Differential Regulation of Endogenous Cadherin Expression in Madin-Darby Canine Kidney Cells by Cell-Cell Adhesion and Activation of β-Catenin Signaling

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin–coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin–coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.

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Myosin-1c regulates the dynamic stability of E-cadherin–based cell–cell contacts in polarized Madin–Darby canine kidney cells

Molecular Biology of the Cell ◽

10.1091/mbc.e12-12-0884 ◽

2013 ◽

Vol 24 (18) ◽

pp. 2820-2833 ◽

Cited By ~ 15

Author(s):

Hiroshi Tokuo ◽

Lynne M. Coluccio

Keyword(s):

Dynamic Stability ◽

Motor Function ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

Cell Contacts ◽

Cell Adhesions ◽

Darby Canine Kidney ◽

Canine Kidney ◽

E Cadherin ◽

Cell Cell

Cooperation between cadherins and the actin cytoskeleton controls the formation and maintenance of cell–cell adhesions in epithelia. We find that the molecular motor protein myosin-1c (Myo1c) regulates the dynamic stability of E-cadherin–based cell–cell contacts. In Myo1c-depleted Madin–Darby canine kidney cells, E-cadherin localization was disorganized and lateral membranes appeared less vertical with convoluted edges versus control cells. In polarized monolayers, Myo1c-knockdown (KD) cells were more sensitive to reduced calcium concentration. Myo1c separated in the same plasma membrane fractions as E-cadherin, and Myo1c KD caused a significant reduction in the amount of E-cadherin recovered in one peak fraction. Expression of green fluorescent protein (GFP)–Myo1c mutants revealed that the phosphatidylinositol-4,5-bisphosphate–binding site is necessary for its localization to cell–cell adhesions, and fluorescence recovery after photobleaching assays with GFP-Myo1c mutants revealed that motor function was important for Myo1c dynamics at these sites. At 18°C, which inhibits vesicle recycling, Myo1c-KD cells accumulated more E-cadherin–positive vesicles in their cytoplasm, suggesting that Myo1c affects E-cadherin endocytosis. Studies with photoactivatable GFP–E-cadherin showed that Myo1c KD reduced the stability of E-cadherin at cell–cell adhesions. We conclude that Myo1c stabilizes E-cadherin at adherens junctions in polarized epithelial cells and that the motor function and ability of Myo1c to bind membrane are critical.

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

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00286.2011 ◽

2012 ◽

Vol 302 (2) ◽

pp. R300-R311 ◽

Cited By ~ 15

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.

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Par1b Promotes Hepatic-type Lumen Polarity in Madin Darby Canine Kidney Cells via Myosin II- and E-Cadherin–dependent Signaling

Molecular Biology of the Cell ◽

10.1091/mbc.e07-02-0095 ◽

2007 ◽

Vol 18 (6) ◽

pp. 2203-2215 ◽

Cited By ~ 36

Author(s):

David Cohen ◽

Yuan Tian ◽

Anne Müsch

Keyword(s):

Cell Adhesion ◽

Mdck Cells ◽

Myosin Ii ◽

Apical Surface ◽

Madin Darby Canine Kidney ◽

Luminal Compartment ◽

Darby Canine Kidney ◽

Canine Kidney ◽

E Cadherin ◽

Cell Cell

Kidney-derived Madin Darby canine kidney (MDCK) cells form lumina at their apices, and target luminal proteins to an intracellular vacuolar apical compartment (VAC) when prevented from polarizing. Hepatocytes, by contrast, organize their luminal surfaces (the bile canaliculi; BC) between their lateral membranes, and, when nonpolarized, they display an intracellular luminal compartment that is distinct from the VACs of MDCK cells. Overexpression of the serine/threonine kinase Par1b/EMK1/MARK2 induces BC-like lateral lumina and a hepatic-type intracellular luminal compartment in MDCK cells, suggesting a role for Par1b in the branching decision between kidney- and hepatic-type epithelial phenotypes. Here, we report that Par1b promotes lateral lumen polarity in MDCK cells independently of Ca2+-mediated cell–cell adhesion by inhibiting myosin II in a rho kinase-dependent manner. Polarization was inhibited by E-cadherin depletion but promoted by an adhesion-defective E-cadherin mutant. By contrast, apical surface formation in control MDCK cells required Ca2+-dependent cell–cell adhesion, but it occurred in the absence of E-cadherin. We propose that E-cadherin, when in an adhesion-incompetent state at the lateral domain, serves as targeting patch for the establishment of lateral luminal surfaces. E-cadherin depletion also reverted the hepatic-type intracellular luminal compartment in Par1b-MDCK cells to VACs characteristic of control MDCK cells, indicating a novel link between E-cadherin and luminal protein targeting.

