α-catenin, vinculin, and F-actin in strengthening E-cadherin cell-cell adhesions and mechanosensing.

Classical cadherins play a crucial role in establishing intercellular adhesion, regulating cortical tension, and maintaining mechanical coupling between cells. The mechanosensitive regulation of intercellular adhesion strengthening depends on the recruitment of adhesion complexes at adhesion sites and their anchoring to the actin cytoskeleton. Thus, the molecular mechanisms coupling cadherin-associated complexes to the actin cytoskeleton are actively being studied, with a particular focus on α-catenin and vinculin. We have recently addressed the role of these proteins by analyzing the consequences of their depletion and the expression of α-catenin mutants in the formation and strengthening of cadherin-mediated adhesions. We have used the dual pipette assay to measure the forces required to separate cell doublets formed in suspension. In this commentary, we briefly summarize the current knowledge on the role of α-catenin and vinculin in cadherin-actin cytoskeletal interactions. These data shed light on the tension-dependent contribution of α-catenin and vinculin in a mechanoresponsive complex that promotes the connection between cadherin and the actin cytoskeleton and their requirement in the development of adhesion strengthening.

Forming and Strengthening E-Cadherin Cell–Cell Adhesions

E-cadherin is a calcium-dependent cell adhesion protein that controls interactions between epithelial cells. This protein is the prototypical classical cadherin composed of an extracellular region (ecto-domain) carrying five “cadherin repeats”, a transmembrane domain, and a cytoplasmic region. Classical cadherins form one of the six clusters evident in the phylogenetic tree of the superfamily of cadherins. This tree comprises several hundred proteins across 30 species characterized by the presence of at least two cadherin repeats., Classical cadherins, herein referred to as “cadherins”, play crucial roles during morphogenesis, and are involved in epithelial-mesenchymal transition, epithelial sheet maintenance, cell migration, tissue rearrangement, cell sorting, and synaptogenesis. Their functional deregulation leads to several pathologies including various cancers, neuropsychiatric disorders and skin defects.-

In epithelia, junctional, and extra-junctional E-cadherin molecules connect neighboring cells, make a bridge between their cortical actin cytoskeleton and contribute to mechanical coupling within cells and mechanical resistance of epithelia to stretch. The ecto-domain of E-cadherin expressed at the cell–cell surface is responsible for homophilic interactions. Despite interacting in trans, E-cadherin ectodomains are actually thought to cluster by cis-interaction. Upon these ecto-domain interactions, the conserved cytoplasmic domain of E-cadherin recruits partners that contribute to its connection to the actin cytoskeleton, leading to the formation of adhesion plaques that mature in adherens junctions in epithelia. The degree of growth or maturation of these plaques, which is a mechanosensitive process, is thought to be related to the strength of the intercellular contacts.- In addition, cadherin-mediated adhesion activates signaling cascades that regulate gene expression and cytoskeleton remodeling.- However, coupling of the cadherin tail to the actin cytoskeleton at the cell surface remains the major factor contributing to junction stabilization (reviewed in ref. 21). The regulation of this coupling is also required for cell–cell adhesion strengthening, collective cell migration, and tissue rearrangement.

The molecular interactions involved in E-cadherin-based adhesion strengthening and mechanosensing are studied mostly in cultured cells but also in vivo. Cadherin–actin coupling is not direct and requires interactions with cytoplasmic partners. The binding of the protein β-catenin at the catenin-binding domain of the cadherin tail is essential but not sufficient for cadherin-based adhesion. β-catenin recruits the vinculin-related protein, α-catenin, a multi-domain F-actin-binding protein with homology to vinculin crucial for strong cell–cell adhesion., It was initially proposed that the cadherin/β-catenin complex is already assembled at the reticulum surface and exported at the cell membrane, and then associated to α-catenin, which directly binds the complex to F-actin. Later, biochemical and structural studies argued for a more complex sequence of molecular interactions and the participation of additional partners such as vinculin, to the extent that α-catenin can interact with various partners and recruit them at cell–cell adhesion sites, in addition to its binding to F-actin (see below). However, very recent functional in vivo studies in Drosophila have re-established the tripartite cadherin/β-catenin/α-catenin complex as the central unit of cadherin adhesions, leaving a central open question as to the mode of association of this unit to the actin cytoskeleton. In vertebrates, two isoforms of α-catenin coexist, αE- and αN-catenins, encoded by two separate genes.

