Fig. 8: The role of αABD oligomerization.

a Diagram of αABD mutants (depicted as in Fig. 1a) of α-catenin. b Fluorescence microscopy (projections of all optical slices) of the cells (the mutant names are on the left) stained for GFP (green) and F-actin (red). The left micrographs show merged images (Scale bar, 20 μm). The separate staining of the representative contacts (in dashed boxes) is zoomed on the right (Scale bar, 10 μm). c Western blotting probed for GFP of cells with GFP-αCat-C805/971 (αCatC), GFP-αCatV870S-C805/871 (V870S), and GFP-αCatI792A-C805/871 (I792A) without (Ctrl) or after cross-linking (XL). Right: Quantification (based on 8 experiments) of the adduct, a1 intensities relative to it in GFP-αCat-C805/871 normalized to the monomers. The means +/- SD are indicated by bars. d Time-lapse microscopy (at 10 s temporal resolution) of the cells expressing α-catenin mutants (abbreviated as in c). Isolated frames of the movies (Supplementary Movies 1, 2) are shown in the top row. The representative line scans along the white lines of 10 consecutive frames (spanning 1.5 min) were combined and differently colorized (color code is on the bottom). Right: Fluorescence intensity of AJs (the value of brightest pixel after background subtraction, the means +/- SD, n = 12 taken from two independent movies). e Dispase-based assay of cells expressing GFP-αCat-C805/971 (αCatC), GFP-αCatV870S-C805/871 (V870S), and GFP-αCatI792A-C805/871 (I792A). Left: Representative images showing the cell sheets detached from the dishes before and after mechanical stress (shaking). Right: Quantification (n = 4) of the size (in pixels) of the sheets before shaking (top) and the number of fragments obtained after shaking (bottom). The cells expressing GFP-αCat (αCat) and GFP-αCatV870A (V870A) are also shown in the graphs. Data are presented as mean values +/- SD. f Left: Vinculin and FBLIM1 staining of the representative cell-cell contacts (abbreviations as in e). Bar, 10 μm. Right: Quantification of peak mean intensities +/- SD of vinculin and FBLIM1 in AJs (15 AJs for each cell line were determined). Statistical significance was calculated using a two tailed Student’s t-test: ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.