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Figure 7.
EGFR activity regulates cortical TRPV3 localization. (a) Dorsal skin sections from shCtr; TRPV3-GFP–transduced E17.5 embryos treated with DMSO or gefitinib immunolabeled for αII-spectrin and pEGFR. (b and c) Quantification of TRPV3-GFP and αII-spectrin cortical enrichment from the data shown in a. Mean ± SD from 30 individual cells from n = 3 embryos per condition. Bars: TRPV3-GFP cortex/cytoplasm ratio; dots: individual cells. **P = 0.0029 for TRPV3-GFP and ***P = 0.0001 for αII-spectrin with Kolmogorov-Smirnov. (d and f) Primary mouse keratinocytes cultured in high-calcium (1.5 mM) medium treated with DMSO, gefitinib, or TGF-α and immunolabelled for p-EGFR, TRPV3, and αII-spectrin. Boxes indicate the location of the magnified area. (e and g) Quantification of TRPV3 and αII-spectrin intensity from the data shown in d and f. Mean ± SD from ∼200 mature junctions from n = 3 experiment per condition. Bars: mean normalized intensity; dots: individual junctions. Nuclei were stained with DAPI. **P = 0.001 and ****P > 0.0001 for TRPV3 intensity. ****P > 0.0001 and ***P = 0.0001 for αII-spectrin intensity with Kolmogorov–Smirnov. (h) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO and gefitinib. Sections were processed for transglutaminase 1 (TGM1) activity assay. (i) Quantification of crosslinked TGM substrate intensity. Mean ± SD of 30 ROIs from n = 3 embryos per condition. Bars: mean normalized intensity; dots: individual microscopy fields. NS: P = 0.3876 with Kolmogorov–Smirnov. (j) Quantification of cortical enrichment of crosslinked TGM substrate. Mean ± SD from 60 individual cells from n = 3 embryos per condition. Bars: means of cortex/cytoplasm ratio; dots: individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. Nuclei were stained with DAPI; dashed lines indicate the dermal-epidermal border. (k) Model - High-tension spectrin-actomyosin cortices regulate TGM activity. Illustration of junction and cytoskeleton distribution across epidermal layers, with spectrin most enriched in SG3 and F-actin most enriched in SG1. Lower left: illustration of spectrin and myosin-dependent organization of cortical F-actin. Upper left: Working model of how lattice organization and myosin tension regulate EGFR/TRPV3 signaling complexes, resulting in Ca2+ influx and cortical TGM activation. ROI, region of interest.

EGFR activity regulates cortical TRPV3 localization. (a) Dorsal skin sections from shCtr; TRPV3-GFP–transduced E17.5 embryos treated with DMSO or gefitinib immunolabeled for αII-spectrin and pEGFR. (b and c) Quantification of TRPV3-GFP and αII-spectrin cortical enrichment from the data shown in a. Mean ± SD from 30 individual cells from n = 3 embryos per condition. Bars: TRPV3-GFP cortex/cytoplasm ratio; dots: individual cells. **P = 0.0029 for TRPV3-GFP and ***P = 0.0001 for αII-spectrin with Kolmogorov-Smirnov. (d and f) Primary mouse keratinocytes cultured in high-calcium (1.5 mM) medium treated with DMSO, gefitinib, or TGF-α and immunolabelled for p-EGFR, TRPV3, and αII-spectrin. Boxes indicate the location of the magnified area. (e and g) Quantification of TRPV3 and αII-spectrin intensity from the data shown in d and f. Mean ± SD from ∼200 mature junctions from n = 3 experiment per condition. Bars: mean normalized intensity; dots: individual junctions. Nuclei were stained with DAPI. **P = 0.001 and ****P > 0.0001 for TRPV3 intensity. ****P > 0.0001 and ***P = 0.0001 for αII-spectrin intensity with Kolmogorov–Smirnov. (h) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO and gefitinib. Sections were processed for transglutaminase 1 (TGM1) activity assay. (i) Quantification of crosslinked TGM substrate intensity. Mean ± SD of 30 ROIs from n = 3 embryos per condition. Bars: mean normalized intensity; dots: individual microscopy fields. NS: P = 0.3876 with Kolmogorov–Smirnov. (j) Quantification of cortical enrichment of crosslinked TGM substrate. Mean ± SD from 60 individual cells from n = 3 embryos per condition. Bars: means of cortex/cytoplasm ratio; dots: individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. Nuclei were stained with DAPI; dashed lines indicate the dermal-epidermal border. (k) Model - High-tension spectrin-actomyosin cortices regulate TGM activity. Illustration of junction and cytoskeleton distribution across epidermal layers, with spectrin most enriched in SG3 and F-actin most enriched in SG1. Lower left: illustration of spectrin and myosin-dependent organization of cortical F-actin. Upper left: Working model of how lattice organization and myosin tension regulate EGFR/TRPV3 signaling complexes, resulting in Ca2+ influx and cortical TGM activation. ROI, region of interest.

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