molecular analysis of rapid endocytosis and plasma membrane recycling in the model eukaryote Trypanosoma brucei

非洲锥虫是引起人类昏睡病的细胞外寄生虫。他们成功的关键是能够从哺乳动物宿主中内吞必需的大分子，而不会暴露免疫系统的注意，这导致了一个非凡的膜运输系统的进化。独特的是，锥虫的内吞作用是高度极化的，并且只发生在膜运输的胞外旋臂的末端：鞭毛袋。虽然这个位点只占总表面的不到5%，但这里的膜流量远远超过任何其他真核生物。与此同时，该途径允许大量糖基磷酸肌醇锚定蛋白的差异分类、内化和再循环。这种最近才被描述的途径的分子基础仍然难以捉摸。我们提出了一种系统的方法来研究布氏体核内体的分子特征，该方法利用RNA干扰、高分辨率3D显微镜、分离核内体的新方法、同位素标记方法和质谱法。这个强大的屏幕专注于关于内吞作用和脂质锚定蛋白循环的重要，一般问题。锥虫内吞机制的分子定量不仅提供了一个独特的机会来加深我们对膜交通的基本理解，而且还可能挑战致命的病原体。

African trypanosomes are extracellular parasites that cause human sleeping sickness. Key to their success has been the ability to endocytose essential macromolecules from the mammalian host without exposing the machinery involved to the attention of the immune system, which has led to the evolution of an extraordinary system for membrane traffic. Uniquely, endocytosis in trypanosomes is highly polarized and occurs solely at the terminus of the exocytotic arm of membrane traffic: the flagellar pocket. Although this site represents less than 5% of the total surface, membrane traffic here far exceeds that of any other eukaryote. Concurrent with this huge traffic the pathway allows the differential sorting, internalization and recycling of abundant glycosylphosphoinositol anchored proteins. The molecular basis for this only recently described pathway remains elusive. We propose a systematic approach to the molecular characterization of the T. brucei endosome that exploits RNA interference, high-resolution 3D microscopy, novel methodology for isolation of endosomes, isotope-tagging methods and mass spectrometry. This powerful screen focuses on important, general questions regarding endocytosis and recycling of lipid-anchored proteins. The molecular quantification of the trypanosome endocytosis machinery not only presents a unique opportunity to deepen our understanding of membrane traffic at a fundamental level, but may also challenge a fatal pathogen.