The focus of this project is on the contractile apparatus responsible for cytokinesis in animal cells. The goals are to understand how actin and myosin are assembled into the contractile units that form the cleavage furrow, and to determine how these units are attached to the cell membrane. Fluorescent analogues of actin, myosin, talin, and vinculin will be injected into living cells and their incorporation into cleavage furrows analyzed. The kinetics of assembly and disassembly of actin and myosin in the furrow will be measured and specific inhibitors introduced to test the hypothesis that the two proteins are recruited co-temporaneously but independently of one another. Short term incorporation of fluorescent actin and myosin analogues during mitosis will be measured to determine if actin and myosin are incorporated preferentially into the equatorial cortex. Inhibitors of talin and vinculin will be introduced into mitotic cells to test the idea that these proteins play a role in attaching actin filaments to the cleavage furrow membrane. Photoactivation and photobleaching of actin and myosin analogues will be used to determine if there is a cortical flow of actin and myosin from the poles to the equatorial cortex to form the cleavage furrow. The significance of the proposed experiments lies in their potential for describing how actin and myosin are assembled into the contractile apparatus that is essential for one of the most basic of biological processes: cell division.
