shotgun: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - shotgun

Synonyms - DE-cadherin

Cytological map position - 57B13-14

Function - cell adhesion

Keyword(s) - epithelial junction protein, tumor suppressor

Symbol - shg

FlyBase ID:FBgn0003391

Genetic map position -

Classification - E-cadherin

Cellular location - surface transmembrane protein

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Nishiguchi, S., Yagi, A., Sakai, N. and Oda, H. (2016). Divergence of structural strategies for E-cadherin homophilic binding among bilaterians. J Cell Sci [Epub ahead of print]. PubMed ID: 27422100
Homophilic binding of E-cadherins through their ectodomains is fundamental to epithelial cell-cell adhesion. Despite this, E-cadherin ectodomains have evolved differently in the vertebrate and hexapod lineages. Of the five rod-like, tandemly aligned extracellular cadherin domains (ECs) of vertebrate E-cadherin, the tip EC plays a pivotal role in binding interactions. Comparatively, the N-terminal six consecutive ECs of Drosophila E-cadherin, DE-cadherin, can mediate adhesion; however, the underlying mechanism is unknown. This study reports atomic force microscopy imaging of DE-cadherin ECs. A tightly folded globular structure formed by the four N-terminal-most ECs stabilized by the subsequent two ECs was identified. Analysis of hybrid cadherins of different hexapods indicated association of the E-cadherin globular portion with the determinants of homophilic binding specificity. The second to fourth ECs were identified as the minimal portion capable of mediating exclusive homophilic binding specificity. These findings suggested that the N-terminal-most four ECs of hexapod E-cadherin are functionally comparable with the N-terminal-most single EC of vertebrate E-cadherin, but that their mechanisms might significantly differ. This work illuminates the divergence of structural strategies for E-cadherin homophilic binding among bilaterians.
Teng, X., Qin, L., Le Borgne, R. and Toyama, Y. (2016).. Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium. Development [Epub ahead of print]. PubMed ID: 27888195
Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, the contraction of actomyosin cables formed in apoptotic and neighboring cells drive cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. A model according which modulation of tissue tension represents a mechanism of apoptotic cell extrusion, and would further influence biochemical signals of neighboring non-apoptotic cells.
Doyle, S. E., Pahl, M. C., Siller, K. H., Ardiff, L. and Siegrist, S. E. (2017). Neuroblast niche position is controlled by PI3-kinase dependent DE-Cadherin adhesion. Development [Epub ahead of print]. PubMed ID: 28126840
Correct positioning of stem cells within their niche is essential for tissue morphogenesis and homeostasis. Yet how stem cells acquire and maintain niche position remains largely unknown. This study shows that a subset of brain neuroblasts (NBs) in Drosophila utilize PI3-kinase and DE-cadherin to build adhesive contact for NB niche positioning. NBs remain within their native microenvironment when levels of PI3-kinase activity and DE-cadherin are elevated in NBs. This occurs through PI3-kinase dependent regulation of DE-Cadherin mediated cell adhesion between NBs and neighboring cortex glia, and between NBs and their GMC daughters. When levels of PI3-kinase activity and/or DE-Cadherin are reduced in NBs, NBs lose niche position and relocate to a non-native brain region that is rich in neurosecretory neurons, including those that secrete some of the Drosophila insulin-like peptides. Linking levels of PI3-kinase activity to strength of adhesive attachment could provide cancer stem cells and hematopoietic stem cells a means to cycle from trophic-poor to trophic-rich microenvironments.
Simoes, S., Oh, Y., Wang, M. F., Fernandez-Gonzalez, R. and Tepass, U. (2017). Myosin II promotes the anisotropic loss of the apical domain during Drosophila neuroblast ingression. J Cell Biol [Epub ahead of print]. PubMed ID: 28363972
Epithelial-mesenchymal transitions play key roles in development and cancer and entail the loss of epithelial polarity and cell adhesion. This study used quantitative live imaging of ingressing neuroblasts (NBs) in Drosophila melanogaster embryos to assess apical domain loss and junctional disassembly. Ingression is independent of the Snail family of transcriptional repressors and down-regulation of Drosophila E-cadherin (DEcad) transcription. Instead, the posttranscriptionally regulated decrease in DEcad coincides with the reduction of cell contact length and depends on tension anisotropy between NBs and their neighbors. A major driver of apical constriction and junctional disassembly are periodic pulses of junctional and medial myosin II that result in progressively stronger cortical contractions during ingression. Effective contractions require the molecular coupling between myosin and junctions and apical relaxation of neighboring cells. Moreover, planar polarization of myosin leads to the loss of anterior-posterior junctions before the loss of dorsal-ventral junctions. It is concluded that planar-polarized dynamic actomyosin networks drive apical constriction and the anisotropic loss of cell contacts during NB ingression.
