Figure 4.
Spectrin determines junctional actomyosin network structure and stability. (a) Newborn epidermal whole-mount immunofluorescence analysis for phalloidin (F-actin) and non-muscle myosin heavy chain IIa (myosin-IIa). Minimal max. projections of the denoted layers are shown. (b) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO or Y27632 (40 µM) immunolabeled for αII-spectrin. (c) Quantification of basal (left graph) and suprabasal (right graph) layer αII-spectrin intensity from data shown in b. Data are the mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: microscopy fields. *P ≤ 0.05; NS: P = 0.3876 with Kolmogorov–Smirnov. (d) Immunofluorescence analysis for αII-spectrin, F-actin, and non-muscle myosin heavy chain IIa (myosin-IIa) (48 h high Ca2+) in Ctr and αII-spectrin–deficient (KO) cells. (e) Immunofluorescence analysis for αII-spectrin and F-actin (48 h high Ca2+) upon αII-spectrin (Sptan1) knockdown and treatment with either DMSO or low-dose blebbistatin (5 µM). Representative images of n = 3 biological replicates each. (f) Immunofluorescence analysis for F-actin (48 h high Ca2+) upon αII-spectrin (Sptan1) knockdown at low tension (low-dose blebbistatin 5 µM) showing streak-like defects similar to in vivo. Representative images of n = 3 biological replicates each. (g) Illustration of laser ablation in multilayered keratinocytes. (h) Laser ablation: Still images from live imaging of SiR-actin (F-actin)-labeled Ctr cells (48 h high Ca2+) at indicated time points after 17 µm line ablation showing progressive elliptical cortical openings. (i) Quantification of the elliptical opening area (red line) i.e., recoil over time. Line: mean ± SD opening area of 13 ablations from N = 4 biological replicates. (j) Cortical laser ablation: Still images from live imaging of SiR-actin (F-actin)–labeled Ctr (as shown h) and αII-spectrin knockdown keratinocytes at the time point of analysis (60 s after a linear laser cut [yellow line]) after 48 h high Ca2+. The opening in the F-actin cortex is seen as black area around the yellow line. (k) Quantification of opening areas in the apical F-actin cortex upon linear laser ablation as shown in j. Lines represent means, dots represent single openings/cells pooled from n = 3 independent experiments/biological replicates. *P = 0.0215 with Kolmogorov–Smirnov. (l) Laser ablation (as described for j) of αII-spectrin knockdown keratinocytes treated with DMSO or low dose blebbistatin (5 µM) 1 h prior to ablation. (m) Quantification of opening areas in the apical F-actin cortex upon linear laser ablation as shown in l. Lines represent means; dots represent single openings/cells pooled from n = 3 independent experiments/biological replicates. ****P < 0.0001 with Kolmogorov–Smirnov. (n) Laser ablation (as described for j) of E-cadherin−/− keratinocytes after 48 h high Ca2+. (o) Quantification of opening areas in the apical F-actin cortex upon linear laser ablation as shown in N. Lines represent means; dots represent single openings/cells pooled from n = 3 independent experiments/biological replicates. ****P < 0.0001 with Kolmogorov–Smirnov. ROI, region of interest.

Spectrin determines junctional actomyosin network structure and stability. (a) Newborn epidermal whole-mount immunofluorescence analysis for phalloidin (F-actin) and non-muscle myosin heavy chain IIa (myosin-IIa). Minimal max. projections of the denoted layers are shown. (b) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO or Y27632 (40 µM) immunolabeled for αII-spectrin. (c) Quantification of basal (left graph) and suprabasal (right graph) layer αII-spectrin intensity from data shown in b. Data are the mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: microscopy fields. *P ≤ 0.05; NS: P = 0.3876 with Kolmogorov–Smirnov. (d) Immunofluorescence analysis for αII-spectrin, F-actin, and non-muscle myosin heavy chain IIa (myosin-IIa) (48 h high Ca2+) in Ctr and αII-spectrin–deficient (KO) cells. (e) Immunofluorescence analysis for αII-spectrin and F-actin (48 h high Ca2+) upon αII-spectrin (Sptan1) knockdown and treatment with either DMSO or low-dose blebbistatin (5 µM). Representative images of n = 3 biological replicates each. (f) Immunofluorescence analysis for F-actin (48 h high Ca2+) upon αII-spectrin (Sptan1) knockdown at low tension (low-dose blebbistatin 5 µM) showing streak-like defects similar to in vivo. Representative images of n = 3 biological replicates each. (g) Illustration of laser ablation in multilayered keratinocytes. (h) Laser ablation: Still images from live imaging of SiR-actin (F-actin)-labeled Ctr cells (48 h high Ca2+) at indicated time points after 17 µm line ablation showing progressive elliptical cortical openings. (i) Quantification of the elliptical opening area (red line) i.e., recoil over time. Line: mean ± SD opening area of 13 ablations from N = 4 biological replicates. (j) Cortical laser ablation: Still images from live imaging of SiR-actin (F-actin)–labeled Ctr (as shown h) and αII-spectrin knockdown keratinocytes at the time point of analysis (60 s after a linear laser cut [yellow line]) after 48 h high Ca2+. The opening in the F-actin cortex is seen as black area around the yellow line. (k) Quantification of opening areas in the apical F-actin cortex upon linear laser ablation as shown in j. Lines represent means, dots represent single openings/cells pooled from n = 3 independent experiments/biological replicates. *P = 0.0215 with Kolmogorov–Smirnov. (l) Laser ablation (as described for j) of αII-spectrin knockdown keratinocytes treated with DMSO or low dose blebbistatin (5 µM) 1 h prior to ablation. (m) Quantification of opening areas in the apical F-actin cortex upon linear laser ablation as shown in l. Lines represent means; dots represent single openings/cells pooled from n = 3 independent experiments/biological replicates. ****P < 0.0001 with Kolmogorov–Smirnov. (n) Laser ablation (as described for j) of E-cadherin−/− keratinocytes after 48 h high Ca2+. (o) Quantification of opening areas in the apical F-actin cortex upon linear laser ablation as shown in N. Lines represent means; dots represent single openings/cells pooled from n = 3 independent experiments/biological replicates. ****P < 0.0001 with Kolmogorov–Smirnov. ROI, region of interest.

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