The zonula adherens matura redefines the apical junction of intestinal epithelia

Abstract

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction (zonula occludens), the zonula adherens (ZA), and the macula adherens. The prevailing dogma is that at the ZA, E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura. We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia.

Keywords: actin cytoskeleton; adhesive complexes; epithelial cells; small intestine; zonula adherens.

Fig. 1.

Segregation of adhesive complexes and their relative position compared to the F-actin belt in human ileum biopsies. (A) Side-view images of junctional complexes proteins (magenta) and F-actin (green). (B) Quantification of the position of adhesive complexes with respect to the F-actin belt on the apico-basal axis. The distributions represent the average intensity of a given protein labeling (plain color) and the average added with their SD (lighter color). Bars on distribution indicate the maxima of the distributions. Methodological details are given in the Materials and Methods. (C) Segregation of occludin (tight junction), afadin, and E-cad. (Scale bars 1 µm.) Details of replicates are given in SI Appendix, Table S1.

Fig. 2.

Segregation of adhesive complexes and their relative position compared to the F-actin belt in Caco-2 cells. (A) Side-view images of junctional proteins (magenta) and F-actin (green). (B) Quantification of the position of adhesive complexes with respect to the F-actin belt on the apico-basal axis. The distributions represent the average intensity of a given protein labeling (plain color) and the average added with their SD (lighter color). Bars on distribution indicate the maxima of the distributions. Methodological details are given in the Materials and Methods. (C) Segregation of the tight junction (ZO-1), afadin, and β-catenin. (D) Side-view images of apical junctions comparing the tight junction (ZO-1) position to the position of E-cad, F-actin, and afadin at 6, 9, and 14 d. At 6 and 9 d, most of the F-actin signal is concentrated in microvilli. (E) Quantification of the position of the previous proteins with respect to ZO-1 on the apico-basal axis at 6, 9, and 14 d. (F) Afadin (magenta) and β-catenin (green) segregate as cells mature. (Scale bars 1 µm.) Details of replicates are given in SI Appendix, Table S2.

Fig. 3.

Effect of the afadin KO on junctional proteins organization in Caco-2 cells. (A, Left) side-view images of E-cad, afadin, and F-actin (green) and the tight junction (ZO-1, magenta). (Scale bars 1 µm.) (Right) quantification of the position of the previous proteins with respect to ZO-1 on the apico-basal axis. The distributions represent the average intensity of a given protein labeling (plain color) and the average added with their SD (lighter color). (B, Left) top-view images of F-actin (green) and afadin (magenta) at the apical surface of wild-type and afadin-KO Caco-2 cell. (Bottom row) zooms of framed areas. (Scale bars, Top rows 5 µm, Bottom row 1 µm.) (Right) quantification of the position of F-actin perpendicular to the junction in the apical plane. Details of replicates are given in SI Appendix, Table S3. Cells were grown for 14 d. (C) Model of the enterocyte apical junction in afadin KO. Actin filaments in the terminal web and microvilli are not represented for clarity.

Fig. 4.

Effect of the afadin KO on the apical mechanics in Caco-2 cells. (A) Shapes of WT/afadin-KO cells junctions. Left, apical junctions of mixed WT and afadin-KO cells. (Top Left) F-actin (green), afadin (magenta), (Bottom Left) afadin (gray). (Bottom Left) areas are generated from the junction that would be hypothetically straight and the actual junction. When the area leans toward a WT cell, it is colored in cyan, whereas when it leans toward an afadin-KO cell, it is colored in red. (Top Right) distribution of the previously defined areas; positive areas are for areas toward WT cells and negative areas toward afadin-KO cells. (Scale bars 10 µm.) (Bottom Right) shapes of individual junctions; shapes toward WT cells in cyan, and afadin-KO cells in red. (B, Left) examples of terminal web laser ablation in WT and afadin-KO cells expressing GFP-occludin grown for 14 d. Location of ablation in light purple. The junction at t = 0 is represented by a full line (kept on all images), and the one at later time points is represented by a dashed line. (Scale bars 5 µm.) Movies of these examples are given in Movies S1 and S2. (Top Right) quantification of the evolution of the normalized apical surface area over time after ablation (t = 0) in WT (cyan) and afadin-KO (red) cells. Thin line, individual cell, thick line averaged over one cell type. For normalization, all cell areas are scaled to 1 at t = 0. (Bottom Right) relative value of initial area spreading measured as the relative area difference over time between t = 0 and t = 2.6 s. Details of replicates are given in SI Appendix, Table S4.

Fig. 5.

Model of the immature and mature enterocyte apical junction. Actin filaments in the terminal web and microvilli are not represented for clarity.