Lai, C. M., Lin, K. Y., Kao, S. H., Chen, Y. N., Huang, F. and Hsu, H. J. (2017). Hedgehog signaling establishes precursors for germline stem cell niches by regulating cell adhesion. J Cell Biol [Epub ahead of print]. PubMed ID: 28363970
Stem cells require different types of supporting cells, or niches, to control stem cell maintenance and differentiation. However, little is known about how those niches are formed. This study reports that in the development of the Drosophila melanogaster ovary, the Hedgehog (Hh) gradient sets differential cell affinity for somatic gonadal precursors to specify stromal intermingled cells, which contributes to both germline stem cell maintenance and differentiation niches in the adult. Traffic Jam (an orthologue of a large Maf transcription factor in mammals) is a novel transcriptional target of Hh signaling to control cell-cell adhesion by negative regulation of E-cadherin expression. These results demonstrate the role of Hh signaling in niche establishment by segregating somatic cell lineages for differentiation.
Cristo, I., Carvalho, L., Ponte, S. and Jacinto, A. (2018). Novel role for Grainy head in the regulation of cytoskeletal and junctional dynamics during epithelial repair. J Cell Sci 131(17). PubMed ID: 30131442
Tissue repair is critical for the maintenance of epithelial integrity and permeability. Simple epithelial repair relies on a combination of collective cell movements and the action of a contractile actomyosin cable at the wound edge that together promote the fast and efficient closure of tissue discontinuities. The Grainy head family of transcription factors (Grh in flies; GRHL1-GRHL3 in mammals) are essential proteins that have been implicated both in the development and repair of epithelia. However, the genes and the molecular mechanisms that it controls remain poorly understood. This study shows that Grh knockdown disrupts actomyosin dynamics upon injury of the Drosophila pupa epithelial tissue. This leads to the formation of an ectopic actomyosin cable away from the wound edge and impaired wound closure. It was also uncovered that E-Cadherin is downregulated in the Grh-depleted tissue around the wound, likely as a consequence of Dorsal (an NF-kappaB protein) misregulation, which also affects actomyosin cable formation. This work highlights the importance of Grh as a stress response factor and its central role in the maintenance of epithelial characteristics necessary for tissue repair through regulating cytoskeleton and E-Cadherin dynamics.
Roper, J. C., Mitrossilis, D., Stirnemann, G., Waharte, F., Brito, I., Fernandez-Sanchez, M. E., Baaden, M., Salamero, J. and Farge, E. (2018). The major beta-catenin/E-cadherin junctional binding site is a primary molecular mechano-transductor of differentiation in vivo. Elife 7. PubMed ID: 30024850
In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. This study finds the molecular stretching of the highly conserved Y654-beta-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the beta-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two alpha-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of beta-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the beta-catenin pathway process.
Vanderleest, T. E., Smits, C. M., Xie, Y., Jewett, C. E., Blankenship, J. T. and Loerke, D. (2018). Vertex sliding drives intercalation by radial coupling of adhesion and actomyosin networks during Drosophila germband extension. Elife 7. PubMed ID: 29985789
Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cell-cell interfaces, while the function of tricellular vertices has remained unaddressed. This study identifies a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation. Through systematic analysis, it was found that the motion of vertices connected by contracting interfaces is not physically coupled, but instead possess strong radial coupling. E-cadherin and Myosin II exist in previously unstudied populations at cell vertices and undergo oscillatory cycles of accumulation and dispersion that are coordinated with changes in cell area. Additionally, peak enrichment of vertex E-cadherin/Myosin II coincides with interface length stabilization. These results suggest a model in which asymmetric radial force balance directs the progressive, ratcheted motion of individual vertices to drive intercalation.
