PROJECT SUMMARY/ABSTRACT Our goal is to determine the molecular mechanisms of myosin-3 motors, unique kinase-myosin hybrids in the myosin superfamily, that are involved in fundamental cellular processes including cargo transport and the organization of the actin cytoskeleton in sensory cells. Despite their critical role in human biology, the molecular mechanisms of myosin-3 motors are not well understood. We propose that different modes of regulation together with different kinase substrates and binding partners determine the molecular mechanisms of myosin-3 motors. An integrated and innovative biochemical, biophysical, cell biological and high-resolution structural approach will be used to (i) determine the enzymatic profile and regulation, (ii) the unique structure (iii) and pathways that control myosin-3 function and regulation in vitro and in cells. Collectively, these studies will reveal novel mechanistic insights into the regulation of myosin-3 motors and have broad implications in the understanding of how different myosins are tuned to organize actin networks in cells.