Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.

ZO-1, originally identified by mAb techniques, is the first protein shown to be specifically associated with the tight junction. Here we describe and compare the physical characteristics of ZO-1 from mouse liver and the Madin-Darby canine kidney (MDCK) epithelial cell line. The ZO-1 polypeptide has an apparent size of 225 kD in mouse tissues and 210 kD in canine-derived MDCK cells as determined by SDS-PAGE/immunoblot analysis. ZO-1 from both sources is optimally solubilized from isolated plasma membranes by either 6 M urea or high pH conditions; partial solubilization occurs with 0.3 M KCl. The nonionic detergents, Triton X-100 and octyl-beta-D-glucopyranoside, do not solubilize ZO-1. These solubility properties indicate that ZO-1 is a peripherally associated membrane protein. ZO-1 was purified to electrophoretic homogeneity from [35S]methionine metabolically labeled MDCK cells by a combination of gel filtration and immunoaffinity chromatography. Purified ZO-1 has an s20,w of 5.3 and Stokes radius of 8.6 nm. These values suggest that purified ZO-1 is an asymmetric monomeric molecule. Corresponding values for mouse liver ZO-1, characterized in impure protein extracts, were 6 s20,w and 9 nm. ZO-1 was shown to be a phosphoprotein in MDCK cells metabolically labeled with [32P]orthophosphate; analysis of phosphoamino acids from purified ZO-1 revealed only phosphoserine. ZO-1 epitope number was determined by Scatchard analysis of competitive and saturable binding of two different 125I-mAbs to SDS-solubilized proteins from liver and MDCK cells immobilized on nitrocellulose. Saturation binding occurs at 26 ng mAb/mg liver and 63 ng/mg of MDCK cell protein. This is equivalent to 30,000 ZO-1 molecules per MDCK cell assuming a single epitope/ZO-1 molecule.

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Betaine and inositol reduce MDCK cell glycerophosphocholine by stimulating its degradation

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.1.c200 ◽

1996 ◽

Vol 270 (1) ◽

pp. C200-C207 ◽

Cited By ~ 41

Author(s):

E. D. Kwon ◽

K. Zablocki ◽

E. M. Peters ◽

K. Y. Jung ◽

A. Garcia-Perez ◽

...

Keyword(s):

Mdck Cell ◽

Mdck Cells ◽

Phospholipase Activity ◽

Madin Darby Canine Kidney ◽

Data Support ◽

Compatible Osmolytes ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Medullary Cells

The amount of glycerophosphocholine (GPC) in renal medullary cells in vivo and in cultured renal [Madin-Darby canine kidney (MDCK)] cells varies with extracellular NaCl and urea. We previously showed that this is largely due to modulation of GPC degradation catalyzed by GPC:choline phosphodiesterase (GPC: PDE). GPC also varies inversely with the levels of other compatible osmolytes, the accumulation of which is induced by high tonicity. We tested whether GPC:PDE activity and GPC degradation are affected by accumulation of compatible osmolytes other than GPC. We find that MDCK cell GPC content decreases when the cells take up betaine and/or inositol from the medium. The effect is considerably greater for cells in isosmotic or high-NaCl medium than in high-urea medium. This difference is associated with suppression of betaine and inositol accumulation with high urea. We then measured GPC:PDE activity with a novel chemiluminescent assay. Addition of inositol and/or betaine to the medium greatly increases GPC:PDE activity in cells in isosmotic or high-NaCl media, but the increase is much less in high-urea medium. The increases in GPC:PDE activity, associated with the presence of betaine, are accompanied by commensurate increases in absolute rates of endogenous GPC degradation by cells in isosmotic or high-NaCl medium. We found previously that, in MDCK cells incubated for 2 days in high-NaCl medium, the rate of GPC synthesis from phosphatidylcholine is increased, correlated with an increase in phospholipase activity. However, in the present experiments, betaine accumulation has no effect on phospholipase activity under those conditions and, thus, presumably does not affect GPC synthesis. Collectively, these data support the conclusion that betaine and/or inositol reduces GPC by increasing GPC degradation catalyzed by GPC:PDE. This mechanism enables GPC to be reciprocally regulated relative to other compatible osmolytes, thus maintaining an appropriate total osmolyte content.

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Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer.

