affect CLS, we measured the CLS (the length of time that a mother cell can survive in a non-dividing, quiescence-like state) of scz1~14 single and zds1∆ sc 1 14 double mutants. In contrast to RLS, some mutants extended CLS. One of the long-lived scz mutants was the cnb1∆ strain, which encodes the regulatory subunit of CN. It has been reported that CN-deficient animals have an extended lifespan through the induction of autophagy in C. elegans. We examined whether autophagy is affected by the deletion of CN. However, cnb1∆ did not induce autophagy in yeast, indicating that cnb1∆ extends lifespan in a different manner. We are currently investigating how the CN affects lifespan.

TUESDAY-POSTER PRESENTATIONS

P2260 Board Number: B889

Nicotinamide reverses senescent cell phenotype: possible differential role of phosphatidylinositol 3-kinase subtype enzymes. K. Matuoka1, K. Sasaki1, K. Nakanishi1, T. IIda1, K.Y. Chen2; 1 Chiba Institute of Science, Chiba-ken, Japan, 2Rutgers, State University of New Jersey, Piscataway, NJ Phosphatidylinositol 3-kinase (PI3K) is an enzyme family functioning in energy metabolism and intracellular signaling pathways for cell survival, proliferation and aging. When cells age, they express a set of senescence-specific phenotypes, including enlarged cell shape, decrease in proliferation and saturation density and expression of senescence-associated β-galactosidase (SA-gal). We have observed that nicotinamide (NAM), an NAD+ precursor, or PI3K inhibitors can reduce certain aging cell phenotypes. We wonder what could be the causes that lead to this phenomenon. With the recognition of the age-dependent decrease in cellular motility, we wonder whether this decrease may be related to other senescence-specific events. We found that treatment of senescent human fibroblastic cells with NAM (4-10 mM) for 10-20 h led to changes in cell morphology and motility, which were then followed by initiation of cell division and a decrease in SA-gal. Similar effects were also observed with PI3K inhibitors, albeit with different potency: BEZ235 was the most effective one, followed by ZSTK474, LY294002 and then wortmannin. As both NAM and PI3K are known to be involved in energy metabolism, we also examined the effect of sirtuin- and AMP kinase-modulating chemicals, including resveratrol, splitomicin, AICAR and dorsomorphin, on senescence-specific phenotypes. None of these chemicals could fully reproduce the effect of NAM on aging, showing that the action of NAM in reversing the senescent phenotypes may be related to the PI3K system, but not to pathways involving sirtuin or AMP kinase. On the other hand, we also observed that the phosphorylation of Akt, the key signaling molecule downstream of PI3K, was reduced in senescent cells, but tended to be higher after the exposure to NAM. This may imply Akt not playing a direct target of the PI3K inhibitors or NAM. As the PI3K system comprises several classes and subtypes, it is possible that the differential effect of PI3K inhibitors on senescent cells could be related to the different spectrum of their actions on PI3K subtype enzyme species. In this regard we found that class I β mRNA was reduced in senescent cells, whereas the class II α and class II β mRNA levels in senescent cells were higher. We speculate that these different PI3K species may have different roles in regulating age-dependent biological events. They may also have different responses to various known PI3K inhibitors. Current work is aiming at using these inhibitors to elucidate the role of individual PI3K subtypes in aging process.

TUESDAY-POSTER PRESENTATIONS

P2261 Board Number: B890

Cockayne syndrome protein B (CSB) localizes to the nucleolus to regulate rDNA transcription in response to oxidative stress. E.L. Boetefuer1, H. Fan2; 1 Department of Biology, University of Pennsylvania, Philadelphia, PA, 2Department of Genetics, University of Pennsylvania, Philadelphia, PA The object of this study is to determine the role of Cockayne syndrome protein B (CSB) in protecting cells against oxidative stress. Cockayne syndrome is a severe, autosomal recessive disorder characterized by sun sensitivity, neurological and developmental defects, and progeroid features. CSB is an ATP-dependent chromatin remodeler and is and critical for relieving oxidative stress; however, little is known as to how CSB accomplishes its specific functions in this process. Here, we perform a complementation test, which demonstrates the presence of CSB promotes cell survival upon oxidative stress. Furthermore, chromatin remodeling is required for CSB’s protective role. Indirect immunofluorescence was used to examine the cellular localization of CSB, which shows oxidative stress also results in the accumulation of CSB within the nucleolus, the site of rDNA transcription and processing. Mutation analysis indicates this localization requires both the ATPase domain and Cterminus of CSB. RT-qPCR analysis of nascent rDNA transcription indicates CSB functions to suppress rDNA transcription upon oxidative stress. Taken together, we demonstrate a role of CSB in protecting cells during oxidative stress by downregulating nascent rDNA transcription within the nucleolus.

P2262 Board Number: B891

Changes in gene expression profile in human mesenchymal stem cells young and senescent treated with titanium microparticles. J.C. Tavares1, D.A. Cornelio2, G.O. Brasil3, I.T. Job e Meira2, S.R. Batistuzzo de Medeiros2; 1 FACISA/UFRN, Santa Cruz, Brazil, 2DBG, UFRN, Natal, Brazil, 3DBG, UFRN, Natl, Brazil Human mesenchymal stem cells (hMSC) are mutipotent cells and have been used in cell therapy. Replicative senescence or acquisition of tumorigenic features can appear after long time cultivation in vitro. Considering the interference of passages and titanium in the gene expression, we decided to evaluate the transcriptome of hMSC isolated from umbilical cord vein. The cells were cultivated until early passages (young cells) and until late passages (senescent cells) without and with exposition to titanium microparticles at 24-hour and 144-hour time point (50 e 100 microparticles/cell, respectively). The cords were obtained from three donors, two with normal karyotype, identified as hMSC/n, and one with an inversion chromosome (46,XY,inv(3)(p13-p25)), called hMSC/inv. Using microarray analysis, we demonstrated a range of differentially expressed genes (DEG) between the young hMSC/inv and young hMSC/n. Interestingly, there were more DEG when they become senescent. There was no DEG between

TUESDAY-POSTER PRESENTATIONS hMSC/n untreated and treated with titanium for all doses and time tested during the treatment. When highest titanium dose at 24-hour, we verified more DGE between hMSC/inv young and senescent. Only in senescent hMSC/inv treated with titanium at 144-hour there was DEG in a titanium dose-dependent manner. The data clearly show the presence of possible new biomarkers of cellular senescence in both hMSC untreated and treated: DIO2, IGFBP5, MRVI1, SCUBE3, KRTAP1-5, HAS3, KRT34, GALNT5, FOXE1, KRT19, PLCB4. We also determine new networks for DEG when comparing senescent and young cells, with the bottlenecks MMP1, THBS1 (senescent hMSC/n) and EGF (senescent hMSC/inv). In senescents hMSC/inv, others functional annotations of segregation of chromosomes, mitotic spindle formation and mitosis and proliferation of tumor lines were the most represented within of functional categories. Genes inserted into functional annotations related to tumors were more pronounced in our analysis, some of them associated to giant cell tumor of bone. The functional categories over-represented in senescent cells without titanium particles were related to cellular development, cell growth and proliferation, cell death, cell signaling and interaction and cell movement. In young cells, the functional category most enriched at 50 particles/cell and at 100 particles/cell dose at 24-hour was cellular development. Specifically, when using 100 particles/cell, other functional categories were represented such as: cell cycle, cellular movement, cell death and survival, cell growth and proliferation and cell morphology. In senescent cells, in this same condition of treatment we identified genes classified as functional category of DNA replication, recombination and repair. Taken together, these data is relevant to improve the knowledge concerning molecular mechanism of the cellular senescence, in presence or absence of titanium particle treatment. Lastly, our data reveal particular genes involved in the initial stages of mesenchymal carcinogenesis. Financial support CNPq

P2263 Board Number: B892

Sirt6 deficiency produces intrinsic osteoblast defects leading to low-turnover (age-related) osteoporosis. T. Sugatani1, O. Agapova1, H. Malluche2, K.A. Hruska1; 1 Washington University School of Medicine, Saint Louis, MO, 2Albert B. Chandler Medical Center, University of Kentucky, Lexington, KY Several pathological conditions resembling human aging, including osteopenia, have been observed in sirtuin 6 (Sirt6)-deficient mice. However, the mechanism by which Sirt6 controls bone metabolism is unknown. The original report ascribing osteopenia to decreased circulating IGF1 is likely incorrect. Here, we show that loss of Sirt6 function produces a low-turnover osteoporosis caused by impaired bone formation by osteoblasts and bone resorption by osteoclasts. Micro CT analysis of long bones from Sirt6deficient mice revealed decreased bone volume, trabecular bone mineral density, trabecular number, trabecular thickness, and cortical thickness compared to wild-type mice. Bone histomorphometry revealed impaired bone formation and bone resorption along with decreased osteoblast and osteoclast numbers. Osteoblastogenesis and osteoclastogenesis, which is an osteoblast free system, were impaired in culture. Furthermore, mineralization by Sirt6-null osteoblasts was remarkably suppressed in vitro.

TUESDAY-POSTER PRESENTATIONS Mechanistically, we discovered that Sirt6 interacts with Sp7, one of the essential transcription factors for osteoblastogenesis, and deacetylates histone H3 lysine 9 (H3K9) at the Dickkopf-1 (Dkk1) promoter in wild-type osteoblasts. In fact, Dkk1 was not detected at protein levels in wild-type osteoblasts with BMP-2 treatment. In contrast, Dkk1 protein expression was detected in Sirt6-null osteoblasts under the same conditions. Given that Dkk1 is a potent Wnt antagonist and negatively regulates osteoblastogenesis and mineralization, up-regulated Dkk1 protein expression might impact osteoblastic development and function in Sirt6-deficient mice. We also found enhanced production of osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, in Sirt6-null osteoblasts with BMP-2 treatment, while OPG production is suppressed by BMP-2 in wild-type cells. Therefore, enhanced OPG expression in osteoblasts may be the cause of impaired osteoclast development and function in Sirt6deficient mice. In fact, the numbers of osteoclasts were attenuated in co-culture assays using primary Sirt6-null osteoblasts and Sirt6-null bone marrow mononuclear cells compared to the co-culture assay using the both wild-type cells. Since Sirt6-deficient mice reveal characteristic features of age-related osteoporosis in humans, the cellular and molecular mechanisms of Sirt6 in bone turnover are potential therapeutic targets for age-related bone loss.

P2264 Board Number: B893

Starvation and inhibition of lysosomal function increased tau secretion by primary cortical neurons. N. Mohamed1, V. Plouffe 2, G. Rémillard-Labrosse3, E. Planel 4, N.M. Leclerc5; 1 Pathologie et Biologie Cellulaire, CRCHUM, Montreal, QC, 2Neurosciences, CRCHUM, Montréal , QC, 3 Neurosciences , Université de Montréal, Montréal , QC, 4Neurosciences, CHUL, Québec, QC, 5 Neurosciences, CRCHUM, Montreal, QC Recent studies have demonstrated that human tau can be secreted by neurons and non-neuronal cells, an event linked to the propagation of tau pathology in the brain. In the present study, we confirmed that under physiological conditions, one tau-positive band was detected in the culture medium with an anti-tau antibody recognizing total tau and the Tau-1 antibody directed against unphosphorylated tau. We then examined whether tau secretion was modified upon insults. Tau secretion was increased by starvation [Earle's Balanced Salt Solution (EBSS)], inhibition of lysosomal function (leupeptin) and when both of these conditions were superimposed, this combined treatment having the most important effects on tau secretion. Interestingly, the pattern of tau secretion was distinct from that of control neurons when neurons were treated either with EBSS alone or EBSS + leupeptin. In these conditions, three tau-positive bands were detected in the culture medium. Two of these three bands were immunoreactive to Tau-1 antibody revealing that at least two tau species were released upon these treatments. Collectively, our results indicate that insults such as nutrient deprivation and lysosomal dysfunction observed in neurodegenerative diseases could result in an increase of tau secretion and propagation of tau pathology in the brain.

TUESDAY-POSTER PRESENTATIONS

P2265 Board Number: B894

Earlier onset of mitophagy contributes to the prolonged chronological lifespan of ras2∆ yeast strain. L. Belicova1, A. Pichova1; 1 Institute of Microbiology, The Academy of Sciences of the Czech Republic, Prague, Czech Republic Ras/adenylate cyclase (AC)/PKA pathway is one of the major pathways regulating chronological aging in various model organisms. Deletion of RAS2 gene in yeast Saccharomyces cerevisiae attenuates this pathway and results in prolonged chronological lifespan of a ras2Δ strain. Mitochondria play an important role in cell aging and their quality control involves autophagic removal of mitochondria – mitophagy. We aimed at investigating whether and how mitochondria and their quality control process – mitophagy – contribute to the prolonged lifespan of the ras2Δ strain. Viability, oxygen consumption, reactive oxygen species (ROS) production, and mitochondrial network morphology were assessed in wild-type and ras2Δ strain. To evaluate the importance of mitophagy, a key mitophagy gene ATG11 was deleted in respective strains (thus creating atg11Δ and double knock-out ras2Δ atg11Δ). The results showed that the extended lifespan of the ras2Δ strain is most probably enhanced by earlier removal of damaged mitochondria by mitophagy and by reducing their respiratory metabolism. In our study, a disruption of mitophagy resulted in higher ROS production and loss of viability in the ras2Δ strain. Inability to perform mitophagy did not influence the wild-type strain in any of the studied parameters. The strain atg11Δ therefore compensates the ATG11 deletion by alternative processes. Our results point to a possible interconnection between mitophagy and Ras/AC/PKA signaling pathway. Understanding the interplay of mitophagy and Ras pathway might help to elucidate how cells ensure the mitochondria quality control in the aging process.

