of the project) The proposed studies will continue the investigation of the role of the actin-based cytoskeleton in renal function and disease. We will continue to delineate the functional properties of the multiple actin-based motors expressed in the kidney with an emphasis on understanding the role of myosins in ischemic injury. Kidney ischemia leads to rapid changes in the actin cytoskeleton and a loss of membrane polarity in the proximal tubule cells. The hypothesis to be tested is that the ATP-dependent mechanochemical activity of one or more of the proximal tubule myosins is essential for maintenance of renal proximal tubule epithelial cell polarity, as well as for recovery of polarity following non-lethal ischemic injury. To understand these roles, we propose to purify and enzymatically characterize two proximal tubule expressed myosins, myosin-VI and myosin-Vlla. These studies will also include identification of myosin tail binding proteins that may link these myosins to the membrane cytoskeleton. We also propose to investigate the locations and cytoskeletal associations of an array of renal myosins during renal injury and recovery. These studies will include both immunolocation and biochemical extraction techniques to assess whether association of myosins with the membrane cytoskeleton is regulated during injury. As the characterization of renal myosins is still incomplete, we propose to identify, using a PCR-based screen, new renal myosins. Specifically, we will look for novel glomerular, distal tubule and collecting duct myosins as well as continue our analysis of proximal tubule myosins. Finally, we propose to assess whether mouse mutants with mutations in myosin-VI (Snell's waltzer) and myosin-VIIa (shaker-1) exhibit kidney dysfunction. These mutant mice will be assayed for sensitivity to renal injury as well as for susceptibility to dietary stress. By isolating and characterizing renal myosins we hope to identify which myosins are involved in the cytoskeletal and membrane rearrangements observed during recovery from ischemic injury.
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