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（Myo 6）的分子和功能表征。Myo 6在基于肌动蛋白的分子马达的肌球蛋白超家族的已知成员中是独特的，因为它沿沿着肌动蛋白丝以相反的方向朝向尖端/负端移动。过去的研究已经暗示了Myo 6的几种潜在功能，包括网格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充分证实，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。第一个将是对电机特性进行广泛的表征 天然组织纯化Myo 6。我们的研究表明，天然Myo 6具有与体外表达的Myo 6显著不同的性质。拟议的研究将检查天然Myo 6相对于占空比和钙，磷酸化和负荷调节的运动性。一个主要的焦点将是Myo 6从负端定向马达转换为正端定向马达的分子基础。第二个目标是Myo 6突变小鼠肾脏近端小管上皮细胞（PTE）的细胞和生理缺陷的表型特征。斯内尔的华尔兹。与Myo 6突变的人类一样，这些小鼠由于内耳神经感觉上皮细胞的退化而失聪。虽然这是这些小鼠中唯一明显的功能障碍，但我们的初步研究结果表明，在许多表达Myo 6的组织（包括肾近端小管）中也存在严重的细胞缺陷。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损伤（包括缺血和化学诱导的糖尿病）的反应。第三个目标将集中在PTE细胞系中表达的荧光标记的Myo 6的体内动力学以及细胞功能，包括内吞作用，伤口愈合和肌动蛋白细胞骨架的组织如何受到Myo 6功能的靶向破坏的影响。

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.