Autophagy P2266 Board Number: B895

TRIM family proteins regulate selective autophagy and act as a new class of autophagic receptors. M. Mandell1, T. Kimura1, V. Deretic1; 1 Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM Tripartite-motif containing proteins (TRIMs) constitute a large family with effects on cell cycle control, innate immunity, inflammation, and antiviral defense. We screened the TRIM family for roles in autophagy and found that more than half of TRIMs modulated autophagy. For most TRIMs tested to date (TRIM5α, TRIM6, TRIM17, TRIM22, and TRIM49), this is accomplished by TRIMs serving as

TUESDAY-POSTER PRESENTATIONS platforms for the assembly of autophagy initiation factors ULK1 and Beclin 1 in their activated states, although TRIMs such as TRIM55 also employ additional and/or alternative mechanisms to promote autophagy induction. We next considered whether TRIMs played additional roles as selective autophagy receptors, this being of interest due to a present dearth of the known selective autophagy receptors in mammalian cells. All TRIMs tested interacted with mammalian Atg8s (mAtg8s), a class of proteins functionally associated with autophagosome membranes. To find a putative autophagic cargo for a TRIM, we used TRIM5α, as it has a well-known physiologically relevant target: retroviral capsid. We found that knock-down of autophagy factors in rhesus cells increased retroviral infection with a virus (HIV) known to be restricted by rhesus TRIM5α but had no effect on a retrovirus (SIV) not recognized by rhesus TRIM5α. We furthermore found that autophagic degradation of retroviral capsid by TRIM5α required TRIM5α’s binding to mAtg8s, thus establishing TRIM5α as an autophagic receptor. Whereas the target of TRIM5α is an exogenous protein (retroviral capsid), we have identified several endogenous proteins that interact with and are delivered for autophagic degradation by specific TRIMs. Thus, the function of TRIMs as autophagic receptor—regulators is conserved among the family. Based on our findings and the sheer number of TRIMs (>70 genes in the human genome), we propose that TRIMs direct and conduct selective autophagy of diverse substrates in metazoan cells.

P2267 Board Number: B896

REGULATION OF AGO2 FUNCTION AND DEGRADATION DURING GLUTATHIONE DEPLETION. H. Mancilla 1, J. Slebe1, G. Meister2, I. Concha1,3; 1 Bioquimica y Microbiologia, Universidad Austral De Chile, Valdivia, Chile, 2Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany, 3I. Bioquimica, Universidad Austral De Chile, Valdivia, Chile miRNAs interact with Argonaute (Ago) proteins to form RNA-induced silencing complex (RISC) and guide them to specific target sites located in the 3´-UTR of target mRNAs leading to translational repression and deadenylation-induced mRNA degradation. Autophagy is the major intracellular degradation system by which cytoplasmic proteins and organelles are delivered and degraded in the lysosome. Ago2 is directed for degradation as miRNA-free entities by the selective autophagy receptor NDP52. We have previously described that a glutathione (GSH) deficiency triggers autophagy. The aim of this work was to evaluate the degradation of Ago2 during a glutathione depletion condition. Here, we show that Hela cells treated with BSO, a potent inhibitor of glutathione biosynthesis, decreases the protein level of Ago2. However, when Hela cells were treated with BSO and chloroquine to inhibit autophagy, accumulation of Ago2 was observed. We found that Ago2 localization and its binding site to the 5´-end of the miRNA are important for its degradation during this condition. Finally GSH depletion and autophagy inhibition affect the interaction of Ago2 with Let7a miRNA. These results suggest that GSH and autophagy machinery are important for the normal gene silencing through miRNAs.

TUESDAY-POSTER PRESENTATIONS FONDECYT 1110508 (IC) y 1141033 (JCS). HM: Becario Doctorado CONICYT, DID-UACH 1330-32-06 y Beca Estadía MECESUP AUS 1203

P2268 Board Number: B897

The Small GTPase Rab7 as a Central Regulator of Hepatocellular Lipophagy. B. Schroeder1, R. Schulze1, S. Weller1, A. Sletten1, C.A. Casey2, M.A. McNiven1; 1 Mayo Clinic, Rochester, MN, 2VAMC (151), Omaha, NE Autophagy is a central mechanism by which hepatocytes catabolize lipid droplets (LDs). Currently, the regulatory mechanisms that control this important process are poorly defined. The small GTPase Rab7 has been implicated in the late endocytic pathway and is known to associate with LDs, although its role in LD breakdown has not been tested. In this study, we demonstrate that Rab7 is indispensable for LD breakdown in hepatocytes subjected to nutrient deprivation. Importantly, Rab7 is dramatically activated in cells placed under nutrient stress; this activation is required for the trafficking of both multivesicular bodies (MVBs) and lysosomes to the LD surface during autophagic catabolism. Depletion of Rab7 leads to gross morphological changes of MVBs, lysosomes, and autophagosomes simultaneously reducing the physical interactions between these compartments and therefore attenuating hepatocellular lipophagy. These findings provide additional support for the role of autophagy in hepatocellular LD catabolism while implicating the small GTPase Rab7 as a key regulatory component of this essential process.

P2269 Board Number: B898

Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo. W.E. Dowdle1, B. Nyfeler1, L. Murphy1; 1 Novartis Institutes for Biomedical Research, Cambridge, MA Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use a novel VPS34 inhibitor to screen for new autophagy substrates. The inhibitor acutely blocks autophagy, de novo lipidation of LC3 and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on VPS34 inhibited cells we identified new autophagy substrates including NCOA4 which accumulates in ATG7 deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the ~450 kDa iron-binding ferritin complex to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4-/- mice display a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together this study provides a novel mechanism for selective autophagy of ferritin and reveals a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.

TUESDAY-POSTER PRESENTATIONS

P2270 Board Number: B899

Ubiquitin-dependent ULK1 negative feedback regulation constraints autophagy activity. C. Liu1,2, L. Pang1,2, P. Wu1, M. Lin1, R. Chen1,2,3; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 2Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan, 3Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 1

Autophagy, a cellular self-eating mechanism, is important for maintaining cell survival and tissue homeostasis in response to various stressed conditions, such as starvation. Although the mechanism of autophagy induction has been well studied, it remains elusive how cells modulate the level of autophagy once it has been induced. KLHL20 is a substrate adaptor of Cullin3-family ubiquitin ligase. Here, we show that ULK1, a serine/threonine kinase critical for autophagy initiation, is a substrate of the Cul3-KLHL20 ubiquitin E3 ligase complex. KLHL20-mediated ubiquitination leads to ULK1 proteasomal degradation. In responses to autophagy induction, the activation and autophosphorylation of ULK1 enhances its affinity to KLHL20, which results in an increased ULK1 ubiquitination and degradation. We further show that this KLHL20-dependent ULK1 regulation controls the amplitude and duration of autophagy to prevent prolonged or overly activated autophagy. This study identifies a novel regulatory mechanism for ULK1 and suggests a role of this regulation in autophagy termination.

P2271 Board Number: B900

A Kinase Independent Role for EGF Receptor in Autophagy Initiation. X. Tan1, R.A. Anderson1; 1 Molecular Pharmacology, University of Wisconsin-Madison, Madison, WI Autophagy is a highly conserved self-eating pathway for homeostasis maintenance in all eukaryotic cells. Misregulation of autophagy is linked to human diseases including cancers. It has been generally established that autophagy prevents tumor initiation in normal cells, but in established tumors autophagy may promote tumor progression by facilitating tumor cell survival in metabolically stressed conditions. The Epidermal Growth Factor Receptor (EGFR) is an oncogenic receptor tyrosine kinase overexpressed and/or over-activated in numerous human cancers. EGFR activation suppresses autophagy in tumor cells, and inhibition of EGFR signaling by tyrosine kinase inhibitors (TKIs) is associated with enhanced autophagy in many cancer cell lines. However, the mechanism by which EGFR inactivation upregulates autophagy is not clear. We have shown that EGFR inactivation resulting from either serum starvation or EGFR TKI treatment induces EGFR accumulation at late endosomes. The endosomal oncoprotein LAPTM4B directly and

TUESDAY-POSTER PRESENTATIONS selectively interacts with inactive EGFR and is required for the accumulation of EGFR at late endosomes upon serum starvation. Most significantly, both LAPTM4B and EGFR are required for autophagy initiation, and re-expression of either wild type or kinase dead EGFR rescues autophagy in EGFR knockdown cells. LAPTM4B and inactive EGFR interact with the Sec5 exocyst subcomplex that is also required for autophagy. Together, LAPTM4B, inactive EGFR and Sec5 collaborate to control the disassociation of Rubicon (an autophagy inhibitor) from the Beclin 1 complex to initiate autophagosome formation. These findings reveal a kinase independent role for EGFR in autophagy initiation upon serum starvation or EGFR TKI treatment. This could be a mechanism for the prosurvival role of EGFR in established tumors and for the EGFR TKI resistance in clinical therapies. This study is supported by Howard Hughes Medical Institute (HHMI) International Student Research Fellowship to X.T. and National Institute of Health (NIH) grant CA104708 and GM057549 to R.A.A.

P2272 Board Number: B901

Detecting the Function of the Small GTP-Binding Protein Arl1 in Autophagy. S. Yang1, A.G. Rosenwald1; 1 Department of Biology, Georgetown University, Washington, DC Autophagy is a cellular degradation process that sequesters components such as organelles or proteins into a double-membrane structure called the autophagosome, which then fuses with the lysosome or vacuole for the hydrolysis of those components. Autophagy can be triggered in response to a variety of intracellular or environmental stresses such as pathogen invasion, starvation, or unfolded-protein stress. Once induced, autophagy is performed and regulated by several Atg proteins. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that a small GTPbinding protein named Arl1, which belongs to the Arf/Arl/Sar protein family is involved in starvationinducted autophagy in Saccharomyces cerevisiae, but only under high temperature stress. Arl1 normally controls the retrograde trafficking from the endosome to the trans-Golgi and is important for proteins to be targeted to the vacuole. Using an assay which measures the degradation of GFP-Atg8 to free GFP to monitor bulk autophagy under different temperatures, we found that starved cells lacking the ARL1 gene show defects in bulk autophagy at elevated temperature, 37 but not at 30oC. The same conclusion was reached with assays using fluorescence microscopy to monitor the GFP-Atg8 signal in the cells under different conditions as well as the Pho8Δ60 activity assay to quantify the magnitude of autophagy. Furthermore, experiments with different nucleotide binding alleles of Arl1 demonstrated that the GTP restrictive form of Arl1 (Arl1[Q72L]) can rescue the defect. Finally, we found that loss of Ypt6, another GTP binding protein that is synthetically lethal with Arl1, shows similar defects in autophagy under heat stress. Future work will focus on determining the mechanism for the roles of both Arl1 and Ypt6 in autophagy.

TUESDAY-POSTER PRESENTATIONS

P2273 Board Number: B902

Regulation of Autophagy by ULK3, an Atg1-family kinase. C.R. Braden1, T.P. Neufeld1; 1 GCD, University of Minnesota, Minneapolis, MN Macroautophagy (hereafter autophagy) is a key cellular housekeeping process in which organelles and macromolecules are packaged in a double walled membrane, the autophagosome, and delivered to the lysosome via membrane fusion. Aggregated and long-lived soluble proteins and malfunctioning organelles are all subject to destruction via autophagy, meaning its proper regulation is a requirement for successful maintenance of cellular function. As an aggregate clearance mechanism, autophagy is of interest in aggregate-prone disease states such as Alzheimer’s. As a requirement for clearing damaged mitochondria, it is a key element in the study of Parkinson’s. As a pro-survival pathway in a variety of stress conditions including starvation, autophagy is scrutinized in the development of cancer treatments, and it is a target of inhibition in chemotherapy. While many signaling pathways lead to autophagy, most require the involvement of serine-threonine kinase Atg1 (Drosophila melanogaster) or its mammalian homologues Unc-51-like kinases 1 & 2 (ULK1 & 2). Under beneficial growth conditions, Atg1 is subject to inhibitory phosphorylation by the Target of Rapamycin (TOR). When inhibition is relieved, such as during amino-acid starvation, Atg1 induces autophagy in a manner dependent on its kinase domain, its binding partner Atg13, and a variety of complex members and downstream targets. Recently, stimuli that can induce autophagy independent of Atg1/ULK1/2 have been described, and it is unclear how these signals interact with the core autophagy machinery. The ULK family includes an additional member, ULK3, whose potential role in autophagy is poorly characterized, and we hypothesize that ULK3 may represent this missing link. Here we demonstrate that like Atg1, ULK3 is capable of inducing autophagy in the Drosophila fat body. Induction of autophagy by Ulk3 overexpression does not require Atg1, but does require the Atg1 binding partner Atg13. Sequence analysis of ULK3 shows a pair of MIT domains, reminiscent of the MIT domain structures identified in Atg1 required for Atg13 interaction. However, unlike Atg1, ULK3 is not required for starvation-induced autophagy. Interestingly, Ulk3 mutation does delay onset of autophagy during treatment with the proteasome inhibitor MG132, a relative of the chemotherapy drug bortezomib. Ulk3 mutant animals are viable, but demonstrate reduced survival rates compared to wild type animals. Taken together, our data suggest ULK3 may act as a junction point between the canonical autophagy signaling cascade and a novel upstream pathway, possibly connected to proteasome malfunction.

