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Published online
doi:10.1083/jcb.200901105
The Journal of Cell Biology, Vol. 186, No. 3, 423-436
The Rockefeller University Press, 0021-9525 $30.00
© Winograd-Katz et al.
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Article

Multiparametric analysis of focal adhesion formation by RNAi-mediated gene knockdown



Sabina E. Winograd-Katz, Shalev Itzkovitz, Zvi Kam, and Benjamin Geiger

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

Correspondence to Benjamin Geiger: benny.geiger{at}weizmann.ac.il

Cell adhesion to the extracellular matrix is mediated by elaborate networks of multiprotein complexes consisting of adhesion receptors, cytoskeletal components, signaling molecules, and diverse adaptor proteins. To explore how specific molecular pathways function in the assembly of focal adhesions (FAs), we performed a high-throughput, high-resolution, microscopy-based screen. We used small interfering RNAs (siRNAs) to target human kinases, phosphatases, and migration- and adhesion-related genes. Multiparametric image analysis of control and of siRNA-treated cells revealed major correlations between distinct morphological FA features. Clustering analysis identified different gene families whose perturbation induced similar effects, some of which uncoupled the interfeature correlations. Based on these findings, we propose a model for the molecular hierarchy of FA formation, and tested its validity by dynamic analysis of FA formation and turnover. This study provides a comprehensive information resource on the molecular regulation of multiple cell adhesion features, and sheds light on signaling mechanisms regulating the formation of integrin adhesions.

© 2009 Winograd-Katz et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

S. Itzkovitz's present address is Dept. of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot 76100, Israel.

Abbreviations used in this paper: EGFR, EGF receptor; FAs, focal adhesions; FAC, FA component; FACA, FA-associated component; MAR, migration- and adhesion-related; OTP, ON-TARGETplus; RF, Risc-free control; ROCK, Rho kinase.


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