α-Catenin harboring an I783P substitution does not bind ZO-1 in vitro or in MDCKII cells. (A) A linear schematic of α-catenin and the fragments of α-catenin used in this study (GST–α-catenin). (B,C) In vitro binding of ZO-1 to various GST–α-catenin proteins. The indicated α-catenin proteins were purified, attached to beads and incubated with purified His-tagged ZO-1 (516–806 for B; 1–806 for C). The levels of GST-tagged α-catenin proteins employed were similar, as represented by the Coomassie-stained blot in the lower panels. Note that in C, ZO-1 fails to bind α-catenin I783P. We consistently obtained some GST in the preparations of the full-length (FL) α-catenin proteins. (D) Endogenous ZO-1 binds WT α-catenin but not I783P α-catenin. GST, GST-tagged WT α-catenin or GST-tagged I783P α-catenin were prebound to glutathione beads and incubated with MDCKII cell lysates. The GST-tagged proteins were recovered and separated using SDS-PAGE. The co-precipitating levels of ZO-1 were examined by immunoblotting. This figure shows that endogenous, full-length ZO-1 binds to WT α-catenin, but fails to bind I783P α-catenin. (E) Expression levels of I783P α-catenin in MDCKII cells. MDCKII cells expressing an shRNA against canine α-catenin were infected a second time with retroviruses encoding GFP–α-catenin with the I783P substitution (I783P α-cat Rescue). Lysates were harvested from cell lines stably expressing these proteins or Control, Knockdown or WT α-cat Rescue cells. Immunoblot analysis was performed using antibodies against α-catenin or the p34-Arc subunit of the Arp2/3 complex as a loading control. (F) ZO-1 fails to co-immunoprecipitate with α-catenin I783P. Confluent monolayers of the indicated cell lines were Ca²⁺-starved overnight and lysed 1 hour post Ca²⁺ addition. ZO-1 was immunoprecipitated, and the bound proteins were washed, resolved using SDS-PAGE, and immunoblotted with an antibody against ZO-1 or α-catenin. (G) Substitution of I783P does not impair α-catenin binding to other ligands. WT α-cat Rescue or I783P α-cat Rescue cells were grown to confluence, lysed and full-length α-catenin and α-catenin I783P were immunoprecipitated using an antibody against GFP. Proteins were recovered, resolved using SDS-PAGE, and immunoblotted with antibodies against vinculin, β-catenin or EPLIN to show the levels of each protein recovered, or GFP to show the levels of each α-catenin protein recovered.

Tight junctions are altered in cells expressing α-catenin with the I783P substitution. (A) The establishment of an epithelial barrier is disrupted in cells expressing I783P α-catenin. Confluent cultures of the indicated cells lines were incubated overnight in Ca²⁺-free medium (0 h). Ca²⁺ was restored to the cultures for the indicated times and resistance was measured using a voltometer on quadruplicate filters. The graph displays the mean±s.d. expressed in Ohms*cm². The right panel shows the early time points (0–4 h) so that the initial delay in barrier establishment can be visualized. *P≤0.01 and **P≤0.001 compared with control. (B,C) The integrity of the tight junction is altered in cells expressing the α-catenin I783P point mutant that doesn't bind ZO-1. WT α-cat Rescue and I783P α-cat Rescue cells were examined using immunofluorescence and antibodies against ZO-1 (B) or occludin (C). Representative confocal images are shown in inverted grayscale. Scale bars: 10 µm. The intensity of ZO-1 or occludin at cell–cell contacts was determined using ImageJ and is shown below the images as the percentage intensity compared with WT α-cat Rescue. *P≤0.01. (D,E) The maintenance of the epithelial barrier is disrupted in I783P-expressing cells. (D) The indicated cell lines were grown to confluence, and resistance across the cell monolayer was measured daily until a consistent reading was made for three consecutive days. The graphed data represents the mean±s.e.m. for four independent experiments. The paracellular flux of 3 kDa FITC-conjugated dextran in the same cultures was measured (E) and the graph represents the mean±s.e.m. for four independent experiments. ^#P≤0.05; *P≤0.01. (F) Expression of tight and adherens junction proteins are unaltered in I783P α-cat Rescue cells. Lysates from the indicated cell lines were harvested and immunoblotting was performed to analyze the expression levels of ZO-1, occludin, actin, afadin and E-cadherin. p34-Arc serves as a loading control.

ZO-1 mobility and actin organization is altered by proline substitution at I783. (A) ZO-1 mobility is increased in I783P α-cat Rescue cells. ZO-1 mobility at cell–cell contacts was examined using FRAP. Cells were transfected with mCherry-tagged ZO-1 and FRAP was performed on adjacent transfected cells. The images presented are a representation of cells prior to bleaching (Prebleach), immediately after bleaching (Bleach) and 5 minutes after bleaching (Postbleach). The box indicates the bleached area. The amount of ZO-1 that recovered was quantified and the mobile fraction of ZO-1 was calculated and is shown in the right panel. (B) Actin organization is disrupted in Knockdown and I783P α-cat Rescue cells. Cells were grown to confluence and incubated overnight in Ca²⁺-free medium. Ca²⁺ was restored for the indicated times and actin organization was analyzed using immunofluorescence. Representative images are presented in inverted grayscale. Scale bars: 10 µm.

The proline substitution at I783 has no effect on adherens junction assembly or cadherin-mediated adhesion. (A) Cadherin-mediated adhesion is established in the presence of I783P α-catenin. The adhesion of the indicated cell lines to immobilized cadherin extracellular domains was examined as described in Fig. 1F. The data presented in the graph are the mean±s.e.m. cells adhered from four independent experiments. (B) E-cadherin localization is unaffected by I783P substitution. WT α-cat Rescue and I783P α-cat Rescue cells were grown to confluence, incubated in Ca²⁺-free medium overnight, and placed in Ca²⁺-containing medium for 0, 1, 2, 4 or 24 hours. The cells were then fixed, and examined by immunofluorescence using a Zeiss 510 confocal microscope. Images are presented in inverted grayscale. Scale bar: 10 µm.