Untersuchung von Endozytose und Aktinzytoskelett in Säugerzellen

哺乳动物细胞内吞作用和肌动蛋白细胞骨架的研究

• 批准号: 25167550
• 负责人: Dr. Barbara Pauly
• 金额: --
• 依托单位: Department of Molecular and Cell Biology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2006
• 资助国家: 德国
• 起止时间: 2005-12-31 至 2008-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/25167550?language=en
• 关键词: Untersuchung; von; Endozytose; und; Aktinzytoskelett

Cells internalize extracellular material by a process called endocytosis. Clathrinmediated endocytosis involves the assembly of a clathrin coat, formation and release of the vesicle into the cytoplasm, and targeting of the vesicle to specific sites within the cell. Besides the key player clathrin, a large number of accessory proteins have been identified that are essential for endocytosis. In addition, it is now well established that the actin cytoskeleton plays a critical role in this process. Actin and actin-regulating proteins like the Arp2/3 complex, cortactin, and N-WASP are localized at endocytic sites. We have recently shown that cortactin and its binding partner Hip1R, a clathrin and actin binding protein, negatively regulate actin assembly at endocytic sites through formation of an actin filament capping complex. On the other hand, cortactin alone can activate the Arp2/3 complex which then leads to assembly of actin into filaments. An important question is therefore how cortactin’s capping activity and its Arp2/3-activating activity are regulated to ensure proper timing and spacing of actin assembly at endocytic sites. Proteins of the WIP (Wasp-interacting protein) family are potential regulators of cortactin. To elucidate the role of these important actin-regulating proteins I will therefore study the role of WIP proteins in controlling cortactin activity during endocytosis. In addition, I will continue to develop an in vitro system for endocytosis in which endocytic vesicles form on the cytoplasmic surface of biological membranes exposed to the medium by “unroofing” cells. I will use this system to investigate the biochemical requirements for endocytosis. I will also investigate the effect of actin polymerization and the influence of actin-regulating proteins like Hip1R, cortactin and WIP on actin assembly and vesicle budding in this in vitro system to complement the in vivo studies described above. The usefulness of such an in vitro system, however, is not limited to studying the proteins proposed here. In the future, it will also allow us to study the role of other proteins known or believed to be involved in endocytosis.

细胞通过内吞作用将细胞外物质内化。网格蛋白介导的内吞作用涉及网格蛋白外壳的组装、囊泡的形成和释放到细胞质中以及囊泡靶向细胞内的特定位点。除了主要参与者网格蛋白外，已经鉴定了大量对内吞作用至关重要的辅助蛋白。此外，现在已经确定肌动蛋白细胞骨架在这一过程中起着关键作用。肌动蛋白和肌动蛋白调节蛋白，如Arp 2/3复合物，corneum和N-WASP定位于内吞位点。我们最近发现，corneum和它的结合伙伴Hip 1 R，网格蛋白和肌动蛋白结合蛋白，负调节肌动蛋白组装在内吞位点通过形成的肌动蛋白丝帽复合物。另一方面，单独的corneum可以激活Arp 2/3复合物，然后导致肌动蛋白组装成细丝。因此，一个重要的问题是如何调节corneumn的加帽活性和Arp 2/3激活活性，以确保在内吞位点的肌动蛋白组装的适当时间和间隔。黄蜂相互作用蛋白（Wasp interacting protein，WASP）家族的蛋白质是coronin的潜在调节因子。为了阐明这些重要的肌动蛋白调节蛋白的作用，我将因此研究的作用，β蛋白在控制corpegrin活性在胞吞作用。此外，我将继续开发一种用于内吞作用的体外系统，其中内吞囊泡通过“去屋顶”细胞在暴露于介质的生物膜的细胞质表面上形成。我将用这个系统来研究内吞作用的生化要求。我还将研究肌动蛋白聚合的影响和肌动蛋白调节蛋白如Hip 1 R，corneumn和actin在这个体外系统中的肌动蛋白组装和囊泡出芽的影响，以补充上述的体内研究。然而，这种体外系统的有用性并不限于研究这里提出的蛋白质。在未来，它也将使我们能够研究其他已知或被认为参与内吞作用的蛋白质的作用。