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Nectin-4 Mutations Causing Ectodermal Dysplasia with Syndactyly Perturb the Rac1 Pathway and the Kinetics of Adherens Junction Formation  Paola Fortugno,

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Presentation on theme: "Nectin-4 Mutations Causing Ectodermal Dysplasia with Syndactyly Perturb the Rac1 Pathway and the Kinetics of Adherens Junction Formation  Paola Fortugno,"— Presentation transcript:

1 Nectin-4 Mutations Causing Ectodermal Dysplasia with Syndactyly Perturb the Rac1 Pathway and the Kinetics of Adherens Junction Formation  Paola Fortugno, Emmanuelle Josselin, Konstantinos Tsiakas, Emanuele Agolini, Gianluca Cestra, Massimo Teson, René Santer, Daniele Castiglia, Giuseppe Novelli, Bruno Dallapiccola, Ingo Kurth, Marc Lopez, Giovanna Zambruno, Francesco Brancati  Journal of Investigative Dermatology  Volume 134, Issue 8, Pages (August 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Altered nectin-4/1 expression and localization in ectodermal-dysplasia-syndactyly syndrome (EDSS1) epidermis and primary keratinocytes. (a, b) Confocal immunofluorescence analysis of double immunostaining with anti-nectin-4 polyclonal antibody (green) and the nectin-1 mAb R1.302 (red) in (a) human epidermis from the EDSS1 patient (242Met-N4) as compared with a healthy control (WT-N4), and (b) in vitro–differentiated keratinocytes from the EDSS1 patient (242Met-N4-HK) as compared with WT-N4-HK. Scale bar=10 μm. Image z-stacks are shown on the side in (a). (c) Immunoblotting analysis of total protein extracts of differentiated keratinocytes. Protein loading was normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels. (d) Relative PVRL4 mRNA expression in differentiated keratinocytes determined by quantitative real-time reverse-transcriptase–PCR. (e) FACS analysis of nectin-4 (left panels) and -1 (right panels) cell surface expression on dissociated 242Met-N4-HK as compared with WT-N4-HK. HK, human keratinocyte; WT, wild type. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Nectin-1/4 trans-dimerization on keratinocyte cell surface. Trans-heterophilic interaction between soluble nectin-1 (N1-Fc) and nectin-4 assessed by FACS analysis on dissociated 242Met-N4-HK as compared with WT-N4-HK (upper panels). N2-Fc was used as a negative binding control (lower panels). HK, human keratinocyte; WT, wild type. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Decreased adhesiveness of epithelial cells expressing mutant nectin-4. (a) MDCK aggregation assay. Cells were allowed to aggregate in suspension overnight. After mechanical dissociation, images were captured by phase-contrast microscopy. Scale bar=100 μm. (b) Time course of adherens junction (AJ) formation in 242Met-N4-HK as compared with WT-N4-HK. Cells, grown in standard medium until confluence (+Ca), were starved in a Ca2+/serum-free medium for 4 hours (-Ca), and then standard medium was added for the indicated times. Assembly of junctions was assessed by immunofluorescence staining with E-cadherin mAb (clone 36). Scale bar=20 μm. (c) AJ stability in MDCK cells. MDCK, WT-N4-MDCK, and 242Met-N4-MDCK cells were grown in standard calcium medium until confluence (+Ca), and then switched to Ca2+-free medium for 4 hours (-Ca). Junction stability was assessed by E-cadherin immunofluorescence staining. Scale bar=20 μm. HK, human keratinocyte; WT, wild type. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Altered Rac1 signaling pathway in cells expressing mutant nectin-4. (a) Active GTP-Rac1 evaluation in HEK-293 cells transfected with pFLAG (mock), pFLAG-WT-N4, pFLAG-242Met-N4, and pFALG-185Met-N4 using pull-down assays. Total Rac1 and nectin-4 expression was assessed on total cell lysates. A representative experiment is shown. Lower panel, densitometric analysis of GTP-Rac1 levels, normalized to the total Rac1. Results represent the mean±s.d. (n=4). P-values are indicated in the graph. (b) Evaluation of Rac1 pathway activation by nectins-1/4 trans-interaction in 242Met-N4-MDCK as compared with WT-N4-MDCK. Cells were stimulated with preclustered N1-Fc for the indicated times. Lower panel, densitometric analysis of GTP-Rac1 levels, normalized to the total Rac1. Results are expressed as percentage of the relative time 0 activity. A representative experiment out of five is shown. HK, human keratinocyte; WT, wild type. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

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