Suisse, A. and Treisman, J. E. (2019). Reduced SERCA function preferentially affects Wnt signaling by retaining E-Cadherin in the endoplasmic reticulum. Cell Rep 26(2): 322-329. PubMed ID: 30625314
Calcium homeostasis in the lumen of the endoplasmic reticulum is required for correct processing and trafficking of transmembrane proteins, and defects in protein trafficking can impinge on cell signaling pathways. This study shows that mutations in the endoplasmic reticulum calcium pump SERCA disrupt Wingless signaling by sequestering Armadillo/beta-catenin away from the signaling pool. Armadillo remains bound to E-cadherin, which is retained in the endoplasmic reticulum when calcium levels there are reduced. Using hypomorphic and null SERCA alleles in combination with the loss of the plasma membrane calcium channel Orai allowed definition of three distinct thresholds of endoplasmic reticulum calcium. Wingless signaling is sensitive to even a small reduction, while Notch and Hippo signaling are disrupted at intermediate levels, and elimination of SERCA function results in apoptosis. These differential and opposing effects on three oncogenic signaling pathways may complicate the use of SERCA inhibitors as cancer therapeutics.
Raza, Q., Choi, J. Y., Li, Y., O'Dowd, R. M., Watkins, S. C., Chikina, M., Hong, Y., Clark, N. L. and Kwiatkowski, A. V. (2019). Evolutionary rate covariation analysis of E-cadherin identifies Raskol as a regulator of cell adhesion and actin dynamics in Drosophila. PLoS Genet 15(2): e1007720. PubMed ID: 30763317
The adherens junction couples the actin cytoskeletons of neighboring cells to provide the foundation for multicellular organization. The core of the adherens junction is the cadherin-catenin complex that arose early in the evolution of multicellularity to link actin to intercellular adhesions. Over time, evolutionary pressures have shaped the signaling and mechanical functions of the adherens junction to meet specific developmental and physiological demands. Evolutionary rate covariation (ERC) identifies proteins with correlated fluctuations in evolutionary rate that can reflect shared selective pressures and functions. This study used ERC to identify proteins with evolutionary histories similar to the Drosophila E-cadherin (DE-cad) ortholog. Core adherens junction components alpha-catenin and p120-catenin displayed positive ERC correlations with DE-cad, indicating that they evolved under similar selective pressures during evolution between Drosophila species. Further analysis of the DE-cad ERC profile revealed a collection of proteins not previously associated with DE-cad function or cadherin-mediated adhesion. The function of a subset of ERC-identified candidates was analyzed by RNAi during border cell (BC) migration, and novel genes were identified that function to regulate DE-cad. Among these, the gene CG42684, which encodes a putative GTPase activating protein (GAP), was found to regulate BC migration and adhesion. CG42684 was name raskol ("to split" in Russian) and it was shown to regulates DE-cad levels and actin protrusions in BCs. It is proposed that Raskol functions with DE-cad to restrict Ras/Rho signaling and help guide BC migration. These results demonstrate that a coordinated selective pressure has shaped the adherens junction and this can be leveraged to identify novel components of the complexes and signaling pathways that regulate cadherin-mediated adhesion.
Iyer, K. V., Piscitello-Gomez, R., Paijmans, J., Julicher, F. and Eaton, S. (2019). Epithelial viscoelasticity is regulated by mechanosensitive E-cadherin turnover. Curr Biol. PubMed ID: 30744966
Studying how epithelia respond to mechanical stresses is key to understanding tissue shape changes during morphogenesis. This study examined the viscoelastic properties of the Drosophila wing epithelium during pupal morphogenesis by quantifying mechanical stress and cell shape as a function of time. A delay of 8 h was found between maximal tissue stress and maximal cell elongation, indicating a viscoelastic deformation of the tissue. This viscoelastic behavior emerges from the mechanosensitivity of endocytic E-cadherin turnover. The increase in E-cadherin turnover in response to stress is mediated by mechanosensitive relocalization of the E-cadherin binding protein p120-catenin (p120) from cell junctions to cytoplasm. Mechanosensitivity of E-cadherin turnover is lost in p120 mutant wings, where E-cadherin turnover is constitutively high. In this mutant, the relationship between mechanical stress and stress-dependent cell dynamics is altered. Cells in p120 mutant deform and undergo cell rearrangements oriented along the stress axis more rapidly in response to mechanical stress. These changes imply a lower viscosity of wing epithelium. Taken together, these findings reveal that p120-dependent mechanosensitive E-cadherin turnover regulates viscoelastic behavior of epithelial tissues.


