Isoforms of caveolin-1 and caveolar structure

2000 ◽  
Vol 113 (19) ◽  
pp. 3509-3517 ◽  
Author(s):  
T. Fujimoto ◽  
H. Kogo ◽  
R. Nomura ◽  
T. Une
Keyword(s):  
Electron Microscopy ◽  
Hepg2 Cells ◽  
Immunofluorescence Microscopy ◽  
Human Fibroblasts ◽  
Freeze Fracture ◽  
Molecular Composition ◽  
Immunogold Electron Microscopy ◽  
Caveolin 1 ◽  
Different Depths ◽  
The Relationship

The relationship between caveolin-1 isoforms alpha and beta and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alphabeta) were not labeled by another antibody specific for the alpha isoform (anti-alpha). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-alpha labeled deep caveolae preferentially over shallow ones, whereas anti-alphabeta labeled both forms with an equivalent frequency and intensity. The presence of the beta isoform in deep caveolae was confirmed by labeling epitope-tagged beta-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.

Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies

1989 ◽  
Vol 93 (3) ◽  
pp. 491-500 ◽  
Author(s):  
A. Woods ◽  
T. Sherwin ◽  
R. Sasse ◽  
T.H. MacRae ◽  
A.J. Baines ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Trypanosoma Brucei ◽  
Immunofluorescence Microscopy ◽  
Complex Mixtures ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Tubulin Isotypes ◽  
Definition Of ◽  
Attachment Zone

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

scholarly journals Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 521-529 ◽  
Author(s):  
MN Fukuda ◽  
G Klier ◽  
J Yu ◽  
P Scartezzini
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Band 3 ◽  
Precursor Cells ◽  
Erythrocyte Membranes ◽  
Immunogold Electron Microscopy ◽  
Glycophorin A ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Cytoplasmic Area

Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

Role of caveolin-1 in thyroid phenotype, cell homeostasis, and hormone synthesis: in vivo study of caveolin-1 knockout mice

2009 ◽  
Vol 297 (2) ◽  
pp. E438-E451 ◽  
Author(s):  
Maximin Senou ◽  
Maria José Costa ◽  
Claude Massart ◽  
Matthieu Thimmesch ◽  
Céline Khalifa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Thyroid Hormone ◽  
Knockout Mice ◽  
Human Thyroid ◽  
Immunogold Electron Microscopy ◽  
Colloid Interface ◽  
Cell Homeostasis ◽  
Hormone Synthesis ◽  
Caveolin 1 ◽  
Thyroid Hormone Synthesis

In human thyroid, caveolin-1 is localized at the apex of thyrocytes, but its role there remains unknown. Using immunohistochemistry, 127I imaging, transmission electron microscopy, immunogold electron microscopy, and quantification of H2O2, we found that in caveolin-1 knockout mice thyroid cell homeostasis was disrupted, with evidence of oxidative stress, cell damage, and apoptosis. An even more striking phenotype was the absence of thyroglobulin and iodine in one-half of the follicular lumina and their presence in the cytosol, suggesting that the iodide organification and binding to thyroglobulin were intracellular rather than at the apical membrane/extracellular colloid interface. The latter abnormality may be secondary to the observed mislocalization of the thyroid hormone synthesis machinery (dual oxidases, thyroperoxidase) in the cytosol. Nevertheless, the overall uptake of radioiodide, its organification, and secretion as thyroid hormones were comparable to those of wild-type mice, suggesting adequate compensation by the normal TSH retrocontrol. Accordingly, the levels of free thyroxine and TSH were normal. Only the levels of free triiodothyronine showed a slight decrease in caveolin-1 knockout mice. However, when TSH levels were increased through low-iodine chow and sodium perchlorate, the induced goiter was more prominent in caveolin-1 knockout mice. We conclude that caveolin-1 plays a role in proper thyroid hormone synthesis as well as in cell number homeostasis. Our study demonstrates for the first time a physiological function of caveolin-1 in the thyroid gland. Because the expression and subcellular localization of caveolin-1 were similar between normal human and murine thyroids, our findings in caveolin-1 knockout mice may have direct relevance to the human counterpart.

