Figure 1.
Expression of the DsRed-tagged E-cadherin cytoplasmic domain in MDCK cells disrupts cell–cell adhesion.
(A) Schematic representation of DsRed-tagged cadherin cytoplasmic domain constructs. DECT is a DsRed-tagged wild-type E-cadherin cytoplasmic domain (ECT). The binding sites for p120 and β-catenin/plakoglobin (β-cat/plako) are shown. DECTEA is an ECT construct with alanine substitutions of the two conserved glutamic acid residues and a conserved aspartic acid residue (Glu-Glu-Asp) in the p120-binding site, which has been shown to eliminate the interaction with p120. DECTSA is an ECT construct with alanine substitutions of the conserved eight serine residues in the catenin-binding site, which has been shown to weaken the interaction with β-catenin. DECTN is a chimeric construct composed of DsRed and the N-terminal region of ECT containing the p120-catenin–binding site. DECTC is a chimera of DsRed and the C-terminal half of ECT containing the catenin–binding site. DNCT is an N-cadherin cytoplasmic domain (NCT) construct. The C-terminus of all constructs, including DsRed, is tagged with the FLAG epitope. (B) Morphology of MDCK cells expressing DsRed, DECT, and Snail. DECT+ and Snail+ cells lose cell–cell contacts. (C) The migration assay. While MDCK cells expressing Snail or DECT show enhanced migration, MDCK cells expressing DsRed do not. The results are represented as the mean ± SD of three independent experiments. (D) Dissociation assays. Cells were incubated with dispase and detached cells were subjected to mechanical stress by pipetting as described in Materials and Methods. (E) Immunoblot analysis revealed that up-regulation of fibronectin, N-cadherin, and vimentin and down-regulation of E-cadherin and occludin occurred in Snail+ cells but not in DECT+ cells. Vinculin was used as a loading control. (F) Invasion assays. Representative photographs of the cells that invaded (Snail+) and not invaded (parental MDCK) Matrigel (upper panels). The results are represented as the mean ± SD of three independent experiments. Bars, 25 µm.
Figure 2.
Expression of the DsRed-tagged E-cadherin cytoplasmic domain inhibits the cell surface localization of endogenous E-cadherin.
(A) Immunofluorescence staining with DECMA-1, an antibody that recognizes the extracellular domain of E-cadherin, revealed that endogenous E-cadherin in DECT+ cells was localized intracellularly. (B) Tryptic digestion of cells with or without free Ca2+. Cells were incubated with 0.01% trypsin for 10 min at 37°C in the presence of 2 mM Ca2+ (TC) or 1 mM EGTA (TE). Then, immunostaining with an anti-E-cadherin mAb showed that a significant percentage of endogenous E-cadherin remained inside DECT+ cells. (C and D) Immunofluorescence staining with an anti-β-catenin (C) or anti-plakoglobin (D) antibodies revealed co-localization of β-catenin and plakoglobin with DECT. By contrast, β-catenin and plakoglobin did not co-localize with DsRed. Bars, 25 µm. (E) β-catenin and plakoglobin co-immunoprecipitated with DECT but not with DsRed, and (F) Reduced amounts of β-catenin and plakoglobin co-immunoprecipitated with endogenous E-cadherin in DECT+ cells as compared with DsRed cells. DECTSA, a DECT-derivative with alanine substitution of the conserved eight serine residues in the catenin-binding site, shows weakened interactions with β-catenin and plakoglobin (E) and did not significantly impair the complex formation of endogenous E-cadherin and β-catenin or plakoglobin (F). An asterisk in (E) indicates the position of the immunoglobin heavy chain.
Figure 3.
The ability to interact with β-catenin/plakoglobin is essential to the potential of the cytoplasmic domains.
(A) Immunoblot detection of the constructs with anti-FLAG antibodies. DECTSA and DECTEA migrated faster than DECT and DNCT. DECTN and DECTC showed similar mobility. (B) Phase contrast (upper panels), β-catenin (middle panels, β-cat), and plakoglobin immunofluorescence (lower panels, plako) images of cells expressing DECT, DECTSA, DECTEA, DNCT, and DECTN, and DECTC. Expression of DECT, DECTEA, and DNCT disrupted cell–cell contacts and induced the intracellular localization of β-catenin and plakoglobin. Expression of DECTC also induced the intracellular localization of β-catenin, but did not affect cell-cell contacts and plakoglobin remained associated with the plasma membrane. Expression of DECTSA did not affect cell–cell contacts and significant amounts of β-catenin remained associated with the plasma membrane. Bars, 25 µm. (C) Quantification of cell dissociation assays. The extent of cell dissociation was represented by the index Np/Nc, where Np and Nc are the total numbers of particles and cells per dish, respectively. Expression of DECT, DECTEA, and DNCT disrupts the mechanical integrity of cell sheets. Cells expressing DsRed, DECTSA, DECTN, and DECTC retain the mechanical integrity of their cell sheets. The results are represented as the mean ± SD of three independent experiments. (D) β-catenin co-immunoprecipitated with DECT or DECTC but not with DsRed or DECTN. Plakoglobin also interacted with DECT and DECTC. However, reduced amounts of plakoglobin co-immunoprecipitated with DECTC, indicating that DECTC shows weakened interactions compared with DECT. An asterisk in indicates the position of the immunoglobin heavy chain. (E) Reduced amounts of β-catenin and plakoglobin co-immunoprecipitated with endogenous E-cadherin in DECT+ cells as compared with DsRed+ or DECTN+ cells. DECTC did not impair the complex formation of endogenous E-cadherin and plakoglobin. Quantification of images revealed increased amounts of plakoglobin co-immunoprecipitated with E-cadherin in DECTC+ cells (see Table 1).
