RESUMO
The flagellum and flagellum attachment zone (FAZ) are important cytoskeletal structures in trypanosomatids, being required for motility, cell division and cell morphogenesis. Trypanosomatid cytoskeletons contain abundant high molecular mass proteins (HMMPs), but many of their biological functions are still unclear. Here, we report the characterization of the giant FAZ protein, FAZ10, in Trypanosoma brucei, which, using immunoelectron microscopy, we show localizes to the intermembrane staples in the FAZ intracellular domain. Our data show that FAZ10 is a giant cytoskeletal protein essential for normal growth and morphology in both procyclic and bloodstream parasite life cycle stages, with its depletion leading to defects in cell morphogenesis, flagellum attachment, and kinetoplast and nucleus positioning. We show that the flagellum attachment defects are probably brought about by reduced tethering of the proximal domain of the paraflagellar rod to the FAZ filament. Further, FAZ10 depletion also reduces abundance of FAZ flagellum domain protein, ClpGM6. Moreover, ablation of FAZ10 impaired the timing and placement of the cleavage furrow during cytokinesis, resulting in premature or asymmetrical cell division.
Assuntos
Citocinese , Flagelos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Posicionamento Cromossômico , Segregação de Cromossomos , Proteínas do Citoesqueleto/metabolismo , Flagelos/ultraestrutura , Técnicas de Silenciamento de Genes , Trypanosoma brucei brucei/ultraestruturaRESUMO
Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.
Assuntos
Endocitose , Eucariotos , Animais , Entamoeba/metabolismo , Entamoeba/fisiologia , Entamoeba/ultraestrutura , Eucariotos/metabolismo , Eucariotos/fisiologia , Eucariotos/ultraestrutura , Giardia/metabolismo , Giardia/fisiologia , Giardia/ultraestrutura , Histocitoquímica , Leishmania/metabolismo , Leishmania/fisiologia , Leishmania/ultraestrutura , Microscopia Eletrônica , Plasmodium/metabolismo , Plasmodium/fisiologia , Plasmodium/ultraestrutura , Trichomonas/metabolismo , Trichomonas/fisiologia , Trichomonas/ultraestrutura , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/fisiologia , Trypanosoma brucei brucei/ultraestrutura , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiologia , Trypanosoma cruzi/ultraestruturaRESUMO
The association of high resolution field emission scanning electron microscopy (FESEM), with a more efficient system of secondary electron (SE) collection and in-lens specimen position, provided a great improvement in the specimen's topographical contrast and in the generation of high-resolution images. In addition, images obtained with the use of the high-resolution backscattered electrons (BSE) detector provided a powerful tool for immunocytochemical analysis of biological material. In this work, we show the contribution of the FESEM to the detailed description of cytoskeletal structures of the protozoan parasites Herpetomonas megaseliae, Trypanosoma brucei and Giardia lamblia. High-resolution images of detergent extracted H. megaseliae and T. brucei showed the profile of the cortical microtubules, also known as sub-pellicular microtubules (SPMT), and protein bridges cross-linking them. Also, it was possible to visualize fine details of the filaments that form the lattice-like structure of the paraflagellar rod (PFR) and its connection with the axoneme. In G. lamblia, it was possible to observe the intricate structure of the adhesive disk, funis (a microtubular array) and other cytoskeletal structures poorly described previously. Since most of the stable cytoskeletal structures of this protozoan rely on tubulin, we used the BSE images to accurately map immunolabeled tubulin in its cytoskeleton. Our results suggest that the observation of detergent extracted parasites using FESEM associated to backscattered analysis of immunolabeled specimens represents a new approach for the study of parasite cytoskeletal elements and their protein associations.