Mechanochemical and functional characterization of myosin VI in kidney.

摘要

Myosin VI (Myo6) is a unique member of the myosin superfamily of actin-based molecular motors that moves toward the minus end of actin filaments, in a direction opposite to all other characterized myosins. Cell culture and in vivo studies have previously implicated Myo6 in clathrin-mediated endocytosis. Myo6 is highly expressed in the intermicrovillar coated pit region of the renal proximal tubular cell brush border. In this thesis, I studied the following aspects of Myo6 at the molecular, cellular, and organ levels: (1) the mechanochemical properties of native Myo6 biochemically isolated from mouse kidney; (2) the functional role of Myo6 in the kidney by examining renal histology and physiology and proximal tubular protein endocytosis in the Myo6 functional null Snell's waltzer (sv) mouse; and (3) the effect of renal ischemia-reperfusion injury on the cellular redistribution of cytoskeletal proteins, including Myo6, in the proximal tubule, using an established mouse model of renal ischemia-reperfusion.; Renal Myo6 exhibited mechanochemical properties that were similar as well as novel compared to those previously reported for baculovirus-expressed Myo6; it was found to be a single-headed monomer, with properties of a relatively high duty ratio motor in both the absence and presence of calcium, and capable of both plus- and minus-end-directed movement on actin filaments with rhodamine actin plus ends.; An endocytic defect in the sv kidney was revealed by observing the uptake of the fluid-phase marker horseradish peroxidase (HRP) by proximal tubule cells in sv mice by light and electron microscopy. Uptake was reduced and delayed compared to wild-type mice at both 5 and 30 minutes after HRP Injection. This is the first direct demonstration of Myo6 involvement in endocytosis in the kidney. Moreover, there was an increase in urinary albumin excretion as assayed by ELISA. Sv kidneys also exhibited pathological changes such as proximal tubular dilation, fibrosis, and signs of cellular proliferation and de-differentiation. Na+ and K + excretions were elevated in sv mice, indicating that Myo6 is also required for efficient salt reabsorption in the proximal tubule.; The cellular re-distribution of cytoskeletal proteins, including Myo6, during ischemia-reperfusion injury was examined using an in vivo mouse model (30 minutes of ischemia followed by 0 to 72 hours of reperfusion). Proteins associated with endocytic machinery but not directly linked to the cytoskeleton (clathrin, megalin) recovered their proper subcellular localizations faster than cytoskeletal proteins (Myo6, Myo7a, villin) during reperfusion, indicating differential response among proteins in the apical microdomains to disruption caused by ischemia.