The Journal of Cell Biology ◽

10.1083/jcb.107.1.221 ◽

1988 ◽

Vol 107 (1) ◽

pp. 221-230 ◽

Cited By ~ 115

Author(s):

B B Finlay ◽

B Gumbiner ◽

S Falkow

Keyword(s):

Mdck Cell ◽

Pathogenic Bacteria ◽

Mdck Cells ◽

Cell Monolayer ◽

Apical Surface ◽

Madin Darby Canine Kidney ◽

Intracellular Parasites ◽

Epithelial Barriers ◽

Darby Canine Kidney ◽

Canine Kidney

Many intracellular parasites are capable of penetrating host epithelial barriers. To study this process in more detail we examined the interactions between the pathogenic bacteria Salmonella choleraesuis and polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells grown on membrane filters. Association of bacteria with the MDCK cell apical surface was an active event, requiring bacterial RNA and protein synthesis, and was blocked by low temperatures. Salmonella were internalized within a membrane-bound vacuole and exhibited penetration through, but not between MDCK cells. A maximum of 14 Salmonella per MDCK cell crossed the monolayer per hour to the basolateral surface yet the monolayer remained viable and impermeable to Escherichia coli. Apical S. choleraesuis infection resulted in an increase in paracellular permeability but the MDCK intercellular contacts were not significantly disrupted. Basolateral S. choleraesuis infection was inefficient, and only small numbers of S. choleraesuis penetrated to the apical medium.

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Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.

The Journal of Cell Biology ◽

10.1083/jcb.103.3.755 ◽

1986 ◽

Vol 103 (3) ◽

pp. 755-766 ◽

Cited By ~ 979

Author(s):

B R Stevenson ◽

J D Siliciano ◽

M S Mooseker ◽

D A Goodenough

Keyword(s):

Molecular Weight ◽

Tight Junction ◽

Membrane Fraction ◽

Mouse Liver ◽

Immunofluorescent Staining ◽

Junctional Complex ◽

Madin Darby Canine Kidney ◽

Complex Region ◽

Darby Canine Kidney ◽

Canine Kidney

A tight junction-enriched membrane fraction has been used as immunogen to generate a monoclonal antiserum specific for this intercellular junction. Hybridomas were screened for their ability to both react on an immunoblot and localize to the junctional complex region on frozen sections of unfixed mouse liver. A stable hybridoma line has been isolated that secretes an antibody (R26.4C) that localizes in thin section images of isolated mouse liver plasma membranes to the points of membrane contact at the tight junction. This antibody recognizes a polypeptide of approximately 225,000 D, detectable in whole liver homogenates as well as in the tight junction-enriched membrane fraction. R26.4C localizes to the junctional complex region of a number of other epithelia, including colon, kidney, and testis, and to arterial endothelium, as assayed by immunofluorescent staining of cryostat sections of whole tissue. This antibody also stains the junctional complex region in confluent monolayers of the Madin-Darby canine kidney epithelial cell line. Immunoblot analysis of Madin-Darby canine kidney cells demonstrates the presence of a polypeptide similar in molecular weight to that detected in liver, suggesting that this protein is potentially a ubiquitous component of all mammalian tight junctions. The 225-kD tight junction-associated polypeptide is termed "ZO-1."

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Presence of a novel influx pathway for Mg2+ in MDCK cells

AJP Cell Physiology ◽

10.1152/ajpcell.1990.259.3.c521 ◽

1990 ◽

Vol 259 (3) ◽

pp. C521-C525 ◽

Cited By ~ 47

Author(s):

G. A. Quamme ◽

L. J. Dai

Keyword(s):

