The proposed experiments are designed to identify the energy transducing enzyme(s) for mitosis and other microtubule-dependent movements through microinjection into living cells of active site specific monoclonal antibodies to various myosins and dyneins, to characterize the molecular interactions responsible for actin filament binding to micro-tubules and to determine the morphological relationships among actin, myosin and micro-tubules in the mitotic spindle and actin and myosin in the contractile ring. We will complete the characterization of monoclonal antibodies to Acanthamoeba myosins-I and II, identify the mechanism by which some interfere with the interaction of purified actin and myosin, use the antibodies to establish that antagonists have been identified for all classes of myosin in the cell and then microinject individual and combinations of these active site specific antibodies into dividing cells to test which myosins (if any) are required for cytokinesis and mitosis. Identical studies will be carried out using human cytoplasmic myosins isolated from platelets to elicit and test monoclonal antibodies that will then be microinjected into dividing HeLa cells. We will complete the characterization of our collection of monoclonal antibodies to sea urchin cytoplasmic dynein in collaboration with Drs. Begg and Pratt (Harvard Medical School) and test them for effects on mitosis by microinjection. We will raise and characterize monoclonal antibodies to Tetrahymena ciliary dynein. They will be used for studies in collaboration with Dr. Ken Johnson (Penn State University) on the mechanism of dynein and to test by microinjection whether protiens with active sites similar to ciliary dynein are required for mitosis. The biochemical studies on actin-microtubule interaction will focus on how the microtubule associated proteins cross-link the two fibers and how this interaction is regulated by phosphorylation and other factors. We will study by electron microscopy the distribution of actin filaments and myosin in dividing HeLa cells using ferritin-labeled monoclonal antibodies to myosin and 2 improved fixation techniques for actin filaments.
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