Vertebrates and invertebrates share common mechanisms for regulation of cell-cell adhesion, including a family of proteins known as cadherins. Cadherins are important in setting up boundaries between epithelial compartments, preventing cell populations with different fates from mixing with one another. They control orderly release of cells from epithelium during epithelial-mesenchymal transitions. shotgun, or DE-cadherin is a homolog of classic vertebrate cadherins. The level of shotgun, expressed from maternal mRNA throughout the entire blastoderm [Image], is high enough to sustain embryonic development except for severe disruption of Malpighian tubules (Uemura, 1996). Zygotic shg is expressed in all epithelial tissue except for presumptive mesoderm cells prior to gastrulation [Image].

The presence of shg expression in neuroectoderm, and its absence in delaminated neuroblasts, suggests an involvement of SHG in the critical process of delamination: the migration of neuroblasts from the ectoderm into the ventral nervous system (Tepass, 1996). Another cadherin, Cadherin-N shows a complementary expression pattern. It is expressed primarily in the nervous system but not in epithelial precursors of neural cells (Iwai, 1997).

E-cadherin is a cell adhesion molecule that provides one constituent of the adherens junction, a cellular junction localized at the apicolateral boundary of an epithelial cell. E-cadherin recruits other proteins to the region of cell-cell contact, such as Crumbs, a transmembrane protein involved in organizing the adherens junction, and Armadillo, a junction associated protein coded for by a pair rule gene involved in wingless signaling.

shotgun is involved in Malpighian tubule morphogenesis. Malpighian tubules are initiated by a local invagination from the hindgut primordia. In shg mutants, the Malpighian tubules are rounded and eventually disintegrate. SHG is concentrated at the apical poles of epithelial cell-cell junctions (Oda, 1994). Interestingly, the apical polarity of the cells is preserved in shg mutants as reflected in Crumbs protein distribution (Uemura, 1996).

Tracheal branch outgrowth and fusion are similarly defective in shotgun mutants. A "stall" phenotype is apparent, exhibiting delayed branch extention. armadillo mutants have similar effects on tracheal development. The role of Armadillo in tracheal morphogenesis is partly separate from that of Wingless, because the formation of dorsal tracheal trunks is only partially impaired in wg mutants. This means that the connection of wingless signaling to occurances at the apical junction is not sufficient to completely disrupt morphogenesis in wingless mutants (Uemura, 1996).

