E-cadherin Clusters Research Articles

Water transport regulates nucleus volume, cell density, Young’s modulus, and E-cadherin expression in tumor spheroids

Cell volume is maintained by the balance of water and solutes across the cell membrane and plays an important role in mechanics and biochemical signaling in cells. Here, we assess the relationship between cell volume, mechanical properties, and E-cadherin expression in three-dimensional cultures for ovarian cancer. To determine the effect of water transport in multi-cellular tumors, ovarian cancer spheroids were subjected to hypotonic and hypertonic shock using water and sucrose mixtures, respectively. Increased osmolality resulted in decreased nucleus volume, increased Young’s modulus, and increased tumor cell density in ovarian cancer spheroids. Next, we looked at the reversibility of mechanics and morphology after 5 min of osmotic shock and found that spheroids had a robust ability to return to their original state. Finally, we quantified the size of E-cadherin clusters at cell-cell junctions and observed a significant increase in aggregate size following 30 min of hypertonic and hypotonic osmotic shocks. Yet, these effects were not apparent after 5 min of osmotic shock, illustrating a temporal difference between E-cadherin regulation and the immediate mechanical and morphology changes. Still, the osmotically induced E-cadherin aggregates which formed at the 30-minute timepoint was reversible when spheroids were replenished with isotonic medium. Altogether, this work demonstrated an important role of osmolality in transforming mechanical, morphology, and molecular states.

Computational model of E-cadherin clustering under force

E-cadherins play a critical role in the formation of cell-cell adhesions for several physiological functions, including tissue development, repair, and homeostasis. The formation of clusters of E-cadherins involves extracellular adhesive (trans-) and lateral (cis-) associations between E-cadherin ectodomains and stabilization through intracellular binding to the actomyosin cytoskeleton. This binding provides force to the adhesion and is required for mechanotransduction. However, the exact role of cytoskeletal force on the clustering of E-cadherins is not well understood. To gain insights into this mechanism, we developed a computational model based on Brownian dynamics. In the model, E-cadherins transit between structural and functional states; they are able to bind and unbind other E-cadherins on the same and/or opposite cell(s) through trans- and cis-interactions while also creating dynamic links with the actomyosin cytoskeleton. Our results show that actomyosin force governs the fraction of E-cadherins in clusters and the size and number of clusters. For low forces (below 10 pN), a large number of small E-cadherin clusters form with less than five E-cadherins each. At higher forces, the probability of forming fewer but larger clusters increases. These findings support the idea that force reinforces cell-cell adhesions, which is consistent with differences in cluster size previously observed between apical and lateral junctions of epithelial tissues.

Computational Model of E-cadherin Clustering under Cortical Tension

E-cadherins are adhesion proteins that play a critical role in the formation of cell-cell junctions for several physiological processes, including tissue development and homeostasis. The formation of E-cadherin clusters involves extracellular trans- and cis- associations between cadherin ectodomains and stabilization through intracellular coupling with the contractile actomyosin cortex. The dynamic remodeling of cell-cell junctions largely depends on cortical tension, but previous modeling frameworks did not incorporate this effect. In order to gain insights into the effects of cortical tension on the dynamic properties of E-cadherin clusters, here we developed a computational model based on Brownian dynamics. The model considers individual cadherins as explicit point particles undergoing cycles of lateral diffusion on two parallel surfaces that mimic the membrane of neighboring cells. E-cadherins transit between functional states, including monomers, X- and S- dimers, that are able to selectively bind and unbind other E-cadherins on the same and/or opposite surface(s), and associate with the actomyosin cytoskeleton to stabilize their interactions and transmit tension. Kinetic rates for binding and unbinding are taken from previous experimental measurements, with unbinding rates reproducing catch or slip bonds lifetimes, depending on the E-cadherin functional state. Results from the model show that cortical tension governs the fraction of E-cadherins in clusters and the distribution of cluster size. For low forces (below 10 pN), a large amount of small clusters form, including less than 5 E-cadherins each, with the majority of E-cadherins in clusters. At higher forces, the probability of forming fewer but larger clusters increases, with most E-cadherins not in clusters. These findings support the general idea of force-dependent reinforcement of cell-cell junctions and are consistent with differences in cluster sizes observed between apical and lateral junctions in epithelial tissues.

