α-cat butterfly-patterned eye dystrophy missense mutants generally show enhanced junctional recruitment of vinculin, comparable to the α-cat R551A salt-bridge disrupting mutant, in MDCK monolayers distal to wound site. (A) Confocal en face views (maximum z-projection of the apical-junction region; 5 steps × 0.15-μm) of α-cat wild-type and mutant -expressing MDCK cells (GFP+) co-cultured with parental MDCK cells (GFP-negative, asterisk [*]) on polycarbonate filters (10 days). Mature monolayers were subjected to “scratch-wounding” with a vacuum-assisted pipette tip to generate wound-proximal versus wound distal regions. Wound distal region is shown here. Filters were immuno-stained for α-cat (cyan), Vinculin (magenta) or GFP (native). Co-culturing GFP-α-cat cells with parental MDCK cells allows for more reliable assessment of small differences in effector recruitment between mutant α-cat lines (yellow arrows) versus wild-type α-cat (white arrows) in the same field of view. Insets (last column) show higher magnification view of Vinculin junctional recruitment between GFP-α-cat (yellow box) versus parental MDCK region (white box). Note that all α-cat mutant cell lines show modestly elevated enrichment of Vinculin to cellular junctions compared to parental MDCK. Scale bars, 20 μm for main panels and 10 μm for insets. (B) Graph re-shows wound distal junctional Vinculin enrichment, with each symbol representing the intensity ratio between paired junction (BCJ) and cytoplasm (Cyto) 0.1 μm ROIs and normalized to α-cat signal (n = 50+/3 FOVs) from panel C of Fig. S4. Data reflects mean ± SD.