Freeze-etching
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Intramembraneous particles represent protein components of the membrane. It is assumed that their number and distribution are in correlation with the level of the metabolic activity of membranes. In the cytoplasmic membrane in the postacrosomal region (PF) near the posterior ring the particles are arranged in form of palisade rows. In the remaining membranes of the head, irregularly distributed particles were observed. Their number at individual places varied. Their size was not equal either. The areas without particles were also found out. The particles in the cytoplasmic membrane of the flagellum (PF) formed clusters and they had circumferential orientation in the direction of mitochondria. At the place of the annulus the number of particles increased. The distribution of particles was irregular in the cytoplasmic membrane of the main section.
- MeSH
- akrozom ultrastruktura MeSH
- buněčná membrána ultrastruktura MeSH
- intracelulární membrány ultrastruktura MeSH
- leptání mrazem MeSH
- skot anatomie a histologie MeSH
- spermie ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot anatomie a histologie MeSH
- zvířata MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
Disintegration of nuclear envelopes is the only ultrastructural change detectable by freeze-etching in dormant spores of the mould Rhizopus nigricans, both dry and swollen, subjected to repeated freezing and thawing. The increase of the number of freeze-inactivated spores corresponds well with the increase of the number of damaged nuclei. This fact led us to formulate a hypothesis that the structure of the nucleus is the primary target of the freezing or thawing damage. As other biomembranes are not damaged it may be assumed that the disintegration of the nuclear membrane is probably secondary. No changes in ultrastructure of metabolically activated spores could be detected, in spite of the fact that the spores lost their germinative ability. Thus, the mechanism of the freeze injury may be different in dormant and growing spores.
- MeSH
- cytoplazma ultrastruktura MeSH
- endoplazmatické retikulum ultrastruktura MeSH
- jaderný obal ultrastruktura MeSH
- leptání mrazem MeSH
- mitochondrie ultrastruktura MeSH
- Rhizopus fyziologie ultrastruktura MeSH
- spory hub fyziologie ultrastruktura MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Monogeneans, in general, show a range of unique adaptations to a parasitic lifestyle, making this group enormously diverse. Due to their unique biological properties, diplozoid monogeneans represent an attractive model group for various investigations on diverse biological interactions. However, despite numerous studies, there are still gaps in our knowledge of diplozoid biology and morphofunctional adaptations. RESULTS: In this study, we provide a comprehensive microscopic analysis of systems/structures involved in niche searching, sensing and self-protection against the host environment, and excretory/secretory processes in Eudiplozoon nipponicum. Freeze-etching enabled us to detect syncytium organisational features not visible by TEM alone, such as the presence of a membrane subjacent to the apical plasma membrane (separated by a dense protein layer) and a lack of basal plasma membrane. We located several types of secretory/excretory vesicles and bodies, including those attached to the superficial membranes of the tegument. Giant unicellular glands were seen accumulating predominantly in the apical forebody and hindbody haptor region. Muscle layer organisation differed from that generally described, with the outer circular and inner longitudinal muscles being basket-like interwoven by diagonal muscles with additional perpendicular muscles anchored to the tegument. Abundant muscles within the tegumentary ridges were detected, which presumably assist in fixing the parasite between the gill lamellae. Freeze-etching, alongside transmission electron and confocal microscopy with tubulin labelling, enabled visualisation of the protonephridia and nervous system, including the peripheral network and receptor innervation. Three types of receptor were identified: 1) uniciliated sensory endings with a subtle (or missing) tegumentary rim, 2) obviously raised uniciliated receptors with a prominent tegumentary rim (packed with massive innervation and muscles) and 3) non-ciliated papillae (restricted to the hindbody lateral region). CONCLUSIONS: This study points to specific morphofunctional adaptations that have evolved in diplozoid monogeneans to confront their fish host. We clearly demonstrate that the combination of different microscopic techniques is beneficial and can reveal hidden differences, even in much-studied model organisms such as E. nipponicum.
- Klíčová slova
- Excretory system, Freeze-etching, Host-parasite interactions, Immunofluorescence, Musculature, Nervous system, Secretion, Sensory structures, Tegument, Ultrastructure,
- Publikační typ
- časopisecké články MeSH
Mutants of Saccharomyces cerevisiae characterized by osmotic fragility showed a marked fibrillar structure on the inner wall surface when studied by two electron microscopic techniques, i.e. freeze-etching of whole native cells and metal shadowing of isolated cell walls. The walls of the mutant cells were more permeable to macromolecules than were those of the wild-type parental strain. The synthesis and assembly of (1----3)-beta-D-glucan wall microfibrils studied in protoplasts of mutant cells were not impaired. It is suggested that the osmotic fragility of the mutant cells is related to the deficiency of the wall structure as a consequence of the srb1 mutation affecting biogenesis of the amorphous (glucan) component.
