scholarly journals Adenosinetriphosphatase and 5-Nucleotidase Activities in the Plasma Membrane of Liver Cells as Revealed by Electron Microscopy

1958 ◽  
Vol 4 (6) ◽  
pp. 711-716 ◽  
Author(s):  
Edward Essner ◽  
Alex B. Novikoff ◽  
Bertha Masek
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Function ◽  
Molecular Transport ◽  
Enzyme Reaction ◽  
Bile Canaliculus ◽  
Nucleotidase Activity ◽  
Reaction Products ◽  
Thin Sections

The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections. These indicate that both ATPase and 5-nucleotidase activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus, and that 5-nucleotidase activity is also present in the microvilli at the sinusoidal aspects of the cells. It is suggested that these enzymes, particularly ATPase, may play a role in molecular transport or in some kind of membrane activity at the cell surface. Of special interest is the apparent differential localization of these enzymes at the absorptive and secretory regions of the plasma membrane of the cell. It may be of interest to study changes in these enzyme localizations in pathologic states, as a sign of changed cell function. Some of the difficulties in the interpretation of enzyme reaction products seen in electron micrographs are discussed.

Lead aspartate: a new en bloc stain for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 34-35
Author(s):  
J.R. Walton
Keyword(s):  
Electron Microscopy ◽  
Calcium Ion ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc ◽  
Thin Sections ◽  
Lead Salts ◽  
Thin Sectioning ◽  
High Alkalinity

In electron microscopy, lead is the metal most widely used for enhancing specimen contrast. Lead citrate requires a pH of 12 to stain thin sections of epoxy-embedded material rapidly and intensively. However, this high alkalinity tends to leach out enzyme reaction products, making lead citrate unsuitable for many cytochemical studies. Substitution of the chelator aspartate for citrate allows staining to be carried out at pH 6 or 7 without apparent effect on cytochemical products. Moreover, due to the low, controlled level of free lead ions, contamination-free staining can be carried out en bloc, prior to dehydration and embedding. En bloc use of lead aspartate permits the grid-staining step to be bypassed, allowing samples to be examined immediately after thin-sectioning.Procedures. To prevent precipitation of lead salts, double- or glass-distilled H20 used in the stain and rinses should be boiled to drive off carbon dioxide and glassware should be carefully rinsed to remove any persisting traces of calcium ion.

scholarly journals Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

1982 ◽  
Vol 94 (3) ◽  
pp. 613-623 ◽  
Author(s):  
J Aggeler ◽  
Z Werb
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Attachment ◽  
High Resolution Electron Microscopy ◽  
Peritoneal Macrophages ◽  
Coated Pits ◽  
Thin Sections ◽  
Particle Uptake ◽  
Freeze Dried

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

scholarly journals Filopodelike projections induced with dimethyl sulfoxide and their relevance to cellular polarity in Dictyostelium.

1983 ◽  
Vol 96 (3) ◽  
pp. 857-865 ◽  
Author(s):  
S Yumura ◽  
Y Fukui
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Dimethyl Sulfoxide ◽  
Cell Polarity ◽  
Cytoplasmic Streaming ◽  
Anterior Part ◽  
Cellular Polarity ◽  
Microtubule Organizing Center ◽  
Cell Organelles ◽  
Thin Sections

When 5% dimethyl sulfoxide (DMSO) was applied to Dictyostelium cells, the cells rounded up in shape and cytoplasmic streaming ceased. The cells resumed both cytoplasmic streaming and locomotion in 20 min. SDS PAGE of isolated plasma membrane fractions showed that actin and myosin apparently became dissociated from the plasma membrane by the action of DMSO. Scanning electron microscopy revealed that many filopodelike projections formed on the surface of cells treated with 5% DMSO for 5 min. Interestingly, the projections were formed on a restricted portion of the cell surface. The phagokinetic track technique of Albrecht-Buehler (1977, Cell, 11: 395-404) showed that the projection region corresponded to the anterior part of a migrating cell. The possible relationship between the DMSO-induced projection region on the cell surface and intracellular organization of cell organelles was investigated using serial thin sections. The DMSO-induced projections contained arrays of microfilaments; and the microtubule organizing center (MTOC), nucleus, and vesicular structure were usually located in this order from the anterior end of the cell. The indirect immunofluorescent study using monoclonal anti-alpha-tubulin antibody was performed with a new fixation technique, which greatly improved the phase as well as immunofluorescent microscopy. It was verified that the intracellular positioning of the MTOC and nucleus had significant correlation with the cell polarity. The results show that DMSO is a powerful tool with which to manipulate the cellular microfilaments and to make visible the differentiation in the cortex layer, which apparently is relevant to the intracellular positioning of cell organelles and cell polarity.

