Cortical actin rearrangements are responsible for a number of indispensable shape changes in non-muscle cells that result in cell motility, chemotaxis, phagocytosis and cytokinesis. Although actin-associated proteins are known to be necessary for remodeling cortical networks of actin filaments, the regulatory events attendant upon reorganizaion and the specific protein associations required are not yet understood. We have isolated and characterized two actin-associated proteins that are localized to active cell cortices. Severin is a 40kD actin filament disassembly protein that localizes to cortical regions undergoing active rearrangements during phagocytosis, pseudopod formation and filipod extension. 34kD is an actin filament bundling protein that is localized exclusively to filipods and forms unipolar bundles characteristic of filipod cores. By the extensive use of fluorescence energy transfer (FET) techniques developed to quantitate the rate and extent of binding of labeled proteins, we will follow specific interactions of regulatory proteins with actin cytoskeletons to establish mechanisms of bundle formation and disassembly in response to controlled manipulatin of metabolic regulatory factors such as Ca+2, calmodulin and severin. Interactins between severin, actin filaments, 34kD bundle forming protein, calmodulin, 30kD bundling protein, and 95kD actin cross- linking protein durng Ca+2-activated cortical actin rearrangements will be mapped. Since other unidentified regulatory proteins are likely, cytoplasmic and cortical regulatory proteins that mediate capping of bundle ends, disassembly of filipod bundles, and reversible inhibition of severin fragmentatin of actin filaments will be identified and isolated. Moreover, cortical actin disassembly and bundle formation in isolated cell cortices will be direcly followed by FET, using acceptor-labeled fluorescent cortical actin networks together with donor-labeled regulatory proteins.
