Effects of siRNA mediated gene knockdown on the formation of focal adhesions Sabina Winograd-Katz, Sabina Winograd-Katz, Zvi Kam and Benjamin Zvi Kam and Benjamin Geiger Geiger Department of Molecular Cell Biology Department of Molecular Cell Biology The Weizmann Institute of Science The Weizmann Institute of Science Rehovot, 76100, ISRAEL Rehovot, 76100, ISRAEL
Effects of siRNA mediated gene knockdown on the formation of focal adhesions Sabina Winograd-Katz, Zvi Kam and Benjamin Geiger Department of Molecular.
Dec 31, 2015
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Effects of siRNA mediated gene knockdown on the formation of
focal adhesions
Sabina Winograd-Katz, Sabina Winograd-Katz, Zvi Kam and Benjamin Zvi Kam and Benjamin GeigerGeiger
Department of Molecular Cell BiologyDepartment of Molecular Cell BiologyThe Weizmann Institute of Science The Weizmann Institute of Science
Rehovot, 76100, ISRAELRehovot, 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
PLA
QU
EP
LA
QU
E
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
CameraMicroscope
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
Paxillin siRNA Talin siRNA
Control
Control
Parameters analyzed in the screen
Focal adhesion parameters
AmountLengthLocalization (periphery or cell center)Intensity
Cell parameters
AmountPolarization Area
SMARTpools screen results*
* Preliminary results. Validation in progress.
Adhesion related genes
KinasesPhosphatases
Total 316 576 193Hits 85 (26.9%)173 (30%) 20 (10.3%)
Impaired cell viability 20 (6.3%)98 (17%) 74 (38.3%)
Adhesion related genes
KinasesPhosphatases
Low amount 25.9 42.2 45.0High amount 0.0 1.7 0.0
Short 15.3 37.6 45.0Long 0.0 1.2 0.0
Central 0.0 0.0 0.0Peripheral 50.6 19.7 35.0
Low intensity 20.0 11.6 20.0High intensity 20.0 8.7 10.0
Round cells 3.5 0.0 0.0Polarized cells 55.3 28.9 35.0Cell area low 10.6 11.6 10.0
Cell area high 25.9 15.0 15.0
Focal adhesions
Cells
Hits phenotypes distribution*
* Preliminary results. Validation in progress.
Summary
We developed an assay for high throughput screening of the effects of siRNA on cell-matrix adhesion.
We screened 1085 siRNAs targeting phosphatases, kinases and adhesion related genes. We recorded focal adhesion and cell shape parameters for all of them.
Further validation is in progress together with deeper investigation of single interesting hits.
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