- MeSH
- buněčná membrána ultrastruktura MeSH
- buněčná stěna chemie ultrastruktura MeSH
- elektronová mikroskopie MeSH
- glukany analýza MeSH
- leptání mrazem MeSH
- mutace MeSH
- osmotická fragilita MeSH
- Saccharomyces cerevisiae genetika fyziologie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glukany MeSH
Various stages of virus and mycoplasma budding indicated that both virus and, most probably some mycoplasma progeny developed by budding. Besides this alternative, binary fission was the mode of mycoplasma reproduction. Mycoplasma-virus and mycoplasma-mycoplasma connections by stems were observed. Circular scars, 40-80 nm in diameter, often in groups, were left in the membrane of mycoplasmas by the budding bodies. cytoplasmic structures seen in cross-fraction are presented. a relatively small number of globular virus-like bodies, not identical with MV-Lg-L 172, were observed budding from mycoplasma cells in the non-infected host culture.
The relationship between the actin cytoskeleton and cell wall synthesis was studied by light and electron microscopy in protoplasts of Saccharomyces cerevisiae DBY 1693 containing the act1-1 allele. Since protoplasting also disturbs the actin cytoskeleton, these mutant protoplasts had a double error in their actin cytoskeletons. In the period between the onset of wall synthesis and completion of the wall, protoplasts grown at the permissive temperature showed an even distribution of actin patches all over the surface on which a new cell wall was being synthesized. After wall completion, actin patches partially disappeared, but then re-appeared, accumulated in growth regions at the start of polarized growth. This was compared with the pattern of actin patches observed in intact temperature-sensitive actin mutant cells cultivated at the permissive temperature. Electron microscopy of freeze-etched replicas revealed finger-like invaginations of the plasma membrane in both the actin mutant cells and their protoplasts. These structures showed a very similar distribution to the actin patches detected by rhodamine phalloidin staining in the fluorescence microscope. A hypothesis is presented, explaining the role of actin patches/finger-like invaginations of the plasma membrane in the synthesis of beta-(1-->3)-D-glucan wall microfibrils in yeast cells.
- MeSH
- aktiny genetika metabolismus MeSH
- buněčná stěna metabolismus ultrastruktura MeSH
- cytoskelet metabolismus ultrastruktura MeSH
- fungální proteiny genetika metabolismus MeSH
- leptání mrazem MeSH
- molekulární modely MeSH
- protoplasty metabolismus ultrastruktura MeSH
- Saccharomyces cerevisiae genetika růst a vývoj ultrastruktura MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aktiny MeSH
- fungální proteiny MeSH
Logarithmic cultures of Saccharomyces cerevisiae strains LBG H 1022, FL-100, X 2180 1A and 1B were studied together with the mutants pep4-3, sec18-1 and sec7-1. The necessary ultrastructural observations showed that, as a rule, juvenile vacuoles were formed de novo from perinuclear endoplasmic reticulum cisternae (ER) packed and inflated with electron-dense (polyanionic) matrix material. This process was disturbed solely in the sec18-1 mutant under non-permissive conditions. The vacuolar marker enzymes adenosine triphosphatase (ATPase) and alkaline phosphohydrolase (ALPase) were assayed by the ultracytochemical cerium precipitation technique. The neutral ATPase was active in vacuolar membranes and in the previously shown (coated) microglobules nearby. ALPase activity was detected in microglobules inside juvenile vacuoles, inside nucleus and in the cytoplasm as well as in the membrane vesicles and in the periplasm. The sites of vacuolar protease carboxypeptidase Y (CPY) activity were assayed using N-CBZ-L-tyrosine-4-methoxy-2-naphthyl-amide (CBZ-Tyr-MNA) as substrate and sites of the amino-peptidase M activity using Leu-MNA as substrate. Hexazotized p-rosaniline served as a coupler for the primary reaction product of both the above proteases (MNA) and the resulting azo-dye was osmicated during postfixation. The CPY reaction product was found in both polar layers of vacuolar membranes (homologous to ER) and in ER membranes enclosing condensed lipoprotein bodies which were taken up by the vacuoles of late logarithmic yeast. Both before and after the uptake into the vacuoles the bodies contained the CPY reaction product in concentric layers or in cavities. Microglobules with CPY activity were also observed. Aminopeptidase was localized in microglobules inside the juvenile vacuoles. These findings combined with the previous cytochemical localizations of polyphosphates and X-prolyl-dipeptidyl (amino)peptidase in S. cerevisiae suggest the following cytologic mechanism for the biosynthetic protein transport: coated microglobules convey metabolites and enzymes either to the cell surface for secretion or enter the vacuoles in all phases of the cell cycle. The membrane vesicles represent an alternative secretory mechanism present in yeast cells only during budding. The homology of the ER with the vacuolar membranes and with the surface membranes of the lipoprotein condensates (bodies) indicates a cotranslational entry of the CPY into these membranes. The secondary transfer of a portion of CPY into vacuoles is probably mediated by the lipoprotein uptake process.