TUESDAY-POSTER PRESENTATIONS

P2274 Board Number: B903

The LRRK2 inhibitor GSK2578215A activates autophagy mediated by mitochondrial fission and mitochondrial-derivated ROS. S. Saez-Atienzar1,2,3, L. Bonet-Ponce1, J. blesa1, F. Romero1, J. Jordan3, M. Galindo2; 1 Dpt Physiology, Universidad Catolica de Valencia, Valencia, Spain, 2Unidad de Neuropsicofarmacología Traslacional, Complejo Hospitalario Universitario de Albacete, Albacete, Spain, 3Grupo de Neurofarmacología, Universidad de Castilla-La Mancha, Albacete, Spain Parkinson´s disease (PD) constitutes the main motor disorder and the second neurodegenerative disease after Alzheimer’s disease. Etiology of PD remains unknown, but both environmental and genetic factors have been implicated. Among the genes associated with PD is the leucine-rich repeat kinase 2 (LRRK2). Mutations in LRRK2 have been described in familial Parkinson´s disease and sporadic disease. However, the role of LRRK2 is still unknown and its inhibition is a promising pharmacological target in PD treatment. We used the LRRK2 inhibitor 2 arylmethyloxy-5-subtitutent-N-arylbenzamide (GSK257815A) at a concentration of 1 nM to study the effects of LRRK2 inhibition on the well-established dopaminergic cell line SH-SY5Y. We focused our study on crucial neurodegenerative features such as mitochondrial dynamics, autophagy and cell death. We found out that LRRK2 inhibition, either by pharmacological inhibition (using GSK2578215A and LRRK2-In-1) or molecularly by siRNA to knockdown LRRK2, activates autophagy since it increased the number of autophagosomes after 9 hours of treatment. Using the lysosome inhibitor chloroquine (CQ) and the mRFP-GFP-LC3 plasmid, we denoted that this increment results from both and increase in autophagosomes synthesis and an impairment of its degradation. LRRK2 inhibition also induced mitochondrial fragmentation mediated by Drp-1, in a time-dependent manner, starting at 6 hours after treatment. The use of MDIVI-1, a mitochondrial division inhibitor, avoided autophagy. This data strongly suggest that mitochondrial fragmentation is indeed an early step preceding autophagy. In order to ascertain the role of autophagy, we used autophagy disruptors that revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria by mitophagy In order to know the role of mitochondrial-derivated ROS, we analyzed 4hydroxy-2-nonenal (4-HNE). We observe accumulation of 4-HNE after GSK2578215A treatment. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphological alterations and autophagy. In summary, our data show how LRRK2 inhibition activates a cellular response where autophagy results in cytoprotection downstream of a disruption of the mitochondrial dynamics balance via a Drp1-dependent process. Our study suggests that drugs which modulate the functions of Drp-1 or mitochondrial-ROS may represent new opportunities to slow down neurodegeneration linked to Parkinson's disease.

TUESDAY-POSTER PRESENTATIONS

P2275 Board Number: B904

Optineurin is an autophagy receptor for damaged mitochondria in parkinmediated mitophagy that is disrupted by an ALS-linked mutation. Y.C. Wong1, E.L. Holzbaur2; 1 Physiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, 2Department of Physiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA Mitophagy is a cellular quality control pathway in which the E3 ubiquitin ligase parkin targets damaged mitochondria for degradation by autophagosomes. We examined the role of optineurin in mitophagy, as mutations in optineurin are causative for glaucoma and the neurodegenerative disease Amyotrophic lateral sclerosis (ALS), diseases in which mitochondrial dysfunction has been implicated. Using live cell imaging, we demonstrate the parkin-dependent recruitment of optineurin to mitochondria damaged by depolarization or reactive oxygen species (ROS). Parkin’s E3 ubiquitin ligase activity is required to ubiquitinate outer mitochondrial membrane proteins, allowing optineurin to stably associate with ubiquitinated mitochondria via its ubiquitin binding domain (UBD); in the absence of parkin, optineurin transiently localizes to damaged mitochondrial tips. Following optineurin recruitment, the omegasome protein DFCP1 (double FYVE-containing protein 1) is transiently recruited to damaged mitochondria to initialize autophagosome formation. Optineurin then recruits autophagosome formation around damaged mitochondria via its LC3 interaction region (LIR) domain. Depletion of endogenous optineurin inhibits LC3 recruitment to mitochondria, leading to increased mtDNA content within cells. This defect in autophagosome formation and mitochondrial degradation is rescued by wildtype optineurin, but not by an ALS-associated UBAN mutant (E478G), or by a LIR mutation in optineurin. Optineurin and the autophagy receptor p62/SQSTM1 are independently recruited to different domains on damaged mitochondria, and p62/SQSTM1 knockdown does not affect either optineurin or LC3-recruitment to damaged mitochondria. Thus, our results demonstrates a critical role for optineurin as an autophagy receptor in parkin-mediated mitophagy. Importantly, as mutations in parkin are linked to Parkinson’s disease, our study further indicates that defects in a single pathway lead to neurodegenerative diseases with distinct pathologies.

P2276 Board Number: B905

L84 interferes with autophagic flux and contributes to Parkinsonism. K. Lee1, S. Namkoong1, I. Jang2, R. Park1, J. Park1; 1 Division of Biological science and technology, Yonsei University, Wonju, Korea, 2kbsi, Daejon, Korea L84 is a antihypertensive drug that has been used to control the high blood pressure, and one of its adverse effects is that long term treatment of L84 often causes Parkinson’s disease in human and animal. However, the precise mechanism of L84 to induce Parkinson’s disease is not clear. Here we report that one possible mechanism of L84 to induce Parkinson’s disease. L84 treatment induced the

TUESDAY-POSTER PRESENTATIONS autophagosome formation by inhibiting the autophagic flux, and also accumulates p62, an autophagy adapter molecule.

P2277 Board Number: B906

Autophagy as a cytoprotective response to ethanol-induced damage in human Retinal Pigment Epithelial cells. L. Bonet-Ponce1, M. Flores-Bellver1, J. Barcia1, N. Martinez-Gil1, S. Saez-Atienzar1,2,3, J. Sancho-Pelluz1, J. Jordan3, M. Galindo2, F. Romero1; 1 Dpt Physiology, Universidad Catolica de Valencia, Valencia, Spain, 2Unidad de Neuropsicofarmacología Traslacional, Complejo Hospitalario Universitario de Albacete, Albacete, Spain, 3Grupo de Neurofarmacología, Universidad de Castilla-La Mancha, Albacete, Spain Retinal Pigment Epithelium (RPE) plays a crucial role in the physiology and pathophysiology of the retina due to its location and metabolism. Oxidative damage has been demonstrated as a pathogenic mechanism in several retinal diseases, and reactive oxygen species are certainly important by-products of ethanol (EtOH) metabolism. Under EtOH exposure, autophagy has been shown to exert a protective effect in different cellular and animal models. Thus we challenged a widely accepted model of human RPE cells (ARPE-19 cells) with 80, 200, 400 and 600 mM EtOH. 24 h after EtOH addition, we found out that EtOH treatment increased GFP-LC3 puncta, starting at 80 mM. LC3-II conversion by western blot was also detected after EtOH treatment, in a concentration dependent-manner. Interestingly, using both the mRFP-GFP-LC3 plasmid and the lysosome inhibitor chloroquine, we observed an increase in autophagy flux, in a concentrationdependent manner. In fact, our data pointed out an implication of the mTOR pathway. Moreover, autophagy activation seemed to be Beclin1 and ATG5-ATG12 dependent, as well. Mitochondrial morphology seemed to be clearly altered under EtOH exposure, leading to an apparent increase in mitochondrial fission. Interestingly, mitochondrial fission appeared to be dynamin-related protein 1 (Drp1) and optic atrophy 1 (OPA1) dependent but Bax independent. Consequently we noticed that mitochondrial autophagy is increased, in a concentration-dependent manner. In fact, we clearly demonstrated that mitochondrial ROS stimulates autophagy. An increase in 2′,7′-dichlorofluorescein fluorescence and accumulation of lipid peroxidation products, such as 4-hydroxy-nonenal (4-HNE), among others, were confirmed. The characterization of these structures confirmed their nature as aggresomes. Furthermore, we observed that autophagy efficiently degrades 4-HNE aggresomes. Finally, after autophagy inhibition, we noticed an over 2 fold increase in cell death, only in EtOH-treated cells. Hence, our data strongly suggest that autophagy plays a cytoprotective role in ARPE-19 cells under EtOH damage, by degrading fragmented mitochondria and 4-HNE aggresomes. Herein, we describe the central implication of autophagy in human RPE cells upon oxidative stress induced by EtOH, with possible implications for other conditions and diseases.

TUESDAY-POSTER PRESENTATIONS

P2278 Board Number: B907

DUAL ROLE OF TFEB IN MICROGLIA. N. Pastore1, T. Huynh1, A. Ballabio2; Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX, 2Telethon Institute of Genetics and Medicine, Naples, Italy

1

Transcription factor EB (TFEB) belongs to the MITF/TFE subfamily of basic helix–loop–helix leucinezipper (bHLH-LZ) transcription factors. In mammalian cells, TFEB is known to exert a transcriptional control of autophagy and lysosomal biogenesis. Recently, it has been reported that TFEB is also important for host defense against infections. Lysosomal activity is essential to autophagy, a cellular pathway that delivers cytoplasmic components to lysosomes for degradation and is involved in many diseases. Autophagy has been extensively implicated in immunity. Mutations in autophagy genes increase susceptibility to infections by intracellular pathogens. Microglia plays a crucial role in maintaining brain tissue homeostasis. In pathological conditions, microglia releases pro-inflammatory cytokines and cytotoxic factors, which aggravate the progression of neurodegenerative diseases. In this study we investigated the role of TFEB in microglia in vitro, in BV2 microglial cells, and in vivo, in LysMCre; TFEB flox/flox and LysM-Cre; TFEB-3xflag mice that have reduced or enhanced expression of TFEB specifically in macrophages. Real time PCR analysis demonstrated that knockdown of TFEB in microglial cells results in increased levels of pro-inflammatory cytokines (IL1β, IL6, TNFα) in basal conditions, while the inflammation is reduced after LPS challenge. Brains from LysM-Cre; TFEB flox/flox mice show increased levels of pro-inflammatory cytokines and inflammation, as demonstrated by NFkB and JNK levels. Serum IL1β levels are also increased in these mice. LPS-treated LysM-Cre; TFEB flox/flox mice show a decrease in inflammatory response compared to control mice. Overexpression of TFEB either in cells or in mice results in a different inflammatory response depending on the levels of TFEB: low levels overexpression result in increased response to LPS treatment, while high levels overexpression result in reduced inflammation after LPS challenge. Our data suggest that TFEB has a dual role in the microglia depending on cell activation status.

TUESDAY-POSTER PRESENTATIONS

P2279 Board Number: B908

Bax inhibitor 1 Enhances Autophagy in Cyclosporine A Treatment and Reduces Renal Dysfunction. R.K. Yadav1, B. Li1, G. Lee1, H. Kim2, H. Chae3,4; 1 Department of pharmacology, Chonbuk National University, Jeonju, Korea, Dem People's Rep, 2School of Dentistry, Wonkwang University, Iksan, Iksan, Korea, Dem People's Rep, 31Department of Pharmacology , Chonbuk National University medical school, Jeonju, Korea, 41Department of Pharmacology , Chonbuk National University medical school, Jeonju, Korea Cyclosporine A (CsA), a calcineurin inhibitor, improves allograft survival in solid organ transplantation. Previous evidence indicates that CsA induces autophagy, and that chronic treatment with CsA results in accumulation of autophagosomes and reduced autophagic clearance. Autophagy is a bulk protein degradation system that likely plays an important role in normal proximal tubule function and recovery from acute kidney injury. In the present study, we used human kidney tubular cells to demonstrate that CsA-induced autophagy is highly stimulated in Bax inhibitor-1 (BI-1)-overexpressing cells (BI-1 cells). BI-1 cells exhibit special activity involving highly maintained lysosome activation that promotes autophagosome and lysosome fusion. In BI-1 cells treated with CsA, transcription and nuclear translocation of TFEB is increased. In addition, treatment of BI-1 knockout mice with CsA resulted in decreased autophagosome and lysosome formation. We also found that BI-1 knockout mice were susceptible to CsA-induced kidney injury. Taken together, BI-1 appears to serve as a protective mechanism against CsA-induced nephrotoxicity through autophagy both in vitro and in vivo.

