Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells

Palovuori, Riitta; Eskelinen, Sinikka

doi:10.1078/0171-9335-00120

Cited by 16 publications

(30 citation statements)

References 43 publications

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“…No major changes in the association with ␣-catenin were detected between the wild-type and E-cadherin variants (data not shown). Therefore, we next examined whether removal of N-glycans affected the interaction between E-cadherin-catenin complexes with the cytoskeletal protein vinculin, because this protein has been shown to have a stabilizing effect on AJs (4,33,34). As shown in Fig.…”

Section: N-glycans Affect the Composition Of E-cadherin Protein Complmentioning

confidence: 99%

N-Glycosylation Affects the Molecular Organization and Stability of E-cadherin Junctions

Liwosz

Lei

Kukuruzinska

2006

Journal of Biological Chemistry

117

147

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Epithelial cell-cell adhesion is mediated by E-cadherin, an intercellular N-glycoprotein adhesion receptor that functions in the assembly of multiprotein complexes anchored to the actin cytoskeleton named adherens junctions (AJs). E-cadherin ectodomains 4 and 5 contain three potential N-glycan addition sites, although their significance in AJ stability is unclear. Here we show that sparse cells lacking stable AJs produced E-cadherin that was extensively modified with complex N-glycans. In contrast, dense cultures with more stable AJs had scarcely N-glycosylated E-cadherin modified with high mannose/hybrid and limited complex N-glycans. This suggested that variations in AJ stability were accompanied by quantitative and qualitative changes in E-cadherin N-glycosylation. To further examine the role of N-glycans in AJ function, we generated E-cadherin N-glycosylation variants lacking selected N-glycan addition sites. Characterization of these variants in CHO cells, lacking endogenous E-cadherin, revealed that site 1 on ectodomain 4 was modified with a prominent complex N-glycan, site 2 on ectodomain 5 did not have a substantial oligosaccharide, and site 3 on ectodomain 5 was decorated with a high mannose/hybrid N-glycan. Removal of complex N-glycan from ectodomain 4 led to a dramatically increased interaction of E-cadherin-catenin complexes with vinculin and the actin cytoskeleton. The latter effect was further enhanced by the deletion of the high mannose/hybrid N-glycan from site 3. In MDCK cells, which produce E-cadherin, a variant lacking both complex and high mannose/hybrid N-glycans functioned like a dominant positive displaying increased interaction with ␥-catenin and vinculin compared with the endogenous E-cadherin. Collectively, our studies show that N-glycans, and complex oligosaccharides in particular, destabilize AJs by affecting their molecular organization.

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Section: N-glycans Affect the Composition Of E-cadherin Protein Complmentioning

confidence: 99%

N-Glycosylation Affects the Molecular Organization and Stability of E-cadherin Junctions

Liwosz

Lei

Kukuruzinska

2006

Journal of Biological Chemistry

117

147

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“…15,19 γ-catenin and vinculin have been shown to play stabilizing roles in AJs. 12,15,[20][21][22] The function of PP2A in AJs is unclear, although several observations indicate that PP2A is critical for the maintenance of E-cadherin-mediated cell-cell contacts. 23 PP2A plays critical roles in cell growth, signaling, and tumor suppression, [24][25][26] and its dysregulation has been linked to the destabilization of MT populations in numerous cell types.…”

Section: Dovepressmentioning

confidence: 99%

<em>N</em>-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs

Jamal

Niță-Lazăr²,

Gao³

et al. 2009

CHC

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N-glycosylation of E-cadherin has been shown to inhibit cell-cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/ threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions. Using the hypoglycosylated E-cadherin variant, V13, we show that V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein. This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that increased association of PP2A with V13-containing AJs promoted their tethering to microtubules. On the other hand, V13/γ-catenin complexes associated more with vinculin, suggesting that they mediated the interaction of AJs with the actin cytoskeleton. N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin. These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through the assembly of distinct β-catenin-and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.

