We apply fast relaxation imaging (FReI) as a novel technique for investigating the folding stability and dynamics of proteins within polyacrylamide hydrogels, which have diverse and widespread uses in biotechnology. FReI detects protein unfolding in situ by imaging changes in fluorescence resonance energy transfer (FRET) after temperature jump perturbations. Unlike bulk measurements, diffraction-limited epifluorescence imaging combined with fast temperature perturbations reveals the impact of local environment effects on protein–biomaterial compatibility. Our experiments investigated a crowding sensor protein (CrH2) and phosphoglycerate kinase (PGK), which undergoes cooperative unfolding. The crowding sensor quantifies the confinement effect of the cross-linked hydrogel: the 4% polyacrylamide hydrogel is similar to aqueous solution (no confinement), while the 10% hydrogel is strongly confining. FRAP measurements and protein concentration gradients in the 4% and 10% hydrogels further support this observation. PGK reveals that noncovalent interactions of the protein with the polymer surface are more important than confinement for determining protein properties in the gel: the mere presence of hydrogel increases protein stability, speeds up folding relaxation, and promotes irreversible binding to the polymer even at the solution–gel interface, whereas the difference between the 4% and the 10% hydrogels is negligible despite their large difference in confinement. The imaging capabilities of FReI, demonstrated to be diffraction limited, further revealed spatially homogeneous protein unfolding across the hydrogels at 500 nm length scales and revealed differences in protein properties at the gel–solution boundary.Keywords: FRET; hydrogels; immobilized proteins; microenvironment; polyacrylamide; protein folding; protein/polymer aggregation;

The vascular endothelium is subject to diverse mechanical cues that regulate vascular endothelial barrier function. In addition to rigidity sensing through integrin adhesions, mechanical perturbations such as changes in fluid shear stress can also activate force transduction signals at intercellular junctions. This study investigated how extracellular matrix rigidity and intercellular force transduction, activated by vascular endothelial cadherin, coordinate to regulate the integrity of endothelial monolayers. Studies used complementary mechanical measurements of endothelial monolayers grown on patterned substrates of variable stiffness. Specifically perturbing VE-cadherin receptors activated intercellular force transduction signals that increased integrin-dependent cell contractility and disrupted cell-cell and cell-matrix adhesions. Further investigations of the impact of substrate rigidity on force transduction signaling demonstrated how cells integrate extracellular mechanics cues and intercellular force transduction signals, to regulate endothelial integrity and global tissue mechanics. VE-cadherin specific signaling increased focal adhesion remodeling and cell contractility, while sustaining the overall mechanical equilibrium at the mesoscale. Conversely, increased substrate rigidity exacerbates the disruptive effects of intercellular force transduction signals, by increasing heterogeneity in monolayer stress distributions. The results provide new insights into how substrate stiffness and intercellular force transduction coordinate to regulate endothelial monolayer integrity.

We describe an equibiaxial cell stretcher and hybrid, elastic membrane platform designed for dynamic imaging of cells on substrates with physiological stiffness undergoing cyclic stretch. Studies enabled by this device revealed that both substrate stiffness and cyclic stretch coordinately protect pulmonary endothelial monolayers against thrombin-induced disruption. The fluorescence imaging possible with the designed hybrid membranes further revealed similarities and differences in actin and cell dynamics during monolayer recovery. The improved live-cell imaging capabilities of this platform, when used in conjunction with fluorescent probes, will have broad applications for investigations of the impact of biochemical stimuli and mechanotransduction mechanisms on mechanically perturbed tissues.

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.

This study investigated the impact of the protein adsorption mechanism(s) on the efficiency of thermally controlled cell adhesion and release from poly(N-isopropyl acrylamide) brushes. Large format polymer gradients were used to screen for grafting densities and substrate chemistries that alter both cell adhesion at 37 °C and rapid cell release at 25 °C. In particular, the grafting conditions investigated allowed protein adsorption to the underlying substrate, penetration of the brush only, or adsorption to the outer edge of the film. At an average molecular weight of 30 kDa (degree of polymerization N ∼ 270), the results show that robust protein adsorption to polymer brushes impairs rapid cell release below the lower critical solution temperature. Conversely, grafting conditions that permit protein penetration of the brush but block strong adsorption to the underlying substrate support cell adhesion above the transition temperature and ensure efficient cell recovery at lower temperature. These findings demonstrate the impact of protein adsorption mechanisms, surface chemistry, and polymer properties on thermally controlled cell capture and release.