The primary role of shotgun is preservation of the integrity of Drosophila epithelium and it is downregulated during critical periods of epithelial-mesenchymal transition such as gastrulation and neurogenesis. As an epithelial cadherin, it is the central element of the adherens junction, a multiprotein complex that holds cells together and transduces adhesion signals across membranes. It is involved as the nucleation site of cytoskeleton to cell junctions. In zygotic shotgun mutants the level of alpha-catenin and Armadillo at the adherens junction is dramatically reduced, pointing to the importance of Drosophila E-cadherin in assembling the adherens junction (Tepass, 1996).

The molecular structures of classic cadherins show clear differences between chordate and nonchordate metazoans. Although nonchordate classic cadherins have cadherin superfamily-specific extracellular repeats (CRs) and a highly conserved cytoplasmic domain (CP), these cadherins have a unique extracellular domain that is absent from vertebrate and ascidian (phylum Urochordata) classic cadherins. This domain is termed here the primitive classic cadherin domain (PCCD). The PCCD consist of three motifs. The first is unique to nonchordate classic cadherins, and has been named the NC (nonchordate motif). The second is rich in cysteine residues and rather resembles EGF repeats, and has been named the CE (cysteine-rich EGF repeat-like motif). The third is similar to the laminin A globular domain, and is named the LAG (laminin A globular domain-like motif). These motifs constitute a domain that constitutes the primitive classic cadherin domain (PCCD). To understand the roles of the PCCD, a series of mutant forms of the Drosophila classic cadherin Shotgun were constructed. Biochemical analyses indicate that the last two CRs and PCCD form a special structure with proteolytic cleavage. Mutations in the PCCD do not eliminate the cell-cell-binding function of Shotgun in cultured cells, but prevent the cadherin from efficiently translocating to the plasma membrane in epithelial cells of the developing embryo. In addition, genetic rescue assays suggest that although CP-mediated control plays a central role in tracheal fusion, the role of the PCCD in efficient recruitment of Shotgun to apical areas of the plasma membranes is also important for dynamic epithelial morphogenesis. It is proposed that there is a fundamental difference in the mode of classic cadherin-mediated cell-cell adhesion between chordate and nonchordate metazoans (Oda, 1999).

When the amino acid sequences of the PCCDs of nonchordate classic cadherins are aligned, the PCCD of Shotgun shows 39.8%, 31.1%, and 27.5% amino acid sequence identity to those of Shotgun, LvG-cadherin (sea urchin), and HMR-1 (C. elegans), respectively. The PCCDs of Shotgun and Drosophila N-cadherin are the most similar to each other in all combinations. A phylogenetic tree was constructed based on the PCCDs, consistent with the tree based on the CPs. The PCCD consists of the NC, CE, and LAG. No significant similarity within the NC is found to sequences of other known proteins. The CEs have 10 or 12 conserved cysteine residues, and the LAGs have 4 conserved cysteine residues. Although sequences similar to the LAGs are found in a variety of proteins, including Drosophila Slit, Fat, Crumbs and rat neurexin I, the first two of the four cysteine residues are not conserved in proteins other than the nonchordate classic cadherins. These amino acid sequence data suggest that the PCCD forms a structure unique to nonchordate classic cadherins (Oda, 1999).

When full-length Shotgun tagged with green fluorescent protein (GFP) at the carboxyl terminus is expressed in Drosophila S2 cells, 150- and 110-kDa polypeptides named distal and proximal polypeptides (DP and PP) are detected in the cell lysates by anti-Shotgun monoclonal antibody DCAD2 and anti-GFP antibody, respectively, in addition to a small amount of the precursor (about 230 kDa). DP corresponds to the polypeptide detected in embryonic lysates by DCAD2, which recognizes the region between CR0 and CR1. A CP-deleted mutant molecule named dCPc3 is separated into a DP of the same size (150-kDa) and a PP of reduced size (80-kDa). Sequence analysis shows that protolytic removal of the signal peptide and proteolytic cleavage between G (aa 1010) and S (aa 1011) residues in the NC result in DP (aa 70-1010) and PP (aa 1011-1507). Deletion analysis indicates that the region covering CR4, CR5, NC, and CE (aa 738-1108) is necessary for NC cleavage, but this is not the case for most other regions, including the transmembrane segment (TM) (Oda, 1999).

