Fig 1: Plexin-B1/Plexin-B2 regulate dynamic crowding-induced changes of cortical stiffness.a, c Confocal images of the epidermal basal layer at E15.5 a stained with phalloidin (green) or c immunostained for anti-phosho-myosin light chain 2 (pMLC-2; Ser19; red). Cell divisions are contoured by dashed lines. Scale bar, 10 µm. b, d Quantification of b phalloidin fluorescence intensities and d pMLC-2 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). b 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. d control: n = 50 dividing cells from three mice, PlexDKO: n = 47 dividing cells from three mice. Two-sided Mann–Whitney U test for b and d p < 0.0001. e, f Representative pMLC-2 (Ser19) immunofluorescence images e and quantification f upon crowding (mean ± s.d., n = 3 independent experiments with >100 cells/condition/experiment; *p = 0.0322, Friedman/Dunn’s; scale bars 20 µm). g Quantification of cell surface areas after static 120% stretch and subsequent release (Tukey’s box and whiskers plot with minimum, first quartile, median, third quartile and maximum; n > 500 cells/condition pooled across three independent experiments; ****p < 0.0001, *p = 0.0131, Kruskal–Wallis/Dunn’s). h Force indentation spectroscopy of cell cortexes from primary mouse keratinocytes adhering on 100 µm circular micropatterns (control: n = 34, PlexDKO: n = 31 micropatterns pooled across three independent experiments). i Confocal images of the epidermal basal layer at E15.5 stained with phalloidin (green). Cell divisions are contoured by dashed lines. Blue: DAPI. Scale bar, 10 µm. j, k Quantification of shape anisotropy of basal cells in early mitosis (i.e. prophase to metaphase) (box plot with minimum, first quartile, median, third quartile and maximum; control: n = 44 cells from three mice, PlexDKO: n = 42 cells from three mice; j p = 0.0035, k p = 0.0033; two-sided Mann–Whitney U test). l Confocal images of the epidermal basal layer at E16.5 immunostained for phospho-histone H3 (green). White dashed line: plane of basal layer; green dashed line: axis of mitosis. Scale bar, 10 µm. m Radial histograms (rose plots) depicting the mitotic spindle angle of dividing control and Plexin-B1/Plexin-B2-deficient basal cells (control: n = 181 cells from six mice, PlexDKO: n = 171 cells from six mice; two-sided Mann–Whitney U test: p = 0.0187).
Fig 2: 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 a-catenin (red), and j a-18 (=a-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 a-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).
Fig 3: Plexin-B1/Plexin-B2 control YAP activity and cell proliferation in basal cell carcinoma.a Schematic illustration of the generation of epidermis-specific tamoxifen-inducible Gas/Plexin-B1/Plexin-B2 triple-deficient mice. b Kaplan–Meier curves representing the percentage of lesion-free mice. Time point “0” indicates the start of tamoxifen treatment (wildtype mice: n = 3, Gas KOind mice: n = 16, Gas KOind;PlexDKOind mice: n = 16; p < 0.0001; Mantel–Cox test for Gas KOind compared to Gas KOind;PlexDKOind). c–h Histological/immunofluorescence stainings (left panels) and respective quantifications (right panels) of adult murine skin of mice with the indicated genotypes 30 days after treatment with tamoxifen. Boxed areas are magnified in the lower rows. Tumors are marked by dashed lines. c Goldner-Elastica stain of histological sections. Scale bar, 100 µm (upper row), 25 µm (lower row). d Quantification of tumor areas (mean ± s.d.; Gas KOind mice: n = 8, Gas KOind;PlexDKOind mice: n = 12; p = 0.0049; two-sided unpaired t-test). e Confocal images of immunostainings using an anti-Ki67 antibody (red). Scale bar, 100 µm (upper row), 25 µm (lower row). f Quantification of Ki67-positive cells (mean ± s.d.; Gas KOind mice: n = 7, Gas KOind;PlexDKOind mice: n = 7; p = 0.0049; two-sided unpaired t-test). g Confocal images of immunostainings using anti-active YAP (red) and anti-K14 antibodies (green). Scale bar, 100 µm (upper row), 25 µm (lower row). h Quantification of cells positive for active YAP (mean ± s.d.; Gas KOind mice: n = 7, Gas KOind;PlexDKOind mice: n = 10; p = 0.0143; two-sided unpaired t-test).
