The E-cadherin/catenins complex

Cadherins are calcium-dependent adhesion proteins involved in the formation of cell-cell contacts in various cell and tissue types and are part of a large multigene family. The classical cadherins are characterized by the presence of five extracellular cadherin repeats (EC domains), a single transmembrane span and a C-terminal cytoplasmic tail (Rudini and Dejana, 2008). They comprise over 20 isoforms and were originally named according to the tissue of their predominant expression, such as E-cadherin and VE-cadherin in epithelial and vascular endothelial cells, respectively, or N-cadherin in the nervous system (Rudini and Dejana, 2008).

E-cadherin is a central protein in the architecture of epithelial AJs, since all AJs contain cadherin-catenin clusters while only a subpopulation additionally have nectin (Indra et al., 2013; Rusu and Georgiou, 2020). E-cadherin is essential for the assembly but is dispensable for the maintenance of cell-cell contacts, since reducing E-cadherin expression by RNAi does not impair AJs and TJs neither cell polarity but disrupts the re-establishment of junctions after their disassembly (Capaldo and Macara, 2007). This central role of E-cadherin in epithelial morphogenesis is confirmed by the embryonic lethality of E-cadherin-KO mice due to failure in the formation of the trophectoderm (Larue et al., 1994; Ohsugi et al., 1997). Upon formation of a cell-cell contact, after nectin-mediated initial adhesion, cadherins cluster at the AJs and then spread laterally, strengthening the contact (Asakura et al., 1999; Adams et al., 1998). Thus, in polarized epithelial cells, E-cadherin is localized at the ZA, associated with a circumferential peri-junctional belt, and also along lateral cell-cell contacts, connected to a more amorphous actin network (Figure 5A,C) (Takeichi, 2014). Cadherins form dimers in cis in each plasma membrane and interact in trans with dimers from the neighboring cell through their extracellular EC domains, which undergo calcium-dependent conformational changes (Takeda et al., 1999;

Yap et al., 1997; Yoshida and Takeichi, 1982; Yoshida-Noro et al., 1984; Pokutta et al., 1994).

The cytoplasmic tail of E-cadherin binds to proteins which stabilize E-cadherin and connect it

to the actin and MT cytoskeletons (Figure 5B), acting in mechanotransduction, and which regulate intracellular signaling and gene transcription. This intracellular part of E-cadherin comprises juxtamembrane and catenin-binding domains, where p120-catenin and ß-catenin link, respectively (Yap et al., 1998; Ozawa et al., 1990; Aberle et al., 1994). ß-catenin interacts with the actin-binding protein α-catenin and thus bridges E-cadherin to actin cytoskeleton (Figure 5B) (Aberle et al., 1994; Rimm et al., 1995; Drees et al., 2005; Yamada et al., 2005;

Sakakibara et al., 2020). p120-catenin interaction stabilizes E-cadherin at AJs by masking the residues implicating in clathrin-mediated endocytosis and Hakai-dependent ubiquitination, thus preventing junction disassembly and stabilizing cell-cell adhesion (Yap et al., 1998;

Miyashita and Ozawa, 2007; Fujita et al., 2002; Troyanovsky et al., 2006; Davis et al., 2003;

Ishiyama et al., 2010). In addition, p120-catenin connects cadherins to MTs through PLEKHA7, which is in turn linked to nezha/CAMSAP3 (Figure 5B), further stabilizing E-cadherin at AJs (Meng et al., 2008). The central role of E-cadherin in cell-cell adhesion, and thus the formation and maintenance of epithelial versus mesenchymal phenotype, supports its function as tumor-suppressor. Accordingly, the loss of E-cadherin is relatively common in cancers of epithelial origin (Kourtidis et al., 2017a). However, E-cadherin can support tumor progression in some cancers, suggesting that E-cadherin-linked signaling processes are involved as well as adhesion-mediated collective cell migration (Rodriguez et al., 2012; Daulagala et al., 2019;

Friedl and Gilmour, 2009).

Four major catenins are localized at the ZA and along lateral contacts, similarly to E-cadherin (Figure 5A,C), and coordinate the dynamics and signaling of AJs. α-, ß- and γ-catenins were first identified as proteins present in E-cadherin immunoprecipitates (Ozawa et al., 1989;

Kemler and Ozawa, 1989). p120-catenin was subsequently identified based on sequence homology with ß-catenin (Reynolds et al., 1992). ß-, γ- and p120-catenins contain homologous armadillo repeats and directly bind E-cadherin, whereas α-catenin has a different structure, is indirectly linked to E-cadherin via ß-catenin and is an actin-binding protein (Rudini and Dejana, 2008).

p120-catenin binds the cytoplasmic juxtamembrane domain of E-cadherin and ensures its stability at AJs by preventing its endocytosis and degradation (Yap et al., 1998; Miyashita and Ozawa, 2007; Fujita et al., 2002; Troyanovsky et al., 2006; Davis et al., 2003; Ishiyama et al., 2010). p120-catenin also bridges E-cadherin to MTs through PLEKHA7 and nezha/CAMSAP3 (Figure 5B), further supporting AJ integrity (Meng et al., 2008). p120-catenin is additionally present in a cytoplasmic pool which interacts directly with MTs (Yanagisawa et al., 2004) and affects cell morphology and motility by regulating the activity of Rho GTPases and thus the organization of actin cytoskeleton (Noren et al., 2000). This regulation of Rho GTPases by p120-catenin also supports AJ assembly by antagonizing Rho and Rac signaling at cell-cell contacts (Wildenberg et al., 2006). In addition, unbound p120-catenin can translocate to the nucleus where it binds the transcription factor Kaiso and releases its repressor activity (Daniel and Reynolds, 1999; Kelly et al., 2004). The fundamental importance of p120-catenin is confirmed by the embryonic lethality of KO mice and strong defects in conditional KO epithelial tissues (Davis and Reynolds, 2006; Perez-Moreno et al., 2006; Smalley-Freed et al., 2010), as well as its dual role in cancers. Indeed, on the one hand, p120-catenin exerts as a tumor suppressor, through the stabilization of E-cadherin at AJs which supports cell-cell adhesion, and through its association with PLEKHA7 at the ZA, which is linked to a RNAi machinery that suppresses the expression of pro-tumorigenic factors (Kourtidis et al., 2013; Kourtidis et al., 2015a; Kourtidis et al., 2017b). On the other hand, a basolateral pool of p120-catenin which is tyrosine-phosphorylated by Src promotes anchorage-independent cell growth and acts thus as a tumor promoter (Kourtidis et al., 2015a; Kourtidis et al., 2015b).

