Structural Basis of Actin Cytoskeleton Dynamics

描述（由申请人提供）：长期目标是了解在细胞运动、内吞作用和细胞内运输等细胞过程中控制肌动蛋白细胞骨架动力学的结构-功能机制。在这一时期的重点将是肌动蛋白丝成核。在细胞中，肌动蛋白以其单体（G-肌动蛋白）和丝状（F-肌动蛋白）形式之间的调节平衡存在，其中F-肌动蛋白占肌动蛋白的大多数细胞功能。成核是肌动蛋白丝组装过程中的速率限制。因此，真核细胞（和某些病原体）使用细丝成核剂来稳定肌动蛋白聚合核（二聚体，三聚体和四聚体）。纤维成核剂不仅控制肌动蛋白聚合的时间和位置，而且控制它们产生的肌动蛋白网络的特定类型。已知的细丝成核剂包括Arp 2/3复合物、formins、Spire、Cobl、VopL/VopF和Lmod。这些分子通常是不相关的，但除了formin外，它们都使用WASP-同源结构域2（WH 2或W），一种小而通用的肌动蛋白结合基序，用于与肌动蛋白相互作用。在此基础上获得的W-肌动蛋白相互作用的深入理解，在此期间的补助金，目前的建议旨在了解W-依赖的肌动蛋白丝成核的结构-功能机制。具体目标是：1.了解NPFs-Arp 2/3复合物对肌动蛋白丝成核的作用机制。Arp 2/3复合物，连同其W-基成核促进因子（NPF）的大家族，在细丝成核剂中是独特的，因为它产生分支肌动蛋白网络。非活性复合物的晶体结构是已知的。在这里，重点将放在激活复合体，一个具有挑战性的结构，其研究将需要设计和实施创新战略。2.目的了解副溶血性弧菌产生的毒力因子和成核因子VopL的成核机制。VopL的研究与其他丝状体成核剂如Cobl和Spire具有普遍相关性，它们与VopL一样基于W结构域的串联重复。3.了解Lmod的成核机制和细胞功能，Lmod是本基金前期发现的一种肌肉特异性肌动蛋白丝成核剂。Lmod与其他纤维成核因子无关，含有三个肌动蛋白结合位点，其中一个是W结构域。大量的初步结果为这些研究奠定了基础。合作研究也在进行中，以了解Lmod的细胞功能，并使用单粒子电子显微镜确定激活的Arp 2/3复合物的结构。这项研究将扩大我们对健康和疾病中肌动蛋白丝成核的分子基础的理解。 公共卫生相关性：肌动蛋白细胞骨架参与大多数细胞功能，包括胞内/胞吐、细胞运动以及细胞形状和极性的维持。这里研究的纤维成核剂在健康和疾病中起着关键作用。因此，Arp 2/3复合物介导的成核是转移性癌细胞迁移的关键因素，VopL是引起病原体副溶血性弧菌的胃肠炎的毒力因子，Lmod参与心肌中的肌原纤维形态发生。在这里获得的知识将是非常相关的理解疾病的分子机制。

DESCRIPTION (provided by applicant): The long-term goal is to understand the structure-function mechanisms that control actin cytoskeleton dynamics during cellular processes such as cell motility, endocytosis and intracellular trafficking. The emphasis during this period will be on actin filament nucleation. In cells, actin exists in regulated equilibrium between its monomeric (G-actin) and filamentous (F-actin) forms, with F-actin accounting for most cellular functions of actin. Nucleation is rate limiting during actin filament assemble. So, eukaryotic cells (and certain pathogens) use filament nucleators to stabilize actin polymerization nuclei (dimers, trimers and tetramers). Filament nucleators control not only the time and location for actin polymerization, but also the specific type of actin networks that they generate. Known filament nucleators include the Arp2/3 complex, formins, Spire, Cobl, VopL/VopF and Lmod. These molecules are generally unrelated, yet with the exception of formins they all use the WASP- Homology Domain 2 (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. Building upon a deep understanding of the W-actin interaction acquired during the previous period of this grant, the current proposal aims to understand the structure-function mechanisms of W-dependent actin filament nucleation. The specific aims are: 1. To understand the mechanism of actin filament nucleation by NPFs-Arp2/3 complex. The Arp2/3 complex, together with its large family of W-based Nucleation Promoting Factors (NPFs), is unique among filament nucleators in that it generates branched actin networks. The crystal structure of the inactive complex is known. Here, the focus will be on the activated complex, a challenging structure whose study will require the design and implementation of innovative strategies. 2. To understand the nucleation mechanism of VopL, a powerful nucleator and virulence factor produced by the gastroenteritis causing pathogen Vibrio parahaemolyticus. The study of VopL has general relevance to other filament nucleators, such as Cobl and Spire, which like VopL are based on tandem repeats of W domains. 3. To understand the nucleation mechanism and cellular function of Lmod, a muscle-specific actin filament nucleator discovered during the previous period of this grant. Lmod, which is unrelated to other filament nucleators, contains three actin-binding sites, one of which is a W domain. Extensive preliminary results lay the groundwork for these studies. Collaborative studies are also underway to understand the cellular function of Lmod, and to determine the structure of activated Arp2/3 complex using single-particle electron microscopy. This research will expand our understanding of the molecular bases of actin filament nucleation in health and disease. PUBLIC HEALTH RELEVANCE: The actin cytoskeleton is involved in most cellular function, including endo/exocytosis, cell motility, and the maintenance of cell shape and polarity. The filament nucleators studied here play critical roles in health and disease. Thus, Arp2/3 complex-mediated nucleation is a crucial factor for the migration of metastatic cancer cells, VopL is a virulence factor of the gastroenteritis causing pathogen Vibrio parahaemolyticus, and Lmod is involved in myofibril morphogenesis in heart muscle. The knowledge gained here will be of great relevance to understanding the molecular mechanisms of disease.