InteractiveFly: GeneBrief

α Catenin: Biological Overview | References

BIOLOGICAL OVERVIEW

The linkage of adherens junctions to the actin cytoskeleton is essential for cell adhesion. The contribution of the cadherin-catenin complex to the interaction between actin and the adherens junction remains an intensely investigated subject that centres on the function of α-catenin, which binds to cadherin through β-catenin and can bind F-actin directly or indirectly. This study delineates regions within Drosophila α-Catenin (α-Cat) that are important for adherens junction performance in static epithelia and dynamic morphogenetic processes. Moreover, whether persistent α-catenin-mediated physical linkage between cadherin and F-actin is crucial for cell adhesion is addressed, and the functions of α-catenin monomers and dimers at adherens junctions is characterized. The data support the view that monomeric α-catenin acts as an essential physical linker between the cadherin-β-catenin complex and the actin cytoskeleton, whereas α-catenin dimers are cytoplasmic and form an equilibrium with monomeric junctional α-catenin (Desai, 2013).

α-catenin is conserved across the eukaryotic kingdom, where it functions broadly in intercellular adhesion during development and differentiation. In Drosophila melanogaster, cell adhesion is disrupted when α-catenin contains a mutation in the binding site for Armadillo, which is the D. melanogastor homologue of β-catenin. Adherens junctions are also present in the nematode Caenorhabditis elegans, which expresses the homologues HMR-1 (cadherin), HMP-1 (α-catenin) and HMP-2 (β-catenin). In mice, there are three α-catenins and one close relative, which all share substantial amino-acid sequence identity: αE-catenin is most prevalent in epithelial tissues; αN-catenin is restricted to neural tissues; αT-catenin is expressed primarily in heart tissue; and α-catulin, which is an α-catenin-like protein, is ubiquitously expressed. A more distant relative is vinculin, which is ubiquitously expressed and localizes to both focal adhesions and adherens junctions (Kobielak, 20044; Desai, 2013 and references therein).

Adherens junctions and their core constituents, the classic cadherin adhesion molecules, contribute significantly to animal development and tissue homeostasis. Adherens junction defects can lead to various human pathologies, including cancer. Adherens junction function relies on the association of cadherins with the microtubule and actin cytoskeleton through their cytoplasmic binding partners, the catenins. Elucidating the function of α-catenin, which operates at the interface of the cadherin- β-catenin complex and F-actin, is a major goal in the field (Desai, 2013).

Studies on mammalian αE-catenin have given rise to two models for α-catenin function: the physical linkage and the allosteric regulation model. αE-catenin can bind both β-catenin and F-actin suggesting that it can physically link the cadherin- β-catenin complex directly to F-actin. This simple model lacks direct experimental support because a quaternary complex between cadherin, β-catenin, αE-catenin and F-actin could not be documented (Yamada, 2005). Complex formation with F-actin could be demonstrated in vitro only in the presence of EPLIN, one of several F-actin-associated proteins that bind to αE-catenin, such as vinculin, α-actinin, afadin, ZO-1 and formin (Maiden, 2011; Kobielak, 2004). Thus, a more complex physical linkage model poses that αE-catenin links the cadherin/β-catenin complex to F-actin indirectly by interacting with actin-binding proteins. A role for αE-catenin as a physical linker between cadherin and actin is consistent with the discovery that αE-catenin acts as a tension sensor that is responsive to actomyosin contraction at adherens junctions (Yonemura, 2010; le Duc, 2010; Taguchi, 2011; Desai, 2013 and references therein).

Alternatively, α-catenin was proposed to regulate actin organization to support adherens junction formation, rather than act as a physical linker (Yamada, 2005; Drees, 2005). αE-catenin binds β-catenin as a monomer but shows high affinity for F-actin only as a homodimer (see Drees, 2005). The β-catenin binding site and homodimerization domain of αE-catenin overlap, suggesting that it cannot interact with β-catenin and F-actin simultaneously. These findings precipitated the view that αE-catenin may act allosterically by binding β-catenin to increase its own local concentration at adherens junctions, which is required to promote αE-catenin dimerization after dissociation from β-catenin (Drees, 2005). This model does not adequately address how adherens junctions are physically linked to actin and resist tensile forces. One question that results from these contradictory models is whether α-catenin dimerization is critical for adherens junction function (Desai, 2013).

This study reports an in vivo structure-function analysis of Drosophila α-Catenin (α-Cat) to assess the roles of its domains in several developmental processes and to distinguish between the physical linkage and allosteric regulation models for α-catenin function (Desai, 2013).

The model that α-catenin acts as a physical linker between cadherin and the actin cytoskeleton seems strongly supported by the ability of cadherin- α-catenin chimaeras to substitute for the cadherin-catenin complex, both in tissue culture assays and during morphogenesis (see Sarpal, 2012). The analysis of these fusion proteins emphasizes that: α-catenin acts in the immediate sub-membranous space, excluding an essential cytosolic function suggested by other studies (Benjamin, 2010) in the tissues this laboratory has examined (Sarpal, 2012). Further, a dynamic interaction between α-catenin and the cadherin-β-catenin complex is not required to support normal adherens junctions. Although recent examples suggest that β-catenin modulation can contribute to the dynamic regulation of the cadherin-catenin complex in certain situations, this is not a basic requirement of adherens junction assembly and function. That cadherin- α-catenin chimaeras can replace the endogenous complex suggests instead that much of the regulation of cadherin-catenin complex function takes place at the interface between α-catenin and the actin cytoskeleton. Physical linkage of α-catenin to cadherin does not interfere with its function. This does not imply, however, that α-catenin normally needs to be physically linked to the cadherin-β-catenin complex to function. Indeed, the estimated dissociation constant (K_d) of the α-catenin β-catenin interaction (~1 µM) is much weaker than the cadherin-β-catenin interaction (Drees, 2005; Shapiro, 2009). The allosteric regulation model for α-catenin suggests that an α-catenin homodimer modulates actin organization through interference with the Arp2/3 complex at adherens junctions (Drees, 2005; Benjamin, 2010; Weis, 2006). Homodimerization and β-catenin binding require the same binding interface and are mutually exclusive. Cadherins can dimerize or cluster (Brasch, 2012) and could therefore promote α-catenin dimerization even in chimaeric proteins that lack the α-catenin dimerization domain (for example, DEcad::αCatΔVH1). Several membrane-bound regulators of actin organization exist, including WASp, indicating that α-catenin could retain its Arp2/3 regulating activity despite its covalent linkage to cadherin. To gain further evidence into the molecular mechanism of α-catenin function this study investigated whether α-catenin function at adherens junctions can be decoupled from β-catenin binding, whether monomeric α-catenin can support adherens junctions and whether α-catenin dimerization promotes or inhibits adherens junction function (Desai, 2013).

To address the first point αCatΔVH1, lacking the first vinculin-homology region (VH1), was fused to either BazOD (BazOD::αCatΔVH1) or Baz (Baz::αCatΔVH1), which recruited αCatΔVH1 to adherens junctions. αCatΔVH1 does not support adherens junctions alone, but did so when fused to DEcad. BazOD::αCatΔVH1 and Baz::αCatΔVH1 showed weak biochemical interactions with Arm and DEcad and little rescue of adherens junctions. These findings suggest that the physical link between α-catenin and β-catenin not only recruits α-catenin to adherens junctions, but needs to persist for normal adherens junction function. Both the physical linkage and allosteric regulation models propose that α-catenin interacts with the actin cytoskeleton at adherens junctions; however, they differ on whether binding of α-catenin to β-catenin is required to physically link cadherin and the actin cytoskeleton. The data argue for persistent physical linkage as a core requirement for α-catenin function. It was also found that localization of αCatΔVH1 to adherens junctions through fusion to Ed did not support adherens junction integrity, in contrast to DEcad::αCatΔVH1, suggesting that cadherins have distinct properties that are important for α-catenin function (Desai, 2013).

Similar to C. elegans (Kwiatkowski, 2010) and D. discoideum (Dickinson, 2011) α-catenin proteins, αN-catenin was found to be monomeric in solution. αN-catenin can functionally replace the Drosophila protein, which formed a large dimer fraction in solution similar to αE-catenin. These results indicate that monomeric α-catenin can support adherens junction function, and that the in vitro monomer/dimer ratio may not correlate with the in vivo function of α-catenins (Desai, 2013).

To address the relationship between α-catenin dimerization and adherens junction function, the effects were tested of enhanced α-catenin dimerization. α-Cat or αCatΔVH1 fusion to BazOD or Baz probably causes enhanced multimerization, including dimerization of α-Cat. Although these chimaeras localize to adherens junctions they show reduced interactions with Arm and perform poorly. Dimerization was also enhanced by removing the N-terminal 56 or 64 amino acids from αN-catenin and α-Cat, respectively. Similar to αEcatΔ57, αCatΔ64 interacted with β-catenin/Arm. However, these constructs performed poorly when compared with their respective full-length proteins, suggesting that α-catenin dimers are inactive in adhesion and may represent a cytoplasmic pool that forms a dynamic equilibrium with α-catenin monomers. Monomers are recruited to adherens junctions through their interaction with β-catenin/ Arm, which probably stabilizes monomeric α-catenin that links cadherin to the actin cytoskeleton (Desai, 2013).

αE-catenin operates as a tension sensor and mechanotransducer at adherens junctions, changing conformation in response to pulling forces exerted by actomyosin. To achieve this, α-catenin needs to be suspended between two anchor points, which could be cadherin- β-catenin and F-actin, consistent with the physical linkage model. However, αE-catenin homodimers contain two actin-binding domains and can bundle actin filaments, raising the possibility that actin filament sliding as a result of myosin activity could apply tension to α-catenin. As the allosteric regulation model poses that α-catenin homodimers form preferentially at adherens junctions (Drees, 2005: Weis, 2006), tension sensing could be restricted to adherens junctions even without α-catenin linking cadherin to actin. However, actomyosin-generated tension also applies to E-cadherin and depends on the presence of αE-catenin (Borghi, 2012), suggesting that at least part of the tension exerted on αE-catenin occurs when it physically links cadherin to the actin cytoskeleton. These data are complementary to the analysis of Drosophila α-Cat. Although it has not been possible to document a quartenary complex of the cadherin-catenin complex with F-actin, the data presented in this study are consistent with α-catenin physically linking cadherin to the actin cytoskeleton as a core requirement of α-catenin and cadherin-catenin complex function (Desai, 2013).

The results on the function of different regions within Drosophila α-Cat are in line with data from tissue culture studies on αE-catenin (Yonemura, 2010; Watabe-Uchida, 1998; Imamura, 1999; Ozawa, 1998). The Arm and actin-binding regions at the N and C termini of α-Cat, respectively, are essential for function. The central region of α-Cat enhances adherens junction stability but does not contribute to α-Cat recruitment to adherens junctions, which relies only on the VH1-dependent binding to Arm. The central region between the VH1 and VH3 domains includes multiple parts that make partly independent contributions to α-Cat function, most likely through interactions with other binding partners. If the central region is activated by actomyosin pulling forces (Yonemura, 2010: Taguchi, 2011), then the tension-mediated conformational change in α-Cat would be expected to facilitate multiple interactions (Desai, 2013).

Although α-catenins can bind directly to F-actin in vitro, whether this occurs in vivo remains unresolved. It is possible that interactions with F-actin are indirect and mediated through F-actin-binding proteins. α-catenin organizes a complex interface between cadherin and the actin cytoskeleton. Uncovering how the multiple interactions between α-catenin and actin-binding proteins such as vinculin, formin, afadin or EPLIN contribute to adherens junction regulation during morphogenesis remains a major challenge for future investigation (Desai, 2013).

RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion

Cdc42-GTP is required for apical domain formation in epithelial cells, where it recruits and activates the Par-6-aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically is unclear. This study used sequence analysis and 3D structural modeling to determine which Drosophila GTPase-activating proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the only high-probability Cdc42GAP required for polarity in the follicular epithelium. RhoGAP19D is recruited by α-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of precancerous breast lesions. Thus, RhoGAP19D couples lateral cadherin adhesion to the apical localization of active Cdc42, thereby suppressing epithelial invasion (Fic, 2021).

The form and function of epithelial cells depends on their polarization into distinct apical, lateral, and basal domains by conserved polarity factors. This polarity is then maintained by mutual antagonism between apical polarity factors such as atypical PKC (aPKC) and lateral factors such as Lethal (2) giant larvae (Lgl) and Par-1. While many aspects of the polarity machinery are now well understood, it is still unclear how the apical domain is initiated and what role cell division control protein 42 (Cdc42) plays in this process. Cdc42 was identified for its role in establishing polarity in budding yeast, where it targets cell growth to the bud tip by polarizing the actin cytoskeleton and exocytosis toward a single site. It has subsequently been found to function in the establishment of cell polarity in multiple contexts. For example, Cdc42 recruits and activates the anterior PAR complex to polarize the anterior-posterior axis in the Caenorhabditis elegans zygote and the apical-basal axis during the asymmetric divisions of Drosophila neural stem cells. Cdc42 also plays an essential role in the apical-basal polarization of epithelial cells, where it is required for apical domain formation. Cdc42 is active when bound to GTP, which changes its conformation to allow it to bind downstream effector proteins that control the cytoskeleton and membrane trafficking. An important Cdc42 effector in epithelial cells is the Par-6-aPKC complex. Par-6 binds directly to the switch 1 region of Cdc42 GTP through its semi-CRIB domain (Cdc42 and Rac interactive binding). This induces a change in the conformation of Par-6 that allows it to bind to the C-terminus of another key apical polarity factor, the transmembrane protein Crumbs, which triggers the activation of aPKC's kinase activity. As a result, active aPKC is anchored to the apical membrane, where it phosphorylates and excludes lateral factors, such as Lgl, Par-1, and Bazooka (Baz). In addition to this direct role in apical-basal polarity, Cdc42 also regulates the organization and activity of the apical cytoskeleton through effectors such as neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin polymerization, and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK; Genghis khan [Gek] in Drosophila), which phosphorylates the myosin regulatory light chain to activate contractility (Fic, 2021).

This crucial role of active Cdc42 in specifying the apical domain raises the question of how Cdc42-GTP itself is localized apically. In principle, this could involve activation by Cdc42 guanine nucleotide exchange factors (Cdc42GEFs) that are themselves apical or lateral inactivation by Cdc42GAPs. The Cdc42GEFs Tuba, intersectin 2, and Dbl3 have been implicated in activating Cdc42 in mammalian epithelia. Only Dbl3 localizes apical to tight junctions, however, as Tuba is cytoplasmic and enriched at tricellular junctions and intersectin 2 localizes to centrosomes. Thus, GEF activity may not be exclusively apical, suggesting that it is more important to inhibit Cdc42 laterally. Although nothing is known about the role of GAPs in restricting Cdc42 activity to the apical domain of epithelial cells, this mechanism plays an instructive role in establishing radial polarity in the blastomeres of the early C. elegans embryo. In this system, the Cdc42GAP PAC-1 is recruited by the cadherin adhesion complex to sites of cell-cell contact, thereby restricting active Cdc42 and its effector the Par-6-aPKC complex to the contact-free surface (Fic, 2021).

This study has analyzed the roles of Cdc42GAPs in epithelial polarity using the follicle cells that surround developing Drosophila egg chambers as a model system. By generating mutants in a number of candidate Cdc42GAPs, this study identified the Pac-1 orthologue, RhoGAP19D, as the GAP that restricts active Cdc42 to the apical domain. In the absence of RhoGAP19D, lateral Cdc42 activity leads to an expansion of the apical domain and a high frequency of epithelial invasion into the germline tissue, a phenotype that mimics the early steps of carcinoma formation (Fic, 2021).

In the absence of RhoGAP19D, both N-WASP and Gek are recruited to the lateral membrane, indicating that Cdc42 is ectopically activated there. This implies that RhoGAP19D is the major Cdc42GAP that represses Cdc42 laterally, because no other GAPs can compensate for its loss. This also suggests that the GEFs that activate Cdc42 are not restricted to the apical domain and can turn it on laterally once this repression is removed. This is consistent with the identification of multiple vertebrate GEFs with different localizations that contribute to apical Cdc42 activation. The current results therefore identify RhoGAP19D as a new lateral polarity factor. This leads to a revised network of polarity protein interactions in which RhoGAP19D functions as the third lateral factor that antagonizes the activity of apical factors, alongside Lgl, which inhibits aPKC, and Par-1, which excludes Baz/Par-3 (Fic, 2021).

The function of RhoGAP19D is very similar to that of its orthologue PAC-1, which inhibits Cdc42 at sites of cell contact in early C. elegans blastomeres to generate distinct apical and basolateral domains. Both RhoGAP19D and PAC-1 are recruited to the lateral domain by E-cadherin complexes, although the exact mechanism is slightly different. RhoGAP19D recruitment is strictly dependent on α-catenin, which links it through β-catenin to the E-cadherin cytoplasmic tail, whereas α-catenin (HMP-1) and p120-catenin (JAC-1) play partially redundant roles in recruiting PAC-1 to E-cadherin (HMR-1) in the worm. Nevertheless, in both cases, the recruitment of the Cdc42GAP translates the spatial cue provided by the localization of cadherin to sites of cell-cell contact into a polarity signal that distinguishes the lateral from the apical domain. Classic work on the establishment of polarity MDCK cells grown in suspension has revealed that the recruitment of cadherin (uvomorulin) to sites of cell-cell contact is the primary cue that drives the segregation of apical proteins from basolateral proteins. Furthermore, the expression of E-cadherin in unpolarized mesenchymal cells is sufficient to induce this segregation, although the mechanisms behind this process are only partially understood. The observation that RhoGAP19D directly links cadherin adhesion to the polarity system in epithelial cells extends the results of Klompstra (2015) in early blastomeres, strongly suggesting that PAC-1/RhoGAP19D plays an important role in the first steps in epithelial polarization (Fic, 2021).

