Dynamic remodeling of the actin cytoskeleton is essential to cell locomotion, cytokinesis, complex morphogenetic and developmental programs in multicellular organisms, and pathological states such as neoplastic transformation and tumor metastasis. All of these processes involve the site specific assembly and disassembly of filamentous actin networks in response to intracellular and extracellular signals. As the association and dissociation of actin monomers occurs exclusively at filament ends, a number of mechanisms have evolved that govern the efficient generation, utilization and subcellular localization of filament ends. Cofilin severs actin filaments by disrupting the noncovalent interactions between adjacent monomers, resulting in the formation of new filament ends that contribute to both assembly and disassembly. Members of the gelsolin family, also sever filaments, but result in stable caps that sequester filament ends; these caps can be released in response to specific signaling cues. Scaffolding proteins of the Ena/VASP/WASP family participate in the formation of multicomponent assemblies that direct the site specific, de novo nucleation of actin filaments, and link these dynamic events with phosphoinositide and small G- protein signaling pathways. Superimposed upon these mechanisms are the nucleotide-dependent properties of actin itself which are essential elements of cytoskeletal dynamics. High resolution X-ray crystallography will identify residues involved in the formation and regulation of the macromolecular assemblies that control cytoskeletal dynamics. The contributions of these residues will be tested by mutagenesis and biochemical characterization. The in vivo importance of specific biochemical properties will be assessed by the expression of biochemically defined mutants in living cells. These studies will result in in vivo structure-function correlations for the molecules that regulate the dynamics of the actin cytoskeleton. This multi- disciplinary approach will allow us to address: 1) The ATP hydrolytic mechanism of actin and its coupling to actin structure and in vivo function. 2) The severing mechanisms utilized by the cofilin and gelsolin families, in particular the determinants responsible for the recognition of both monomeric and filamentous actin. 3) The domain organization of the Ena/VASP/WASP family and the ligand interactions that control site specific actin filament assembly and the integration of these activities into the overall physiology of the cell.
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