Structure and function of Arp 2/3 complex--Subproject 4

利用遗传学方法了解Arp 2/3复合体产生肌动蛋白丝的结构基础; Arp 2/3复合物是一种在进化过程中保守的肌动蛋白成核因子。在体外，Arp 2/3复合物被Wiskott-Aldrich综合征家族蛋白激活，并以与现有细丝不同的角度引发新细丝的聚合，导致独特的肌动蛋白分支结构。这些结构也已在体内观察到，并被认为是细胞运动过程中产生膨胀力的关键。该项目的总体目标是了解Arp 2/3复合物执行其功能的详细结构和生化机制。该项目为Arp 2/3复合物的结构/功能分析提供了遗传学方法。大多数目标涉及使用酵母遗传技术 以在Arp 2/3复合物的各个亚基中产生突变或标签。生化分析和结构表征（通过NMR和电子冷冻显微镜等）突变体或标记复合物的研究将允许测试Arp 2/3复合物激活和肌动蛋白成核的特定模型，以及定义激活过程中发生的结构变化。在最后一个目标中，我们将使用小鼠胚胎干细胞遗传学来产生Arp 2/3复合物亚基突变的细胞系，该突变可能影响肌动蛋白分支的形成、形态或稳定性。 成纤维细胞分支连接的动力学和结构特征 从这些细胞系中分化出来的细胞将提供对Arp 2/3复合物在细胞运动过程中肌动蛋白丝的产生和组织中的作用的关键见解。

Using genetics approaches to understand the structural basis for actin filament generation by the Arp2/3 complex ; The Arp2/3 complex is an actin nucleation factor conserved across evolution. In vitro, the Arp2/3 complex is activated by Wiskott-Aldrich syndrome family proteins and initiates the polymerization of a new filament at a distinct angle from an existing filament, resulting in a unique actin branch structure. These structures have also been observed in vivo and are thought to be critical for protrusive force generation during cell motility. The overall goal of this program project is to understand the detailed structural and biochemical mechanisms by which the Arp2/3 complex carries out its function. This project contributes genetic approaches to structure/function analysis of the Arp2/3 complex. Most of the aims involve using yeast genetic techniques to generate mutations or tags in various subunit of the Arp2/3 complex. Biochemical analysis and structural characterization (by NMR and electron cryo-microscopy, etc.) of the mutant or tagged complexes will allow testing specific models for Arp2/3 complex activation and actin nucleation, as well as defining the structural changes that occur during activation. In the last aim, we will use mouse embryonic stem cell genetics to generate cell lines bearing mutations in an Arp2/3 complex subunit that could affect actin branch formation, morphology or stability. Characterization of the dynamics and structure of branch junctions in fibroblasts differentiated from these cell lines wilt provide key insights into the role of the Arp2/3 complex in the generation and organization of actin filaments during cell motility.