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

项目摘要/摘要 动力蛋白GTP酶通过包裹萌发小泡的颈部，在介导内吞作用中起着关键作用 催化从质膜中释放新生囊泡所必需的膜分裂。 最近，我们发现了Dynamin在捆绑大量肌动蛋白细丝中的一个新角色，这具有一定的意义 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 已经阐明了动力素组装和收缩膜的机制，有几个没有回答 问题仍然存在，包括Dynamin实际上是如何组织的并介导细胞内的分裂，如何激活 形成最终的裂变前状态，在那里它以超收缩状态包裹脂质小管，如何动态化 通过富含脯氨酸结构域(PRD)与底物结合，以及底物结合如何调节动力蛋白活性。这个 这种应用的长期目标是阐明动力蛋白介导的膜分裂的机制。 来自体外和体内的研究，并确定动力蛋白与肌动蛋白和SH3结构域相互作用的机制。 含有能将动力素募集到内吞作用部位的蛋白质。这些目标将由 具体目标如下：(1)确定超缩预裂变态中的动力素原子模型 (2)研究了动力蛋白螺旋聚合物在膜上的组装 以及(3)阐明PRD与肌动蛋白细丝和含有SH3结构域的相互作用的机制 有约束力的伙伴。这些目标的基本原理是：(1)没有原子模型来描述结构 动力素在自发性超收缩状态下将膜收缩到3.4 nm的基础 半分裂和膜分裂发生；(2)动力素在细胞中是如何组织的还没有报道； 以及(3)关键的PRD的结构以及它如何结合动力蛋白底物，包括肌动蛋白、两栖动物蛋白和 交点是未知的。研究设计和方法如下：目标1，应用低温电子 显微镜(冷冻-EM)以确定周围组织的全长Dynamin(包含PRD)的结构 超缩状态下的脂质小管；目标2，获得冷冻电子断层图像和亚断层图像平均值 在细胞内的动力蛋白的GTPase缺陷的动力蛋白突变体，延迟膜分裂和 延长动态蛋白螺旋在细胞膜上的寿命；目标3，获得肌动蛋白的冷冻-EM结构 用动力素中的PRD装饰的长丝，以及动力素/两栖动物素络合物和 动力/交叉点。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