scholarly journals Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity.

1988 ◽  
Vol 107 (6) ◽  
pp. 2363-2376 ◽  
Author(s):  
P J Salas ◽  
D E Vega-Salas ◽  
J Hochman ◽  
E Rodriguez-Boulan ◽  
M Edidin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Fluorescence Recovery ◽  
Apical Surface ◽  
Collagen Gels ◽  
Surface Polarity ◽  
Basal Surface ◽  
Apical Domain ◽  
Cell Biol ◽  
In Cells

We have studied the role of restrictions to lateral mobility in the segregation of proteins to apical and basolateral domains of MDCK epithelial cells. Radioimmunoassay and semiquantitative video analysis of immunofluorescence on frozen sections showed that one apical and three basolateral glycoproteins, defined by monoclonal antibodies and binding of beta-2-microglobulin, were incompletely extracted with 0.5% Triton X-100 in a buffer that preserves the cortical cytoskeleton (Fey, E. G., K. M. Wan, and S. Penman. 1984. J. Cell Biol. 98:1973-1984; Nelson, W. T. and P. J. Veshnock. 1986. J. Cell Biol. 103:1751-1766). The marker proteins were preferentially extracted from the "incorrect" domain (i.e., the apical domain for a basolateral marker), indicating that the cytoskeletal anchoring was most effective on the "correct" domain. The two basolateral markers were unpolarized and almost completely extractable in cells prevented from establishing cell-cell contacts by incubation in low Ca++ medium, while an apical marker was only extracted from the basal surface under the same conditions. Procedures were developed to apply fluorescent probes to either the apical or the basolateral surface of live cells grown on native collagen gels. Fluorescence recovery after photobleaching of predominantly basolateral antigens showed a large percent of cells (28-52%) with no recoverable fluorescence on the basal domain but normal fluorescence recovery on the apical surface of most cells (92-100%). Diffusion coefficients in cells with normal fluorescence recovery were in the order of 1.1 x 10(-9) cm2/s in the apical domain and 0.6-0.9 x 10(-9) cm2/s in the basal surface, but the difference was not significant. The data from both techniques indicate (a) the existence of mobile and immobile protein fractions in both plasma membrane domains, and (b) that linkage to a domain specific submembrane cytoskeleton plays an important role in the maintenance of epithelial cell surface polarity.

scholarly journals Move your microvilli

2014 ◽  
Vol 207 (1) ◽  
pp. 9-11 ◽  
Author(s):  
Robert S. Fischer
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Polarity ◽  
Apical Membrane ◽  
Apical Surface ◽  
Epithelial Cell Polarity ◽  
Cell Biol ◽  
Polarized Epithelial Cells

Polarized epithelial cells create tightly packed arrays of microvilli in their apical membrane, but the fate of these microvilli is relatively unknown when epithelial cell polarity is lost during wound healing. In this issue, Klingner et al. (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201402037) show that, when epithelial cells become subconfluent, actomyosin contractions locally within the apical cortex cause their microvilli to become motile over the dorsal/apical surface. Their unexpected observations may have implications for epithelial responses in wound healing and disease.

scholarly journals Expression of keratin and vimentin intermediate filaments in rabbit bladder epithelial cells at different stages of benzo[a]pyrene-induced neoplastic progression.

1981 ◽  
Vol 90 (1) ◽  
pp. 63-69 ◽  
Author(s):  
I C Summerhayes ◽  
Y S Cheng ◽  
T T Sun ◽  
L B Chen
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Intermediate Filaments ◽  
Epithelial Cell Line ◽  
Neoplastic Progression ◽  
Collagen Gels ◽  
Rabbit Bladder ◽  
Vimentin Filaments ◽  
In Cells

Rabbit bladder epithelium, grown on collagen gels and exposed to the chemical carcinogen benzo[a]pyrene, produced nontumorigenic altered foci as well as tumorigenic epithelial cell lines during 120-180 d in culture. Immunofluorescence studies revealed extensive keratin filaments in both primary epithelial cells and benzo[a]pyrene-induced altered epithelial foci but showed no detectable vimentin filaments. The absence of vimentin expression in these cells was confirmed by two-dimensional gel electrophoresis. In contrast, immunofluorescence staining of the cloned benzo[a]pyrene-transformed rabbit bladder epithelial cell line, RBC-1, revealed a reduction in filamentous keratin concomitant with the expression of vimentin filaments. The epithelial nature of this cell line was established by the observation that cells injected into nude mice formed well-differentiated adenocarcinomas. Frozen sections of such tumors showed strong staining with antikeratins antibodies, but no detectable staining with antivimentin antibodies. These results demonstrated a differential expression of intermediate filament type in cells at different stages of neoplastic progression and in cells maintained in different growth environments. It is apparent that the expression of intermediate filaments throughout neoplastic progression is best studied by use of an in vivo model system in parallel with culture studies.

scholarly journals Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells.

