scholarly journals Newly synthesized claudins but not occludin are added to the basal side of the tight junction

2019 ◽  
Vol 30 (12) ◽  
pp. 1406-1424 ◽  
Author(s):  
Christina M. Van Itallie ◽  
Karin Fredriksson Lidman ◽  
Amber Jean Tietgens ◽  
James Melvin Anderson
Keyword(s):  
Tight Junction ◽  
Protein Turnover ◽  
Madin Darby Canine Kidney ◽  
Strand Breaks ◽  
Lateral Membrane ◽  
Fluorescent Ligands ◽  
Carboxy Terminal ◽  
Fibroblast Model ◽  
Darby Canine Kidney ◽  
Canine Kidney

A network of claudin strands creates continuous cell–cell contacts to form the intercellular tight junction barrier; a second protein, occludin, is associated along these strands. The physiological barrier remains stable despite protein turnover, which involves removal and replacement of claudins both in the steady state and during junction remodeling. Here we use a pulse–block–pulse labeling protocol with fluorescent ligands to label SNAP/CLIP-tags fused to claudins and occludin to identify their spatial trafficking pathways and kinetics in Madin–Darby canine kidney monolayers. We find that claudins are first delivered to the lateral membrane and, over time, enter the junction strand network from the basal side; this is followed by slow replacement of older claudins in the strands. In contrast, even at early times, newly synthesized occludin is found throughout the network. Taking the results together with our previous documentation of the mechanism for claudin strand assembly in a fibroblast model, we speculate that newly synthesized claudins are added at strand breaks and free ends; these are most common in the basalmost edge of the junction. In contrast, occludin can be added directly within the strand network. We further demonstrate that claudin trafficking and half-life depend on carboxy-terminal sequences and that different claudins compete for tight junction localization.

scholarly journals Cdc42-dependent Modulation of Tight Junctions and Membrane Protein Traffic in Polarized Madin-Darby Canine Kidney Cells

2001 ◽  
Vol 12 (8) ◽  
pp. 2257-2274 ◽  
Author(s):  
Raul Rojas ◽  
Wily G. Ruiz ◽  
Som-Ming Leung ◽  
Tzuu-Shuh Jou ◽  
Gerard Apodaca
Keyword(s):  
Tight Junction ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Kidney Cells ◽  
Cellular Polarity ◽  
Membrane Domain ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.

scholarly journals Restoration of Tight Junction Structure and Barrier Function by Down-Regulation of the Mitogen-activated Protein Kinase Pathway in Ras-transformed Madin-Darby Canine Kidney Cells

2000 ◽  
Vol 11 (3) ◽  
pp. 849-862 ◽  
Author(s):  
Yan-hua Chen ◽  
Qun Lu ◽  
Eveline E. Schneeberger ◽  
Daniel A. Goodenough
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Tight Junctions ◽  
Mitogen Activated Protein Kinase ◽  
Madin Darby Canine Kidney ◽  
Mitogen Activated Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell–cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin–mediated cell–cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell–cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell–cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.

scholarly journals Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon

2012 ◽  
Vol 302 (2) ◽  
pp. R300-R311 ◽  
Author(s):  
M. B. Engelund ◽  
A. S. L. Yu ◽  
J. Li ◽  
S. S. Madsen ◽  
N. J. Færgeman ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Atlantic Salmon ◽  
Protein Expression ◽  
Tight Junction ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Junction Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon ( Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na+-K+-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium.

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

1988 ◽  
Vol 106 (4) ◽  
pp. 1141-1149 ◽  
Author(s):  
J M Anderson ◽  
B R Stevenson ◽  
L A Jesaitis ◽  
D A Goodenough ◽  
M S Mooseker
Keyword(s):  
Tight Junction ◽  
Mdck Cell ◽  
Mouse Liver ◽  
Mdck Cells ◽  
Cell Protein ◽  
Scatchard Analysis ◽  
Madin Darby Canine Kidney ◽  
Mouse Tissues ◽  
Darby Canine Kidney ◽  
Canine Kidney

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.

scholarly journals The Effect of Capsaicin Derivatives on Tight-Junction Integrity and Permeability of Madin-Darby Canine Kidney Cells

2016 ◽  
Vol 105 (2) ◽  
pp. 630-638 ◽  
Author(s):  
Mathias Kaiser ◽  
Sudharani Chalapala ◽  
Christian Gorzelanny ◽  
Ramu Sridhar Perali ◽  
Francisco Martin Goycoolea
Keyword(s):  
Tight Junction ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

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

1986 ◽  
Vol 103 (3) ◽  
pp. 755-766 ◽  
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."

aPKC-PAR complex dysfunction and tight junction disassembly in renal epithelial cells during ATP depletion

