ENDOCYTOSIS AND RECYCLLING OF GPI-ANCHORED PROTEINS IN TRYPANOSOMA BRUCEI

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 我们用光镜和电镜观察了T. brucei描述了其对变异表面糖蛋白（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行程，我们的结果提出了一些令人惊讶的结论：（1）内体系统的不同亚区室明显位于鞭毛口袋（FP），溶酶体和高尔基复合体之间的一个微小的体积内。(2)T.布氏杆菌分别通过网格蛋白包被的囊泡和Rab 11阳性的胞吐载体发生。(3)T.布氏杆菌比其他任何生物都快。(4)GPI锚定的蛋白质默认被分选到大池中。(5)高尔基体不参与表面涂层的回收。然而，我们试图解决的3D组织的明确定位的内体亚隔间，被挫败的结构本身的复杂性和光和传统的电子显微镜技术的分辨率限制。 来自连续切片的初步数据表明存在非常大的有孔池参与VSG再循环，我们现在正在更详细地分析该池与RAB 5和RAB 11阳性结构的关系。 这些不同的隔间通过快速的聚变/裂变事件进行通信，或者它们可以是连续的。 我们现在已经收集了10个双轴断层扫描的体积周围的FP在锥虫标记的液相内吞作用和抗体的一些特定的内吞隔间。 这些重建现在正在分析，以帮助我们了解膜交通，使这种寄生虫逃避宿主的免疫反应如此成功。

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.