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“…Microinjections were carried out using an Eppendorf micromanipulator 5171 and microinjector 5246 (Hamburg, Germany) installed on an Axiovert 405 M inverted microscope with a heating stage (Zeiss, Oberkochen, Germany) as described earlier (Palovuori and Eskelinen, 2000). Typically, all of the cells within one or two squares of the etched cover slips were injected within a time period of 10 to 30 minutes for each experiment.…”

Section: Microinjection Of Inactive Racmentioning

confidence: 99%

“…It was dissolved in dimethyl sulfoxide in a concentration of 10 mM and used for experiments in a concentration of 50 M (Brandt et al, 2002). Inactive RacGTPase was purchased from Cytoskeleton (Denver, Colorado), dissolved in distilled water to a concentration of 1 mg/ml, and diluted to 100 g/ml in 100 mM KCl, 5 mM HEPES, pH 7.25 (Palovuori and Eskelinen, 2000). Nocodazole was purchased from Calbiochem, dissolved in dimethyl sulfoxide in a concentration of 1 mg/ml, and diluted in HBSS (HBSS, Gibco) at a concentration of 5 g/ml.…”

Section: Antibodies and Other Reagentsmentioning

confidence: 99%

“…The specimens were viewed by Zeiss 510 confocal laser scanning microscope (Jena, Germany) equipped with an argon laser (488 nm) and HeNe laser (543 nm) and by using a ϫ63 objective as described earlier (Palovuori and Eskelinen, 2000;Rahikkala et al, 2001). Optical sections of the specimens were generated through the cell by scanning with 0.36 m intervals and by using a 505 to 530 nm band pass filter for the green emission and a 560 or 630 nm low-pass filter for the red emission channel.…”

Section: Confocal Fluorescence Microscopymentioning

confidence: 99%

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Src-Induced Disintegration of Adherens Junctions of Madin-Darby Canine Kidney Cells Is Dependent on Endocytosis of Cadherin and Antagonized by Tiam-1

Palovuori

Sormunen

Eskelinen

2003

Laboratory Investigation

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SUMMARY:The effects of Src tyrosine kinase activation in subconfluent temperature sensitive (ts)-Src-transformed MadinDarby canine kidney (MDCK) cells were analyzed by shifting them from nonpermissive (40.5°C) to permissive (35°C) temperature. Already, in 15 minutes, adherens junction components were released from the lateral walls and accumulated to basal surfaces. Simultaneously, membranous actin staining vanished, actin bundles appeared at the basal surface, and the cells flattened. The only component phosphorylated and translocated after the shift to 35°C was p120ctn. The epithelial-mesenchymal transition could be inhibited by a specific inhibitor of Src kinase, PP2, or by inhibiting endocytosis. Therefore, Src activation was responsible for the transition, but not because of phosphorylation of adherens junction components but by way of activation of endocytic machinery and RhoGTPase. Expression of an RacGEF, Tiam-1 (T-lymphoma invasion and metastasis gene 1), prevented flattening of Src-transformed MDCK cells at 35°C and resulted in accumulation of cadherin to lateral membranes. In the case where the Src-MDCK cells were cultivated at 35°C and shifted for short time periods to 40.5°C, cadherin rapidly returned to lateral membranes, whereas actin and p120ctn followed hours afterward. This further supports the view that cadherin internalization is the primary target of Src kinase. We also looked at the cell morphology and distribution of cadherin and Tiam-1 in cells grown in three-dimensional gels composed of collagen and laminin or in Matrigel. At nonpermissive temperature, both Src-MDCK and Tiam-1-transfected Src-MDCK cells exhibited nonpolarized morphology in collagen I, a loose cluster in the mixture of collagen I and laminin, and a differentiated cyst in Matrigel. In growth factor-depleted Matrigel, the Src-MDCK cells grew in nondifferentiated clusters, whereas Tiam-1-transfected cells went to apoptosis. The differentiated phenotype of both cell lines could be rescued by Matrigel-conditioned medium, platelet-derived growth factor, or cholera toxin. Concomitantly, both cadherin and Tiam-1 were recruited to lateral membranes. Therefore, cadherin and Tiam-1 seem to be the key players in the differentiation process of MDCK cells. (Lab Invest 2003, 83:1901-1915.

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Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells

Cited by 16 publications

References 43 publications

N-Glycosylation Affects the Molecular Organization and Stability of E-cadherin Junctions

N-Glycosylation Affects the Molecular Organization and Stability of E-cadherin Junctions

<em>N</em>-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs

Src-Induced Disintegration of Adherens Junctions of Madin-Darby Canine Kidney Cells Is Dependent on Endocytosis of Cadherin and Antagonized by Tiam-1

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Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells

Cited by 16 publications

References 43 publications

N-Glycosylation Affects the Molecular Organization and Stability of E-cadherin Junctions

N-Glycosylation Affects the Molecular Organization and Stability of E-cadherin Junctions

<em>N</em>-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct &beta;-catenin- and &gamma;-catenin-containing AJs

Src-Induced Disintegration of Adherens Junctions of Madin-Darby Canine Kidney Cells Is Dependent on Endocytosis of Cadherin and Antagonized by Tiam-1

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<em>N</em>-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs