Biochemical and genetic approaches will be used to study actin assembly in yeast. The principles established from these studies are likely to apply to more complex eukaryotes where actin dynamics underlie many motile processes and where defective cytoskeleton function contributes to conditions such as muscular dystrophy, certain hereditary anemias, and cancer. The following questions will be addressed (1) What is the relationship between actin structure and actin function? The development of atomic models for the actin monomer and filament provide an opportunity to bring our understanding of the function and regulation of the actin cytoskeleton to a new level. Interaction sites on actin and the orientation of the monomers within the filament model will be tested using an existing collection of 36 actin mutations. A systematic mutagenesis of actin residues implicated in nucleotide binding will be performed to identify mutants with altered nucleotide hydrolysis or exchange rates. These mutations will test the importance of nucleotide exchange and hydrolysis in vivo, and will be used to genetically identify factors that regulate these reactions. (2) What are the functions of the actin-binding protein cofilin, and what is the relationship between cofilin structure and cofilin function? A synoptic set of 15 charged-to-alanine mutations will be isolated in the gene encoding the multi-functional actin-binding protein cofilin and the biochemical and physiological effects will be determined. A collaboration aimed at developing an atomic model of yeast cofilin will be initiated. In total, this will be the most comprehensive in vivo and in vitro study of the structure and function of an actin-binding protein performed to date. (3) How are the activities of the actin-binding proteins cofilin and profilin regulated? The regulatory mechanisms that control the dynamic actin reorganizations that underlie such varied processes as blood clotting and phagocytosis have not been elucidated. Since profilin and cofilin are likely to play central roles in regulating actin dynamics, genetic strategies will be employed to identify regulators of these proteins. Genes that, at increased or decreased dosage, result in an inviability that can be suppressed by overproduction of cofilin or profilin will be isolated. Positive or negative regulators of cofilin or profilin might be among the proteins encoded by these genes. The functions of these proteins will be studies genetically.
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