RESUMO
In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy.
Assuntos
Ambystoma mexicanum/anatomia & histologia , Microscopia Eletrônica/métodos , Xenopus laevis/anatomia & histologia , Ambystoma mexicanum/embriologia , Animais , Embrião não Mamífero/ultraestrutura , Substituição ao Congelamento/métodos , Imuno-Histoquímica/métodos , Microscopia Eletrônica/instrumentação , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos , Xenopus laevis/embriologiaRESUMO
The hydrogenosome, an organelle that produces molecular hydrogen and ATP from the oxidation of pyruvate or malate under anaerobic conditions, presents some characteristics common to mitochondria. The hydrogenosome of Tritrichomonas foetus, a cattle parasite, is a spherical organelle that presents a peripheral vesicle the origin and behavior of which is poorly known. In this article it is reported an ultrastructural and microanalytical study using energy dispersive X-ray analysis, 3D reconstruction and cytochemistry of the hydrogenosome peripheral vesicle and then compare the results with the endoplasmic reticulum and the nuclear envelope of T. foetus. Similarities between the hydrogenosome peripheral vesicle and the ER are presented. This study included: (1) the detection of ER enzymes by cytochemistry, such as glucose-6-phosphatase, IDPase, acid phosphatase and Ca(2+) -ATPase; (2) elemental composition by X-ray microanalysis and the mapping of calcium, phosphorus and oxygen in both ER and hydrogenosome peripheral vesicle; (3) freeze-fracture; (4) TEM of routine and cryofixed cells by high-pressure freezing and freeze-substitution; (5) 3D reconstruction, (6) monoclonal antibody anti-trichomonads ER; and (6) other cytochemical techniques that detects ER, such as the ZIO and lectins. We found a similar composition of the tested enzymes and other elements present in the ER when compared with the hydrogenosome's peripheral vesicle. It was concluded that, like mitochondria, hydrogenosome presents relationships with the ER, especially the peripheral vesicle.
Assuntos
Vesículas Citoplasmáticas/ultraestrutura , Hidrogênio/metabolismo , Animais , Bovinos , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/fisiologia , Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/ultraestrutura , Substituição ao Congelamento , Histocitoquímica , Tritrichomonas foetus/metabolismo , Tritrichomonas foetus/fisiologia , Tritrichomonas foetus/ultraestruturaRESUMO
We present observations on the fine structure and the division process of the Golgi complex in the protists Trichomonas vaginalis and Tritrichomonas foetus, parasites of the urogenital tract of humans and cattle, respectively. The Golgi in trichomonads is a prominent structure, associated with striated parabasal filaments to which this organelle seems to be connected. We followed by immunofluorescence and electron microscopy the Golgi in interphasic and mitotic cells. Ultrastructural studies were performed using fast-freezing fixation, immunocytochemistry using antisera to the known adhesins AP65 and AP51, cytochemistry (acid phosphatase, Ca++-ATPase, zinc iodide-osmium tetroxide technique (ZIO), for analysis of distribution of the endoplasmic reticulum and Golgi complex, and Thiéry's techniques), routine and serial thin-sections. Three-dimensional reconstruction, NBD-ceramide, fluorescent lectin (WGA) and nocodazole treatments were also used. We demonstrate that: (1) the Golgi in trichomonads is a single-copy organelle; (2) presents a fenestrated structure; (3) is formed by 8-12 saccules; (4) is connected to the parabasal filaments by thin filamentous bridges; (5) by cytochemistry, presents a positive reaction for the lectin WGA, Ca++-ATPase, acid phosphatase, ZIO and Thiéry's techniques; (6) does not appear to break down at any point of the cell cycle; (7) elongates during the cell cycle by lateral growth; (8) is labeled by anti-glutamylated tubulin antibodies, but it is not fragmented by nocodazole treatment; (9) before mitosis, the already elongated Golgi ribbon undergoes progressive medial fission, cisternae by cisternae, starting at the cisternae adjacent to the cell surface and ending with the cis-most cisternae; (10) the Golgikinesis originates two small Golgi ribbons; (11) the Golgi is intensely labeled with the antisera to the AP65 and AP51 adhesins in T. vaginalis, thus seeming to be a key station in the production of adhesins.
