Myosin VI in Intracellular Transport/Localization

描述(申请人提供)：肌球蛋白是唯一已知的基于肌动蛋白的马达，许多编码肌球蛋白的基因已被鉴定。很少有人对肌球蛋白进行足够深入的研究，以了解它们在细胞中的功能。一些肌球蛋白沿着肌动蛋白移动，以运输细胞成分或产生张力。肌球蛋白VI是一种不同寻常的肌球蛋白，因为它的运动方向与大多数其他肌球蛋白相反--它朝向肌动蛋白细丝的尖端或生长缓慢的一端移动。它还与肌动蛋白紧密结合，并采取非常大的步骤。这些特征表明，它的功能可能与其他家庭成员非常不同。脊椎动物中的肌球蛋白VI突变会由于感觉上皮细胞的退化而导致耳聋。这个组织的主要缺陷是以肌动蛋白为基础的立体纤毛，如果毛细胞.这些结构对于感知声音很重要。果蝇肌球蛋白VI的突变是致命的，这表明它在体内发挥着独特而重要的作用。在脊椎动物和模式生物如果蝇和线虫中，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，了解肌球蛋白VI在细胞中的作用机制对于了解并最终修复肌球蛋白VI功能丧失所造成的缺陷非常重要。我们正在使用果蝇作为一个模型系统来研究肌球蛋白VI在体内的作用。我们主要研究精子发生过程中肌球蛋白的功能，这对肌动蛋白介导的称为精子细胞个体化的膜重塑过程很重要。 我们有证据表明肌球蛋白VI调节肌动蛋白组装。肌动蛋白组装对于肌动蛋白结构的运动非常重要，肌动蛋白结构介导了膜的重塑，即肌动蛋白锥体。这一运动过程与其他已被充分研究的运动过程既有相似之处，也有不同之处，如运动细胞前缘突起。肌球蛋白VI对肌动蛋白组装的调控是此前意想不到的作用。在这项研究中，我们计划研究肌球蛋白VI调控肌动蛋白组装的机制，以及这种调控如何导致正常的肌动蛋白锥体运动。我们计划使用生化、免疫细胞化学、分子遗传学和经典遗传学方法来确定哪些蛋白质在个体化过程中与肌球蛋白VI起作用。我们已经提出了一个涉及个体化的肌动蛋白结构如何运动的模型，并将测试该模型的各种预测。此外，我们将研究肌球蛋白VI在另一个过程中的功能-唾液腺胶粒的分泌-以确定其作用机制是否相似。我们也有初步证据表明，肌球蛋白VI与与钙调蛋白相关的组织特异性轻链有关。其他肌球蛋白的轻链调节活动。来自肌球蛋白VI体外酶特性分析的一些数据支持这样一种观点，即它也是通过其轻链来调节的。我们建议研究轻链相互作用。从长远来看，我们希望研究肌球蛋白VI的轻链、磷酸化、运动性和其他特性之间的关系，以了解它们在体内的重要性。肌球蛋白VI在许多细胞类型的肌动蛋白过程中很可能是重要的。由于肌球蛋白VI和肌动蛋白细胞骨架的其他元件在多细胞动物中是保守的，我们所了解的应该是广泛适用的。

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.