Molekulare Analyse von schneller Endozytose und Plasmamembran-Recycling im Modell-Eukaryoten Trypanosoma brucei
Zusammenfassung der Projektergebnisse
Trypanosomes are unicellular parasites that infect humans and cattle. The flagellate protozoa have evolved a battery of defence strategies against immune attack by the mammalian host. The extremely high rate of endocytosis is a prime example and has been in the focus of the studies that have been supported by DFG. We have worked on all seven key-questions that had been identified in the original proposal. (1) We asked how the fast endocytic traffic in trypanosomes can be exclusively handled by clathrin. Our working hypothesis was that negative regulators of clathrin vesicle assembly and budding could either be missing in trypanosomes or have lost its functionality. We found that the clathrin light chain, although expressed in trypanosomes, in fact does not fulfil a negative regulatory role as in other eukaryotes. Furthermore, the adapter protein AP-2 is missing and AP180 and epsin have redundant functions in the parasites. We have generated a comprehensive quantitative proteome of clathrin vesicles in T. brucei, which sheds light on various aspects of endocytic control. (2) All endocytic traffic has to pass a tiny invagination on the trypanosome cell surface, the flagellar pocket. We postulated that the pocket membrane must be functionally polarized. In fact, the exocytic carriers that constitute the recycling branch of the endocytic apparatus, exclusively fuse to a specific membrane area of the pocket. Likewise, molecular markers for recycling endosomes, such as Rab11, were specifically detectable here. The experiments were backed up by electron tomography and RNAi depletion of trafficking regulators. (3) Highresolution electron tomography and correlative microscopy (CLEM) also allowed addressing the problem of how the variant surface glycoproteins (VSGs) can be passaged through endosomal subcompartments with a kinetics that is not compatible with successive membrane fission and fusion steps. In trypanosomes early and recycling endosomes are part of the same giant and complex endomembrane system that mainly consist of flat, fenestrated membrane sheets and cisternae. (4) The diameter of endosomal cisternae is very small, with only a few nanometres between the opposing VSG layers. This explains why large cargo accumulates at the rims of the endosome, where it is packed into lysosome-bound clathrin vesicles. (5) We also found that VSGs are optimally structured for fast diffusion on the cell surface, allowing randomization of the recycled VSGs within the cell surface coat. (6) Randomization and free mobility of VSG on the cell surface is crucial for antibody clearance, a unique process, by which the parasites exploit hydrodynamic drag generated by the incessantly swimming parasites to remove host antibodies. We found that trypanosome motility is optimally suited for guaranteeing efficient redistribution of antibody-complexed VSG on the trypananosome cell surface. (7) Lastly, we considered the problem that the shielding function of the highly dynamic VSG coat has to be maintained during the cell division cycle and in the event of an antigenic switch. We found that in trypanosomes a complete new endosome is synthesized, however, remains functionally silent until very late in the the cell cycle. Only when the VSG coat has been duplicated, the new endosome is activated and the cell divides. In summary, we have made significant progress along all line of he original proposal. One major aim was to provide an unbiased blueprint of the trypanosome endocytosis apparatus. This goal has not been achieved, in part because of limiting resources available for the project, but also because a set of quantitative proteomes were published soon after the project was launched.
Projektbezogene Publikationen (Auswahl)
- (2005) Binding affinity and capacity of putative adaptor-mediated sorting of a Type I membrane protein in Leishmania mexicana. Mol Biochem Parasitol 142: 203–211
Weise F, Thilo L, Engstler M, Wiese M, Benzel I, Kühn C, Bühring H-J, Overath P
- (2005) The membrane-bound histidine acid phosphatase TbMBAP1 is essential for endocytosis and membrane recycling in Trypanosoma brucei. J Cell Sci 118: 2105–2118
Engstler M, Weise F, Bopp K, Grünfelder CG, Günzel M, Heddergott N, Overath P
- (2005) Trypanosomes change their transferrin receptor expression to allow effective uptake of host transferrin. Mol Microbiol 58: 151–165
van Luenen HGAM, Kieft R, Mussmann R, Engstler M, ter Riet B, Borst P
- (2006) Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest. J Cell Sci 119: 2968–2974
Chanez A-L, Hehl AB, Engstler M, Schneider A
- (2007) Hydrodynamic flow-mediated protein sorting on the cell surface of trypanosomes. Cell 131: 505–515
Engstler M, Pfohl T, Herminghaus S, Boshart M, Wiegertjes G, Heddergott N, Overath P
- (2008) Role of protein translocation pathways across the endoplasmic reticulum in Trypanosoma brucei. J Biol Chem 283: 32085–32098
Goldshmidt H, Sheiner L, Bütikofer P, Roditi I, Uliel S, Günzel M, Engstler M, Michaeli S
- (2009) Macromolecular trafficking and immune evasion in African trypanosomes. Int Rev Cell Mol Biol 278: 1–67
Field MC, Lumb JH, Adung’a VO, Jones NG, Engstler M
- (2010) Depletion of 14-3-3 proteins in bloodstream-form Trypanosoma brucei inhibits variant surface glycoprotein recycling. Int J Parasitol 40: 629–634
Benz C, Engstler M, Hillmer S, Clayton C
- (2011) Impact of microscopic motility on the swimming behavior of parasites: straighter trypanosomes are more directional. PLoS Comput Biol 7: e1002058
Uppaluri S, Nagler J, Stellamanns E, Heddergott N, Herminghaus S, Engstler M, Pfohl T
- (2012) Trypanosome motion represents an adaptation to the crowded environment of the vertebrate bloodstream. PLoS Pathog 8: e1003023
Heddergott N, Krüger T, Babu SB, Wei A, Stellamanns E, Uppaluri S, Pfohl T, Stark H, Engstler M
(Siehe online unter https://doi.org/10.1371/journal.ppat.1003023)
