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、肌肉收缩和胞吞作用）。 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中受损，特别是那些与心脏、肺和血液研究相关的疾病。 人WASp（Wiskott Aldrich综合征蛋白）（肌动蛋白相关蛋白（阿普）2/3复合物介导的肌动蛋白组装的刺激物）中的突变导致中性粒细胞运动性的缺陷和免疫功能的丧失。 在哮喘中，钙水平响应于组胺释放而升高，并诱导肺呼吸道中的平滑肌收缩。 肌动蛋白本身的缺陷与扩张型心肌病和心力衰竭以及关键肌动蛋白调节因子（例如，cofilin）导致肌肉去神经支配和营养不良。 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正常功能的重要第一步。 对芽殖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，因为可以使用遗传和生物化学相结合的方法。 该研究职业奖（RCA）的目的是通过引入两种新的显微镜工具来研究Arp 2/3复合物的结构和作用机制，从而扩展现有R 01（芽殖酵母中肌动蛋白组装的调节）的具体目标。 全内反射荧光（TIRF）显微镜将被用来研究肌动蛋白丝聚合和分支的Arp 2/3复合物和WASp，Abp 1，和冠蛋白在真实的时间的调节。 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Arp 2/3复合物的结构， 体内缺陷。 这项建议的拨款将大大提高首席研究员的能力，借着减少他的教学和行政职务，以达致这些目的。 这项研究计划的跨学科性质需要PI在他们没有经验的技术和领域的学生和博士后培训的中心参与。 PI将直接参与TIRF显微镜和单粒子成像到他的研究计划的整合。

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.