Our goals are to understand the mechanisms of cytokinesis and of motility contributed by myosin-I. To reach these goals, we propose to use biochemistry, genetics and microscopy, largely with the fission yeast, Schizosaccharomyces pombe, as our experimental organism. Project 1. Regulation of cytokinesis. We will determine genetic interactions between the two isoforms of fission yeast myosin-Il and other gene products known to participate in cytokinesis. We will use genetic screens to identify new components that regulate the time and position of furrow formation, with the long term goal of tracing the signaling pathway from the mitotic spindle to the cleavage furrow, where the contractile ring of actin filaments and myosin-Il divides the two daughter cells. To understand the mechanisms and functions of the products of these genes, we will purify and characterize the isoforms of myosin-Il and accessory proteins that participate in cytokinesis. We will test hypotheses arising from our biochemical studies and genetic analysis at the cellular level using live cell imaging and photobleaching to study the dynamics of the proteins. To understand the assembly of myosin-Il in more detail, we will complete our work on the crystal structure of the C-terminal assembly domain of the tail of Acanthamoeba myosin-Il. Project 2. Structure and functions of myosin-I in fission yeast. Our previous studies implicated the product of the single S. pombe myosin-I gene in the organization of actin filament networks, so we will study the biochemical interactions of the various domains of myosin-I with the WASp homologue (Wsp1p), verprolin (Vrp1p) and Arp2/3 complex to learn how these proteins control actin filament assembly. We will also seek new genetic interactions of these components with other components of the actin system and study the dynamics of these proteins in live cells. The combination of genetics, biochemistry and microscopy of fission yeast offers great advantages for establishing the molecular pathways controlling actin assembly.
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