The topography of poly (N-isopropyl acrylamide) brushes end-grafted from initiator-terminated monolayers was imaged by atomic force microscopy, as a function of the area per chain and of solvent quality. Measurements were done in air and in water, below and above the lower critical solution temperature. At low grafting densities and molecular weights, area-averaged ellipsometry measurements did not detect changes in the volume of water-swollen, end-grafted polymer films above the lower critical solution temperature. However, atomic force microscopy images revealed surface features that suggest the formation of lateral aggregates or “octopus micelles”. At high grafting densities and molecular weights, the films collapsed uniformly, as detected by both AFM imaging and ellipsometry. These findings reconcile in part prior results suggesting that some poly(N-isopropyl acrylamide) chains do not collapse in poor solvent, and they also reveal more complex collapse behavior above the lower critical solution temperature than is commonly assumed. This behavior would influence the ability to tune the functional properties of poly(N-isopropyl acrylamide) coatings.

Force–distance measurements have been used to examine differences in the interaction of the dendritic cell glycan-binding receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR (L-SIGN) with membranes bearing glycan ligands. The results demonstrate that upon binding to membrane-anchored ligand, DC-SIGNR undergoes a conformational change similar to that previously observed for DC-SIGN. The results also validate a model for the extracellular domain of DC-SIGNR derived from crystallographic studies. Force measurements were performed with DC-SIGNR variants that differ in the length of the neck that result from genetic polymorphisms, which encode different numbers of the 23-amino acid repeat sequences that constitute the neck. The findings are consistent with an elongated, relatively rigid structure of the neck repeat observed in crystals. In addition, differences in the lengths of DC-SIGN and DC-SIGNR extracellular domains with equivalent numbers of neck repeats support a model in which the different dispositions of the carbohydrate-recognition domains in DC-SIGN and DC-SIGNR result from variations in the sequences of the necks.

The protein resistance of poly(N-isopropylacrylamide) brushes grafted from silicon wafers was investigated as a function of the chain molecular weight, grafting density, and temperature. Above the lower critical solution temperature (LCST) of 32 °C, the collapse of the water-swollen chains, determined by ellipsometry, depends on the grafting density and molecular weight. Ellipsometry, radio assay, and fluorescence imaging demonstrated that, below the lower critical solution temperature, the brushes repel protein as effectively as oligoethylene oxide-terminated monolayers. Above 32 °C, very low levels of protein adsorb on densely grafted brushes, and the amounts of adsorbed protein increase with decreasing brush-grafting-densities. Brushes that do not exhibit a collapse transition also bind protein, even though the chains remain extended above the LCST. These findings suggest possible mechanisms underlying protein interactions with end-grafted poly(N-isopropyl acrylamide) brushes.

This report describes an approach to extend indefinitely the duration of traction force measurements with cells cultured on soft polyacrylamide gels. Typical observation times in traction force measurements on similar substrates have been limited to 24–48 h, but cell differentiation or responses to external stimuli often occur over much longer periods of several days or weeks. This study describes a method for covalently linking fluorescent marker beads to a polyacrylamide matrix that renders the hydrogels useful for traction force measurements over several days. This approach was validated by comparing the contractility of C2C12 murine skeletal muscle cells prior to myotube formation, after one day in culture, with that of myotubes after 7 days in culture. Measured tractions increased concurrent with the differentiation of C2C12 cells to the contractile, myotube phenotype. Covalent bead linkage thus extends the useful period during which traction force data can be obtained with cells cultured on optically transparent polyacrylamide hydrogels with controlled elastic moduli.

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.

These studies quantified the relative effects of E-cadherin expression and homophilic ligation on the integrin-mediated motility of epithelial cells. Micropatterned proteins were used to quantitatively titrate the ligation of E-cadherin and integrin receptors in order to assess their coordinate influence on the migration velocities of MDA-MB-231 breast tumor epithelial cells. Fibronectin, E-cadherin, and mixtures of fibronectin and E-cadherin were covalently patterned on solid surfaces at defined compositions and mass coverages. The migration velocities of parental epithelial cells and of cells engineered to express E-cadherin under tetracycline control show that E-cadherin expression reduces cell motility by both adhesion-dependent and adhesion-independent mechanisms. Increasing E-cadherin expression levels also suppresses the dependence of cell velocity on the fibronectin coverage. On E-cadherin-containing substrata, the cell velocity decreases both with the E-cadherin expression level and with the immobilized E-cadherin surface density. These studies thus identified conditions under which E-cadherin preferentially suppresses cell migration by adhesion-independent versus adhesion-dependent mechanisms.