2007 ◽  
Vol 292 (3) ◽  
pp. C1094-C1102 ◽  
Author(s):  
Shobha Gopalakrishnan ◽  
Mark A. Hallett ◽  
Simon J. Atkinson ◽  
James A. Marrs
Keyword(s):  
Tight Junction ◽  
Atp Depletion ◽  
Cell Junctions ◽  
Cell Contact ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Rac Gtpase ◽  
Signaling Complex ◽  
Darby Canine Kidney ◽  
Canine Kidney

Renal ischemia and in vitro ATP depletion result in disruption of the epithelial tight junction barrier, which is accompanied by breakdown of plasma membrane polarity. Tight junction formation is regulated by evolutionarily conserved complexes, including that of atypical protein kinase C (aPKC), Par3, and Par6. The aPKC signaling complex is activated by Rac and regulated by protein phosphorylation and associations with other tight junction regulatory proteins, for example, mLgl. In this study, we examined the role of aPKC signaling complex during ATP depletion and recovery in Madin-Darby canine kidney cells. ATP depletion reduced Rac GTPase activity and induced Par3, aPKCζ, and mLgl-1 redistribution from sites of cell-cell contact, which was restored following recovery from ATP depletion. Zonula occludens (ZO)-1 and Par3 phosphorylation was reduced and association of aPKCζ with its substrates Par3 and mLgl-1 was stabilized in ATP-depleted Madin-Darby canine kidney cells. ATP depletion also induced a stable association of Par3 with Tiam-1, a Rac GTPase exchange factor, which explains how aPKCζ and Rac activities were suppressed. Experimental inhibition of aPKCζ during recovery from ATP depletion interfered with reassembly of ZO-1 and Par3 at cell junctions. These data indicate that aPKC signaling is impaired during ATP depletion, participates in tight junction disassembly during cell injury and is important for tight junction reassembly during recovery.

scholarly journals Phosphorylation of the tight-junction protein ZO-1 in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance

1989 ◽  
Vol 263 (2) ◽  
pp. 597-599 ◽  
Author(s):  
B R Stevenson ◽  
J M Anderson ◽  
I D Braun ◽  
M S Mooseker
Keyword(s):  
Tight Junction ◽  
Specific Protein ◽  
Resistance Strain ◽  
Transepithelial Resistance ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Junction Protein ◽  
Junctional Permeability ◽  
Darby Canine Kidney ◽  
Canine Kidney

A comparison was made of the phosphate content of the tight-junction-specific protein ZO-1 in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance, a parameter reflective of tight-junctional permeability. Analysis revealed that the ZO-1 from the low-resistance strain contained approximately twice as much phosphate as that from the high-resistance strain.

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

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.

scholarly journals E-cadherin Polarity Is Determined by a Multifunction Motif Mediating Lateral Membrane Retention through Ankyrin-G and Apical-lateral Transcytosis through Clathrin

2013 ◽  
Vol 288 (20) ◽  
pp. 14018-14031 ◽  
Author(s):  
Paul M Jenkins ◽  
Chirag Vasavda ◽  
Janell Hostettler ◽  
Jonathan Q. Davis ◽  
Khadar Abdi ◽  
...  
Keyword(s):  
High Fidelity ◽  
Partial Loss ◽  
Madin Darby Canine Kidney ◽  
Lateral Membrane ◽  
Ankyrin G ◽  
Restricted Mobility ◽  
And Function ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin

We report a highly conserved motif in the E-cadherin juxtamembrane domain that determines apical-lateral polarity by conferring both restricted mobility at the lateral membrane and transcytosis of apically mis-sorted protein to the lateral membrane. Mutations causing either increased lateral membrane mobility or loss of apical-lateral transcytosis result in partial mis-sorting of E-cadherin in Madin-Darby canine kidney cells. However, loss of both activities results in complete loss of polarity. We present evidence that residues required for restricted mobility mediate retention at the lateral membrane through interaction with ankyrin-G, whereas dileucine residues conferring apical-lateral transcytosis act through a clathrin-dependent process and function in an editing pathway. Ankyrin-G interaction with E-cadherin is abolished by the same mutations resulting in increased E-cadherin mobility. Clathrin heavy chain knockdown and dileucine mutation of E-cadherin both cause the same partial loss of polarity of E-cadherin. Moreover, clathrin knockdown causes no further change in polarity of E-cadherin with dileucine mutation but does completely randomize E-cadherin mutants lacking ankyrin-binding. Dileucine mutation, but not loss of ankyrin binding, prevented transcytosis of apically mis-sorted E-cadherin to the lateral membrane. Finally, neurofascin, which binds ankyrin but lacks dileucine residues, exhibited partial apical-lateral polarity that was abolished by mutation of its ankyrin-binding site but was not affected by clathrin knockdown. The polarity motif thus integrates complementary activities of lateral membrane retention through ankyrin-G and apical-lateral transcytosis of mis-localized protein through clathrin. Together, the combination of retention and editing function to ensure a high fidelity steady state localization of E-cadherin at the lateral membrane.

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