Dynamics of connexins, E-cadherin and alpha-catenin on cell membranes during gap junction formation

1997 ◽  
Vol 110 (3) ◽  
pp. 311-322 ◽  
Author(s):  
K. Fujimoto ◽  
A. Nagafuchi ◽  
S. Tsukita ◽  
A. Kuraoka ◽  
A. Ohokuma ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Gap Junction ◽  
Immunofluorescence Microscopy ◽  
Immunogold Electron Microscopy ◽  
Cell Contact ◽  
Contact Sites ◽  
Junction Formation ◽  
Intramembrane Proteins ◽  
Junction Proteins ◽  
E Cadherin

We examined the dynamics of connexins, E-cadherin and alpha-catenin during gap-junction disassembly and assembly in regeneration hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or alpha-catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.

scholarly journals Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 521-529 ◽  
Author(s):  
MN Fukuda ◽  
G Klier ◽  
J Yu ◽  
P Scartezzini
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Band 3 ◽  
Precursor Cells ◽  
Erythrocyte Membranes ◽  
Immunogold Electron Microscopy ◽  
Glycophorin A ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Cytoplasmic Area

Abstract Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

Intra- and submembrane particle densities during CO2 stimulation of H+ secretion in turtle bladder

1997 ◽  
Vol 272 (4) ◽  
pp. F491-F497 ◽  
Author(s):  
O. F. Kohn ◽  
A. R. Hand ◽  
P. P. Mitchell ◽  
P. R. Steinmetz
Keyword(s):  
Electron Microscopy ◽  
Apical Cell ◽  
Freeze Fracture ◽  
Alpha Cells ◽  
Transmission Electron ◽  
Wide Range ◽  
Freeze Fracture Electron Microscopy ◽  
The One ◽  
Apical Membranes ◽  
The Relationship

The apical cell membranes of the H+ secreting, alpha-intercalated cells of turtle urinary bladder (TB) are characterized by studs (cytoplasmic domains of V-adenosinetriphosphatase) on thin-section transmission electron microscopy and by intramembrane particles (spherical units, SPUs) occurring as rod-shaped particles on freeze-fracture electron microscopy. To examine the relationship between studs and SPUs, morphometric studies were carried out on bladders maintained in 5% CO2 and in the absence of exogenous CO2. The stud density per square micrometer of apical membrane was 3,909 +/” 352 (+/”SE) in four TBs (29 alpha-cells) at 5% CO2 and 3,667 +/” 448 (+/”SE) in the paired halves of the same bladders without CO2 (25 alpha-cells). Corresponding densities of SPUs counted on apical membranes of the same bladders (n = 4) were 3,941 +/” 545 in 5% CO2 and 3,599 +/” 511 without CO2. The similarity of the densities of studs and SPUs under both conditions indicates that each SPU within the membrane is matched by one stud projecting into the cytoplasm. The one-for-one relationship between studs and SPUs was preserved over a wide range of transport rates. Addition of CO2 caused only inconsistent increments in the densities of studs and SPUs despite substantial increases in H+ transport rate. Slight variations in spacing of studs were consistent with patterns of distribution of SPUs on fracture surfaces.

scholarly journals Human Coronavirus 229E Binds to CD13 in Rafts and Enters the Cell through Caveolae

2004 ◽  
Vol 78 (16) ◽  
pp. 8701-8708 ◽  
Author(s):  
Ryuji Nomura ◽  
Asuka Kiyota ◽  
Etsuko Suzaki ◽  
Katsuko Kataoka ◽  
Yoshihide Ohe ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Interference ◽  
Human Fibroblasts ◽  
Cross Linking ◽  
Subsequent Infection ◽  
Human Coronavirus ◽  
Caveolin 1 ◽  
Membrane Microdomain ◽  
Triton X ◽  
Human Coronavirus 229E

ABSTRACT CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37°C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37°C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37°C. The depletion of plasmalemmal cholesterol with methyl β-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.

scholarly journals The polarized distribution of an apical cell surface glycoprotein is maintained by interactions with the cytoskeleton of Madin-Darby canine kidney cells.

