α-cat M-domain butterfly-patterned eye dystrophy missense mutants show wound-front enhanced junctional recruitment of LZTS2. (A) Confocal en face views (maximum z-projection) 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) using a Nikon AXR microscope with Apo TIRF 60× Oil DIC N2 objective. Mature monolayers were subjected to “scratch-wounding” with a vacuum-assisted pipette tip to generate wound-proximal versus wound distal regions. Wound proximal region is shown (left leading edge of images, black). Filters were immuno-stained for α-cat (cyan), LZTS2 (magenta) or GFP (native). Dashed white line demarks 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. Insets (last column) show higher magnification view of LZTS2 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 LZTS2 to cellular junctions compared to parental MDCK. Scale bars, 20 μm for main panels and 10 μm for insets. (B) Graph shows wound proximal 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). Error bars reflect ± SD. α-cat mutants show significantly enhanced recruitment by ANOVA (****P < 0.0001, α-cat-E307K; *P = 0.0260, α-cat-R551A; Kruskal Wallis) or unpaired t test (**P = 0.0060, α-cat-H0-ABD⁺; *P = 0.0179, α-cat-L436P; Mann Whitney). (C) Graph shows wound distal junctional LZTS2 enrichment quantified as above and in Materials and methods. Fewer α-cat mutants show significantly enhanced LZTS2 recruitment by ANOVA (****P < 0.0001, α-cat-H0-ABD⁺; **P = 0.0017, α-cat-E307K; Kruskal Wallis). α-cat-R551A and α-cat-L436P show no significant recruitment of LZTS2 by any test. Wound distal images corresponding to C are shown in Fig. S6.