Molecular Mechanisms of Actin Cytoskeleton Dynamics

 描述（由申请人提供）：无法获得肌动蛋白细胞骨架上的原子结构严重阻碍了我们对这种最丰富的真核蛋白及其通过与数百种肌动蛋白结合蛋白相互作用执行无数细胞功能（如细胞运动、胞质分裂和形态发生）的动态周转的理解。本申请描述了一种创新的双突变策略，以克服这一长期存在的障碍，在肌动蛋白细胞骨架领域。事实上，这种新颖的双突变策略已经使PI成功地确定了肌动蛋白-成核剂复合物的第一个原子晶体结构 （肌动蛋白-Cobl复合物）。此外，类似的方法已被用于解决肌动蛋白的结构与细菌效应，VopL。肌动蛋白-Cobl结构中所观察到的非丝状构象和肌动蛋白-VopL结构中所观察到的非丝状构象一起表明，通过双突变策略获得的肌动蛋白复合物完全可接近这两种类型的构象，因此其真实构象最有可能被保留。在本申请中提出的实验将应用双突变体的策略，以最新的类肌动蛋白成核剂，其特征在于存在串联肌动蛋白结合位点的三个最不同的成员。我们的目标是破译它们的肌动蛋白成核的分子机制，ATP水解在其功能周期中的作用，以及它们如何与特定的细胞组分合作以实现其功能。这一目标将在三个具体目标中使用组合的结构和功能方法来实现：目标1：Cobl介导的肌动蛋白成核机制;目标2：Lmod的肌动蛋白成核机制研究;目标3：APC介导的肌动蛋白成核机制研究。本申请中提出的广泛的初步研究表明，所提出的研究很有希望取得成功。这项研究的结果不仅在阐明它们各自在神经发生、肌肉发育和肿瘤发生中的作用的分子机制方面具有重要意义，而且在揭示一些长期以来一直寻求的从头肌动蛋白成核的一般基础方面也具有重要意义，这是哺乳动物发育的每个阶段以及许多类型的病原性感染的基础过程。通过提供一个详细的原子画廊如何从头肌动蛋白成核完成和调节，这项研究将刺激更深层次的机制调查，这些成核剂以及发现和表征新的肌动蛋白成核剂。更重要的是，该应用将验证对具有和不具有肌动蛋白成核活性的蛋白质/蛋白质片段的双突变策略，从而提供 足够的证明的原则，这种方法扩展到许多其他肌动蛋白参与的生物过程以外的肌动蛋白成核。最终，沿着这条线的研究预计将直接受益于许多形式的人类疾病的治疗，由于肌动蛋白细胞骨架功能障碍，包括神经退行性疾病，肌营养不良症，肿瘤发生和转移。

 DESCRIPTION (provided by applicant): The inability to obtain atomic structures on actin cytoskeleton has severely hindered our understanding of this most abundant eukaryotic protein and its dynamic turnovers in performing a myriad of cellular functions such as cell motility, cytokinesis and morphogenesis through interactions with hundreds of actin-binding proteins. This application describes an innovative double-mutant strategy to overcome this longstanding barrier in the actin cytoskeleton field. Indeed, this novel double-mutant strategy has enabled the PIs' successful determination of the very first atomic crystal structure of actin-nucleator complex (actin-Cobl complex). In addition, a similar approach has been used to solve the structure of actin with a bacterial effector, VopL. The observed non-filament-like conformation in actin-Cobl structure and filament-like conformation in actin-VopL structure together suggest that both types of conformation are fully accessible to an actin complex obtained via the double-mutant strategy, thus its true conformation is most likely preserved. The experiments proposed in this application will apply the double-mutant strategy to three most divergent members of the latest class actin nucleators characterized by the presence of tandem actin-binding sites. The goal is to decipher their molecular mechanisms of actin nucleation, the roles of ATP hydrolysis in their functional cycle, and how they collaborate with specific cellular components to fulfill their functions. This goal will be achieved in three Specific Aims using combined structural and functional approaches: Aim 1: Mechanisms of Cobl-mediated actin nucleation; Aim 2: Mechanistic study of actin nucleation by Lmod; and Aim 3: Mechanistic study of APC-mediated actin nucleation. The extensive preliminary studies presented in this application suggest a high promise of success for the proposed research. Results from this study will be significant not only in elucidating the molecular mechanisms of their respective roles in neurogenesis, muscle development and tumor initiation, but also in unraveling some long-sought-after general underpinnings for de novo actin nucleation, a process underlying every stage of mammalian development as well as many types of pathogenic infection. By providing a detailed atomic gallery of how de novo actin nucleation is accomplished and regulated, this study will stimulate deeper mechanistic investigations on these nucleators as well as discovery and characterization of new actin nucleators. More importantly, this application will validate the double-mutant strategy on proteins/protein fragments with and without actin nucleation activities, thus providing sufficient proof-of-principle for extending this approach to many other actin-involved biological processes beyond actin nucleation. Ultimately, the research along this line is expected to directly benefit the treatment of many forms of human diseases due to actin cytoskeleton malfunctions including neurodegenerative disorders, muscular dystrophy, tumorigenesis and metastasis.