1988 ◽  
Vol 107 (6) ◽  
pp. 2377-2387 ◽  
Author(s):  
G K Ojakian ◽  
R Schwimmer
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Immunofluorescence Microscopy ◽  
Immunogold Electron Microscopy ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Surface Distribution ◽  
Membrane Contacts ◽  
Darby Canine Kidney ◽  
Canine Kidney

A monoclonal antibody made against a 135-kD glycoprotein (gp135) on the plasma membrane of Madin-Darby canine kidney (MDCK) cells was used to study the development and maintenance of epithelial cell surface polarity. Immunofluorescence microscopy and immunogold electron microscopy of confluent monolayers demonstrated that gp135 had a polarized cell surface distribution and was only localized on the apical surface. The role of membrane contacts in establishing gp135 polarity was determined by plating cells in low Ca++-medium to prevent the formation of intercellular junctions. Quantitative immunogold electron microscopy demonstrated that gp135 had a polarized distribution on cells lacking membrane contacts and was observed on the apical surface at a density 24 times that of the basal membrane contacting the substratum. The possibility that gp135 was associated with components of the apical cytoskeleton was investigated using cytoskeleton-disrupting drugs. Incubation in cytochalasin D produced a clustering of both actin and gp135, and double-label fluorescence microscopy demonstrated that these proteins were colocalized. Experiments using nocodazole had no effect, suggesting that gp135 could be interacting with actin microfilaments, but not microtubules. Treatment with Triton X-100 extracted approximately 50% of the gp135 and immunofluorescence microscopy indicated that the gp135 which remained associated with the detergent-insoluble cytoskeleton had a distribution identical to that of control cells. Experiments demonstrating that gp23, a nonpolarized glycoprotein, was preferentially extracted from the apical membrane suggested that the improperly sorted apical gp23 did not interact with the cytoskeleton. These results provided evidence that the polarized cell surface distribution of gp135 was maintained through its interaction with actin in the apical cytoskeleton.

scholarly journals Tyrosine phosphorylation in desmosomes and hemidesmosomes of the corneal epithelium.

1996 ◽  
Vol 44 (1) ◽  
pp. 19-25 ◽  
Author(s):  
K Amino ◽  
M Takahashi ◽  
Y Honda ◽  
T Fujimoto
Keyword(s):  
Electron Microscopy ◽  
Signal Transduction ◽  
Cell Membrane ◽  
Tyrosine Phosphorylation ◽  
Corneal Epithelium ◽  
Immunofluorescence Microscopy ◽  
Immunogold Electron Microscopy ◽  
Frozen Sections ◽  
Normal Rat ◽  
Modified Proteins

We examined the localization of phosphotyrosine (p-Tyr)-modified proteins in the normal corneal epithelium using affinity-purified rabbit anti-p-Tyr antibody. Normal rat cornea was fixed and semi-thin and ultra-thin frozen sections were prepared. Immunofluorescence microscopy showed that p-Tyr was distributed along the cell membrane of the corneal epithelium. Immunogold electron microscopy revealed that the labeling is exclusively localized in the desmosomes and hemidesmosomes. A fraction enriched with desmosomes was extracted from the bovine corneal epithelium and examined by Western blotting. Immunoblotting for p-Tyr showed eight prominent bands (290, 200, 190, 115, 85, 62, 50, and 47 kD) in the desmosomal fraction. The enrichment of p-Tyr in desmosomes and hemidesmosomes of the corneal epithelium suggests that these cell-to-cell and cell-to-substrate junctions are involved in signal transduction.

Studies on Leukemogenic and Sarcomagenic Viruses

1970 ◽  
Vol 28 ◽  
pp. 156-157
Author(s):  
Leon Dmochowski
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Morphological Properties ◽  
Mode Of Development ◽  
Relationship Of ◽  
The Relationship

Electron microscopy has proved to be an invaluable discipline in studies on the relationship of viruses to the origin of leukemia, sarcoma, and other types of tumors in animals and man. The successful cell-free transmission of leukemia and sarcoma in mice, rats, hamsters, and cats, interpreted as due to a virus or viruses, was proved to be due to a virus on the basis of electron microscope studies. These studies demonstrated that all the types of neoplasia in animals of the species examined are produced by a virus of certain characteristic morphological properties similar, if not identical, in the mode of development in all types of neoplasia in animals, as shown in Fig. 1.

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