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 a-catenin. Activation of a-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 a-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 a-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 a-catenin. Taken together, these results indicate that although the initial activation of a-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining a-catenin in the active state.

Supported lipid bilayer platforms to probe cell mechanobiology

“…Low diffusivity promoted clustering and active nucleation, allowing junction formation to proceed. Within junctions, FRAP revealed that cadherins had low turnover and instead were immobilized within stable junctions [27]. Surprisingly, mechanical resistance alone was insufficient to form adherens junctions.…”

“…SLBs with reduced fluidity by the addition of high density poly-His E-cadherin ectodomain also supported AJ formation and were used to study α-catenin mechanobiology in cadherin-mediated adhesions [27]. Cells spread on these SLBs exhibited two populations of cadherins that clustered during filapodial retraction: AJs at the cell periphery and cadherins loosely clustered in “central adhesions” underneath the cell.…”

“…Cells spread on SLBs with widely spaced chromium grids exhibited normal cluster formation, but spreading and cluster formation by filapodia, along with α18 binding, was reduced on narrow grids (Figure 6E). Thus, α-catenin activation required mechanotransduction during cell spreading and retraction to activate, but sustained forces were not necessary for it to maintain its open conformation in cadherin clusters [27]. …”

“…Recently, several studies have attempted to bridge this gap by using supported lipid bilayer (SLB) technologies to spatially control the generation of mechanical forces [22–27]. SLBs are biomimetic phospholipid membranes that self-assemble on planar glass substrates (Figure 1A).…”

Techniques to stimulate and interrogate cell–cell adhesion mechanics

“…In a recent study, the desmosome/IF linkage at cell–cell junctions has also been shown to regulate not only traction forces at cell–ECM adhesions, but also modulate forces at cell–cell junctions [21]. Using a combined approach of micropost arrays and AFM, epithelial cells with genetic mutation of a desmosome/IF linker molecule, DP, which lacks the IF-binding domain, exhibited significantly reduced traction forces and potentially increased tension or conformational changes in α -catenin of AJs [21,116]. This evidence lends support to the notion that abundant molecular communication exists between the two mechanically active junctional complexes [117].…”