The CR domain, PCCD, and CP are essential for the morphogenetic function of Shotgun. The morphogenetic abilities of Shotgun mutant molecules are not necessarily correlated with their adhesive abilities in S2 cells. Various extracellular-domain deletion mutants, dCR3h, dCR4h, and dEx, which have no cell-cell-binding ability, can produce positive morphogenetic effects on tracheal fusion, but dCPc3, dNc, dCL3, and inNcHA show no positive morphogenetic effects, despite having cell-cell-binding ability in S2 cells. These results indicate that CP-mediated control plays a central role in tracheal fusion. The CP binds to the catenins, which may mediate interaction with the actin cytoskeleton. This coupling is expected to translate the force of actin bundle contraction into cell shape changes. Even if cell-cell binding function is removed from Shotgun, such mechanical forces may induce incomplete tracheal fusion. Despite the intact CP, some mutations in the PCCD lead to complete loss of the morphogenetic ability of Shotgun. These mutant molecules fail in prompt translocation to the plasma membrane when expressed in epithelial cells of the developing embryo. Efficient recruitment of Shotgun to appropriate sites of the plasma membrane may be important for dynamic epithelial morphogenesis during development (Oda, 1999).

Shotgun protein must be translocated from the ER to the contact sites of the plasma membrane to participate in cell-cell binding. Exogenously expressed normal Shotgun immediately accumulates at apicolateral borders of the plasma membrane in tracheal cells and many other epithelial cells. Mutational analyses reveal important aspects of Shotgun translocation involving the last two CRs, PCCD, and CP. Mutations in the PCCD lead to strong accumulation of the molecules in subdomains of the cytoplasm probably corresponding to the ER. Deletion of the large extracellular region encompassing CRs 1-5 and the PCCD also reduce the efficiency of translocation. These observations favor the idea that the extracellular region, including the PCCD, has an active role in translocation out of the ER to the plasma membrane. Deficiencies in deletion mutants dCR3h or dCR4h have little effect on translocation, but dCR4 exhibits a defect similar to that of the PCCD mutant molecules. These observations suggested that the last two CRs are functionally linked with the PCCD, and that this set is required for normal translocation. It is intriguing that NC cleavage, which presumably takes place in the ER, also requires the same region. However, the results from mNcGSP and dCL3 indicate that NC cleavage is not a prerequisite for translocation and occurs even under conditions in which translocation is blocked. It is speculated that CRs 4 and 5 and the PCCD are an overlapping region required for NC cleavage and for translocation, but these two events occur independently. There are three possible explanations for the mechanism of the involvement of the PCCD in translocation. (1) The unique structure of the last two CRs and PCCD may interact in the ER lumen with some machinery that carries out protein sorting and directional transport. (2) The PCCD structure may be involved in a lateral interaction (between the synthesized cadherin proteins) that facilitates translocation. (3) The PCCD may have a role in folding the extracellular part of the synthesized cadherin in the ER lumen to form the compact structure necessary for efficient translocation. It is hypothesized that the common ancestor of chordate classic cadherins adopted an alternative translocation mechanism to the PCCD-mediated mechanism coincident with the structural change to small size (Oda, 1999).

The CP is another part essential for normal translocation of Shotgun. Although the CP-deleted molecule tends to translocate to the apical plasma membrane, it is not so actively concentrated into the sites of cell-cell contact. Despite the reduced efficiency of translocation, the mutant molecule lacking the large extracellular region appears to be recruited to the apical zones of cell-cell contact once it leaves the ER. It seems that the CP-mediated control plays an important role in efficient and directional translocation of Shotgun from the ER to the contact sites of the plasma membrane. Shotgun mutant molecules accumulating in the cytoplasm due to the lack of translocation appear to bind to Armadillo and alpha-catenin. This raises the possibility that normal Shotgun joins Armadillo and alpha-catenin before arriving at the plasma membrane (Oda, 1999).

