scholarly journals Insulin-induced receptor loss in the cultured human lymphocyte: quantitative morphological perturbations in the cell and plasma membrane

1979 ◽  
Vol 39 (1) ◽  
pp. 77-88
Author(s):  
P. Gordon ◽  
J.L. Carpentier ◽  
E. Van Obberghen ◽  
P. Barazzone ◽  
J. Roth ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Human Lymphocyte ◽  
Human Lymphocytes ◽  
Experimental Conditions ◽  
Peroxidase Uptake

When cultured human lymphocytes (IM-9) are exposed to 10(−6) M procine insulin for 6 h, washed, and incubated with 125I-insulin, the ability of the cell to bind the labelled hormone is reduced by a mean of 78%. Under these experimental conditions that induce insulin-receptor loss in this cell there is a mean 95% increase in microinvaginations in the plasma membrane revealed by electron microscopy on freez-fractured replicas of the cell. At the same time, horseradish peroxidase uptake, a marker of endocytosis, is increased in the cells incubated with insulin. Coupled with our recent EM autoradiographic evidence that labelled insulin is acutely internalized by this cell, these studies are consistent with the possibility that endocytosis represents a mechanism by which receptor is removed from the cell surface.

scholarly journals Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver

2000 ◽  
Vol 279 (2) ◽  
pp. E266-E274 ◽  
Author(s):  
Paul G. Drake ◽  
Alejandro Balbis ◽  
Jiong Wu ◽  
John J. M. Bergeron ◽  
Barry I. Posner
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Kinase Activity ◽  
Glycogen Synthase ◽  
Metabolic Effects ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK β-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

scholarly journals Inhibition of pinocytosis in rat embryo fibroblasts treated with monensin.

1982 ◽  
Vol 92 (3) ◽  
pp. 859-864 ◽  
Author(s):  
D K Wilcox ◽  
R P Kitson ◽  
C C Widnell
Keyword(s):  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Reversible Inhibitor ◽  
Membrane Recycling ◽  
Rat Embryo ◽  
Galactosyl Transferase ◽  
Maximum Inhibition ◽  
Embryo Fibroblasts ◽  
Peroxidase Uptake ◽  
Pinocytic Vesicles

Rat embryo fibroblasts cultured in the presence of monensin exhibited an inhibited uptake of horseradish peroxidase. The inhibition was detected after 3 h, after which time the cells became increasingly vacuolated; the concentration of monensin required to inhibit pinocytosis (0.4 microM for half-maximum inhibition at 18 h) was similar to that found by others to inhibit secretion. Both the exchange of 5'-nucleotidase between the membranes of cytoplasmic organelles and the cell surface and the internalization of anti-5'-nucleotidase bound to the cell surface were inhibited by approximately 90% in monensin-treated cells. The effects of monensin were reversible: cells cultured first with monensin, and then in fresh medium, exhibited control levels of horseradish peroxidase uptake, exchange of 5'-nucleotidase, and internalization of anti-5'-nucleotidase bound to the cell surface. After monensin treatment, the median density of both galactosyl transferase and 5'-nucleotidase increased from 1.128 to 1.148, and the median density of both N-acetyl-beta-glucosaminidase and horseradish peroxidase taken up by endocytosis decreased from 1.194 to 1.160. The results indicate that monensin is a reversible inhibitor of pinocytosis and, presumably, therefore, of membrane recycling. They suggest that the inhibition of membrane recycling occurs at a step other than the fusion of pinocytic vesicles with lysosomes and is perhaps a consequence of an effect of the ionophore on the Golgi complex.

scholarly journals Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages.

1976 ◽  
Vol 24 (8) ◽  
pp. 948-955 ◽  
Author(s):  
P E McKeever ◽  
A J Garvin ◽  
S S Spicer
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Immune Complex ◽  
Immune Complexes ◽  
External Surface ◽  
Binding Capacity ◽  
Ultrastructural Localization ◽  
Soluble Complex ◽  
Receptor Sites

A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.

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.

scholarly journals Neutrophils adherent to a nonphagocytosable surface (glomerular basement membrane) produce oxidants only at the site of attachment

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 161-166 ◽  
Author(s):  
MC Vissers ◽  
WA Day ◽  
CC Winterbourn
Keyword(s):  
Superoxide Dismutase ◽  
Plasma Membrane ◽  
Cytochrome C ◽  
Basement Membrane ◽  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Oxygen Uptake ◽  
Glomerular Basement Membrane ◽  
Cerium Chloride ◽  
Cytochrome C Reduction

Abstract Adherence of neutrophils to glomerular basement membrane containing immunoglobulin G aggregates was accompanied by a marked increase in oxygen uptake (eightfold). Very little of the O2 consumed was recovered as superoxide, measured by cytochrome c reduction, or as H2O2, measured with horseradish peroxidase and scopoletin. When neutrophils were incubated with the basement membrane preparation in the presence of cerium chloride to detect H2O2, electron micrographs showed cerium perhydroxide deposits in the contact area between the cells and the basement membrane, but not on the remainder of the cell surface. The results imply that superoxide is produced only where the plasma membrane is in contact with the basement membrane matrix, and that it mostly breaks down to H2O2 or undergoes other reactions at this site. The longer lifetime of H2O2 compared with that of superoxide allows some of the H2O2 produced to be detected in the medium. The results also suggest that the area of contact between the neutrophil and surfaces such as basement membrane is inaccessible to proteins in the medium, eg, cytochrome c. Circulating scavengers such as superoxide dismutase or catalase, or proteolytic inhibitors, may therefore be unable to control events occurring at this site.