- MeSH
- adenosintrifosfatasy analýza MeSH
- alkalická fosfatasa analýza MeSH
- aminopeptidasy analýza MeSH
- elektronová mikroskopie MeSH
- endoplazmatické retikulum metabolismus MeSH
- histocytochemie MeSH
- karboxypeptidasy analýza MeSH
- kathepsin A MeSH
- leptání mrazem MeSH
- lipoproteiny metabolismus MeSH
- mutace MeSH
- Saccharomyces cerevisiae - proteiny MeSH
- Saccharomyces cerevisiae enzymologie genetika ultrastruktura MeSH
- vakuoly enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adenosintrifosfatasy MeSH
- alkalická fosfatasa MeSH
- aminopeptidasy MeSH
- karboxypeptidasy MeSH
- kathepsin A MeSH
- lipoproteiny MeSH
- PRC1 protein, S cerevisiae MeSH Prohlížeč
- Saccharomyces cerevisiae - proteiny MeSH
- serine carboxypeptidase MeSH Prohlížeč
- MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- buněčná stěna metabolismus MeSH
- elektronová mikroskopie MeSH
- fosfolipasy metabolismus MeSH
- glykosidhydrolasy metabolismus MeSH
- hlemýždi enzymologie MeSH
- leptání mrazem MeSH
- lipasa metabolismus MeSH
- lysofosfatidylcholiny metabolismus MeSH
- mikroskopie fázově kontrastní MeSH
- multienzymové komplexy metabolismus MeSH
- papain metabolismus MeSH
- polysacharidy biosyntéza MeSH
- pronasa metabolismus MeSH
- proteasy metabolismus MeSH
- protoplasty metabolismus ultrastruktura MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- Saccharomyces ultrastruktura MeSH
- trypsin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fosfolipasy MeSH
- glykosidhydrolasy MeSH
- lipasa MeSH
- lysofosfatidylcholiny MeSH
- multienzymové komplexy MeSH
- papain MeSH
- polysacharidy MeSH
- pronasa MeSH
- proteasy MeSH
- trypsin MeSH
Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment-protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II.
- Publikační typ
- časopisecké články MeSH
In this study we present an innovative method for the preparation of fully hydrated samples of microbial biofilms of cultures Staphylococcus epidermidis, Candida parapsilosis and Candida albicans. Cryo-scanning electron microscopy (cryo-SEM) and high-pressure freezing (HPF) rank among cutting edge techniques in the electron microscopy of hydrated samples such as biofilms. However, the combination of these techniques is not always easily applicable. Therefore, we present a method of combining high-pressure freezing using EM PACT2 (Leica Microsystems), which fixes hydrated samples on small sapphire discs, with a high resolution SEM equipped with the widely used cryo-preparation system ALTO 2500 (Gatan). Using a holder developed in house, a freeze-fracturing technique was applied to image and investigate microbial cultures cultivated on the sapphire discs. In our experiments, we focused on the ultrastructure of the extracellular matrix produced during cultivation and the relationships among microbial cells in the biofilm. The main goal of our investigations was the detailed visualization of areas of the biofilm where the microbial cells adhere to the substrate/surface. We show the feasibility of this technique, which is clearly demonstrated in experiments with various freeze-etching times.
- Klíčová slova
- Biofilm, Candida, Freeze-fracturing, High-pressure freezing (HPF), Staphylococcus, cryo-SEM,
- MeSH
- biofilmy MeSH
- Candida albicans ultrastruktura MeSH
- Candida parapsilosis ultrastruktura MeSH
- elektronová kryomikroskopie metody MeSH
- extracelulární matrix ultrastruktura MeSH
- mikroskopie elektronová rastrovací metody MeSH
- mrazové lámání metody MeSH
- Staphylococcus epidermidis ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
