scholarly journals Zonula Occludens-1 Function in the Assembly of Tight Junctions in Madin-Darby Canine Kidney Epithelial Cells

2006 ◽  
Vol 17 (4) ◽  
pp. 1922-1932 ◽  
Author(s):  
Elizabeth McNeil ◽  
Christopher T. Capaldo ◽  
Ian G. Macara
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Point Mutations ◽  
Sh3 Domain ◽  
Madin Darby Canine Kidney ◽  
Zonula Occludens ◽  
Junction Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney

Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of ∼3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.

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 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 NZO-3 Expression Causes Global Changes to Actin Cytoskeleton in Madin-Darby Canine Kidney Cells: Linking a Tight Junction Protein to Rho GTPases

2003 ◽  
Vol 14 (5) ◽  
pp. 1757-1768 ◽  
Author(s):  
Erika S. Wittchen ◽  
Julie Haskins ◽  
Bruce R. Stevenson
Keyword(s):  
Tight Junction ◽  
Actin Cytoskeleton ◽  
Tight Junction Protein ◽  
Junctional Complex ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
P120 Catenin ◽  
Junction Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney

We previously demonstrated that exogenous expression of a truncated form of the tight junction protein ZO-3 affected junctional complex assembly and function. Current results indicate that this ZO-3 construct influences actin cytoskeleton dynamics more globally. We show that expression of the amino-terminal half of ZO-3 (NZO-3) in Madin-Darby canine kidney cells results in a decreased number of stress fibers and focal adhesions and causes an increased rate of cell migration in a wound healing assay. We also demonstrate that RhoA activity is reduced in NZO-3–expressing cells. We determined that ZO-3 interacts with p120 catenin and AF-6, proteins localized to the junctional complex and implicated in signaling pathways important for cytoskeleton regulation and cell motility. We also provide evidence that NZO-3 interacts directly with the C terminus of ZO-3, and we propose a model where altered interactions between ZO-3 and p120 catenin in NZO-3–expressing cells affect RhoA GTPase activity. This study reveals a potential link between ZO-3 and RhoA-related signaling events.

scholarly journals Autocrine Transforming Growth Factor-β1 Activation Mediated by Integrin αVβ3 Regulates Transcriptional Expression of Laminin-332 in Madin-Darby Canine Kidney Epithelial Cells

2010 ◽  
Vol 21 (21) ◽  
pp. 3654-3668 ◽  
Author(s):  
Jose V. Moyano ◽  
Patricia G. Greciano ◽  
Mary M. Buschmann ◽  
Manuel Koch ◽  
Karl S. Matlin
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Integrin Αvβ3 ◽  
Type I ◽  
Madin Darby Canine Kidney ◽  
Epithelial Regeneration ◽  
Tgf Β1 ◽  
Darby Canine Kidney ◽  
Canine Kidney

Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-β1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-β type I receptor (TβR-I) and the Smad2–Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-β1 secretion and activation mediated by integrin αVβ3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-β1 is secreted apically, whereas TβR-I and integrin αVβ3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-β1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.

Cytoskeletal involvement in the modulation of cell-cell junctions by the protein kinase inhibitor H-7

1994 ◽  
Vol 107 (3) ◽  
pp. 683-692 ◽  
Author(s):  
S. Citi ◽  
T. Volberg ◽  
A.D. Bershadsky ◽  
N. Denisenko ◽  
B. Geiger
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Immunofluorescence Microscopy ◽  
Kinase Inhibitor ◽  
Cytochalasin D ◽  
Extracellular Calcium ◽  
Protein Kinase Inhibitor ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The protein kinase inhibitor H-7 has been shown to block junction dissociation induced by low extracellular calcium in Madin Darby canine kidney epithelial cells (S. Citi, J. Cell Biol. (1992) 117, 169–178). To understand the basis of this effect, we have examined how H-7 affects the organization of junctions and the actin cytoskeleton in different types of epithelial cells in culture. Immunofluorescence microscopy showed that H-7 confers Ca2+ independence on cultured epithelial lens cells, which lack tight junctions and desmosomes but have microfilament-associated adherens junctions. In these cells, H-7 did not protect N-cadherin from trypsin digestion at low extracellular calcium, suggesting that H-7 does not stabilize the ‘active’ cadherin conformation. In cultured Madin Darby canine kidney cells, H-7 partially prevented the fall in transepithelial resistance induced by cytochalasin D, either alone or in conjunction with calcium chelators. Double-immunofluorescence microscopy showed that H-7 inhibits both the fragmentation of labeling for the tight junction protein cingulin and the condensation of actin into cytoplasmic foci induced by cytochalasin D. Taken together, these observations indicate that H-7 inhibits junction dissociation by affecting the contractility of the adherens junction-associated microfilaments following treatment with calcium chelators or cytochalasin D.

Sign in / Sign up

Export Citation Format

Share Document