Trypanosoma cruzi: Contribution to the study of the dependent and independent endocytosis of clatrina in epimastigote forms.
Endocytosis in eukaryotic cells is the incorporation process ofmacromolecules, through different pathways with different associated proteins,through vesicles budding at the plasma membrane and addressing of these vesiclesto cytoplasmic endosomal compartments. Trypanosomatids are pathogenic flagellateprotozoa of great medical and veterinary importance that present different evolutiveforms along their life cycle. These parasites are structurally organized inside a cageof subpelicular microtubules, what makes it difficult to form membrane invaginationalong most part of the cell body. However, this cage is missing at the flagellar pocketregion, where the flagellum emerges from the cell. Epimastigote forms ofTrypanosoma cruzi present besides the flagellar pocket a second membraneinvagination, the cytostome/cytopharynx. This structure is sustained by specializedmicrotubules that actively participate in the endocytic process. This thesis hasinvestigated the endocytic pathways at the two competent sites for nutrient uptake(flagellar pocket and cytostome) of T. cruzi epimastigotes. After parasites incubationat 28oC with gold-conjugated albumine, transferrin or LDL, followed by processing fortransmission electron microscopy (TEM), it was possible to observe coated endocyticvesicles loaded with albumin, budding off from the flagellar pocket. Analysis of theproteic content of epimastigote forms detected the expression of clathrin, confirmedby flow cytometry and in silico analysis of the T. cruzi genomic database. Confocalmicroscopy allowed the visualization of the clathrin expression at the flagellar pocket.As transferrin was seen in uncoated vesicles located mainly in the cytostome,detergent-resistant membrane fractions were purified by cell fractioning and thelipidic contents was analyzed by chromatography, as well as the protein andcholesterol contents in the fractions. Dot blots of such membrane fractions werepositive for lipid raft universal labels (flotillin-1 and cholera B toxin). Byimmunofluorescence microscopy, transferrin and flotillin were co-localized at thecytostome. Thus we propose that endocytosis of transferrin occurs mainly throughthe cytostome via detergent-resistant membrane domains. Epimastigote forms werepre-treated to specifically hinder clathrin-mediated and caveolae-mediatedendocytosis, by using drugs that impair these pathways and interfere with the cellcytoskeleton, followed by incubation with transferrin. Data from TEM and flowcytometry showed that the endocytosis of transferrin occurred normally in sampleswhere budding of clathrin-coated vesicles was hindered, but it was not observed insamples where caveolae-mediated endocytosis was inhibited. Analysis of growthcurves of treated parasites showed a relation between interference with detergentresistantmembrane domains and cell surviving. Our data allowed to elaborate amodel depicting the endocytic mechanisms in Trypanosoma cruzi epimastigotes.
Advisor:Maurilio José Soares
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Trypanosoma cruzi Endocitose Clatrina Endocytosis Transferrin Membrane Lipids Clathrin Eukaryotic Cells
Date of Publication:09/18/2007