P2280 Board Number: B909

The autophagosome marker LC3 undergoes regulated targeting to the nucleus and nucleolus. L.J. Kraft1,2, J. Dowler1, A.K. Kenworthy1; 1 Mol Physiol Biophys, Vanderbilt University Medical Center, Nashville, TN, 2Harvard University, Boston, MA Autophagy is a lysosomal degradation pathway that is important for the maintenance of cellular homeostasis. Although the formation of autophagosomes occurs in the cytoplasm, it is increasingly evident that several key proteins in the autophagy pathway shuttle in and out of the nucleus. We previously reported that LC3, a protein that participates in autophagosome formation and cargo selection, is enriched in the nucleus in a slowly diffusing form. However, the mechanisms that retain LC3 in the nucleus and control its nuclear dynamics remain poorly understood. To address this, we used a combination of fluorescence microscopy and FRAP to study Venus-LC3 in HeLa cells. We find that mutating residues involved in binding of LC3 to other proteins and RNA moderately decreases its

TUESDAY-POSTER PRESENTATIONS nucleocytoplasmic ratio, but disrupting LC3 lipid modification does not change its nucleocytoplasmic ratio. This suggests that the majority of LC3 in the nucleus is soluble, and that it is retained in the nucleus by interactions with either proteins or RNA. Consistent with this, soluble nuclear LC3 diffuses more slowly than predicted for a monomer. Perturbing the autophagy pathway with rapamycin or chloroquine has little effect on LC3’s nuclear localization, but does change the apparent size of LC3associated complexes. This implies that soluble nuclear LC3 associates with complexes with functions possibly related to autophagy. Unexpectedly, we also find that nuclear LC3 is enriched within the nucleolus. We show that the triple arginine motif of LC3, a region previously shown to bind to both proteins and RNA, shares similarities with a consensus nucleolar detention sequence. Furthermore, perturbation of LC3’s triple arginine motif completely abolishes its localization to the nucleolus. Together, these findings reveal a potential role for both protein-protein and protein-RNA interactions in targeting LC3 to the nucleus and nucleolus.

P2281 Board Number: B910

ATG5 and ATG7 fulfill essential and non-redundant roles in human hematopoietic stem/progenitor cells. M. Gomez Puerto1, H. Folkerts2, A. Wierenga2, K. Schepers1, J.J. Schurunga2, E. Vallenga2, P. Coffer1; 1 Cell biology , University medical center Utrecht, Utrecht, Holland, 2Department of Experimental Hematology, University medical center Groningen, Groningen, Holland Autophagy is a homeostatic mechanism that is crucial in the degradation of damaged organelles and misfolded proteins. This catabolic process is characterized by the formation of a double membrane vesicle called autophagosome that after engulfing cellular components will fuse with a lysosome for degradation of the cargo. Autophagy can be triggered in response to stress conditions such as starvation, energy limitation, hypoxia and DNA damage. Surprisingly, the role of autophagy in the regulation human hematopoietic stem cell (HSC) function remains unclear. Besides their ability to differentiate into all blood cell lineages, HSCs have the capacity to self renew and remain quiescence for the lifelong maintenance of hematopoiesis. Thus, HSCs require stringent housekeeping mechanisms safeguarding their integrity and protecting them from accumulation of damage. Currently little is known concerning the regulation of autophagy in hematopoietic stem and progenitor cells, or how this may change during myeloid differentiation. Autophagy was analyzed by Western blotting and FACS. In addition colony forming cell (CFC) assays, long term culture-initiating cell (LTC-IC) assays, apoptosis measurements, cell-cycle analysis and reactive oxygen species (ROS) measurements were performed. We demonstrated that autophagy is functional in human hematopoietic stem/progenitor cells (HSPCs). Robust accumulation of the autophagy markers LC3-II and p62 were observed in HSPCs treated with bafilomycin-A1 (BafA1) or hydroxychloroquine (HCQ). In addition, CD34+CD38- stem cell-enriched cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BafA1 and HCQ treatment. To study the functional consequences of autophagy,

TUESDAY-POSTER PRESENTATIONS ATG5 or ATG7 were knockdown in CD34+ cells, using lentiviral shRNAs. Downregulation of ATG5 and ATG7 resulted in a marked decrease in progenitor frequencies, which coincided with a strong reduction in expansion of CD34+ cells. In addition, a 3-fold reduction in stem cell frequencies was observed. The reduction in HSPCs was not due to impaired differentiation, but was at least in part due to reduced cell cycle progression and an increase in apoptosis. The changes in cell cycle and increased apoptosis coincided with increased expression of p53 and downstream proapoptotic target genes BAX, PUMA and PHLDA3 and cell cycle inhibitor p21. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny.

P2282 Board Number: B911

Autophagy protects against neurodegeneration in a Drosophila model of C9orf72mediated ALS. K. Zhang1, K. Cunningham2, J.B. Machamer1, T.E. Lloyd1,2,3; 1 Department of Neurology, Johns Hopkins University, School of medicine, Baltimore, MD, 2Graduate Program in Cellular and Molecular Medicine, Johns Hopkins University, School of medicine, Baltimore, MD, 3The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, School of medicine, Baltimore, MD A recently identified GGGGCC hexanucleotide repeat expansion (HRE) mutation in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS). In a forward genetic modifier screen of HRE toxicity in Drosophila, we discovered that the loss of function of p62 strongly suppresses HREinduced neurodegeneration. p62 plays a critical role in autophagy, a self-degradation process that prevents the accumulation of toxic misfolded proteins in many neurodegenerative disorders. p62 binds and delivers ubiquitinylated proteins to the autophagosome, eventually leading to lysosomal degradation of both p62 and its cargos. Defects in this process cause accumulation of p62 as well as its cargos. Interestingly, we find that flies expressing the HRE display increased p62 protein levels, intracellular p62 protein aggregates in motor neurons, and impaired synaptic transmission at the neuromuscular junction, consistent with the findings reported in C9orf72 ALS patient autopsies. Furthermore, we find that genetic and/or pharmacological activation of the autophagic pathway significantly suppresses neurodegeneration concomitantly with p62 aggregation, strongly suggesting that autophagic clearance of p62-positive aggregates can rescue C9orf72-related neurotoxicity. Hence, we propose that C9orf72 HRE causes cytotoxic protein aggregations containing p62, which can be rescued by increasing autophagy. Our study suggests that activation of autophagy may be a therapeutic strategy for C9orf72-mediated ALS.

TUESDAY-POSTER PRESENTATIONS

P2283 Board Number: B912

BNIP3 Induces Full-length PINK1 Accumulation to Promote Mitophagy. T. Zhang1, L. Xue2, L. Li1, C. Tang1, Z. Wan1, R. Wang1, Z. Zeng1, W. Andy Tao2, Z. Zhang3; 1 Central South University, State Key Lab of Medical Genetics, Changsha, China, 2Purdue University, Department of Biochemistry, West Lafayette, IN, 3 Central South University, State Key Laboratory of Medical Genetics, Changsha, China Mutations in PINK1 cause early onset familial Parkinson’s disease (PD). PINK1 is accumulated on followed by recruiting parkin to the outer membrane of damaged mitochondria to promote mitophagy. Here, we demonstrate that BNIP3, a mitochondrial BH3-only protein, interacts with PINK1 to promote stabilization and accumulation of full length PINK1 on outer membrane of damaged mitochondria, therefore, to facilitate parkin recruitment to mitochondria. The results suggest that BNIP3 plays an essential role in PINK1/parkin-mediated mitophagy and likely contributes to PD pathogenesis. Keywords: Parkinson’s disease, PINK1, BNIP3, mitophagy

Systems and Synthetic Cell Biology P2284 Board Number: B914

Measuring fast gene dynamics in single cells with timelapse luminescence microscopy. A. Mazo-Vargas1, H. Park1, M. Aydin1, N. Buchler1; 1 Biology, Duke University, Durham, NC Timelapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular auto-fluorescence or photo-toxicity that arise from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. However, the photon flux per luciferase is significantly lower than fluorescent proteins. To date, timelapse luminescence microscopy has been successfully used to track gene dynamics in larger organisms and for slower processes, where more total photons can be collected in one exposure. Here, we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining timelapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with sub-minute time resolution over many generations. Our protocol is significantly faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 20 minutes, which is consistent with its known rate of chromophore maturation in yeast. Our work shows that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

TUESDAY-POSTER PRESENTATIONS

P2285 Board Number: B915

Two sequential decisions commit human cells to start the cell cycle. S.D. Cappell1, S.L. Spencer1, M. Chung1, A. Jaimovich1, T. Meyer1; Department of Chemical and Systems Biology, Stanford University, Stanford, CA

1

One of the most fundamental decision processes in biology is the commitment of cells to enter the cell cycle and generate two daughter cells. Cell-cycle commitment in mammalian cells is thought to be regulated by a single decision point in G1 called the Restriction Point, after which growth factors are no longer required for the cell cycle to proceed. Using live-cell microscopy of cell-cycle sensors and singlecell analysis, we have identified the moment at which cells cross the Restriction Point and find that more than three hours pass between the Restriction Point and the actual start of DNA replication. Furthermore, we find that cell stress after the Restriction Point but before the start of DNA replication results in cell-cycle exit and a return to a pre-Restriction Point state, providing evidence for another later decision to commit to the cell cycle. We present evidence for a second bistable switch regulating Sphase entry. The trigger for this second switch is the CDK2/Cyclin E-mediated partial inactivation of APCCdh1, an E3-ligase that controls the degradation of proteins necessary for S-phase progression. We show that the switch is driven by a double negative feedback between the regulatory protein Emi1 and APCCdh1. Markedly, Emi1 functions as both an inhibitor and a substrate of the APC, which generates a bistable and irreversible switch regulating S-phase entry. Thus, cells commit to the cell cycle by making two sequential decisions. They first monitor mitogen signals to decide whether to cross the Restriction Point, and then monitor stress signals to decide whether to irreversibly inactivate APCCdh1, start DNA replication, and fully commit to the cell cycle.

P2286 Board Number: B916

Multiplexed transcriptional analysis of p53 targets in single cells. J.R. Porter1, B.E. Fisher1, E. Batchelor1; 1 Laboratory of Pathology, NCI/NIH, Bethesda, MD The transcription factor p53 is activated in response to a wide range of biological stresses. To cope with different stresses, p53 regulates the expression of over a hundred different target genes in a stressspecific manner. Understanding how stresses are relayed by p53 into distinct regulation of such a large number of genes is a major challenge. The problem is confounded by the fact that common transcriptomic methods often rely on measurements of populations of cells, which can obscure information regarding single-cell heterogeneity and the coregulation of specific targets. To address these concerns, we used a microfluidic platform to analyze the expression of nearly one hundred wellcharacterized p53 target genes simultaneously in individual cells. We focused on p53’s dynamic response to DNA double strand breaks, which we had previously characterized as an undamped pulsatile

TUESDAY-POSTER PRESENTATIONS dynamical response. We identified distinct classes of downstream target dynamics, including oscillatory and delayed target gene expression. We also characterized target genes based on expression noise as a measure of single-cell heterogeneity. From our co-expression measurements, we identified gene regulatory sub-networks operating downstream of p53. Our findings provide new mechanistic insights into how p53 coordinates stress-specific responses in individual cells, and may provide novel therapeutic strategies for altering cell fates.

P2287 Board Number: B917

Constant Growth Rate Can Be Supported by Decreasing Energy Flux and Increasing Aerobic Glycolysis. N. Slavov1,2, A. van Oudenaarden3; 1 Massachusetts Institute of Technology, Cambridge, MA, 2Systems Biology, Harvard University, Cambridge, MA, 3Hubrecht Institute, Utrecht, Netherlands Fermenting glucose in the presence of enough oxygen to support respiration, known as aerobic glycolysis, is believed to maximize growth rate. We observed increasing aerobic glycolysis during exponential growth, suggesting additional physiological roles for aerobic glycolysis. We investigated such roles in yeast batch cultures by quantifying O2 consumption, CO2 production, amino acids, mRNAs, proteins, posttranslational modifications, and stress sensitivity in the course of nine doublings at constant rate. During this course, the cells support a constant biomass-production rate with decreasing rates of respiration and ATP production but also decrease their stress resistance. As the respiration rate decreases, so do the levels of enzymes catalyzing rate-determining reactions of the tricarboxylic-acid cycle (providing NADH for respiration) and of mitochondrial folate-mediated NADPH production (required for oxidative defense). The findings demonstrate that exponential growth can represent not a single metabolic/physiological state but a continuum of changing states and that aerobic glycolysis can reduce the energy demands associated with respiratory metabolism and stress survival. http://www.cell.com/cell-reports/abstract/S2211-1247(14)00254-X

P2288 Board Number: B918

Variable stoichiometry among core ribosomal proteins. N. Slavov1,2, A. van Oudenaarden3; 1 Massachusetts Institute of Technology, Cambridge, MA, 2Systems Biology, Harvard University, Cambridge, MA, 3Hubrecht Institute, Utrecht, Netherlands Understanding the regulation and structure of the eukaryotic ribosome is essential to understanding protein synthesis and its deregulation in disease. While ribosomes are believed to have a fixed

TUESDAY-POSTER PRESENTATIONS stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Reconciling these views requires direct and precise quantification of RPs. We used massspectrometry to directly quantify RPs across monosomes and polysomes of budding yeast and mouse embryonic stem cells (ESC). Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in ribosomes bound to multiple mRNAs. Together, these findings support the existence of ribosomes with distinct protein composition and physiological function. http://biorxiv.org/content/early/2014/05/26/005553

P2289 Board Number: B919

How do bacterial growth rates relate to evolutionary fitness landscapes for energy-efficiency?. A. Maitra1, K.A. Dill1; 1 Laufer Center for Physical and Quant. Biology, Stony Brook University, Stony Brook, NY We are interested in energy flows in bacteria, and how energy gets trafficked between producing ribosomal and nonribosomal proteins under different growth conditions. We describe an analytical model that leverages extensive data on experimental growth laws to infer the underlying fitness landscape in E. coli. This model gives insight into some of the complex nonlinear relationships between energy utilization and ribosomal and non-ribosomal production as a function of cell growth conditions. We draw inferences about what evolution has optimized in E. coli. Is E. coli optimized for growth speed or for energy efficiency? Experimental data shows that at its replication speed limit, E. coli produces 4 mass equivalents of non-ribosomal proteins for every mass equivalent of ribosomes. The model shows that this ratio is expected if the bacterial fitness function is the energy efficiency of fast-growing cells. We conclude that a principal evolutionary driving force for bacteria is the energy efficiency of the fastest growing cells.

