Mechanical disruption of E-cadherin complexes with epidermal growth factor receptor actuates growth factor-dependent signaling.

Increased intercellular tension is associated with enhanced cell proliferation and tissue growth. Here, we present evidence for a force-transduction mechanism that links mechanical perturbations of epithelial (E)-cadherin (CDH1) receptors to the force-dependent activation of epidermal growth factor receptor (EGFR, ERBB1)-a key regulator of cell proliferation. Here, coimmunoprecipitation studies first show that E-cadherin and EGFR form complexes at the plasma membrane that are disrupted by either epidermal growth factor (EGF) or increased tension on homophilic E-cadherin bonds. Although force on E-cadherin bonds disrupts the complex in the absence of EGF, soluble EGF is required to mechanically activate EGFR at cadherin adhesions. Fully quantified spectral imaging fluorescence resonance energy transfer further revealed that E-cadherin and EGFR directly associate to form a heterotrimeric complex of two cadherins and one EGFR protein. Together, these results support a model in which the tugging forces on homophilic E-cadherin bonds trigger force-activated signaling by releasing EGFR monomers to dimerize, bind EGF ligand, and signal. These findings reveal the initial steps in E-cadherin-mediated force transduction that directly link intercellular force fluctuations to the activation of growth regulatory signaling cascades.

Computational and experimental approaches to quantify protein binding interactions under confinement.

Crowded environments and confinement alter the interactions of adhesion proteins confined to membranes or narrow, crowded gaps at adhesive contacts. Experimental approaches and theoretical frameworks were developed to quantify protein binding constants in these environments. However, recent predictions and the complexity of some protein interactions proved challenging to address with prior experimental or theoretical approaches. This perspective highlights new methods developed by these authors that address these challenges. Specifically, single-molecule fluorescence resonance energy transfer and single-molecule tracking measurements were developed to directly image the binding/unbinding rates of membrane-tethered cadherins. Results identified predicted cis (lateral) interactions, which control cadherin clustering on membranes but were not detected in solution. Kinetic Monte Carlo simulations, based on a realistic model of cis cadherin interactions, were developed to extract binding/unbinding rate constants from heterogeneous single-molecule data. The extension of single-molecule fluorescence measurements to cis and trans (adhesive) cadherin interactions at membrane junctions identified unexpected cooperativity between cis and trans binding that appears to enhance intercellular binding kinetics. Comparisons of intercellular binding kinetics, kinetic Monte Carlo simulations, and single-molecule fluorescence data suggest a strategy to bridge protein binding kinetics across length scales. Although cadherin is the focus of these studies, the approaches can be extended to other intercellular adhesion proteins.

Cadherin cis and trans interactions are mutually cooperative.

Cadherin transmembrane proteins are responsible for intercellular adhesion in all biological tissues and modulate tissue morphogenesis, cell motility, force transduction, and macromolecular transport. The protein-mediated adhesions consist of adhesive trans interactions and lateral cis interactions. Although theory suggests cooperativity between cis and trans bonds, direct experimental evidence of such cooperativity has not been demonstrated. Here, the use of superresolution microscopy, in conjunction with intermolecular single-molecule Förster resonance energy transfer, demonstrated the mutual cooperativity of cis and trans interactions. Results further demonstrate the consequent assembly of large intermembrane junctions, using a biomimetic lipid bilayer cell adhesion model. Notably, the presence of cis interactions resulted in a nearly 30-fold increase in trans-binding lifetimes between epithelial-cadherin extracellular domains. In turn, the presence of trans interactions increased the lifetime of cis bonds. Importantly, comparison of trans-binding lifetimes of small and large cadherin clusters suggests that this cooperativity is primarily due to allostery. The direct quantitative demonstration of strong mutual cooperativity between cis and trans interactions at intermembrane adhesions provides insights into the long-standing controversy of how weak cis and trans interactions act in concert to create strong macroscopic cell adhesions.

Protein Folding Stability and Kinetics in Alginate Hydrogels.

