ESTUDO DA PARTICIPAÇÃO DE CLATRINA NO TRÁFEGO INTRACELULAR DE VESÍCULAS EM TRYPANOSOMA CRUZI

The vesicular traffic mediated by clathrin - mechanism by which proteins and lipids are transported between membrane bound organelles - is responsible for a large proportion of internalization of plasma membrane (endocytosis) and transport from the Golgi Network Trans to the endosomal system. Clathrin-mediated events are not well known in Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In addition, studies of genome sequences show an extreme degree of divergence between trypanosomatids, animals and fungi, with about 1/3 of the predicted proteins with unknown function. In this study, gene expression and protein localization of the clathrin heavy (TcCHC) and light (TcCLC) chains were investigated in different T. cruzi developmental forms (epimastigotes, trypomastigotes and amastigotes), using polyclonal and monoclonal antibodies produced against T. cruzi recombinant proteins. Cellular localization of epsin (TcEpsin), an important protein associated with clathrin, was investigated through its fusion with GFP and subsequent immunolocalization. Analysis by confocal microscopy revealed an accumulation of TcEpsin, TcCHC and TcCLC in the anterior portion of the cells, where the flagellar pocket and Golgi complex are located. TcCLC partially co-localized with the Golgi marker TcRAB7-GFP and ingested albumin, but not co-located with transferrin, a protein endocytosed mainly via uncoated vesicles formed in the cytostome/cytopharynx complex. The clathrin light and heavy chains typically located at the anterior region of the kinetoplast and flagellar pocket, close to the Golgi apparatus. Our data indicate that in T. cruzi epimastigotes clathrin mediated endocytosis of albumin occurs in the flagellar pocket, while clathrin independent endocytosis of transferrin occurs in the cytostome/cytopharynx complex. For proteomics analysis of protein complexes associated with clathrin and epsin, the clathrin light chain was fused to proteins A and C and epsin was fused to GFP. Both proteins were then subjected to cryogrinding followed by immunoprecipitation of associated complexes. This methodology allowed the identification of associated proteins by mass spectrometry, such Adaptor Complexes AP-1 and AP-4, vSNAREs, Rab4, Rab11, epsin, tepsin, AP180, Auxilin and Scamp. Putative proteins were also demonstrated in this analysis. This methodology allowed us to suggest the way by which the vesicular traffic and the maturation of reservosomes occurs in T. cruzi. Reservosomes may accumulate functions of early endosome, late endosome and lysosome. Further analyzes are needed to confirm these hypothesis.