1988 ◽  
Vol 107 (5) ◽  
pp. 1717-1728 ◽  
Author(s):  
D E Vega-Salas ◽  
P J Salas ◽  
E Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Cell Contact ◽  
Apical Surface ◽  
Sequence Of Events ◽  
Cell Biol ◽  
Intercellular Contacts ◽  
Apical Compartment ◽  
Cell Cell

The vacuolar apical compartment (VAC) is an organelle found in Madin-Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo.

Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells

1998 ◽  
Vol 111 (6) ◽  
pp. 825-832 ◽  
Author(s):  
U. Vogel ◽  
K. Sandvig ◽  
B. van Deurs
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Epithelial Cell ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Wild Type ◽  
Caveolin 1 ◽  
Endogenous Level ◽  
Epithelial Cell Lines ◽  
Different Levels

We have studied caveolin-1 expression and the frequency and distribution of typical invaginated caveolae as they are identified by electron microscopy in the polarized epithelial cell lines MDCK II and Caco-2. In wild-type MDCK II cells caveolin expression is high and more than 400 caveolae/mm filter were observed at the basolateral membrane. No caveolae were found at the apical surface. By contrast, wild-type Caco-2 cells do not express caveolin-1 and have extremely few, if any caveolae. Caco-2 cells were stably transfected with the gene for caveolin-1 in order to investigate if the formation of caveolae is polarized also in these cells. We have isolated Caco-2 clones expressing different levels of caveolin-1, where the level of expression varies from 10–100% of the endogenous level in MDCK II cells. Caveolin-1 expression in Caco-2 cells gives rise to a marked immunofluorescense labeling mainly at the lateral plasma membrane. By electron microscopy an increase from less than 4 caveolae/mm filter in wild-type Caco-2 cells to 21–76 caveolae/mm filter in Caco-2 clones transfected with caveolin-1 was revealed and these caveolae were exclusively localized to the basolateral membrane. Thus expression of heterologous caveolin-1 in Caco-2 cells leads to polarized formation of caveolae, but there is a lack of correlation between the amount of caveolin expressed in the cells and the number of caveolae, suggesting that factors in addition to caveolin are required for generation of caveolae.

scholarly journals Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

2008 ◽  
Vol 183 (4) ◽  
pp. 625-633 ◽  
Author(s):  
Aron B. Jaffe ◽  
Noriko Kaji ◽  
Joanne Durgan ◽  
Alan Hall
Keyword(s):  
Cell Division ◽  
Epithelial Cell ◽  
Three Dimensional ◽  
Epithelial Morphogenesis ◽  
Spindle Orientation ◽  
Epithelial Cell Line ◽  
Epithelial Tissue ◽  
Apical Surface ◽  
Apical Domain ◽  
Underlying Mechanisms

The establishment of apical–basal polarity within a single cell and throughout a growing tissue is a key feature of epithelial morphogenesis. To examine the underlying mechanisms, the human intestinal epithelial cell line Caco-2 was grown in a three-dimensional matrix to generate a cystlike structure, where the apical surface of each epithelial cell faces a fluid-filled central lumen. A discrete apical domain is established as early as the first cell division and between the two daughter cells. During subsequent cell divisions, the apical domain of each daughter cell is maintained at the center of the growing structure through a combination of mitotic spindle orientation and asymmetric abscission. Depletion of Cdc42 does not prevent the establishment of apical–basal polarity in individual cells but rather disrupts spindle orientation, leading to inappropriate positioning of apical surfaces within the cyst. We conclude that Cdc42 regulates epithelial tissue morphogenesis by controlling spindle orientation during cell division.

scholarly journals Studies on the development and maintenance of epithelial cell surface polarity with monoclonal antibodies.

1984 ◽  
Vol 98 (5) ◽  
pp. 1777-1787 ◽  
Author(s):  
D A Herzlinger ◽  
G K Ojakian
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Electrical Resistance ◽  
Mdck Cells ◽  
Continuous Process ◽  
Surface Polarity ◽  
Confluent Monolayer ◽  
Epithelial Cell Surface

We examined epithelial cell surface polarity in subconfluent and confluent Madin-Darby canine kidney (MDCK) cells with monoclonal antibodies directed against plasma membrane glycoproteins of 35,000, 50,000, and 60,000 mol wt. The cell surface distribution of these glycoproteins was studied by immunofluorescence and immunoelectron microscopy. At the ultrastructural level, the electron-dense reaction product localizing all three glycoproteins was determined to be uniformly distributed over the apical and basal cell surfaces of subconfluent MDCK cells as well as on the lateral surfaces between contacted cells; however, after formation of a confluent monolayer, these glycoproteins could only be localized on the basal-lateral plasma membrane. The development of cell surface polarity was followed by assessing glycoprotein distribution with immunofluorescence microscopy at selected time intervals during growth of MDCK cells to form a confluent monolayer. These results were correlated with transepithelial electrical resistance measurements of tight junction permeability and it was determined by immunofluorescence that polarized distributions of cell surface glycoproteins were established just after electrical resistance could be detected, but before the development of maximal resistance. Our observations provide evidence that intact tight junctions are required for the establishment of the apical and basal-lateral plasma membrane domains and that development of epithelial cell surface polarity is a continuous process.

scholarly journals Myosin-1a powers the sliding of apical membrane along microvillar actin bundles