Although PAC-1 and RhoGAP19D perform equivalent functions in early blastomeres and epithelial cells, there is one important difference between their mutant phenotypes. In pac-1 mutants, Par-6 and aPKC are mislocalized to the contacting surfaces of C. elegans blastomeres where Cdc42 is ectopically active. By contrast, Par-6 and aPKC are not mislocalized laterally in rhogap19d mutant Drosophila epithelial cells, even though lateral Cdc42-GTP does recruit two other Cdc42 effectors, N-WASP and Gek. Thus, lateral Cdc42 activity is sufficient to recruit Par-6/aPKC to the lateral domain in early blastomeres, but not in epithelial cells. Instead, it was observed that lateral Cdc42 activity in rhogap19d mutant follicle cells acts at a distance to expand the size of the apical domain. A likely explanation for this difference is the presence of Crumbs in epithelial cells. The interaction between Cdc42-GTP and Par-6 alters the conformation of Par-6 so that it can bind to Crumbs, which anchors the Par-6-aPKC complex to the apical membrane and activates aPKC's kinase activity. Although Par-6 presumably binds to Cdc42 laterally in rhogap19D mutants and undergoes the conformational change, it cannot be anchored laterally in the absence of Crumbs. This activated Par-6-aPKC complex can then diffuse until it is captured by Crumbs in the apical domain, thereby increasing apical aPKC activity, providing an explanation for why the apical domain expands in rhogap19d mutant cells. C. elegans has three Crumbs orthologues, but removal of all three simultaneously has no effect on viability or polarity. Thus, in contrast to Drosophila epithelial cells, C. elegans Crumbs proteins are not required for Par-6/aPKC localization and activation, suggesting that some other mechanism, such as Cdc42 binding, is sufficient to activate aPKC (Fic, 2021).

If the failure of active Cdc42 to recruit aPKC laterally in rhogap19d mutant cells is due to the absence of Crumbs in this region, there must be a mechanism to exclude Crumbs from the lateral domain. One proposed mechanism depends on Yurt (Moe and EPB41L5 in vertebrates), which is restricted to the lateral domain by aPKC and binds to Crumbs to antagonize its activity. However, no lateral recruitment of aPKC was observed in rhogap19d;yurt double-mutant cells. Thus, there must be some parallel mechanism that excludes Crumbs, Par-6, and/or aPKC from the lateral domain (Fic, 2021).

Although loss of RhoGAP19D only leads to a partial disruption of polarity, it causes the follicular epithelium to invade the adjacent germline tissue with 40% penetrance. This invasive behavior is not driven by an epithelial-to-mesenchymal transition, because the cells retain their apical adherens junctions and epithelial organization. Instead, the deformation of the epithelium seems to be driven by the combination of an increase in lateral contractility and an expansion of the apical domain, because reducing the dosage of Gek, which activates myosin II to drive the contractility, significantly reduces the frequency of this phenotype, as does halving the dosage of any of the apical polarity factors. The expansion of the apical domain makes the domain too long for the cells to adopt the lowest-energy conformation, giving them a tendency to become wedge shaped, which could drive the evagination. It is also possible that buckling of the epithelium contributes to invasion. Recent work has shown that epithelial monolayers under compressive stress and constrained by a rigid external scaffold have a tendency to buckle inward. The follicular cell layer is surrounded by an ECM that constrains the shape of the egg chamber and that should therefore resist expansion. In addition, the pulses of lateral contractility are likely to generate compressive stress because transiently reducing cell height while maintaining a constant volume will increase the cells' cross-sectional area, thereby exerting a pushing force on the neighboring cells. This compression coupled to the tendency to become wedge shaped due to apical expansion could therefore trigger the rare buckling events that initiate invasion. In support of this view, lateral contractility has been shown to drive the folding of the imaginal wing disc between the prospective hinge region and the pouch. This phenotype provides an example of how a partial disruption of polarity can induce cell shape changes that lead to major alterations in tissue morphogenesis (Fic, 2021).

The rhogap19d phenotype resembles the defects earliest observed in the development of ductal carcinoma in situ. In flat epithelial atypia (FEA), the ductal cells are still organized into an epithelial layer, but they display apical protrusions that are strongly labeled by the apical polarity factor Par-6. This suggests that the apical domain has expanded and bulges out of the cell, just as was observed in the rhogap19d mutant follicular cells. In the next stage, atypical ductal hyperplasia (ADH), the ductal cells start to invade the lumen of the duct while retaining aspects of normal apical-basal polarity. This again resembles the invasive phenotype of rhogap19d mutants, although overproliferation of the ductal cells probably also contributes to invasion in this case. Thus, these abnormalities, which can sometimes progress to ductal carcinoma in situ and breast cancer, mirror the effects of lateral Cdc42 activation. The RhoGAP19D human orthologues, ARHGAP21 and ARHGAP23, have been shown to bind directly to α-catenin and localize to cell-cell junctions. Furthermore, low expression of ARHGAP21 or ARHGAP23 correlates with reduced survival rates in several cancers of epithelial origin. It would therefore be interesting to determine whether these orthologues perform the same functions in epithelial polarity as RhoGAP19D and if their loss contributes to tumor development (Fic, 2021).

Recruitment of Jub by alpha-catenin promotes Yki activity and Drosophila wing growth

The Hippo signaling network controls organ growth through YAP family transcription factors, including the Drosophila Yorkie protein. YAP activity is responsive to both biochemical and biomechanical cues, with one key input being tension within the F-actin cytoskeleton. Several potential mechanisms for biomechanical regulation of YAP proteins have been described, including tension-dependent recruitment of Ajuba family proteins, which inhibit kinases that inactivate YAP proteins, to adherens junctions (AJ). This study investigated the mechanism by which the Drosophila Ajuba family protein, Ajuba LIM protein (Jub) is recruited to adherens junctions, and the contribution of this recruitment to the regulation of Yorkie. Alpha-catenin is identifed as the mechanotransducer responsible for tension-dependent recruitment of Jub by identifying a region of α-catenin that associates with Jub, and by identifying a region, which when deleted, allows constitutive, tension-independent recruitment of Jub. Increased Jub recruitment to alpha-catenin is associated with increased Yorkie activity and wing growth, even in the absence of increased cytoskeletal tension. These observations establish alpha-catenin as a multi-functional mechanotransducer and confirm Jub recruitment to alpha-catenin as a key contributor to biomechanical regulation of Hippo signaling (Alegot, 2019).

To evaluate the role of α-catenin in the recruitment of Jub to AJ, Jub localization was examined in wing discs isolated from animals expressing truncated forms of α-catenin. This was achieved by using RNAi to knock-down expression of endogenous α-catenin and expressing RNAi-insensitive Venus- or V5-tagged α-catenin transgenes under UAS-Gal4 control. Transgenes were expressed in posterior cells using an en-Gal4 driver, so that anterior cells could serve as a control for Jub localization. Full-length α-catenin expressed under UAS-Gal4 control rescued both the lethality associated with knockdown of α-catenin, and Jub localization. α-catenin has similarity to Vinculin in N-terminal, middle, and C-terminal regions termed VH1, VH2, and VH3. An initial series of constructs deleted regions of α-catenin centered around these Vinculin homology regions, as well as separate deletions of either the N-terminal or C- terminal half of VH2. Constructs lacking either the N- or C-terminus of α-catenin (ΔVH1 or ΔVH3) could not rescue the lethality associated with α-catenin knockdown. Thus, to enable visualization of the consequences of these deletions on Jub, conditional knockdown and expression of α-catenin was achieved using a temperature sensitive-allele of Gal80 (Gal80ts) that represses Gal4-driven expression at 18°C but not at 29°C. After a 30 hour shift to higher temperature, Jub was mostly lost from apical cell junctions. However, E-cadherin was also mostly lost, indicating that these regions of α-catenin are required to maintain AJ. This is consistent with the roles of F-actin and α-catenin in stabilizing AJ, as the VH1 region is required for association with β-catenin and thus localization to AJ, and the VH3 region is required for association with F-actin. The ΔVH1 and ΔVH3 constructs also did not themselves localize to junctions. The loss of Jub from cell junctions under these conditions is consistent with studies indicating its association with AJ, but does not provide specific information about its interactions with α-catenin (Alegot, 2019).

Conversely, deletions within the central region of α-catenin increased Jub recruitment to AJ. Deletion of the entire VH2 region increased Jub recruitment but was also associated with abnormal cell morphology and E-cadherin localization. However, two smaller deletions comprising the N- and C-terminal halves of the VH2 region (ΔVH2-N and ΔVH2-C) rescued knock-down of α-catenin, resulting in cells that appear to have normal localization of E-cadherin and α-catenin. The ΔVH2-C deletion also appears to have normal Jub localization. In contrast, the ΔVH2-N deletion is associated with increased Jub localization at AJ. Moreover, in cells with the ΔVH2-N deletion, Jub is distributed relatively uniformly around cell junctions, whereas in cells with wild-type α-catenin, Jub localization to junctions is often punctate. The ΔVH2-N deletion was not only associated with increased Jub recruitment when expressed in place of endogenous α-catenin; it could also promote Jub recruitment even when expressed in the presence of endogenous α-catenin. Thus, expression of this isoform dominantly increases Jub recruitment to AJ (Alegot, 2019).

To further investigate the influence of α-catenin on Jub, smaller deletion constructs were created. To aid in the design of these additional constructs, Phyre2 was used to predict the structure of Drosophila α-catenin, based on its sequence similarity to mammalian α-catenin proteins with experimentally-determined structures. Mammalian α-catenin consists largely of a series of α-helical bundles: two N-terminal 4-helix bundles (N1 and N2) that share one long α-helix; three central 4-helix bundles (M1, M2, and M3), and a C-terminal 5-helix bundle. These structural features are also predicted for Drosophila α-catenin (Alegot, 2019).

The ΔVH2-N deletion that increases Jub recruitment to AJ deletes both the M1 and M2 helical bundles. Thus, separate deletions were created of either the M1 or M2 bundles. Deletion of the M2 bundle (ΔM2) slightly increased Jub localization to AJ, but a much stronger increase in Jub binding was detected when the M1 bundle was deleted (ΔM1a or ΔM1b). Recruitment of Jub to AJ is normally promoted by cytoskeletal tension. To examine the possibility that M1 deletions increase Jub recruitment by increasing tension, levels were examined of junctional myosin (using a GFP-tagged myosin light chain) that correlate with junctional tension, but no difference was observed. F-actin levels were similarly unaffected. Staining was carried out specifically for the phosphorylated (activated) form of myosin regulatory light chain (pMLC) in discs expressing Jub:GFP. This revealed similar levels of pMLC in control cells and cells expressing α-catenin ΔM1b, whereas expression of rok RNAi or an activated form of Rok visibly decreased or increased, respectively, junctional levels of both pMLC and Jub. Since Jub associates with α-catenin, the observation that deletion of the M1 bundle increases Jub localization to AJ without increasing tension suggests that deletion of this region alters the structure of α-catenin in a way that makes a Jub-binding region more accessible. The M1 bundle might thus act as an 'inhibitory' region that prevents Jub binding, with this inhibition normally alleviated in wild-type α-catenin by a conformational change induced by high tension. Alternatively, deletion of the M1 bundle might destabilize a non-Jub binding conformation of α-catenin and thereby indirectly increase accessibility of a Jub-binding region (Alegot, 2019).

While both Jub and Vinculin exhibit tension-dependent association with α-catenin, the results suggest that their interactions are distinct. A region initially identified as inhibitory for Vinculin binding, corresponds to the M3 helical bundle and the linker between VH2 and VH3. Moreover, the M1 helical bundle includes the Vinculin binding interface. To directly compare the influence of α-catenin deletions on Jub and Vinculin binding, advantage was taken of a Drosophila genomic GFP-tagged Vinculin (Vinc:GFP). Both of the M1 deletions substantially reduced, but did not eliminate, Vinculin localization to AJ, which fits with observations that Vinculin interacts with sequences in the M1 bundle, but implies that additional mechanisms also contribute to Vinculin localization. Deletion of the M2 bundle increased Vinculin localization to AJ, consistent with observations that M2 participates in interactions that stabilize a conformation of α-catenin that is 'closed' with respect to Vinculin binding (Alegot, 2019).

If M1 deletions mimic the influence of cytoskeletal tension on the ability of α-catenin to bind to Jub, the increased recruitment of Jub in M1 deletion isoforms could occur even in the absence of tension. To test this, Jub localization was examined in flies expressing α-catenin constructs and with cytoskeletal tension decreased by RNAi of Rho-associated protein kinase (Rok), a promoter of myosin activity. In the presence of full-length α-catenin, rok RNAi decreases Jub recruitment to junctions. Conversely, in the presence of deletions that include the M1 bundle, Jub recruitment to junctions remains elevated. Quantitation of Jub at junctions, normalized to E-cadherin, revealed a decrease in Jub recruitment when tension is lowered even in cells expressing M1 deletions, but Jub recruitment nonetheless remains above that in control cells. Thus, these deletions recruit Jub even under low-tension conditions, which implicates α-catenin as the key mechanotransducer responsible for tension-dependent recruitment of Jub (Alegot, 2019).

Cytoskeletal tension promotes Yki activity, and Yki activity is suppressed by knockdown of Jub. However, other mechanisms by which tension might increase Yki activity have also been suggested, such as an influence on spectrins. The observation that deletions of the M1 bundle lead to increased Jub at AJ without increasing myosin identifies a condition under which it was possible to determine whether recruitment of Jub to junctions is sufficient to increase Yki activity, and thus distinguish the contribution of Jub from other potential influences of cytoskeletal tension (Alegot, 2019).

Yki activity was evaluated by examining ex-lacZ expression, which is a reporter for the Yki target gene expanded (ex). Expression of α-catenin with deletion of the M1 bundle increased ex-lacZ, whereas expression of full length α-catenin did not. Increased Yki activity is associated with increased accumulation of Yki in the nucleus, and nuclear levels of Yki were slightly increased in wing disc cells expressing M1 region deletions. To further examine the influence of the M1 deletion, adult wing size was examined, as wing growth is promoted by Yki activity. Expression of M1 deletion isoforms throughout the developing wing under nub-Gal4 control increased wing size as compared to wings expressing wild-type α-catenin. The increased Yki activity and wing growth in M1 deletion mutants are Jub-dependent, because they are reversed by RNAi of jub. As mutation or knock-down of Vinculin has only minor phenotypic consequences in Drosophila, the effects of M1 deletion on Yki activity and wing growth cannot be attributed to loss of Vinculin. Altogether, these observations establish that increased Jub recruitment to junctions can be sufficient to elevate Yki activity. This strongly supports the conclusion that Jub recruitment to AJ is a key component of the biomechanical response linking cytoskeletal tension to Yki activity (Alegot, 2019).

However, analysis of the larger ΔVH2N deletion suggests that the role of α-catenin in promoting Yki activity is more complex. Although expression of ΔVH2N α-catenin increased Jub recruitment at AJ, it did not detectably increase ex-lacZ expression or wing size. Similarly, even though deletion of the M2 helical bundle modestly increased Jub recruitment to AJ, it did not detectably increase ex-lacZ expression or wing size. Thus, it is inferred that additional features of α-catenin may contribute to Yki regulation (Alegot, 2019).

Jub requires α-catenin to localize to junctions. To map regions of α-catenin that mediate association with Jub, co-immunoprecipitation experiments were performed in Drosophila S2 cells expressing transfected constructs. Initially, co- immunoprecipitation of Jub with α-catenin full length, VH1, VH2, and VH3 region constructs was compared. Significant association of Jub and a VH1 region construct was detected. Conversely, association of Jub with full length, VH2, or VH3 constructs was weaker, and close to the non- specific background. The association with the VH1 region of α-catenin was not as strong as binding of Jub to Warts, which was included as a positive control. Association of Jub with the VH1 region is consistent with reports that the mammalian Ajuba protein can associate with an N- terminal fragment of α-catenin, corresponding roughly to VH1. The stronger association of Jub with a VH1 fragment as compared to full length α-catenin is consistent with normal binding depending upon junctional tension, as S2 cells are not epithelial, so it would be expected that the transfected α-catenin would be in a low-tension conformation. Moreover, co-immunoprecipitation experiments comparing M1 bundle deletion constructs to full length α-catenin revealed that M1 deletions significantly increased association of Jub with α-catenin (Alegot, 2019).