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Faculty Opinions recommendation of Myosin-1c regulates the dynamic stability of E-cadherin-based cell-cell contacts in polarized Madin-Darby canine kidney cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718040393.793529402 ◽

2017 ◽

Author(s):

Vivian Tang

Keyword(s):

Dynamic Stability ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

Cell Contacts ◽

Darby Canine Kidney ◽

Canine Kidney ◽

E Cadherin ◽

Cell Cell

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Enhancement of cell-cell contact by claudin-4 in renal epithelial madin-darby canine kidney cells

Journal of Cellular Biochemistry ◽

10.1002/jcb.23373 ◽

2012 ◽

Vol 113 (2) ◽

pp. 499-507 ◽

Cited By ~ 13

Author(s):

Akira Ikari ◽

Kosuke Atomi ◽

Ayumi Takiguchi ◽

Yasuhiro Yamazaki ◽

Hisayoshi Hayashi ◽

...

Keyword(s):

Cell Contact ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Cell Cell

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PAR1b Promotes Cell–Cell Adhesion and Inhibits Dishevelled-mediated Transformation of Madin-Darby Canine Kidney Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-03-0193 ◽

2006 ◽

Vol 17 (8) ◽

pp. 3345-3355 ◽

Cited By ~ 36

Author(s):

Maya Elbert ◽

David Cohen ◽

Anne Müsch

Keyword(s):

Cell Adhesion ◽

Epithelial Cells ◽

Wnt Signaling ◽

Mdck Cells ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney ◽

E Cadherin ◽

Cell Cell

Mammalian Par1 is a family of serine/threonine kinases comprised of four homologous isoforms that have been associated with tumor suppression and differentiation of epithelial and neuronal cells, yet little is known about their cellular functions. In polarizing kidney epithelial (Madin-Darby canine kidney [MDCK]) cells, the Par1 isoform Par1b/MARK2/EMK1 promotes the E-cadherin–dependent compaction, columnarization, and cytoskeletal organization characteristic of differentiated columnar epithelia. Here, we identify two functions of Par1b that likely contribute to its role as a tumor suppressor in epithelial cells. 1) The kinase promotes cell–cell adhesion and resistance of E-cadherin to extraction by nonionic detergents, a measure for the association of the E-cadherin cytoplasmic domain with the actin cytoskeleton, which is critical for E-cadherin function. 2) Par1b attenuates the effect of Dishevelled (Dvl) expression, an inducer of wnt signaling that causes transformation of epithelial cells. Although Dvl is a known Par1 substrate in vitro, we determined, after mapping the PAR1b-phosphorylation sites in Dvl, that PAR1b did not antagonize Dvl signaling by phosphorylating the wnt-signaling molecule. Instead, our data suggest that both proteins function antagonistically to regulate the assembly of functional E-cadherin–dependent adhesion complexes.

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AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture

The Journal of Cell Biology ◽

10.1083/jcb.200307098 ◽

2003 ◽

Vol 164 (1) ◽

pp. 133-144 ◽

Cited By ~ 120

Author(s):

Christelle Benaud ◽

Benoît J. Gentil ◽

Nicole Assard ◽

Magalie Court ◽

Jerome Garin ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Adhesion ◽

Small Interfering Rna ◽

Kidney Cells ◽

Cortical Actin ◽

Madin Darby Canine Kidney ◽

Annexin 2 ◽

Interfering Rna ◽

Darby Canine Kidney ◽

Canine Kidney

Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell–cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca2+-dependent cell–cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2–specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

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Formation of the apical pole of epithelial (Madin-Darby canine kidney) cells: polarity of an apical protein is independent of tight junctions while segregation of a basolateral marker requires cell-cell interactions

The Journal of Cell Biology ◽

10.1083/jcb.104.4.905 ◽

1987 ◽

Vol 104 (4) ◽

pp. 905-916 ◽

Cited By ~ 136

Author(s):

DE Vega-Salas ◽

PJ Salas ◽

D Gundersen ◽

E Rodriguez-Boulan

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Tight Junctions ◽

Cell Interactions ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

Apical Pole ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Cell Cell

The time course of development of polarity of an apical (184-kD) and a basolateral (63-kD) plasma membrane protein of Madin-Darby canine kidney cells was followed using semiquantitative immunofluorescence on semithin (approximately 0.5-micron) frozen sections and monoclonal antibody probes. The 184-kD protein became highly polarized to the apical pole within the initial 24 h both in normal medium and in 1-5 microM Ca2+, which results in well-spread, dome-shaped cells, lacking tight junctions and other lateral membrane interactions. In contrast, the basolateral 63-kD membrane protein developed full polarity only after incubation in normal Ca2+ concentrations for greater than 72 h, a time much longer than that required for the formation of tight junctions (approximately 18 h) and failed to polarize in 1-5 microM Ca2+. These results demonstrate that intradomain restriction mechanisms independent of tight junctions, such as self-aggregation or specific interactions with the submembrane cytoskeleton, participate in the regionalization of at least some epithelial plasma membrane proteins. The full operation of these mechanisms depends on the presence of normal cell-cell interactions in the case of the basolateral 63-kD antigen but not in the case of the apical 184-kD protein.

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