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

项目总结/摘要 动力蛋白GTP酶通过包裹出芽囊泡的颈部在介导内吞作用中起关键作用 以催化从质膜释放新生囊泡所必需的膜分裂。 最近，我们发现了发动蛋白在捆绑众多肌动蛋白丝中的一个新作用， 用于肌动蛋白介导的过程，如细胞-细胞融合和迁移。虽然结构和生物物理研究 已经阐明了发动蛋白组装和膜收缩的机制，有几个未回答的 问题仍然存在，包括发动蛋白实际上是如何组织和介导细胞内的裂变，发动蛋白如何 形成最终的裂变前状态，它以超收缩状态包裹在脂质小管周围，发动蛋白是如何形成的？ 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发动蛋白活性。的 本申请的长期目标是阐明动力蛋白介导的膜分裂的机制 从体外和体内研究，并确定动力蛋白与肌动蛋白和SH 3结构域相互作用的机制， 含有将发动蛋白募集到内吞作用位点的蛋白质。这些目标将由 具体目标如下：（1）确定超压缩裂变前态动力素的原子模型 并确定了PRD的结构;（2）研究了动力蛋白螺旋聚合物在膜上的组装 （3）阐明PRD与肌动蛋白丝和含SH 3结构域的 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 发动蛋白在超收缩状态下将膜收缩到3.4 nm的基础， （2）发动蛋白在细胞中是如何组织的，还没有报道; 和（3）关键PRD的结构以及它如何结合动力蛋白底物，包括肌动蛋白、两性蛋白和 交叉口未知。研究设计和方法如下：目的1、应用低温电子 显微镜（cryo-EM），以确定组织在周围的全长发动蛋白（包含PRD）的结构 脂质小管处于超收缩状态;目的2，获得冷冻电子断层图像和亚断层图像平均值 用GTP酶缺陷型发动蛋白突变体转染的细胞内的发动蛋白， 目的3、获得肌动蛋白的冷冻电镜结构 用来自发动蛋白的PRD装饰的细丝，以及发动蛋白/两性物理蛋白和 发动蛋白/交叉蛋白这项工作是

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