P2290 Board Number: B920

Engineering a modular synthetic cyclic AMP generator. M. Ritt1, S. Sivaramakrishnan1; 1 Cell and Developmental Biology, University of Michigan, Ann Arbor, MI Adenylyl cyclases (ACs) are key amplifiers of GPCR signaling. Mammalian cells express nine distinct AC isoforms, all of which convert ATP to the second messenger cyclic AMP (cAMP). While much is known

TUESDAY-POSTER PRESENTATIONS about the structure of ACs and the signaling pathways they control, the differential function of AC isoforms and their spatio-temporal regulation remains poorly understood. Here we describe the detailed characterization of a genetically encoded synthetic adenylyl cyclase (SYNAC) that can be generalized to any AC isoform and targeted independent of the native AC. SYNAC activity can be monitored orthogonally using FRET, and is controlled by forskolin and Gαs in a manner similar to native AC. We also describe a modular extension of SYNAC that can be used to modify its activity independent of endogenous G proteins.

P2291 Board Number: B921

Cell based high throughput assay to evaluate cardiovascular safety profile of newly synthesized compounds to be nominated for clinical development. C.R. Sharma1,2, J.M. Harris2, M.R. Sharma2, M.B. Navel2, N. Amezcua2, R.R. Rodriguez2, S. Satish2, J.R. Martinez2, J. Collins3, J. Jani2, H. Wong3, J.P. Sharma2; 1 Celprogen Inc., Torrance, CA, 2Stem Cell Biology, Celprogen Inc, Torrance, CA, 3Biopico, UCI, CA The optimal cell based lineage for cardiac-regeneration still remains a mystery. Here we have developed a novel cell based assay to screen novel synthetic compounds for cardiovascular safety and regenerative profiles for clinical development. The human cardiomyocytes were isolated from adult ventricular and atrial appendages, characterized, and electrically profiled for functional physiological cardiac evaluations. The electrophysiology of adult human cardiomyocyte consists of 4X4 array of platinum microelectrodes and electronic measurement system based on a 1mV rms noise level amplifier and 16 channel 16bit data acquisition card. This High throughput ElectroPhysiological Assay (HEPA) cardiac chip technology was developed by Biopico Systems Inc., for characterization of the human cardiomyocytes electrophysiology functional index for cardiovascular safety and regenerative profiles. Prior to utilizing the human cardiomyocytes they were tested positive with Real time PCR, IHC and Western Blot Analysis for the following biomarkers: ckit, Actinin, ANP, Connexin 43, Desmin, KDR, Nkx2.5, GATA-binding protein 4 (GATA4) and SERCA2. The in-vitro assay procedure was established to determine cardiovascular safety and regenerative index of newly identified led molecules. In this study we tested CEP1430, a novel anti-tumor agent targeting pancreatic stem cells for cardiovascular safety; our lead molecule had no adverse cardiovascular liability. In-vivo daily administration of CEP1430 for 20 days had no cardiovascular adverse effect. From this study we were able to conclude that we have identified a lead compound for targeting pancreatic cancer stem cells that has no adverse cardiovascular effect when monitored for 20 days with once daily administration of the compound by intraperitoneal injections. This in-vitro cell based cardiac assay enabled us to determine the cardiac safety value help us determine the molecule to be a potential candidate for clinical development.

TUESDAY-POSTER PRESENTATIONS

P2292 Board Number: B922

Biosynthesis of Silver Nanoparticles using Biden frondosa and its Tyrosinase Activity. Q. Abbas1, S. Seo1; 1 Department of Biology, College of Natural Sciences, Kongju National University, Kongju, Korea Herbal Nanoparticles gain lot of attention because of their novel drug delivery system. In this study the silver nanoparticles (AgNPs) were synthesized by using aqueous extract of Biden frondosa. The appearance of brown color indicates the formation of Biden frondosa-AgNPs. The Formation and stability of AgNPs were determined by UV–Vis spectroscopy; Fourier Transform Infrared (FTIR) data confirm the presence of various aromatic and –OH moieties. The formation of herbal AgNPs further assured by energy dispersive X-ray spectroscopy (EDX) and Scanning electron microscopy (SEM) size ranging30-50nm. The mushroom tyrosinase inhibitory activity of synthesized AgNPs was evaluated and it was found that they exhibited potent tyrosinase inhibitory activity compared to control. The IC50 value of AgNPs was found to be 9 and that of kojic acid is 15μg/mL. AgNPs of Biden frondosa may be considered as potential candidate for the production of medical and cosmetic products.

P2293 Board Number: B923

Bacterial Cell Adhesion to Switchable Polymer Brushes. T. Babar1, A. Sidorenko1, M. Bruist2; 1 Department of Chemistry and Biochemistry, University of the Sciences, Philadelphia, PA, 2Chemistry Biochemistry, University of the Sciences, Philadelphia, PA Polymer brushes have the potential to be used for medical supplies and as new materials for industry that require a reduction or elimination of bacterial growth. We propose that the modification of solid surfaces with switchable polymer brushes allows adherence of bacteria to a hydrophobic surface or the controlled detachment of the bacteria from a hydrophilic polymer surfaces. Based on the research by Cunliffe, et. al (1991), we analyzed the physical adhesion properties of Escherichia coli and Salmonella typhimurium1 when applied to switchable hybrid molecular brush (HMB) systems. HMB’s are composed of a chitosan backbone with added polystyrene (PS) and polyacrylamide (PAAm) side chains. Upon optimization of production of the brushes, with respect to the polymer thickness and wettability, the bacterial response to each surface polymer was characterized using Atomic Force Microscopy (AFM). Through AFM, the initial stage of bacterial adherence, in which the bacteria recognize the surface using responding extracellular extensions, topographical images were acquired. The images show the cellular appendages, fimbria, extended from the cell’s surface while adhered to polystyrene. There is an absence of fimbria when cells are on polyacrylamide. Further experiments will be conducted to observe the surface characteristics of cells when they are allowed to grow on the brushes for longer periods of times; along with comparisons of cell adherence and surface characteristics on modified HMB's.

TUESDAY-POSTER PRESENTATIONS [1]

Cunliffe, D., Smart, C.A., Alexander, C., Vulfson, E.N. Bacterial Adhesion at Synthetic Surfaces. Appl. Environ. Microbiol. 1991, 65, 4995.

P2294 Board Number: B924

Reconstitution of regulated FtsZ localization by Min protein gradients in vitro. K. Zieske1, P. Schwille2; 1 Max Planck Institute of Biochemistry, Munich, Germany, 2Max Planck Institute for Biochemistry, Martinsried, Germany The controlled placement of the division apparatus plays a key role for cell development. During cell division in Escherichia coli the oscillatory self-assembly of the MinCDE proteins directs the assembly of the division machinery. To investigate the minimal requirements for division site localization in a controlled environment we engineered a biomimetic system by reconstituting the cell division protein FtsZ and the Min protein system in membrane clad soft-polymer compartments. This bottom-up system system allows studying complex organizational principles, such as the self-assembly of Min protein concentration gradients and controlled localization of FtsZ to the middle of a cell-like. Furthermore the role of geometric boundary conditions and minimal biochemical requirements for division site localization can be addressed.

Tissue Development and Morphogenesis 3 P2295 Board Number: B926

Actomyosin network dynamics in epithelial morphogenesis. K.L. Ong1, S. DiNardo2; 1 Cell Molecular Biology Graduate Program, University of Pennsylvania, Philadelphia, PA, 2Department of Cell Developmental Biology, University of Pennsylvania , Philadelphia, PA The lowest energy state of a simple epithelium is a hexagonal packing composed of 3-cell junctions. To generate cell geometries away from equilibrium, two types of molecular machines are required: (1) force-generating networks to remodel or stabilize cell-cell contacts and (2) polarity determinants to control the pattern of cell shape changes. Coordination of cell shape changes in a group of cells is required for proper tissue morphology and function. Dysregulated tissue morphogenesis underlies pathologies such as developmental defects and cancer. As a model of epithelial morphogenesis, we study a dramatic alignment in the Drosophila embryonic epidermis that produces columns of aligned, rectangular cells. These columns give rise to lines of hooks required for larval mobility, thus the geometry of this epithelium is key to its function. Past work in our

TUESDAY-POSTER PRESENTATIONS lab has suggested that actomyosin-mediated junction remodeling events that convert 3-cell junctions to 4-cell junctions produce this rectilinear arrangement. Recently, we have characterized the cell shape changes in this epithelium at higher temporal resolution. We find that the straightening of DorsalVentral (DV) interfaces between cell columns does not always temporally coordinate with junction remodeling events. By live imaging of fluorescently tagged myosin light chain, we observe two separate pools of myosin with different behaviors. First, actomyosin cables stably co-localize with the adherens junctions of straightening DV interfaces throughout alignment. Second, an apical pool of myosin located medially in aligning cells undergoes rapid cycles of contraction and disassembly. Based on these findings, we hypothesize that two distinct actomyosin networks, regulated in sequence or parallel, exert different forces on the cells in this tissue to mediate alignment: (1) actomyosin cables generate tension across all DV contacts to straighten interfaces between columns and (2) medial-apical myosin elevates force on select contacts to induce junction remodeling events. In the future, we hope to determine the polarity mechanisms that temporally regulate these two pools of actomyosin, quantitate the contractile dynamics of the medial-apical actomyosin population with respect to junction remodeling, and dissect the contribution of these distinct actomyosin pools to collective cell shape changes.

P2296 Board Number: B927

Paradoxical sleep deprivation and its consequences on the morphophysiology of ventral prostate. R.A. Fochi1, J.Q. Antoniassi2, R.M. Goes3, S.R. Taboga4; 1 Department of Biology, São Paulo State University, IBILCE/UNESP, São José Do Rio Preto, Brazil, 2 Department of Structural and Functional Biology, State University of Campinas, IB/UNICAMP, Campinas, Brazil, 3Biology, São Paulo State University, IBILCE/UNESP, São José Do Rio Preto, Brazil, 4 Biology, São Paulo State University, IBILCE/UNESP, S J do Rio Preto, Brazil This study aimed to evaluate possible morphological and functional changes in the ventral prostate of the adults (90 days) Mongolian gerbil (Meriones unguiculatus) after the paradoxical sleep deprivation (also known as rapid eye movement phase – R.E.M.). The REM sleep deprivation was induced over a period of four consecutive days using the modified multiplatform method. Through this technique, the animals of treated group (PSD) remained in cages containing 18 fixed platforms, allowing their movement and access to food and water ad libitum, filled with water up to 1 cm below the surface of the platforms. When animals of the PSD group enter in the REM sleep, they lose muscle tone and fall in the water, disrupting the sleep cycle. The animals of control group (NC) remained in polypropylene cages filled with shaving woods during the whole experiment, receiving water and food ad libitum. The serum dosage for testosterone and corticosterone was performed by ELISA. Prostatic fragments were analyzed by morphometry and stereological techniques, as well as immunohistochemistry for proliferating cell nuclear antigen (PCNA) and TUNEL for apoptosis evaluation. In the prostate of PSD animals occurred an evident regression of the glandular epithelium besides some foci of hyperplasia, while the muscular stroma also becomes thinner and disorganized when compared to NC. These

TUESDAY-POSTER PRESENTATIONS morphological changes occurred without testosterone serum levels fluctuations, but an important increase in corticosterone serum levels was noted (5.37 ng / mL to 19.99 ng / ml). The animals subjected to sleep deprivation also showed a significant reduction in body weight (62.50 g to 52.75 g), however, the ventral prostate weight remained unchanged. Furthermore, there was also a decrease of PCNApositive cells in the epithelial and stromal compartments, which was accompanied by an increase in apoptosis, particularly in the stromal region. Based on these results, it can be concluded that sleep disruption triggers important hormonal oscillations that negatively reflect in the morphophysiology of prostate gland and this condition could be considered a promoter factor of prostatic lesions.

