This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Special Emphasis Panel (ZRG1-CB-J (40))
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University of Colorado at Boulder
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Giddings Jr, Thomas H; Morphew, Mary K; McIntosh, J Richard (2017) Preparing Fission Yeast for Electron Microscopy. Cold Spring Harb Protoc 2017:pdb.prot091314
Zhao, Xiaowei; Schwartz, Cindi L; Pierson, Jason et al. (2017) Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium ""Candidatus Pelagibacter ubique"". Appl Environ Microbiol 83:
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