To further refine the Jub-binding region, the predicted Drosophila α-catenin structure was used to design constructs corresponding to the N1 or N2 helical bundles. No significant co-immunoprecipitation with the N1 bundle was detected, whereas co- immunoprecipitation with N2 was comparable to that for the VH1 region construct. Thus, it is inferred that the primary site of association of Jub with α-catenin is within N2. While the simplest model would be that Jub directly binds to this region of α-catenin, it remains possible that their association is mediated through other proteins. The N2 bundle is near the M1 bundle, but they do not directly contact each other in the predicted structure. Thus, the effect of M1 deletion on Jub binding may be an indirect consequence of a conformational change in α-catenin when this region is deleted, or reduced stability of a more closed conformation, rather than the M1 bundle directly obscuring a binding site within N2. Attempts were also mad to examine the consequences of loss of Jub recruitment to junctions by expressing α-catenin with N2 deleted in vivo. However, this protein failed to rescue the viability of wing disc cells expressing α-catenin RNAi, so this could not be examined. Moreover, when expressed in wild-type cells, the ΔN2 construct failed to localize to adherens junctions (Alegot, 2019).

These results establish key features of α-catenin mechanotransduction and tension- dependent regulation of Yki activity. They implicate α-catenin as a multi-functional mechanotransducer that associates with both Vinculin and Jub upon a tension-induced conformational change in α-catenin, but through distinct sites. The complete structure of the open conformation of α-catenin has not been determined, but it is inferred that it makes Jub- associating regions accessible. M1 deletions also make the Jub-associating region more accessible, but it's not clear whether they do so in a manner similar to, or distinct from, the effect of tension. The observation that M1 deletions increase Yki activity also supports the crucial role of Jub recruitment to AJ in promoting Yki activity. This does not exclude the possibility that of other consequences of cytoskeletal tension contribute to Yki regulation, but clearly implicates recruitment of Jub as a key factor (Alegot, 2019).

Role of alpha-Catenin and its mechanosensing properties in regulating Hippo/YAP-dependent tissue growth

alpha-Catenin is a key protein of adherens junctions (AJs) with mechanosensory properties. It also acts as a tumor suppressor that limits tissue growth. This study analyzed the function of Drosophila alpha-Catenin (alpha-Cat) in growth regulation of the wing epithelium. Different alpha-Cat levels led to a differential activation of Hippo/Yorkie or JNK signaling causing tissue overgrowth or degeneration, respectively. alpha-Cat can modulate Yorkie-dependent tissue growth through recruitment of Ajuba, a negative regulator of Hippo signaling to AJs but also through a mechanism independent of Ajuba recruitment to AJs. Both mechanosensory regions of alpha-Cat, the M region and the actin-binding domain (ABD), contribute to growth regulation. Whereas M is dispensable for alpha-Cat function in the wing, individual M domains (M1, M2, M3) have opposing effects on growth regulation. In particular, M limits Ajuba recruitment. Loss of M causes Ajuba hyper-recruitment to AJs, promoting tissue-tension independent overgrowth. Although M binds Vinculin, Vinculin is not responsible for this effect. Moreover, disruption of mechanosensing of the alpha-Cat ABD affects tissue growth, with enhanced actin interactions stabilizing junctions and leading to tissue overgrowth. Together, these findings indicate that alpha-Cat acts through multiple mechanisms to control tissue growth, including regulation of AJ stability, mechanosensitive Ajuba recruitment, and dynamic direct F-actin interactions (Sarpal, 2019).

One key factor that determines the impact of α-Cat on tissue growth, leading either to overgrowth or tissue degeneration, is the amount of α-Cat at AJs. Analysis of phenotypic defects resulting from the differential reduction of gene function revealed that Drosophila α-Cat is a negative regulator of tissue growth. Moderate α-Cat overexpression or moderate depletion of α-Cat or DEcad caused Yki activation and overproliferation. Moreover, consistent with these findings, a previous analysis of strong loss-of-function conditions for α-Cat, DEcad, or Arm led to the conclusion that a loss of the cadherin-catenin complex (CCC) reduces Yki activity and causes JNK-meditated tissue degeneration. This conclusion was at odds with some mammalian studies where the loss of E-cadherin or αE-catenin causes YAP or TAZ-dependent overgrowth in a number of different tissues and cell lines. The current data showing that α-Cat and DEcad limit Yki activity and tissue growth in Drosophila similar to mammalian tissues suggest a conserved functional relationship between AJs and the regulation of Yki/YAP/TAZ activity. Only when the Drosophila CCC is strongly depleted does a substantive activation of JNK signaling override Yki activation, causing tissue degeneration. This could potentially involve a direct inhibition of Yki by JNK (Sarpal, 2019).

α-Cat loss-of-function conditions in conjunction with a block of cell death defined three distinct phenotypic classes that broadly align with the progression sequence of epithelial cancer from adenoma (epithelial overgrowth), to adenocarcinoma (overgrowth associated with a partial loss of epithelial integrity), to carcinoma (loss of epithelial integrity with cells showing protrusive activity). Similarly, in mammalian cancer, such as human colon cancer is the down-regulation of αE-catenin associated with an increased propensity for cells to become invasive. On the other hand, loss of αE-catenin was reported to suppress colorectal adenomas induced by APC loss-of-function, an effect that could be mediated by the Rho-Rho kinase signalling-dependent cell death elicited by the loss of the CCC in an APC mutant background. Also in Drosophila, it was found that Rho1 is required for cell death resulting from the loss of α-Cat, likely mediated by the Rho1 signalling dependent activation of the JNK pathway (Sarpal, 2019).

Loss of α-Cat leads to a corresponding decline of DEcad and Arm at AJs. Quantification of CCC proteins in the late stage embryonic epidermis of zygotic α-Cat null mutants suggested that AJs can be retained and support normal tissue architecture when CCC levels are reduced to less than 10% of normal. As heterozygous α-Cat animals are normal, it is anticipated that reduction of α-Cat to somewhere between 50% and 10% of wild-type levels will cross a threshold that will increase Yki activity without activating JNK signaling sufficiently to cause cell death. How strongly α-Cat and DEcad have to be reduced to cause an activation of Yki remains to be explored. For example, reducing α-Cat could compromise the interactions between the AJ protein Echinoid and Salvadore, a Hippo binding partner important for normal Hippo activity, or compromise the interactions between Crb and Ex that could deregulate Hippo signaling. Loss of α-Cat could also affect actin polymerization as mature AJs suppress actin polymerization, whereas enhanced actin polymerization is a known activator of Yki. Reducing α-Cat could therefore directly promote actin polymerization prior to an overt defect in cell adhesion, and consequently stimulate Yki activity and tissue growth. Finally, loss of α-Cat may affect Yki not through the Hippo pathway as was documented for αE-catenin in mammalian keratinocytes (Sarpal, 2019).

Mammalian studies raised the possibility that αE-catenin could act independently of AJs to regulate tissue growth. In contrast, several observations argue that Drosophila α-Cat acts as an AJ component to limit tumorigenesis: (1) The mechanosensory properties of α-Cat regulate the recruitment of Jub to AJs, which stimulates Yki activity and tissue growth and implies that α-Cat is suspended between the E-cadherin/β-catenin complex and actomyosin. In particular, α-Cat mutants that disrupt mechanosensing and cause overgrowth (α-CatR-ΔM1 and α-CatR-H1) are effectively recruited to AJs where they support normal epithelial organization, suggesting that a specific junctional defect disrupts growth regulation. (2) A reduction of DEcad is also associated with hyperplastic tissue overgrowth similar to the partial loss of α-Cat. (3) The expression of a DEcad::αCat fusion protein restores both the epithelial organization and normal growth of α-Cat compromised wing discs, and rescues α-Cat null mutant clones which normally fail to develop in wing discs. Together, these data suggest that α-Cat acts as a component of the CCC at AJs to regulate tissue growth (Sarpal, 2019).

The mechanosensitive interactions between Jub and α-Cat are thought to transmit tissue tension into growth regulatory signals. Cytoskeletal tension enhances recruitment of Jub to junctional α-Cat where Jub forms a complex with Wts, preventing it from phosphorylating and hence deactivating Yki. Supporting this model, Jub and Yki-dependent tissue overgrowth was observed that correlates with an enhanced recruitment of Jub to AJs. In particular, the data suggest that the M1 domain acts as a gatekeeper for Jub recruitment. In the absence of M1, junctional Jub levels become strikingly high, suggesting that M region mechanosensing has become ineffective in limiting Jub recruitment, causing Yki activation and overgrowth. Tension is thought to cause a conformational change in the M region and an unfurling of the M1 domain exposing a Vinc binding site. However, Vinc is not required for limiting Jub recruitment and deletion of M1 causes a reduction of junctional Vinc but not a complete loss. This leaves the mechanism of how M1 moderates Jub recruitment unresolved. A second unknown binding partner of M1 may be involved or an intramolecular interaction between M1 and the Jub binding site in the N domain could control Jub binding (Sarpal, 2019).

This Jub recruitment-dependent model of how mechanotransduction by the α-Cat M region regulates tissue growth does not explain all the observations and therefore needs to be extended to incorporate additional mechanisms of how AJs can control tissue growth. First, this model cannot explain the Yki-dependent overgrowth precipitated by low α-Cat levels, and corresponding low Jub levels at AJs. Second, it was observed that two fusion proteins between DEcad and α-Cat, one containing both full-length proteins (DEcad::αCat) and one lacking the Jub binding domain in α-Cat (DEcadΔβ::αCatΔN), can both support normal growth in α-Cat KD tissue but only DEcad::αCat restored normal junctional Jub levels whereas DEcadΔβ::αCatΔN did not. Third, expression of α-CatR in α-Cat KD tissue restores α-Cat to approximately 63% of normal levels. However, Jub increases to 119% without a noticeable increase in tissue size. One possibility is that mechanical force distributed over fewer α-Cat molecules enhances the mechanosensory response of individual α-Cat molecules in a non-linear manner resulting in higher Jub recruitment. Evidence for such a mechanism was recently reported for the recruitment of Vinc to AJs in the Drosophila germband, and may be similar for Jub recruitment to AJ in that tissue. Fourth, removing the entire M region results in an α-Cat protein that can support normal wing development, implying that M region mechanosensing is not an essential aspect of regulating tissue growth. In light of the results with α-CatR-ΔM1, this can only be explained by assuming that removing M2 and M3 in addition to M1 has a compensatory effect. Whereas α-CatR and α-CatR-ΔM expression in α-Cat depleted PC tissue enhances Jub recruitment above AC control levels, loss of M2 or M3 causes Jub levels to remain below AC levels suggesting that these two domains somehow normally support the ability of α-Cat to recruit Jub, possibly by supporting the stability of the CCC at AJs. Collectively, these findings suggest that M region mechanosensing contributes to Jub recruitment and Hippo/Yki pathway regulation but that the actual mechanisms involved have considerable complexity and require further analysis to be resolved (Sarpal, 2019).

Increased levels of Jub were also observed in response to disrupting the mechanosensory properties of the α-Cat ABD. Disruption of the α1-helix of ABD did not only enhance F-actin binding in vitro but also stabilized the CCC at AJs in the wing epithelium as suggested by the current observations. A corresponding increase in Jub levels at AJs could account for the persistent overgrowth observed in α-Cat KD tissue expressing α1-helix compromised α-Cat (α-CatR-H1). Thus, the mechanosensory properties of the M region and the α-Cat ABD are both important for regulating Jub recruitment to AJs and growth regulation through the Hippo/Yki pathway. As changes in tissue tension are thought to modulate the Hippo/Yki pathway through junctional recruitment of Jub it was asked whether α-Cat mutants that compromise mechanosensing cause changes in tissue tension or viscoelasticity that could indirectly affect Jub recruitment to AJs. We did not observe such changes after laser ablation of cell-cell junction. This is consistent with the finding that replacing α-Cat with α-CatR-ΔM1 did not change junctional myosin II levels, the major tension generator in the wing disc epithelium. Taken together, these data suggest a direct molecular role of α-Cat in Jub recruitment and strongly argue that α-Cat operates as a crucial mechanosensor to regulate tissue growth (Sarpal, 2019).

In summary, it is concluded that α-Cat uses multiple mechanisms to act as an important regulator of tissue growth in the Drosophila wing disc epithelium. It is doing so at least in part by operating as a mechanotransducer, engaging both M region and ABD mechanosensing, to relay cytoskeletal tension into growth regulatory signals. One of these mechanisms involves the recruitment of Jub to the N domain of α-Cat that can be modulated by both mechanosensing mechanisms. However, α-Cat also engages mechanisms that are independent of the junctional recruitment of Jub to control tissue growth, which remain to be explored further (Sarpal, 2019).

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

This study investigated how cell contractility and adhesion are functionally integrated during epithelial morphogenesis. To this end, the role of α-Catenin, a key molecule linking E-Cadherin-based adhesion and the actomyosin cytoskeleton, was analyzed during Drosophila embryonic dorsal closure, by studying a newly developed allelic series. α-Catenin was found to regulate pulsatile apical contraction in the amnioserosa, the main force-generating tissue driving closure of the embryonic epidermis. α-Catenin controls actomyosin dynamics by stabilising and promoting the formation of actomyosin foci, and also stabilises DE-Cadherin (Drosophila E-Cadherin, also known as Shotgun) at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, a genetic interaction was uncovered between α-Catenin and Vinculin, and a tension-dependent recruitment of Vinculin to amniosersoa apical cell membranes, suggesting the existence of a mechano-sensitive module operating in this tissue (Jurado, 2016).

How adhesion and actomyosin contractility are integrated at junctions is a fundamental question in morphogenesis. To tackle this, the role of α-Catenin, a key protein linking adherens junctions and the actin cytoskeleton, was analyzed in the context of Drosophila embryogenesis and in particular during dorsal closure. α-Catenin was found to regulates pulsatile actomyosin dynamics in apically contracting cells by stabilising and promoting actomyosin contractions. α-Catenin also stabilises DE-Cadherin at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, the results reveal an interaction between α-Catenin and Vinculin that could be important for DE-Cadherin stabilisation (Jurado, 2016).

Live imaging of mutant embryos shows a strong requirement for α-Catenin in the migration of the dorsal ridge primordia towards the dorsal midline, preventing the formation of the dorsal ridge and thus affecting both dorsal closure and head involution. These results reveal that the dorsal ridge is particularly sensitive to the levels of α-Catenin and suggest it is a key region that could mechanically coordinate both processes. Although it is clear that some of the defects observed during dorsal closure are a consequence of the defective dorsal ridge morphogenesis, this analysis shows that other cellular processes more specific to dorsal closure are affected. In particular, it was observed that the actin cable is disorganised and that the pulsatile apical contraction of the amnioserosa is abnormal (Jurado, 2016).

The defects observed at the level of amnioserosa apical cell oscillations could be a consequence of a defective actin cable, which would be acting as a ratchet and thus progressively restricting the expansion of apical cell area. However, several lines of evidence suggest that a ratchet mechanism stabilising the contracted state of amnioserosa cells is acting at the level of individual cells. In particular, the analysis performed in this study of actin oscillatory dynamics in α-Cat mutants suggests that the increase in the expansion half-cycle of amnioserosa apical cell oscillations could be due to an increase in the time interval between the appearance of consecutive foci. Thus, the results favour the idea that the Cadherin–Catenin complex has a role in promoting actomyosin oscillatory dynamics. How α-Catenin promotes actomyosin contractility remains to be elucidated, but it is likely to involve both direct and indirect (through other actin-binding proteins) interactions with the actin cytoskeleton. For example, an antagonistic interaction between α-Catenin and the Arp2/3 complex has been observed both in cell systems and in Drosophila embryos, raising the possibility that the actin-bundling activity of α-Catenin at adherens junctions, rather than the formation of Arp2/3-dependent networks, could be important for apical contraction (Jurado, 2016).

Interestingly, it was found that with the α-Cat²⁰⁴⁹ allele, adhesion dynamics are also defective, suggesting that medioapical actomyosin dynamics promote adherens junction stabilisation. In contrast, with the α-Cat⁴²¹ allele, which would bind constitutively to Vinculin in a context of defective medioapical actomyosin dynamics, DE-Cadherin stabilisation is recovered. This result suggests that the stabilisation of DE-Cadherin could be mediated by the binding of Vinculin to α-Catenin. This is in agreement with what has been observed in cell systems, where forms of α-Catenin that constitutively bind to Vinculin have decreased mobility. It was further shown that, although DE-Cadherin is stabilised in α-Cat⁴²¹ mutants, possibly due to the Vinculin–α-Catenin interaction, this stabilisation is not able to rescue normal medioapical actin dynamics. Thus, it is suggestd that direct binding of α-Catenin to actin through its actin-binding domain promotes the formation of medioapical actomyosin foci, whereas indirect binding to actin through Vinculin would promote junction stabilisation. Taken together, these data suggest that α-Catenin domains, through their interactions with other actin-binding proteins and actin, might differentially regulate actin dynamics (Jurado, 2016).

Finally, the results show that there is a tension-dependent recruitment of Vinculin at the membranes of amnioserosa cells, which could be mediated by α-Catenin. Interestingly, it has recently been found, in experiments using a heat-shock inducible Vinculin reporter, that the rate of change of Vinculin levels correlates with junctional tension. The results also suggest that Vinculin is able to perform an adhesive function when α-Catenin function is compromised. This could result from an α-Catenin-independent binding of Vinculin to E-Cadherin or from an interaction between Vinculin and other junctional proteins such as ZO-1 (also known as TJP1), which has been shown to recruit Vinculin to VE-cadherin junctions and increase cell–cell tension. However, given that ZO-1 can also interact with α-Catenin, it remains to be investigated whether the mechano-sensitivity of Vinculin is completely dependent on α-Catenin. Thus, it is likely that Vinculin is able to perform different functions depending on its developmental context. Interestingly, different mechanisms for Vinculin binding to Talin in integrin-mediated adhesion have recently been uncovered in different morphogenetic processes, meaning that Talin can sense different force vectors. Given that a role for Talin and integrin-mediated adhesion during dorsal closure has been uncovered, it would be interesting to investigate whether Vinculin is also involved in integrin-mediated adhesion at this stage. The results suggest that a tension-dependent module involving Vinculin is present in amnioserosa cells. An exciting avenue will be to identify the mechanisms and function of such module in the context of morphogenesis (Jurado, 2016).