F-Actin Binding Proteins α-Catenin and Vinculin

Vinculin is a scaffolding protein recruited at integrin-associated complexes that contributes to mechanotransduction at cell–matrix adhesions. It is composed mostly of helical bundles that are organized toward a globular head at its N-terminus comprising four vinculin-homology (VH) domains that carry binding sites for talin, α-actinin, IpaA, α-catenin, and β-catenin. A short linker connects the vinculin head to the rod-shaped tail domain (VH5) located at the C-terminus, which carries binding sites regulating vinculin binding to actin and cytoskeletal remodeling proteins, including VASP, vinexin, ponsin, Arp2/3, as well for binding of PKC, paxillin, PiP2, and F-actin (reviewed in refs. 32 and 33). Vinculin exists in an auto-inhibitory form where the head and tail domains interact with high affinity. In this closed conformation state, vinculin cannot bind to its partners or to F-actin. The activation of vinculin by talin or its additional partners in a combinatorial activation model disrupts this auto-inhibitory interaction and exposes the head and tail domains for their binding to F-actin.- This protein is also recruited at cell–cell adhesion sites.

α-catenins are members of the vinculin family of proteins, characterized by three successive VH domains from its N- to C-terminus., The VH1 carries the β-catenin binding site, and a homodimerization site partly overlapping with the β-catenin, ajuba, and spectrin binding sites. The VH2 is subdivided into two parts: VH2a and VH2b. VH2a carries vinculin-, formin-, and α-actinin-binding sites whereas VH2b comprises a modulation domain that overlaps with the afadin-binding site. Finally, the VH3 domain interacts with eplin, ZO-1, and F-actin. Each of these domains is structured in α-helical bundles, as in the vinculin molecule. In vitro, purified αE-catenin is mostly monomeric but can homodimerize. Only the monomeric form can bind to E-cadherin/β-catenin complexes, likely because the dimerization domain and the β-catenin-binding site partly overlap. In contrast, αE-catenin homodimers bind to F-actin with high affinity., Moreover, a recent study analyzing the crystal structure of full-length αE-catenin revealed that the αE-catenin dimer is asymmetric and that β-catenin binding to αE-catenin hinders the recognition of F-actin at the C-terminal site of αE-catenin.

Thus, the tripartite cadherin/β-catenin/αE-catenin complex initially thought to directly connect cadherin to actin appears insufficient to tether cadherins to F-actin, even transiently. The interaction of α-catenin with actin-binding proteins such as vinculin and eplin could fulfill the function to connect the cadherin–catenin complex to F-actin.,, Alternatively, it was proposed that the transient dissociation of αE-catenin from the tripartite complex induces a local increase in monomers that favors the formation of homodimers in the vicinity of the cadherin-mediated adhesion sites, the latter negatively regulating Arp2/3 complex-dependent actin assembly and promoting actin bundling., This assumption on the modulation of αE-catenin monomer vs. dimer concentration near adhesion sites was tested using a mathematical modeling based on experimental data. The findings suggested that the local increase in homodimers near cell–cell adhesion sites depends on the transient homodimerization of αE-catenin at the cadherin/β-catenin/αE-catenin complex. However, this does not provide any molecular cues for the tethering of cadherin-associated complexes to F-actin. Moreover, it has been recently shown that while neither C. elegans α-catenin nor vertebrate αN-catenin can assemble into homodimers, both proteins can sustain adherens junction assembly in a similar tripartite cadherin/β-catenin/α-catenin complex., These findings suggest that both functions of αE-catenin (i.e., tethering the tripartite adhesion complex and regulating actin dynamics) could be uncoupled.

