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The cadherin-catenin complex is a conserved, calcium-dependent cell-cell adhesion module that is necessary for normal development and the maintenance of tissue integrity in bilaterian animals. Despite longstanding evidence of a deep ancestry of calcium-dependent cell adhesion in animals, the requirement of the cadherin-catenin complex to coordinate cell-cell adhesion has not been tested directly in a non-bilaterian organism. Here, we provide the first analysis of classical cadherins and catenins in the Starlet Sea Anemone, Nematostella vectensis. Gene expression, protein localization, siRNA-mediated knockdown of α-catenin, and calcium-dependent cell aggregation assays provide evidence that a bonafide cadherin-catenin complex is present in the early embryo, and that α-catenin is required for normal embryonic development and the formation of cell-cell adhesions between cells dissociated from whole embryos. Together these results support the hypothesis that the cadherin-catenin complex was likely a complete and functional cell-cell adhesion module in the last common cnidarian-bilaterian ancestor. SUMMARY STATEMENT: Embryonic manipulations and ex vivo adhesion assays in the sea anemone, Nematostella vectensis, indicate that the necessity of the cadherin-catenin complex for mediating cell-cell adhesion is deeply conserved in animal evolution.

Introduction Mechanical forces regulate many facets of cell and tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories. Methods We show how to fabricate a simple, low-cost, uniaxial stretching device, with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, as well as a custom MATLAB code to quantify individual cell strains. Results As an application note using this device, we show that uniaxial stretch slows down cellular movements in a mammalian epithelial monolayer in a cell density-dependent manner. We demonstrate that the effect on cell movement involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK). Conclusions This mechanical device provides a platform for broader involvement of engineers and biologists in this important area of cell and tissue biology. We used this device to demonstrate the mechanical regulation of collective cell movements in epithelia.

This year's Canada Gairdner International Prize is shared by Rolf Kemler and Masatoshi Takeichi for the discovery of the cadherin family of Ca2+-dependent cell-cell adhesion proteins, which play essential roles in animal evolution, tissue development, and homeostasis, and are disrupted in human cancers.

Mechanical forces play important roles in the biological function of cells and tissues. While numerous studies have probed the force response of cells and measured cell-generated forces, they have primarily focused on tensile, but not shear forces. Here, we describe the design, fabrication, and application of a silicon micromachined device that is capable of independently applying and sensing both tensile and shear forces in an epithelial cell monolayer. We integrated the device with an upright microscope to enable live cell brightfield and fluorescent imaging of cells over many hours following mechanical perturbation. Using devices of increasing stiffness and the same displacement input, we demonstrate that epithelia exhibit concomitant higher maximum resistive tensile forces and quicker force relaxation. In addition, we characterized the force response of the epithelium to cyclic shear loading. While the maximum resistive forces of epithelia under cyclic shear perturbation remained unchanged between cycles, cyclic loading led to faster relaxation of the resistive forces. The device presented here can be applied to studying the force response of other monolayer-forming cell types and is compatible with pharmacological perturbation of cell structures and functions.

The cilium, once considered a vestigial structure, is a conserved, microtubule-based organelle critical for transducing extracellular chemical and mechanical signals that control cell polarity, differentiation, and proliferation. The cilium undergoes cycles of assembly and disassembly that are controlled by complex inter-relationships with the cytoskeleton. Microtubules form the core of the cilium, the axoneme, and are regulated by post-translational modifications, associated proteins, and microtubule dynamics. Although actin and septin cytoskeletons are not major components of the axoneme, they also regulate cilium organization and assembly state. Here, we discuss recent advances on how these different cytoskeletal systems­ affect cilium function, structure, and organization.

