Vinculin anchors contractile actin to the cardiomyocyte adherens junction

FIGURE 1:. Vinculin and afadin recruitment to cardiomyocyte AJs is tension dependent. (A–E) Mouse neonatal cardiomyocytes were treated for 1 h with DMSO (A, D) or 100 μM blebbistatin (B, E) before fixation. Cells were stained for F-actin (A, B, D, and E), vinculin, and plakoglobin (A, B), or afadin and αE-catenin (D, E). Individual and merged vinculin (green) and plakoglobin (magenta) channels shown in A and B. Individual and merged afadin (green) and αE-catenin (magenta) channels shown in D and E. Far right column is a higher magnification of the boxed contact in merge. (C, F) Quantification of vinculin (C) or afadin (F) intensity at cell–cell contacts. Vinculin or afadin signal intensity was measured in cells treated with DMSO or blebbistatin for 10, 30, and 60 min before fixation. All data points are plotted. Middle horizontal bar is the median and error bars represent the quartile range. One-way ANOVA, n ≥ 60 images from at least three independent experiments. Images are maximum projections of 5 µm stacks. Scale bar is 10 μm in lower-magnification images, 5 μm in higher-magnification images.

FIGURE 2:. Loss of N-cadherin disrupts adhesion protein localization. (A) Cartoon schematic of desmosome, AJ, and gap junction proteins at cardiomyocyte cell–cell contacts. (B–F) Neonatal cardiomyocytes from Ncadfx/fx mice were uninfected (B) or infected with adenovirus expressing Cre recombinase (C–F) and fixed over 96 h to assess N-cadherin expression. Cells were stained for F-actin (left panel) and N-cadherin (N-cad, right panel). (G–R) Control and Cre-infected neonatal cardiomyocytes from Ncadfx/fx mice were fixed 96 h postinfection and stained for AJ proteins β-catenin and αE-catenin (G–J); AJ adapter proteins vinculin and afadin (K–N); connexin 43 (Cx43; O, P), and desmoglein 2 (Dsg2; Q, R). Images are maximum projections of 2–3 μm deconvolved stacks. Scale bar is 10 μm.

FIGURE 3:. N-cadherin-GFP rescues cardiomyocyte junctional complexes. (A–F) Neonatal Ncadfx/fx cardiomyocytes infected sequentially with adenoviruses expressing Cre and N-cadherin-GFP, fixed, and stained for AJ-associated proteins (A–D), gap junctions (E), and desmosomes (F). Individual and merged N-cadherin-GFP (green) and ICD components (magenta) channels are shown. Far right columns are higher magnifications of the boxed contact in the merge. Individual and merged channels are shown. Images are maximum projections of 2–3 μm deconvolved stacks. Scale bar is 10 μm in lower-magnification images, 5 μm in higher-magnification images.

FIGURE 4:. N-cadherin-GFP-αE-catenin fusions selectively recruit ligands to cardiomyocyte cell–cell contacts. (A) Table of N-cadherin-GFP-αE-catenin fusion constructs used in this study. Nomenclature, domain schematic, and actin linkage cartoon are shown. (B) Quantification of N-cadherin-GFP-αE-catenin fusion cell–cell contact morphology. Mean contact length plotted against mean contact circularity. Error bars represent SEM. Data are from at least 180 individual cell–cell contacts from two independent infections with each fusion construct. (C–F) Cardiomyocytes infected with Cre and N-cadherin-GFP (C) or Cre and fusion adenoviruses (D–F), fixed, and stained for vinculin and afadin. Individual and merged GFP (green), vinculin (red), and afadin (blue) channels are shown. Images are a maximum projection of 2–3 μm deconvolved stacks. Bottom image is a higher magnification of boxed contact. (G–J) Quantification of vinculin and afadin intensities at cell–cell contacts. Signal intensity at contacts was divided by the average cytoplasmic intensity and a scatter plot of all data points is shown. The black horizontal line is the median and the error bars define the interquartile range. The shaded gray…

FIGURE 5:. N-cadherin-GFP-αE-catenin fusion dynamics at cardiomyocyte cell–cell contacts. (A–D) Representative prebleach, postbleach, and recovery images from FRAP studies of Ncadfx/fx cardiomyocytes infected with Cre and N-cadherin-GFP (A) or Cre and N-cadherin-GFP-αE-catenin fusion adenoviruses (B–D). Orange arrowhead marks the FRAP region at a cell–cell contact. (E–H) Plots of mean ± SD FRAP recovery fraction over 15 min. The mean is represented by a black circle, and the SD is shown as a black line. The data were fitted to a double-exponential curve (orange line). The numbers of FRAP regions measured for each fusion construct are listed in gray. FRAP data were collected from at least two independent infections for each fusion. (I) Summary of the mobile fraction (percentage) and recovery halftime (seconds). Scale bar is 5 μm in A–D.

FIGURE 6:. Vinculin recruitment is required to couple myofibrils to the AJ. Ncadfx/fx cardiomyocytes uninfected (A, B), infected with Cre (C, D), infected with Cre and Ncad-GFP (E, F), or infected with Cre and N-cadherin-GFP-αE-catenin fusion adenoviruses (G–L) were fixed and processed for immunostaining or thin-section TEM. (A, C, E, G, I, K) Immunofluorescence lower-magnification (40×) images of control and infected cardiomyocytes. Uninfected control (A) and Cre-infected (C) cardiomyocytes were stained for N-cadherin and F-actin. Ncad-GFP (E) and N-cadherin-GFP-αE-catenin fusion adenovirus-infected (G, I, K) cardiomyocytes were stained for F-actin. Individual and merged N-cadherin/GFP (green) and F-actin (magenta) channels shown. Images are maximum projections of 5 μm stacks. (B, D, F, H, J, L) Representative TEM image of a cell–cell contact from >60 images from at least three independent experiments. Cell–cell junctions (J) are highlighted in yellow, Z-discs (Z) are colored orange, and a terminal myofibril sarcomere (S) is defined by a double arrow line. In L, the arrowhead marks electron density along the cell–cell junction and the asterisks mark poorly organized F-actin. Scale bar is…

FIGURE 7:. Model for linking contractile actin to cardiomyocyte AJs. (1) The cadherin-catenin complex makes initial contact with F-actin through α-catenin. Mechanical force is required to promote strong binding of the αE-catenin ABD to F-actin. (2) Increased tension induces conformational changes in the central M-region of α-catenin to promote recruitment of vinculin and afadin. (3) Vinculin recruitment stabilizes the AJ-actin interface under mechanical load to promote myofibril coupling and mechanical continuity between cardiomyocytes. The role of afadin at cardiomyocyte AJs is not clear.