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.

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.

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.

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.

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.

Cadherin point mutations alter cell sorting and modulate GTPase signaling.

This study investigated the impact of cadherin binding differences on both cell sorting and GTPase activation. The use of N-terminal domain point mutants of Xenopus C-cadherin enabled us to quantify binding differences and determine their effects on cadherin-dependent functions without any potential complications arising as a result of differences in cytodomain interactions. Dynamic cell-cell binding measurements carried out with the micropipette manipulation technique quantified the impact of these mutations on the two-dimensional binding affinities and dissociation rates of cadherins in the native context of the cell membrane. Pairwise binding affinities were compared with in vitro cell-sorting specificity and ligation-dependent GTPase signaling. Two-dimensional affinity differences greater than five-fold correlated with cadherin-dependent in vitro cell segregation, but smaller differences failed to induce cell sorting. Comparison of the binding affinities with GTPase signaling amplitudes further demonstrated that differential binding also proportionally modulates intracellular signaling. These results show that differential cadherin affinities have broader functional consequences than merely controlling cell-cell cohesion.

Recapitulating cell-cell adhesion using N-cadherin biologically tethered to substrates.

Intercellular adhesion modulated by cadherin molecules plays an important role in diverse cellular functions including tissue morphogenesis, regeneration, and pathogenesis. However, it is a challenging task to decipher the effects of cell-cell adhesion in vitro because of difficulty in controlling the extent and numbers of cell-cell contacts. In this study, we hypothesize that tethering recombinant extracellular domains of neural cadherin with a C-terminal immunoglobulin Fc domain (N-Cad-Fc) to a substrate with an immobilized anti-Fc antibody (Fc-antibody) and a bifunctional polymer, which is reactive to both protein and substrate, would allow us to recapitulate cell-cell adhesion, independent of the number of cells plated on the substrate. To examine this hypothesis, we first immobilized Fc-antibody to a polyacrylamide hydrogel and a methacrylate-substituted glass using poly(amino-2-hydroxyethyl-co-2-methacryloxyethyl aspartamide)-g-poly(ethylene glycol)-N-hydroxysuccinimide ester (PHMAA-g-PEGNHS) and then incubated the gel in medium containing defined concentrations of the recombinant N-Cad-Fc. The resulting N-Cad-conjugated substrate enabled us to modulate adhesion of bone marrow stromal cells to the gel surface by varying the surface density of N-Cad-Fc. In contrast, direct chemical conjugation of N-Cad-Fc to the gel surface did not support cell adhesion. Additionally, the glass substrate biologically tethered with N-Cad-Fc promoted neuronal adhesion significantly more than substrates coated with poly-l-lysine. We suggest that this novel biological tethering method could be broadly applicable for modifying substrates with a variety of classical cadherins to enable the systematic study of the effects of cadherin-modulated cell-cell adhesion on cellular activities.

A computational study for understanding the impact of p120-catenin on the cis-dimerization of cadherin.

A prototype of cross-membrane signal transduction is that extracellular binding of cell surface receptors to their ligands induces intracellular signaling cascades. However, much less is known about the process in the opposite direction, called inside-out signaling. Recent studies show that it plays a more important role in regulating the functions of many cell surface receptors than we used to think. In particular, in cadherin-mediated cell adhesion, recent experiments indicate that intracellular binding of the scaffold protein p120-catenin can promote extracellular clustering of cadherin and alter its adhesive function. The underlying mechanism, however, is not well understood. To explore possible mechanisms, we designed a new multiscale simulation procedure. Using all-atom molecular dynamics simulations, we found that the conformational dynamics of the cadherin extracellular region can be altered by the intracellular binding of p120-catenin. More intriguingly, by integrating all-atom simulation results into coarse-grained random sampling, we showed that the altered conformational dynamics of cadherin caused by the binding of p120-catenin can increase the probability of lateral interactions between cadherins on the cell surface. These results suggest that p120-catenin could allosterically regulate the cis-dimerization of cadherin through two mechanisms. First, p120-catenin controls the extracellular conformational dynamics of cadherin. Second, p120-catenin oligomerization can further promote cadherin clustering. Our study, therefore, suggests a mechanistic foundation for the inside-out signaling in cadherin-mediated cell adhesion, while the computational framework can be generally applied to other cross-membrane signal transduction systems.

Binding-site geometry and flexibility in DC-SIGN demonstrated with surface force measurements.

