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

Arp2/3 复合体及其调节因子协调分支肌动蛋白网络的组装和拆卸。

• 批准号: 8961427
• 负责人: Bradley J Nolen
• 金额: $ 28.6万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8961427
• 关键词: Actins; Address; Architecture; Bacterial Infections; Bacteriophages; Binding; Biochemical; Biological Assay; Biological Process; Cancerous; Cell physiology; Cells; Complex; Crystallography; Cytoskeleton; Data; Endocytosis; Ensure; Face; Family; Filament; Fission Yeast; Fluorescence Microscopy; Foundations; Generations; Goals; Grant; Growth Cones; Human; Image; In Vitro; Leucine; Life; Malignant Neoplasms; Mediating; Microfilaments; Molecular; Molecular Conformation; Movement; Neoplasm Metastasis; Organelles; Play; Process; Protein Family; Proteins; Publishing; Role; Seeds; Signal Transduction; Source; Testing; Total Internal Reflection Fluorescent; WASP protein; Work; base; cell motility; fluorescence imaging; improved; in vivo; insight; mathematical model; member; novel; pathogen; polymerization; public health relevance; research study; response; single molecule

 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.

 描述（由申请人提供）：这项工作的长期目标是了解 Arp2/3 复合体及其调节因子如何控制肌动蛋白网络的组装和拆卸，因为 Arp2/3 复合体是肌动蛋白组装机器的重要组成部分。它响应细胞信号而使分支肌动蛋白丝成核的能力涉及健康细胞中发生的许多过程，例如内吞作用和生长运动。因此，对 Arp2/3 复合物产生的分支肌动蛋白网络的细胞控制的了解的进展将为推进细菌感染和癌症的治疗奠定基础。 Arp2/3 复合物的活性在体内受到严格调控，绝大多数关于 Arp2/3 复合物激活的研究都集中在 WASP 家族蛋白上。 WASP 蛋白需要预先形成的丝来激活 Arp2/3 复合物并增殖。分支网络，但很少有研究解决启动分支的预形成丝的来源，我们最近发现 WISH/DIP/SPIN90 (WDS) 蛋白是关键的分支肌动蛋白网络启动子，可以通过激活 Arp2/3 复合物来播种 WASP 介导的分支。尽管事实上引发可能是最重要的一步。 控制分支网络组装，WDS 蛋白如何激活复合物以产生种子丝，目前尚不清楚 WDS 蛋白的活性如何与 WASP 等其他激活剂协调，或者 WDS 蛋白本身如何受到调节。确定 WDS 蛋白单独激活 Arp2/3 复合物以及与 WASP 协同作用的分子机制。此外，我们将阐明 WDS 蛋白是如何调节的，这将使我们能够了解分支肌动蛋白网络的分子基础。这些研究的完成将为我们对肌动蛋白细胞骨架的细胞控制的理解提供重大进展，我们将通过以下三个具体目标来实现这些目标：1.) 定义 Arp2/3 复合物激活所需的 WDS 蛋白的结构特征。 ; 2.) 确定 WDS 蛋白如何与 Arp2/3 复合物相互作用并影响其构象以刺激激活；以及 3.) 确定 WDS 蛋白如何受到调节，我们将使用以下组合。 X 射线晶体学、生物化学和生物物理测定、数学模型、单分子全内反射荧光显微镜和活的粟酒裂殖酵母细胞的荧光成像。