In addition to the subcellular distributions of the classic cadherins, there are marked differences between chordates and nonchordates in junctional complexes. Adherens junctions (AJs) and septate junctions (SJs) are observed in a wide range of nonchordate metazoans by electron microscopy. The AJ is located at the apical-most portion of the cell, followed by the SJ. In chordates (including vertebrates and ascidians) tight junctions (TJs), in addition to AJs, are frequently found, but SJs have not been found. Exceptionally, in Amphioxus cephalochordata, neither TJs nor SJs have been observed. The TJ is positioned at the apical end of the lateral plasma membrane in chordate epithelial cells, followed by the AJ. These observations indicate that there are fundamental differences between chordate and nonchordate metazoans not only in cadherin-based cell-cell adhesion but also in other mechanisms of cell-cell connection (Oda, 1999).

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, 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. 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).

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).

Adherens junction length during tissue contraction is controlled by the mechanosensitive activity of actomyosin and junctional recycling

During epithelial contraction, cells generate forces to constrict their surface and, concurrently, fine-tune the length of their adherens junctions to ensure force transmission. While many studies have focused on understanding force generation, little is known on how junctional length is controlled. This study shows that, during amnioserosa contraction in Drosophila dorsal closure, adherens junctions reduce their length in coordination with the shrinkage of apical cell area, maintaining a nearly constant junctional straightness. This study reveals that junctional straightness and integrity depend on the endocytic machinery and on the mechanosensitive activity of the actomyosin cytoskeleton. On one hand, upon junctional stretch and decrease in E-cadherin density, actomyosin relocalizes from the medial area to the junctions, thus maintaining junctional integrity. On the other hand, when junctions have excess material and ruffles, junction removal is enhanced, and high junctional straightness and tension are restored. These two mechanisms control junctional length and integrity during morphogenesis (Sumi, 2018).

This study has shown that adherens junction length is actively controlled during epithelial contraction, leading to a constant junctional straightness upon cell contraction. It is proposed that two different cellular mechanisms are at the origin of this length control. First, a modulation of actomyosin localization from the medial areas toward junctions allows for integrity maintenance by increasing actomyosin contraction along the junctions upon junctional stretch. Possibly, E-cadherin dilution triggered by junctional stretching is responsible for the myosin recruitment, a possibility supported by the anti-correlation between junctional myosin and E-cadherin. Second, the junction removal rate depends on junctional straightness, maintaining a preferred junctional straightness. Presumably, the endocytosis machinery is at the origin of this maintenance. As junctional tension is dependent on junctional straightness, maintaining junctional straightness could also allow it to maintain junctional tension, which remains approximately constant during DC (Sumi, 2018).

These mechanisms rely on a complex interplay between junctional E-cadherin, actomyosin, and tension. Indeed, this study finds that (1) higher junctional E-cadherin levels are associated with lower myosin levels and (2) decreased junctional straightness is associated with lower junctional tension. These observations suggest that E-cadherin is an essential component for the control of adherens junction length and for actomyosin localization in the cell. In cell culture experiments, E-cadherin has already been determined to be a key player in cell-cell mechanical interplay and shown to interact closely with the actomyosin cortex. In the case of the AS tissue, an enrichment of actomyosin was observed at junctions when E-cadherin levels are reduced. A similar interaction has been previously identified in the context of cytokinesis. Ghd results indicate a general interplay between E-cadherin and myosin that could play a role in several morphogenetic rearrangements. Such an interplay is likely to involve actin-associated proteins that could respond to changes in mechanical states of the adherens junction. Consistent with this idea, previous studies identified dynamic enrichment of the formin Diaphanous or of vinculin specifically at adherens junctions (Sumi, 2018).