The evolution of cell-adhesion mechanisms in animals facilitated the assembly of organized multicellular tissues. Studies in traditional animal models have revealed two predominant adhesion structures, the adherens junction (AJ) and focal adhesions (FAs), which are involved in the attachment of neighboring cells to each other and to the secreted extracellular matrix (ECM), respectively. The AJ (containing cadherins and catenins) and FAs (comprising integrins, talin, and paxillin) differ in protein composition, but both junctions contain the actin-binding protein vinculin. The near ubiquity of these structures in animals suggests that AJ and FAs evolved early, possibly coincident with multicellularity. However, a challenge to this perspective is that previous studies of sponges—a divergent animal lineage—indicate that their tissues are organized primarily by an alternative, sponge-specific cell-adhesion mechanism called “aggregation factor.” In this study, we examined the structure, biochemical properties, and tissue localization of a vinculin ortholog in the sponge Oscarella pearsei (Op). Our results indicate that Op vinculin localizes to both cell–cell and cell–ECM contacts and has biochemical and structural properties similar to those of vertebrate vinculin. We propose that Op vinculin played a role in cell adhesion and tissue organization in the last common ancestor of sponges and other animals. These findings provide compelling evidence that sponge tissues are indeed organized like epithelia in other animals and support the notion that AJ- and FA-like structures extend to the earliest periods of animal evolution.

Cell-cell junctions, acting as 'secret handshakes', mediate cell-cell interactions and make multicellularity possible. Work over the previous century illuminated key players comprising these junctions including the cadherin superfamily, nectins, CAMs, connexins, notch/delta, lectins, and eph/Ephrins. Recent work has focused on elucidating how interactions between these complex and often contradictory cues can ultimately give rise to large-scale organization in tissues. This effort, in turn, has enabled bioengineering advances such as cell-mimetic interfaces that allow us to better probe junction biology and to develop new biomaterials. This review details exciting, recent developments in these areas as well as providing both historical context and a discussion of some topical challenges and opportunities for the future.

Intercellular epithelial junctions formed by classical cadherins, β-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the β-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds β-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.

Listeria monocytogenes invades epithelial cells by binding to the host cell receptor E-cadherin, a component of the adherens junction. E-cadherin serves primarily as an adhesive to mediate bacterial invasion; the canonical E-cadherin/catenin/F-actin complex is not required for this process., The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell–cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin–mediated coupling of the bacterium to F-actin is not required.

The primary cilium is a central signaling hub in cell proliferation and differentiation, and is built and disassembled every cell cycle in most animal cells. Disassembly is critically important: misregulation or delay of disassembly leads to cell cycle defects. The physical means by which cilia are disassembled are poorly understood, and thought to involve resorption of disassembled components into the cell body. To investigate cilium disassembly in mammalian cells, we used rapid live-cell imaging to comprehensively characterize individual disassembly events. The predominant mode of disassembly was rapid cilium loss via deciliation, in which the membrane and axoneme of the cilium was shed from the cell. Gradual resorption was also observed, as well as events in which a period of gradual resorption ended with rapid deciliation. Deciliation resulted in intact shed cilia that could be recovered from culture medium and contained both membrane and axoneme proteins. We modulated levels of katanin and intracellular calcium, two putative regulators of deciliation, and found that excess katanin promotes disassembly by deciliation, independently of calcium. Together, these results demonstrate that mammalian ciliary disassembly involves a tunable decision between deciliation and resorption.

Cell shape and the spatial distributions of cell–cell and cell–ECM adhesions govern the force balance in cell pairs. Cell–ECM adhesions at the distal ends of cell–cell junctions regulate junction length and the balance of forces across the junction, while molecular tension in E-cadherin remains constant., Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.

Stretching cell sheets promotes proliferation Mechanical strain regulates the development, organization, and function of multicellular tissues. But how? Cadherins mechanically couple neighboring epithelial cells through extracellular interactions and sequester the transcription factors β-catenin and Yap1. To find out more, Benham-Pyle et al. stretched epithelial cell sheets. This mechanical strain induced rapid cell cycle reentry, DNA synthesis by sequential nuclear accumulation, and transcriptional activation of Yap1 and β-catenin. Thus, cell-cell junctions are mechanically responsive structural scaffolds providing signaling centers that coordinate transcriptional responses to externally applied force. Science , this issue p. 1024

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites.

Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of E-cadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.

Cell-cell junctions link cells to each other in tissues, and regulate tissue homeostasis in critical cell processes that include tissue barrier function, cell proliferation, and migration. Defects in cell-cell junctions give rise to a wide range of tissue abnormalities that disrupt homeostasis and are common in genetic abnormalities and cancers. Here, we discuss the organization and function of cell-cell junctions primarily involved in adhesion (tight junction, adherens junction, and desmosomes) in two different epithelial tissues: a simple epithelium (intestine) and a stratified epithelium (epidermis). Studies in these tissues reveal similarities and differences in the organization and functions of different cell-cell junctions that meet the requirements for the specialized functions of each tissue. We discuss cell-cell junction responses to genetic and environmental perturbations that provide further insights into their roles in maintaining tissue homeostasis.