The fine structure of Sphaerotilus nutans

1973 ◽  
Vol 19 (3) ◽  
pp. 309-313 ◽  
Author(s):  
Judith F. M. Hoeniger ◽  
H.-D. Tauschel ◽  
J. L. Stokes
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Fine Structure ◽  
Lateral Position ◽  
Thin Sections ◽  
Liquid Cultures ◽  
Sphaerotilus Natans ◽  
Stationary Liquid ◽  
Basal Disk ◽  
Polar Organelle

Sphaerotilus natans developed sheathed filaments in stationary liquid cultures and motile swarm cells in shaken ones. Electron microscopy of negatively stained preparations and thin sections showed that the sheath consists of fibrils. When the filaments were grown in broth with glucose added, the sheath was much thicker and the cells were packed with granules of poly-β-hydroxybutyrate.Swarm cells possess a subpolar tuft of 10 to 30 flagella and a polar organelle which is usually inserted in a lateral position and believed to be ribbon-shaped. The polar organelle consists of an inner layer joined by spokes to an accentuated plasma membrane. The flagellar hook terminates in a basal disk, consisting of two rings, which is connected by a central rod to a second basal disk.

scholarly journals Further Observations on the Fine Structure of Chrysochromulina Ericina Parke & Manton

1961 ◽  
Vol 41 (1) ◽  
pp. 145-155 ◽  
Author(s):  
I. Manton ◽  
G. F. Leedale
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Cell Surface ◽  
Light Microscopy ◽  
Flat Plate ◽  
Outer Layer ◽  
Living Cells ◽  
Layered Plates ◽  
Thin Sections ◽  
Micro Anatomy

C. ericina Parke & Manton has been re-investigated to add salient features of micro-anatomy from the electron microscopy of thin sections and also to add photographs of living cells taken with anoptral contrast light microscopy.The most important new observations concern the scales which are shown to be essentially two-layered plates in which the layers in the very large spined scales have become separated except at their edges, with the outer layer greatly hypertrophied to produce a hollow spine with a flared base closed at the bottom by a flat plate. The patterns of external marking on the two layers are very similar in both plate-scales and spines in this species and the orientation of both with respect to the cell surface has been demonstrated by a section of the scales in situ.

scholarly journals Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells

2002 ◽  
Vol 83 (3) ◽  
pp. 611-621 ◽  
Author(s):  
Gaie Brown ◽  
James Aitken ◽  
Helen W. McL. Rixon ◽  
Richard J. Sugrue
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
Confocal Microscopy ◽  
Cell Surface ◽  
Filamentous Form ◽  
Caveolin 1 ◽  
Immuno Electron Microscopy ◽  
Infected Cells ◽  
Syncytial Virus

We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.

scholarly journals Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells.

1987 ◽  
Vol 35 (8) ◽  
pp. 809-816 ◽  
Author(s):  
R Pakkanen ◽  
K Hedman ◽  
O Turunen ◽  
T Wahlström ◽  
A Vaheri
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Polyclonal Antibodies ◽  
Apical Cell ◽  
Antigenic Site ◽  
Electron Microscopic ◽  
Permeabilized Cells ◽  
Choriocarcinoma Cells

We have previously purified from cultured JEG-3 choriocarcinoma cells an Mr 75,000 protein, originally detected using antibodies to a retrovirus-related synthetic peptide. Using polyclonal antibodies, we have now localized this protein immunocytochemically in JEG-3 cells at both light and electron microscopic levels. In immunofluorescence microscopy of saponin-permeabilized cells, the antigen appeared as dots and short strands at the apical cell surface. In pre-embedding immunoperoxidase electron microscopy, the Mr 75,000 protein was specifically localized to microvilli on the apical cell surface. Immunoferritin electron microscopy was used to assess more quantitatively the antigen distribution in the plane of the plasma membrane, and to define the position of the antigenic site(s) with respect to the membrane. The immunoferritin results confirmed the microvillus specificity of the Mr 75,000 protein and showed that the antigenic portion of the protein is within a few nanometers from, and on the cytoplasmic side of, the lipid bilayer. In detergent extraction experiments, the Mr 75,000 antigen was highly enriched in the soluble fractions. These results demonstrate that the Mr 75,000 protein is a membrane protein highly specific for microvilli.

