Matrix-dependent Tiam1/Rac Signaling in Epithelial Cells Promotes Either Cell–Cell Adhesion or Cell Migration and Is Regulated by Phosphatidylinositol 3-Kinase

We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin–mediated cell–cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin–mediated cell–cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin–mediated cell– cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase– PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin–mediated cell– cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.

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Recruitment of E-cadherin associated with α- and β-catenins and p120ctn to the nectin-based cell-cell adhesion sites by the action of 12-O-tetradecanoylphorbol-13-acetate in MDCK cells

Genes to Cells ◽

10.1111/j.1365-2443.2005.00846.x ◽

2005 ◽

Vol 10 (5) ◽

pp. 435-445 ◽

Cited By ~ 21

Author(s):

Ryoko Okamoto ◽

Kenji Irie ◽

Akio Yamada ◽

Tatsuo Katata ◽

Atsunori Fukuhara ◽

...

Keyword(s):

Cell Adhesion ◽

Mdck Cells ◽

Adhesion Sites ◽

E Cadherin ◽

Cell Cell

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Actin protrusions push on E-cadherin to maintain epithelial cell-cell adhesion

10.1101/644302 ◽

2019 ◽

Author(s):

John Xiao He Li ◽

Vivian W. Tang ◽

William M. Brieher

Keyword(s):

Cell Adhesion ◽

Actin Polymerization ◽

Mdck Cells ◽

Myosin Ii ◽

Cell Junctions ◽

Apical Junctions ◽

Actin Protrusions ◽

Adhesive Contacts ◽

E Cadherin ◽

Cell Cell

AbstractCadherin mediated cell-cell adhesion is actin dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough together to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized MDCK cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells while reformation of cadherin clusters across the cell-cell boundary triggers microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as three actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNAi results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

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TGF -induced downregulation of E-cadherin-based cell-cell adhesion depends on PI3-kinase and PTEN

Journal of Cell Science ◽

10.1242/jcs.02594 ◽

2005 ◽

Vol 118 (20) ◽

pp. 4901-4912 ◽

Cited By ~ 107

Author(s):

R. Vogelmann

Keyword(s):

Cell Adhesion ◽

Pi3 Kinase ◽

E Cadherin ◽

Cell Cell

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Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.5.c1472 ◽

2000 ◽

Vol 279 (5) ◽

pp. C1472-C1482 ◽

Cited By ~ 10

Author(s):

Ingrid Marschitz ◽

Judith Lechner ◽

Irene Mosser ◽

Martina Dander ◽

Roberto Montesano ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Adhesion ◽

Mdck Cells ◽

Cell Types ◽

Mek Inhibitor ◽

Mitogen Activated Protein Kinase ◽

Cyclin D ◽

Invasive Phenotype ◽

E Cadherin ◽

Cell Cell

Overexpression of a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (caMEK1) in epithelial Madin-Darby canine kidney (MDCK)-C7 cells disrupts morphogenesis, induces an invasive phenotype, and is associated with a reduced rate of cell proliferation. The role of cell-cell adhesion molecules and cell cycle proteins in these processes, however, has not been investigated. We now report loss of E-cadherin expression as well as a marked reduction of β- and α-catenin expression in transdifferentiated MDCK-C7 cells stably expressing caMEK1 (C7caMEK1) compared with epithelial mock-transfected MDCK-C7 (C7Mock1) cells. At least part of the remaining α-catenin was coimmunoprecipitated with β-catenin, whereas no E-cadherin was detected in β-catenin immunoprecipitates. In both cell types, the proteasome-specific protease inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to a time-dependent accumulation of β-catenin, including the appearance of high-molecular-weight β-catenin species. Quiescent as well as serum-stimulated C7caMEK1 cells showed a higher cyclin D expression than epithelial C7Mock1 cells. The MEK inhibitor U-0126 inhibited extracellular signal-regulated kinase phosphorylation and cyclin D expression in C7caMEK1 cells and almost abolished their already reduced cell proliferation rate. We conclude that the transdifferentiated and invasive phenotype of C7caMEK1 cells is associated with a diminished expression of proteins involved in cell-cell adhesion. Although β-catenin expression is reduced, C7caMEK1 cells show a higher expression of U-0126-sensitive cyclin D protein.

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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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Inhibiting cadherin function by dominant mutant E-cadherin expression increases the extent of tight junction assembly

Journal of Cell Science ◽

10.1242/jcs.113.6.985 ◽

2000 ◽

Vol 113 (6) ◽

pp. 985-996 ◽

Cited By ~ 1

Author(s):

M.L. Troxell ◽

S. Gopalakrishnan ◽

J. McCormack ◽

B.A. Poteat ◽

J. Pennington ◽

...

Keyword(s):

Cell Adhesion ◽

Tight Junction ◽

Mdck Cells ◽

Freeze Fracture ◽

Junctional Complex ◽

Negative Effects ◽

Tight Junction Formation ◽

Freeze Fracture Electron Microscopy ◽

E Cadherin ◽

Cell Cell

Previous studies have shown that induction of cadherin-mediated cell-cell adhesion leads to tight junction formation, and that blocking cadherin-mediated cell-cell adhesion inhibits tight junction assembly. Here we report analysis of tight junction assembly in MDCK cells overexpressing a mutant E-cadherin protein that lacks an adhesive extracellular domain (T151 cells). Mutant E-cadherin overexpression caused a dramatic reduction in endogenous cadherin levels. Despite this, tight junction assembly was extensive. The number of tight junction strands observed by freeze-fracture electron microscopy significantly increased in T151 cells compared to that in control cells. Our data indicate that the hierarchical regulation of junctional complex assembly is not absolute, and that inhibition of cadherin function has both positive and negative effects on tight junction assembly.

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Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells

The Journal of Cell Biology ◽

10.1083/jcb.139.4.1047 ◽

1997 ◽

Vol 139 (4) ◽

pp. 1047-1059 ◽

Cited By ~ 387

Author(s):

Kenji Takaishi ◽

Takuya Sasaki ◽

Hirokazu Kotani ◽

Hideo Nishioka ◽

Yoshimi Takai

Keyword(s):

Cell Adhesion ◽

Tight Junction ◽

Cell Lines ◽

Actin Filaments ◽

Mdck Cells ◽

Wild Type ◽

Cell Functions ◽

Adhesion Sites ◽

E Cadherin ◽

Cell Cell

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell–cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell–cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell–cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell–cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell– cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell–cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell–cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell– cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell– cell adhesion.

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Actin protrusions push at apical junctions to maintain E-cadherin adhesion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1908654117 ◽

2019 ◽

Vol 117 (1) ◽

pp. 432-438 ◽

Cited By ~ 12

Author(s):

John Xiao He Li ◽

Vivian W. Tang ◽

William M. Brieher

Keyword(s):

Cell Adhesion ◽

Actin Polymerization ◽

Mdck Cells ◽

Myosin Ii ◽

Cell Junctions ◽

Apical Junctions ◽

Actin Protrusions ◽

Adhesive Contacts ◽

E Cadherin ◽

Cell Cell

Cadherin-mediated cell–cell adhesion is actin-dependent, but the precise role of actin in maintaining cell–cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized Madin-Darby canine kidney (MDCK) cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells, while reformation of cadherin clusters across the cell–cell boundary correlates with microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as 3 actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNA interference (RNAi) results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell–cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

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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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