Table 1.
Relative amounts of catenins co-precipitated with endogenous E-cadherin isolated from MDCK cells expressing different constructs.
Figure 4.
E-cadherin–α-catenin chimeric molecules restore cell–cell adhesion and junctional assembly.
(A) Schematic representation of E-cadherin and its three derivatives. E-cadherin associates with catenins (α-cat and β-cat/plako) and p120. ELA is a mutant E-cadherin in which two leucine residues at positions 587 and 588, which are close to the p120-binding site, were substituted with two alanine residues. This substitution improves the cell surface localization of E-cadherin. ELAαM and ELAαC are ELA-α-catenin chimeric proteins consisting of (a) the entire extracellular and transmembrane domains of E-cadherin as well as the first 80 amino acids of its cytoplasmic domain, excluding the region required for β-catenin or plakoglobin-binding, and (b) α-catenin regions encompassing either amino acids 157–381 or 612–906, which include the domains necessary for association with formin/vinculin or ZO-1/actin, respectively, but not the domain essential for association with β-catenin (α-catenin residues 48–163). Thus, ELAαM and ELAαC could not associate with β-catenin but could still interact with p120. All constructs were tagged with HA. (B) Immunoblot detection of ELA, ELAαM, and ELAαC chimeras expressed in DECT+ cells. Cell lysates prepared from DECT+ cells and DECT+ cells expressing ELA (+ELA), ELAαM (+ELAαM), or ELAαC (+ELAαC) were analyzed. Blots were stained with anti-HA antibodies. (C) Immunofluorescence staining of MDCK cells expressing ELA, DECT, or the indicated combinations of proteins: DECT and ELA (DECT+ELA), DECT and the ELAαM chimera (DECT+ELAαM), or DECT and the ELAαC chimera (DECT+ELAαC). The expression of DECT in MDCK cells induced the intracellular accumulation of not only β-catenin (β-cat) and plakoglobin (plako), but also p120, desmoplakin (DP), and ZO-1. Significant amounts of ELAαM and ELAαC, but not ELA, were observed at the cell surface as detected by anti-HA. The expression of ELAαM or ELAαC, but not ELA, in DECT+ cells induced the redistribution of p120, desmoplakin (DP), and ZO-1 to the cell surface. β-catenin and plakoglobin in the same cells remained in the cytoplasm. (D) Dissociation assays. Quantified data were shown under each panel. The results are represented as the mean ± SD of three independent experiments. Expression of ELAαM and ELAαC in DECT+ cells restored the mechanical integrity of cell sheets. Bars, 25 µm.
Figure 5.
Reversibility of the cadherin cytoplasmic domain activity.
(A) Dox-repressible expression of DNCT in MDCK cells. An MDCK derivative (T23), expressing the tet repressor, was transfected with an expression vector encoding DNCT under the control of the tet promoter. Clones showing tet-repressible expression of DNCT were isolated. The cells of a representative clone were cultured for 4 days with (+) or without (−) Dox, and subjected to immunoblot analysis with the indicated antibodies. DNCT was detected with an anti-FLAG antibody. The addition of Dox repressed DNCT expression and induced a slight increase in E-cadherin expression, but did not affect the expression of other proteins. Vinculin was used as a loading control. (B) Expression of DNCT inhibited the cell surface localization of endogenous E-cadherin (E-cad); its associated proteins, β-catenin (β-cat), plakoglobin (plako), and p120 (p120); the desmosomal protein, desmoplakin (DP); and the tight junction protein, ZO-1. The addition of Dox induced the cell surface localization of these components. (C) Dissociation assays. Cells were cultured in the presence (+) or the absence of Dox (−) for 4 days, and then subjected to the assay. Cells treated with Dox retained the mechanical integrity of their cell sheets, but untreated cells did not. Detached cell sheets became dissociated in the presence of EGTA. Thus, the mechanical integrity depended on the presence of Ca2+ in the medium. (D) Quantification of cell dissociation assays. The results are represented as the mean ± SD of three independent experiments. Bars, 25 µm.