RhoA Mediates Epithelial Cell Shape Changes via Mechanosensitive Endocytosis.

Epithelial remodeling involves ratcheting behavior whereby periodic contractility produces transient changes in cell-cell contact lengths, which stabilize to produce lasting morphogenetic changes. Pulsatile RhoA activity is thought to underlie morphogenetic ratchets, but how RhoA governs transient changes in junction length, and how these changes are rectified to produce irreversible deformation, remains poorly understood. Here, we use optogenetics to characterize responses to pulsatile RhoA in model epithelium. Short RhoA pulses drive reversible junction contractions, while longer pulses produce irreversible junction length changes that saturate with prolonged pulse durations. Using an enhanced vertex model, we show this is explained by two effects: thresholded tension remodeling and continuous strain relaxation. Our model predicts that structuring RhoA into multiple pulses overcomes the saturation of contractility and confirms this experimentally. Junction remodeling also requires formin-mediated E-cadherin clustering and dynamin-dependent endocytosis. Thus, irreversible junction deformations are regulated by RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling.

Actin protrusions push at apical junctions to maintain E-cadherin adhesion

Cadherin-mediated cell-cell adhesion is actin-dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized Madin-Darby canine kidney (MDCK) cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells, while reformation of cadherin clusters across the cell-cell boundary correlates with microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as 3 actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNA interference (RNAi) results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

Myosin II isoforms play distinct roles in adherens junction biogenesis.

Adherens junction (AJ) assembly under force is essential for many biological processes like epithelial monolayer bending, collective cell migration, cell extrusion and wound healing. The acto-myosin cytoskeleton acts as a major force-generator during the de novo formation and remodeling of AJ. Here, we investigated the role of non-muscle myosin II isoforms (NMIIA and NMIIB) in epithelial junction assembly. NMIIA and NMIIB differentially regulate biogenesis of AJ through association with distinct actin networks. Analysis of junction dynamics, actin organization, and mechanical forces of control and knockdown cells for myosins revealed that NMIIA provides the mechanical tugging force necessary for cell-cell junction reinforcement and maintenance. NMIIB is involved in E-cadherin clustering, maintenance of a branched actin layer connecting E-cadherin complexes and perijunctional actin fibres leading to the building-up of anisotropic stress. These data reveal unanticipated complementary functions of NMIIA and NMIIB in the biogenesis and integrity of AJ.

Non-junctional E-Cadherin Clusters Regulate the Actomyosin Cortex in the C. elegans Zygote

Non-junctional E-Cadherin Clusters Regulate the Actomyosin Cortex in the C. elegans Zygote

Receptor Nucleation and Clustering in Cellular Adhesion and Mechanical Signal Transduction

E-cadherin-based cell-cell adhesions are key to development and maintenance of the epithelial tissue, and a loss of these adhesions may contribute to cancer development. These are mechanosensitive structures in that they are strengthened under tension. Mechanotransduction in these adhesions has been postulated to be mediated, in part, by a force-dependent conformational activation of α-catenin, which allows it to interact with vinculin, in addition to F-actin, resulting in strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions are formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central depending on their relative location at the cell-bilayer adhesion. While F-actin, vinculin and phosphorylated myosin light chain associate only with the peripheral assemblies, activated α-catenin is present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes, by confining bilayer bound E-cadherin extracellular domain movement on nanopatterned substrates, reduced levels of activated α-catenin. Taken together, although the initial activation of α-catenin requires micron-scale clustering that may allow development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state.