Interestingly, this study observed that when junctions are stretched and E-cadherin density decreased, the contractile pulses of the AS tissue are arrested, possibly due to the observed myosin recruitment to junctions. Such pulses of contraction have been identified in several tissues during Drosophila, chicken, and mouse embryo development. They have been associated with global tissue contraction and epithelial remodeling. This study has shown that the pulsatile activity can be tuned by junctional stretch and E-cadherin levels. The results therefore uncover a direct coupling between cell-cell adhesion and the contractile activity of the cell (Sumi, 2018).

Finally, the rate of junction removal appears to depend on junctional straightness. How is junction straightness sensed to dictate junction removal? Laser ablation experiments indicate that junctions with lower straightness also have lower mechanical tension. In single cells, it is known that the endocytic rate is modulated by the tension of the cell membrane. In a developmental context, endocytosis could also be tension sensitive, resulting in the control of junctional straightness through junctional tension. Alternatively, E-cadherin density could play a role in the control of the junction turnover rate. Indeed, an increase of E-cadherin density associated to a decrease in junction straightness could trigger an enhanced endocytic rate, therefore controlling junction straightness and tension. The mechanisms regulating junction length and integrity identified here involve the activity of two essential cellular machineries. They are therefore likely to act during many morphogenetic events that involve tissue contraction, such as neural tube closure, optic cup morphogenesis, or wound healing (Sumi, 2018).


Amino Acids - 1507

Structural Domains

The Drosophila homolog of classic vertebrate cadherins is DE-cadherin. The extracellular domain of DE-cadherin has six cadherin-specific repeats, although the first repeat seems to be cleaved off upon maturation, and the cytoplasmic domain shows significant identity to that of classic vertebrate cadherins. DE-cadherin is distinguishable from its vertebrate counterparts by a large insertion between the last cadherin repeat and the transmembrane domain, with local sequence similarity to Fat, Laminin A chain, Slit, and Neurexin I at the proximal region of the extracellular domain. This domain has two motifs, one a cysteine rich domain similar to Fat EGF repeat 3. The other is a laminin A globular-domain (G-domain) repeat (Oda, 1994).

The enormous size of Cadherin-N is primarily due to the presence of 15 cadherin repeats in the extracellular region, presenting a contrast to the 4 repeats in all vertebrate classic cadherins and the 6 repeats in Shotgun, the Drosophila E-cadherin. Both Drosophila cadherins have insertions of similar sequences between the last extracellular cadherin repeat and the membrane-spanning segment. The insert contains a series of subdomains: an Fcc box (fly classic cadherin box), a cysteine-rich segment (C-rich 1), a laminin A (See Drosophila Laminin A) globular segment (LmA-G), and another cysteine-rich segment (C-rich 2). The Fcc box, comprising 170 amino acids is defined as such because database searches with its sequences identify only the comparable regions of Shotgun as a relative. Similar sequences are not found in vertebrate cadherins. The whole Cadherin-N LmA-G displays 25% sequence identity to mouse laminin A and to the presynaptic transmembrane protein Neurexin. The Cadherin-N cytoplasmic domain is much more similar to those domains of Shotgun and vertebrate classic cadherins with respect to both size and sequence. The intracellular domains of the two Drosophila cadherins and mouse N-cadherin range between 157 and 160 amino acids in length, and have 37% and 46% sequence identity in any combination among them, with the higher figure representing the Cadherin-N identity to murine N-cadherin, and the lower representing Shotgun identity to murine N-cadherin. The degree of sequence conservation between the two Drosophila cadherins (41% identity) is lower than the 63% identity between N- and E-cadherin in the same vertebrate species, as for example, in mice (Iwai, 1997).

shotgun: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised:  20 APR 97

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