Proteins are commonly encapsulated in alginate gels for drug delivery and tissue-engineering applications. However, there is limited knowledge of how encapsulation impacts intrinsic protein properties such as folding stability or unfolding kinetics. Here, we use fast relaxation imaging (FReI) to image protein unfolding in situ in alginate hydrogels after applying a temperature jump. Based on changes in the Förster resonance energy transfer (FRET) response of FRET-labeled phosphoglycerate kinase (PGK), we report the quantitative impact of multiple alginate hydrogel concentrations on protein stability and folding dynamics. The gels stabilize PGK by increasing its melting temperature up to 18.4 °C, and the stabilization follows a nonmonotonic dependence on the alginate density. In situ kinetic measurements also reveal that PGK deviates more from two-state folding behavior in denser gels and that the gel decreases the unfolding rate and accelerates the folding rate of PGK, compared to buffer. Phi-value analysis suggests that the folding transition state of an encapsulated protein is structurally similar to that of folded protein. This work reveals both beneficial and negative impacts of gel encapsulation on protein folding, as well as potential mechanisms contributing to altered stability.

Protein Stabilization by Alginate Binding and Suppression of Thermal Aggregation.

Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely established. Alginate is widely used to encapsulate proteins for drug delivery and tissue regeneration despite limited knowledge of its impact on protein stability. Here, we present evidence that alginate can both increase protein folding stability and suppress the aggregation of unfolded protein through direct interactions without crowding. We used a fluorescence-based conformational reporter of two proteins, the metabolic protein phosphoglycerate kinase (PGK) and the hPin1 WW domain to monitor protein stability and aggregation as a function of temperature and the weight percent of alginate in solution. Alginate stabilizes PGK by up to 14.5 °C, but stabilization is highly protein-dependent, and the much smaller WW domain is stabilized by only 3.5 °C against thermal denaturation. Stabilization is greatest at low alginate weight percent and decreases at higher alginate concentrations. This trend cannot be explained by crowding, and ionic screening suggests that alginate stabilizes proteins through direct interactions with a significant electrostatic component. Alginate also strongly suppresses aggregation at high temperature by irreversibly associating with unfolded proteins and preventing refolding. Both the beneficial and negative impacts of alginate on protein stability and aggregation have important implications for practical applications.

Stabilization and Kinetics of an Adsorbed Protein Depends on the Poly(N-isopropylacrylamide) Grafting Density.

The solubility transition at the lower critical solution temperature (LCST, 32 °C) of poly(N-isopropylacrylamide) (PNIPAM) is widely used as a thermal switch to rapidly and reversibly capture and release proteins and cells. It is generally assumed that proteins adsorbed to PNIPAM above the LCST are unaffected by polymer interactions. Here we show that the folding stability of the enzyme phosphoglycerate kinase (PGK) is increased by interactions with end-grafted PNIPAM films above the LCST. We systematically compare two protein mutants with different stabilities. The stabilization mirrors the degree of protein adsorption under grafting conditions studied previously. Maximum stabilization occurs when proteins adsorb to low density, collapsed polymer "mushrooms". In the denser polymer "brush" regime, protein stabilization decreases back to a value indistinguishable from the bulk solution, consistent with low protein adsorption on dense, collapsed brushes. The temperature-dependent kinetics measured by Fast Relaxation Imaging reveals that PNIPAM does not affect the overall folding/unfolding mechanism. Based on the different stabilizations of two mutants and the relaxation kinetics, we hypothesize that the polymer acts mainly by increasing the conformational entropy of the folded protein by interacting with the protein surface and less by crowding the unfolded state of PGK.

Epidermal growth factor receptor and integrins control force-dependent vinculin recruitment to E-cadherin junctions.

This study reports novel findings that link E-cadherin (also known as CDH1)-mediated force-transduction signaling to vinculin targeting to intercellular junctions via epidermal growth factor receptor (EGFR) and integrins. These results build on previous findings that demonstrated that mechanically perturbed E-cadherin receptors activate phosphoinositide 3-kinase and downstream integrins in an EGFR-dependent manner. Results of this study show that this EGFR-mediated kinase cascade controls the force-dependent recruitment of vinculin to stressed E-cadherin complexes - a key early signature of cadherin-based mechanotransduction. Vinculin targeting requires its phosphorylation at tyrosine 822 by Abl family kinases (hereafter Abl), but the origin of force-dependent Abl activation had not been identified. We now present evidence that integrin activation, which is downstream of EGFR signaling, controls Abl activation, thus linking E-cadherin to Abl through a mechanosensitive signaling network. These findings place EGFR and integrins at the center of a positive-feedback loop, through which force-activated E-cadherin signals regulate vinculin recruitment to cadherin complexes in response to increased intercellular tension.This article has an associated First Person interview with the first author of the paper.

Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation.