Non-junctional E-Cadherin Clusters Regulate the Actomyosin Cortex in the C. elegans Zygote

Classical cadherins are well known for their essential function in mediating cell-cell adhesion via their extra-cellular cadherin domains and intra-cellular connections to the actin cytoskeleton [1-3]. There is evidence, however, of adhesion-independent cadherin clusters existing outside of cell-cell junctions [4-6]. What function, if any, these clusters have is not known. HMR-1, the sole classical cadherin in Caenorhabditis elegans, plays essential roles during gastrulation, blastomere polarity establishment, and epidermal morphogenesis [7-11]. To elucidate the physiological roles of non-junctional cadherin, we analyzed HMR-1 in the C.elegans zygote, which is devoid of neighbors. We show that non-junctional clusters of HMR-1 form during the one-cell polarization stage and associate with F-actin at the cortex during episodes of cortical flow. Non-junctional HMR-1 clusters downregulate RHO-1 activity and inhibit accumulation of non-muscle myosin II (NMY-2) at the anterior cortex. We found that HMR-1 clusters impede cortical flows and play a role in preserving the integrity of the actomyosin cortex, preventing it from splitting in two. Importantly, we uncovered an inverse relationship between the amount of HMR-1 at the cell surface and the rate of cytokinesis. The effect of HMR-1 clusters on cytokinesis is independent of their effect on NMY-2 levels, and is also independent of their extra-cellular domains. Thus, in addition to their canonical role in inter-cellular adhesion, HMR-1 clusters regulate RHO-1 activity and NMY-2 level at the cell surface, reinforce the stability of the actomyosin cortex, and resist its movement to influence cell-shape dynamics.

Force Bistability in Adhesion Switch

The lateral junctions of epithelial cells must display mechanical stability to form barriers, but also significant plasticity to allow for reorganization during development and repair. In this issue, Biswas et al. (1) extend the use of fluid supported-lipid bilayers (SLBs) to reconstitute function of the key adhesion molecule that initiates lateral junctions of epithelial cells, E-cadherin, and to further study the requirements for mechanical forces in activating the cytoplasmic complex that links E-cadherin to the cytoskeleton (1).

Sustained α-catenin Activation at E-cadherin Junctions in the Absence of Mechanical Force

Mechanotransduction at E-cadherin junctions has been postulated to be mediated in part by a force-dependent conformational activation of α-catenin. Activation of α-catenin allows it to interact with vinculin in addition to F-actin, resulting in a strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions were formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central assemblies depending on their relative location at the cell-bilayer adhesion. Whereas F-actin, vinculin, and phosphorylated myosin light chain associated only with the peripheral assemblies, activated α-catenin was present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes by confining the movement of bilayer-bound E-cadherin on nanopatterned substrates reduced the levels of activated α-catenin. Taken together, these results indicate that although the initial activation of α-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state.

Balancing spatially regulated β-actin translation and dynamin-mediated endocytosis is required to assemble functional epithelial monolayers.

Regulating adherens junction complex assembly/disassembly is critical to maintaining epithelial homeostasis in healthy epithelial tissues. Consequently, adherens junction structure and function is often perturbed in clinically advanced tumors of epithelial origin. Some of the most studied factors driving adherens junction complex perturbation in epithelial cancers are transcriptional and epigenetic down-regulation of E-cadherin expression. However, numerous reports demonstrate that post-translational regulatory mechanisms such as endocytosis also regulate early phases of epithelial-mesenchymal transition and metastatic progression. In already assembled healthy epithelia, E-cadherin endocytosis recycles cadherin-catenin complexes to regulate the number of mature adherens junctions found at cell-cell contact sites. However, following de novo epithelial cell-cell contact, endocytosis negatively regulates adherens junction assembly by removing E-cadherin from the cell surface. By contrast, following de novo epithelial cell-cell contact, spatially localized β-actin translation drives cytoskeletal remodeling and consequently E-cadherin clustering at cell-cell contact sites and therefore positively regulates adherens junction assembly. In this report we demonstrate that dynamin-mediated endocytosis and β-actin translation-dependent cadherin-catenin complex anchoring oppose each other following epithelial cell-cell contact. Consequently, the final extent of adherens junction assembly depends on which of these processes is dominant following epithelial cell-cell contact. We expressed β-actin transcripts impaired in their ability to properly localize monomer synthesis (Δ3'UTR) in MDCK cells to perturb actin filament remodeling and anchoring, and demonstrate the resulting defect in adherens junction structure and function is rescued by inhibiting dynamin mediated endocytosis. Therefore, we demonstrate balancing spatially regulated β-actin translation and dynamin-mediated endocytosis regulates epithelial monolayer structure and barrier function.