Cell Line ◽

Mdck Cell ◽

Mdck Cells ◽

Cell Types ◽

Madin Darby Canine Kidney ◽

Entry Pathway ◽

Electrical Gradient ◽

Free Media ◽

Darby Canine Kidney ◽

Canine Kidney

Basal free Mg2+ concentration was 0.49 +/- 0.03 mM in normal single Madin-Darby canine kidney (MDCK) cells as measured by fluorescence with the aid of mag-fura-2. Accordingly, Mg2+ may enter the cell down a transmembrane electrical gradient. The present study describes some aspects of Mg2+ entry into the established MDCK cell line. MDCK cells were Mg2(+)-depleted (0.26 +/- 0.01 mM) by culturing in Mg2(+)-free media for 16-20 h. Cells were subsequently exposed to 5 mM MgCl2, and intracellular Mg2+ concentration ([Mg2+]i) was monitored with fluorescence. [Mg2+]i returned to normal basal levels, 0.56 +/- 0.05 mM, with a refill rate of 272 +/- 39 nM/s, n = 4. Mg2+ entry was not changed by 5.0 mM external Ca2+ but was completely inhibited with 5.0 mM La3+. Intracellular Ca2+ concentration was not altered by Mg2+ depletion or during Mg2+ repletion. Mg2+ uptake was inhibited by verapamil (0 +/- 27 nM/s, n = 3), was inhibited less so by diltiazem (141 +/- 34 nM/s, n = 3), and was not affected by nifedipine (300 +/- 53 nM/s, n = 6). These inhibitors were fully reversible on removal, and [Mg2+]i returned to normal levels. These data indicate the presence of a unique Mg2+ entry pathway in MDCK cells that may be important in Mg2+ homeostasis. The model of Mg2+ refill into Mg2(+)-depleted cells may be useful in other cell types.

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Evidence for ERK1/2 phosphorylation controlling contact inhibition of proliferation in Madin-Darby canine kidney epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00020.2004 ◽

2004 ◽

Vol 287 (2) ◽

pp. C432-C439 ◽

Cited By ~ 20

Author(s):

Shixiong Li ◽

Edward R. Gerrard ◽

Daniel F. Balkovetz

Keyword(s):

Cell Proliferation ◽

Cyclin D1 ◽

Mdck Cell ◽

Mdck Cells ◽

High Density ◽

Contact Inhibition ◽

Low Density ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney

Increasing cell density arrests epithelial cell proliferation by a process termed contact inhibition. We investigated mechanisms of contact inhibition using a model of contact-inhibited epithelial cells. Hepatocyte growth factor (HGF) treatment of contact-inhibited Madin-Darby canine kidney (MDCK) cells stimulated cell proliferation and increased levels of phosphorylated ERK1/2 (phospho-ERK1/2) and cyclin D1. MEK inhibitors PD-98059 and U0126 inhibited these HGF-dependent changes, indicating the dependence on phosphorylation of ERK1/2 during HGF-induced loss of contact inhibition. In relation to contact-inhibited high-density cells, low-density MDCK cells proliferated and had higher levels of phospho-ERK1/2 and cyclin D1. PD-98059 and U0126 inhibited low-density MDCK cell proliferation. Trypsinization of high-density MDCK cells immediately increased phospho-ERK1/2 and was followed by a transient increase in cyclin D1 levels. Reformation of cell junctions after trypsinization led to decreases in phospho-ERK1/2 and cyclin D1 levels. High-density MDCK cells express low levels of both cyclin D1 and phospho-ERK1/2, and treatment of these cells with fresh medium containing HGF but not fresh medium alone for 6 h increased phospho-ERK1/2 and cyclin D1 levels compared with cells without medium change. These data provide evidence that HGF abrogates MDCK cell contact inhibition by increasing ERK1/2 phosphorylation and levels of cyclin D1. These results suggest that in MDCK cells, contact inhibition of cell proliferation in the presence of serum occurs by cell density-dependent regulation of ERK1/2 phosphorylation.

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Apical Actin-myosin Network Regulates the Tight Junction of Polarized Madin-Darby Canine Kidney Cells

10.1101/2021.05.30.446323 ◽

2021 ◽

Author(s):

Chia-hsuan Lu ◽

Fu-lai Wen ◽

Shawn Ching-Chung Hsueh ◽

Wen-hsiu Wu ◽

Yu-Fang Lin ◽

...