This review addresses the cellular and molecular mechanisms of cadherin-based tissue morphogenesis. Tissue physiology is profoundly influenced by the distinctive organizations of cells in organs and tissues. In metazoa, adhesion receptors of the classical cadherin family play important roles in establishing and maintaining such tissue organization. Indeed, it is apparent that cadherins participate in a range of morphogenetic events that range from support of tissue integrity to dynamic cellular rearrangements. A comprehensive understanding of cadherin-based morphogenesis must then define the molecular and cellular mechanisms that support these distinct cadherin biologies. Here we focus on four key mechanistic elements: the molecular basis for adhesion through cadherin ectodomains, the regulation of cadherin expression at the cell surface, cooperation between cadherins and the actin cytoskeleton, and regulation by cell signaling. We discuss current progress and outline issues for further research in these fields.

E-cadherin-mediated force transduction signals regulate global cell mechanics.

This report elucidates an E-cadherin-based force-transduction pathway that triggers changes in cell mechanics through a mechanism requiring epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), and the downstream formation of new integrin adhesions. This mechanism operates in addition to local cytoskeletal remodeling triggered by conformational changes in the E-cadherin-associated protein α-catenin, at sites of mechanical perturbation. Studies using magnetic twisting cytometry (MTC), together with traction force microscopy (TFM) and confocal imaging identified force-activated E-cadherin-specific signals that integrate cadherin force transduction, integrin activation and cell contractility. EGFR is required for the downstream activation of PI3K and myosin-II-dependent cell stiffening. Our findings also demonstrated that α-catenin-dependent cytoskeletal remodeling at perturbed E-cadherin adhesions does not require cell stiffening. These results broaden the repertoire of E-cadherin-based force transduction mechanisms, and define the force-sensitive signaling network underlying the mechano-chemical integration of spatially segregated adhesion receptors.

Soluble Zwitterionic Poly(sulfobetaine) Destabilizes Proteins.

The widespread interest in neutral, water-soluble polymers such as poly(ethylene glycol) (PEG) and poly(zwitterions) such as poly(sulfobetaine) (pSB) for biomedical applications is due to their widely assumed low protein binding. Here we demonstrate that pSB chains in solution can interact with proteins directly. Moreover, pSB can reduce the thermal stability and increase the protein folding cooperativity relative to proteins in buffer or in PEG solutions. Polymer-dependent changes in the tryptophan fluorescence spectra of three structurally-distinct proteins reveal that soluble, 100 kDa pSB interacts directly with all three proteins and changes both the local polarity near tryptophan residues and the protein conformation. Thermal denaturation studies show that the protein melting temperatures decrease by as much as ∼1.9 °C per weight percent of polymer and that protein folding cooperativity increases by as much as ∼130 J mol-1 K-1 per weight percent of polymer. The exact extent of the changes is protein-dependent, as some proteins exhibit increased stability, whereas others experience decreased stability at high soluble pSB concentrations. These results suggest that pSB is not universally protein-repellent and that its efficacy in biotechnological applications will depend on the specific proteins used.

Local VE-cadherin mechanotransduction triggers long-ranged remodeling of endothelial monolayers.

In this study, we present results demonstrating that mechanotransduction by vascular endothelial cadherin (VE-cadherin, also known as CDH5) complexes in endothelial cells triggers local cytoskeletal remodeling, and also activates global signals that alter peripheral intercellular junctions and disrupt cell-cell contacts far from the site of force application. Prior studies have documented the impact of actomyosin contractile forces on adherens junction remodeling, but the role of VE-cadherin in force sensation and its ability to influence endothelial cell and tissue mechanics globally have not been demonstrated. Using mechanical manipulation of VE-cadherin bonds and confocal imaging, we demonstrate VE-cadherin-based mechanotransduction. We then demonstrate that it requires homophilic VE-cadherin ligation, an intact actomyosin cytoskeleton, Rho-associated protein kinase 1 (ROCK1) and phosphoinositide 3-kinase. VE-cadherin-mediated mechanotransduction triggered local actin and vinculin recruitment, as well as global signals that altered focal adhesions and disrupted peripheral intercellular junctions. Confocal imaging revealed that VE-cadherin-specific changes appear to propagate across cell junctions to disrupt distant inter-endothelial junctions. These results demonstrate the central role of VE-cadherin adhesions and the actomyosin cytoskeleton within an integrated, mechanosensitive network that both induces local cytoskeletal remodeling at the site of force application and regulates the global integrity of endothelial tissues.

α-Catenin phosphorylation promotes intercellular adhesion through a dual-kinase mechanism.

