CYTOPLASMIC MYOSIN FUNCTION IN VITRO AND IN VIVO

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Our goals are to examine the biological processes that require myosin function , identify the components and architecture of the supramolecular complex that contributes to force generation for cell shape change and investigate the molecular mechanism by which myosin functions. We focus on the function of nonmuscle (cytoplasmic) myosin in Drosophila for two reasons. First, a diverse array of interesting movements provide the structural basis for spatial differentiation of the embryo and are characteristic of various movements seen during the development of other species throughout phylogeny. They include nuclear divisions and migrations, pole cell formation and cellularization, the complex cellular migrations and shape changes of gastrulation and later stages of embryo, larval and pupal morphogenesis. Second, these movements are amenable to study by a range of powerful approaches. We used classical protein biochemical and immunological methods to purify and characterize cytoplasmic myosin from Drosophila cells in culture, then showed that in developing embryos that myosin is localized in a pattern consistent with its role in cellularization and cell sheet movements. The real advantage of Drosophila is its accessibility to genetic, molecular biological and modem molecular genetic manipulation, so we cloned the genes that encode its 3 polypeptide subunits. We used reverse genetic methods to recover mutations in the heavy chain and have collaborated on a mutation in the regulatory light chain. The work establishes that the myosin is required for cell shape change, both in cell sheet morphogenesis during development and for cytokinesis. The experiments provide the first link between a known chemomechanical force producing protein, or motor, and morphogenesis and further suggests that cell shape changes for cytokinesis and morphogenesis are intimately and mechanistically related. This proposal requests funds to continue a multidisciplinary analysis of the nonmuscle myosin heavy chain encoded by the zipper locus at 6OE9 and a new myosin heavy chain gene at 35B,C. Our emphasis will be on genetic strategies designed to establish which movements that require these myosins for their proper execution and cell biological approaches to ascertaining the molecular mechanism by which myosin is targeted to a discrete domain of the submembraneous actin cortex, how its activity is regulated and how it contributes to cell shape change during development. Together these studies forge a comprehensive investigation of the mechanism of conventional nonmuscle myosin function and its role in cellular movements.