The long range goals of this research program are to (1) establish the structural changes in the actomyosin complex that produce motile force coupled to ATP hydrolysis, (2) characterize the mechanism of organelle movements by myosin-1 and (3) determine the mechanisms that control the accumulation of myosin and its activation in the cleavage furrow during cytokinesis. To reach these goals, we propose the following studies: Project 1, Electron microscopy of intermediate in the actomyosin cycle. We will use a novel rapid-mixing/stopped-flow rapid- freezing device to capture the myosin intermediates that are weakly bound to actin filaments in the actomyosin ATPase cycle. After freeze-fracturing, the structure of these intermediates will be studied directly by electron microscopy to establish the structural changes that produce force and motion. Project 2. Assembly and function of myosin-II. To characterize the molecular events in the assembly and function of myosin filaments in non-muscle cells, we will use a variety of biophysical methods to establish the mechanism of assembly and its regulation by other proteins. This will include the effects of monoclonal antibodies on assembly, ATPase activity and in vitro assays for motility. cDNA's for the molecule will be expressed in bacteria to map precisely the binding sites for more than 40 monoclonal antibodies, to provide antigens for the production of new monoclonal antibodies and to produce myosins modified by deletions or point mutations in vitro for biochemical tests for function. Project 3. Interaction of myosin-1 with membranes. We will carry out quantitative assays to characterize the binding of myosin-1 to the membranes of isolated organelles and test the reconstituted membranes for their ability to move along actin filaments. To establish the membrane binding region of the myosin-1, we will test well defined segments of the protein expressed in bacteria from cloned cDNAs for their ability to bind to membranes. We will also carry out in vitro assays of actin motility with myosin-1 to establish the regulatory functions of 2 classes of heavy chain kinases. Project 4. Regulation of cytokinesis. We will produce monoclonal antibodies to the phosphorylated sites of the myosin light chain and use them to map out the times and places where myosin is phosphorylated during cell division. In parallel experiments we will modify isolated myosin light chains with a fluorescent dye so that after they are microinjected into live cells (where they will recombine with myosin heavy chains) we can follow the movements of the myosin during cell division. By also modifying these fluorescent light chains with thiophosphate on either the protein kinase C site or the myosin light chain kinase site, we will establish whether either or both of these modifications influence the movement of the myosin into the cleavage furrow at cytokinesis.
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