Fig 4: Plexin-B1/Plexin-B2 inhibit YAP activity in response to mechanical forces.a Confocal images of murine epidermis at E15.5 and E16.5 immunostained for active YAP (red) and K14 (green). Scale bar, 25 µm. b, c Quantification of the data in a (percentage of basal cells) (mean ± s.d.; E15.5: n = 5 mice per genotype, E16.5: n = 4 mice per genotype; b p = 0.0047, c p = 0.0078; two-sided unpaired t-test). d, e Primary mouse keratinocytes on d circular or e square micropatterns immunostained for Yap (green) after 3 h of 1.8 mM Ca2+. Representative images are shown on the left, quantifications of Yap localization are shown on the right (mean ± s.d.; circles: control: n = 73 micropatterns from 5 mice, PlexDKO: n = 73 micropatterns from four mice; N > C: p = 0.000008, N = C: p = 0.012038, N < C: p = 0.000008; squares: control: n = 69 micropatterns from five mice, PlexDKO: n = 69 micropatterns from four mice; N > C: p < 0.000001, N = C: p < 0.000001, N < C: p = 0.000050; two-sided unpaired t-test). Blue: DAPI. Scale bars, 25 µm. f Schematic illustration of abrupt crowding experiments. Confluent monolayers were exposed to 120% static uniaxial stretch, which was abruptly released. Cells were analyzed at the indicated time points (R5’: 5 min after release; R15’: 15 min after release). g, h Representative YAP immunofluorescence images g and quantification of nuclear YAP h upon crowding (mean ± s.d.; n = 5 independent experiments with >200 cells/condition/experiment; *p = 0.0358, ***p = 0.0004, one-way ANOVA, Tukey test; values are normalized to control 120% stretch; statistics have been done from the non-normalized values; scale bars 20 µm). i–l Pregnant females were injected at E15.5 with vehicle control (DMSO) or verteporfin (i.p., 100 µg/g body weight). Embryos were harvested at E16.5. Shown are in i representative H&E-stained histological sections, in j the quantification of epidermal thickness (mean ± s.e.m.; control DMSO-treated: n = 4 embryos, PlexDKO DMSO-treated: n = 4 embryos, control verteporfin-treated: n = 7 embryos, PlexDKO verteporfin-treated: n = 8 embryos; p = 0.011 for DMSO, p = 0.165 for verteporfin; two-sided unpaired t-test), in k representative images of immunohistochemical stainings of the embryonic epidermis using an anti-BrdU antibody (brown), and in l the quantification of BrdU-positive cells (mean ± s.e.m.; control DMSO-treated: n = 4 embryos, PlexDKO DMSO-treated: n = 4 embryos, control verteporfin-treated: n = 7 embryos, PlexDKO verteporfin-treated: n = 7 embryos; p = 0.016 for DMSO, p = 0.345 for verteporfin; two-sided unpaired t-test).
Fig 5: B-plexins suppress YAP activity in human basal cell carcinoma cells.a Human primary epidermal keratinocytes (hPEKS) and human primary basal cell carcinoma cells (hPBCCs) were lysed, and proteins were detected using the indicated antibodies. b, c hPBCCs engineered to express GFP (“control”) or myc-tagged Plexin-B2 (“Plexin-B2”) and cultured at dense conditions were immunostained for YAP (red) and myc (green). Blue: DAPI. Representative confocal images are shown in b with dashed boxed areas depicted in the right panel. A quantification of YAP nuclear intensity is shown in c (mean ± s.d.; n = 3 independent experiments; p = 0.0495; two-sided unpaired t-test). d hPBCCs were engineered to express GFP (“control”) or myc-tagged Plexin-B2 (“Plexin-B2”), and cultured at dense conditions. Relative mRNA expression levels of the YAP target genes ctgf and cyr61 were determined by quantitative RT-PCR (mean ± s.d.; n = 5 independent experiments; p = 0.0123 for ctgf, p = 0.0002 for cyr61; two-sided unpaired t-test).
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