Untersuchung von Endozytose und Aktinzytoskelett in Säugerzellen

点击翻译按钮获取中文摘要

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.