Mechanisms of regulation of Arp2/3 complex

描述（由申请人提供）：这项工作的长期目标是了解Arp 2/3复合物调节的分子基础。Arp 2/3复合物是肌动蛋白组装机制的重要组成部分，因为它能够响应细胞信号使分支的肌动蛋白丝成核。Arp 2/3复合物不仅参与健康细胞的内吞、生长锥的运动等过程，而且在病原菌感染宿主细胞、肿瘤细胞转移等过程中发挥重要作用。Arp 2/3复合物的活性在体内受到严格的调节，已经发现了许多该复合物的激活剂和抑制剂，包括WASp/Scar家族蛋白和冠蛋白家族蛋白。尽管Arp 2/3复合物的核心重要性，其激活的分子机制仍然是未知的，由于缺乏部分或完全激活状态的复合物的生化和高分辨率结构信息。在这里，我们建议使用结构，生物化学和细胞生物学的方法来确定一个精确的分子机制，Arp 2/3复合物及其调节蛋白创建分支肌动蛋白网络。我们将通过以下三个具体目标来实现这一目标：1。确定允许Arp 2/3复合物与肌动蛋白的母丝和子丝结合的关键结构元件; 2.）确定WASp/Scar家族蛋白如何激活Arp 2/3复合物;以及3.）确定冠状蛋白如何抑制Arp 2/3复合物。我们的方法将是使用突变分析和生化测定来精确地绘制S。粟酒裂殖酵母Arp 2/3复合物，具有肌动蛋白的母丝和子丝。我们将使用质谱，X射线晶体学和突变分析来确定WASp/Scar家族蛋白与Arp 2/3复合物结合的位置以及它们如何为子丝募集第一个肌动蛋白单体。最后，我们将使用生物化学和结构的方法来确定如何酵母冠蛋白抑制Arp 2/3复合物，并影响其与肌动蛋白丝的相互作用。

DESCRIPTION (provided by applicant): The long term goal of this work is to understand the molecular basis for the regulation of Arp2/3 complex. 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. The activity of Arp2/3 complex is tightly regulated in vivo, and numerous activators and inhibitors of the complex have been discovered, including WASp/Scar family proteins and coronin family proteins. Despite the central importance of the Arp2/3 complex, the molecular mechanism of its activation is still unknown due to the lack of biochemical and high resolution structural information of partially or fully activated states of the complex. Here we propose to use structural, biochemical and cell biological approaches to determine a precise molecular mechanism by which Arp2/3 complex and its regulatory proteins create branched actin networks. We will pursue this goal through the following three specific aims: 1.) Determine the key structural elements that allow Arp2/3 complex to bind to mother and daughter filaments of actin; 2.) Determine how WASp/Scar family proteins activate Arp2/3 complex; and 3.) Determine how Coronin proteins inhibit Arp2/3 complex. Our approach will be to use mutational analysis and biochemical assays to precisely map the interactions of S. pombe Arp2/3 complex with mother and daughter filaments of actin. We will use mass spectrometry, x-ray crystallography and mutational analysis to determine where WASp/Scar family proteins bind to Arp2/3 complex and how they recruit the first actin monomer for the daughter filament. Finally, we will use biochemical and structural methods to determine how yeast coronin proteins inhibit Arp2/3 complex and influence its interactions with actin filaments.