The research proposed in this application is directed to an understanding of the mechanisms of formation and the structure of filamentous networks of actin, which is the most abundant protein in the peripheral cytoplasm of many cell types. Changes in the structure of actin networks in the cell cortex locomotion, secretion, and cell activation, and so are involved in such varied processes as the immune response, wound healing and blood coagulation. Motile cells must posses a mechanism for the recognition of appropriate signals as well as the machinery to enable them to translocate large volumes of cytoplasm. Reversible remodeling of the actin network brought about by actin-associated proteins and metabolites could be involved in both of these features. Four particular aspects of actin polymers and the regulation of their assembly will be studied. 1) These functionally distinct actin-binding proteins, gelsolin, profilin, and ABP/filamin will be isolated from platelets and macrophages and their effects on the kinetics and extent of actin polymerization measured by light scattering or fluorescence assays using a fluorescent label attached to actin. Special attention will be paid to the effects of phosphoinositides on these interactions, since this class of phospholipids has recently been shown to affect some aspects of actin assembly in vitro. 2) The interaction of gelsolin, profilin, and ABP in variously prepared cell extracts will be examined by immunoaffinity adsorption and fluorescence measurements of labeled analogues of these proteins, and compared to their function observed with purified actin. 3) The viscoelastic properties of actin networks composed of filaments of various lengths and with different degrees of cross-linking will be examined to describe the response of these materials to imposed deforming stresses. 4) The structure of actin filament networks, prepared under various conditions, will be examined by quasielastic light scattering (QLS) and electron microscopy. The structural and dynamic information, on a microscopic scale, obtained from such measurements will be compared to the macroscopic viscoelastic properties of these networks. These optical measurements will also be preformed on the cytoplasm of living cells to attempt to observe the changes on actin filament structure predicted from experiments performed in vitro. The information should lead to a better descriptive understanding of the structure and function of actin and its associated regulatory proteins, and will also serve as an experimental test of theories that describe rodlike polymer systems in general.
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