Fig. 3: p73 regulation of the actin cytoskeleton and NMII activation is essential for the formation of sub-apical actin lattices and BB docking in ECs.

a, b Detection of activated NMII and actin cytoskeleton in WT vs. p73KO-iPSC colonies (a), or after ectopic TAp73 expression in p73KO-iPSCs (b). a Orthogonal projections from WT and p73KO-iPSC where arrows indicate F-actin and p-MLC colocalization at intercellular junctions. Scale bar: 10 µm. b p-MLC colocalization with cortical actin bundles is recovered (arrows) upon TAp73 transfection, but not vector control. Scale bar: 10 µm. c–f Confocal images of WT (c, e) and p73KO (d, f) P15 WMs displaying as indicated: actin cytoskeleton (Phalloidin), p-MLC (green), and BBs alone (γ-tubulin, red) or with the cell membrane (β-catenin and γ-tubulin, blue). The white arrows point to cortical actin; the white arrowheads indicate apical actin lattices and blue arrowheads mark the sub-apical actin filaments. Scale bar: 5 µm. e, f p-MLC puncta associated with actin lattice around BBs or with plasma membrane are marked by white arrowheads and white arrows, respectively. f Yellow arrowheads and yellow arrows indicate the lack of such p-MLC association. Scale bar: 5 µm. (g, g′) BB docking was analyzed in WT and p73KO (g) or TAp73KO (g′) ECs. Lateral views of WM confocal images labeled with anti-γ-tubulin (red) and β-catenin (blue). Scale bar: 5 µm. Graphs represent the percentage of cells with correct docking (green bars) versus cells with incomplete alignment (purple bars) within the indicated genotypes. Contingency analysis (Fisher’s exact test) was performed to evaluate statistical differences (g: n = 125 WT cells and 140 p73KO cells; g': n = 117 WT cells and 127 TAp73KO cells; images from three mice per genotype were analyzed ; ***p < 0.001)