ß-catenin is linked to the C-terminal cytoplasmic catenin-binding domain of E-cadherin via its central armadillo repeats and to the actin-binding protein α-catenin through its N-terminal region (Ozawa et al., 1990; Aberle et al., 1994). ß-catenin promotes cadherin-mediated cell-cell adhesion by participating in E-cadherin trafficking from the endoplasmic reticulum (ER) to the plasma membrane (Chen et al., 1999) and by bridging E-cadherin to the actin cytoskeleton through α-catenin (Figure 5B) (Rimm et al., 1995; Drees et al., 2005). Afadin reinforces this

bridging by binding to α-catenin complexed with ß-catenin, enhancing its F-actin-binding activity and eventually promoting the correct actomyosin organization at AJs (Figure 6) (Rimm et al., 1995; Drees et al., 2005; Yamada et al., 2005; Sluysmans et al., 2017; Sakakibara et al., 2020). Under normal conditions, the majority of ß-catenin is immobilized by E-cadherin at AJs, and the excess cytoplasmic unbound pool is phosphorylated, ubiquitinated and degraded (Nelson and Nusse, 2004; Valenta et al., 2012; MacDonald et al., 2009; Orford et al., 1997).

Wnt signaling inhibits this degradation and triggers the translocation of ß-catenin to the nucleus, where it associates with transcription factors to activate gene expression and regulate crucial cellular processes including proliferation and migration (Nelson and Nusse, 2004;

MacDonald et al., 2009). Imbalance in the distribution and signaling of ß-catenin often results in dysregulated growth connected to cancer and metastasis (Valenta et al., 2012; Shang et al., 2017), and its KO in mouse is embryonic lethal due to gastrulation defects (Haegel et al., 1995).

γ-catenin is homologous to ß-catenin and also binds to the C-terminal cytosolic catenin-binding domain of cadherins. Its role in cell-cell adhesion is mainly due to its high enrichment at desmosomes, even if it is mostly found at AJs in endothelial cells which are devoid of desmosomes (Knudsen and Wheelock, 1992; Rudini and Dejana, 2008; Aktary et al., 2017).

Unlike the other catenins, α-catenin is not directly linked to E-cadherin, but indirectly through ß-catenin. It ensures the connection of AJs to the actin circumferential belt by its binding and bundling capacity of actin filaments (Figure 5B) (Rimm et al., 1995; Drees et al., 2005; Yamada et al., 2005; Sakakibara et al., 2020). The observation that the binding of α-catenin to F-actin and ß-catenin was mutually exclusive in vitro has been recently solved by showing that the interaction of afadin with α-catenin in complex with ß-catenin enhances its F-actin-binding activity and eventually promotes the correct actomyosin organization at ZA (Drees et al., 2005;

Yamada et al., 2005; Sakakibara et al., 2020). The association of AJs to actomyosin cytoskeleton is crucial for their stabilization and the organization of the AJC, since epithelial cells deficient for α-catenin fail to adhere to each other and mice KO for the epithelial isoform die at embryonic stage (Watabe et al., 1994; Watabe-Uchida et al., 1998; Hirano et al., 1992;

Vasioukhin et al., 2001; Torres et al., 1997). Through its tension-dependent conformational change, α-catenin is also central in the mechanotransduction of forces exerted on cadherin-based junctions and on the actomyosin cytoskeleton, coupling the response to tension between neighboring cells, which is crucial for tissue morphogenesis, homeostasis and plasticity (Maitre et al., 2012; Ganz et al., 2006; Ladoux et al., 2010; le Duc et al., 2010;

Yonemura et al., 2010). Indeed, force induces a transient binding of the α-catenin–β-catenin–

E-cadherin complex to F-actin, and the conformationally open form of α-catenin binds to afadin, which increases the F-actin binding activity of α-catenin and stabilizes the connection to actin cytoskeleton (Figure 6) (Buckley et al., 2014; Ishiyama et al., 2018; Sakakibara et al., 2020).

In addition, the tension-dependent conformational change of α-catenin ensures mechanosensing and dynamically strengthens the link of E-cadherin complexes to actin cytoskeleton via the recruitment of vinculin and EPLIN, which reinforce α-catenin open conformation and bundle and stabilize actin filaments (Figure 6) (Yonemura et al., 2010; Abe and Takeichi, 2008; Taguchi et al., 2011; Sluysmans et al., 2017).

Figure 6. F-actin linkage and mechanotransduction at cadherin/catenins complex of epithelial cells.

Tension induces transient binding of the α-catenin–ß-catenin–E-cadherin complex to F-actin. Afadin binds to the conformationally open α-catenin and increases its F-actin-binding activity, stabilizing the linkage to actin cytoskeleton. The tension-induced change in α-catenin conformation unmasks its vinculin-binding domain, allowing the recruitment of vinculin and EPLIN to reinforce the linkage to F-actin.