Ultrastructure of the in situ adherence of Mobiluncus to vaginal epithelial cells

1988 ◽  
Vol 34 (6) ◽  
pp. 757-766 ◽  
Author(s):  
Jan M. De Boer ◽  
Friso H. F. Plantema
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Bacterial Vaginosis ◽  
Vaginal Fluid ◽  
Thin Sections ◽  
Epithelial Cell Surface ◽  
Curved Rods

From patients with bacterial vaginosis motile, anaerobic, comma-shaped bacteria can be isolated, which have recently been placed into the new genus Mobiluncus. In this study, electron microscopy was used to examine the in situ adherence of these motile curved rods to detached epithelial cells (comma cells) in vaginal fluid from two patients with bacterial vaginosis. Thin sections showed that the curved rods attached both directly to the epithelial cell surface and at various distances from it. It is concluded that after initial attachment these motile bacteria can grow at the epithelial cell surface in sessile microcolonies. Ruthenium red staining demonstrated a coating of precipitated glycocalyx material both on the surface of the curved rods and on their flagella. This may indicate that in situ the adherent curved rods were enclosed in a very hydrated matrix of exopolysaccharides. Conspicuous was the ability of the curved rods to attach to the epithelial cell surface via their cell tips. However, in situ no specialized bacterial cell surface structures were seen that might explain this polar attachment. Electron microscopy of pure cultures demonstrated that both Mobiluncus curtisii subsp. curtisii and Mobiluncus mulieris can produce a glycocalyx in vitro.

scholarly journals Lead asparate, an en bloc contrast stain particularly useful for ultrastructural enzymology.

1979 ◽  
Vol 27 (10) ◽  
pp. 1337-1342 ◽  
Author(s):  
J Walton
Keyword(s):  
Electron Microscopy ◽  
Aspartic Acid ◽  
Lead Nitrate ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.

The Surface Structure of Cultured Rabbit Kidney Cells as Revealed by Electron Microscopy

1970 ◽  
Vol 7 (3) ◽  
pp. 719-737
Author(s):  
ELIZABETH DIMMOCK
Keyword(s):  
Electron Microscopy ◽  
Surface Layer ◽  
Cell Surface ◽  
Blood Group ◽  
Potassium Permanganate ◽  
Ruthenium Red ◽  
Rabbit Kidney ◽  
Dense Layer ◽  
Kidney Cells ◽  
Thin Sections

An epithelioid line of rabbit kidney cells (RK 13), in which the distribution of blood group antigen A had previously been investigated by mixed agglutination, was chosen for studies of the structure of the cell surface. Cells were grown in monolayer culture, and thin sections were examined by electron microscopy after fixation in glutaraldehyde and osmium tetroxide and staining of the sections with lead, or uranyl and lead. Cells were also treated with ruthenium red incorporated in the osmium fixative. Other cells were fixed in lanthanum or potassium permanganate, and both stained and unstained sections were examined. The morphology of RK 13 cell surfaces is described. There is apparently no great degree of cellular specialization. The treatment with ruthenium red resulted in dense staining of a layer of the cell surface that is not visible in conventional preparations, and sometimes in staining of the surfaces of intracellular organelles. Lanthanum permanganate fixation also revealed a dense layer on the surface of the plasma membrane; a less dense surface layer was distinguished in many cells fixed in potassium permanganate. The reaction of ruthenium red with the cell surface is probably due to the presence of acidic glycoproteins, but the chemical specificity of the staining method is not yet clear. The nature of the material revealed by lanthanum and potassium permanaganates is also undefined. However, these staining methods reveal that the cell surface is more complex than is apparent in cells prepared by conventional techniques. The additional surface layer is probably the site of many blood group substances and other compounds involved in the physiological reactions of the cell surface.

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