Structural basis of dynamin-mediated membrane fission actin bundling and interaction with binding partners.

动力介导的膜裂变肌动蛋白捆绑和与结合伙伴相互作用的结构基础。

• 批准号: 10525706
• 负责人: John Jimah
• 金额: $ 24.9万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-16 至 2025-01-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10525706
• 关键词: Actins; Address; Affect; Arginine; Binding; Biological Assay; Biomedical Research; Biophysics; Bundling; Caliber; Cell fusion; Cell membrane; Cell physiology; Cells; Cellular Membrane; Cellular biology; Charge; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Cytoskeleton; Defect; Development; Dynamin; Electrons; Elements; Endocytosis; Foundations; GTP Binding; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; In Vitro; Length; Lipids; Liposomes; Malignant Neoplasms; Mediating; Membrane; Membrane Lipids; Mentors; Microfilaments; Mission; Modeling; Molecular; Movement; Neck; Neuropathy; Organism; Play; Polymers; Process; Proline; Proline-Rich Domain; Protein Region; Proteins; Regulation; Reporting; Research; Research Design; Research Methodology; Research Personnel; Role; SH3 Domains; Signal Transduction; Site; Structure; System; Tertiary Protein Structure; Testing; Tomogram; Training; Tube; United States National Institutes of Health; Vesicle; Work; amphiphysin; base; biophysical analysis; career; cell motility; constriction; in vivo; insight; intersectin 1; mechanical properties; mutant; novel; programs; public health relevance; reconstitution; reconstruction; recruit

Project Summary/Abstract Dynamin GTPases have critical roles in mediating endocytosis by wrapping around the neck of budding vesicles to catalyze membrane fission necessary for the release of nascent vesicles from the plasma membrane. Recently, we discovered a novel role for dynamin in bundling numerous actin filaments, which has implications for actin-mediated processes, such as cell-cell fusion and migration. While structural and biophysical studies have elucidated the mechanism of dynamin assembly and constriction of membranes, several unanswered questions remain including how dynamin is actually organized and mediates fission within cells, how dynamin forms the final pre-fission state where it wraps around lipid tubules in a superconstricted state, how dynamin binds substrates via the proline rich domain (PRD), and how substrate binding regulates dynamin activity. The long-term objective of this application is to elucidate the mechanism of dynamin-mediated membrane fission from in vitro and in vivo studies, and to define the mechanism of dynamin interaction with actin and SH3 domain- containing proteins that recruit dynamin to sites of endocytosis. These objectives will be addressed by the following specific aims: (1) determine the atomic model of dynamin in the superconstricted prefission state and define the structure of the PRD; (2) investigate the assembly of the dynamin helical polymer on membranes within cells; and (3) Elucidate the mechanism of PRD interaction with actin filaments and SH3 domain-containing binding partners. The rationale for these aims is that: (1) there is no atomic model describing the structural basis by which dynamin constricts membranes to 3.4 nm in the superconstricted state where spontaneous hemifission and membrane fission occurs; (2) how dynamin is actually organized in cells has not been reported; and (3) the structure of the critical PRD and how it binds dynamin substrates including actin, amphiphysin and intersectin is unknown. The research design and methods are as follows : Aim 1, Apply cryo-electron microscopy (cryo-EM) to determine the structure of full-length dynamin (containing the PRD) organized around lipid tubules in the superconstricted state; Aim 2, obtain cryo-electron tomograms and subtomogram averages of dynamin within cells transfected with a GTPase-deficient dynamin mutant which delays membrane fission and extends the lifetime of dynamin helices on cellular membranes; Aim 3, obtain the cryo-EM structure of actin filaments decorated with PRD from dynamin, as well as complexes of dynamin/amphiphysin and dynamin/intersectin. This work is of

项目概要/摘要 动态 GTP 酶通过包裹出芽囊泡的颈部，在介导内吞作用中发挥关键作用 催化从质膜释放新生囊泡所必需的膜裂变。 最近，我们发现了动力蛋白在捆绑大量肌动蛋白丝方面的新作用，这具有重要意义 用于肌动蛋白介导的过程，例如细胞间融合和迁移。结构和生物物理研究 已经阐明了动力组装和膜收缩的机制，一些尚未解答的问题 仍然存在的问题包括动力如何实际组织并介导细胞内的裂变，动力如何 形成最终的裂变前状态，以超收缩状态包裹脂质小管，动力如何 通过富含脯氨酸的结构域 (PRD) 结合底物，以及底物结合如何调节动力活性。这 该应用的长期目标是阐明动力介导的膜裂变的机制 来自体外和体内研究，并定义动力蛋白与肌动蛋白和 SH3 结构域相互作用的机制 - 含有将动力招募到内吞作用位点的蛋白质。这些目标将通过 具体目标如下：（1）确定超收缩预裂变态动力的原子模型 并定义PRD的结构； (2) 研究动力螺旋聚合物在膜上的组装 细胞内； (3) 阐明PRD与肌动蛋白丝和含有SH3结构域的相互作用的机制 具有约束力的伙伴。这些目标的基本原理是：（1）没有描述结构的原子模型 动力在超收缩状态下将膜收缩至 3.4 nm 的基础，其中自发 发生半裂和膜裂； (2)动力蛋白在细胞中实际上是如何组织的尚未有报道； (3) 关键 PRD 的结构以及它如何结合动力底物，包括肌动蛋白、两性蛋白和 相交蛋白未知。研究设计和方法如下： 目的1、应用低温电子 显微镜（冷冻电镜）以确定围绕周围组织的全长动力（包含 PRD）的结构 脂质小管处于超收缩状态；目标 2，获得冷冻电子断层图和次断层图平均值 转染 GTP 酶缺陷型动力突变体的细胞内动力的增加，延迟了膜裂变和 延长细胞膜上动力螺旋的寿命；目标3，获得肌动蛋白的冷冻电镜结构 用来自动力的 PRD 装饰的细丝，以及动力/两性蛋白的复合物和 动力/相交。这部作品是