We designed, fabricated, and deployed a silicon MEMS device to strain a sheet of epithelial (2D and skin-like) cells in tension and/or shear simultaneously. We deployed the device with upright bright-field and fluorescence microscopy in live cell experiments spanning 42 hours. For the first time, our MEMS-based assay enabled quantification of the mechanics and dynamics of epithelial monolayers under shear deformation. In-plane shear forces are important in biological processes such as development, growth, collective migration, or cancer metastasis.

Both cell–cell adhesion and oriented cell division play prominent roles in establishing tissue architecture, but it is unclear how they might be coordinated. Here, we demonstrate that the cell–cell adhesion protein E-cadherin functions as an instructive cue for cell division orientation. This is mediated by the evolutionarily conserved LGN/NuMA complex, which regulates cortical attachments of astral spindle microtubules. We show that LGN, which adopts a three-dimensional structure similar to cadherin-bound catenins, binds directly to the E-cadherin cytosolic tail and thereby localizes at cell–cell adhesions. On mitotic entry, NuMA is released from the nucleus and competes LGN from E-cadherin to locally form the LGN/NuMA complex. This mediates the stabilization of cortical associations of astral microtubules at cell–cell adhesions to orient the mitotic spindle. Our results show how E-cadherin instructs the assembly of the LGN/NuMA complex at cell–cell contacts, and define a mechanism that couples cell division orientation to intercellular adhesion., Cell–cell adhesion and oriented cell division play key roles in tissue architecture, but how they are coordinated is not known. Here, the authors show that E-cadherin interacts with LGN, and thereby provides a cortical cue that serves to stabilize cortical attachment of astral microtubules at cell–cell adhesions, thus orienting the mitotic spindle.

Plk1 regulates Nek2 activity in stabilizing β-catenin at mitotic centrosomes and in promoting centrosome separation. Nek2 phosphorylates the same regulatory sites (S33/S37/T41) as GSK3β in β-catenin, as well as additional sites, and inhibits binding of the E3 ligase β-TrCP to β-catenin, thereby preventing β-catenin ubiquitination and degradation., β-Catenin is a multifunctional protein with critical roles in cell–cell adhesion, Wnt signaling, and the centrosome cycle. Whereas the regulation of β-catenin in cell–cell adhesion and Wnt signaling are well understood, how β-catenin is regulated at the centrosome is not. NIMA-related protein kinase 2 (Nek2), which regulates centrosome disjunction/splitting, binds to and phosphorylates β-catenin. Using in vitro and cell-based assays, we show that Nek2 phosphorylates the same regulatory sites in the N-terminus of β-catenin as glycogen synthase kinase 3β (GSK3β), which are recognized by a specific phospho-S33/S37/T41 antibody, as well as additional sites. Nek2 binding to β-catenin appears to inhibit binding of the E3 ligase β-TrCP and prevents β-catenin ubiquitination and degradation. Thus β-catenin phosphorylated by Nek2 is stabilized and accumulates at centrosomes in mitosis. We further show that polo-like kinase 1 (Plk1) regulates Nek2 phosphorylation and stabilization of β-catenin. Taken together, these results identify a novel mechanism for regulating β-catenin stability that is independent of GSK3β and provide new insight into a pathway involving Plk1, Nek2, and β-catenin that regulates the centrosome cycle.

Epithelial monolayers undergo self-healing when wounded. During healing, cells collectively migrate into the wound site, and the converging tissue fronts collide and form a stable interface. To heal, migrating tissues must form cell-cell adhesions and reorganize from the front-rear polarity characteristic of cell migration to the apical-basal polarity of an epithelium. However, identifying the "stop signal" that induces colliding tissues to cease migrating and heal remains an open question. Epithelial cells form integrin-based adhesions to the basal extracellular matrix (ECM) and E-cadherin-mediated cell-cell adhesions on the orthogonal, lateral surfaces between cells. Current biological tools have been unable to probe this multicellular 3D interface to determine the stop signal. We addressed this problem by developing a unique biointerface that mimicked the 3D organization of epithelial cell adhesions. This "minimal tissue mimic" (MTM) comprised a basal ECM substrate and a vertical surface coated with purified extracellular domain of E-cadherin, and was designed for collision with the healing edge of an epithelial monolayer. Three-dimensional imaging showed that adhesions formed between cells, and the E-cadherin-coated MTM resembled the morphology and dynamics of native epithelial cell-cell junctions and induced the same polarity transition that occurs during epithelial self-healing. These results indicate that E-cadherin presented in the proper 3D context constitutes a minimum essential stop signal to induce self-healing. That the Ecad:Fc MTM stably integrated into an epithelial tissue and reduced migration at the interface suggests that this biointerface is a complimentary approach to existing tissue-material interfaces.

