EM & TIRF analysis of Arp2/3 complex and actin assembly

描述(由申请人提供)： 在所有真核生物中，肌动蛋白被组装成高度动态的细丝，这些细丝被组织成网络，提供极性和力来驱动不同的细胞过程(例如，细胞迁移、胞质分裂、肌肉收缩和内吞作用)。肌动蛋白组装和肌动蛋白细胞骨架功能的适当调节在许多疾病状态下都受到损害，特别是那些与心脏、肺和血液研究相关的疾病。人类黄蜂(Wiskott Aldrich综合征蛋白)是肌动蛋白相关蛋白(Arp)2/3复合药物组装的刺激物，其突变会导致中性粒细胞运动性缺陷和免疫功能丧失。在哮喘患者中，组胺释放会导致钙水平升高，并导致肺部呼吸道的平滑肌收缩。肌动蛋白本身的缺陷直接与扩张型心肌病和心力衰竭有关，而关键肌动蛋白调节因子(如cofilin)的突变会导致肌肉失神经和营养不良。因此，了解肌动蛋白组装的生化基础是确定这些病理过程如何破坏这些组织的正常功能的重要第一步。 对萌芽酵母酿酒酵母的研究有助于剖析关键肌动蛋白调节因子的功能，因为可以结合使用遗传和生物化学方法。这个研究生涯奖(RCA)的目标是通过引入两种新的显微镜工具来研究Arp2/3复合体的结构和作用机制，从而扩展现有的R01(芽殖酵母中肌动蛋白组装的调节)的特定目标。全内反射荧光(TIRF)显微镜将被用来实时研究Arp2/3复合体对肌动蛋白细丝聚合和分支以及黄蜂、Abp1和胡萝卜素对其的调节。电子显微镜和单粒子图像分析将用于研究活性受损的突变Arp2/3复合体的结构和 体内缺陷。这项提议的经费将通过减少他的教学和行政职责，大大提高他实现这些目标的能力。这一研究计划的跨学科性质要求PI集中参与对学生和博士后在他们缺乏经验的技术和领域的培训。PI将直接参与将TIRF显微镜和单粒子成像整合到他的研究计划中。

DESCRIPTION (provided by applicant): In all eukaryotes, actin is assembled into highly dynamic thin filaments, which are organized into networks that provide polarity and force to drive different cellular processes (e.g., cell migration, cytokinesis, muscle contraction, and endocytosis). The proper regulation of actin assembly and actin cytoskeletal function is impaired in many disease states, particularly those relevant to heart, lung, and blood research. Mutations in human WASp (Wiskott Aldrich Syndrome protein), a stimulator of actin related protein (Arp) 2/3 complex-medicated actin assembly, cause defects in neutrophil cell motility and loss of immune function. In asthma, calcium levels rise in response to histamine release and induce smooth muscle contraction in respiratory tracts of the lungs. Defects in actin itself are linked directly to dilated cardiomyopathy and heart failure, and mutations in key actin regulators (e.g., cofilin) lead to muscle denervation and dystrophy. Thus, understanding the biochemical basis of actin assembly is an important first step in defining how these pathological processes disrupt normal function in these tissues. Studies in the budding yeast Saccharomyces cerevisiae have been instrumental in dissecting the functions of key actin regulators, because combined genetic and biochemical approaches can be used. The objectives of this research career award (RCA) are to expand the specific aims of an existing R01 (Regulation of Actin Assembly in Budding Yeast) by introducing two new microscopy tools to study the structure and mechanism of action of Arp2/3 complex. Total internal reflection fluorescence (TIRF) microscopy will be used to study actin filament polymerization and branching by Arp2/3 complex and its regulation by WASp, Abp1, and coronin in real time. Electron microscopy and single particle image analysis will be used to study the structures of mutant Arp2/3 complexes with impaired activities and in vivo defects. The funding of this proposal would significantly enhance the ability of the PI to accomplish these goals by reducing his teaching and administrative duties. The interdisciplinary nature of this research program necessitates central involvement of the PI in training of students and postdocs in techniques and areas in which they are inexperienced. The PI will be directly involved in the integration of TIRF microscopy and single particle imaging into his research program.