scholarly journals Cell shape and plasma membrane alterations after static magnetic fields exposure

10.4081/840 ◽  
2009 ◽  
Vol 47 (4) ◽  
pp. 299 ◽  
Author(s):  
A Chionna ◽  
M Dwikat ◽  
E Panzarini ◽  
B Tenuzzo ◽  
EC Carlà ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Magnetic Fields ◽  
Cell Surface ◽  
Cell Shape ◽  
Biological Effects ◽  
Human Lymphocytes ◽  
Cell Types ◽  
Continuous Exposure ◽  
U937 Cells ◽  
Static Magnetic Fields

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneosly exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.

scholarly journals Glycosylation of proteins from sugar nucleotides by whole cells. Effect of ammonium chloride treatment on mouse thymocytes

1983 ◽  
Vol 216 (3) ◽  
pp. 681-686 ◽  
Author(s):  
R Cecchelli ◽  
R Cacan ◽  
B Hoflack ◽  
A Verbert
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Trypan Blue ◽  
Sugar Nucleotides ◽  
Whole Cells ◽  
Acetylneuraminic Acid ◽  
Intracellular Vesicles ◽  
Chloride Treatment ◽  
Stimulation Of

When thymocytes are treated with iso-osmotic NH4Cl, the sugar incorporation into endogenous acceptors from labelled sugar nucleotides is largely increased compared with that in control thymocytes. This effect was obtained with labelled GDP-mannose, UDP-galactose and CMP-N-acetylneuraminic acid. The stimulation observed with NH4Cl-treated thymocytes does not involve the glycosylation of exogenous acceptors, and it was proved that the NH4Cl treatment (1) does not stimulate glycosyltransferase activities themselves, (2) does not lead to the release of soluble glycosyltransferases as the result of an extensive lysis of the thymocytes and (3) does not cause the emergence of glycosyltransferases at the cell surface. In fact, electron-microscopy observations showed that, although marked changes had occurred in the cytoplasm, the plasma membrane is sufficiently maintained to allow the cell to keep roughly its original shape and to retain the intracellular vesicles. We thus demonstrate that this stimulation is due to an enhancement of the entry of sugar nucleotides into the cell. As demonstrated by the inclusion of Trypan Blue within the cells, and the non-stimulation of glycosylation of exogenous large-molecular-mass acceptors, the effect of NH4Cl seems to be limited to the penetration of small-molecular-sized compounds through the plasma membrane. Thus NH4Cl treatment allows the labelled sugar nucleotides to penetrate the cell and to behave as the cellular pool to be utilized for glycosylation by intracellular vesicles.

scholarly journals Multiple endosomal recycling pathways in rat adipose cells

1998 ◽  
Vol 331 (3) ◽  
pp. 829-835 ◽  
Author(s):  
Konstantin V. KANDROR ◽  
Paul F. PILCH
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Sedimentation Coefficient ◽  
Membrane Vesicles ◽  
Average Diameter ◽  
Subcellular Fractionation ◽  
Adipose Cells

Adipose and skeletal-muscle cells can translocate several membrane proteins from intracellular compartment(s) to the cell surface in an insulin-dependent fashion. Among these proteins is Glut4, a physiologically important glucose transporter which mediates insulin's effect on blood glucose clearance. Under basal conditions, Glut4 is localized in uniform, intracellular membrane vesicles with an average diameter of 50–70 nm and a sedimentation coefficient of 100–120 S. The nature of this compartment and its trafficking pathway to the plasma membrane is still unresolved. We show here that, in addition to Glut4, the aminopeptidase gp160 or insulin-responsive aminopeptidase (‘IRAP’), sortilin, and an acutely recycling population of the insulin-like growth factor-II/mannose 6-phosphate receptor, this compartment includes 60% of the intracellular population of the transferrin receptor. We used subcellular fractionation, cell-surface biotinylation, and radioactive-ligand (125I-transferrin) uptake to demonstrate that the transferrin receptor recycles between this compartment and the plasma membrane in response to insulin along with Glut4 and other protein components of these vesicles. The co-localization of Glut4 and several endosomal markers in the terminally differentiated fat-cells during several stages of their cycling pathways suggests that the ‘Glut4 pathway ’ may derive from the hormone-insensitive endosomes of undifferentiated preadipocytes. The insulin receptor is excluded from Glut4-containing vesicles in both insulin-stimulated and unstimulated adipocytes, and thus it is likely to traffic independently from Glut4 through different intracellular compartments. Our data show that, in adipose cells, the ligand-dependent recycling pathway of the insulin receptor is structurally separated from the ligand-independent pathway of the transferrin receptor, and that Glut4 is specifically targetted to the latter.

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