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

项目摘要/摘要 Dynamin GTPase通过包裹在萌芽囊泡的脖子上，在介导内吞作用中具有关键作用 催化从质膜释放新生囊泡所需的膜裂变。 最近，我们发现了Dynamin在捆绑众多肌动蛋白丝中的新作用，这具有含义 用于肌动蛋白介导的过程，例如细胞 - 细胞融合和迁移。而结构和生物物理研究 已经阐明了动力蛋白组装和膜收缩的机制，几个未得到解答 问题仍然存在，包括如何实际组织动力蛋白并介导细胞内的裂变，如何动态 形成最终的前膜前状态，该状态在超收缩状态下围绕脂质小管包裹，如何动态 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蛋白活性。这 该应用的长期目的是阐明动力蛋白介导的膜裂变的机理 从体外和体内研究，并定义动力蛋白与肌动蛋白和SH3结构域的相互作用的机理 含有促进动力蛋白到内吞作用部位的蛋白质。这些目标将由 以下特定目的：（1）确定超收缩的预分发状态中元素的原子模型 并定义PRD的结构； （2）研究膜上动力蛋白螺旋聚合物的组装 在细胞内； （3）阐明与肌动蛋白丝和含SH3结构域的pRD相互作用的机制 约束伙伴。这些目标的理由是：（1）没有描述结构的原子模型 在超收缩状态下，动力学将膜收缩至3.4 nm的基础 半叶和膜裂变发生； （2）尚未在细胞中实际组织动力蛋白； （3）关键PRD的结构及其如何结合肌动蛋白，两亲蛋白和 相交是未知的。研究设计和方法如下：AIM 1，应用冷冻电子 显微镜（冷冻EM），以确定周围组织的全长动力蛋白（包含PRD）的结构 超收缩状态的脂质小管； AIM 2，获得冷冻电子断层图和亚图平均图 用GTPase缺陷型动力蛋白突变体转染细胞内的动力蛋白，该突变蛋白突变体延迟膜裂变和 扩展细胞膜上动力蛋白螺旋的寿命；目标3，获取肌动蛋白的冷冻EM结构 用Dynamin的PRD装饰的细丝以及Dynamin/Amphiphysin和 动力蛋白/间替蛋白。这项工作是