Vinculin only recently became an important candidate in asserting the link between cadherin complexes and F-actin. Biochemical and crystal structure analyses of the interaction of the VH2a domain of αE-catenin with the VH1 domain of vinculin revealed that an αE-catenin–vinculin complex can form and bind to F-actin by the C-terminal actin binding domains of either protein.,, The binding of α-catenin on vinculin, playing the role of talin binding, may induce the head-tail opening of the molecule, increasing its affinity for F-actin. These studies further reported the existence of a vinculin–α-catenin heterotetramer, at least in vitro, which may contain a total of four functional binding sites for F-actin. As such, αE-catenin may exist within cells in at least three forms (monomeric, dimeric, and heterotetrameric) that could theoretically be diffusive or bound to cadherin–catenin complexes or F-actin with multiple valences. Whether this tetramer or the αE-catenin–vinculin heterodimers are bound to F-actin, associated to cadherin complexes, or both is unknown so far. A demonstration of the involvement of these F-actin adaptors in cadherin adhesion strengthening in cellulo have been hampered, in part, because α-catenin and vinculin also play biological roles unrelated to cadherin-based adhesions.

α-Catenin and Vinculin in Mechanosensing and Strengthening of Intercellular Adhesions

The formation of cadherin adhesions depends on the environmental stiffness. This has been previously demonstrated by culturing C2 myoblast cells on polyacrylamide gels or on flexible polydimethylsiloxane (PDMS) pillars coated with Ncad-Fc. However, only on rigid surfaces do cells form cadherin adhesions connected to actin cables that transmit myosin II-generated forces. Cadherin adhesion formation following increased environmental stiffness correlates with an increase in cell traction forces at these sites, indicating that cadherins contribute to the distribution of intercellular contractility through a myosin II-dependent mechanism., This process shares similarities with the mechanism of mechanosensing described for integrin-based adhesions, which involve acto-myosin cables and mechanosensors located at the interface between adhesion receptors and the cytoskeleton. Borghi and coworkers showed recently that the tension transmitted to cadherin-based adhesion through the actin cytoskeleton requires α-catenin. Independently, α-catenin was proposed to act as an acto-myosin-dependent mechanotransducer at adherens junctions. Structural and biochemical analyses of the αE-catenin VH2 domain suggest that this domain may switch between a closed and open conformation in a force-dependent manner. In a closed conformation, the vinculin-binding domain is masked by the modulation domain, whereas in open conformation, this site is accessible for interaction with vinculin.,, Furthermore, vinculin recruitment at adherens junctions is inhibited by blebbistatin, suggesting that the α-catenin–vinculin interaction is increased upon force at adhesion sites., Thus, vinculin may contribute to cadherin-based mechanosensing and junctional remodeling.- However, there has been no direct demonstration of the contribution of the force-dependent conformational switch of α-catenin or of the recruitment of vinculin in mechanosensitive contact reinforcement at intercellular junctions since this point has been mostly investigated in the cases of cell–cell junctions relaxation using myosin II inhibition- or endothelial junction remodeling using thrombin treatment.