Mutational analysis supports a core role for Drosophila α-catenin in adherens junction function

α-catenin associates the cadherin-catenin complex with the actin cytoskeleton. α-catenin binds to β-catenin, which links it to the cadherin cytoplasmic tail, and F-actin, but also to a multitude of actin-associated proteins. These interactions suggest a highly complex cadherin-actin interface. Moreover, mammalian αE-catenin has been implicated in a cadherin-independent cytoplasmic function in Arp2/3-dependent actin regulation, and in cell signaling. The function and regulation of individual molecular interactions of α-catenin, in particular during development, are not well understood. This study has generated mutations in Drosophila α-Catenin (α-Cat) to investigate α-Catenin function in this model, and to establish a setup for testing α-Catenin-related constructs in α-Cat-null mutant cells in vivo. This analysis of α-Cat mutants in embryogenesis, imaginal discs and oogenesis reveals defects consistent with a loss of cadherin function. Compromising components of the Arp2/3 complex or its regulator SCAR ameliorate the α-Cat loss-of-function phenotype in embryos but not in ovaries, suggesting negative regulatory interactions between α-Catenin and the Arp2/3 complex in some tissues. It was also shown that the α-Cat mutant phenotype can be rescued by the expression of a DE-cadherin::α-Catenin fusion protein, which argues against an essential cytosolic, cadherin-independent role of Drosophila α-Catenin (Sarpal, 2012).

Drosophila is used intensively to investigate the function of adherins junctions (AJs) in development. This analysis is facilitated by the availability of mutations in three of the four core components of the cadherin-catenin complex: the classic cadherin (DEcad or DN-cadherin) and the catenins Arm/β-catenin and p120 catenin. This study reports the isolation of null mutations in Drosophila α-Cat, expanding the fly tool kit to address AJ function. α-Cat mutants display defects that are indicative of loss of cadherin-mediated adhesion in late embryos, imaginal discs and ovaries. Zygotic α-Cat mutant embryos display defects in head morphogenesis, which resemble those caused by weak mutations affecting shg/DEcad. This mild phenotype is probably due to the presence of maternal α-Catenin, which is still detectable in late embryos and supports most DEcad-dependent processes during embryogenesis (Sarpal, 2012).

Analysis of head morphogenesis revealed two defects in α-Cat mutants. First, tissue breaks in the head epithelium are apparent in close proximity to clusters of cells that undergo apical constriction. It is likely that the reduced levels of the cadherin-catenin complex in α-Cat mutants do not provide sufficient adhesive strength to withstand the mechanical force exerted by those apically constricting cells. A correlation between the degree of morphogenetic stress that the epithelium is exposed to and the level of expression of the cadherin-catenin complex needed to maintain tissue integrity has been well established in embryos with reduced DEcad and in other systems. The second defect seen in α-Cat mutants is a failure in head involution. Despite any obvious defects in epithelial integrity, the dorsal fold fails to move forward and to envelope the anterior-dorsal head. As the mechanisms of head involution are unclear, there is currently no basis for speculation on how α-Catenin might contribute to this process. Breaks in the head epithelium are variable in frequency and strength, whereas the failure in head involution is a robust defect, suggesting that the breaks in the epithelium might not be the immediate cause for the problems in head involution (Sarpal, 2012).

Quantification of fluorescent intensities of α-Catenin at AJs of α-Cat zygotic mutant embryos suggested that the levels of α-Catenin had dropped to a small percentage of levels in wild-type embryos by late embryogenesis (early stage 17). DEcad and Arm were also strongly reduced but not to the same degree as α-Catenin. That the loss of α-Catenin does not lead to an immediate corresponding loss of other components of the cadherin-catenin complex was also seen during mesoderm formation in early Drosophila embryos (Oda, 1998). This suggests that the cadherin-β-catenin complex has significant α-catenin-independent membrane stability, perhaps mediated by the association with p120catenin, which regulates cadherin endocytosis. By contrast, normal protein levels of Ed and Baz were retained at AJs in α-Cat mutants, suggesting that the concentrations of these proteins at AJs do not depend on the levels of the cadherin-catenin complex. This is consistent with work in early Drosophila embryos that suggested that Baz acts upstream of the cadherin-catenin complex in AJ assembly. Ultrastructurally, AJs can retain normal appearance and size even though the level of the cadherin-catenin complex is strongly depleted. Collectively, these findings suggest that although the adhesive strength of AJs correlates with the cadherin-catenin complex content, AJ maintenance is largely unaffected by variations in cadherin-catenin complex concentration (Sarpal, 2012).

α-Cat mutant cell clones in the adult ovary display several defects previously seen in shg/DEcad or arm mutant cell clones. These include a mis-localization of the oocyte, a block of border cell migration, and lack of follicle formation and separation. α-Cat mutant cells in the follicular epithelium (FE) flatten and detach from each other and their wild-type neighbors, indicating a reduction or loss in lateral cell adhesion. Even in very large cell clones, α-Cat mutant cells retain a monolayered arrangement, being sandwiched between the germline cells and the basement membrane. Interestingly, α-Cat mutant cells display apical-basal polarity, as indicated by a tuft of microvilli positive for the microvillus cadherin Cad99C. However, apical-basal axis orientation between cells appears uncoordinated, suggesting that the cadherin-catenin complex is not required for all aspects of apical-basal polarity in follicle cells but is required for intercellular adhesion and axis alignment of neighboring cells to form a proper epithelium (Sarpal, 2012).

One interesting feature of α-Cat mutant follicle cells is the formation of prominent cytoplasmic clusters of α-Spectrin, probably the result of a collapse of the lateral cytocortex. Previous work with S2 cells suggested that the cadherin-catenin complex might not be engaged in recruiting and stabilizing cytocortical spectrin in Drosophila. However, the current data and observations made in Drosophila embryos depleted of α-Catenin with RNAi (Magie, 2002) suggest that the cadherin-catenin complex plays a crucial role in the formation of the spectrin-based lateral cytocortex, similar to its role in mammalian cells. Cytoplasmic spectrin clusters also become enriched in other basolateral and apical markers including DEcad and Crb. Notably, these clusters are also enriched in the recycling endosome GTPase Rab11. Rab11 and its effector, the exocyst, are required for surface delivery of DEcad and Crb. AJs can act as docking stations for exocyst-mediated membrane delivery. These observations raise the possibility that DEcad and Crb are retained in Rab11-positive compartments that fail to fuse with the plasma membrane due to the lack of a cadherin- or catenin-dependent docking mechanism (Sarpal, 2012).

Mammalian αE-catenin and the Arp2/3 complex can act as competitive regulators of actin polymerization. Three modes of interaction between the cadherin-catenin complex and Arp2/3 have been proposed. Model 1 proposes that the observed enrichment of αE-catenin at AJs, resulting from the interaction of αE-catenin with β-catenin, fosters local αE-catenin dimerization. Dimer formation and binding to β-catenin are mutually exclusive due to overlapping binding sites, so that dimerization of αE-catenin is thought to occur after dissociation from β-catenin. αE-catenin dimers then bind to F-actin and prevent Arp2/3 interaction with F-actin (Drees, 2005; Benjamin, 2010). αE-catenin therefore promotes the formation of F-actin bundles that are normally associated with AJs, rather than Arp2/3-dependent actin networks, because αE-catenin itself has actin bundling activity and can interact with other actin bundling proteins such as formin (Kobielak, 2004). Model 2 proposes the existence of independent junctional and cytosolic pools of αE-catenin, and that the cytosolic pool of αE-catenin shows negative regulatory interactions with Arp2/3 and, consequently, counteracts lamellipodia formation and cell motility (Benjamin, 2010). A third model for functional interactions between Arp2/3 and the cadherin-catenin complex is based on evidence suggesting that Arp2/3 is required for cadherin endocytosi. Compromised Arp2/3 activity could enhance surface abundance of the cadherin-catenin complex and therefore counteract a genetic reduction of cadherin or catenin levels (Sarpal, 2012).

Each of these models predicts that defects arising from a reduction in α-Catenin function could be suppressed by a concurrent reduction in Arp2/3 activity. This genetic interaction was observed in embryos mutant for α-Cat in which Arp2/3 or SCAR function was reduced. This study also observed that reduced Arp2/3 activity ameliorates defects seen in embryos mutant for intermediate arm alleles. This finding is consistent with models 1 and 3 but difficult to reconcile with model 2 because the loss of Arm should enhance the cytosolic pool of α-Catenin as less α-Catenin is recruited to the junction. However, the observed genetic interactions could be reconciled with model 2 by assuming that the Arm-α-Catenin interaction is required to make α-Catenin competent to interact with F-actin, either by promoting dimerization as suggested (Drees, 2005), or through promoting a post-translational modification of α-Catenin such as phosphorylation (Sarpal, 2012 and references therein).

To explore potential interactions between α-Catenin and Arp2/3 in follicle cells, advantage was taken of RNAi, and α-Cat-RNAi was coexpressed with either Sop2-RNAi or SCAR-RNAi in follicle cell clones. Sop2 or SCAR mutant or knockdown cells showed defects similar to those reported for cells in the pupal wing disc epithelium that lacked Arp2/3 function, and which apparently disrupts cadherin endocytosis. Defects in follicle cells with compromised Arp2/3 are seen only at late stages of follicle development, suggesting that the early FE does not require Arp2/3 function. α-Cat-RNAi expression caused defects similar to those observed in mutant cells, characterized by a loss of cell contact and the formation of spectrin aggregates. Expression of either Sop2-RNAi or SCAR-RNAi in α-Cat-RNAi cells did not noticeably modify the α-Cat phenotype. One potential explanation for the lack of interactions in this context might be that the disruption of adhesion by α-Cat-RNAi is too strong to be overcome significantly by lowering Arp2/3 activity, assuming that the interaction between α-Catenin and Arp2/3 is one among several α-Catenin activities. Alternatively, α-Catenin might not functionally interact with the Arp2/3 complex in follicle cells (Sarpal, 2012).

To reveal a potential function for a cytosolic pool of α-Catenin, a DEcad::αCat fusion protein was expressed in α-Cat mutants. Previously, it was shown that a mammalian E-cadherin:: αE-catenin fusion protein could function in cell adhesion (Nagafuchi, 1994), and that in Drosophila the DEcad::αCat protein can fully substitute for the loss of DEcad or Arm in all cadherin-dependent processes during oogenesis (Pacquelet, 2005), and for the loss of DEcad (but not Arm) during dorsal closure of the embryo (Gorfinkiel, 2007). However, the Drosophila rescue experiments were carried out in the presence of endogenous α-Catenin, leaving open the possibility that α-Catenin has a cytosolic function or interacts with the DEcad::αCat protein through the α-Catenin dimerization domain, which could create a dimer that interacts with actin. It was found that expression of DEcad::αCat rescued head morphogenesis and embryonic lethality of α-Cat mutants, α-Cat mutant cell clones in imaginal discs, the FE, and border cell migration. These effects were similar to those for expression of exogenous α-Catenin. It was suggested that the expression of DEcad::αCat increases the surface abundance of cadherin, which could restore cell adhesion (Weis, 2006). However, overexpression of DEcad in α-Cat mutants did not show any rescue activity, arguing against this notion. The possibility was also considered that DEcad::αCat undergoes proteolytic cleavage to release α-Catenin. However, no cleavage product was detected on immunoblots. Moreover, whereas expression of a truncated form of α-Catenin that lacks the N-terminal β-catenin-binding and dimerization domain (deletion of amino acids 1-233) does not result in any rescue of α-Cat mutant defects, expression of the same truncated form of α-Catenin fused to DEcad rescues α-Cat mutants similarly to DEcad::αCat (Sarpal, 2012).

Collectively, these data indicate that a cytosolic form of α-Catenin is not required for α-Catenin function in several Drosophila tissues that were have investigated, and that all essential aspects of α-Catenin function during morphogenesis are executed in the immediate vicinity of the plasma membrane. These data do not rule out the possibility that α-Catenin directly interferes with the Arp2/3-actin interaction, but confines the potential for this interaction to the immediate submembranous space (Sarpal, 2012).

CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions in the mouse

Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. This paper reports that the MT plus end-binding protein CLASP2 localizes to adherens junctions (AJs) via direct interaction with p120-catenin (p120) in primary basal mouse keratinocytes. Reductions in the levels of p120 or CLASP2 decreased the localization of the other protein to cell-cell contacts and altered AJ dynamics and stability. These features were accompanied by decreased MT density and altered MT dynamics at intercellular junction sites. Interestingly, CLASP2 was enriched at the cortex of basal progenitor keratinocytes, in close localization to p120. These findings suggest the existence of a new mechanism of MT targeting to AJs with potential functional implications in the maintenance of proper cell-cell adhesion in epidermal stem cells (Shahazi 2013).

alpha-Catenin interacts with APC to regulate beta-catenin proteolysis and transcriptional repression of Wnt target genes

Mutation of the adenomatous polyposis coli (APC) tumor suppressor stabilizes beta-catenin and aberrantly reactivates Wnt/beta-catenin target genes in colon cancer. APC mutants in cancer frequently lack the conserved catenin inhibitory domain (CID), which is essential for beta-catenin proteolysis. This study shows that the APC CID interacts with alpha-catenin, a Hippo signaling regulator and heterodimeric partner of beta-catenin at cell:cell adherens junctions. Importantly, alpha-catenin promotes beta-catenin ubiquitylation and proteolysis by stabilizing its association with APC and protecting the phosphodegron. Moreover, beta-catenin ubiquitylation requires binding to alpha-catenin. Multidimensional protein identification technology (MudPIT) proteomics of multiple Wnt regulatory complexes reveals that alpha-catenin binds with beta-catenin to LEF-1/TCF DNA-binding proteins in Wnt3a signaling cells and recruits APC in a complex with the CtBP:CoREST:LSD1 histone H3K4 demethylase to regulate transcription and beta-catenin occupancy at Wnt target genes. Interestingly, tyrosine phosphorylation of alpha-catenin at Y177 disrupts binding to APC but not beta-catenin and prevents repression of Wnt target genes in transformed cells. Chromatin immunoprecipitation studies further show that alpha-catenin and APC are recruited with beta-catenin to Wnt response elements in human embryonic stem cells (hESCs). Knockdown of alpha-catenin in hESCs prevents the switch-off of Wnt/beta-catenin transcription and promotes endodermal differentiation. These findings indicate a role for alpha-catenin in the APC destruction complex and at Wnt target genes (Choi, 2013).

Adhesion disengagement uncouples intrinsic and extrinsic forces to drive cytokinesis in epithelial tissues

Cytokinesis entails cell invagination by a contractile actomyosin ring. In epithelia, E-cadherin-mediated adhesion connects the cortices of contacting cells; thus, it is unclear how invagination occurs, how the new junction forms, and how tissue integrity is preserved. Investigations in Drosophila embryos first show that apicobasal cleavage is polarized: invagination is faster from the basal than from the apical side. Ring contraction but not its polarized constriction is controlled by septin filaments and Anillin. Polarized cleavage is due instead to mechanical anchorage of the ring to E-cadherin complexes. Formation of the new junction requires local adhesion disengagement in the cleavage furrow, followed by new E-cadherin complex formation at the new interface. E-cadherin disengagement depends on the tension exerted by the cytokinetic ring and by neighboring cells. This study uncovers intrinsic and extrinsic forces necessary for cytokinesis and presents a framework for understanding how tissue cohesion is preserved during epithelial division (Guillot, 2013).

Epithelial cells divide in the plane of the tissue, allowing the equal partitioning of polarity proteins. This study delineated two major events during epithelial cytokinesis that shed light on how this is controlled. Cleavage progresses along the apicobasal axis and is polarized, as it is faster from basal to apical. This is not due to polarized contraction of the ring but to apical anchoring of the ring to E-cad complexes. Second, cleavage occurs in the plane of junction and involves local adhesion disengagement. In contrast to standard cytokinesis, this study delineated intrinsic and extrinsic mechanical processes operating during epithelial cytokinesis. Contractility of the ring itself is dependent on septins and Anillin. Ring contraction is resisted by intercellular adhesion mediated by E-cadherin complexes and by tension from neighboring cells transmitted by adhesion. Thus, E-cad-based adhesion plays a pivotal role in epithelial cytokinesis by anchoring the contractile ring, while its disengagement uncouples intrinsic and extrinsic tensile activity (Guillot, 2013).