2007 ◽  
Vol 177 (4) ◽  
pp. 671-681 ◽  
Author(s):  
Russell E. McConnell ◽  
Matthew J. Tyska
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Apical Membrane ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Intestinal Lumen ◽  
Apical Surface ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Membrane Protrusions

Microvilli are actin-rich membrane protrusions common to a variety of epithelial cell types. Within microvilli of the enterocyte brush border (BB), myosin-1a (Myo1a) forms an ordered ensemble of bridges that link the plasma membrane to the underlying polarized actin bundle. Despite decades of investigation, the function of this unique actomyosin array has remained unclear. Here, we show that addition of ATP to isolated BBs induces a plus end–directed translation of apical membrane along microvillar actin bundles. Upon reaching microvillar tips, membrane is “shed” into solution in the form of small vesicles. Because this movement demonstrates the polarity, velocity, and nucleotide dependence expected for a Myo1a-driven process, and BBs lacking Myo1a fail to undergo membrane translation, we conclude that Myo1a powers this novel form of motility. Thus, in addition to providing a means for amplifying apical surface area, we propose that microvilli function as actomyosin contractile arrays that power the release of BB membrane vesicles into the intestinal lumen.

scholarly journals Hemidesmosomes in the epithelial cell line 804G: their fate during wound closure, mitosis and drug induced reorganization of the cytoskeleton

1992 ◽  
Vol 103 (2) ◽  
pp. 475-490 ◽  
Author(s):  
K.S. Riddelle ◽  
S.B. Hopkinson ◽  
J.C. Jones
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Ultrastructural Level ◽  
Cytochalasin D ◽  
Epithelial Cell Line ◽  
Cell Periphery ◽  
Basal Surface ◽  
Drug Induced ◽  
In Cells

Recently, we identified a novel epithelial cell line, 804G, derived from rat bladder, which readily forms hemidesmosomes in vitro. One of the major structural components of the plaques of 804G cell hemidesmosomes is a 230 kDa antigen recognized by autoantibodies in the sera of patients with bullous pemphigoid (BP). An additional polypeptide of 180 kDa also localizes to the hemidesmosome plaque of 804G cells as determined by immunoelectron microscopy. Using confocal fluorescence/phase microscopy, we have employed both 230 kDa and 180 kDa antibody probes to monitor the fate of hemidesmosomes following closure of in vitro wounds, during mitosis, and following drug induced disruption of the cytoskeleton. The punctate cell-substratum associated staining generated by the hemidesmosomal antibodies in stationary unwounded 804G cell cultures is greatly diminished or even lost in cells which enter wound sites, presumably in response to enhanced cell motility. Few, if any hemidesmosomes are observed at the ultrastructural level in cells which have migrated into the wound area. However, as closure of the wound becomes complete, staining along the substratum attached surface of cells returns. During mitosis, there is no obvious loss of hemidesmosomal antigens along the basal surface of 804G cells, and formed hemidesmosomes can be observed in mitotic cells at the ultrastructural level. In 804G cells treated with colchicine, the typical subnuclear pattern of distribution of hemidesmosomal antigens is unaffected. In contrast, following treatment of 804G cells with cytochalasin D, hemidesmosomal antigens become concentrated at the cell periphery and no longer appear in the subnuclear region. Furthermore, formed hemidesmosomes are observed at the cell periphery of cytochalasin D-treated cells by electron microscopy. We suggest that hemidesmosomal plaques are mobile within the plasma membrane. We speculate that hemidesmosomal interactions with extracellular ligands are dynamic and we discuss a possible mechanism by which cytochalasin D induces reorganization of hemidesmosomes along the basal surface of 804G cells.

scholarly journals ARF1 at the crossroads of podosome construction and function

2016 ◽  
Vol 216 (1) ◽  
pp. 13-15
Author(s):  
Elisabeth Genot
Keyword(s):  
Plasma Membrane ◽  
Membrane Traffic ◽  
Cell Biol ◽  
Guanosine Triphosphatase ◽  
And Function ◽  
In Cells

Podosomes are actin-based proteolytic microdomains of the plasma membrane found in cells that travel across tissues. In this issue, Rafiq et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201605104) reveal that the small guanosine triphosphatase ARF1, a well-known orchestrator of membrane traffic at the Golgi, regulates podosome formation, maintenance, and function.

scholarly journals Strength in numbers: Phosphofructokinase polymerization prevails in the liver

2017 ◽  
Vol 216 (8) ◽  
pp. 2239-2241 ◽  
Author(s):  
Elma Zaganjor ◽  
Jessica B. Spinelli ◽  
Marcia C. Haigis
Keyword(s):  
Plasma Membrane ◽  
Spatial Organization ◽  
Metabolic Enzymes ◽  
Cell Biol ◽  
Regulatory Functions ◽  
In Cells

Numerous metabolic enzymes assemble into filamentous structures, which are thought to serve additional regulatory functions. In this issue, Webb et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201701084) show that the liver-specific isoform of phosphofructokinase-1 forms filaments in vitro and localizes as puncta in cells along the plasma membrane. This suggests spatial organization of glycolysis in higher organisms.

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