In a recent study, we directly analyzed the effect of the force exerted at cell–cell adhesions and showed that stretching of cell doublets increases vinculin recruitment at intercellular adhesion sites and induces a change of αE-catenin from a closed to an open conformation, as revealed by the recognition by a specific antibody directed against an epitope accessible only in the open conformation. This finding points to a force-driven, α-catenin-dependent mechanism for the recruitment of vinculin at cadherin-based adhesions. Furthermore we directly addressed the roles of αE-catenin and vinculin in cadherin-based intercellular adhesion strength using the dual pipette assay. In this assay, the force required to separate cell doublets is used as a read-out of adhesion strength. We have shown in earlier studies a time-dependent adhesion strengthening that depends on cadherin expression levels, actin cytoskeleton dynamics, and cadherin–actin coupling., Indeed, cytoplasmic domain-deleted E-cadherin does not allow cell–cell adhesion strengthening, whereas a chimeric E-cadherin–αE-catenin can rescue this process as efficiently as wild-type cadherin. In addition, defective adhesions were described in α-catenin-depleted MDCK or CHO cells, and in DLD-1 cell variant (R2/7) lacking this protein., Other studies showed that vinculin depletion produces defective adhesion in F9 and MDCK cells., However, no one has tested whether αE-catenin and vinculin, as well as αE-catenin–vinculin interactions, were involved in cadherin adhesion strengthening. Thus, we further investigated the abilities of various αE-catenin mutants and fusion proteins to rescue E-cadherin adhesion strengthening. For this we used first α-catenin-depleted DLD-1-R2/7 cells that express E-cadherin but failed to strengthen their intercellular adhesions over time. We expressed in these cells the wild-type αE-catenin or truncated proteins lacking actin-binding binding domain (aa 1–670 αE-catenin) or both this domain and the modulation domain (aa 1–510 αE-catenin) or an αE-catenin mutated in its vinculin-binding site by inverting the charge of aa residues at 12 positions between aa 326 and 348. We also expressed membrane-targeted αE-catenin such as chimeric E-cadherin fused after its transmembrane domain with wild-type αE-catenin or to a αE-catenin lacking the VH1 domain. The defective adhesion strengthening in these cells was rescued by the expression of soluble or membrane-targeted αE-catenin (E-cadherin αE-catenin chimera), the latter bearing or not the VH1 domain. This indicated that the constitutive recruitment of α-catenin at the cadherin-tail could fulfill the α-catenin function in adhesion strength independent of the α-catenin homodimer regulation of the actin cytoskeleton, as confirmed since then in vivo in Drosophila embryo epithelial cells.

We further showed that αE-catenin mutant lacking both the modulation and actin-binding domains (aa 1–510 αE-catenin) rescued E-cadherin-mediated adhesion, the recruitment of vinculin at cadherin adhesions and allowed cells to spread on Ecad-Fc surfaces, while the mutant lacking only the actin-binding binding domain (aa 1–670 αE-catenin) failed to do so, reinforcing the requirement of binding of vinculin to an open form of αE-catenin to trigger effective cell contact formation. However, the 1–510 mutant failed to rescue strong adhesion strength; indicating that vinculin recruitment at those sites is not sufficient for mechanosensitivity adhesion strengthening. αE-catenin mutated in the vinculin-binding domain similarly triggered cell–cell contact formation but fails to rescue strong adhesion strength. Furthermore, the vinculin depletion in E-cadherin expression expressing S180 cells that also express α-catenin or the expression of αE-catenin modulation domain (aa 491–660 αEcat) also leads to reduced adhesion strength within cell doublets. Together, these findings demonstrate for the first time that vinculin recruitment at cadherin-based adhesions contributes to intercellular adhesion strengthening and supports the hypothesis that both α-catenin and vinculin are required for and cooperate in intercellular contact maturation and strengthening, although the exact temporal and spatial details of this molecular cooperation is not understand so far and in particular cannot be directly linked to the available structural data.

In conclusion, a large body of very recent work using various cellular models, biophysical approaches and in vivo studies illustrate that intercellular adhesion strengthening is dependent on α-catenin and vinculin and their cooperation to promote mechanoresponsive cadherin-actin linkage. However, the complexity of the interactome at cadherin-mediated adhesion sites suggests that many other players could also contribute to intercellular adhesion development. Indeed, the presence of the F-actin-binding protein, eplin, regulates mechanosensing at adherens junctions. Additional studies are still required to identify new catenin partners, and characterize their function and dynamics at those sites to help decipher the molecular mechanisms controlling adhesion development and mechanosensing at adherens junctions. Further investigations at the structural, cellular, and functional levels are also needed to understand the exact sequence of molecular interactions and conformational changes operating between the cadherin/β-catenin/α-catenin tripartite complex and vinculin and F-actin during cadherin adhesion formation, strengthening, and tension-dependent remodeling in cell collectives and tissues.