Assuntos
Complexo de Golgi/ultraestrutura , Trichomonas vaginalis/ultraestrutura , Tritrichomonas foetus/ultraestrutura , Fosfatase Ácida/metabolismo , Animais , ATPases Transportadoras de Cálcio/metabolismo , Bovinos , Ciclo Celular/fisiologia , Simulação por Computador , Técnica de Fratura por Congelamento , Substituição ao Congelamento , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Humanos , Trichomonas vaginalis/metabolismo , Tritrichomonas foetus/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
The hydrogenosome, an organelle that produces molecular hydrogen and ATP from the oxidation of pyruvate or malate under anaerobic conditions, presents some characteristics common to mitochondria. It is found in several trichomonad species, protists living in oxygen-poor environments, as well as certain free-living ciliates, rumen ciliates, and some fungi. We performed a comparative microanalytical study (energy dispersive X-ray analysis and electron spectroscopic imaging) of different fixation methods for electron microscopy analyzing hydrogenosomes of the bovine parasite Tritrichomonas foetus. The study included the elemental composition and the mapping of calcium, phosphorus, and oxygen. A preparation of T. foetus cells, based on cryoimmobilization by high-pressure freezing and freeze-substitution, was compared to a second preparation based on chemical fixation followed by dehydration and routine processing. The ultrastructural preservation achieved by the cryotechnique was far superior to the chemical fixation, since it allowed the successful cryoimmobilization of intracellular ion contents. The detection of several cations (Al, Mg, Co, Ca, Fe) by X-ray microanalysis inside the peripheral vesicle of the hydrogenosome was only possible in cryofixed cells. The presence of aluminum and cobalt ion in the hydrogenosomal vesicle was established for the first time. Electron-spectroscopic images of calcium showed that this element, in addition to the vesicle compartment, is present in the hydrogenosome's membrane in varying concentrations, which might reflect changes in the physiology of this organelle.
Assuntos
Hidrogênio/metabolismo , Organelas/ultraestrutura , Tritrichomonas foetus/ultraestrutura , Animais , Criopreservação/métodos , Microanálise por Sonda Eletrônica/métodos , Substituição ao Congelamento , Microscopia Eletrônica/métodos , Organelas/química , Tritrichomonas foetus/fisiologiaRESUMO
We have applied three-dimensional helical reconstruction techniques to images of myosin filaments of tarantula leg muscle obtained from rapidly frozen, freeze-substituted specimens. Computed Fourier transforms of filaments selected from longitudinal sections show up to six layer lines indexing on the 43.5-nm helical repeat of myosin crossbridges. The three-dimensional reconstruction, performed after separation of overlapped Bessel functions, shows four continuous strands of density on the surface of the filament, modulated by density at 14.5-nm intervals, corresponding to the myosin heads aligned approximately along the helical strands. In transverse viw, the reconstruction shows four projections and is similar in profile to myosin filaments seen in thin transverse sections of rapidly frozen muscle. The reconstruction is similar to that of negatively stained, isolated tarantula filaments except that in the latter there is an additional modulation of the helix density, which better resolves the two heads of each myosin crossbridge. Thus, the general arrangement of the myosin heads in the freeze-substituted specimens is preserved, although finer details of structure such as individual myosin heads are lost.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Processamento de Imagem Assistida por Computador , Aranhas/ultraestrutura , Animais , Substituição ao Congelamento , Microscopia EletrônicaRESUMO
Tritrichomonas foetus was studied using different physical and chemical fixation methods such as fast-freezing (by high pressure, "slam-freezing," and jet-propane), freeze-substitution, conventional freeze-fracture and deep-etching, cryoultramicrotomy, and routine preparation for transmission electron microscopy. The use of fast-freezing fixation (FFF) proved to be superior in terms of structural preservation due to the rapidity of this fixation compared to that obtained using conventional chemical fixation. The low temperature techniques used here were useful to confirm data already obtained by conventional freeze-fracture using chemical fixation and cryoprotection, such as the presence of flagellar rosettes and costa structure. Cryoultramicrotomy and slam-freezing also demonstrated the presence of hair-like structures projecting out from the protozoan surface. New aspects of organelles of T. foetus were demonstrated.