Specific inhibition of myosin-Va and - Vb

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DESCRIPTION (provided by applicant): Myosins are essential for intracellular transport, and mutations in the genes encoding them have been shown to cause several inherited diseases. The characterization of the specific cellular functions of unconventional myosins has lagged behind their identification for several reasons. First, there is a great deal of redundancy within families of related myosins. Several groups have identified specific functions by expressing the cargo-binding, or tail, domains of myosins; however, this approach fails to detect any functions dependent upon prior positioning by the motor domain and can yield nonspecific effects from high levels of expression. Second, the study of mutants has been employed. Although genetic ablation approaches are very powerful, null mutants often only illuminate the initial process for which a given myosin is essential during development, precluding the identification of later physiological functions. The ultimate goal of this research is to understand the roles of myosin-Va and -Vb in multiple cellular functions. To this end, a chemical-genetic strategy has been developed that achieves selectivity, specificity, and temporal control by enlarging the ATP-binding site of the myosin using site-directed mutagenesis. This sensitizes the myosin to inhibition by a specific ADP analog while still allowing it to hydrolyze cellular ATP. The acute application of the analog then causes tight binding to actin, arresting the movement of the sensitized mutant myosin. The experiments proposed in this application will test the hypothesis that myosin-Va and myosin-Vb function in initiation, maintenance, antagonism, or termination of microtubule-based motility, using specific inhibition in HeLa, PC 12, MDCK, and B16 cell lines as well as primary cells from transgenic and knock-in mutant mice.