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

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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Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells.

The Journal of Cell Biology ◽

10.1083/jcb.107.6.2389 ◽

1988 ◽

Vol 107 (6) ◽

pp. 2389-2399 ◽

Cited By ~ 98

Author(s):

J D Siliciano ◽

D A Goodenough

Keyword(s):

Plasma Membrane ◽

Extracellular Calcium ◽

Cell Contact ◽

Suspension Cells ◽

Madin Darby Canine Kidney ◽

Low Calcium ◽

Normal Calcium ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Cell Cell

Using the monoclonal antibody R26.4, we have previously identified a approximately 225-kD peripheral membrane protein, named ZO-1, that is uniquely associated with the tight junction (zonula occludens) in a variety of epithelia including the Madin-Darby canine kidney (MDCK) epithelial cell line (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). In this study we have analyzed the effects of cell-cell contact and extracellular calcium on the localization and the solubility of ZO-1. In confluent monolayers under normal calcium conditions, ZO-1 immunoreactivity is found exclusively at the plasma membrane in the region of the junctional complex. If MDCK cells are maintained in spinner culture under low calcium conditions, ZO-1 is diffusely organized within the cytoplasm. After the plating of suspension cells at high cell density in medium with normal calcium concentrations, ZO-1 becomes localized to the plasma membrane at sites of cell-cell contact within 5 h in a process that is independent of de novo protein synthesis. However, if suspension cells are plated at high density in low calcium medium or if suspension cells are plated at low cell density in normal calcium growth medium, ZO-1 remains diffusely organized. ZO-1 localization also becomes diffuse in monolayers that have been established in normal calcium medium and then subsequently switched into low calcium medium. These results suggest that both extracellular calcium and cell-cell contact are necessary for normal localization of ZO-1 to the plasma membrane. An analysis of the solubility properties of ZO-1 from suspension cells and monolayers revealed that high salt, nonionic detergent, and a buffer containing chelators were somewhat more effective at solubilizing ZO-1 from suspension cells than from monolayers.

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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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Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells.

The Journal of Cell Biology ◽

10.1083/jcb.104.6.1527 ◽

1987 ◽

Vol 104 (6) ◽

pp. 1527-1537 ◽

Cited By ~ 80

Author(s):

W J Nelson ◽

P J Veshnock

Keyword(s):

Membrane Proteins ◽

Epithelial Cells ◽

Mdck Cells ◽

Membrane Skeleton ◽

Cell Contact ◽

Madin Darby Canine Kidney ◽

The Stability ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Cell Cell

During growth of Madin-Darby canine kidney (MDCK) epithelial cells, there is a dramatic change in the stability, biophysical properties, and distribution of the membrane skeleton (fodrin) which coincides temporally and spatially with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral domain of the plasma membrane. These changes occur maximally upon the formation of a continuous monolayer of cells, indicating that extensive cell-cell contact may play an important role in the organization of polarized MDCK cells (Nelson, W. J., and P. J. Veshnock, 1986, J. Cell Biol., 103:1751-1766). To directly analyze the role of cell-cell contact in these events, we have used an assay in which the organization of fodrin and membrane proteins is analyzed in confluent monolayers of MDCK cells in the absence or presence of cell-cell contact by adjusting the concentration Ca++ in the growth medium. Our results on the stability and solubility properties of fodrin reported here show directly that there is a positive correlation between cell-cell contact and increased stability and insolubility of fodrin. Furthermore, we show that fodrin can be recruited from an unstable pool of protein to a stable pool during induction of cell-cell contact; significantly, the stabilization of fodrin is not affected by the addition of cyclohexamide, indicating that proteins normally synthesized during the induction of cell-cell contact are not required. Together these results indicate that cell-cell contact may play an important role in the development of polarity in MDCK cells by initiating the formation of a stable, insoluble matrix of fodrin with preexisting (membrane) proteins at the cell periphery. This matrix may function subsequently to trap proteins targeted to the membrane, resulting in the maintenance of membrane domains.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m000467200 ◽

2000 ◽

Vol 275 (27) ◽

pp. 20707-20716 ◽

Cited By ~ 34

Author(s):

Daniel B. Stewart ◽

Angela I. M. Barth ◽

W. James Nelson

Keyword(s):

Cell Adhesion ◽

Differential Regulation ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Cell Cell

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

AJP Cell Physiology ◽

10.1152/ajpcell.00099.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. C1094-C1102 ◽

Cited By ~ 30

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.

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Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis.

The Journal of Cell Biology ◽

10.1083/jcb.106.3.677 ◽

1988 ◽

Vol 106 (3) ◽

pp. 677-685 ◽

Cited By ~ 88

Author(s):

M Pasdar ◽

W J Nelson

Keyword(s):

Epithelial Cells ◽

Junctional Complex ◽

Cell Contact ◽

Major Protein ◽

Madin Darby Canine Kidney ◽

Kidney Epithelial Cells ◽

Kinetics Of ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Cell Cell

The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.

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