Role of dynamin-auxilin interactions in endocytosis

描述(由申请人提供)：在质膜上形成被膜蛋白包裹的囊泡是由膜蛋白介导的内吞过程的基础，细胞通过该过程内化受体、跨膜通道、转运体、激素、生长因子和营养物质。内吞作用的错误调节可导致肿瘤转化。因此，胞苷介导的内吞作用的机制和调控对人类健康具有重要意义。内吞作用的早期事件包括形成包被网状蛋白的小坑、小坑内陷、收缩和游离囊的萌发。内吞作用所需的基本因子之一是动力蛋白，这是一种不寻常的GTP酶，其作用机制尚不清楚。最近的证据表明，Dynamin是内吞作用的主要调节者，利用其GTPase循环招募执行内陷囊泡收缩的蛋白质机械。这个实验室现在已经确定Hsc70和它的辅助伴侣生长素是两种与Dynamin特异相互作用的蛋白质：GTP。已经绘制了与动力蛋白相互作用的生长素中的两个结构域，从体外内吞试验中免疫耗竭生长素可以抑制收缩小泡的形成。这些结果与Hsc70/Axlin是动力蛋白效应器的结果一致。重要的是，已知Hsc70/avinin可以催化从游离囊泡中去除网状蛋白涂层。因此，生长素和Hsc70在内吞作用的早期(囊泡形成)和晚期(囊泡去涂层)起作用。结果表明，在该模型中，胞吞作用的许多阶段或全部阶段是由Hsc70/生长素介导的网状蛋白外壳重排驱动的。可以预见，Hsc70/avinin对笼状蛋白涂层的特定影响是由与Dynamin的相互作用调节的。这一建议解决了以下问题：特殊目的1使用电子显微镜和依赖于生长素的体外内吞试验相结合的方法来确定需要生长素的笼蛋白包裹的囊泡形成中的所有步骤(起始、内陷、收缩、分裂)。在特定的目标2中，缺乏生长素结合的动力素突变体将被用来证实生长素是动力素效应器的模型。具体目的3通过测试动力素的严重组装突变体是否支持内吞作用来检查动力素自组装的功能。突变体的组装缺陷将在体内使用荧光寿命成像显微镜(Flim)进行确认。具体目标4建立在发现生长素与脂质结合的基础上，并将测试生长素是否作为一种脂类磷酸酶。在内吞作用中，将使用半通透性细胞试验来检测由生长素引起的脂质结合/磷酸酶活性的作用。

DESCRIPTION (provided by applicant): Clathrin-coated vesicle formation at the plasma membrane underlies the process of clathrin-mediated endocytosis by which the cell internalizes receptors, transmembrane channels, transporters, hormones growth factors and nutrients. Mis-regulation of endocytosis can cause neoplastic transformation. Therefore, the mechanism and regulation of clathrin-mediated endocytosis are of critical importance for human health. The early events of endocytosis involve formation of clathrin-coated pits, pit invagination, constriction and budding of the free vesicle. One of the essential factors required for endocytosis is dynamin, an unusual GTPase whose mechanism of action remains uncertain. Recent evidence suggests that dynamin is a master regulator of endocytosis, using its GTPase cycle to recruit the protein machinery that executes the constriction of invaginated vesicles. This laboratory has now identified Hsc70 and its co-chaperone auxilin as two proteins that specifically interact with dynamin:GTP. Two domains within auxilin that interact with dynamin have been mapped, and immunodepletion of auxilin from an in vitro endocytosis assay inhibits formation of constricted vesicles. These results are consistent with Hsc70/auxilin being dynamin effector. Importantly, Hsc70/auxilin was already known to catalyze removal of the clathrin coat from the free vesicle. Therefore, auxilin and Hsc70 act at early (vesicle formation) and late (vesicle uncoating) stages of endocytosis. The results suggest a model in which many, or perhaps all, stages of enocytosis are driven by Hsc70/auxilin-mediated rearrangements of the clathrin coat. It is envisioned that the specific consequences of Hsc70/auxilin on the clathrin coat are regulated by interactions with dynamin. This proposal addresses the following questions: Specific Aim 1 uses a combination of electron microscopy and an auxilin-dependent in vitro endocytosis assay to identify all the steps (initiation, invagination, constriction, fission) in clathrin-coated vesicle formation that require auxilin. In Specific Aim 2, mutants of dynamin that are deficient in auxilin binding will be used to corroborate the model that auxilin is a dynamin effector. Specific Aim 3 examines the function of dynamin self-assembly by testing whether severe assembly mutants of dynamin support endocytosis. The assembly defects of the mutants will be confirmed in vivo using fluorescence lifetime imaging microscopy (FLIM). Specific Aim 4 builds on the finding that auxilin binds lipids and will test whether auxilin acts as a lipid phosphatase. The function of lipid binding/phosphatase activity by auxilin in endocytosis will be examined using a semi-permeable cell assay.