The general objective of the proposed studies is to continue the molecular and functional characterization of myosin-VI (Myo6). Myo6 is unique among known members of the myosin superfamily of actin based molecular motors in that it moves in the opposite direction along the actin filament, toward the pointed/minus end. Past studies have implicated several potential functions for Myo6 including clathrin mediated endocytosis, Golgi traffic and cellular motility. The health relevance of the proposed studies has been well established as this myosin is a target for mutations that lead to inherited deafness and cardiac myopathies. It also has been shown to be a key contributor to the invasiveness of ovarian tumor cells. The goals of this project are three fold. The first will be to conduct an extensive characterization of the motor properties of native tissue purified Myo6. Our studies indicate that native Myo6 has properties significantly different from in-vitro expressed Myo6. Proposed studies will examine the motility of native Myo6 with respect to duty ratio and regulation by calcium, phosphorylation and load. A major focus will be the molecular basis for the conversion of Myo6 from a minus- to a plus- end directed motor. The second goal will be the phenotypic characterization of the cellular and physiologic defects in the proximal tubule epithelial (PTE) cells of the kidney in the Myo6 mutant mouse?Snell's waltzer. Like humans with Myo6 mutations, these mice are deaf due to degeneration of the neurosensory epithleum of the inner ear. Although this is the only overt dysfunction in these mice, our preliminary findings indicate that there are also serious cellular deficiencies in a number of tissues that express Myo6 including the renal proximal tubule. Studies will include assessment of defects in PTE architecture, endocytosis of glomerular filtrate, and responses to renal injuries including ischemia and chemically induced diabetes. The third goal will focus on the in vivo dynamics of fluorescently tagged Myo6 expressed in a PTE cell line and how cellular functions including endocytosis, wound healing and organization of the actin cytoskeleton are affected by targeted disruption of Myo6 function.
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