Effects of siRNA mediated gene knockdown on the formation of focal adhesions Sabina Winograd-Katz, Zvi Kam and Benjamin Geiger Department of Molecular.

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Effects of siRNA mediated gene knockdown on the formation of focal adhesions Sabina Winograd-Katz, Zvi Kam and Benjamin Geiger Department of Molecular Cell Biology The Weizmann Institute of Science Rehovot, 76100, ISRAEL

Cell adhesion to the extracellular matrix occurs by the assembly of multi-protein complexes that create "contact-points" developing into stress fibers-anchored focal adhesions. Focal adhesions Regulation of cell shape Response to stress Intracellular trafficking Cell proliferationCell migration Cells survival

Focal adhesion are very complex structures that by different pathways link the integrins to the actin cytoskeleton Integrin PLAQUE Geiger et al. (2001)

Aims of the project To reveal the involvement of individual proteins in the regulation of cell adhesion Validation and enrichment of the cell- matrix adhesion network

193 phosphatases 576 kinases 316 adhesion related genes Dharmacon SMARTpools screened

HeLa JW (Cervical cancer cells) Reporter Cell Line Paxillin-YFP Focal adhesions

The Assay Cell plating on fibronectin (384 wells plate) Transfection with siRNAs Fixation Screen in an automatic microscope at high-resolution (Objective 60/0.9) 24h 72h

The Screening System Camera Microscope Automatic Stage Computer Autofocus Inverted fluorescent microscope (high NA air objectives, correction for plate-bottom thickness). Automated stage and focus control (x,y,z). Computer-controlled filters and illumination shutters. Fast laser auto-focus system. High quantum efficiency CCD camera. Environmental control for live cell imaging.

Image Acquisition and Visualization Single image Plate 384 wells Montage of images Single well

Paxillin siRNA Talin siRNA Control

Parameters analyzed in the screen Focal adhesion parameters Amount Length Localization (periphery or cell center) Intensity Cell parameters Amount Polarization Area

SMARTpools screen results * * Preliminary results. Validation in progress.

Hits phenotypes distribution * * Preliminary results. Validation in progress.

Summary 3 We developed an assay for high throughput screening of the effects of siRNA on cell-matrix adhesion. 3 We screened 1085 siRNAs targeting phosphatases, kinases and adhesion related genes. We recorded focal adhesion and cell shape parameters for all of them. 3 Further validation is in progress together with deeper investigation of single interesting hits.