Fig. 4: Plexin-B1/Plexin-B2 mediate mechanosensation through stabilization of adhesive cell–cell junctions.

a Primary mouse keratinocytes were cultured with 70 µM Ca²⁺. 1.8 mM Ca²⁺ was added (“high calcium”). Shown are confocal images of immunostainings for Plexin-B1 (green) and Plexin-B2 (red). Scale bar, 25 µm. b, f Specific adhesion of primary mouse keratinocytes to the recombinant extracellular portions of b Plexin-B1 or Plexin-B2, and f E-cadherin (mean ± s.d.; n = 4 mice per genotype; p = 0.0124 for Plexin-B1, p = 0.0332 for Plexin-B2; p = 0.0006 for E-cadherin; two-sided unpaired t-test). c Live cell imaging of primary mouse keratinocytes expressing E-cadherin-mRuby. Shown are representative epifluorescence still images of cell divisions. Arrows: dividing cells; arrowheads: cell–cell contacts. Time: hours:minutes format. Scale bar, 10 µm. d, h, j Confocal images of the epidermal basal layer at E15.5 immunostained for d E-cadherin (red), h α-catenin (red), and j a-18 (=α-catenin tension-sensitive epitope antibody; green). Cell divisions are contoured by dashed lines. Scale bar, 10 µm. e, i, k Quantification of e E-cadherin, i α-catenin, and k a-18 immunofluorescence intensities at cell–cell contacts between a dividing cell and its immediate neighbors (box plot with minimum, first quartile, median, third quartile and maximum). e control: n = 207 cell–cell contacts of 34 dividing cells from three mice, PlexDKO: n = 209 cell–cell contacts of 34 dividing cells from three mice. i control: n = 228 cell–cell contacts of 37 dividing cells from three mice, PlexDKO: n = 244 cell–cell contacts of 39 dividing cells from three mice. k control: n = 222 cell–cell contacts of 36 dividing cells from three mice, PlexDKO: n = 219 cell-cell contacts of 37 dividing cells from three mice. Two-sided Mann–Whitney U test for e, i, and k: p < 0.0001. g Super-resolution structured illumination microscopy images of the epidermal basal layer at E15.5 immunostained for Plexin-B2 (red) and E-cadherin (green). Scale bar, 2 µm. The dashed boxed area is magnified on the right. Scale bar, 1 µm. l Representative a-18 (magenta) and nuclear (DAPI; cyan) immunofluorescence confocal images of primary mouse keratinocyte monolayers exposed to uniaxial static stretch and subsequent release. Scale bars, 20 µm. m Quantification of l showing the orientation angle of cell major axes perpendicular to the stretch direction (frequency distribution of >500 cells/condition pooled across three independent experiments; K² = 366.4 (0% contr.), 5.804 (120% contr.) 164.2 (0% PlexDKO), 215.2 (120% PlexDKO); D’Agostino–Pearson Omnibus test).