Considering autophagy, β-Catenin and E-Cadherin as innovative therapy aspects in AML.

Considering autophagy, β-Catenin and E-Cadherin as innovative therapy aspects in AML.

Actin-Delimited Adhesion-Independent Clustering of E-Cadherin Forms the Nanoscale Building Blocks of Adherens Junctions

E-cadherin is the major adhesion receptor in epithelial adherens junctions, which connect cells to form tissues and are essential for morphogenesis and homeostasis. The mechanism by which E-cadherin monomers cluster and become organized in adherens junctions remains poorly understood. Here, using superresolution microscopy techniques in combination with structure-informed functional mutations, we found that loosely organized clusters of approximately five E-cadherin molecules that form independently of cis or trans interactions, and that are delimited by the cortical F-actin meshwork, are the precursors of trans-ligated adhesive clusters that make up the adherens junction. The density of E-cadherin clusters was wide ranged, and notably, we could detect densities consistent with the crystal lattice structure at the core of adhesive clusters, which were dependent on extracellular domain interactions. Thus, our results elucidate the nanoscale architecture of adherens junctions, as well as the molecular mechanisms driving its assembly.

Actomyosin bundles serve as a tension sensor and a platform for ERK activation.

Tensile forces generated by stress fibers drive signal transduction events at focal adhesions. Here, we report that stress fibers per se act as a platform for tension-induced activation of biochemical signals. The MAP kinase, ERK is activated on stress fibers in a myosin II-dependent manner. In myosin II-inhibited cells, uniaxial stretching of cell adhesion substrates restores ERK activation on stress fibers. By quantifying myosin II- or mechanical stretch-mediated tensile forces in individual stress fibers, we show that ERK activation on stress fibers correlates positively with tensile forces acting on the fibers, indicating stress fibers as a tension sensor in ERK activation. Myosin II-dependent ERK activation is also observed on actomyosin bundles connecting E-cadherin clusters, thus suggesting that actomyosin bundles, in general, work as a platform for tension-dependent ERK activation.

Self-Organizing Actomyosin Patterns on the Cell Cortex at Epithelial Cell-Cell Junctions

The behavior of actomyosin critically determines morphologically distinct patterns of contractility found at the interface between adherent cells. One such pattern is found at the apical region (zonula adherens) of cell-cell junctions in epithelia, where clusters of the adhesion molecule E-cadherin concentrate in a static pattern. Meanwhile, E-cadherin clusters throughout lateral cell-cell contacts display dynamic movements in the plane of the junctions. To gain insight into the principles that determine the nature and organization of these dynamic structures, we analyze this behavior by modeling the 2D actomyosin cell cortex as an active fluid medium. The numerical simulations show that the stability of the actin filaments influences the spatial structure and dynamics of the system. We find that in addition to static Turing-type patterns, persistent dynamic behavior occurs in a wide range of parameters. In the 2D model, mechanical stress-dependent actin breakdown is shown to produce a continuously changing network of actin bridges, whereas with a constant breakdown rate, more isolated clusters of actomyosin tend to form. The model qualitatively reproduces the dynamic and stable patterns experimentally observed at the junctions between epithelial cells.

