Sustained α-catenin Activation at E-cadherin Junctions in the Absence of Mechanical Force

Abstract

Mechanotransduction at E-cadherin junctions has been postulated to be mediated in part by a force-dependent conformational activation of α-catenin. Activation of α-catenin allows it to interact with vinculin in addition to F-actin, resulting in a strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions were formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central assemblies depending on their relative location at the cell-bilayer adhesion. Whereas F-actin, vinculin, and phosphorylated myosin light chain associated only with the peripheral assemblies, activated α-catenin was present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes by confining the movement of bilayer-bound E-cadherin on nanopatterned substrates reduced the levels of activated α-catenin. Taken together, these results indicate that although the initial activation of α-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state.

Figure 1

E-cadherin adhesion on a supported lipid bilayer. (A) Schematic representation of a cell forming an adhesion on an E-cad-ECD-functionalized bilayer. (B) Graph showing a comparison of the number of cells adhering to control (without E-cad-ECD) versus E-cad-ECD-functionalized bilayers. The data shown are from multiple independent experiments and the p-value was obtained from an unpaired t-test. (C) Bright-field (BF) and TIRF microscopy images of E-cadherin in cells seeded on control (without E-cad-ECD) or on E-cad-ECD-functionalized bilayers in the absence and presence of 2 mM CaCl₂, respectively. Note the extensive zone of cellular E-cadherin enrichment on E-cadherin bilayers in the presence of CaCl₂. (D) Plot showing multiple individual radial E-cadherin intensity profiles obtained from confocal images of adhesions formed by cells on E-cad-ECD-functionalized bilayers, revealing a peak at the cell periphery and random distribution of E-cadherin clusters within the adhesions. Original individual intensity profiles were smoothed with the average of three data points. The lower x axis panel shows the average cell contact radius, revealing micron-scale features of E-cadherin in individual radial profiles. The dotted black curve shows the average profile of all the 15 cells shown individually. (E) Schematic of a micropatterned substrate containing 2 μm discs of supported lipid bilayers functionalized with E-cad-ECD on a PEG surface. BF, epifluorescence (Epi), and TIRF images of an adhering cell on the micropatterned substrate. Bilayer discs are shown in red. Note that the enrichment of cellular E-cadherin coincides with regions of the substrate containing E-cad-ECD-functionalized bilayer discs. (F) BF, RICM, and TIRF images of an adhering cell on micropatterned substrate containing 2 μm discs of bilayers functionalized with E-cad-ECD, showing loss of RICM intensity due to interference at regions containing E-cadherin clusters. (G) TIRF and RICM images of a cell forming an adhesion on an E-cad-ECD bilayer, showing the addition of E-cadherin clusters formed by retracting filopodia, leading to the formation of large assemblies of E-cadherin at the adhesion. Scale bar, 5 μm.

Figure 2

Interaction between E-cadherin assemblies and the actin cytoskeleton. (A) BF and confocal images of E-cadherin and F-actin (phalloidin) in cells adhering to E-cad-ECD bilayers. A volume and orthoslice reconstruction of the actin cytoskeleton and E-cadherin assemblies, performed using z-scan confocal imaging of the cell, shows a remodeled actin cytoskeleton. Note that the peripheral E-cadherin assemblies associate with F-actin, but the central assemblies do not (although intermittent patches of F-actin are present in the central part of the cell). (B and C) BF and confocal images of E-cadherin and either β-catenin (B) or α-catenin (C) in cells adhering to E-cad-ECD bilayers, showing colocalization of E-cadherin with β-catenin (B) or α-catenin (C), respectively. (D) Graph showing Pearson’s R-value of E-cadherin colocalization with F-actin, β-catenin, and α-catenin in the peripheral and central clusters. Scale bar, 5 μm.

Figure 3

α-catenin is sustained in the active conformation irrespective of actomyosin tension. (A–D) BF and confocal images of cells forming junctions on E-cad-ECD-functionalized bilayers stained for stained for vinculin (A) or phosphorylated myosin light chain (B), and cells expressing the vinculin-head domain fused to mCherry (vinc-head) (C) and stained for the open conformation of α-catenin using the α18 antibody (11) (D). (G–J) BF and confocal images of 50 μM Y-27632-treated cells stained for vinculin (G) or phosphorylated myosin light chain (H), and cells expressing the vinculin-head domain fused to mCherry (vinc-head) (I) and stained for the open conformation of α-catenin using the α18 antibody (J) (11). (E and K) BF and confocal images of control (E) and Y-27632-treated (K) cells adhering to E-cad-ECD bilayers. Cells were stained for total α-catenin using an antibody that binds to the C-terminus of the protein independently of its conformation, and for the conformationally activated α-catenin using the α18 antibody. The lower-right panels in (E) and (K) are ratiometric images of α18 and total α-catenin staining for the respective cells. (F and L) BF and confocal images of cells forming junctions in monolayers and stained for total α-catenin and the conformationally activated α-catenin visualized with α18 antibody in control (F) or 50 μm Y-27632-treated (L) cells. (M) Graph showing the ratio of pMLC and E-cadherin-GFP intensities at the cell periphery in control and Y-27632-treated cells, revealing a reduction in the actomyosin tension upon Y-27632 treatment of the cells. (N) Graph showing the ratio of α18 and total α-catenin staining intensities in control and Y-27632-treated cells on bilayers or monolayer cultures. No significant differences are observed in the levels of activated α-catenin in control and Y-27632-treated cells. Scale bar, 5 μm.

Figure 4

Nucleation and micron-scale assembly of E-cadherin clusters regulate the conformational activation of α-catenin. (A) Schematic representation of a nanopatterned supported lipid bilayer, BF and confocal images of cells adhering to the respective nanopatterned bilayers stained for total α-catenin, the open conformation of α-catenin using α18 antibody (11), and the ratio of α18 and total α-catenin intensities. Scale bar, 5 μm. (B) Graph showing the ratio of α18 and total α-catenin intensities (mean ± SD) obtained from multiple cells from a representative experiment. (C) Schematic representation of the conformational regulation of α-catenin by micron-scale assembly of E-cadherin clusters. A cell seeded on an E-cad-ECD-functionalized bilayer clusters E-cadherin by retracting filopodia. α-catenin is activated during the process of assembly of micron-scale E-cadherin clusters due to its association with the actin cytoskeleton. Once activated, α-catenin stays in that form even in the absence of its association with actin cytoskeleton as seen with the central E-cadherin assemblies, or in the absence of mechanical force as seen with the Y-27623 treatment.