P2297 Board Number: B928

Scarb2a gene expression is required for Notochord Development in Zebrafish. A. Diaz Tellez1; 1 IFC, UNAM, DF, Mexico Scarb2 is a glycoprotein, with two transmembrane sites, several sites for N-glycosylation and a Cterminal di-leucine motif . In humans, mutations in Scarb2 were described as causing of Action Myclonus Renal Failure Syndrome (AMRF), which is diagnosed as a progressive myoclonus epilepsy. Zebrafish has three homologous genes for scarb2 (scarb2a, scarb2b and scarbc). Scarb2a insertional-mutant was obtained in a large-scale forward genetic screening, this mutant is characterized by the presence of vesicular bodies in the brain at 1 dpf and hypopigmentation at 2 dpf both phenotypes are restored by itself at 3 dpf . Furthermore, since 1 dpf scarb2a mutant show a particular defect in the notochord formation, in which notochord vacuoles never reach their normal size. Whole mount in situ hybridization revealed that scarb2a is expressed in the brain and in the notochord at early stages. Interestingly, as development progress, scarb2a expression is dynamically displaced in an anterioposterior direction. We also notice, by immunostaining, that laminin is not arranged properly at the notochord basal membrane. Through electronic microscopy, we identified that specific basal membrane defects are specifically localized at the intermedial zone, however internal and external layers seems unaffected.

P2298 Board Number: B929

Dachsous, Fat and actin modulators contribute to planar cell polarity in the Drosophila ventral epidermis. G.E. Osborn1, D. Ly1, K. Lawlor1, S. DiNardo1; 1 Department of Cell Developmental Biology, University of Pennsylvania , Philadelphia, PA Morphogenesis is orchestrated by the cooperation of many cells: for proper execution, cells must respond to directional information and transmit these cues to neighboring cells. To accomplish this,

TUESDAY-POSTER PRESENTATIONS epithelial cells tend to establish polarity within the plane of the epithelium, in addition to apicobasal polarity. Such planar cell polarization (PCP) allows cells to define their position within a field, thereby allowing cells to coordinately produce asymmetric responses essential for tissue function. The molecules that control planar cell polarity have been identified and well studied; polarizing systems such as the conserved Frizzled (Fz) and the Dachsous/Fat (Ds/Fat) systems allow cellular neighbors to establish and maintain polarity. Less understood is how these systems integrate with downstream effectors to choreograph asymmetric responses. Here we study the Drosophila ventral epidermis, an epithelium in which cells produce a stereotyped template for the production of denticles, critical for larval motility. This template is comprised of actin-based protrusions (ABPs), which are asymmetrically polarized within these cells. The Ds/Fat system is essential for the proper localization of ABPs; the current model is that Ds signals through the Fat receptor to impose polarity. We are performing functional domain analysis of Fat, in order to determine critical motifs of this molecule and to predict downstream effectors of the Ds/Fat system. In addition, through live imaging and genetic techniques, we have begun analysis of candidate cytoskeletal molecules (actin modulators, myosin II) to identify the machinery that engages in the construction and placement ABPs at the posterior edge of these cells. By working from receptor to cytoskeleton, we hope to understand how molecular polarity determinants integrate with structural effectors to functionally polarize tissue, a process critical in both development and disease.

P2299 Board Number: B930

Claudin localization to tight junctions is required for neural tube morphogenesis. A. Baumholtz1, A. Simard1, A. Ryan2; 1 Human Genetics, McGill University, Montreal, QC, 2Pediatrics and Human Genetics, McGill University, Montreal, QC Neurulation is a critical developmental process that converts the flat neural plate into a closed neural tube. Throughout this process the neural plate epithelium undergoes extensive remodeling including cell intercalation and apical constriction. These events require the maintenance of cell interactions within the neural epithelium through intercellular junctions. The most apical of these junctions are tight junctions (TJs), where members of the claudin family regulate apical-basal cell polarity and cell adhesion, and link the TJ to the actin cytoskeleton. We hypothesize that claudins are required for coordinating the morphogenetic events that drive neural tube closure. To test our hypothesis, we used the C-terminal domain of Clostridium perfringens enterotoxin (cCPE) to remove Claudin-4 and-8, and Claudin-3 and -4 from TJs of the neural and non-neural ectoderm of chick embryos respectively. Disrupting the localization of these claudins to tight junctions led to folate-resistant NTDs in 100% of embryos. In situ hybridization analysis of gene expression in the ectoderm and along anterior-posterior boundaries of the neural tube suggested that NTDs were not due to defects in ectoderm differentiation or patterning. cCPE-treated embryos displayed defects consistent with a disruption of convergent extension: a shortened anterior-posterior axis, a reduced length-to-width ratio, misshapen somites, and a broadened notochord. Transmission electron microscopy analysis of cCPE-treated embryos showed that midline

TUESDAY-POSTER PRESENTATIONS cells failed to undergo apical constriction. Furthermore, we showed reduced apical accumulation of RhoA and pMLC, an activated form of myosin II that is phosphorylated by Rho kinase (ROCK), in cCPEtreated embryos. These data indicate that claudins function upstream of RhoA/ROCK signalling to regulate actin-myosin contraction and thus, cell intercalation and apical constriction. This is the first time that claudins have been shown to directly regulate neural tube closure.

P2300 Board Number: B931

Slug/Snail2 expression during the development of squamous metaplasia in airways of patients with chronic obstructive pulmonary disease (COPD). C. Ben Brahim1, C. Courageux 1, N. Ferhani 1, J. Brard1, M. Pretolani 1, M. Aubier1, P.A. Leroy1; 1 INSERM UMR1152, Université Paris Diderot, Faculté de Médecine Bichat, Paris, France Chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide, is clinically characterized by a slow and progressive airflow obstruction and decline in lung function, resulting from a remodeling of the airways that includes several epithelium abnormalities. Inhaled agents, mainly cigarette smoke, are the main cause of COPD. The airway epithelium, first in line during exposure to inhaled environmental factors, is often injured and need to be repaired. The normal bronchial epithelium is pseudostratified, consisting of ciliated, goblet and basal cells. The latter are stem/progenitor cells that can self-renew and/or differentiate into ciliated and goblet cells in a well controled balance to reconstitute a functional epithelium. Chronic toxic injury can repetitively perturb this balance and lead to epithelium abnormalities such as mucous cell hyperplasia or squamous metaplasia (SM). SM is frequent in the bronchial epithelium of smokers but its extent is increased in smokers with COPD compared to smokers without COPD. In COPD there is also a loss of normal pseudo stratified epithelium with transition into a fully squamous epithelium (Wilson et al, ATS2013). The objective of our work is to understand the molecular mechanisms underlying the abnormal airway epithelial repair that are specific to COPD. Slug was found to be one of the most expressed genes in mouse bronchial basal cells when compared to non-basal cells (Rock et al, 2009). We hypothesized that Slug is a key regulator of basal cell renewal and differentiation and could be involved in the development of SM. We found, by immunohistochemical labeling of bronchial biopsies, that Slug is expressed in the nuclei of cells in zones of SM in both control and COPD patients; however, lower levels are observed in biopsies from COPD. To follow the expression of Slug during the regeneration of a bronchial epithelium, we used primary human bronchial epithelial cells (HBECs) cultured in air-liquid interface (ALI). This culture system allows regeneration of a normal epithelium at high concentration of ATRA, while in conditions depleted in ATRA, SM is induced as shown by involucrin increase and cytokeratin 18 decrease of expression. Using this model, we found that stable SM is associated with a decrease of Slug protein expression. When cells are treated with TGFβ, a factor previously reported to induce SM, we observed a decrease of Slug associated with SM in COPD and this correlates with a decrease in pAKT. In summary, our work shows that the more stable and mature SM found in COPD correlates with a decrease of Slug expression and that Slug may be involved in the reversibility of SM.

TUESDAY-POSTER PRESENTATIONS

P2301 Board Number: B932

Male offspring of diabetic rats exhibit increased activity of MMP-2 associated to higher content of TGF-β, androgen receptor and cell proliferation in the ventral prostate. A.A. Damasceno1, C.P. Carvalho1, F.A. Araujo1, F.V. Botelho1, R.M. Goes2, D.L. Ribeiro1; 1 Histology, Institute of Biomedical Sciences/ Federal University of Uberlândia- UFU, Uberlândia, Brazil, 2 Biology, São Paulo State University, IBILCE/UNESP, São José Do Rio Preto, Brazil This study presents a comprehensive view of histological and functional status in the prostate of adult rat offspring of mothers subjected to gestational diabetes. Diabetes was induced in pregnant female Wistar rats by alloxan (i.p. 100 mg/kg b.w.). The ventral prostate of male adult offspring of diabetic (DP) or normal mothers (CP) was evaluated for collagen fibres, cell death (TUNEL), immunohistochemistry (fibroblasts, smooth muscle cells, cell proliferation), western blotting (PCNA, metalloproteinases, androgen receptor-AR), TGFβ-1 (ELISA), catalase and total antioxidant activity. The prostates of DP animals showed lower weight compared to the CP group and these animals also exhibited hyperglycaemia and hypotestosteronaemia. Cell proliferation and AR were higher in the diabetic offspring. The DP group also showed a reduction in α-actin, which may interfere with the reproductive function of the prostate. They also had enhanced activity of MMP-2, although the absolute content of this MMP was lower in this group. This finding was associated with an increase in TGFβ-1 and a decrease in collagen distribution. The prostates of DP rats also exhibited reductions in catalase and total antioxidant activity. These data indicate that rats developing in a diabetic intrauterine environment have glycemic and hormonal changes that impact on the structure and physiology of prostate in adulthood. They exhibit increased AR expression, which may be responsible for the elevated cell proliferation. There was also stromal remodelling characterized by enhanced activity of MMP-2 and collagen degradation, even with increased TGF β-1 activation. These changes associated with increased oxidative stress may interfere with tissue architecture and glandular homeostasis, favouring the development of proliferative disorders in the prostate.

TUESDAY-POSTER PRESENTATIONS

P2302 Board Number: B933

Analysis of the different functional domains relevant for chicken Scratch2 function. T.Y. Nakamura Kanno1, M. Soares Fogo1, F. Monteleone Vieceli1, C. Yan1; 1 Department of Cell and Developmental Biology, Universidade de São Paulo, São Paulo, Brazil cScratch2 (cScrt2) is an evolutionarily conserved transcription factor that belongs to the Snail/Slug superfamily. This superfamily is involved in different developmental processes including neural differentiation, cell division, cell survival and left–right identity. As a norm, members of this family act through transcriptional repression. The repressor activity of these transcription factors depends both on the amino-terminal SNAG and carboxi-terminal Zinc-Finger domains. cScrt2 is expressed in the chick embryonic neural tube. Specifically, it is found in early postmitotic neural progenitors. Consistent with its cellular function, MYC-cScrt2 localizes to the nucleus of chick embryonic neural progenitors and HEK293T cells. An in silico search for subcellular localization regulatory sites revealed that residues tyrosine77 (Y77) and serine78 (S78) are possible targets for tyrosine or serine/threonine kinases. The sequence containing these residues is completely conserved between the homologues of cScrt2 in chicken, human and mice. Overexpression of cScrt2Y77F, cScrt2Y77E, cScrt2S78A and cScrt2S78D in HEK293T and chicken embryos remain in the nucleus as observed by immunofluorescence and Western Blot. The truncated protein with only the C-terminal domain is also restricted to the nucleus. Thus, it is possible that the nuclear localization of cScrt2 is mainly controlled by the Zinc-Finger domain. We thus investigated if phosphorylation of Y77 and S78 is relevant for repressor activity. All of the different Y77 and S78 point mutants decreased repressor ability as seen by luciferase assays. Given that we observe alterations in cScrt2 repressor activity independently of the charge value of the residue that substituted the endogenous Y77 or S78. We propose that all of the above mutations alter protein conformation and, as a consequence, the ability to recruit the nuclear partners involved in the cScrt2mediated transcriptional regulation. In other words, our results suggest that Y77 and S78 are relevant for the correct conformation of interaction domains involved in recruiting nuclear repressor machinery partners while the subcellular localization of cScrt2 could be controlled by the DNA-binding Zinc-Finger domain.

