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: 9283993
• 负责人: Bradley J Nolen
• 金额: $ 18.12万
• 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/9283993
• 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; Health; Human; 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; microscopic imaging; novel; pathogen; polymerization; 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Arp 2/3复合物及其调节因子如何控制肌动蛋白网络的组装和拆卸。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。因此，了解Arp 2/3复合物产生的分支肌动蛋白网络的细胞控制的进展将为推进细菌感染和癌症的治疗提供基础。Arp 2/3复合物的活性在体内受到严格调控，并且绝大多数关于Arp 2/3复合物活化的研究集中在WASP家族蛋白。WASP蛋白需要预先形成的细丝来激活Arp 2/3复合物并传播分支网络，但很少有研究解决预先形成的细丝的来源来启动分支。我们最近发现，WISH/DIP/SPIN 90（WDS）蛋白是关键的分支肌动蛋白网络的启动子，可以通过激活Arp 2/3复合物而无需预先形成的细丝来播种WASP介导的分支。尽管启蒙可能是最重要的一步， 由于控制分支网络组装，WDS蛋白如何激活复合物以产生种子丝尚不清楚。此外，目前还不清楚WDS蛋白的活性如何与其他激活剂如WASP协调，或者WDS蛋白本身是如何调节的。在这里，我们建议确定的分子机制，WDS蛋白激活Arp 2/3复合物，单独和协调与WASP。此外，我们将阐明WDS蛋白是如何调节的，这将使我们能够理解分支肌动蛋白网络起始的分子基础。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一个重要的进展。我们将通过以下三个具体目标来实现这些目标：1。定义Arp 2/3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Arp 2/3复合物相互作用并影响其构象以刺激活化;和3.）确定WDS蛋白质是如何调节的。为了实现这些目标，我们将结合使用X射线晶体学，生物化学和生物物理测定，数学建模，单分子全内反射荧光显微镜和荧光成像的活S。粟酒裂殖酵母细胞