MYOSIN VI FUNCTION AND MECHANISM

描述（由申请人提供）：本竞争性修订补充申请是对NOT-OD-09-058的回应，根据ARRA NIH公告：NIH宣布竞争性修订申请的恢复法案资金的可用性。在这个申请中，我们建议扩大我们目前资助的项目R01 GM060494的范围，题为“Myosin VI的功能和机制”。肌动蛋白结构在多细胞生物的细胞中除了参与细胞运动外，还起着许多重要的作用。它们在产生分化细胞形态和介导特化细胞功能中起着至关重要的作用。这些结构是基于在运动细胞中发现的结构基序组织的，例如网状和平行束，但通常具有非常不同的形成和周转动力学，并且执行非常不同的功能，需要不同的机械和物理特性。我们正在研究一个有趣的例子，一个特殊的肌动蛋白结构在细胞分化中很重要，这是在果蝇精细胞个体化过程中发生的细胞重塑。在这一过程中，一组由细胞质桥连接的64个精子分离成单个精子。锥形肌动蛋白结构沿着高度拉长的细胞的长度移动，重塑细胞膜，将细胞内容物和大部分细胞质推出。这种肌动蛋白结构非常致密，因此它可以将细胞质内容物推出细胞外。肌凝蛋白VI是一种基于肌动蛋白的运动蛋白，在形成锥体前部的密集肌动蛋白网络的构建中起着关键作用。当它缺失时，肌动蛋白积累不足，视锥细胞不能正常工作，细胞质和细胞器的移除有缺陷。Myosin VI的作用是稳定肌动蛋白网络。在构建这个有趣的肌动蛋白结构时，肌凝蛋白VI与arp 2/3复合物合作，这对网状结构的形成很重要。剖面蛋白也是必需的，它对形成锥体后部的束的组装很重要，可能是与孔蛋白合作形成的。其他蛋白质位于肌动蛋白锥上，包括束状蛋白（烤），绒毛蛋白（鹌鹑），接触蛋白和动力蛋白。在我们目前资助的工作（R01GM060494）中，我们主要研究myosin VI在肌动蛋白稳定作用中的作用机制。然而，我们假设其他蛋白质在产生这种有趣的结构中起着重要作用，我们建议通过筛选现有的雄性不育突变体来扩展我们的工作范围，以识别这些其他蛋白质。我们将目视筛选具有肌动蛋白锥体结构缺陷和肌凝蛋白VI定位缺陷的突变体。一旦确定了在这一过程中重要的基因集合，我们将研究蛋白质如何协同工作以产生这种结构，并询问是否在其他细胞和过程中使用类似的机制。由于在其他情况下，密集的肌动蛋白结构对于定位细胞成分或结构支持是重要的，因此我们获得的关于这一过程的信息可能不仅仅与果蝇精子发生有关。这里提出的工作将扩大我们资助的工作范围，导致雇用额外的人员，购买设备和用品，涉及生成和分析DNA序列数据的服务合同，并加快科学发现的步伐。

DESCRIPTION (provided by applicant): This application for Competitive Revision Supplement is in response to NOT-OD-09-058, under the ARRA NIH announcement: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. In this application we propose to expand the scope of our currently funded project R01 GM060494 entitled "Myosin VI function and mechanism." Actin structures play many important roles in cells of multi-cellular organisms that go beyond participating in cell motility. They are crucial in generating differentiated cell form and mediating specialized cell function. Such structures are organized based on structural motifs found in motile cells, such as meshwork and parallel bundles, but often have very different kinetics of formation and turnover and perform very different functions, requiring different mechanical and physical properties. We are studying interesting example of a specialized actin structure important in cellular differentiation is the cellular remodeling that occurs during spermatid individualization in Drosophila. During this process a group of 64 spermatids interconnected by cytoplasmic bridges are separated into individual sperm. Cone-shaped actin structures move along the length of the highly elongated cell, remodeling the membrane and pushing out cellular contents and the bulk of the cytoplasm. This actin structure is extremely dense, so it can push the cytoplasmic contents out of the cell. Myosin VI, an actin-based motor protein, plays a key role in building the dense actin meshwork that forms the cone front. When it is absent, insufficient actin accumulates, the cones don't function properly, and the removal of cytoplasm and organelles is defective. Myosin VI's role is to stabilize the actin meshwork. In building this interesting actin structure, myosin VI cooperates with the arp 2/3 complex, which is important for meshwork formation. Profilin also is required, being important for assembling the bundles that form the rear of the cones, probably in collaboration with a formin. Other proteins are located on the actin cones, including fascin (singed), villin (quail), cortactin, and dynamin. In our currently funded work (R01GM060494), we focus on understanding myosin VI's mechanism of action in its actin stabilization role. However, we hypothesize that other proteins play important roles in generating this interesting structure and we propose to extend the scope of our work by screening an existing collection of male sterile mutants to identify these other proteins. We will visually screen for mutants with defects in actin cone structure and myosin VI localization. Once a collection of genes important in this process is identified, we will examine how the proteins work together to generate this structure and ask whether similar mechanisms are used in other cells and processes. Since in other situations dense actin structures are important for localizing cellular components or structural support, the information we obtain about this process may be relevant beyond Drosophila spermatogenesis. The work proposed here will extend the scope of our funded work, result in the hiring of additional personnel, the purchase of equipment and supplies, involve the contracting of services for generation and analysis of DNA sequence data, and accelerate the pace of scientific discovery. . PUBLIC HEALTH RELEVANCE: Actin structures play important roles in all cells and are the key cellular components that give differentiated cells their shape and organization. Defects in these structures and in actin organization and dynamics underlie many aspects of disease. Our studies will identify important regulators of actin assembly and organization in particular specialized cell types. By studying these regulators and how they help in formation of the correct structures at the correct time, in the correct place and with the correct form, a better understanding of the basic cellular processes underlying normal cell function and disease will emerge.