Intercellular epithelial junctions formed by classical cadherins, β-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the β-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds β-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.

It is unknown whether homologs of the cadherin·catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin·β-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that Danio rerio and Mus musculus αE-catenin have striking functional differences. We demonstrate that D. rerio αE-catenin is monomeric by size exclusion chromatography, native PAGE, and small angle x-ray scattering. D. rerio αE-catenin binds F-actin in cosedimentation assays as a monomer and as an α/β-catenin heterodimer complex. D. rerio αE-catenin also bundles F-actin, as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of α-catenin function, F-actin and β-catenin binding, are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.

In the adherens junction (AJ), cadherin and catenin proteins form a cell–cell adhesion complex that is indispensable for tissue morphogenesis and homeostasis. The complex mechanically couples neighboring cells through intercellular binding by cadherins, and actin binding and regulation by the cytoplasmic catenins. In addition, the cadherin–catenin complex participates in signaling pathways that direct cellular organization, proliferation, and motility. Some of these signaling pathways can be regulated by mechanical stimulation or posttranslational modification of the components of the AJ. In light of these findings, we discuss our current understanding of how AJ signaling and mechanical functions are regulated by phosphorylation and force, and speculate on the mechanisms underlying the coordination between these two types of modifications.

A simple epithelium consists of a two-dimensional sheet of structurally and functionally polarized cells that are often organized into a tube, and is a defining feature of animal body plans. An epithelium (the trophectoderm) is the first differentiated tissue formed during embryogenesis, and many adult tissues are composed of epithelia [1, 2]. Epithelial cells have a polarized organization of the plasma membrane, cytoskeleton, and cytoplasmic organelles [2, 3]. The apical plasma membrane faces the lumen of an organ or the outside of the organism, while the basal (or basolateral) membrane contacts the underlying tissue. Polarized epithelial sheets regulate the directional absorption and secretion of proteins and other solutes, an essential physiological function that is often disrupted in human disease [1]. In addition, the polarized organization of cytoskeletal components in epithelial cells contributes to tissue shape changes during embryonic morphogenesis [4]. Despite the diversity of body plans and lifestyles, all metazoans share an epithelial tissue as their basic unit of organization, suggesting that the development of epithelial cell polarity was a very early event in metazoan evolution [5, 6]. Genetic, cell biological and molecular studies in a variety of metazoans have shown that the formation and maintenance of polarized epithelial cells require cell-cell adhesion mediated by the cadherin-catenin complex [7]. Cadherins are transmembrane receptors that form homophilic adhesive interactions with cadherins on adjacent cells, providing a spatial cue that initiates cell polarity [8]. β-Catenin and α-catenin are cyotosolic binding partners of cadherin that transduce this adhesive cue and mediate cell polarity, in part by directing the reorganization of the cytoskeleton [8, 9]. Consistent with its fundamental role in epithelial organization, the cadherin-catenin complex is conserved in all metazoans. Phylogenetically, metazoans belong to the unikonts, a group that also includes fungi, social amoebae and a number of unicellular or colonial protists (see Figure 2) [10, 11]. Historically, it was thought that multicellularity evolved independently in animals, fungi and social amoebae, and that epithelial tissue was a unique feature of animals [11–14]. However, two recent studies have established the existence of polarized epithelial tissue in the non-metazoan social amoeba Dictyostelium discoideum [15, 16]. This finding calls into question the notion of an independent origin of multicellularity in animals and social amoebae. Here, we propose and discuss the alternative hypothesis that animals evolved from an ancestor with a simple multicellular organization. Figure 2 Conservation of epithelial characters in unikonts

The adherens junction (AJ) comprises multi-protein complexes required for cell-cell adhesion in embryonic development and adult tissue homeostasis. Mutations in key proteins and mis-regulation of AJ adhesive properties can lead to pathologies such as cancer. In recent years, the zebrafish has become an excellent model organism to integrate cell biology in the context of a multicellular organization. The combination of classical genetic approaches with new tools for live imaging and biophysical approaches has revealed new aspects of AJ biology, particularly during zebrafish gastrulation. These studies have resulted in progress in understanding the relationship between cell-cell adhesion, cell migration and plasma membrane blebbing.

