Figure 3.
Cortical F-actin and spectrin organization are mutually dependent. (a) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO or latrunculin B (2.5 µM) immunolabeled for αII-spectrin. (b) Quantification of basal (left graph) and suprabasal (right graph) 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. **P = 0.0072 (basal, latrunculin B), **P = 0.0165 (suprabasal, latrunculin B) with Kolmogorov–Smirnov. (c) Dorsal skin sections from shCtr and mosaic shSptan1 0595-transduced E17.5 embryos labeled for F-actin. Upper insets show the transduced cells (H2B−GFP+). (d) Quantification of basal (left) and suprabasal (right) layer F-actin intensity from data shown in c. Mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: individual microscopy fields. NS: P = 0.1344 (shSptan1 0595, basal), P = 0.354 (shSptan1 9753, basal), **P = 0.001 (shSptan1 0595, suprabasal), **P = 0.001 (shSptan1 9753, suprabasal) by unpaired t-test. Nuclei were stained with DAPI; dotted lines indicate the dermal-epidermal border. NS, not significant. (e) Newborn epidermal whole-mount immunofluorescence analysis for cortical F-actin organization upon loss of αII-spectrin. Note streak-like (arrow) and spot-like reorganization of F-actin upon loss of αII-spectrin. Representative images of four biological replicates (mice). (f) Quantification of the percentage of cells in the granular layer showing either normal (lattice-like) or abnormal (streak-like or spot-like) F-actin organization. *P < 0.05 with Mann–Whitney for the mean of n = 4 biological replicates, including 466 (Ctr) and 385 (Sptan1epi−/−) analyzed cells. (g) Immunofluorescence analysis for αII-spectrin and F-actin after 6 h or 48 h in high Ca2+ at cell–cell interfaces (arrow: actin-spectrin lattice, arrowhead: TJ-supporting apical F-actin ring) and apical surface. Representative images of n ≥ 3 biological replicates each. (h) Schematic representation of the image position in the mono or multilayered keratinocytes. (i) Immunofluorescence analysis for αII-spectrin and F-actin after 48 h in high Ca2+ at cell–cell interfaces with and without latrunculin B treatment (1 h, 0.1 µM). (j) Immunofluorescence analysis for αII-spectrin and F-actin (48 h high Ca2+) upon αII-spectrin knockdown. Representative images of n = 3 biological replicates each. ROI, region of interest.

Cortical F-actin and spectrin organization are mutually dependent. (a) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO or latrunculin B (2.5 µM) immunolabeled for αII-spectrin. (b) Quantification of basal (left graph) and suprabasal (right graph) 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. **P = 0.0072 (basal, latrunculin B), **P = 0.0165 (suprabasal, latrunculin B) with Kolmogorov–Smirnov. (c) Dorsal skin sections from shCtr and mosaic shSptan1 0595-transduced E17.5 embryos labeled for F-actin. Upper insets show the transduced cells (H2B−GFP+). (d) Quantification of basal (left) and suprabasal (right) layer F-actin intensity from data shown in c. Mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: individual microscopy fields. NS: P = 0.1344 (shSptan1 0595, basal), P = 0.354 (shSptan1 9753, basal), **P = 0.001 (shSptan1 0595, suprabasal), **P = 0.001 (shSptan1 9753, suprabasal) by unpaired t-test. Nuclei were stained with DAPI; dotted lines indicate the dermal-epidermal border. NS, not significant. (e) Newborn epidermal whole-mount immunofluorescence analysis for cortical F-actin organization upon loss of αII-spectrin. Note streak-like (arrow) and spot-like reorganization of F-actin upon loss of αII-spectrin. Representative images of four biological replicates (mice). (f) Quantification of the percentage of cells in the granular layer showing either normal (lattice-like) or abnormal (streak-like or spot-like) F-actin organization. *P < 0.05 with Mann–Whitney for the mean of n = 4 biological replicates, including 466 (Ctr) and 385 (Sptan1epi−/−) analyzed cells. (g) Immunofluorescence analysis for αII-spectrin and F-actin after 6 h or 48 h in high Ca2+ at cell–cell interfaces (arrow: actin-spectrin lattice, arrowhead: TJ-supporting apical F-actin ring) and apical surface. Representative images of n ≥ 3 biological replicates each. (h) Schematic representation of the image position in the mono or multilayered keratinocytes. (i) Immunofluorescence analysis for αII-spectrin and F-actin after 48 h in high Ca2+ at cell–cell interfaces with and without latrunculin B treatment (1 h, 0.1 µM). (j) Immunofluorescence analysis for αII-spectrin and F-actin (48 h high Ca2+) upon αII-spectrin knockdown. Representative images of n = 3 biological replicates each. ROI, region of interest.

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