TUESDAY-POSTER PRESENTATIONS

P2303 Board Number: B934

8-Nitro-guanosine 3’,5’-cyclic monophosphate mediates elongation of the growth plate cartilage in mice. M. Hoshino1,2, Y. Miyamoto1, K. Yoshimura1, D. Suzuki1, T. Akaike3, K. Mishima4, K. Baba2, R. Kamijo1; 1 Department of Biochemistry, Showa University School of Dentistry, Tokyo, Japan, 2Department of Prosthodontics, Showa University School of Dentistry, Tokyo, Japan, 3Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Miyagi, United States, 4Division of Pathology, Department of Oral Diagnostic Sciences, Showa University School of Dentistry, Tokyo, Japan The majority of our bones develop through endochondral ossification, except for the collarbones and flat bones such as such as the skull bones and the jawbones that are formed through membranous ossification. Endochondral bone formation is regulated by growth and differentiation of chondrocytes in growth plates. It is known that nitric oxide (NO) plays important roles in controlled elongation of growth plates and hypertrophic differentiation of chondrocytes. It is also reported that C-type natriuretic peptide (CNP) stimulates proliferation and hypertrophic differentiation of chondrocytes through elevation of intracellular level of guanosine 3’,5’-monophosphate (cGMP). Recently, 8-nitro-cGMP was identified in various types of cells including macrophages, glia cells, and cardiomyocytes as a novel signaling molecule. Therefore, we hypothesized that 8-nitro-cGMP may function as a mediator of proliferation and/or differentiation of chondrocytes in the growth plates. In this study, we investigated the formation and function of 8-nitro-cGMP in the growth plate cartilage in mice. Tibias excised from mice on embryonic day 16 were cultured for 4 days in the presence of CNP and either an NO synthase inhibitor, L-NAME, or its control D-NAME, the optical isomer of L-NAME. Tibias grew 1087 ± 188.2 m in the direction of the long axis in 4-day culture with CNP and D-NAME, whereas they grew only 766.2 ± 181.0 m in the culture with CNP and L-NAME. Histochemical analyses revealed that L-NAME caused suppressed elongation of the growth plate cartilage induced by CNP, indicating NO was involved in the proliferation of chondrocytes in the growth plates. Formation of 8-nitro-cGMP was detected immunohistochemically in the growth plates after cultivation with CNP, while that was inhibited by LNAME. In addition, chemically synthesized 8-nitro-cGMP induced the ex-vivo growth of tibias in a concentration-dependent manner. Hence we examined the effect of chemically synthesized 8-nitrocGMP on the proliferation and differentiation of mouse primary chondrocytes in vitro. 8-Nitro-cGMP induced the proliferation of chondrocytes in a concentration-dependent manner, while it did not have little effect on the expression of marker genes of hypertrophic differentiation. Collectively, it is suggested that 8-nitro-cGMP is a novel signaling molecule that facilitates proliferation of chondrocytes and elongation of growth plate cartilage.

TUESDAY-POSTER PRESENTATIONS

P2304 Board Number: B935

The Role of Twist1 in endothelial cell sprouting in the lung. T. Mammoto1, A. Mammoto2; 1 Vascular Biology Program, Boston Children's Hospital/Harvard Medical School, Boston, MA, 2Boston Children's Hosp/Harvard Med Sch, Boston, MA Recent significant advances in stem cell research and bioengineering techniques made a great progress in utilizing biomaterials to regenerate or repair damages in simple tissues in the orthopedic and periodontal fields. However, attempts to bioengineer the structures and functions of more complex three-dimensional organs such as lungs have not been very successful. Angiogenesis –the formation of new blood capillaries- plays an important role in organ development, homeostasis, and regeneration and deregulated angiogenesis contributes to various disease states. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components, but also provide instructive signals to surrounding cells in tissues. Thus, in order to engineer functional organs such as lungs, we need to understand the mechanisms that govern angiogenesis. The transcription factor, Twist1, controls mouse embryonic development and the epithelial-mesenchymal transition that occurs in lung fibrosis and lung cancer, diseases in which angiogenesis is deregulated. Here we demonstrate that Twist1 controls the expression of angiogenic factor receptor, Tie2 and endothelial cell sprouting in lung human microvascular endothelial (L-HMVE) cells. Furthermore, we recently developed a unique method to implant fibrin gel on the mouse lung, which enables the precise analysis of the biological process of host lung-derived angiogenesis and interaction between the newly formed blood vessels and other cellular (e.g., alveolar cells, immune cells) and non-cellular components in mice. When the fibrin gel supplemented with angiogenic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) (0, 10 and 100 ng/ml each) is implanted on the surface of living mouse lung, hostderived CD31-positive vascular networks are constructed inside the gels 7 days after implantation in a VEGF/bFGF dose-dependent way. Importantly, angiogenesis is inhibited in fibrin gels implanted on lungs of Tie2-specific Twist1 conditional knockout mice. These results suggest that Twist1 controls angiogenesis in vitro and in the mouse lung in vivo and that Twist1 may be one of the key molecules to control lung organ morphogenesis.

TUESDAY-POSTER PRESENTATIONS

P2305 Board Number: B936

Prenatal exposure to estrogen disrupts development of male and female prostate in Mongolian gerbil. B.C. Zani1, J.S. Maldarine2, J.A. Vieira2, M.M. Jesus3, F.A. Santos4, M.F. Biancardi4, B.D. Sanches3, S.R. Taboga5; 1 Biology, UNESP, São José Do Rio Preto, Brazil, 2Biology, UNESP, S J do Rio Preto, Brazil, 3Biology, UNICAMP, Campinas, Brazil, 4Biological Sciences, Federal University of Goias, Goiania, Brazil, 5Biology, São Paulo State University, IBILCE/UNESP, S J do Rio Preto, Brazil Prenatal estrogenization disrupts development of prostate gland in male rodents, causing alterations in the gland growth, as well, deep changes in urethral structure. Despite much is known about the effects in male prostate, there is little research on intrauterine estrogenization in the female prostate and the differences between genders. The aim of this study was to evaluate the effects of prenatal estrogenization on the ventral prostate of Mongolian gerbils (Meriones unguiculatus), this species is relevant for this type of research, since the presence of functional prostate in female is almost ubiquitous. Developing prostate complexes were collected from animals at 20, 22, 24 days of postnatal life exposed to high dosages of estrogen (500 mg/kg BW/day) during perinatal life, fixed and processed for paraffin embedding and cut in series. The slides were stained with HE and immunohistochemistry essays, as well three-dimensional reconstructions were performed. Results suggest gender differences: females were more susceptible to developmental disruptions, as a decrease in the number of prostatic ducts and a increase in their thickness. Acknowledgements: FAPESP, CNPq and CAPES for financial support and scholarships.

P2306 Board Number: B937

Evaluation of Intrauterine oestrogenic exposure on female prostate development in Mongolian Gerbil. B.D. Sanches1, J.S. Maldarine2, M.F. Biancardi3, B.C. Zani4, F.A. Santos3, M.M. Jesus1, J.A. Vieira2, S.R. Taboga5; 1 Biology, UNICAMP, Campinas, Brazil, 2Biology, UNESP, S J do Rio Preto, Brazil, 3Biological Sciences, Federal University of Goias, Goiania, Brazil, 4Biology, UNESP, São José Do Rio Preto, Brazil, 5Biology, São Paulo State University, IBILCE/UNESP, S J do Rio Preto, Brazil Much is known about the effects of prenatal exposure to estrogen in the prostate gland in male rodents, however, there is little research on this process in the female prostate and the differences between genders. The aim of this study was to evaluate the effects of prenatal estrogenization on the ventral prostate of Mongolian gerbils (Meriones unguiculatus), this species is promising for this type of research, since the presence of functional prostate in female is almost ubiquitous. Developing prostate

TUESDAY-POSTER PRESENTATIONS complexes were collected from animals at 7, 14, 30 days of postnatal life exposed to high dosages of estrogen (500 mg/kg BW/day) during the prenatal life , fixed and processed for paraffin embedding and cut in series. The slides were submitted to HE staining technique and immunohistochemistry essays, as well three-dimensional reconstructions were performed. Results suggest gender differences: females were more susceptible to alterations in the urethra and prostate gland in early postnatal life, showing profound histologic changes in urethra and decreased cell proliferation in the prostate. Acknowledgements: FAPESP, CNPq and CAPES for financial support and scholarships.

P2307 Board Number: B938

A CLEM approach to studying the membrane repair process in zebrafish myofibers in vivo. Effect of high pressure freezing on fluorescence emission by fluorescent proteins. C. Antony1, V. Middel2, T. Beil3, U. Straehle4; 1 IGBMC, CNRS, Strasbourg, France, 2ITG, Karlsruhe Institute of Technology , Karlsruhe, Germany, 3ITG, KIT, karlsruhe, Germany, 4ITG, KIT, Karlsruhe, Germany In order to study the process of membrane repair in myofibers in zebrafish embryos after sarcolemma injury, we designed a workflow to track the fluorescent positive myofibers (labelled with proteins of interest involved in the repair process) by LM so as to be able to eventually find them in the EM scope for high resolution investigation. For this purpose which involves the vitrification of 3-days-old embryos by HPFreezing (with a HPF device, Compact 02, Wohlwend, CH) we first tested that the fluorescence signal was preserved right after HPFreezing of our samples. We discovered that some fluorescent proteins stand the effect of the high pressure (2100 bar) and the freezing to liquid nitrogen temperature, as seen with samples visualized under liquid nitrogen with a macroscope (UV illumination), while some others do not and, as a consequence, loose their ability to emit fluorescence. Later on, after freeze substitution (FS) and plastic embedding in HM20 Lowicryl resin, the fluorescence was preserved within 250-300 nm thick sections in cases where the fluorescence was intact after the HPFreezing step, while it was extinguished in case where if was no longer detected after the vitrification step. This shows that fluorescent proteins (FPs) display a differential sensitivity to the vitrification process, probably depending on the molecular structure of FPs and/or their environment. We currently investigate which FPs are optimally suitable for CLEM studies.

TUESDAY-POSTER PRESENTATIONS

P2308 Board Number: B939

Cell Rearrangement and Rosette Formation During Epithelial Morphogenesis of the Xenopus laevis Neural Plate. D.S. Vijayraghavan1, L.A. Davidson1,2; 1 Bioengineering, University of Pittsburgh, Pittsburgh, PA, 2Developmental Biology, and Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA During neurulation cells within the planar neural plate must coordinate their behaviors in order to deform into an elongate tubular structure. Neural tube formation is comprised of several steps including cell shape patterning of the early neural plate, neural fold formation at the lateral edge of the plate, bending at and fold convergence towards the dorsal midline, and finally fold fusion. Much of the focus of neural tube mechanics have been on the bending of the neural plate and formation of hinge points both at the midline and mediolaterally at the folds. Researchers have identified coordinated apical constriction of epithelial cells are crucial to these bending processes. However, even before bending occurs, the neural plate, as well as the whole embryo, undergoes a large convergence mediolaterally and extension in the anterior posterior direction. Convergence and extension (CE) of the neuroepithelium is thought to be driven in part by cell rearrangements through planar polarized actomyosin contractility. We have undertaken a stage-by-stage analysis of cell shape changes and local geometric patterning within the dorsal apical surface of fixed Xenopus laevis embryos. Cells within neural plate undergo directed cell rearrangement and form large multi-cellular rosettes where 5 or more cells share a single vertex. Directed formation and resolution of rosettes have been implicated as a driver of convergence and extension in Drosophila germ band epithelial morphogenesis and has been observed in later stage chick neural tube formation. Preliminary analysis of live imaging confocal data of labeled actin in the neural plate demonstrates cells frequently change neighbor both through rosette formation and more general T1 transitions. Rosettes resolution typically occurs within 1 hour after formation. High magnification time-lapses reveal a dense and highly contractile actin cortex within the neural epithelium. These observations support the notion that actively rearranging cells coincide with convergence and extension. However, it remains to be seen whether rearrangements provide the motive force driving CE or whether rearrangements merely serve to dissipate stresses acting within the epithelium.

TUESDAY-POSTER PRESENTATIONS

P2309 Board Number: B940

α integrin cytoplasmic tails can rescue the loss of Rho-family GTPase signaling in the C. elegans somatic gonad. C. Meighan1, V. Kelly1, E. Krahe1, A. Gaeta1; 1 Christopher Newport Univ, Newport News, VA Integrin signaling impacts a diverse set of cell behaviors through multiple, distinct pathways. The Rho family of GTPases are integrin signaling partners that regulate cell polarity, actin dynamics, and cell migration. Though the connection between integrins and the Rho family has been studied extensively, there is still much that is unknown about the complex interplay between these protein families. To further resolve this issue, we used the nematode C. elegans as a simple model for integrin and Rho family function. C. elegans has two α integrins, ina-1 and pat-2, that pair with β integrin pat-3. Our previous work developed chimeric α integrins which link the extracellular domain of α integrin ina-1 to the cytoplasmic tail of α integrin pat-2 and the extracellular domain of pat-2 to the cytoplasmic tail of ina-1. These constructs showed the α integrin cytoplasmic tails have tissue specific functions during development. Here, we present evidence showing the cytoplasmic tails of α integrins ina-1 and pat-2 have a cell type dependent ability to bypass signaling from members of the Rho family of GTPases.