The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian αE-catenin (also known as catenin α-1) and Drosophila α-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of α-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal α-catenin function during development and collective cell migration, although no obvious defects were found in cadherin-catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of α-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of α-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the α-catenin P-linker are required for normal cadherin-catenin complex function in Drosophila and mammalian cells.

Cadherin Diffusion in Supported Lipid Bilayers Exhibits Calcium-Dependent Dynamic Heterogeneity.

Ca2+ ions are critical to cadherin ectodomain rigidity, which is required for the activation of adhesive functions. Therefore, changes in Ca2+ concentration, both in vivo and in vitro, can affect cadherin conformation and function. We employed single-molecule tracking to measure the diffusion of cadherin ectodomains tethered to supported lipid bilayers at varying Ca2+ concentrations. At a relatively high Ca2+ concentration of 2 mM, cadherin molecules exhibited a fast diffusion coefficient that was identical to that of individual lipid molecules in the bilayer (Dfast ≈ 3 µm2/s). At lower Ca2+ concentrations, where cadherin molecules were less rigid, the ensemble-average cadherin diffusion coefficient was systematically smaller. Individual cadherin trajectories were temporally heterogeneous, exhibiting alternating periods of fast and slow diffusion; the periods of slow diffusion (Dslow ≈ 0.1 µm2/s) were more prevalent at lower Ca2+ concentration. These observations suggested that more flexible cadherin ectodomains at lower Ca2+ concentration alternated between upright and lying-down conformations, where the latter interacted with more lipid molecules and experienced greater viscous drag.

A Computational Model for Kinetic Studies of Cadherin Binding and Clustering.

Cadherin is a cell-surface transmembrane receptor that mediates calcium-dependent cell-cell adhesion and is a major component of adhesive junctions. The formation of intercellular adhesive junctions is initiated by trans binding between cadherins on adjacent cells, which is followed by the clustering of cadherins via the formation of cis interactions between cadherins on the same cell membranes. Moreover, classical cadherins have multiple glycosylation sites along their extracellular regions. It was found that aberrant glycosylation affects the adhesive function of cadherins and correlates with metastatic phenotypes of several cancers. However, a mechanistic understanding of cadherin clustering during cell adhesion and the role of glycosylation in this process is still lacking. Here, we designed a kinetic model that includes multistep reaction pathways for cadherin clustering. We further applied a diffusion-reaction algorithm to numerically simulate the clustering process using a recently developed coarse-grained model. Using experimentally measured rates of trans binding between soluble E-cadherin extracellular domains, we conducted simulations of cadherin-mediated cell-cell binding kinetics, and the results are quantitatively comparable to experimental data from micropipette experiments. In addition, we show that incorporating cadherin clustering via cis interactions further increases intercellular binding. Interestingly, a two-phase kinetic profile was derived under the assumption that glycosylation regulates the kinetic rates of cis interactions. This two-phase profile is qualitatively consistent with experimental results from micropipette measurements. Therefore, our computational studies provide new, to our knowledge, insights into the molecular mechanism of cadherin-based cell adhesion.

Structural Determinants of the Mechanical Stability of α-Catenin.

α-Catenin plays a crucial role in cadherin-mediated adhesion by binding to ß-catenin, F-actin, and vinculin, and its dysfunction is linked to a variety of cancers and developmental disorders. As a mechanotransducer in the cadherin complex at intercellular adhesions, mechanical and force-sensing properties of α-catenin are critical to its proper function. Biochemical data suggest that α-catenin adopts an autoinhibitory conformation, in the absence of junctional tension, and biophysical studies have shown that α-catenin is activated in a tension-dependent manner that in turn results in the recruitment of vinculin to strengthen the cadherin complex/F-actin linkage. However, the molecular switch mechanism from autoinhibited to the activated state remains unknown for α-catenin. Here, based on the results of an aggregate of 3 µs of molecular dynamics simulations, we have identified a dynamic salt-bridge network within the core M region of α-catenin that may be the structural determinant of the stability of the autoinhibitory conformation. According to our constant-force steered molecular dynamics simulations, the reorientation of the MII/MIII subdomains under force may constitute an initial step along the transition pathway. The simulations also suggest that the vinculin-binding domain (subdomain MI) is intrinsically much less stable than the other two subdomains in the M region (MII and MIII). Our findings reveal several key insights toward a complete understanding of the multistaged, force-induced conformational transition of α-catenin to the activated conformation.

