Only α-cat H0-ABD and α-cat E307K mutants show enhanced junctional recruitment of LZTS2 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), LZTS2 (magenta) or GFP (native). Dashed white line marks border between GFP-α-cat expressing versus parental MDCK cells. 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. Note that all α-cat mutant cell lines show modestly enhanced enrichment of LZTS2 to cellular junctions compared to control MDCK. Scale bar, 10 μm. (B) Graph re-shows wound distal junctional LZTS2 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. 6. Error bars reflect ± SD.