Arrhythmogenic cardiomyopathy related DSG2 mutations affect desmosomal cadherin binding kinetics

Figures

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  Figure 1. (A) Crystal structure of Dsg2 homodimer shown in ribbon representation [12, pdbID: 5ERD]. Coordinated Ca2+ are shown as blue spheres. (B) Schematic of cantilever tip and sample substrate functionalized with Dsg2 for AFM single molecule force spectroscopy. Both, cantilever and substrate are modified with covalently immobilized Dsg2 fragments. (C) SMFS working principle and typical force-distance cycle exposing a specific dissociation event. The striated area under the curve represents the dissipated work to break a Dsg2 homodimer. (D) Principle of dispase assay for measuring cell-cell adhesion. Cells grown to confluency (a) are lifted up by dispase treatment and exposed to mechanical stress by shaking of cell cultures. Resulting islets of the monolayer are counted and recombinant cell cultures of mutant and wildtype are compared (b).

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  Figure 2. (A) Force loading rate graph showing the most probable dissociation force \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${F}^{\ast }$$\end{document}F⁎ plotted versus the logarithmic loading rate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{ln}(r)$$\end{document}ln(r). Colors represent Dsg2 wildtype in green, Dsg2- p.D105E (p.D154E) in red and Dsg2-p.V343I (p.V392I) in yellow. The error bars of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${F}^{\ast }$$\end{document}F⁎ are estimated by the standard error of the mean (SEM) and were used for a weighted fit of the Evans and Ritchie model (Eq. 2). Inset: The uncertainty of the…

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  Figure 3. (A) Series of control experiments for different Ca2+ concentrations and sample substrates. The dissociation probability distributions are scaled for the absolute binding frequency. Experiments with Dsg2 modified AFM cantilever and sample substrate in 2 mM calcium-buffer (green) and EDTA-buffer (orange) expose a clear dependency of the binding frequency on the calcium concentration. The background of unspecific adhesion was tested in an experiment conducted on substrates lacking Dsg2 in calcium (red) and EDTA-buffer (gray). (B) Evolution of the Jarzynski equality. The exponentially averaged work \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${W}_{n}$$\end{document}Wn plotted versus the number of dissociation events \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n$$\end{document}n. For wildtype Dsg2 and mutations thereof…

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  Figure 4. Confluent monolayers of transfected cells were subjected to dispase assay. Decreased number of fragments indicates relatively enhanced cell-cell adhesion. Box and whiskers plots represent the number of fragments in six independent experiments for Dsg2 wildtype, p.D105E (p.D154E; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p < 0.001$$\end{document}p<0.001 versus wildtype) and p.V343I (p.V392I). Box and whiskers plot: boxes extend from the 25th–75th, whiskers from the 5th–95th percentiles, the medians are shown as lines; outliers are given as dots. Means are indicated as crosses.