Allosteric Regulation of E-Cadherin Adhesion.

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation.

α-catenin cytomechanics--role in cadherin-dependent adhesion and mechanotransduction.

The findings presented here demonstrate the role of α-catenin in cadherin-based adhesion and mechanotransduction in different mechanical contexts. Bead-twisting measurements in conjunction with imaging, and the use of different cell lines and α-catenin mutants reveal that the acute local mechanical manipulation of cadherin bonds triggers vinculin and actin recruitment to cadherin adhesions in an actin- and α-catenin-dependent manner. The modest effect of α-catenin on the two-dimensional binding affinities of cell surface cadherins further suggests that force-activated adhesion strengthening is due to enhanced cadherin-cytoskeletal interactions rather than to α-catenin-dependent affinity modulation. Complementary investigations of cadherin-based rigidity sensing also suggest that, although α-catenin alters traction force generation, it is not the sole regulator of cell contractility on compliant cadherin-coated substrata.

A genetic variant of cortactin linked to acute lung injury impairs lamellipodia dynamics and endothelial wound healing.

Inflammatory mediators released in acute lung injury (ALI) trigger the disruption of interendothelial junctions, leading to loss of vascular barrier function, protein-rich pulmonary edema, and severe hypoxemia. Genetic signatures that predict patient recovery or disease progression are poorly defined, but recent genetic screening of ALI patients has identified an association between lung inflammatory disease and a single nucleotide polymorphism (SNP) in the gene for the actin-binding and barrier-regulatory protein cortactin. This study investigated the impact of this disease-linked cortactin variant on wound healing processes that may contribute to endothelial barrier restoration. A microfabricated platform was used to quantify wound healing in terms of gap closure speed, lamellipodia dynamics, and cell velocity. Overexpression of wild-type cortactin in endothelial cells (ECs) improved directional cell motility and enhanced lamellipodial protrusion length, resulting in enhanced gap closure rates. By contrast, the cortactin SNP impaired wound closure and cell locomotion, consistent with the observed reduction in lamellipodial protrusion length and persistence. Overexpression of the cortactin SNP in lung ECs mitigated the barrier-enhancing activity of sphingosine 1-phosphate. These findings suggest that this common cortactin variant may functionally contribute to ALI predisposition by impeding endothelial wound healing.

Cadherin-dependent mechanotransduction depends on ligand identity but not affinity.

This study investigates the relationship between classical cadherin binding affinities and mechanotransduction through cadherin-mediated adhesions. The mechanical properties of cadherin-dependent intercellular junctions are generally attributed to differences in the binding affinities of classical cadherin subtypes that contribute to cohesive energies between cells. However, cell mechanics and mechanotransduction may also regulate intercellular contacts. We used micropipette measurements to quantify the two-dimensional affinities of cadherins at the cell surface, and two complementary mechanical measurements to assess ligand-dependent mechanotransduction through cadherin adhesions. At the cell surface, the classical cadherins investigated in this study form both homophilic and heterophilic bonds with two-dimensional affinities that differ by less than threefold. In contrast, mechanotransduction through cadherin adhesions is strongly ligand dependent such that homophilic, but not heterophilic ligation mediates mechanotransduction, independent of the cadherin binding affinity. These findings suggest that ligand-selective mechanotransduction may supersede differences in cadherin binding affinities in regulating intercellular contacts.

N-glycosylation alters cadherin-mediated intercellular binding kinetics.

We present direct evidence that the N-glycosylation state of neural cadherin impacts the intrinsic kinetics of cadherin-mediated intercellular binding. Micropipette manipulation measurements quantified the effect of N-glycosylation mutations on intercellular binding dynamics. The wild-type protein exhibits a two-stage binding process in which a fast, initial binding step is followed by a short lag and second, slower transition to the final binding stage. Mutations that ablate N-glycosylation at three sites on the extracellular domains 2 and 3 of neural cadherin alter this kinetic fingerprint. Glycosylation does not affect the affinities between the adhesive N-terminal domains, but instead modulates additional cadherin interactions, which govern the dynamics of intercellular binding. These results, together with previous findings that these hypo-glycosylation mutations increase the prevalence of cis dimers on cell membranes, suggest a binding mechanism in which initial adhesion is followed by additional cadherin interactions, which enhance binding but are modulated by N-glycosylation. Given that oncogene expression drives specific changes in N-glycosylation, these results provide insight into possible mechanisms altering cadherin function during tumor progression.