Structural Basis of Actin Cytoskeleton Dynamics

描述(由申请人提供)：长期目标是了解在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，如细胞运动、内吞作用和细胞内运输。在此期间，重点将放在肌动蛋白细丝的成核上。在细胞中，肌动蛋白以其单体形式(G-肌动蛋白)和丝状肌动蛋白(F-肌动蛋白)之间的调节平衡状态存在，其中F-肌动蛋白占据了肌动蛋白的大部分细胞功能。在肌动蛋白细丝组装过程中，成核是速度限制的。因此，真核细胞(和某些病原体)使用细丝核子来稳定肌动蛋白聚合核(二聚体、三聚体和四聚体)。微丝核子不仅控制肌动蛋白聚合的时间和位置，而且还控制它们产生的特定类型的肌动蛋白网络。已知的细丝成核剂包括Arp2/3复合体、福尔明斯、尖顶、COBL、VopL/VopF和Lmod。这些分子通常是无关的，但除了福尔马林外，它们都使用WASP-同源结构域2(WH2或W)与肌动蛋白相互作用，这是一个小而多功能的肌动蛋白结合基序。建立在对W-肌动蛋白相互作用的深入理解的基础上，本提案旨在了解W依赖肌动蛋白细丝成核的结构-功能机制。具体目的是：1.了解NPFS-Arp2/3复合体对肌动蛋白微丝的成核机制。Arp2/3复合体及其以W为基础的成核促进因子(NPF)大家族在细丝核苷中是独一无二的，因为它能产生分支肌动蛋白网络。非活性络合物的晶体结构是已知的。在这里，重点将放在激活的综合体上，这是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.了解副溶血性弧菌引起的胃肠炎所产生的强核素和毒力因子VopL的成核机制。VopL的研究与其他丝状核子，如COBL和Spire具有普遍的相关性，与VopL一样，VopL也是基于W结构域的串联重复。3.为了了解Lmod的成核机制和细胞功能，Lmod是在本资助的前一阶段发现的一种肌肉特异性肌动蛋白细丝核心剂。Lmod与其他丝状核酸体无关，它含有三个肌动蛋白结合位点，其中一个是W结构域。广泛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，并利用单粒子电子显微镜确定激活的Arp2/3复合体的结构。这项研究将扩大我们对肌动蛋白细丝成核在健康和疾病中的分子基础的理解。 与公共健康相关：肌动蛋白细胞骨架参与大多数细胞功能，包括内吞/胞吐、细胞运动以及维持细胞形状和极性。这里研究的细丝核素在健康和疾病中起着至关重要的作用。因此，Arp2/3复合体介导的核化是转移性癌细胞迁移的关键因素，VopL是引起副溶血性弧菌的胃肠炎的毒力因子，Lmod参与心肌肌原纤维的形态发生。在这里获得的知识将对理解疾病的分子机制具有重要意义。

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.