Mechanical strain regulates the development, organization, and function of multicellular tissues, but mechanisms linking mechanical strain and cell-cell junction proteins to cellular responses are poorly understood. Here, we showed that mechanical strain applied to quiescent epithelial cells induced rapid cell cycle reentry, mediated by independent nuclear accumulation and transcriptional activity of first Yap1 and then β-catenin. Inhibition of Yap1- and β-catenin-mediated transcription blocked cell cycle reentry and progression through G1 into S phase, respectively. Maintenance of quiescence, Yap1 nuclear exclusion, and β-catenin transcriptional responses to mechanical strain required E-cadherin extracellular engagement. Thus, activation of Yap1 and β-catenin may represent a master regulator of mechanical strain-induced cell proliferation, and cadherins provide signaling centers required for cellular responses to externally applied force.

Endothelial cells form cell-cell adhesive structures, called adherens and tight junctions, which maintain tissue integrity, but must be dynamic for leukocyte transmigration during the inflammatory response and cellular remodeling during angiogenesis. This review will focus on Vascular Endothelial (VE)-cadherin, an endothelial-specific cell-cell adhesion protein of the adherens junction complex. VE-cadherin plays a key role in endothelial barrier function and angiogenesis, and consequently VE-cadherin availability and function are tightly regulated. VE-cadherin also participates directly and indirectly in intracellular signaling pathways that control cell dynamics and cell cycle progression. Here we highlight recent work that has advanced our understanding of multiple regulatory and signaling mechanisms that converge on VE-cadherin and have consequences for endothelial barrier function and angiogenic remodeling.

The ternary complex of cadherin, β-catenin, and α-catenin regulates actin-dependent cell–cell adhesion. α-Catenin can bind β-catenin and F-actin, but in mammals α-catenin either binds β-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of α-catenin is evolutionarily conserved. The Caenorhabditis elegans α-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1–HMP-2(β-catenin)–HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of α-catenin and suggests that multiple mechanisms regulate α-catenin binding to F-actin.

Adenomatous polyposis coli is a cytoskeletal organizer and a scaffold for mediating degradation of the Wnt effector β-catenin. We uncouple these different APC functions and show that GSK3β/CKI phosphorylation regulates APC clusters and cell migration independently of cell–cell adhesion and β-catenin transcriptional activity., Adenomatous polyposis coli (APC), a tumor suppressor commonly mutated in cancer, is a cytoskeletal organizer for cell migration and a scaffold for GSK3β/CKI-mediated phosphorylation and degradation of the Wnt effector β-catenin. It remains unclear whether these different APC functions are coupled, or independently regulated and localized. In primary endothelial cells, we show that GSK3β/CKI-phosphorylated APC localizes to microtubule-dependent clusters at the tips of membrane extensions. Loss of GSK3β/CKI-phosphorylated APC from these clusters correlates with a decrease in cell migration. GSK3β/CKI-phosphorylated APC and β-catenin at clusters is degraded rapidly by the proteasome, but inhibition of GSK3β/CKI does not increase β-catenin–mediated transcription. GSK3β/CKI-phosphorylated and -nonphosphorylated APC also localize along adherens junctions, which requires actin and cell–cell adhesion. Significantly, inhibition of cell–cell adhesion results in loss of lateral membrane APC and a concomitant increase in GSK3β/CKI-phosphorylated APC in clusters. These results uncouple different APC functions and show that GSK3β/CKI phosphorylation regulates APC clusters and cell migration independently of cell–cell adhesion and β-catenin transcriptional activity.

