Dynamic Visualization of α-Catenin Reveals Rapid, Reversible Conformation Switching between Tension States

Abstract

Summary The cytosolic protein α–catenin is a postulated force-transducer at cadherin complexes [1]. The demonstration of force activation, identification of consequent downstream events in live cells, and development of tools to study these dynamic processes in living cells are central to elucidating the role of α–catenin in cellular mechanics and tissue function [2–10]. Here we demonstrate that α–catenin is a force-activatable mechano-transducer at cell-cell junctions, using an engineered α-catenin conformation sensor, based on fluorescence resonance energy transfer (FRET). This sensor reconstitutes α-catenin-dependent functions in α-catenin depleted cells, and recapitulates the behavior of the endogenous protein. Dynamic imaging of cells expressing the sensor demonstrated that α-catenin undergoes immediate, reversible conformational switching, in direct response to different mechanical perturbations of cadherin adhesions. Combined magnetic twisting cytometry with dynamic FRET imaging [11] revealed rapid, local conformational switching, upon the mechanical stimulation of specific cadherin bonds. At acutely stretched cell-cell junctions, the immediate, reversible conformational change further reveals that α-catenin behaves like an elastic spring in series with cadherin and actin. The force-dependent recruitment of vinculin—a principal α-catenin effector—to junctions requires the vinculin-binding-site of the α–catenin sensor [1, 12–16]. In cells, the relative rates of force-dependent α–catenin conformation switching and vinculin recruitment reveal that α–catenin activation and vinculin recruitment occur sequentially rather than in a concerted process, with vinculin accumulation being significantly slower. This engineered α-catenin sensor revealed that α–catenin is a reversible, stretch-activatable sensor that mechanically links cadherin complexes and actin, and is an indispensable player in cadherin-specific mechano-transduction at intercellular junctions.

Structural Determinants of the Mechanical Stability of α-Catenin

“…Its activation and recruitment to force-activated talin (and likely to ␣-catenin) involve phosphorylation and allosteric interactions with cytosolic binding partners (34,61,63,64). Conversely, recent experimental data demonstrated that force directly activates ␣-catenin (30,42), and the present simulations suggest that this occurs by a process involving salt-bridge disruption within the M region. Despite sharing a core structural motif, these proteins differ in their activation mechanisms, functional binding partners, and roles in force transduction.…”

“…As a force transducer in the mechanical chain between cadherins and the cytoskeleton, ␣-catenin is postulated to adopt an autoinhibited conformation in the absence of junctional tension, but mechanical stress alters the conformation to expose a cryptic binding site for the actin-binding protein, vinculin (18,30). At cadherin complexes, vinculin is an ␣-catenin effector that binds the exposed vinculin-binding site (VBS) 3 in the force-activated ␣-catenin conformation (15, 19, 20, 30 -32).…”

α-Catenin stabilises Cadherin–Catenin complexes and modulates actomyosin dynamics to allow pulsatile apical contraction

“…Vinculin is then recruited to adherens junctions and becomes associated with more actin filaments thus reinforcing cell-cell adhesion (Kim et al, 2015;le Duc et al, 2010;Yao et al, 2014;Yonemura et al, 2010). Thus, we decided to investigate whether Vinculin and α-Catenin also interacted during Drosophila embryogenesis.…”

“…Although biochemical studies had challenged the notion that the Cadherin-Catenin complex binds directly to F-actin Yamada et al, 2005), recent experimental findings using an optical trap assay have shown that strong and stable bonds between the Cadherin-Catenin complex and an actin filament form under force, probably requiring a conformational change of α-Catenin (Buckley et al, 2014). Force-dependent conformational changes in vertebrate αE-Catenin (also known as CTNNA1) regulate its binding to Vinculin, an actin-binding protein, and reinforce intercellular adhesion (Kim et al, 2015;le Duc et al, 2010;Yao et al, 2014;Yonemura et al, 2010). Moreover, using a fluorescence resonance energy transfer (FRET) tension sensor, it has been shown that the actomyosin cytoskeleton exerts tensile forces on E-Cadherin in an α-Catenin-dependent manner (Borghi et al, 2012).…”

E-cadherin-mediated force transduction signals regulate global cell mechanics

“…The rudiments of intercellular mechanotransduction mechanisms have been identified in only a few cases (Barry et al, 2014;Collins et al, 2012;Kim et al, 2015;le Duc et al, 2010;Tzima et al, 2005;Yonemura et al, 2010). E-cadherin complexes at epithelial intercellular junctions are force sensitive (Barry et al, 2014;le Duc et al, 2010;Thomas et al, 2013;Yonemura et al, 2010), and α-catenin is an identified force-transducing protein in these complexes (Yonemura et al, 2010).…”