CYTOPLASMIC MYOSIN FUNCTION IN VITRO AND IN VIVO

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Our goal is to determine which cellular movements require myosin for force production and to understand, on the molecular level, the mechanisms of myosin function in vitro and in vivo. Our approach has been to produce and characterize monoclonal, site-specific antibodies to cytoplasmic myosin as probes of myosin function in Acanthamoeba. We will analyze force production by myosin with purified proteins, in cell free model systems and in living cells through microinjection. Acanthamoeba contains two classes of myosin that are morphologically distinct. The role these two myosins play in cell movement is not known. In vitro, we have and will map antibody binding sites by electron microscopy of antibody-myosin complexes and by antibody binding to myosin peptides. We have and will analyze the interaction of antibodies with purified contractile proteins to determine how distinct domains in the myosin molecule contribute to myosin filament formation, actin binding and actin-activated ATPase activity. We have and will apply the antibodies to cell free model systems of contractility. To date we have characterized 23 monoclonal antibodies directed against at least 15 unique sites distributed from the myosin-II head to the tip of its tail. Two antibodies block polymerization, 12 block actomyosin-II ATPase activity and 14 inhibit contraction of gelled extracts of amoeba cytoplasm. Our data shows that regions near the tip of the myosin tail are required for polymerization and that another domain on the myosin tail, close to the myosin head, is essential for ATPase activity and force production. Finally, we have and will probe myosin function in whole cells. Fluorescent antibody staining of fixed cells, electron microscopy of antibody stained frozen thin sections, and the distribution of labeled myosin microinjected into living cells will localize myosin. Most significantly, we will microinject the antibodies into living cells to determine which motilities (locomotion, cytokinesis, etc.) require myosin function. We are encouraged by preliminary microinjection studies on Acanthamoeba, and by our antibody-microinjection studies on myosin function in starfish eggs, which showed that myosin is necessary for cytokinesis but not chromosome movement. By analyzing antibody effects on biochemical function, on contractility in vitro, on the location of myosin in whole cells, and on motility in vivo, we expect to elucidate the molecular mechanism of myosin function in living cells.