scholarly journals Characterization of a 36,000-dalton protein from the surface of Madin-Darby canine kidney cells involved in cell attachment and spreading.

1985 ◽  
Vol 100 (6) ◽  
pp. 2001-2007 ◽  
Author(s):  
M Sabanero ◽  
A Gonzalez-Robles ◽  
I Meza
Keyword(s):  
Tight Junctions ◽  
Cell Attachment ◽  
External Surface ◽  
Polyclonal Antibodies ◽  
Inhibitory Effect ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

We have identified and immunochemically characterized a 36,000-dalton membrane glycoprotein from Madin-Darby canine kidney cells. This protein is surface-labeled by lactoperoxidase-mediated iodination and metabolically labeled by [35S]methionine. It binds to Concanavalin A and incorporates 2-D-3H-mannose residues, thus indicating it is a glycoprotein. Rabbit polyclonal antibodies against this protein evenly decorate the external surface of trypsinized, unpolarized cells. The external apical surface of confluent monolayers, grown under culture conditions in which the tight junctions are closed and the cells have acquired polarity, is also evenly stained. The basolateral aspects of the external surface are stained only when the tight junctions are opened by removal of Ca++ or when the antibody has access to the monolayer from the basal side, which indicates an even distribution of this antigen on the surface of polarized cells. The antibody has no inhibitory effect on the opening and resealing of tight junctions in dense cultures, but does inhibit the attachment and spreading of cells on a substrate, which then blocks the establishment of a confluent functional monolayer.

scholarly journals Similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized Madin–Darby canine kidney cells

2013 ◽  
Vol 24 (8) ◽  
pp. 1196-1207 ◽  
Author(s):  
Steeve Boulant ◽  
Megan Stanifer ◽  
Comert Kural ◽  
David K. Cureton ◽  
Ramiro Massol ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Endocytic Pathway ◽  
Cell Entry ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin–Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.

scholarly journals Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. II. Immunological quantitation.

1983 ◽  
Vol 97 (3) ◽  
pp. 638-643 ◽  
Author(s):  
M Pesonen ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Binding Assay ◽  
Protein A ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The envelope of vesicular stomatitis virus was fused with the apical plasma membrane of Madin-Darby canine kidney cells by low pH treatment. The fate of the implanted G protein was then followed using a protein A-binding assay, which was designed to quantitate the amount of G protein in the apical and the basolateral membranes. The implanted G protein was rapidly internalized at 31 degrees C, whereas at 10 degrees C no uptake was observed. Already after 15 min at 31 degrees C, a fraction of the G protein could be detected at the basolateral membrane. After 60 min 25-48% of the G protein was basolateral as measured by the protein A-binding assay. At the same time, 25-33% of the implanted G protein was detected at the apical membrane. Internalization of G protein was not affected by 20 mM ammonium chloride or by 10 microM monensin. However, the endocytosed G protein accumulated in intracellular vacuoles and redistribution back to the plasma membrane was inhibited. We conclude that the implanted G protein was rapidly internalized from the apical surface of Madin-Darby canine kidney cells and a major fraction was routed to the basolateral domain.

Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells

1990 ◽  
Vol 96 (1) ◽  
pp. 143-149
Author(s):  
J.M. Wilson ◽  
N. Fasel ◽  
J.P. Kraehenbuhl
Keyword(s):  
Plasma Membrane ◽  
Common Mechanism ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Transfected Cells ◽  
Glycosyl Phosphatidylinositol ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 micron frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool. Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail. In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.

scholarly journals The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium.

1989 ◽  
Vol 109 (6) ◽  
pp. 2817-2832 ◽  
Author(s):  
R Bacallao ◽  
C Antony ◽  
C Dotti ◽  
E Karsenti ◽  
E H Stelzer ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Golgi Complex ◽  
Single Cells ◽  
Junctional Complex ◽  
Apical Region ◽  
Kidney Cells ◽  
Basal Region ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4-nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.

scholarly journals Involvement of the Interaction of Afadin with ZO-1 in the Formation of Tight Junctions in Madin-Darby Canine Kidney Cells

2009 ◽  
Vol 285 (7) ◽  
pp. 5003-5012 ◽  
Author(s):  
Takako Ooshio ◽  
Reiko Kobayashi ◽  
Wataru Ikeda ◽  
Muneaki Miyata ◽  
Yuri Fukumoto ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

scholarly journals Involvement of Nectin-activated Cdc42 Small G Protein in Organization of Adherens and Tight Junctions in Madin-Darby Canine Kidney Cells

2003 ◽  
Vol 278 (51) ◽  
pp. 51885-51893 ◽  
Author(s):  
Atsunori Fukuhara ◽  
Kazuya Shimizu ◽  
Tomomi Kawakatsu ◽  
Tatsuro Fukuhara ◽  
Yoshimi Takai
Keyword(s):  
Tight Junctions ◽  
G Protein ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Small G Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney

scholarly journals Intracellular transport of influenza virus hemagglutinin to the apical surface of Madin-Darby canine kidney cells.

