Abstract
The structural organization of Trypanosoma cruzi has been intensely investigated by different microscopy techniques. At the electron microscopy level, bi-dimensional analysis of thin sections of chemically fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation. In contrast, more sophisticated and elaborate techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. In addition, the 3D characterization of such cells has been carried out mostly using 3D reconstruction from serial sections, currently considered a low resolution technique when compared to electron tomography (ET). In this work, we re-visited the 3D ultrastructure of T. cruzi using a combination of two approaches: (1) analysis of both conventionally processed and cryofixed and freeze substituted cells and (2) 3D reconstruction of large volumes by serial electron tomography. The analysis of high-pressure frozen and freeze substituted parasites showed novel characteristics in a number of intracellular structures, both in their structure and content. Organelles generally showed a smooth and regular morphology in some cases presenting a characteristic electron dense content. Ribosomes and new microtubule sets showed an unexpected localization in the cell body. The improved preservation and imaging in 3D of T. cruzi cells using cryopreparation techniques has revealed some novel aspects of the ultrastructural organization of this parasite.
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Acknowledgments
We thank Lissa Catherine Reignault and Emile Barrias for the amastigote and trypomastigote forms of T. cruzi, Thiago Luiz de Barros Moreira for technical assistance, Dirceu Esdras for helping with the videos, and Ricardo Correia and Camilly Andrade for helping with the 3D models. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Financiadora de Estudos e Projetos (FINEP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Brazil).
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Supplementary Fig. 1 Serial electron tomography of an anterior region of an epimastigote in the G2 phase of the cell cycle. a-c Virtual sections at different planes showing the Golgi (G), multivesicular bodies (MB) and components of the CVC, such as vesicles, spongiome (Sp) and central vacuole (CV). The electron dense region between the CV and flagellar pocket could be observed (arrow in c). d-f 3D model showing the different organelles in the vicinity of the flagellar pocket. The trans Golgi was in close proximity with the vesicles (arrowheads in e) that connected the CV (arrow in e). The tubules of spongiome could be observed concentrated in the anterior region of the CV (arrows in f). The asterisks show an additional vacuole that connects to the CVC. Scale bars: 200 nm (TIFF 2938 kb)
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Supplementary Fig. 2 Multivesicular bodies in epimastigotes. a Virtual section showing internal vesicles (arrows) in a multivesicular body (MB). c-d 3D models of the multivesicular body membrane (yellow) and its internal vesicles (red). Scale bar: 100 nm (TIFF 1575 kb)
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Supplementary Fig. 3 Microtubules in chemically fixed epimastigotes. a Virtual section from a tomogram of a CF epimastigote used for segmentation. b-e 3D model and virtual slices showing single microtubules (arrows) in different locations and the microtubule quartet (arrowheads) around the flagellar pocket. The numbers are correspondent to microtubules seen in Fig. 6. Mitochondrion (green), single microtubules (red), microtubule quartet (dark blue), Nucleus and kinetoplast DNA (blue), surface membrane (white) and flagellum (brown). (F) flagellum, (K) kinetoplast, (G) Golgi. Scale bars: 200 nm (TIFF 12715 kb)
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Supplementary Fig. 4 Morphological details in the mitochondrion of cryofixed cells. a-d 3D model and virtual slices of a tomogram showing a single microtubule (arrowheads) passing through an opening in the mitochondrion (arrow) of an amastigote. The numbers are correspondent to the microtubules seen in Fig. 6. Mitochondrion (green), single microtubules (red), array of microtubules (yellow), subpellicular microtubules (purple), microtubule quartet (dark blue), Nucleus and kinetoplast DNA (blue), surface membrane (white) and flagellum (brown). Scale bars: 200 nm (TIFF 4649 kb)
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Supplementary Video 1 Tomogram showing a region of the Golgi cisternae in a 200 nm thick section, completely filled by an electron dense content as seen through the virtual sections (MPG 2010 kb)
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Supplementary Video 2 3D model of some clustered ribosomes (pink) in the anterior region of an epimastigote (MPG 2895 kb)
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Supplementary Video 5 Tomogram and 3D model (yellow) showing the array of microtubules running through the cell body (arrows) (MPG 2970 kb)
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Supplementary Video 6 3D model of different organelles in amastigotes showing a single microtubule crossing through a tunnel formed in the mitochondrion. Mitochondrion (green), single microtubules (red), array of microtubules (yellow), subpellicular microtubules (purple), microtubule quartet (dark blue), Nucleus and kinetoplast DNA (blue), surface membrane (white) and flagellum (brown) (MPG 3194 kb)
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Girard-Dias, W., Alcântara, C.L., Cunha-e-Silva, N. et al. On the ultrastructural organization of Trypanosoma cruzi using cryopreparation methods and electron tomography. Histochem Cell Biol 138, 821–831 (2012). https://doi.org/10.1007/s00418-012-1002-8
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DOI: https://doi.org/10.1007/s00418-012-1002-8