Staying in Place The primary cilium is found on nearly all mammalian cells and is a key regulatory organelle for proper signal transduction throughout development and in adults. Extracellular signal transduction, such as that promoted by Sonic hedgehog (Shh), requires the enrichment of receptors and downstream signaling components in the ciliary membrane. Intraflagellar transport is involved in selective trafficking of proteins into the cilium, but it is not known how these proteins are retained in the cilium. It has been speculated that a diffusion barrier exists at the base of the ciliary membrane. Now, Hu et al. (p. 436 , published online 17 June) demonstrate directly that a membrane diffusion barrier is indeed present at the base of the ciliary membrane. SEPT2, a member of the septin family that also forms a diffusion barrier in budding yeast and mammalian sperm membranes, localizes to the base of the ciliary membrane and is required for ciliogenesis, ciliary membrane protein localization, and cilium-dependent Shh signaling.

The cadherin family of Ca 2+ -dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell–cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 stages. First, the functional unit of cadherin adhesion is a cis dimer formed by the binding of the extracellular regions of 2 cadherins on the same cell surface. Second, formation of low-affinity trans interactions between cadherin cis dimers on opposing cell surfaces initiates cell–cell adhesion. Third, lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Evidence of these cadherin binding states during adhesion is, however, contradictory, and evidence for cooperativity is lacking. We used single-molecule structural (fluorescence resonance energy transfer) and functional (atomic force microscopy) assays to demonstrate directly that cadherin monomers interact via their N-terminal EC1 domain to form trans adhesive complexes. We could not detect the formation of cadherin cis dimers, but found that increasing the density of cadherin monomers cooperatively increased the probability of trans adhesive binding.

Septins are filamentous GTPases that associate with cell membranes and the cytoskeleton and play essential roles in cell division and cellular morphogenesis. Septins are implicated in many human diseases including cancer and neuropathies. Small molecules that reversibly perturb septin organization and function would be valuable tools for dissecting septin functions and could be used for therapeutic treatment of septin-related diseases. Forchlorfenuron (FCF) is a plant cytokinin previously shown to disrupt septin localization in budding yeast. However, it is unknown whether FCF directly targets septins and whether it affects septin organization and functions in mammalian cells. Here, we show that FCF alters septin assembly in vitro without affecting either actin or tubulin polymerization. In live mammalian cells, FCF dampens septin dynamics and induces the assembly of abnormally large septin structures. FCF has a low level of cytotoxicity, and these effects are reversed upon FCF washout. Significantly, FCF treatment induces mitotic and cell migration defects that phenocopy the effects of septin depletion by small interfering RNA. We conclude that FCF is a promising tool to study mammalian septin organization and functions.

Cadherins are transmembrane adhesion molecules that mediate homotypic cell-cell contact. In adherens junctions, the cytoplasmic domain of cadherins is functionally linked to the actin cytoskeleton through a series of proteins known as catenins. E-cadherin binds to beta-catenin, which in turn binds to alpha-catenin to form a ternary complex. alpha-Catenin also binds to actin, and it was assumed previously that alpha-catenin links the cadherin-catenin complex to actin. However, biochemical, structural and live-cell imaging studies of the cadherin-catenin complex and its interaction with actin show that binding of beta-catenin to alpha-catenin prevents the latter from binding to actin. Biochemical and structural data indicate that alpha-catenin acts as an allosteric protein whose conformation and activity changes depending on whether or not it is bound to beta-catenin. Initial contacts between cells occur on dynamic lamellipodia formed by polymerization of branched actin networks, a process controlled by the Arp2/3 (actin-related protein 2/3) complex. alpha-Catenin can suppress the activity of Arp2/3 by competing for actin filaments. These findings lead to a model for adherens junction formation in which clustering of the cadherin-beta-catenin complex recruits high levels of alpha-catenin that can suppress the Arp2/3 complex, leading to cessation of lamellipodial movement and formation of a stable contact. Thus alpha-catenin appears to play a central role in cell-cell contact formation.

