Coordinated assembly and disassembly of branched actin networks by Arp2/3 complex and its regulators.

 描述(申请人提供)：这项工作的长期目标是了解Arp2/3复合体及其调控因子如何控制肌动蛋白网络的组装和拆解。Arp2/3复合体是肌动蛋白组装机械的重要组成部分，因为它能够响应细胞信号而使分枝的肌动蛋白细丝成核。Arp2/3复合体不仅参与了健康细胞的内吞和生长锥体运动等过程，还参与了细菌感染宿主细胞和肿瘤细胞转移的过程。因此，了解Arp2/3复合体形成的分支肌动蛋白网络的细胞控制机制的进展将为进一步治疗细菌感染和癌症提供基础。Arp2/3复合体的活性在体内受到严格的调控，绝大多数关于Arp2/3复合体激活的研究都集中在WASP家族蛋白上。黄蜂蛋白需要预成型的细丝来激活Arp2/3复合体并繁殖分支网络，但很少有研究解决预成型细丝的来源来启动分支。我们最近发现Wish/DIP/SPIN90(WDS)蛋白是重要的分支肌动蛋白网络启动子，它可以通过激活Arp2/3复合体而不需要预先形成细丝来启动WASP介导的分支。尽管印心可能是 控制分支网络组装的WDS蛋白如何激活该复合体以创建种子细丝尚不清楚。此外，WDS蛋白的活性如何与WASP等其他激活剂协调，或者WDS蛋白本身是如何调节的，目前还不清楚。在这里，我们建议确定WDS蛋白单独和与WASP协同激活Arp2/3复合体的分子机制。此外，我们还将阐明WDS蛋白是如何被调控的，这将使我们能够理解分支肌动蛋白网络启动的分子基础。这些研究的完成将为我们理解肌动蛋白细胞骨架的细胞控制提供重要的进步。我们将通过以下三个具体目标实现这些目标：1.确定Arp2/3复合体激活所需的WDS蛋白的结构特征；确定WDS蛋白如何与Arp2/3复合体相互作用并影响其构象以刺激激活；确定WDS蛋白是如何被调控的。为了实现这些目标，我们将结合使用X射线结晶学、生化和生物物理分析、数学建模、单分子全内反射荧光显微镜和活的S.pombe细胞的荧光成像。

 DESCRIPTION (provided by applicant): The long-term goal of this work is to understand how Arp2/3 complex and its regulators control the assembly and disassembly of actin networks. Arp2/3 complex is an essential component of the actin assembly machinery because of its ability to nucleate branched actin filaments in response to cellular signals. Arp2/3 complex is involved in a number of processes that occur in healthy cells, such as endocytosis and motility of growth cones, but also plays roles in host cell infection by bacterial pathogens and the metastasis of tumor cells. Therefore, advances in understanding cellular control of branched actin networks created by Arp2/3 complex will provide a foundation to advance treatments of bacterial infections and cancer. The activity of Arp2/3 complex is tightly regulated in vivo, and the vast majority of studies on Arp2/3 complex activation have focused on WASP family proteins. WASP proteins require preformed filaments to activate Arp2/3 complex and propagate branched networks, but few studies have addressed the source of preformed filaments to initiate branching. We recently discovered that WISH/DIP/SPIN90 (WDS) proteins are key branched actin network initiators that can seed WASP-mediated branching by activating Arp2/3 complex without preformed filaments. Despite the fact that initiation is perhaps the most important step in controlling branched network assembly, how WDS proteins activate the complex to create seed filaments is unknown. Furthermore, it is not understood how the activity of WDS proteins is coordinated with other activators like WASP, or how WDS proteins themselves are regulated. Here we propose to determine the molecular mechanisms by which WDS proteins activate Arp2/3 complex, alone and coordinately with WASP. In addition, we will elucidate how WDS proteins are regulated, which will allow us to understand the molecular basis for branched actin network initiation. Completion of these studies will provide a significant advance in our understanding of cellular control of the actin cytoskeleton. We will pursue these goals through the following three specific aims: 1.) Define structural features in WDS proteins required for Arp2/3 complex activation; 2.) Determine how WDS proteins interact with Arp2/3 complex and influence its conformation to stimulate activation; and 3.) Determine how WDS proteins are regulated. To accomplish these aims we will use a combination of x-ray crystallography, biochemical and biophysical assays, mathematical modeling, single molecule total internal reflection fluorescence microscopy and fluorescence imaging of live S. pombe cells.