ON  THE  COVER   
 Vol. 75   No.2   June 2010
 Technical note 

     Tyrosine-phoshorylation in focal adhesion formation

     Rhodamine (red) shows actin fibers, FITC (green) indicates phosphorylated tyrosine in the focal adhesions, and DAPI (blue) shows nuclei. Focal adhesions are large protein complexes which connect the cytoskeleton of a cell and the extracellular matrix. They transmit mechanical force and regulatory signals. They contain a diverse range of molecules such as adapter proteins, kinases and proteins that physically link the integrin receptor to actin, completing the connection to the cytoskeleton. These proteins associate and disassociate continually as signals are transmitted to other parts of the cells. Their assembly and disassembly are important in cell migration. One of the mechanisms regulating the formation and dynamics of focal adhesions involves tyrosine phosphorylation. Focal adhesions contain several tyrosine-specific kinases (focal adhesion kinase (FAK), and pp60src) and phosphatases, as well as their substrates (paxillin, CrkII, CAS, Src and FAK), whose phosphorylation creates docking sites for phosphotyrosine binding proteins. Phosphorylation of these proteins regulates focal adhesion formation and disassembly, actin stress fiber and cytoskeletal organization, and dynamic alterations in cell shape.
     The A7r5 rat smooth muscle cells were grown on a 35-mm glass-bottom dish at 37°C in a humidified atmosphere of 5% C02. The cells werc fixed in 4% paraformaldehyde in PBS for 20min and washed twice with PBS, then incubated for 5 min in 0.2% Triton X-100 in PBS f'or permeabilization. The phosphorylation of tyrosine was detected with primary monoclonal antibody against phosphotyrosine and secondary FITC-conjugated goat anti-mouse IgG. The filamentous actins were stained with Pharoidinrhodamine, overlying the DAPI nuclear stain.


     (Emiko Hayama and Toshio Nakanishi, Pediatric Cardiology, Tokyo Women's  Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan)

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