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的响应，根据ARRA NIH公告：NIH宣布可为竞争性修订申请提供恢复法案资金。在本申请中，我们建议扩大我们目前资助的项目R01 GM060494的范围，该项目的标题为“myosin VI功能和机制”。肌动蛋白结构在多细胞生物体的细胞中扮演着许多重要的角色，而不仅仅是参与细胞的运动。它们在产生分化的细胞形态和调节特化细胞功能方面起着至关重要的作用。这种结构是基于活动细胞中的结构基序组织的，例如网状结构和平行束，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。我们正在研究一种对细胞分化很重要的特殊肌动蛋白结构的有趣例子，那就是在果蝇精子细胞个体化过程中发生的细胞重塑。在这个过程中，一组由细胞质桥连接的精子细胞被分离成单独的精子。锥形肌动蛋白结构沿着高度伸长的细胞的长度移动，重塑细胞膜，并推出细胞内容物和细胞质的大部分。这种肌动蛋白结构极其致密，因此它可以将细胞质内容物推出细胞。肌球蛋白VI是一种以肌动蛋白为基础的运动蛋白，在形成锥体前锋的致密肌动蛋白网络中起着关键作用。当缺乏肌动蛋白时，肌动蛋白积累不足，视锥细胞功能不正常，细胞质和细胞器的移除是有缺陷的。肌球蛋白VI的作用是稳定肌动蛋白网络。在构建这种有趣的肌动蛋白结构时，肌球蛋白VI与ARP2/3复合体合作，ARP2/3复合体对网络的形成至关重要。轮廓蛋白也是必需的，对于组装形成圆锥体后部的束状物很重要，可能是与福尔马林协作。其他蛋白质位于肌动蛋白锥体上，包括筋膜蛋白(烧灼)、绒毛蛋白(鹌鹑)、皮质蛋白和动力素。在我们目前资助的工作(R01GM060494)中，我们专注于了解肌球蛋白VI在其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发挥了重要作用，我们建议通过筛选现有的雄性不育突变体来鉴定这些其他蛋白质，从而扩大我们的工作范围。我们将直观地筛选肌动蛋白锥体结构和肌球蛋白VI定位缺陷的突变。一旦确定了在这一过程中重要的基因集合，我们将研究这些蛋白质是如何共同作用来产生这种结构的，并询问其他细胞和过程是否使用了类似的机制。由于在其他情况下，致密的肌动蛋白结构对于定位细胞成分或结构支撑是重要的，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的范围，导致雇用更多的人员，购买设备和用品，包括签订产生和分析DNA序列数据的服务，并加快科学发现的步伐。。 公共卫生相关性：肌动蛋白结构在所有细胞中发挥重要作用，是赋予分化细胞形态和组织的关键细胞成分。这些结构以及肌动蛋白组织和动力学上的缺陷是疾病的许多方面的基础。我们的研究将确定肌动蛋白组装和组织的重要调节因素，特别是特定的细胞类型。通过研究这些调节因子以及它们如何帮助在正确的时间、正确的位置和正确的形式形成正确的结构，将会更好地理解正常细胞功能和疾病背后的基本细胞过程。

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.