The dendritic cell receptor DC-SIGN mediates pathogen recognition by binding to glycans characteristic of pathogen surfaces, including those found on HIV. Clustering of carbohydrate-binding sites in the receptor tetramer is believed to be critical for targeting of pathogen glycans, but the arrangement of these sites remains poorly understood. Surface force measurements between apposed lipid bilayers displaying the extracellular domain of DC-SIGN and a neoglycolipid bearing an oligosaccharide ligand provide evidence that the receptor is in an extended conformation and that glycan docking is associated with a conformational change that repositions the carbohydrate-recognition domains during ligand binding. The results further show that the lateral mobility of membrane-bound ligands enhances the engagement of multiple carbohydrate-recognition domains in the receptor oligomer with appropriately spaced ligands. These studies highlight differences between pathogen targeting by DC-SIGN and receptors in which binding sites at fixed spacing bind to simple molecular patterns.

Ionic strength dependent forces between end-grafted Poly(sulfobetaine) films and mica.

The molecular surface properties of zwitterionic polymer coatings are central to their ultra-low fouling properties and effectiveness as steric stabilizers in concentrated salt solutions. Here, Surface Force Apparatus measurements quantified the molecular forces between end-grafted poly(sulfobetaine) methacrylate thin films and mica, as a function of the chain grafting density and ionic strength. These results demonstrate that, at the ionic strengths considered, end-grafted poly(sulfobetaine) films can be described by models for polymers in good solvent. Parameters determined from data fits to the Milner-Witten-Cates or Dolan and Edwards models for dense or dilute chains, respectively, varied with ionic strength, in ways that reflect poly(sulfobetaine) swelling and the increased excluded volume strength of chain segments. These force measurements provide new insight into how polymer coverage and salt cooperate to regulate repulsive poly(sulfobetaine) steric barriers. These findings have implications for the design of grafted poly(sulfobetaine) as colloidal stabilizers or nonfouling surface coatings.

Forces between mica and end-grafted statistical copolymers of sulfobetaine and oligoethylene glycol in aqueous electrolyte solutions.

This study quantified the interfacial forces associated with end-grafted, statistical (AB) co-polymers of sulfobetaine methacrylate (SBMA) and oligoethylene glycol methacrylate (OEGMA) (poly(SBMA-co-OEGMA)). Surface force apparatus measurements compared forces between mica and end-grafted copolymers containing 0, 40, or 80 mol% SBMA. Studies compared forces measured at low grafting density (weakly overlapping chains) and at high density (brushes). At high density, the range of repulsive forces did not change significantly with increasing SBMA content. By contrast, at low density, both the range and the amplitude of the repulsion increased with the percentage of SBMA in the chains. The ionic strength dependence of the film thickness and repulsive forces increased similarly with SBMA content, reflecting the increasing influence of charged monomers and their interactions with ions in solution. The forces could be described by models of simple polymers in good solvent. However, the forces and fitted model parameters change continuously with the SBMA content. The latter behavior suggests that ethyene glycol and sulfobetaine behave as non-interacting, miscible monomers that contribute independently to the interfacial forces. The results suggest that molecular scale properties of statistical poly (SBMA-co-OEGMA) films can be readily tuned by simple variation of the monomer ratios.

Extracellular matrix regulates force transduction at VE-cadherin junctions.

Increased tension on VE-cadherin (VE-cad) complexes activates adaptive cell stiffening and local cytoskeletal reinforcement--two key signatures of intercellular mechanotransduction. Here we demonstrate that tugging on VE-cad receptors initiates a cascade that results in downstream integrin activation. The formation of new integrin adhesions potentiates vinculin and actin recruitment to mechanically reinforce stressed cadherin adhesions. This cascade differs from documented antagonistic effects of integrins on intercellular junctions. We identify focal adhesion kinase, Abl kinase, and RhoA GTPase as key components of the positive feedback loop. Results further show that a consequence of integrin involvement is the sensitization of intercellular force transduction to the extracellular matrix (ECM) not by regulating junctional tension but by altering signal cascades that reinforce cell-cell adhesions. On type 1 collagen or fibronectin substrates, integrin subtypes α2ß1 and α5ß1, respectively, differentially control actin remodeling at VE-cad adhesions. Specifically, ECM-dependent differences in VE-cad force transduction mirror differences in the rigidity sensing mechanisms of α2ß1 and α5ß1 integrins. The findings verify the role of integrins in VE-cad force transduction and uncover a previously unappreciated mechanism by which the ECM impacts the mechanical reinforcement of interendothelial junctions.