In Drosophila embryos, epithelial cells exhibit polarized cleavage furrow ingression. This is likely to be general in epithelial cells, albeit at different magnitudes. MDCK cells too divide from the basal side toward the apex, and neuroepithelial cells in vertebrates partition the basal body first before the more apical part of the cell. Polarized cleavage is not a property unique to epithelial cells, however. Embryonic cleavage in several species exhibit a range of patterns, from completely unilateral cleavage, as reported in jellyfish (Clytia and Beroe) and Ctenophores (Pleurobrachia), to partly asymmetric cleavage in the one-cell-stage C. elegans embryos). In the latter case, polarized ingression of the cleavage furrow is stochastic and correlates with heterogeneities in the recruitment of the actin crosslinker Anillin and of septins. In anillin and septin knockdowns, cleavage becomes symmetric. This contrasts with activators of MyoII, such as Rho kinase, which affects the speed of contraction but not its polarity. Thus, in nonepithelial cells, polarized cleavage is a purely autonomous process governed by heterogeneities in regulators of contractility. This study found, however, that in Drosophila embryos, polarized cleavage is not determined by polarized distribution of Anillin and septins or by differential biomechanical properties of the ring. Septins display a marginal yet significant enrichment basally, and Anillin is slightly enriched apically. However, invagination was still normally polarized along the apicobasal axis in both peanut mutants and anillin RNAi embryos, despite strong reduction in constriction rat. Moreover, no significant difference between apical and basal relaxation kinetics was detected following ablation in wild-types. The ablation kinetics reflects the relative effect of stiffness in the ring and friction internal to the ring and with the cytoplasm. With the caveat that the latter cannot be directly measured and and is assumed to be uniform, these ablation experiments indicate the relative stiffness in the ring. The fact that relaxation is faster (<5 s) than turnover of the internal components of the ring, such as MyoII, substantiates the idea that mostly the elastic relaxation of the ring was measured and not a quasi-static relaxation associated with turnover/movements of ring components (Guillot, 2013).

The rate of constriction was monotonic such that big rings and small rings contracted at a constant rate in wild-types but also in anillin or septin mutants, although it was strongly reduced in the latter cases. This contrasts with reports in C. elegans, where constriction was scaling with ring size, suggesting a mechanism based on disassembly of contractile units whose number scales with ring size. This difference may stem from the fact that cytokinesis is especially rapid in Drosophila embryos (about 150 s). Alternatively, it could reflect the epithelial nature of the divisions reported in this study (Guillot, 2013).

The evidence argues instead that polarized ingression depends largely on apical anchoring of the ring to E-cad complexes. First, E-cad complexes colocalize with the contractile ring for the most part of invagination. Second, ingression is symmetric in either e-cad or α-cat RNAi embryos. Although E-cad complexes, in particular α-cat, can recruit regulators of MyoII (Ratheesh, 2012), this cannot explain polarized invagination of the ring, since apical and basal relaxations are not significantly different in wild-types and in α-cat RNAi embryos. E-cad complexes transmit actomyosin tension in epithelia (Lecuit, 2011). Two sets of observation support the idea that junctions exert pulling forces on the ring due to anchoring. The ring is stretched laterally as it constricts, and this requires apical junctions via e-cad and α-cat. The relative deformation of the ring following ablation is larger apically than basally, and this also requires cell junctions. It is striking that extrinsic and intrinsic regulators of the ring contraction have very different effects on ring dynamics. In the absence of Pnut or Anillin, the ring constriction is reduced but it is still polarized. However, following e-cad or α-cat depletion, ring constriction is normal but symmetric. It is concluded that the mechanical connection of E-cad complexes to the contractile ring causes polarized invagination. It is possible that, in other systems, both intrinsic and extrinsic regulation will operate in parallel to increase the cleavage asymmetry. This may be important in highly columnar epithelial cells or when adhesion is lower and unable to resist the ring tension (Guillot, 2013).

Polarized cleavage effectively separates apical and basal cleavage, adhesion complexes being a barrier separating the apical and lateral domains. The central problem becomes: How does cleavage occur at adherens junctions? This study delineated two critical phases in junctional cleavage. First, the adherens junctions invaginate with the actomyosin ring, consistent with the fact that the ring is anchored to the junctions. During this phase, E-cad intercellular adhesion is stable in the face of the tension exerted by the ring, and E-cad colocalizes with the ring at the point of coupling. Invagination of junctions then stops as E-cad levels decrease in this area. However, ring constriction continues and appears to detach from junctions. This is interpreted as a point of adhesion disengagement. Adhesion disengagement marks the formation of the new vertices and of the new junction between daughter cells. Electron microscopy images show this membrane disengagement. Consistent with this, the membrane still invaginates with the actomyosin ring), although E-cad is still not detected. Closer examination shows that E-cad monomers are present at this late stage of cytokinesis but that adhesion complexes form gradually from this stage onward. It is striking that adhesion is very locally (<1 μm out of ∼40 μm of junction perimeter) and transiently (∼200 s) perturbed during division. In the first 150 s, E-cad clusters immediately adjacent to the cleavage furrow remain in position as the junction invaginates. This suggests that the cortex can be extensively remodeled locally. It likely reflects the fact that tension induces membrane flows with respect to the actin-rich cortex and argues that E-cad-mediated adhesion does not prevent membrane flow during disengagement. Interestingly, local disengagement allows local cell deformation without affecting the overall shape of cell contacts. Consistent with the idea that adhesion is locally disengaged, the amount of E-cad has a strong impact on the timing and depth of junctional cleavage. Increasing E-cad delays disengagement (i.e., the formation of the new junction, inducing strong cell deformations. More generally, this implies that increasing adhesion may provide an efficient mechanism to prevent local cell-cell disengagement when internal tension is used to remodel junctions during morphogenesis. In apical constriction in the Drosophila mesoderm, actomyosin cables pull on the junctional cortex and reduce junction lengths. If adhesion was not strong enough, local disengagement would occur and junctions could not remodel. The fact that adhesion disengagement is local and transient during cytokinesis is also probably key to the overall maintenance of cell polarity and adhesion during epithelial division (Guillot, 2013).

It is proposed that adhesion disengagement is mechanically induced by tension in the cytokinetic ring and by tension from neighboring cells. When the cumulated tension is higher that the adhesive force, disengagement occurs. Consistent with this, disengagement and formation of the new junction is strongly delayed in mutants that reduce the constriction of the cytokinetic ring, namely, in septin mutants and in Anillin knockdown embryos. Likewise, ablation of neighboring cells delays disengagement. It is, however, possible that adhesion is also locally disrupted by either E-cad endocytosis or phosphorylation of β-cat/Arm (Guillot, 2013).

Adhesion complexes transmit cell tension exerted by neighboring cells. Surrounding junctions and, more specifically, MyoII cables oriented toward or near the cleavage furrow strongly affect furrow invagination when E-cad is present at high levels. The invagination in this case is very shallow, suggesting a tug of war between intrinsic (ring contraction) and extrinsic tension (MyoII cables in neighbors). This results in asymmetric furrows in the plane of junctions due to the asymmetric distribution of MyoII cables around the cell. When E-cad is expressed at lower levels, even if surrounding junctions are oriented toward the cleavage furrow, invagination is unaffected and symmetric. It is proposed that E-cad complexes sensitize cells to their mechanical environment. This may provide a mechanism for cells to integrate stress coming from the environment. It will be important to explore how E-cad levels may affect cells responsiveness to extrinsic stress during division by affecting the timing of the formation of the new junction by local disengagement and the resulting cell shape and topology (Guillot, 2013).

Distinct Rap1 activity states control the extent of epithelial invagination via α-Catenin

Localized cell shape change initiates epithelial folding, while neighboring cell invagination determines the final depth of an epithelial fold. The mechanism that controls the extent of invagination remains unknown. During Drosophila gastrulation, a higher number of cells undergo invagination to form the deep posterior dorsal fold, whereas far fewer cells become incorporated into the initially very similar anterior dorsal fold. A decrease in α-catenin activity causes the anterior fold to invaginate as extensively as the posterior fold. In contrast, constitutive activation of the small GTPase Rap1 restricts invagination of both dorsal folds in an α-catenin-dependent manner. Rap1 activity appears spatially modulated by Rapgap1, whose expression levels are high in the cells that flank the posterior fold but low in the anterior fold. A model is propsed whereby distinct activity states of Rap1 modulate α-catenin-dependent coupling between junctions and actin to control the extent of epithelial invagination (Wang, 2013).

This study used the dorsal fold system to investigate whether specific cellular mechanisms actively regulate the extent of epithelial invagination. α-catenin was shown to be required for the restricted invagination caused by constitutive activation of Rap1, and Rapgap1 was identified as a locally expressed modulator of Rap1 that is required for the extensive invagination of the posterior fold. These data suggest a model whereby Rap1 regulates dorsal fold invagination through an α-catenin-dependent process and establish that differential regulation of an active, specific cellular mechanism confers distinct properties to the neighboring cells to control the extent of epithelial invagination (Wang, 2013).

Genetic analysis identifies two separate functions of Rap1 during dorsal fold formation. The early function appears to be a general role required in all cells that is important for junctional positioning. This was established via examination of embryos that lack Rap1 activity, such as embryos that are produced by the germline clones of null alleles of Rap1 or dizzy, which encodes the Drosophila homolog of PDZ-GEF, a known guanine nucleotide exchange factor that activates Rap1 or embryos that overexpress a GDP-locked, dominant-negative form of Rap1, Rap1N17 (see Spahn, 2012). These embryos display normal assembly of the adherens junctions, the initial basal shift of junction positioning in the initiating cells, and attempt to form dorsal folds. Subsequently, however, the junctions relocalize to the apical surface in all dorsal cells, reversing these initial attempts of dorsal fold formation and eliminating all folding structures (Wang, 2013).

Rap1 appears to maintain the junctional positioning by maintaining the junctional levels of Bazooka. This notion is supported by the lower levels of junctional Bazooka in the Rap1 mutant embryos and by suppression of the loss-of-function phenotype of Rap1 by Bazooka overexpression, which restores the apical domain in the initiating cells and the dorsal fold structures in the Rap1 mutant embryos. Because Bazooka levels are uniform across the dorsal epithelium), this early function of Rap1 appears broadly required, independently of the levels of Rapgap1 expression, and operates in addition to Rap1's later role during epithelial invagination. The effective suppression of Rap1 loss of function following Bazooka overexpression suggests that the two separate functions of Rap1 (the maintenance of Bazooka levels and the regulation of junction-actin connection during epithelial invagination) could be decoupled, allowing comparison of the effect of loss of Rap1 function to that of constitutively active Rap1V12 (Wang, 2013).

The later, spatially regulated function of Rap1 is independent of Bazooka and is differentially modulated by the spatially restricted expression of Rapgap1. Since active Rap1 appears to act through α-catenin to inhibit invagination, it seems plausible that distinct Rap1 activity states modulate the coupling strength between junctions and actin, thereby conferring distinct properties of junctional restructuring to the neighboring cells of the anterior and posterior folds. The geometric measurements of the neighboring cells suggest a model whereby constitutively active Rap1 inhibits junctional mobility so that the size of the apical domain remains constant in the cells surrounding the anterior fold where Rapgap1 levels are low. In contrast, Rapgap1 expression modulates Rap1 activity to promote junctional mobility in the neighboring cells of the posterior fold so that their apical domain expands. In this view, both the initiation and invagination processes require active remodeling of the junctions, but differ in their underlying cellular mechanisms. During initiation, the junctional shift is induced by a modification of the epithelial apical-basal polarity as a result of the downregulation of Par-1 in the initiating cells (Wang, 2012). During invagination, since Par-1 levels do not decrease, mechanical stress might be the dominant force that causes the junctions to move in the neighboring cells (Wang, 2013).

How Rap1 modulates α-catenin-dependent junction-actin coupling remains unknown. The intensities, localization, and turnover kinetics (as measured by fluorescent recovery after photobleaching) were examined of the core junctional components (E-Cadherin and Armadillo), α-catenin, two junctional proteins that interact with both α-catenin, and actin, but no difference between the neighboring cells of the anterior and posterior folds was detected. Recent work in mammalian tissue culture cells showed that the FRET (fluorescent resonance energy transfer) intensities of an E-Cadherin tension sensor correlate with the actin-coupling states of adherens junctions (Borghi, 2012). The use of such a sensor in the living Drosophila embryo might help to reveal the difference in junction-actin coupling states between the anterior and posterior fold neighboring cells (Wang, 2013).

Recent work suggests that α-catenin undergoes a conformational change upon mechanical stretch at the cell junctions. Such conformational change could in principle relieve α-catenin from an intramolecular inhibition on actin binding, thereby increasing its affinity to, or stabilizing its interaction with, the junctional actin (Choi, 2012). Changes in α-catenin conformation thus may determine its ability to mediate the physical coupling between junctions and actin. It is of note that expression of a mutant form of α-catenin that lacks the domain that modulates its conformational change can support static junctional function, but fails to effectively rescue the loss-of-α-catenin phenotype in dynamic morphogenetic processes (Desai, 2013). It is possible that mechanical forces during morphogenesis dynamically modulate the conformational states of α-catenin, the maintenance of which may require distinct Rap1 activity states. The dynamic changes of the α-catenin conformations and the actin-coupling states of adherens junctions that they confer might be crucial for morphogenetic processes that involve extensive restructuring of cell-cell adhesion (Wang, 2013).

If Rapgap1 dictates the spatial extent of cell invagination, one simple model would envision that elevating the levels of Rapgap1 expression in the anterior region could promote anterior fold invagination. This possibility was explored using a UAS transgene to uniformly express Rapgap1 under the control of a maternal Gal4 driver. Two classes of phenotypes were observed: either a complete loss of dorsal fold formation or a limited degree of invagination similarly in both dorsal folds. The former class suggests that the level of expression may be too high to permit the normal function of Rap1, while the latter class suggests that a reversal of the expression pattern (high in the anterior fold, but low in the posterior fold) might be necessary. Attempts were made to express Rapgap1 in the cells anterior to the anterior fold using a Gal4 driver localized through the 3' UTR of the bicoid gene, and the enhancer of the Kr gene was also used to direct the expression of Rapgap1 in cells that are posterior to the anterior fold. In neither case was there an effect on anterior fold invagination. It is possible that driving extensive invagination for the anterior fold would require that Rapgap1 be expressed only in the surrounding cells of the anterior fold in a manner that mimics the endogenous pattern of Rapgap1 expression in the region of posterior fold. Currently, no cis-regulatory element or a Gal4 driver has been found that can drive gene expression in such a specific pattern. Thus, it remains unresolved whether ectopic expression in the anterior fold region would be sufficient to cause extensive invagination (Wang, 2013).

In summary, the data suggest an exciting conceptual framework in which regulated coupling between junctions and actin has a profound impact on the levels of tissue reorganization and on the cellular responses to mechanical stresses that arise during tissue reorganization. This study has defined a specific molecular pathway that produces drastically different epithelial structures from a morphogenetic process whose initiation mechanism appears similar. The regulatory principles that were unveil for Rap1 and α-catenin might be employed in other contexts of morphogenesis in which a tissue undergoes dramatic remodeling, while unperturbed tissue integrity and cell adhesion must be maintained (Wang, 2013).

Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules

Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), homologs of Drosophila Yorkie. However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. This study dissects how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, these results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation. (Yang, 2014).

This report addresses the effects of AJs and basolateral cell polarity determinants on the activity of the Hippo pathway in Drosophila imaginal discs. Knockdown of AJs and basolateral components both induced ectopic activation of Yki. However, knockdown of AJs and basolateral proteins had strikingly different effects on Yki. Disruption of the basolateral module induced mainly a cell-autonomous increase in Yki activity, whereas knockdown of AJs caused non-autonomous induction of Yki reporters. Therefore, these data identify and genetically uncouple multiple different molecular pathways from AJs and the basolateral module that regulate Yki activity (Yang, 2014).

These studies further show that knockdown of AJs induces cell-autonomous reduction of Yki activity and causes cell death and decreased size of Drosophila imaginal discs. Likewise, E-cad and :alpha;-cat mutant clones do not survive in imaginal discs. This effect may be mediated by LIM domain proteins of the Zyxin and Ajuba subfamilies, which regulate Hippo signaling by directly inhibiting Wts/Lats kinases and by interacting with Salvador (Sav), an adaptor protein that binds to the Hpo/MST kinases. A recent report shows that α-Cat recruits Ajuba and indirectly Wts to AJs and loss of Ajuba leads to activation of Wts and hence phosphorylation and inhibition of Yki and diminished tissue size. Thus, α-cat mutant cells may inactivate Yki because they lose Ajuba function (Yang, 2014).

In contrast, in mammalian systems, several in vivo and in vitro studies have shown the opposite effect on Hippo signaling upon AJ disruption; knockdown of E-cad or α-cat caused an increase in cell proliferation and nuclear accumulation of YAP, and conditional knockout of α-cat in mouse skin cells caused tumor formation and elevated nuclear YAP staining. This suggests that AJ components have a tumor suppressor function in mammals. The observation that Scrib is mislocalized upon disruption of AJs in several different mammalian cell lines suggested that YAP activation could be due to the concomitant disruption of the basolateral module. However, the finding that acute disruption of AJs can cause YAP activation without disrupting Scrib localization and vice versa indicates that AJs and the basolateral module also act independently on the Hippo pathway in mammalian cells. In mammalian cells, α-Cat forms a complex with YAP and 14-3-3 proteins, thereby sequestering phosphorylated YAP at the plasma membrane. However, α-Cat may function as a tumor suppressor only in epidermal stem cells, as conditional deletion of α-cat in differentiated cells only caused a mild phenotype with no overgrowth and tumor formation. Therefore, it is possible that the negative regulation of YAP by α-Cat is cell type-specific, although further testing is required to fully address this issue (Yang, 2014).