Ca2+-dependent cell–cell adhesion is regulated by the cadherin family of cell adhesion proteins. Cadherins form trans-interactions on opposing cell surfaces which result in weak cell–cell adhesion. Stronger cell–cell adhesion occurs by clustering of cadherins and through changes in the organization of the actin cytoskeleton. Although cadherins were thought to bind directly to the actin cytoskeleton through cytoplasmic proteins, termed α- and β-catenin, recent studies with purified proteins indicate that the interaction is not direct, and instead an allosteric switch in α-catenin may mediate actin cytoskeleton reorganization. Organization and function of the cadherin–catenin complex are additionally regulated by phosphorylation and endocytosis. Direct studies of cell–cell adhesion has revealed that the cadherin–catenin complex and the underlying actin cytoskeleton undergo a series of reorganizations that are controlled by the Rho GTPases, Rac1 and RhoA, that result in the expansion and completion of cell–cell adhesion. In the present article, in vitro protein assembly studies and live-cell studies of de novo cell–cell adhesion are discussed in the context of how the cadherin–catenin complex and the actin cytoskeleton regulate cell–cell adhesion.

The cadherin/catenin complex, comprised of E-cadherin, beta-catenin and alpha-catenin, is essential for initiating cell-cell adhesion, establishing cellular polarity and maintaining tissue organization. Disruption or loss of the cadherin/catenin complex is common in cancer. As the primary cell-cell adhesion protein in epithelial cells, E-cadherin has long been studied in cancer progression. Similarly, additional roles for beta-catenin in the Wnt signaling pathway has led to many studies of the role of beta-catenin in cancer. Alpha-catenin, in contrast, has received less attention. However, recent data demonstrate novel functions for alpha-catenin in regulating the actin cytoskeleton and cell-cell adhesion, which when perturbed could contribute to cancer progression. In this review, we use cancer data to evaluate molecular models of alpha-catenin function, from the canonical role of alpha-catenin in cell-cell adhesion to non-canonical roles identified following conditional alpha-catenin deletion. This analysis identifies alpha-catenin as a prognostic factor in cancer progression.

In epithelial cells, polarized growth and maintenance of apical and basolateral plasma membrane domains depend on protein sorting from the trans-Golgi network (TGN) and vesicle delivery to the plasma membrane. Septins are filamentous GTPases required for polarized membrane growth in budding yeast, but whether they function in epithelial polarity is unknown. Here, we show that in epithelial cells septin 2 (SEPT2) fibers colocalize with a subset of microtubule tracks composed of polyglutamylated (polyGlu) tubulin, and that vesicles containing apical or basolateral proteins exit the TGN along these SEPT2/polyGlu microtubule tracks. Tubulin-associated SEPT2 facilitates vesicle transport by maintaining polyGlu microtubule tracks and impeding tubulin binding of microtubule-associated protein 4 (MAP4). Significantly, this regulatory step is required for polarized, columnar-shaped epithelia biogenesis; upon SEPT2 depletion, cells become short and fibroblast-shaped due to intracellular accumulation of apical and basolateral membrane proteins, and loss of vertically oriented polyGlu microtubules. We suggest that septin coupling of the microtubule cytoskeleton to post-Golgi vesicle transport is required for the morphogenesis of polarized epithelia.

While much is known of the molecular machinery involved in protein sorting during exocytosis, less is known about the spatial regulation of exocytosis at the plasma membrane (PM). This study outlines a novel method, dual substrate display, used to formally test the hypothesis that E-cadherin-mediated adhesion directs basolateral vesicle exocytosis to specific sites at the PM. We show that vesicles containing the basolateral marker protein VSV-G preferentially target to sites of adhesion to E-cadherin rather than collagen VI or a control peptide. These results support the hypothesis that E-cadherin adhesion initiates signaling at the PM resulting in targeted sites for exocytosis.

Spatiotemporal coordination of cell–cell adhesion involving lamellipodial interactions, cadherin engagement, and the lateral expansion of the contact is poorly understood. Using high-resolution live-cell imaging, biosensors, and small molecule inhibitors, we investigate how Rac1 and RhoA regulate actin dynamics during de novo contact formation between pairs of epithelial cells. Active Rac1, the Arp2/3 complex, and lamellipodia are initially localized to de novo contacts but rapidly diminish as E-cadherin accumulates; further rounds of activation and down-regulation of Rac1 and Arp2/3 occur at the contacting membrane periphery, and this cycle repeats as a restricted membrane zone that moves outward with the expanding contact. The cortical bundle of actin filaments dissolves beneath the expanding contacts, leaving actin bundles at the contact edges. RhoA and actomyosin contractility are activated at the contact edges and are required to drive expansion and completion of cell–cell adhesion. We show that zones of Rac1 and lamellipodia activity and of RhoA and actomyosin contractility are restricted to the periphery of contacting membranes and together drive initiation, expansion, and completion of cell–cell adhesion.

