Benny Geiger's Lab

Focal adhesions are highly complex protein networks, consisting of more than 150 different proteins that display more than 700 interactions between them. The formation and fate of focal adhesions are regulated in concert by integrin-mediated interactions with the extracellular matrix, as well as by the activation of signaling events, including phosphorylation and dephosphorylation. In order to reveal the involvement of individual proteins in the regulation of cell adhesion, we designed a high-throughput screening strategy, based on siRNA (small interference RNA) technology, in which we analyze the effects of gene knockdown  on the formation and morphogenesis of focal adhesions. Three siRNA libraries were used for that purpose (Fig. 1), targeting 196 phosphatases, 580 kinases and 318 additional genes, including small GTPases and their regulators, and known structural components of focal adhesions and the actin cytoskeleton (“SMARTpools”, Dharmacon, USA). A reporter cell line was developed consisting of HeLa JW cells expressing YFP (yellow fluorescent protein)-tagged paxillin (Fig. 2). For the screening, the cells were seeded on fibronectin-coated 384-well plates, and transfected with various siRNAs. After 72 hours, the cells were fixed and screened using a microscope-based automatic system equipped with an auto-focusing device, and a high-resolution image acquisition capacity (Fig. 3). The protein function was deduced from the effect on cell viability, morphology and the changes in focal adhesion quantity, size, shape, intensity and organization. The results show several effects, including cell rounding, cell elongation, decrease in focal adhesion number, and changes in their distribution throughout the cell, among others (Fig. 4). Analysis of the screen results and further validation will lead us to the identification of novel proteins and pathways involved in the regulation of cell shape and cell adhesion.

This work was conducted in collaboration with Dr. Shalev Itzkovitz and Prof. Zvi Kam from the Department of Molecular Cell Biology, Weizmann Institute of Science.

Figure 1. The siRNA libraries assayed in the screen. Each siRNA pool consists of four duplexes targeting the same coding sequence.		Figure 2. Preparation of reporter cell line A. HeLa JW cells were infected with a retroviruses expressing Paxillin-YFP fusion protein. B. A stable cell line was developed from a single cell clone that expressed high levels of Paxillin-YFP in focal adhesions. The arrow indicates the fluorescently labeled focal adhesions.
Figure 3. Workflow of the screen.		Figure 4. Examples of images obtained in the screen. Control cells, cells transfected with siRNA targeting paxillin, leading to knockdown of Paxillin-YFP and cells transfected with siRNA targeting Talin, a protein that links integrins to the actin cytoskeleton.