The non-cell-autonomous effect of AJ knockdown on the Hippo pathway is an intriguing phenomenon. Several groups reported non-autonomous effects on the Hippo pathway in Drosophila in other mutant conditions. Disrupting the expression gradients of the atypical Cadherin Dachsous or that of its regulator Four-jointed, clones of cells mutant for the tumor suppressor genes vps25 or hyperplastic discs (hyd) , clones of cells overexpressing Src64, or overexpression of the proapoptotic gene reaper or the JNK signaling ligand eiger all cause non-autonomous activation of Yki. This non-autonomous activation of Yki may be part of a regenerative response that stimulates cell proliferation in cells neighboring tissue defects. The signals that activate Yki in these situations are not known, nor is it known whether these mutant conditions activate the same or different signaling mechanisms. The non-autonomous activation of Yki around cells with AJ knockdown may be mediated by changes in mechanical forces. AJs are important for maintaining tension between cells across epithelia, and disruption of AJs leads to an imbalance of apical tension. Mechanical forces are known to regulate the Hippo pathway, and YAP/TAZ act as mediators of mechanical cues from the cellular microenvironment such as matrix stiffness. In particular, the Zyxin and Ajuba family LIM domain proteins can act as sensors of mechanical forces and may be involved in the non-autonomous activation of Yki. The effects on Hippo signaling of solely changing Zyxin and Ajuba may not be as strong as those described here, and these proteins may thus cooperate with other molecular conduits to regulate the activity of the Hippo pathway in response to changes in AJ strength. Unraveling these mechanisms will provide important new insights into understanding how cells interact with neighboring cells to regulate proliferation, apoptosis, and the Hippo pathway (Yang, 2014).

It is currently unknown whether AJs also exert non-autonomous effects on the Hippo pathway in mammalian tissues. Amphiregulin, an EGF ligand, is a downstream target of YAP and can induce non-cell-autonomous cell proliferation through EGFR signaling. However, it is not known whether YAP itself is activated non-cell-autonomously to contribute to the hyper-proliferation phenotypes observed upon disruption of AJs in vivo and in vitro. It will be interesting to determine whether AJs and other cell-cell signaling mechanisms also have non-cell-autonomous effects on the activity of YAP in mammalian tissues, for example during regeneration (Yang, 2014).

Finally, the apical proteins aPKC and Crb modulate the activity of the Hippo pathway, and many Hippo pathway components are apically localized, which is important for their activity. The data presented in this study add to these findings. Disruption of AJs causes reduced Yki activity, despite the fact that Crb and Mer are mislocalized. Thus, AJs and cell polarity components regulate Yki activity through multiple, genetically separable inputs. It will be interesting to decipher all of the different underlying molecular mechanisms of how AJs and basolateral proteins regulate the Hippo pathway and how these mechanisms evolved in Drosophila and in mammals (Yang, 2014).

Centralspindlin and alpha-catenin regulate Rho signalling at the epithelial zonula adherens

The biological impact of Rho depends critically on the precise subcellular localization of its active, GTP-loaded form. This can potentially be determined by the balance between molecules that promote nucleotide exchange or GTP hydrolysis. However, how these activities may be coordinated is poorly understood. This study reports a molecular pathway that achieves exactly this coordination at the epithelial zonula adherens. An extramitotic activity is reported of the centralspindlin complex, better understood as a cytokinetic regulator, which localizes to the interphase zonula adherens by interacting with the cadherin-associated protein, alpha-catenin. Centralspindlin (the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4) recruits the RhoGEF, ECT2, to activate Rho and support junctional integrity through myosin IIA. Centralspindlin also inhibits the junctional localization of p190 B RhoGAP, which can inactivate Rho. Thus, a conserved molecular ensemble that governs Rho activation during cytokinesis is used in interphase cells to control the Rho GTPase cycle at the zonula adherens (Ratheesh, 2012).

alpha-Catenin as a tension transducer that induces adherens junction development

Adherens junctions (AJs), which are organized by adhesion proteins and the underlying actin cytoskeleton, probably sense pulling forces from adjacent cells and modulate opposing forces to maintain tissue integrity, but the regulatory mechanism remains unknown at the molecular level. Although the possibility that alpha-catenin acts as a direct linker between the membrane and the actin cytoskeleton for AJ formation and function has been minimized, this study shows that alpha-catenin recruits vinculin, another main actin-binding protein of AJs, through force-dependent changes in alpha-catenin conformation. Regions were identified in the alpha-catenin molecule that are required for its force-dependent binding of vinculin by introducing mutant alpha-catenin into cells and using in vitro binding assays. Fluorescence recovery after photobleaching analysis for alpha-catenin mobility and the existence of an antibody recognizing alpha-catenin in a force-dependent manner further supported the notion that alpha-catenin is a tension transducer that translates mechanical stimuli into a chemical response, resulting in AJ development (Yonemura, 2010).

Planar polarized actomyosin contractile flows control epithelial junction remodelling

Force generation by Myosin-II motors on actin filaments drives cell and tissue morphogenesis. In epithelia, contractile forces are resisted at apical junctions by adhesive forces dependent on E-cadherin, which also transmits tension. During Drosophila embryonic germband extension, tissue elongation is driven by cell intercalation, which requires an irreversible and planar polarized remodelling of epithelial cell junctions. This study investigate how cell deformations emerge from the interplay between force generation and cortical force transmission during this remodelling in Drosophila melanogaster. The shrinkage of dorsal-ventral-oriented ('vertical') junctions during this process is known to require planar polarized junctional contractility by Myosin II. This study shows that this shrinkage is not produced by junctional Myosin II itself, but by the polarized flow of medial actomyosin pulses towards 'vertical' junctions. This anisotropic flow is oriented by the planar polarized distribution of E-cadherin complexes, in that medial Myosin II flows towards 'vertical' junctions, which have relatively less E-cadherin than transverse junctions. The evidence suggests that the medial flow pattern reflects equilibrium properties of force transmission and coupling to E-cadherin by alpha-Catenin. Thus, epithelial morphogenesis is not properly reflected by Myosin II steady state distribution but by polarized contractile actomyosin flows that emerge from interactions between E-cadherin and actomyosin networks (Rauzi, 2010).

The data suggest that the anisotropic actomyosin flow may largely depend on the distribution of junctional anchoring points. This requires E-cadherin/β-Catenin complexes at AJs and depends on α-Catenin (Cavey, 2008; Yonemura, 2010). E-cadherin/β-Catenin/α-Catenin complexes are planar polarized, such that medial pulses flow towards regions with lower amounts of E-cadherin complexes. The level of E-cadherin along 'vertical' relative to adjacent junctions (E-cadherin anisotropy) is also fluctuating. Moreover, the onset of medial pulses coincided with the time when E-cadherin anisotropy reached a local maximum raising the possibility that E-cadherin anisotropy may orient the actomyosin flow. Reduction of E-cadherin by RNAi causes the disappearance of medial Myo-II. The junctional Myo-II level is consequently strongly reduced and no longer planar polarized. It was reasoned that reducing the levels of α-Catenin by RNAi should attenuate coupling more subtly. α-Catenin RNAi reduces the number of E-cadherin clusters at AJs and disrupts interactions with junctional F-actin. Moreover, the distribution of E-cadherin is no longer planar polarized in α-CateninRNAi embryos. This is associated with a loss of medial and junctional Myo-II planar polarity. Thus, the planar polarized distribution of E-cadherin/β-Catenin/α-Catenin complexes biases the flow of medial Myo-II and junctional polarization (Rauzi, 2010).

In addition to Myo-II contractility, flow requires (1) crosslinkers between filaments to transmit tension within the medial meshwork, and (2) coupling at the cortex to E-cadherin/β-Catenin/α-Catenin complexes. Increased levels of E-cadherin in 'transverse' junctions may change properties of the actin network (for example, crosslinking/viscosity) and inhibit internal transmission of contractile forces and hence prevent D-V oriented flow. To test this, the force balance within the medial actomyosin network was disrupted by focal ablation, and the redistribution of medial clusters was imaged. If increased E-cadherin levels at transverse junctions inhibit tension transmission along the D-V axis, then medial pulses should not flow in this direction following ablation. However, it was observed that Myo-II medial clusters flowed radially and away from the point of ablation towards the junctions in 100% of cases, even towards transverse junctions. Focal ablation of the actin meshwork produces a local hole, which expands radially. This argues that transverse junctions do not inhibit flow per se and that flow directionality emerges from the properties of the actomyosin meshwork integrated over the entire apical surface (Rauzi, 2010).

The mechanical properties of the medial actomyosin network are locally defined by Myo-II contractility (concentration, affinity, duty cycle), tension transmission within the network (crosslinking), and viscous resistance to deformations (interactions between filaments). Moreover, these properties fluctuate owing to protein turnover and interactions. E-cadherin is known to anchor and modify actin dynamics. The results suggest that the polarized distribution of E-cadherin may control the actomyosin flow pattern by spatially modulating mechanical properties of the actin network (Rauzi, 2010).

Current models of epithelial morphogenesis centre on Myo-II steady state distribution and associated contractile forces. The current data show however that cell deformations cannot be simply derived from the Myo-II distribution itself, but from two central features of actomyosin dynamics, namely concentration (pulses) and movement (flow). Pulsed dynamics defines the rhythm and possibly the speed of deformation. Flow pattern, which in the case of intercalation is anisotropic, dictates the orientation of cell deformation. Flows of Myo-II foci have been reported in the one-cell stage C. elegans embryo, pointing to a more general property of actomyosin networks. An important future avenue of research will be to investigate what properties of actin networks control Myo-II flow dynamics in different systems (Rauzi, 2010).

Requirements for adherens junction components in the interaction between epithelial tissues during dorsal closure in Drosophila

Dynamic interactions between epithelial sheets are a regular feature of morphogenetic processes. Dorsal closure in Drosophila relies on the coordinated movements of two epithelia, the epidermis and the amnioserosa, and provides an excellent model system for a genetic and cell biological approach. This study analyzed the contribution of junctional organization of these epithelia to dorsal closure. A stringent requirement was observed for adherens junctions at the leading edge, the interface between the amnioserosa and the epidermis, for the transmission of the forces generated during the process. It was also found that interactions between Armadillo and E-cadherin play an important role in maintaining the adhesion at the leading edge, revealing the particular dynamics of this interface. These results show that regulated cell adhesion is a crucial element of the interactions that shape epithelial sheets in morphogenetic processes (Gorfinkiel, 2007).

The dynamics of the AJs at the LE is highlighted by the observation that E-cadherin requires interactions with Armadillo even when it is directly linked to α-catenin. Whereas a full-length E-cadherin molecule fused to α-catenin can rescue DC in shg mutants, a similar fusion lacking the Armadillo-binding domain cannot. This indicates that the rescue of the full-length molecule is mediated, in part, by binding of Armadillo - a surprising result in the context of the classical model in which the function of Armadillo and β-catenin is to link cadherin to the actin cytoskeleton via α-catenin. This view has recently been challenged by the observation that α-catenin has two mutually exclusive states. As a monomer it can bind β-catenin, whereas as a dimer it can bind actin (Drees, 2005; Yamada, 2005). This observation could help explain the current results. If interactions between α-catenin and Armadillo/β-catenin are dynamic, β-catenin could act to bring α-catenin to the junctions where it would dissociate to promote actin polymerization. In the absence of an Armadillo-binding site, the only junctional α-catenin would be the one in the chimera which is clearly not sufficient for full function, probably because it cannot dissociate from cadherin. Whereas this provides an explanation for the current results, like the biochemical experiments, it cannot explain how the cytoskeleton is linked to cadherin based at AJs. One possibility is that the link is not a static structure but rather it is maintained through a dynamic equilibrium based on the ability of β-catenin to recruit α-catenin, which in turn can recruit actin polymers to the LE. A second possibility is that, within this context, there are other proteins that provide the stable link between E-cadherin, α-catenin and actin. In either case it might be that dynamic structures such as the LE exploit these properties of the AJ components, because the chimera can rescue adhesion in other circumstances. It will be important to compare the dynamics and activity of the AJs in different contexts to gain some insight into how the regulatory properties of AJs are used in the modulation of morphogenetic processes (Gorfinkiel, 2007).

Regulatory mechanisms required for DE-cadherin function in cell migration and other types of adhesion

Cadherin-mediated adhesion can be regulated at many levels, as demonstrated by detailed analysis in cell lines. This study examines the requirements for Drosophila epithelial (DE) cadherin regulation in vivo. Investigating Drosophila oogenesis as a model system allowed the dissection of DE-cadherin function in several types of adhesion: cell sorting, cell positioning, epithelial integrity, and the cadherin-dependent process of border cell migration. Multiple fusions were generated between DE-cadherin and alpha-catenin as well as point-mutated ß-catenin and the ability of these fusion proteins to support these types of adhesion was analyzed. It was found that (1) although linking DE-cadherin to alpha-catenin is essential, regulation of the link is not required in any of these types of adhesion; (2) ß-catenin is required only to link DE-cadherin to alpha-catenin, and (3) the cytoplasmic domain of DE-cadherin has an additional specific function for the invasive migration of border cells, which is conserved to other cadherins. The nature of this additional function is discussed (Pacquelet, 2005).

Classic cadherin proteins have multiple essential roles during animal development both in keeping tissues/epithelia intact and in allowing dynamic cell rearrangements. One dramatic example of the latter is the invasive migration of border cells during oogenesis, for which DE-cadherin is essential. This study investigates which features of DE-cadherin are required for migration, and these features are compared with features that are required more generally for other adhesion functions. Cadherin proteins are well conserved from fly to man; the cytoplasmic domain, in particular, is well conserved, and it interacts with the cytoskeleton. Therefore, the in vivo genetic analyses focused on dissecting the functions of DE-cadherin cytoplasmic domain. In the type of in vivo replacement experiments performed, clear conclusions could be made about what is and is not required under physiological conditions. This is the strength of the analysis, and it is thought to be important to further the understanding of the much-studied cadherin molecules. Generally speaking, the idea cannot be excluded that a type of regulation that is not genetically required does, in fact, occur under normal conditions and contribute somewhat to regulation (e.g., to make the system more robust) (Pacquelet, 2005).

Focus was initially placed on a conserved tyrosine of ß-catenin, the phosphorylation of which may induce ß-catenin to dissociate from cadherin, resulting in a decrease of adhesion. This conserved tyrosine (and, hence, its phosphorylation) is not essential even during border cell migration. The idea that phosphorylation of this tyrosine residue happens or that it may induce some dissociation of ß-catenin from DE-cadherin cannot be excluded. What these results show is that such phosphorylation is not an essential mechanism for adhesion regulation in any of the tested types of cadherin-dependent adhesion in vivo. Significant emphasis has been put in the literature on the putative regulatory role of this conserved tyrosine of ß-catenin. However, much of this emphasis is based on correlations between ß-catenin tyrosine phosphorylation and adhesion down-regulation. It is not clear whether ß-catenin phosphorylation is really the cause of adhesion down-regulation. In addition, the tyrosine kinase Src causes a decrease of adhesion in L cells expressing the fusion protein E-cadherin/alpha-catenin. Thus, Src-induced adhesion down-regulation can be independent of ß-catenin phosphorylation. Therefore, the ability to regulate adhesion without phosphorylating ß-catenin tyrosine may be more general (Pacquelet, 2005).

Next, it was found that neither the link between DE-cadherin and ß-catenin nor that between ß- and alpha-catenin need be regulated at all for DE-cadherin function in vivo. A fusion between DE-cadherin-FL and alpha-catenin fully substitutes for endogenous DE-cadherin during oogenesis even in the absence of endogenous ß-catenin. It was surprising to find that there is no need to regulate the link between DE-cadherin and alpha-catenin, since earlier studies using similar fusion proteins had concluded that regulation was required for mouse E-cadherin to support 'intercellular migration'. There are two main explainations for this discrepancy. (1) The previous study did not fuse alpha-catenin to E-cadherin-FL but fused to a truncated E-cadherin (analogous to DE-cadherinΔß/alpha-catenin). As was found in the current study, this not only affects the ability to regulate the link to alpha-catenin but also removes additional functionality from cadherin. It was not directly investigated in Nagafuchi (1994) whether the defects were caused by ß-catenin regulation as proposed. (2) Different cell types were analyzed; the previous study overexpressed E-cadherin in mouse fibroblasts that normally have very little of the protein, whereas this study investigated cells that normally depend on DE-cadherin for biological function(Pacquelet, 2005).

It is possible that the link between DE-cadherin and the actin cytoskeleton does need to be regulated but that it occurs downstream of alpha-catenin. More studies of alpha-catenin and of how its interactions are regulated will be of interest, in particular in a physiological context. Alternatively, regulation of adhesion may primarily occur by the turning over of DE-cadherin and/or DE-cadherin complexes via endocytosis. A Cbl-related E3 ligase called Hakai has been identified as a specific regulator of mammalian E-cadherin endocytosis. It is recruited to specific phosphorylated tyrosines on E-cadherin. No evidence was found that the homologous D. melanogaster protein (CG10263) affects DE-cadherin or border cell migration, and the key docking tyrosines are not conserved. However, other regulators may play an analogous role. Finally, adhesive strength could be regulated by lateral clustering of cadherin complexes; for example, by the binding of additional regulatory proteins to the intracellular domain (Pacquelet, 2005).

