Untersuchung von Endozytose und Aktinzytoskelett in Säugerzellen

细胞通过内吞作用使细胞外物质内化。网格蛋白介导的内吞作用包括网格蛋白外壳的组装，囊泡的形成和释放到细胞质中，以及将囊泡靶向到细胞内的特定位置。除了关键的网格蛋白外，大量的辅助蛋白已被确定为内吞作用所必需的。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Arp2/3复合物、内联蛋白和N-WASP定位于内吞位点。我们最近发现，cortacn及其结合伙伴Hip1R（一种网格蛋白和肌动蛋白结合蛋白）通过形成肌动蛋白丝盖复合体负向调节肌动蛋白在内吞位点的组装。另一方面，单独接触可以激活Arp2/3复合体，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环的封顶活性及其arp2 /3激活活性，以确保肌动蛋白在内吞位点组装的适当时间和间隔。WIP（黄蜂相互作用蛋白）家族的蛋白质是潜在的接触调节因子。为了阐明这些重要的肌动蛋白调节蛋白的作用，我将研究WIP蛋白在内吞过程中控制接触活性的作用。此外，我将继续开发一种体外系统的内吞作用，其中内吞囊泡形成的生物膜的细胞质表面上暴露在介质中“脱顶”的细胞。我将使用这个系统来研究内吞作用的生化要求。我还将在体外系统中研究肌动蛋白聚合的作用以及肌动蛋白调节蛋白如Hip1R、cortinn和WIP对肌动蛋白组装和囊泡出芽的影响，以补充上述体内研究。然而，这种体外系统的有用性并不局限于研究这里提出的蛋白质。在未来，它还将使我们能够研究已知或被认为参与内吞作用的其他蛋白质的作用。

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.