Myosin VI in Intracellular Transport/Localization

描述（由申请人提供）：肌凝蛋白是唯一已知的基于肌动蛋白的马达，许多编码肌凝蛋白的基因已经被确定。很少有肌凝蛋白被深入研究以了解它们在细胞中的功能。一些肌凝蛋白沿着肌动蛋白移动以运输细胞成分或产生张力。肌凝蛋白VI是一种不寻常的肌凝蛋白，因为它的运动方向与大多数其他肌凝蛋白相反——它向肌动蛋白丝的尖端或缓慢生长的末端移动。它也与肌动蛋白紧密结合，并采取非常大的步骤。这些特征表明，它可能与其他家庭成员有非常不同的功能。脊椎动物的肌球蛋白VI突变由于感觉上皮的变性而引起耳聋。该组织的主要缺陷是毛细胞的肌动蛋白基立体纤毛。这些结构对于感知声音很重要。肌球蛋白VI在果蝇体内的突变是致命的，表明它在体内起着独特而重要的作用。在脊椎动物和模式生物（如果蝇和秀丽隐杆线虫）中，肌球蛋白VI已被证明与几种膜相关蛋白结合，并参与许多需要肌动蛋白-膜相互作用的过程。目前尚不清楚肌球蛋白VI的缺失是如何导致观察到的缺陷的。然而，了解肌凝蛋白VI在细胞中的作用机制对于理解和最终修复由肌凝蛋白VI功能丧失引起的缺陷非常重要。我们使用果蝇作为模型系统来研究肌球蛋白VI在体内的作用。我们主要研究的是在精子发生过程中肌凝蛋白的功能，它对于肌动蛋白介导的膜重塑过程（称为精子个体化）非常重要。

DESCRIPTION (provided by applicant): Myosins are the only known actin-based motors and many genes encoding myosins have been identified. Few myosins have been studied in enough depth to understand their functions in cells. Some myosins move along actin to transport cellular components or create tension. Myosin VI is an unusual myosin because in moves in a direction opposite to most other myosins - it moves toward the pointed or slow growing end of an actin filament. It also binds tightly to actin and takes very large steps. The characteristics suggest it may have a very different function than other family members. Myosin VI mutants in vertebrates cause deafness due to degeneration of the sensory epithelia. The primary defect in this tissue is in the actin-based stereocilia if the hair cells. These structures are important for sensing sound. Mutations in myosin VI in Drosophila are lethal, suggesting it plays a unique and important role in vivo. Myosin VI has been shown to bind to several membrane-associated proteins and be involved in a number of processes that require actin-membrane interaction in vertebrates and model organisms such as Drosophila and C. elegans. It is not clear how loss of myosin VI causes the observed defects. However, understanding the mechanism by which myosin VI works in cells is important to understanding and eventually repairing defects caused by myosin VI loss of function. We are using Drosophila as a model system to investigate myosin VI's role in vivo. We are primarily examining myosin function during spermatogenesis, where it is important for an actin-mediated membrane remodeling process called spermatid individualization. We have evidence that myosin VI regulates actin assembly. Actin assembly is important for movement of the actin structure that mediated membrane remodeling, the actin cone. This motility process has some similarities to and differences from other wellstudied motility processes, like motile cell leading edge protrusion. Actin assembly regulation is a previously unexpected role for myosin VI. In this proposal we plan to investigate the mechanism by which myosin VI regulates actin assembly and how this regulation leads to normal actin cone movement. We plan to use biochemical, immunocytochemical, molecular genetic and classical genetic methods to determine which proteins work with myosin VI during individualization. We have proposed a model for how the actin structures involved in individualization move and will test various predictions of this model. In addition, we will examine myosin VI function in another process - secretion of salivary gland glue granules - to determine if its mechanism of action is similar. We also have preliminary evidence that myosin VI associates with tissue-specific light chain related to calmodulin. Light chains of other myosins regulate activity. Some data from assays of myosin VI's enzymatic properties in vitro support the idea that it, too, is regulated through its light chain. We propose to investigate light chain interactions. In the long term, we hope to examine associations with light chains, phosphorylation, motility and other properties of myosin VI to understand their importance in vivo. Myosin VI is likely to be important in many cell types for actin-based processes. Since myosin VI and other elements of the actin cytoskeleton are conserved across multicellular animals, what we learn should be widely applicable.