The full functionality of DE-cadherin-FL/alpha-catenin in the absence of ß-catenin also indicates that ß-catenin has no essential adhesive function other than linking DE-cadherin to alpha-catenin. Based on the abnormal localization of various DE-cadherin mutants, it had been proposed that ß-catenin was required for proper translocation of cadherin to the plasma membrane. However, the relatively normal subcellular localization of DE-cadherin-FL/alpha-catenin that was observed in the absence of ß-catenin suggests that this is not generally the case. It remains possible that ß-catenin also contributes to modifying cadherin localization in D. melanogaster cells, but in a more subtle, nonessential way. This study suggests that parts of the cadherin tail that bind ß-catenin may also have ß-catenin-independent functions. This would complicate the interpretation of how modified cadherin molecules behave unless it is also investigated by ß-catenin loss-of-function experiments (Pacquelet, 2005).

In contrast with DE-cadherin-FL/alpha-catenin, a fusion protein between DE-cadherin and alpha-catenin lacking the DE-cadherin cytoplasmic tail (DE-cadherinΔCyt/alpha-catenin) could not substitute for DE-cadherin during border cell migration. It was targeted to the cell surface and was functional in all other contexts. This indicates that the DE-cadherin cytoplasmic tail has a specific function during invasive migration in addition to the basic ß-catenin/alpha-catenin linkage. The function could not be provided by an unrelated cytoplasmic linker (CD2) but could be provided by the corresponding region from mouse E-cadherin or D. melanogaster N-cadherin. Most likely, one or more interactions that are specific to cadherin tails have a critical function in this context. These results raise two questions: (1) why is DE-cadherin tail specifically important for border cell migration and (2) what is the molecular nature of the required function (Pacquelet, 2005)?

With regard to the specific requirement in border cells, the role of DE-cadherin in their migration needs to be considered. Given the absolute requirement for this particular cell-cell interaction to achieve invasive border cell movement, it is likely to be force bearing. DE-cadherin-mediated adhesion between the front of border cells and the attachment point on nurse cells needs to be strong enough to allow border cells to pull themselves into the compact germ line tissue. As the border cell cluster initiates migration using a long, slender cellular extension, the local force application at the tip may be quite high. As an illustration of the forces involved, it was found that mutant border cells with impaired cortical cytoskeleton will break apart when they attempt to invade, whereas other follicle cells (including centripetal cells) with the same defect appear to be relatively normal. It is suggested that the DE-cadherin tail may be required to allow a build-up of sufficiently strong adhesion to withstand forces that are involved in migration (Pacquelet, 2005).

Another important aspect of adhesion during cell movement is that it may need to be effectively down-regulated at the rear of the cells to allow cell translocation along the substrate. Experiments indicated that the primary defect for DE-cadherinΔCyt/alpha-catenin in border cells is not a lack of down-regulation; in other words, it is not caused by an excess of adhesion. However, an inability of DE-cadherinΔCyt/alpha-catenin to provide sufficient adhesion for migration as discussed above could mask possible additional (migration specific) defects of the fusion protein such as the ability to be down-regulated (Pacquelet, 2005).

The molecular nature of the DE-cadherin tail requirement in migration is in need of further investigation. The function does not simply map to any previously known signal or interaction, suggesting involvement of a novel interaction and/or a redundancy of interactions. The DE-cadherinΔß/alpha-catenin fusion results indicate that the most COOH-terminal domain contributes to DE-cadherin function in border cells independently of ß-catenin binding. However, this domain is not essential on its own nor when coexpressed with p120 catenin RNA interference constructs, indicating that additional important signals are located in the more proximal region of the DE-cadherin cytoplasmic domain. A mutant form of Xenopus laevis C-cadherin lacking the 94 proximal amino acids of its cytoplasmic domain can mediate some adhesion but is unable to support strong adhesion. This seems to be caused by its inability to form lateral clusters. Similarly, an absence of the proximal region in DE-cadherinΔ-Cyt/alpha-catenin could prevent its clustering and, thereby, prevent adhesion strengthening (Pacquelet, 2005).

In conclusion, this structure/function analysis of DE-cadherin in different types of cell adhesion has given new information about cadherin regulation in vivo. Several previously defined potential points of regulation that were established through detailed work in tissue culture were found not to be essential for functionality in vivo. The cytoplasmic tail of cadherin was found to have a unique role in the demanding process of invasive cell migration, possibly through a novel interaction (Pacquelet, 2005).

Rho1 interacts with p120^ctn and α-catenin, and regulates cadherin-based adherens junction components in Drosophila

Rho GTPases are important regulators of cellular behavior through their effects on processes such as cytoskeletal organization. Interactions have been studied between Drosophila Rho1 and the adherens junction components α-catenin and p120^ctn. While Rho1 protein is present throughout the cell, it accumulates apically, particularly at sites of cadherin-based adherens junctions. Cadherin and catenin localization is disrupted in Rho1 mutants, implicating Rho1 in their regulation. p120^ctn has recently been suggested to inhibit Rho activity through an unknown mechanism. This study found that Rho1 accumulates in response to lowered p120^ctn activity. Significantly, Rho1 was found to bind directly to α-catenin and p120^ctn in vitro, and these interactions map to distinct surface-exposed regions of the protein not previously assigned functions. In addition, both α-catenin and p120^ctn co-immunoprecipitate with Rho1-containing complexes from embryo lysates. These observations suggest that α-catenin and p120^ctn are key players in a mechanism of recruiting Rho1 to its sites of action (Magie, 2002).

The results indicate that in addition to Rho1's ubiquitous cytoplasmic expression, it accumulates at adherens junctions and is involved in regulating the proper localization of AJ components. Further, direct physical interactions occur between Rho1 and the catenins, p120^ctn and α-catenin. Isoprenylation at the C-terminal CAAX motif is involved in regulating the subcellular localization of Rho, however, binding to the catenins may represent another mechanism of recruiting Rho1 to its sites of action (Magie, 2002).

Rho1 activity is required to properly localize DE-cadherin during development, consistent with data from mammalian cell culture experiments implicating Rho and Rac in cadherin assembly and maintenance. The defects observed in cadherin localization are most prevalent in and around the leading edge (LE) cells undergoing dorsal closure. Previously Rho1 had been implicated in dorsal closure via its regulation of the LE actin cytoskeleton in cells flanking the segment borders. However, the disruption observed in cadherin distribution suggests that regulation of cell-cell adhesion may play a role in the dorsal closure phenotype observed in these embryos. Thus Rho1's effects on cadherin localization could be the result of a direct role in DE-cadherin clustering, or an indirect effect on the cortical actin cytoskeleton. The process of AJ formation in keratinocytes has been shown to require actin polymerization and the interdigitation of filopodia from neighboring cells. A similar interdigitation of filopodia is seen during dorsal closure in Drosophila and is likely involved in forming adhesive contacts between the two epithelial fronts. Since Rho and Cdc42 have been shown to act antagonistically in the formation of cellular processes in neurons, it is possible that disrupting the balance of Rho1 and Cdc42 function in LE cells results in inappropriate regulation of filopodial extensions. This could partially explain the disruption of DE-cadherin localization observed in Rho1 mutants. Alternatively, Rho1's primary role could be in directly regulating the adhesion of cells near the LE, with Rac and Cdc42 acting as the major organizers of the acto-myosin network (Magie, 2002).

In addition to the accumulation of Rho1 protein at sites of cadherin localization, a direct physical interaction was observed between Rho1 and both p120^ctn and α-catenin. The catenin family of proteins is important in regulating cadherin-based adhesion and linking cadherins to the actin cytoskeleton. ß-catenin binds to the catenin-binding domain of the cadherin molecule as well as to α-catenin. α-catenin, in turn, acts as a link to the actin cytoskeleton, either by directly binding actin filaments or through association with other actin-binding proteins. α-catenin also has been shown to bind spectrin, a major component of the membrane skeleton underlying the plasma membrane involved in stabilizing it and determining cell shape. Human colon carcinoma Clone A cells that contain mutant α-catenin have defects in spectrin assembly. Consistent with this, a breakdown of the α-spectrin cytoskeleton was oberved in embryos injected with α-catenin dsRNA, especially in morphogenetically active cells early in gastrulation. α-catenin protein is enriched at adherens junctions, but is not as strictly localized to them as is DE-cadherin. Binding of α-catenin to Rho1 may be a general mechanism through which Rho1 is recruited to the plasma membrane (Magie, 2002).

p120^ctn regulates the adhesive properties of cadherin complexes through its binding to the juxtamembrane domain of the cadherin molecule, although the precise mechanisms underlying this function are not known. p120^ctn also acts in the cytoplasm where it has been proposed to negatively regulate Rho activation in a manner similar to the GDI proteins, which prevent Rho from exchanging GDP for GTP, although it shares no sequence homology with them. The binding of p120^ctn to cadherins and its effects on Rho function have been shown to be mutually exclusive, such that once p120^ctn binds a cadherin molecule, it is no longer capable of inhibiting Rho activity or function. Rho would then be accessible to activating regulatory proteins such as GEFs, and could carry out its downstream functions. The physical interaction observed between Rho1 and p120^ctn suggests that this negative regulation of Rho1 is due to direct binding of p120^ctn to GDP-Rho1. Interestingly, this is the same face of the Rho protein that has been shown to bind to classical GDIs, consistent with the idea that despite the lack of sequence homology, p120^ctn may be acting in a similar way. Overexpression of p120^ctn in mammalian cells leads to an inhibition of Rho activity. Overexpression of p120^ctn in the system used in this study enhances the Rho1 mutant phenotype, as would be expected for a negative regulator. Embryos homozygous for a deficiency uncovering the p120^ctn locus show an accumulation of Rho1 protein at the leading edge and exhibit a severe dorsal open phenotype. A similar accumulation of Rho1 protein is observed in embryos injected with p120^ctn dsRNA. A positive feedback mechanism may be functioning whereby the relief of p120^ctn-mediated regulation in those cells results in the upregulation of Rho1 protein or an increase in Rho1 stability. It has recently been shown that overexpression of a RhoGDI in the hearts of mouse embryos results in the upregulation of RhoA expression, suggesting the existence of a negative feedback mechanism in the regulation of RhoA levels, although there are no other instances in which a positive feedback mechanism has been linked to Rho expression. Excess Rho activity disrupts cellular migration; cells at the leading edge in embryos that lack p120^ctn function remain cuboidal, rather than elongating as they would during normal dorsal closure, suggesting that Rho1 may be involved in regulating these cell shape changes. Alternatively, p120^ctn has been suggested to activate Rac and Cdc42 in the cytoplasm through an interaction with the GEF Vav2, and this could account for some of its effects on cell morphology. The observation that Rho1 can bind both p120^ctn and α-catenin and that their binding sites are not overlapping suggests that either could be involved in recruiting Rho1 to AJs or the plasma membrane in general. The data indicating that overexpression of α-catenin enhances the Rho1 mutant phenotype to a greater degree than p120^ctn suggests an important role for α-catenin in Rho1 function, perhaps as a factor generally involved in localizing Rho1 to its sites of action, while p120^ctn plays a more specific role at AJs (Magie, 2002).

The data suggest a model in which p120^ctn or α-catenin or both are involved in recruiting Rho1 to sites of cadherin localization, where it can then be activated and carry out its functions, including proper AJ formation. If Rho1 is not recruited properly, as in the case of a Rho1 mutant, this results in mislocalization of AJ components. The binding of p120^ctn to Rho1, either in the cytoplasm or while Rho1 is tethered at AJs through its interaction with α-catenin, inhibits the exchange of GDP for GTP and keeps Rho1 in an inactive state. The binding of p120^ctn to the juxtamembrane domain may release Rho1, allowing it to be activated by GEFs. GTP-Rho1 could then bind its downstream effectors and either directly regulate DE-cadherin assembly or maintenance, or indirectly affect AJ formation through its effects on the actin cytoskeleton. Rho1 localization at AJs could then be mediated either through continued association with α-catenin or through isoprenylation and insertion into the plasma membrane. Mutational analysis aimed at distinguishing between these models will provide further insight into this important feature of Rho1 function during morphogenesis (Magie, 2002).

Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dalpha-catenin-GFP fusion protein

Cell-cell adherens junctions (AJs), comprised of the cadherin-catenin adhesion system, contribute to cell shape changes and cell movements in epithelial morphogenesis. However, little is known about the dynamic features of AJs in cells of the developing embryo. In this study, Dalpha-catenin fused with a green fluorescent protein (Dalpha-catenin-GFP) was constructed, and found to be targeted to apically located AJ-based contacts but not other lateral contacts in epithelial cells of living Drosophila embryos. Using time-lapse fluorescence microscopy, the dynamic performance of AJs containing Dalpha-catenin-GFP in epithelial morphogenetic movements was examined. In the ventral ectoderm of stage 11 embryos, concentration and deconcentration of Dalpha-catenin-GFP occurs concomitantly with changes in length of AJ contacts. In the lateral ectoderm of embryos at the same stage, dynamic behavior of AJs is concerted with division and delamination of sensory organ precursor (SOP) cells. Moreover, changes in patterns of AJ networks during tracheal extension can be followed. Finally, Dalpha-catenin-GFP was used to precisely observe the defects in tracheal fusion in shotgun mutants. Thus, the Dalpha-catenin-GFP fusion protein is a helpful tool to simultaneously observe morphogenetic movements and AJ dynamics at high spatio-temporal resolution (Oda, 1999).

The lateral ectoderm of living stage 11 embryos of arm-GAL4:UAS-DalphaC-GFP was studied. At early stage 11, many cell divisions occur in the lateral ectoderm. Dividing epithelial cells show dynamic behavior of Dalpha-catenin-GFP. Before cytokinesis, Dalpha-catenin-GFP is distributed in a line at the equator of spherical cells. During cytokinesis, Dalpha-catenin GFP-positive lines of contact between the dividing cell and surrounding cells can be seen. At the end of cytokinesis, new AJ contacts where Dalpha-catenin-GFP has begun to be concentrated appear to be established between the daughter cells. Pairs of divided cells that had delaminated from the surface ectodermal layer were followed. Considering their positions and behavior, these cells seemed to be sensory organ precursor (SOP) cells. After cytokinesis, two daughter cells of a primary SOP cell recover features characteristic of epithelial cells. The cells are indistinguishable in the Dalpha-catenin-GFP distribution pattern from the other ectodermal cells. About 30 min later, the paired SOP cells simultaneously begin to reduce their apical surface area. They constrict their apices smoothly once the constriction begins. It takes 5-10 min for completion of the apical constriction phase. Before entering the constriction phase, Dalpha-catenin-GFP is relatively evenly distributed at apical cell-cell contacts. During the constriction phase, however, Dalpha-catenin-GFP is unevenly distributed at the apical portions of delaminating SOP cells. After SOP cells disappear from the embryo surface, high concentrations of Dalpha-catenin-GFP are left between epithelial cells which have surrounded SOP cells. Eventually, the remaining epithelial cells appear to recover characteristic mesh patterns of Dalpha-catenin-GFP on the apical surface (Oda, 1999).

These observations seem to reveal a typical series of cellular events in the development of the early embryonic peripheral nervous system (PNS). Division of an SOP cell precedes delamination from the surface ectodermal layer. Although SOP delamination seems to be closely correlated with mitosis, the two events are temporally separate. The delamination is coincident with the apical constriction presumably caused by dynamic functions of AJs. This kind of cellular behavior is commonly observed in a variety of morphogenetic events. For example, the behavior of delaminating SOP cells is reminiscent of that of invaginating presumptive mesodermal cells at the beginning of gastrulation. The invagination process of the mesoderm takes less than 10 min. The time course of mesoderm invagination is comparable to that of SOP delamination. During mesoderm invagination, apically located AJs are broken coinciding with apical constriction. It is possible that a similar AJ disruption event occurs in SOP cells during the apical constriction phase (Oda, 1999).

Tracheal morphogenesis shows dynamic aspects of DE-cadherin-based cell-cell adhesion. GFP fluorescence of btl-GAL4;UAS-DaC-GFP#3 embryos were observed to directly investigate AJ dynamics during tracheal extension. Concentration of GFP fluorescence can be detected at portions corresponding to AJs in tracheal primordia from stage 11, although signals are also seen uniformly in the cytoplasm but not in nuclei. Tracheal development is not affected by expression of Dalpha-catenin-GFP. Tracheal tissues are located on the inside of the embryo and therefore it has been difficult to examine changes in cell arrangements in living embryos. However, targeted expression of Dalpha-catenin-GFP using btl-GAL4 overcomes this difficulty. Time-lapse observation reveals successive changes in patterns of AJ networks containing Dalpha-catenin-GFP during tracheal extension, although it was still difficult to precisely follow changes in the three-dimensional patterns of AJ networks. Each tracheal primordium elongates along the dorso-ventral axis and the distance between the neighboring tracheal primordia is seen to shorten while the germband is retracting. Tracheal branches extend, coinciding with elongation of Dalpha-catenin GFP-labelled lines. Notably, relatively intense signals for Dalpha-catenin-GFP are often seen around the tips of extending branches. Contact of dorsal trunk (DT) cells between neighboring segments occurs shortly before completion of germband retraction. Soon after the occurrence of this contact, lines of weak Dalpha-catenin-GFP signals can be detected that run through areas of intersegmental DT contact. A previous study has shown that DE-cadherin and Dalpha-catenin are concentrated in tip cells, contributing to the generation of new AJ-based contacts between the cells that give rise to a pore that connects DT lumina. Although it could not be directly determine whether the lines of Dalpha-catenin-GFP accumulation are in tip cells, it is possible that Dalpha-catenin-GFP signals are primary signs of establishing contacts between tip cells of DT (Oda, 1999).

