Functions for Myosin VI in the kidney proximal tubule

拟议研究的总体目标是继续肌球蛋白VI(Myo6)的分子和功能特征。Myo6在肌球蛋白超家族肌动蛋白分子马达的已知成员中是独一无二的，因为它沿着肌动蛋白细丝朝着尖端/负端相反的方向移动。过去的研究已经暗示了Myo6的几个潜在功能，包括网状蛋白介导的内吞作用、高尔基体运输和细胞运动。拟议研究的健康相关性已经得到很好的证实，因为这种肌球蛋白是导致遗传性耳聋和心脏肌病的突变的靶点。它也被证明是卵巢肿瘤细胞侵袭性的关键因素。这个项目的目标有三个方面。第一个将是对马达特性进行广泛的表征 天然组织纯化的Myo6。我们的研究表明，天然Myo6具有与体外表达的Myo6显著不同的特性。拟议的研究将检查天然Myo6的运动性，涉及占空比以及钙、磷酸化和负荷的调节。一个主要的焦点将是Myo6从负端定向马达转变为正端定向马达的分子基础。第二个目标将是对Myo6突变小鼠的肾脏近端小管上皮(PTE)细胞和生理缺陷的表型特征？斯内尔华尔兹。像带有Myo6基因突变的人类一样，这些小鼠由于内耳神经感觉上皮的退化而耳聋。虽然这是这些小鼠唯一明显的功能障碍，但我们的初步发现表明，在许多表达Myo6的组织中也存在严重的细胞缺陷，包括肾脏近端小管。研究将包括评估 PTE结构缺陷、肾小球滤液的内吞作用以及对肾脏损伤的反应，包括缺血和化学诱导的糖尿病。第三个目标将集中在PTE细胞系中表达的荧光标记的Myo6的体内动力学，以及靶向干扰Myo6功能如何影响细胞功能，包括内吞作用、伤口愈合和肌动蛋白细胞骨架的组织。

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.