ENDOCYTOSIS AND RECYCLLING OF GPI-ANCHORED PROTEINS IN TRYPANOSOMA BRUCEI

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们用光学和电子显微镜描述了布氏毛滴虫对变异型表面糖蛋白(VSG)的内吞作用，VSG是这种寄生微生物的主要表面抗原。我们发表的工作描述了这种蛋白质内化的动力学和细胞内路线，我们的结果提出了一些令人惊讶的结论：(1)内体系统的不同亚室明显位于鞭毛袋(FP)、溶酶体和高尔基复合体之间的一个很小的体积内。(2)布氏毛滴虫的内吞作用和胞吐作用分别通过笼蛋白包裹的囊泡和Rab11阳性的胞外载体发生。(3)布氏毛滴虫的纹孔形成速度快于其他任何生物。(4)默认情况下，GPI锚定的蛋白质被分类到大池中。(5)高尔基体不参与面层回收。然而，由于结构本身的复杂性以及光和传统电子显微镜技术的分辨率限制，我们试图解决明显定位的内体亚室的3D组织的努力受挫。来自连续切片的初步数据表明，在VSG循环中存在非常大的有窗池，我们现在正在努力更详细地分析这种池与RAB5和Rab11阳性结构的关系。这些不同的隔间要么通过快速聚变/裂变事件进行通信，要么它们可能是连续的。我们现在已经收集了10张锥虫体内FP周围体积的双轴断层图像，这些图像标记了液相内吞作用的标志物和一些特定的内吞间隔的抗体。现在正在分析这些重建，以帮助我们了解使这种寄生虫如此成功地逃避宿主免疫反应的膜交通。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have used light and electron microscopy of T. brucei to describe its endocytosis of the variant surface glycoprotein (VSG), the major surface antigen of this parasitic microorganism. Our published work describes the kinetics and intracellular itinerary of this protein internalization, and our results suggest some surprising conclusions: (1) Different sub-compartments of the endosomal system are distinctly located within a tiny volume that lies between the flagellar pocket (FP), the lysosome and the Golgi complex. (2) Endoyctosis and exocytosis in T. brucei occur exclusively via clathrin-coated vesicles and Rab11-positive exocytic carriers, respectively. (3) Formation of clathrin-coated pits in T. brucei is faster than in any other organism. (4) GPI-anchored proteins are sorted by default into large cisternae. (5) The Golgi is not involved in surface coat recycling. Our attempts to resolve the 3D organization of the distinctly located endosomal sub-compartments has, however, been frustrated by the complexity of the structures themselves and the resolution limits of both light and conventional electron microscopic techniques. Preliminary data from serial sections has indicate the existence of very large fenestrated cisterna involved in VSG recycling, and we are now working to analyze the relationship of this cisterna with RAB5- and RAB11-positive structures in more detail. These different compartments either communicate by rapid fusion/fission events, or they may be continuous. We have now collected 10 dual-axis tomograms of the volume around the FP in trypanosomes labeled with markers for fluid-phase endocytosis and with antibodies to some specific endocytic compartments. These reconstructions are now being analyzed to help us understand the membrane traffic that allows this parasite to evade the host's immune response so successfully.