Figure 2.
E-cadherin controls cell shape upstream of spectrin. (a) Sagittal views of dorsal skin sections from shCtr and shAnk3 6780-transduced E17.5 embryos immunolabeled for αII-spectrin. Insets show the transduced cells (H2B−GFP+). (b) Quantification of basal (left) and suprabasal (right) layer αII-spectrin intensity from data shown in a. Mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: microscopy fields. NS: P = 0.586 (basal), NS: P = 0.9525 with Kolmogorov–Smirnov. (c) Dorsal skin sections from shCtr and mosaic shSptan1 0595-transduced E17.5 embryos immunolabeled for Ankyrin-3. Upper insets show the transduced cells (H2B−GFP+). (d) Left graph: Quantification of Ankyrin-3 intensity from data shown in c. Mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: microscopy fields. NS: P = 0.235 with Kolmogorov–Smirnov. Right graph: Quantification of Ankyrin-3 cortical enrichment from the data shown in c. Mean ± SD from 90 individual cells from n = 3 embryos per condition. Bars: Ankyrin-3 cortex/cytoplasm intensity ration mean; dots: individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. (e) αII-Spectrin staining on Ctr and E-cadherin–deficient newborn epidermis sections showing impaired cortical recruitment of αII-Spectrin upon loss of E-cadherin. Representative images of n = 3 biological replicates. (f) Immunofluorescence analysis for shape using combined staining for desmoglein1,2,3 (Dsg1,2,3) on Ctr and E-cadherin–deficient newborn epidermis sections. (g) Quantification of cell sagittal area and shape (perimeter/√area)/layer using stainings as shown in f. **P < 0.005, *P < 0.03; cells: n = 420 (basal), n = 445 (spinous), 277 (granular) with Kolmogorov–Smirnov per layer. (h) Phalloidin staining of single cells isolated from the granular layer of Ctr or E-cadherin–deficient newborn epidermis. (i) Quantification of the cell top-view area of isolated SG cells from Ctr and E-cadherin–deficient newborn epidermis as shown in h and treated with latrunculin B (0.1 µM). Dots represent individual cells isolated from three mice. ****P < 0.0001; n ≥ 98 cells with Kruskal–Wallis, Dunn’s multiple comparison test. All images: Nuclei were stained with DAPI (blue). ROI, region of interest.

E-cadherin controls cell shape upstream of spectrin. (a) Sagittal views of dorsal skin sections from shCtr and shAnk3 6780-transduced E17.5 embryos immunolabeled for αII-spectrin. Insets show the transduced cells (H2B−GFP+). (b) Quantification of basal (left) and suprabasal (right) layer αII-spectrin intensity from data shown in a. Mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: microscopy fields. NS: P = 0.586 (basal), NS: P = 0.9525 with Kolmogorov–Smirnov. (c) Dorsal skin sections from shCtr and mosaic shSptan1 0595-transduced E17.5 embryos immunolabeled for Ankyrin-3. Upper insets show the transduced cells (H2B−GFP+). (d) Left graph: Quantification of Ankyrin-3 intensity from data shown in c. Mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: microscopy fields. NS: P = 0.235 with Kolmogorov–Smirnov. Right graph: Quantification of Ankyrin-3 cortical enrichment from the data shown in c. Mean ± SD from 90 individual cells from n = 3 embryos per condition. Bars: Ankyrin-3 cortex/cytoplasm intensity ration mean; dots: individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. (e) αII-Spectrin staining on Ctr and E-cadherin–deficient newborn epidermis sections showing impaired cortical recruitment of αII-Spectrin upon loss of E-cadherin. Representative images of n = 3 biological replicates. (f) Immunofluorescence analysis for shape using combined staining for desmoglein1,2,3 (Dsg1,2,3) on Ctr and E-cadherin–deficient newborn epidermis sections. (g) Quantification of cell sagittal area and shape (perimeter/√area)/layer using stainings as shown in f. **P < 0.005, *P < 0.03; cells: n = 420 (basal), n = 445 (spinous), 277 (granular) with Kolmogorov–Smirnov per layer. (h) Phalloidin staining of single cells isolated from the granular layer of Ctr or E-cadherin–deficient newborn epidermis. (i) Quantification of the cell top-view area of isolated SG cells from Ctr and E-cadherin–deficient newborn epidermis as shown in h and treated with latrunculin B (0.1 µM). Dots represent individual cells isolated from three mice. ****P < 0.0001; n ≥ 98 cells with Kruskal–Wallis, Dunn’s multiple comparison test. All images: Nuclei were stained with DAPI (blue). ROI, region of interest.

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