Dalpha-catenin-GFP was used to analyse tracheal phenotypes of zygotic shg mutants. Mutant embryos could be unambiguously identified by GFP fluorescence in tracheal primordia without any staining. Moreover, targeted expression of Dalpha-catenin-GFP allows clear visualization of morphological defects in the trachea of shg mutants. Epithelial integrity and extension of the tracheal primordia are relatively normal at earlier stages of tracheal development (stages 11-13) probably because of the contribution of maternally supplied functional DE-cadherin molecules. Consistently, considerable amounts of Da-catenin-GFP are detected at apical cell-cell contacts even in shg zygotic mutants although the levels of its accumulation at cell contact sites are reduced compared to those in normal embryos. In normal DT fusion, DE-cadherin-based cell-cell contacts are newly established between tip cells, giving rise to ring-like patterns of cadherin-based junctions, which are visualized by Dalpha-catenin-GFP. In contrast, the process of DT fusion is specifically blocked in shg mutants. At fusion points, tube structures are reduced in diameter or are not constructed at all. Dalpha-catenin-GFP does not accumulate between tip cells of DT in shg mutants probably because a lack of zygotic DE-cadherin causes failure in establishment of new AJ-based contacts between the cells. These results indicate that the accumulation of Dalpha-catenin-GFP at apical contact sites between tip cells is dependent on zygotic expression of normal DE-cadherin. Tracheal fusion is a process in which new apical surface domains facing the lumen are established (Oda, 1999).

These observations suggested that DE-cadherin is required not only for establishment of AJ-based contacts but also for generation and definition of apical cell domains. In summary, Dalpha-catenin-GFP is a new tool that enables simultaneous visualization of morphogenetic movements and behavior of AJs or the cadherin-based adhesion system in living wild-type and mutant Drosophila embryos. These observations revealed the dynamic performance of AJ-based cell contacts. The methods used in this study will facilitate analysis of the dynamics of cell-cell adhesion at high spatio-temporal resolution in living animals (Oda, 1999).

Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation

During Drosophila gastrulation, morphogenesis occurs as a series of cell shape changes and cell movements which probably involve adhesive interactions between cells. This study examined the dynamic aspects of cadherin-based cell-cell adhesion in the morphogenetic events to assess its contribution to morphogenesis. DE- and DN-cadherin show complementary expression patterns in the presumptive ectoderm and mesoderm at the mRNA level. It was found that switching of cadherin expression from the DE- to the DN-type in the mesodermal germ layer occurred downstream of the mesoderm-determination genes twist and snail. However, examination of their protein expression patterns showed that considerable amounts of DE-cadherin remained on the surfaces of mesodermal cells during invagination, while DN-cadherin did not appear on the cell surfaces at this stage. Further immunocytochemical analysis of the localizations of DE-cadherin and its associated proteins Armadillo (beta-catenin) and Dalpha-catenin revealed dynamic changes in their distributions which were accompanied by changes in cell morphology in the neuroectoderm and mesoderm. Simultaneously, adherens junctions (AJs), based on the cadherin-catenin system, were shown to change their location, size, and morphology. These dynamic aspects of cadherin-based cell-cell adhesion appeared to be associated with the following: (1) initial establishment of the blastoderm epithelium, (2) acquisition of cell motility in the neuroectoderm, (3) cell sheet folding, and (4) epithelial to mesenchymal conversion of the mesoderm. These observations suggest that the behavior of the DE-cadherin-catenin adhesion system may be regulated in a stepwise manner during gastrulation to perform successive cell-morphology conversions. Moreover, the processes responsible for loss of epithelial cell polarity and elimination of preexisting DE-cadherin-based epithelial junctions during early mesodermal morphogenesis are discussed (Oda, 1998).

Drosophila alpha-catenin and E-cadherin bind to distinct regions of Drosophila Armadillo

Adherens junctions are multiprotein complexes mediating cell-cell adhesion and communication. They are organized around a transmembrane cadherin, which binds a set of cytoplasmic proteins required for adhesion and to link the complex to the actin cytoskeleton. Three components of Drosophila adherens junctions, analogous to those in vertebrates, have been identified: Armadillo (homolog of beta-catenin), Drosophila E-cadherin (DE-cadherin), and α-catenin. This study describes the first analysis of the interactions between these proteins using in vitro binding assays, the yeast two-hybrid system, and in vivo assays. A 76-amino acid region of Armadillo was identified that is necessary and sufficient for binding alpha-catenin and it was found that the N-terminal 258 amino acids of alpha-catenin interact with Armadillo. A large region of Armadillo, spanning six central Armadillo repeats, is required for DE-cadherin binding, whereas only 41 amino acids of the DE-cadherin cytoplasmic tail are sufficient for Armadillo binding. These data complement and extend results obtained in studies of vertebrate adherens junctions, providing a foundation for understanding how junctional proteins assemble and a basis for interpreting existing mutations and creating new ones (Pai, 1996).

Identification of a Drosophila homologue of alpha-catenin and its association with the armadillo protein

The cadherin cell adhesion system plays a central role in cell-cell adhesion in vertebrates, but its homologues are not identified in the invertebrate. alpha-Catenins are a group of proteins associated with cadherins, and this association is crucial for the cadherins' function. This study reports the cloning of a Drosophila alpha-catenin gene by low stringent hybridization with a mouse alpha E-catenin probe. Isolated cDNAs encoded a 110-kD protein with 60% identity to mouse alpha E-catenin, and this protein was termed D alpha-catenin. The gene of this protein was located at the chromosome band 80B. Immunostaining analysis using a mAb to D alpha-catenin revealed that it was localized to cell-cell contact sites, expressed throughout development and present in a wide variety of tissues. When this protein was immunoprecipitated from detergent extracts of Drosophila embryos or cell lines, several proteins co-precipitated. These included the armadillo product which was known to be a Drosophila homologue of beta-catenin, another cadherin-associated protein in vertebrates, and a 150-kD glycoprotein. These results strongly suggest that Drosophila has a cell adhesion machinery homologous to the vertebrate cadherin-catenin system (Oda, 1993).

Search PubMed for articles about αCatenin

Alegot, H., Markosian, C., Rauskolb, C., Yang, J., Kirichenko, E., Wang, Y. C. and Irvine, K. D. (2019). Recruitment of Jub by alpha-catenin promotes Yki activity and Drosophila wing growth. J Cell Sci. PubMed ID: 30659113

Benjamin, J. M., Kwiatkowski, A. V., Yang, C., Korobova, F., Pokutta, S., Svitkina, T., Weis, W. I. and Nelson, W. J. (2010). αE-catenin regulates actin dynamics independently of cadherin-mediated cell-cell adhesion. J Cell Biol 189: 339-352. PubMed ID: 20404114

Borghi, N., Sorokina, M., Shcherbakova, O. G., Weis, W. I., Pruitt, B. L., Nelson, W. J. and Dunn, A. R. (2012). E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch. Proc Natl Acad Sci U S A 109: 12568-12573. PubMed ID: 22802638

Brasch, J., Harrison, O. J., Honig, B. and Shapiro, L. (2012). Thinking outside the cell: how cadherins drive adhesion. Trends Cell Biol 22: 299-310. PubMed ID: 22555008

Cavey, M., Rauzi, M., Lenne, P. F. and Lecuit, T. (2008). A two-tiered mechanism for stabilization and immobilization of E-cadherin. Nature 453: 751-756. PubMed ID: 18480755

Choi, H. J., Pokutta, S., Cadwell, G. W., Bobkov, A. A., Bankston, L. A., Liddington, R. C. and Weis, W. I. (2012). alphaE-catenin is an autoinhibited molecule that coactivates vinculin. Proc Natl Acad Sci U S A 109: 8576-8581. PubMed ID: 22586082

Choi, S. H., Estaras, C., Moresco, J. J., Yates, J. R. and Jones, K. A. (2013). alpha-Catenin interacts with APC to regulate beta-catenin proteolysis and transcriptional repression of Wnt target genes. Genes Dev 27: 2473-2488. PubMed ID: 24240237

Desai, R., Sarpal, R., Ishiyama, N., Pellikka, M., Ikura, M. and Tepass, U. (2013). Monomeric α-catenin links cadherin to the actin cytoskeleton. Nat Cell Biol 15: 261-273. PubMed ID: 23417122

Dickinson, D. J., Nelson, W. J. and Weis, W. I. (2011). A polarized epithelium organized by beta- and α-catenin predates cadherin and metazoan origins. Science 331: 1336-1339. PubMed ID: 21393547

Drees, F., Pokutta, S., Yamada, S., Nelson, W. J. and Weis, W. I. (2005). α-catenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament assembly. Cell 123: 903-915. PubMed ID: 16325583

Fic, W., Bastock, R., Raimondi, F., Los, E., Inoue, Y., Gallop, J. L., Russell, R. B. and St Johnston, D. (2021). RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion. J Cell Biol 220(4). PubMed ID: 33646271

Gorfinkiel, N. and Arias, A. M. (2007). Requirements for adherens junction components in the interaction between epithelial tissues during dorsal closure in Drosophila. J Cell Sci 120: 3289-3298. PubMed ID: 17878238

Guillot, C. and Lecuit, T. (2013). Adhesion disengagement uncouples intrinsic and extrinsic forces to drive cytokinesis in epithelial tissues. Dev Cell 24: 227-241. PubMed ID: 23410938

Imamura, Y., Itoh, M., Maeno, Y., Tsukita, S. and Nagafuchi, A. (1999). Functional domains of α-catenin required for the strong state of cadherin-based cell adhesion. J Cell Biol 144: 1311-1322. PubMed ID: 10087272

Jurado, J., de Navascues, J. and Gorfinkiel, N. (2016). α-Catenin stabilises Cadherin-Catenin complexes and modulates actomyosin dynamics to allow pulsatile apical contraction. J Cell Sci 129: 4496-4508. PubMed ID: 27831494

Klompstra, D., Anderson, D. C., Yeh, J. Y., Zilberman, Y. and Nance, J. (2015). An instructive role for C. elegans E-cadherin in translating cell contact cues into cortical polarity. Nat Cell Biol 17(6): 726-735. PubMed ID: 25938815

Kobielak, A. and Fuchs, E. (2004). α-catenin: at the junction of intercellular adhesion and actin dynamics. Nat Rev Mol Cell Biol 5: 614-625. PubMed ID: 15366705

Kwiatkowski, A. V., Maiden, S. L., Pokutta, S., Choi, H. J., Benjamin, J. M., Lynch, A. M., Nelson, W. J., Weis, W. I. and Hardin, J. (2010). In vitro and in vivo reconstitution of the cadherin-catenin-actin complex from Caenorhabditis elegans. Proc Natl Acad Sci U S A 107: 14591-14596. PubMed ID: 20689042

Lecuit, T., Lenne, P. F. and Munro, E. (2011). Force generation, transmission, and integration during cell and tissue morphogenesis. Annu Rev Cell Dev Biol 27: 157-184. PubMed ID: 21740231

le Duc, Q., Shi, Q., Blonk, I., Sonnenberg, A., Wang, N., Leckband, D. and de Rooij, J. (2010). Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II-dependent manner. J Cell Biol 189: 1107-1115. PubMed ID: 20584916

Magie, C. R., Pinto-Santini, D. and Parkhurst, S. M. (2002). Rho1 interacts with p120ctn and alpha-catenin, and regulates cadherin-based adherens junction components in Drosophila. Development 129: 3771-3782. PubMed ID: 12135916

Maiden, S. L. and Hardin, J. (2011). The secret life of α-catenin: moonlighting in morphogenesis. J Cell Biol 195: 543-552. PubMed ID: 22084304

Nagafuchi, A., Ishihara, S. and Tsukita, S. (1994). The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-alpha catenin fusion molecules. J Cell Biol 127: 235-245. PubMed ID: 7929566

Oda, H., Uemura, T., Shiomi, K., Nagafuchi, A., Tsukita, S. and Takeichi, M. (1993). Identification of a Drosophila homologue of alpha-catenin and its association with the armadillo protein. J Cell Biol 121: 1133-1140. PubMed ID: 8501118

Oda, H., Tsukita, S. and Takeichi, M. (1998). Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation. Dev Biol 203: 435-450. PubMed ID: 9808792

Oda, H. and Tsukita, S. (1999). Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dalpha-catenin-GFP fusion protein. Dev Genes Evol 209: 218-225. PubMed ID: 10079365

Ozawa, M. (1998). Identification of the region of α-catenin that plays an essential role in cadherin-mediated cell adhesion. J Biol Chem 273: 29524-29529. PubMed ID: 9792660

Pacquelet, A. and Rorth, P. (2005). Regulatory mechanisms required for DE-cadherin function in cell migration and other types of adhesion. J Cell Biol 170: 803-812. PubMed ID: 16129787

Pai, L. M., Kirkpatrick, C., Blanton, J., Oda, H., Takeichi, M. and Peifer, M. (1996). Drosophila alpha-catenin and E-cadherin bind to distinct regions of Drosophila Armadillo. J Biol Chem 271: 32411-32420. PubMed ID: 8943306

Ratheesh, A., Gomez, G. A., Priya, R., Verma, S., Kovacs, E. M., Jiang, K., Brown, N. H., Akhmanova, A., Stehbens, S. J. and Yap, A. S. (2012). Centralspindlin and alpha-catenin regulate Rho signalling at the epithelial zonula adherens. Nat Cell Biol 14: 818-828. PubMed ID: 22750944

Rauzi, M., Lenne, P. F. and Lecuit, T. (2010). Planar polarized actomyosin contractile flows control epithelial junction remodelling. Nature 468: 1110-1114. PubMed ID: 21068726

Sarpal, R., Pellikka, M., Patel, R. R., Hui, F. Y., Godt, D. and Tepass, U. (2012). Mutational analysis supports a core role for Drosophila α-catenin in adherens junction function. J Cell Sci 125: 233-245. PubMed ID: 22266901

Sarpal, R., Yan, V., Kazakova, L., Sheppard, L., Yu, J. C., Fernandez-Gonzalez, R. and Tepass, U. (2019). Role of alpha-Catenin and its mechanosensing properties in regulating Hippo/YAP-dependent tissue growth. PLoS Genet 15(11): e1008454. PubMed ID: 31697683

Shahbazi, M. N., Megias, D., Epifano, C., Akhmanova, A., Gundersen, G. G., Fuchs, E. and Perez-Moreno, M. (2013). CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions. J Cell Biol 203: 1043-1061. PubMed ID: 24368809

Shapiro, L. and Weis, W. I. (2009). Structure and biochemistry of cadherins and catenins. Cold Spring Harb Perspect Biol 1: a003053. PubMed ID: 20066110

Spahn, P., Ott, A. and Reuter, R. (2012). The PDZ-GEF protein Dizzy regulates the establishment of adherens junctions required for ventral furrow formation in Drosophila. J Cell Sci 125: 3801-3812. PubMed ID: 22553205

Taguchi, K., Ishiuchi, T. and Takeichi, M. (2011). Mechanosensitive EPLIN-dependent remodeling of adherens junctions regulates epithelial reshaping. J Cell Biol 194: 643-656. PubMed ID: 21844208

Wang, Y. C., Khan, Z., Kaschube, M. and Wieschaus, E. F. (2012). Differential positioning of adherens junctions is associated with initiation of epithelial folding. Nature 484: 390-393. PubMed ID: 22456706

Wang, Y. C., Khan, Z. and Wieschaus, E. F. (2013). Distinct Rap1 activity states control the extent of epithelial invagination via α-Catenin. Dev Cell 25: 299-309. PubMed ID: 23623612

Watabe-Uchida, M., Uchida, N., Imamura, Y., Nagafuchi, A., Fujimoto, K., Uemura, T., Vermeulen, S., van Roy, F., Adamson, E. D. and Takeichi, M. (1998). α-Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells. J Cell Biol 142: 847-857. PubMed ID: 9700171

Weis, W. I. and Nelson, W. J. (2006). Re-solving the cadherin-catenin-actin conundrum. J Biol Chem 281: 35593-35597. PubMed ID: 17005550

Yamada, S., Pokutta, S., Drees, F., Weis, W. I. and Nelson, W. J. (2005). Deconstructing the cadherin-catenin-actin complex. Cell 123: 889-901. PubMed ID: 16325582

Yang, C.C., Graves, H.K., Moya, I.M., Tao, C., Hamaratoglu, F., Gladden, A.B. and Halder, G. (2015). Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules. Proc Natl Acad Sci USA 112(6):1785-90. PubMed ID: 25624491

Yonemura, S., Wada, Y., Watanabe, T., Nagafuchi, A. and Shibata, M. (2010). α-Catenin as a tension transducer that induces adherens junction development. Nat Cell Biol 12: 533-542. PubMed ID: 20453849

date revised: 25 April 2024

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