ENDOCYTOSIS AND RECYCLLING OF GPI-ANCHORED PROTEINS IN TRYPANOSOMA BRUCEI

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们利用光镜和电镜观察了布鲁氏菌对其主要表面抗原变异表面糖蛋白（VSG）的内吞。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)内体系统的不同亚室明显位于鞭毛袋（FP），溶酶体和高尔基复合体之间的微小体积内。(2)布鲁氏体的胞吞作用和胞吐作用分别只通过网格蛋白包被的囊泡和rab11阳性的胞吐载体发生。(3)布氏体中网格蛋白包覆坑的形成速度比其他任何生物都要快。(4) gpi锚定蛋白默认被分类到大池中。(5)高尔基体不参与表面涂层的回收。然而，由于结构本身的复杂性以及光学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。来自连续切片的初步数据表明，存在参与VSG回收的非常大的开孔池，我们现在正在更详细地分析该池与RAB5-和rab11阳性结构的关系。这些不同的隔室要么通过快速的聚变/裂变事件进行通信，要么它们可能是连续的。我们现在收集了10张双轴断层图，显示锥虫FP周围的体积，这些体积被标记为液相内吞作用的标记物和一些特定内吞区室的抗体。现在正在分析这些重建，以帮助我们了解允许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.