MOLECULAR ANALYSIS OF A MUSCLE ACTIN-CAPPING PROTEIN

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Actin is a ubiquitous protein that is involved in numerous structural and motile processes in a variety of cells. We have postulated that proteins that bind to the ends of actin filaments are important in the formation, orientation and attachment of actin filaments to other structures. To explore this hypothesis, we have isolated a protein from chicken skeletal muscle (Cap Z) that binds with high affinity to the barbed end of actin filaments and is localized to the Z-line. The effect of Cap Z on actin and its location in muscle suggest that Cap Z may be important in the formation and attachment of actin filaments to the Z-line. We have recently identified cDNAs encoding proteins that are homologous to both subunits of Cap Z (two for the alpha subunit, one for the beta) . Other studies have shown that Cap Z is highly homologous to proteins from Dictyostelium, Xenopus laevis and C. elegans that are biochemically distinct from other actin-capping proteins. One of these proteins (from Xenopus laevis) has a nuclear localization. It has also been suggested that Cap Z is related to a protein that binds to the opposite, or pointed, end of actin filaments and that Cap Z itself may have a nuclear localization. The goal of this project is to define the biologic activities of the chicken Cap Z homologues. The expression of Cap Z during embryogenesis will be studied using Northern and Western blots to see if there is differential expression of the homologous subunits. The subunits of Cap Z will be expressed in vitro using bacterial expression systems and their effect on actin and DNA-binding characteristics will be examined in detail. The expressed proteins will be used to generate antibodies that recognize each subunit specifically for use in studying their expression and tissue localization. We will also determine the binding site for actin by generating a series of deletion or insertion mutant proteins and studying proteolytic cleavage fragments of Cap Z. These studies should provide important information about the structure and function of actin-capping proteins that will be of relevance to a number of fundamental processes, including the development of normal skeletal and cardiac muscle, cell motility and the related cellular processes that involve actin.