CYTOCHALASIN AND PROTEINS THAT AFFECT ENDS OF F-ACTIN

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The long term goal of this project is to understand the regulation of the assembly of actin filaments and the attachment of the filaments to other cellular structures for force generation and transmission. In the past several years, the focus of the research has been on the structure and function of tensin, a type of protein implicated in the linkage of actin filaments to membranes and other structures, and in signal transduction. This protein has the capability to bind to the fast- growing ends (barbed ends) of actin filaments in vitro, inhibiting polymerization and depolymerization at those ends. It is the only cytoskeletal protein known to have the SH2 domain found in many proteins involved in signal transduction, and is phosphorylated at tyrosine residues in cells stimulated with growth factor or transformed by oncogenic virus. Immunofluorescence experiments showed that this type of protein is present in many different types of locations where the ends of actin filaments are anchored to the cell membrane or to other structures (e.g., z-lines of muscle, dense plaques of smooth muscle, intercalated discs of cardiac muscle, adhesion plaques of fibroblasts, zonula adherens of epithelial cells etc.). The proposed project is divided into the following components: (a) The study of the structure and function of sub-molecular domains of tensin involved in interaction with actin, phospholipids, signal transduction proteins, protein kinases, cytoskeletal proteins, etc., and to determine the possible interplay among these domains. (b) Characterization and comparison of the structure and function of different forms of muscle and nonmuscle tensin, including molecular cloning and immunolocalization of an abundant form in smooth muscle with no apparent actin binding activity. (c) Identification and characterization of tensin-binding components other than actin with the use of binding assays, cross-linking reactions, and selective elution followed by incorporation of labelled tensin into isolated adhesion plaques, z-discs, etc. (d) Study the modifications in tensin structure (e.g., phosphorylation, proteolysis, etc.) and correlate these modifications to changes in cytoskeletal organization in response to growth factor stimulation, neoplastic transformation, drug treatment, etc. The results of the proposed studies in this project will increase our general understanding of diseases relating to muscle tissue and to various types of nonmuscle cells.