Synapses are specialized adhesive contacts characteristic of many types of cell-cell interactions involving neurons, immune cells, epithelial cells, and even pathogens and host cells. Cell-cell adhesion is mediated by structurally diverse classes of cell-surface glycoproteins, which form homophilic or heterophilic interactions across the intercellular space. Adhesion proteins bind to a cytoplasmic network of scaffolding proteins, regulators of the actin cytoskeleton, and signal transduction pathways that control the structural and functional organization of synapses. The themes of this review are to compare the organization of synapses in different cell types and to understand how different classes of cell adhesion proteins and cytoplasmic protein networks specify the assembly of functionally distinct synapses in different cell contexts.

Coordinated movement of large groups of cells is required for many biological processes, such as gastrulation and wound healing. During collective cell migration, cell-cell and cell-extracellular matrix (ECM) adhesions must be integrated so that cells maintain strong interactions with neighboring cells and the underlying substratum. Initiation and maintenance of cadherin adhesions at cell-cell junctions and integrin-based cell-ECM adhesions require integration of mechanical cues, dynamic regulation of the actin cytoskeleton, and input from specific signaling cascades, including Rho family GTPases. Here, we summarize recent advances made in understanding the interplay between these pathways at cadherin-based and integrin-based adhesions during collective cell migration and highlight outstanding questions that remain in the field.

Cell-cell adhesion plays critical roles in establishing and maintaining tissue architecture and function (1, 2). In these roles, cell-cell adhesion must be adaptable and (depending on the biological circumstance) weak, dynamic, or strong. Different adhesion structures (adherens junctions, tight junctions, desmosomes) regulate cell-cell adhesion, and each comprises distinct membrane-bound adhesion proteins that interact with the cytoskeleton. Of the proteins that form these adhesion structures, the Ca2+-dependent classical cadherins found in adherens junctions have critical roles in controlling the specificity, organization, and dynamics of cell-cell adhesions. Cadherins are required for the formation of the first overt forms of tissue differentiation in early vertebrate and invertebrate development (3, 4). The specificity of adhesion among different cell types depends upon the strength of binding between and surface concentration of cadherins (5, 6). During Ca2+-dependent cell-cell adhesion, cadherins rapidly concentrate at sites of cell-cell contact through an active process involving the actin cytoskeleton (7), which reorganizes as cell-cell contacts form (8, 9). Actomyosin contractility may also play a role in cell-cell adhesion (10) and remodeling of cell and tissue structures in development (11, 12).

Septins are conserved GTP-binding proteins that associate with cellular membranes and the actin and microtubule cytoskeletons. They polymerize to form filamentous structures that act as diffusion barriers between different membrane domains and as molecular scaffolds for membrane- and cytoskeleton-binding proteins. In yeast, septins are central to the spatio-temporal coordination of membrane polarity and cell division, but the roles of their mammalian counterparts have remained poorly understood. However, recent findings have shed light on the dynamics and regulation of mammalian septin assembly and our understanding of septin functions in cytoskeleton and membrane organization. The mammalian septins appear to form a novel network of hetero-polymers that are multi-functional, inter-changeable and respond dynamically to signals that coordinate events at the interface between cytoskeleton and membrane biology. Hence, studies of these molecules might provide new insights not only into how cells coordinate their functions, but also into the pathogenesis of cancer and other diseases in which septins are abnormally expressed.

Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through beta-catenin and alpha-catenin, although the quaternary complex has never been demonstrated. We test this hypothesis and find that alpha-catenin does not interact with actin filaments and the E-cadherin-beta-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and alpha-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, beta-catenin, and alpha-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated.

SummaryEpithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that α-catenin links the adhesion protein E-cadherin through β-catenin to the actin cytoskeleton, in the accompanying paper we report that α-catenin does not bind simultaneously to both E-cadherin-β-catenin and actin filaments. Here we demonstrate that α-catenin exists as a monomer or a homodimer with different binding properties. Monomeric α-catenin binds more strongly to E-cadherin-β-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that α-catenin is an allosteric protein. Significantly, α-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for α-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.