Antagonistic Regulation of Intercellular Cohesion by Plakophilins 1 and 3  René Keil, Katrin Rietscher, Mechthild Hatzfeld  Journal of Investigative Dermatology  Volume 136, Issue 10, Pages 2022-2029 (October 2016) DOI: 10.1016/j.jid.2016.05.124 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Expression of PKP1 and PKP3 is regulated during in vitro differentiation of mouse keratinocytes. (a) WB of HaCaT cells (H) and mouse keratinocytes (M) grown for 24 h in HCM showing the expression of PKP1–3. (b) Representative WB of mouse keratinocytes grown for 1 day and 2 days in LCM or HCM, respectively, showing Ca2+-dependent changes in the expression of PKP1 and PKP3, DSP, DSG1/2, DSC2, and E-cadherin. α-Tubulin was used as loading control. (c) Quantification of protein (white; normalized against α-tubulin) and mRNA (gray; normalized against POLR2A) levels depicted as fold change in HCM (mean ± SD, n ≥ 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. DSC, desmocollin; DSG, desmoglein; DSP, desmoplakin; HCM, high calcium medium; LCM, low calcium medium; PKP, plakophilin, SD, standard deviation; WB, western blot. Journal of Investigative Dermatology 2016 136, 2022-2029DOI: (10.1016/j.jid.2016.05.124) Copyright © 2016 The Authors Terms and Conditions

Figure 2 PKP1 and PKP3 form distinct cell-cell contacts during differentiation of mouse keratinocytes. Mouse keratinocytes were incubated in HCM for the indicated time and immunostained for PKP1, PKP3, and DSP (anti-DSP1/2-mouse). Depicted are confocal images of single optical sections. To highlight the partial colocalization of PKP3 and DSP at 0.5 and 2 hours in HCM, brightness and contrast of the DSP channel (red) have been adapted in the merged images. Scale bars: 20 μm. DSP, desmoplakin; HCM, high calcium medium; PKP, plakophilin. Journal of Investigative Dermatology 2016 136, 2022-2029DOI: (10.1016/j.jid.2016.05.124) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Loss of either PKP1 or PKP3 alters the appearance of cell-cell contacts. WT, PKP1-KO, and PKP3-KO keratinocytes grown for 24 hours in HCM were immunostained for PKP1, PKP3, and DSP (anti-DSP1/2-mouse). (a) Maximum intensity projections of at least 15 optical sections are depicted. Scale bars: 20 μm. (b) DSP fluorescence intensities at cell-cell contacts were normalized against cytoplasmic DSP and the respective ratios are shown as box plot of three independent experiments with n = 180 cells. The whiskers extend to the 5th and 95th percentile. ∗∗∗P < 0.001. DSP, desmoplakin; HCM, high calcium medium; KO, knockout; PKP, plakophilin; WT, wild type. Journal of Investigative Dermatology 2016 136, 2022-2029DOI: (10.1016/j.jid.2016.05.124) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Antagonistic regulation of intercellular cohesion by PKP1 and PKP3. (a) Keratinocytes grown for 24 hours in HCM were incubated in LCM-EGTA for the indicated time and stained for DSP. (b) Quantification of the resulting intercellular gaps. Depicted are the exposed areas in percent per image (mean ± SD, n = 3; 20 images each). (c) Dispase assay of the depicted cell lines (1+1, PKP1-KO+PKP1-GFP; 1+3, PKP1-KO+PKP3-GFP; 3+3, PKP3-KO+PKP3-GFP) and its quantification (d; mean number of fragments ± SD; n = 4). (e) The indicated cell lines were grown for 3 days in HCM without (DMSO) or with Gö6976. Hyperadhesion was addressed by incubation of detached monolayers for 90 minutes in LCM-EGTA, subsequent application of mechanical stress and counting of the resulting fragments (f; mean number of fragments ± SD; n = 4). (g) WB analysis of the respective cell lines. α-Tubulin was used as loading control. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. DSP, desmoplakin; GFP, green fluorescent protein; HCM, high calcium medium; KO, knockout; LCM, low calcium medium; PKP, plakophilin; SD, standard deviation; WB, western blot. Journal of Investigative Dermatology 2016 136, 2022-2029DOI: (10.1016/j.jid.2016.05.124) Copyright © 2016 The Authors Terms and Conditions

Figure 5 PKP3 contributes to desmosome dynamics. PKP1-KO and PKP3-KO keratinocytes expressing either PKP1- or PKP3-GFP were grown for 24 hours in HCM and subsequently used for FRAP experiments. (a) Representative images of FRAP series depict the recovery of PKP1- or PKP3-GFP in the respective cell lines. Circles point out bleached areas (approximately 200–300 μm2). Scale bars: 20 μm. (b) Quantification of mobile fractions (mean ± SD, n ≥ 10 cells) of desmosomal PKP1-GFP and PKP3-GFP in the respective cell lines. Only membrane-associated PKP1-GFP and PKP3-GFP was used to calculate mobile fractions. (c) Model illustrating antagonistic functions of PKP1 and PKP3 in remodeling desmosomal properties. ∗P < 0.05, ∗∗∗P < 0.001. FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; HCM, high calcium medium; KO, knockout; PKP, plakophilin; SD, standard deviation. Journal of Investigative Dermatology 2016 136, 2022-2029DOI: (10.1016/j.jid.2016.05.124) Copyright © 2016 The Authors Terms and Conditions