Pulsatile contractility of actomyosin networks organizes the cellular cortex at lateral cadherin junctions

The physical properties of cells reflect how the structure and dynamics of the actomyosin cortex are coupled to the plasma membrane. In epithelia, adhesive E-cadherin clusters associate with the cell cortex to assemble the junctional actomyosin that participates in epithelial morphogenesis. E-cadherin is present not only at the apical zonula adherens (ZA), but also distributed throughout the lateral adherens junction (LAJ) below the ZA. However, the organizational dynamics of the actomyosin network at the LAJs remains elusive. To address this, we used quantitative real-time imaging to characterize the dynamics of actomyosin contractility at lateral cadherin contacts. Here, we report that contractility is coordinated into smaller actomyosin rings that link cadherin clusters together within the larger cortical network at the lateral junctions. We conclude that Myosin II activity determines the contractility of actomyosin cables between cadherin clusters to propagate pulsatility across lateral cell–cell contacts.

Principles of E-Cadherin Supramolecular Organization In Vivo

E-cadherin plays a pivotal role in tissue morphogenesis by forming clusters that support intercellular adhesion and transmit tension. What controls E-cadherin mesoscopic organization in clusters is unclear. We use 3D superresolution quantitative microscopy in Drosophila embryos to characterize the size distribution of E-cadherin nanometric clusters. The cluster size follows power-law distributions over three orders of magnitude with exponential decay at large cluster sizes. By exploring the predictions of a general theoretical framework including cluster fusion and fission events and recycling of E-cadherin, we identify two distinct active mechanisms setting the cluster-size distribution. Dynamin-dependent endocytosis targets large clusters only, thereby imposing a cutoff size. Moreover, interactions between E-cadherin clusters and actin filaments control the fission in a size-dependent manner. E-cadherin clustering depends on key cortical regulators, which provide tunable and local control over E-cadherin organization. Our data provide the foundation for a quantitative understanding of how E-cadherin distribution affects adhesion and might regulate force transmission in vivo.

Oscillation and Polarity of E-Cadherin Asymmetries Control Actomyosin Flow Patterns during Morphogenesis

Actomyosin flows are involved in a variety of cellular processes, including cytokinesis, cell migration, polarization, and morphogenesis. In epithelia, flow polarization orients cell deformations. It is unclear, however, how flows are polarized and how global patterns of junction remodeling emerge from flow polarization locally. We address this question during intercalation-driving extension of the Drosophila germband. Intercalation is associated with polarized junction remodeling, whereby actomyosin pulses flow anisotropically toward dorsal-ventral junctions and shrink them. Here, we show that planar polarization of flows emerges from polarized fluctuations in the levels of E-cadherin clusters that produce transient and oscillating asymmetries of coupling. These fluctuations are triggered by polarized E-cadherin endocytosis and are amplified by flow itself. This work suggests that fluctuations and mechanical instability are not the consequences of limited control over the systems key parameters, but rather that they define the axis of symmetry breaking.

ROCK1 and ROCK2 regulate epithelial polarisation and geometric cell shape

Cell-cell adhesion and contraction play an essential role in the maintenance of geometric shape and polarisation of epithelial cells. However, the molecular regulation of contraction during cell elongation leading to epithelial polarisation and acquisition of geometric cell shape is not clear. Upon induction of cell-cell adhesion, we find that human keratinocytes acquire specific geometric shapes favouring hexagons, by re-modelling junction length/orientation and thus neighbour allocation. Acquisition of geometric shape correlates temporally with epithelial polarisation, as shown by an increase in lateral height. ROCK1 and ROCK2 are important regulators of myosin II contraction, but their specific role in epithelial cell shape has not been addressed. Depletion of ROCK proteins interferes with the correct proportion of hexagonal cell shapes and full elongation of lateral domain. Interestingly, ROCK proteins are not essential for maintenance of circumferential thin bundles, the main contractile epithelial F-actin pool. Instead, ROCK1 or ROCK2 regulates thin bundle contraction and positioning along the lateral domain, an important event for the stabilisation of the elongating lateral domain. Mechanistically, E-cadherin clustering specifically leads to ROCK1/ROCK2-dependent inactivation of myosin phosphatase and phosphorylation of myosin regulatory light chain. These events correlate temporally with the increase in lateral height and thin bundle compaction towards junctions. We conclude that ROCK proteins are necessary for acquisition of elongated and geometric cell shape, two key events for epithelial differentiation.