Classical cadherins are the principle adhesive proteins at cohesive intercellular junctions, and are essential proteins for morphogenesis and tissue homeostasis. Because subtype-dependent differences in cadherin adhesion are at the heart of cadherin functions, several structural and biophysical approaches have been used to elucidate relationships between cadherin structures, biophysical properties of cadherin bonds, and cadherin-dependent cell functions. Some experimental approaches appeared to provide conflicting views of the cadherin binding mechanism. However, recent structural and biophysical data, as well as computer simulations generated new insights into classical cadherin binding that increasingly reconcile diverse experimental findings. This review summarizes these recent findings, and highlights both the consistencies and remaining challenges needed to generate a comprehensive model of cadherin interactions that is consistent with all available experimental data.

Cell-to-cell junctions are crucial mechanical and signaling hubs that connect cells within tissues and probe the mechanics of the surrounding environment. Although the capacity of cell-to-extracellular-matrix (ECM) adhesions to sense matrix mechanics and proportionally modify cell functions is well established, cell–cell adhesions only recently emerged as a new class of force sensors. This finding exposes new pathways through which force can instruct cell functions. This review highlights recent findings, which demonstrate that protein complexes associated with classical cadherins, the principal architectural proteins at cell–cell junctions in all soft tissues, are mechanosensors. We further discuss the current understanding of the rudiments of a cadherin-based mechanosensing and transduction pathway, which is distinct from the force sensing machinery of cell–ECM adhesions.Highlights► The role of force at cell-to-cell junctions in development and disease. ► Recent advances in quantitative measurements of intercellular tension. ► Recent results demonstrating that cadherin complexes are tension sensors at cell-to-cell junctions. ► Recent findings indicate that alpha catenin is a tension sensor at cadherin-mediated cell–cell adhesions. ► Initial mechanistic studies implicating vinculin in the alpha-catenin-dependent force-transduction pathway at cadherin adhesions.

Atomic force microscopy and surface force apparatus measurements determined the functional impact of the cadherin point mutation W2A and domain deletion mutations on C-cadherin binding signatures. Direct comparison of results obtained using both experimental approaches demonstrates that C-cadherin ectodomains form multiple independent bonds that require different structural regions. The results presented reveal significant interdomain cross talk. They further demonstrate that the mutation W2A not only abolishes adhesion between N-terminal domains, but allosterically modulates other binding states that require functional domains distal to the N-terminal binding site. Such allosteric effects may play a prominent role in modulating adhesion by Type I classic cadherins, cadherin oligomerization at junctional contacts, and propagation of binding information to the cytoplasmic region.

Living cells are being extensively studied to build functional tissues that are useful for both fundamental and applied bioscience studies. Increasing evidence suggests that cell–cell adhesion controlled by the intercellular cadherin junction plays important roles in the quality of the resulting engineered tissue. These findings prompted efforts to interrogate biological effects of cadherin at a molecular scale; however, few efforts were made to harness the effects of cadherin on cells cultured in an in vivo-like three dimensional matrix. To this end, this study reports a hydrogel matrix three dimensionally functionalized with a controlled number of Fc-tagged recombinant N-cadherins (N-Cad-Fc). To retain the desired conformation of N-Cad, these cadherins were immobilized and oriented to the gel by anti-Fc-antibodies chemically coupled to gels. The gels were processed to present N-Cad-Fc in uniaxially aligned microchannels or randomly oriented micropores. Culturing cortical cells in the functionalized gels generated a large fraction of neurons that are functional as indicated by increased intracellular calcium ion concentrations with the microchanneled gel. In contrast, direct N-Cad-Fc immobilization to microchannel or micropore walls of the gel limited the growth of neurons and increased the glial to neuron ratio. The results of this study will be highly useful to organize a wide array of cadherin molecules in a series of biomaterials used for three-dimensional cell culture and to regulate phenotypic activities of tissue-forming cells in an elaborate manner.