Analysis of Ena/VASP Anti-Capping Activity

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DESCRIPTION (provided by applicant): Cell migration plays an integral role in many processes important for human health, including embryonic development, immunological response and metastasis. Ena/VASP proteins are key regulators of cell migration. Ena/VASP proteins bind to the barbed ends of filamentous actin and induce changes in the geometry of the actin cytoskeleton that include increased actin filament length. The overall goal of this proposal is to determine if Ena/VASP functions as an "anti-capping" protein, causing alteration of the actin network by shielding the barbed ends of actin from capping protein. To test this hypothesis, levels of capping protein activity will be altered in cells to determine if reciprocal changes in actin cytoskeleton geometry can be achieved. In vitro actin polymerization experiments will be performed to directly measure antagonism between Ena/VASP and capping protein. Microscopy will be used to detect binding of Ena/VASP to barbed ends and to determine if the anti-capping mechanism involves exclusion of capping protein from the barbed ends. Finally, Ena/VASP mutants will be used to determine domains necessary for anti-capping function and to test if phosphorylation of Ena/VASP by PKA regulates anti-capping activity.