1984 ◽  
Vol 98 (1) ◽  
pp. 308-319 ◽  
Author(s):  
E Rodriguez-Boulan ◽  
K T Paskiet ◽  
P J Salas ◽  
E Bard
Keyword(s):  
Influenza Virus ◽  
Golgi Apparatus ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Surface Appearance ◽  
Influenza Virus Hemagglutinin ◽  
Intracellular Pathway ◽  
Darby Canine Kidney ◽  
Canine Kidney

The intracellular pathway followed by the influenza virus hemagglutinin (HA) to the apical surface of Madin-Darby canine kidney cells was studied by radioimmunoassay, immunofluorescence, and immunoelectron microscopy. To synchronize the migration, we used a temperature-sensitive mutant of influenza WSN, ts61, which, at the nonpermissive temperature, 39.5 degrees C, exhibits a defect in the HA that prevents its exit from the endoplasmic reticulum. Upon transfer to permissive temperature, 32 degrees C, the HA appeared in the Golgi apparatus after 10 min, and on the apical surface after 30-40 min. In the presence of cycloheximide, the expression was not inhibited, indicating that the ts defect is reversible; a wave of HA migrated to the cell surface, where it accumulated with a half time of 60 min. After passage through the Golgi apparatus the HA was detected in a population of smooth vesicles, about twice the size of coated vesicles, located in the apical half of the cytoplasm. These HA-containing vesicles did not react with anti-clathrin antibodies. Monensin (10 microM) delayed the surface appearance of HA by 2 h, but not the transport to the Golgi apparatus. Incubation at 20 degrees C retarded the migration to the Golgi apparatus by approximately 30 min and blocked the surface appearance by acting at a late stage in the intracellular pathway, presumably at the level of the post-Golgi vesicles. The initial appearance of HA on the apical surface was in the center; no preference was observed for the tight-junctional regions.

scholarly journals Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses.

1985 ◽  
Vol 100 (1) ◽  
pp. 136-151 ◽  
Author(s):  
M J Rindler ◽  
I E Ivanov ◽  
H Plesken ◽  
D D Sabatini
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Plasma Membranes ◽  
Temperature Sensitive ◽  
Kidney Cells ◽  
Apical Surface ◽  
Nonpermissive Temperature ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The intracellular route followed by viral envelope glycoproteins in polarized Madin-Darby canine kidney cells was studied by using temperature-sensitive mutants of vesicular stomatitis virus (VSV) and influenza, in which, at the nonpermissive temperature (39.5 degrees C), the newly synthesized glycoproteins (G proteins) and hemagglutinin (HA), respectively, are not transported out of the endoplasmic reticulum. After infection with VSV and incubation at 39.5 degrees C for 4-5 h, synchronous transfer of G protein to the plasma membrane was initiated by shifting to the permissive temperature (32.5 degrees C). Immunoelectron microscopy showed that under these conditions the protein moved to the Golgi apparatus and from there directly to a region of the lateral plasma membrane near this organelle. G protein then seemed to diffuse progressively to basal regions of the cell surface and, only after it had accumulated in the basolateral domain, it began to appear on the apical surface near the intercellular junctions. The results of these experiments indicate that the VSV G protein must be sorted before its arrival at the cell surface, and suggest that passage to the apical domain occurs only late in infection when tight junctions are no longer an effective barrier. In complementary experiments, using the temperature-sensitive mutant of influenza, cultures were first shifted from the nonpermissive temperature (39.5 degrees C) to 18.5 degrees C, to allow entrance of the glycoprotein into the Golgi apparatus (see Matlin, K.S., and K. Simons, 1983, Cell, 34:233-243). Under these conditions HA accumulated in Golgi stacks and vesicles but did not reach the plasma membrane. When the temperature was subsequently shifted to 32.5 degrees C, HA rapidly appeared in discrete regions of the apical surface near, and often directly above, the Golgi elements, and later diffused throughout this surface. To ensure that the anti-HA antibodies had access to lateral domains, monolayers were treated with a hypertonic medium to dilate the intercellular spaces. Some labeling was then observed in the lateral plasma membranes soon after the shift, but this never increased beyond 1.0 gold particle/micron, whereas characteristic densities of labeling in apical surfaces soon became much higher (approximately 10 particles/micron). Our results suggest that the bulk of HA follows a direct pathway leading from the Golgi to regions of the apical surface close to trans-Golgi cisternae.

scholarly journals Isolation and partial characterization of human platelet vinculin.

1985 ◽  
Vol 100 (3) ◽  
pp. 669-676 ◽  
Author(s):  
G C Rosenfeld ◽  
D C Hou ◽  
J Dingus ◽  
I Meza ◽  
J Bryan
Keyword(s):  
Actin Polymerization ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Adhesion Plaques ◽  
End Capping ◽  
Darby Canine Kidney ◽  
Canine Kidney

A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE-Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.

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