P2310 Board Number: B941

Role of shootin1 during embryonic development in zebrafish. A. Urasaki1, T. Matsui1, K. Kawakami2, Y. Bessho1, N. Inagaki3; 1 Bioscience, Nare Institute of Science and Technology, Nara, Japan, 2National Institute of Genetics, Mishima, Japan, 3Grad. Sch. Bio. Sci., Nara Institute of Science and Technology, Ikoma, Japan Axon outgrowth is essential for the formation of neuronal networks. Shootin1 was identified as a protein involved in the polarization of primary cultured hippocampal neurons. Shootin1 functions as a linker “clutch” molecule that couples treadmilling F-actin and the adhesive substrate at the growth cones to promote axon outgrowth. However, little is known about the in vivo functions of shootin1. Here we identified three shootin family members ( shootin1, shootin2 and shootin3 ) in the zebrafish genome and analyzed the functions of shootin1 in zebrafish. Zebrafish is widely used to study vertebrate development. Their external fertilization, rapid development and optical clarity make them well-suited for studying important cellular processes during embryonc development such as cell proliferation, migration and rearrangement. To see whether shootin1 is expressed in zebrafish embryos, we performed whole-mount in situ hybridization. shootin1 was expressed in the ventral forebrain and eyes. Next, to understand whether shootin1 is required for forebrain and eye formation, we performed knockdown experiments. The shootin1 knockdown embryos exhibited defects in the forebrain and eyes. Our data suggest that shootin1 is involved in the forebrain and eye formation. Furthermore, shootin1 was also expressed in the posterior lateral line primordia, thereby suggesting that shootin1 may be

TUESDAY-POSTER PRESENTATIONS involved in collective cell migration of these cells. To confirm these possibilities, we are currently generating the shootin1 mutant by using the CRISPR system in zebrafish.

P2311 Board Number: B942

RNA-binding proteins regulate dendrite morphogenesis in C. elegans. S. Antonacci1, D. Forand2, E.C. Olesnicky2, D.J. Killian1; 1 Molecular Biology, Colorado College, Colorado Springs, CO, 2Biology, University of Colorado Colorado Springs, Colorado Springs, CO Neurons are polarized cells with complex morphology that enables processing and transmission of information to and from the neural network and surrounding environment. The growth and maintenance of neuronal processes such as dendrites are key to establishing sensory fields and the synaptic connections that control cognition, behavior, and adaptive responses to the environment. A growing body of evidence highlights mRNA transport and local translational control as key processes in generating dendritic branches, maintaining cell structure, and promoting synaptic plasticity. Therefore it is important to investigate the role of RNA-binding proteins (RBPs), which are involved in mRNA processing, transport, localization, stability, and translational control, in the regulation of dendrite form and function. To determine the extent to which RNA-binding proteins influence dendrite morphology, Olesnicky et al. (2014) conducted a genetic screen of all RBP-encoding genes in the Drosophila genome and found 63 RBPs required for normal dendrite morphology in dendritic arborization (da) sensory neurons. An in silico analysis showed a high sequence-similarity among diverse species for the RBPs that affect da dendrite structure in Drosophila, including 54 homologous genes in C. elegans. This suggests that conserved RBPs may regulate dendrite morphology in diverse animal species. To test this hypothesis we screened these 54 RBP-encoding genes in C. elegans for dendrite defects using the multidendritic PVD sensory neuron as a model. Using a combination of mutant alleles and RNA interference, our screen identified 12 conserved RBPencoding genes that produce a reduction in the number of dendritic termini upon loss or reduction of gene function. To determine if the reduction of dendritic termini is caused by a failure to form branches, a delay in formation, or a loss of initially established branches, we performed a time-course analysis. Our results suggest that most RBPs are required for dendritic branch formation but two are required for branch maintenance and one influences the timing of branch formation. Reporter transgenes confirm that candidate genes are actively transcribed in the PVD neuron and RBP::GFP fusion proteins were used to determine the subcellular localization of each RBP. Subcellular localization and gene ontology information suggest that nuclear RBPs are involved in mRNA processing such as splicing and that cytoplasmic RBPs are likely involved in mRNA transport and translational regulation. Taken together, our results highlight a fundamental role for RBPs in dendrite morphogenesis, and suggest that these

TUESDAY-POSTER PRESENTATIONS evolutionarily conserved RBPs participate in molecular mechanisms that contribute to neurite development and maintenance in diverse animal species

P2312 Board Number: B943

Role of BMP signaling in adult tissue homeostasis. P. Nigam1, P. Parashar1, A. Nag1, A. Bandyopadhyaya1; 1 Biological Sciences Bioengineering, Indian Institute of Technology, Kanpur, India Background &Aim: Bone morphogenetic protein (BMP) signaling, in co-ordination with other signaling pathways control key events in organogenesis. Though BMP signaling pathway has primarily been investigated for its role during embryonic development, the identification of Bmpr1a and Smad4 mutations in patients with Juvenile Polyposis Syndrome (JPS), suggests that BMP signaling may be an important regulator of adult tissue homeostasis as well. Here we report our initial observation following depleting Bmp2 and Bmp4 proteins specifically in adult mice which suggests that BMP signaling is required for adult tissue homeostasis. Methods: We have created a conditional Bmp2/4 double knockout mouse mutant (genotype - Bmp2fl/fl; Bmp4fl/fl;ROSA26::CreER-T2). Eight weeks old control and test mice were administered tamoxifen for seven consecutive days. Results: Fifteen days after initiating tamoxifen injection we observed signs of mild diarrhea in these mutant mice. Molecular and histological analyses of BMP signaling depleted intestine suggests that BMP loss triggers activation of Wnt signaling in intestinal stem cell niche (crypt) resulting in remarkable increase in cell proliferation. The resultant crypt-villus morphology (ectopic, misplaced & elongated crypts with short villi), following Bmp depletion, resembles conditions similar to JPS. Loss of BMP signaling also resulted in improper nuclear positioning which is suggestive of loss of the baso-apical enterocyte polarity. Bmp ablation also causes detachment of enterocytes from the basal lamina of the villi. In our study, Bmp depletion is also associated with impaired terminal differentiation of cells from secretory lineage. This perturbance of the normal process of tissue homeostasis causes apoptosis of the cells. Interestingly, these mutant mice also exhibited hair fall following Bmp depletion. The molecular changes observed in mutant follicles is reminiscent of the changes observed in the intestine. Taken together, our results suggest that BMP signaling is an important regulator of epithelial tissue homeostasis in adult animals. Conclusion: We strongly suggests that BMP signaling have a major regulatory role in the homeostasis of an adult epithelial tissue and a better understanding of all of this will help to utilize novel therapeutic strategies to treat conditions like intestinal polyps and cancers.

TUESDAY-POSTER PRESENTATIONS

P2313 Board Number: B944

Discovery of CEP1410, a small molecule inducer that promote proliferation of pancreatic beta cells using novel micro-3D histo-culture assay procedure. S. Satish1, C.R. Sharma1,2, J.M. Harris1, R.R. Rodriguez1, M.R. Sharma1, M.B. Navel1, N. Amezcua1, J.R. Martinez1, J. Jani1, J.P. Sharma1; 1 Stem Cell Biology, Celprogen Inc, Torrance, CA, 2Celprogen Inc., Torrance, CA Identification of novel small molecule inducers that promote proliferation of pancreatic beta-cell is an important approach to treat diabetes. This unique approach represents a potential regeneration strategy for the treatment of type 1 diabetes. Unfortunately, the lack of availability of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted and successfully developed the human islets micro-3D histo-cultures for high-throughput cell based assays. This micro-3D histo-culture system of culturing human islet for high-throughput screening utilizing a thymidine analog, EdU, to detect beta-cell replication during screening. This culture system allows simultaneous monitoring of Ki67, EdU incorporation and beta cell numbers provides robust assay for beta-cell replication. Importantly, this micro-3D histo-culture system preserved the beta-cell physiological function, as measured by glucose-stimulated insulin secretion. We then performed a pilot screen of 384 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds. From this screen we were able to characterize 10 lead compounds CEP1410,1412,1413,1414,1415,1416,1417,1418,1419 and 1420 that were able to increase beta cells mass and responded to glucose –stimulated insulin secretion. CEP1410, our lead compound was tested in-vivo. In diabetic rat model, once a day oral administration of CEP1410 increased the mass of Beta cells. . The gene expression profile of pancreatic Beta-cells revealed high gene expression of Insulin, glucagon, somatostatin, Glut-2 and Isl-1 on Day 20 when study was terminated.

Germ Cells, Gameogenesis, and Fertilization P2314 Board Number: B945

ISOLATION,CHARACTERIZATION, AND GENE EXPRESSION PROFILE OF RAT EPIDIDYMAL BASAL CELLS. M. Mandon1, L. Hermo2, D.G. Cyr1; 1 INRS-Institut Armand-Frappier, Laval, QC, 2McGill University, Montreal, QC Epididymal basal cells are localized to the basal compartment of the epithelium. These cells have thin processes that extend along the base of the epithelium and that can also extend apically and reach the lumen of the epididymis. There is little information on the function and role of epididymal basal cells.

TUESDAY-POSTER PRESENTATIONS Various studies have suggested that these may be stem cells, be part of the immune system, regulate other epithelial cells via the production of hormones and secondary messengers, and may be regulators of the tight junctions of the blood-epididymis barrier. The objective of this study was to isolate a relatively pure fraction of epididymal basal cells and characterize their function. Immunolocalization studies indicated that integrin-α6 was specifically localized to basal cells in all regions of the rat epididymis. Epididymides from 42 day old rat, an age when there are no sperm in the epididymis, were enzymatically digested and cells isolated. Using a magnetic microbeads and integrin-α6 antibody, basal cells were isolated. Immunofluorescence using a specific marker of basal cells, cytokeratin 5 (KRT5), revealed a 90% enrichment of basal cells. Flow cytometry performed with another marker of basal cells, cyclooxygenase 1 (Cox1), confirmed this result. Electron microscopy of isolated cells also revealed morphological characteristics of basal cells. Total cellular RNA was isolated from the enriched basal cell fracture and subjected to RT-PCR using specific markers of basal cells, principle cells, dendritic cells, and clear cells. Microarray analysis indicated the basal cell fraction contained 1359 differentially expressed genes relative to other epididymal epithelial cells. Among the differentially expressed genes, numerous integrins and proto-oncogenes were expressed in the epididymis. Furthermore, the tight junction protein, claudin1, highly expressed as were connexin 43, 31.1, 30.3 and P-cadherin. Pathways analysis predicted the expression of several intracellular signaling pathways including those implicated in EGFR signaling, integrin signaling, molecular mechanisms of cancer, inflammatory response and canonical WNT signaling. These studies provide novel information on function of the epididymal basal cells and their role in the regulation of the blood-epididymis barrier. Supported by CIHR and FQRNT.

P2315 Board Number: B946

Polyglucosan Molecules Induce Testis Degeneration And Apoptosis In Germ Cells Without Affecting The Integrity And Functionality Of Sertoli Cells. F. Villarroel-Espíndola1, I.I. Concha1, J. Slebe2, J.J. Guinovart3,4; 1 Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile, 2Bioquimica y Microbiologia, Universidad Austral De Chile, Valdivia, Chile, 3Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain, 4University of Barcelona, Barcelona, Spain Glycogen is the main storage form of glucose for most tissues; however, the accumulation of aberrant polyglucosan molecules can lead to degeneration and death in some cell types. Previously, we reported that the accumulation of glycogen in testis of transgenic animals overexpressing a constitutively active form of glycogen synthase (KIN-GS) enhances the apoptosis of premeiotic cells in seminiferous tubules. Similarly, the activation of endogenous glycogen synthase (GS) in a germ cell line (GC-1) stimulates the deposition of glycogen and triggers the activation of caspase 3. Here we sought to further identify the effects of glycogen storage in GC-1 and Sertoli cells (42GPA9 cell line) and the mechanism behind the pro-apoptotic activity induced. By spectrophotometric analysis, we found that glycogen synthesized in both cell lines—by expression of a superactive form of GS or by activation of endogenous GS by PTG (Protein Targeting to Glycogen) expression—is poorly branched. In addition, the immunodetection of

TUESDAY-POSTER PRESENTATIONS cleaved caspase 3/9 suggests that cellular death induced by polyglucosan molecules affects GC-1 but not 42GPA9 cells. The former cells showed changes in intracellular ATP and cytochrome C content after polyglucosan accumulation, thereby suggesting mitochondrial impairment and activation of an intrinsic apoptotic pathway. Furthermore, we analyzed the effects of glycogen deposition during the establishment of an in vitro blood-testis barrier. The results using a non-permeable fluorescent molecule (Evans blue) showed that, in conditions of over-synthesis of glycogen, 42GPA9 cells do not lose their capacity to generate an impermeable barrier. In the same cell line, immunodetection showed that the levels of connexin43 (Cnx43), occludin (Occl), and ZO1 proteins were not affected by glycogen accumulation. Similarly, in the KIN-GS mice, the distribution and intensity of signals for Cnx43, Occl, and ZO1 point to a Sertoli cell-only syndrome in this model, affecting the viability of male germ cells but not the viability or stability of Sertoli cells, as shown by confocal microscopy analysis. These results confirm that the accumulation of polyglucosan molecules has a selective effect—triggered by the intrinsic activation of the apoptotic pathway—in germ cells. Supported by grants: FONDECYT 3130449 (FVE), 1110508 (ICC) and 1141033 (JCS)

P2316 Board Number: B947

UNDERSTANDING THE REGULATION OF CONNEXIN 26 IN THE EPIDIDYMIS. C. Adam1, D.G. Cyr1; 1 INRS-Institut Armand-Frappier, Laval, QC Connexins (Cxs) are proteins that form gap junctions allowing neighboring cells to communicate by the diffusion of ions and small molecules (