Keyword(s):

Epithelial Cells ◽

Tight Junction ◽

Mdck Cell ◽

Cell Interactions ◽

Apical Surface ◽

Actin Network ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Cell Cell

The tight junction outlines the apicolateral border of epithelial cells like a belt, sealing the paracellular space when cells form contacts with each other. The permeability and morphology of tight junction are regulated by actomyosin contractility, which has been conventionally thought from the purse-string-like circumferential actomyosin belt along tight junction. Spatially, the tight junction is close to the apical actin network, which exerts inward contractions orthogonal to the tight junction. To test the contributions from apical actin network, we laser-ablated spots on the apical surface of polarized Madin-Darby Canine Kidney (MDCK) epithelial cells. Laser ablation severed the apical cytoskeleton network, decreased in-plane tension, increased the apical surface area, and rendered the tight junction less tortuous in shape. Consistent with these observations, changes in MDCK cell sheet morphology due to cell proliferation, or perturbation with the ROCK inhibitor Y27632 increased the density of the apical actin network and decreased tight junction tortuosity. The morphological analysis revealed scutoids in flat MDCK cell sheets, contrary to predictions from a previous model that only considered cell-cell interactions as line tension. Additional cell-cell interactions from apical in-plane tension provides probable cause for the occurrence of scutoids on flat geometry. Taken together, our findings identify the importance of the apical actin network exerting in-plane apical tension to regulate tight-junction mechanobiology and epithelial cell shape.

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Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance.

The Journal of Cell Biology ◽

10.1083/jcb.107.6.2401 ◽

1988 ◽

Vol 107 (6) ◽

pp. 2401-2408 ◽

Cited By ~ 166

Author(s):

B R Stevenson ◽

J M Anderson ◽

D A Goodenough ◽

M S Mooseker

Keyword(s):

Tight Junction ◽

Mdck Cells ◽

Freeze Fracture ◽

Transepithelial Resistance ◽

Madin Darby Canine Kidney ◽

Junctional Permeability ◽

Tight Junction Permeability ◽

Junction Structure ◽

Darby Canine Kidney ◽

Canine Kidney

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219-232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).

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Differential sensitivity of Madin-Darby canine kidney (MDCK) cells to epinephrine

The journal of nutrition health & aging ◽

10.1007/s12603-015-0604-y ◽

2015 ◽

Vol 20 (5) ◽

pp. 486-493 ◽

Cited By ~ 4

Author(s):

P. Muthuraman ◽

P. C. Nagajyothi ◽

M. Chandrasekaran ◽

G. Enkhtaivan ◽

B. Venkitasamy ◽

...

Keyword(s):

Mdck Cells ◽

Differential Sensitivity ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney

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Accumulation of natural and synthetic betaines by a mammalian renal cell line

Biochemistry and Cell Biology ◽

10.1139/o96-030 ◽

1996 ◽

Vol 74 (2) ◽

pp. 283-287 ◽

Cited By ~ 12

Author(s):

K. Randall ◽

M. Lever ◽

B. A. Peddie ◽

S. T. Chambers

Keyword(s):

Osmotic Stress ◽

Glycine Betaine ◽

Mdck Cells ◽

Intracellular Accumulation ◽

Madin Darby Canine Kidney ◽

Renal Cell Line ◽

High Concentrations ◽

Inhibition Studies ◽

Darby Canine Kidney ◽

Canine Kidney

Intracellular accumulation of different betaines was compared in osmotically stressed Madin Darby canine kidney (MDCK) cells to model the betaine accumulation specificity of the mammalian inner medulla and to show how this accumulation differed from that of bacteria. All betaines accumulated less than glycine betaine. Arsenobetaine (the arsenic analogue of glycine betaine) accumulated to 12% of the glycine betaine levels and the sulphur analogue dimethylthetin accumulated to >80%. Most substituted glycine betaine analogues accumulated to 2–5% of intracellular glycine betaine concentrations, however, serine betaine accumulated to <0.5% of glycine betaine levels. Inhibition studies to distinguish the betaine ports were performed by the addition of proline. Butyrobetaine and carnitine accumulation was not proline sensitive, whereas that of omer betaines was. As with glycine betaine, the accumulation of propionobetaine and dimethylthetin was proline sensitive and osmoregulated. Pyridinium betaine was accumulated by both proline-sensitive and -insensitive systems, with a small increase under osmotic stress. High concentrations (10 times that of glycine betaine) of the dietary betaines proline betaine and trigonelline inhibited total betaine accumulation. Because α-substituted betaines are accumulated by bacteria and not by MDCK cells, these betaines may be the basis for design of antimicrobial agents.Key words: MDCK cells, betaine accumulation, osmolytes, betaine analogues.

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