Strengthening E-cadherin adhesion via antibody-mediated binding stabilization.

E-cadherins (Ecads) are a crucial cell-cell adhesion protein with tumor suppression properties. Ecad adhesion can be enhanced by the monoclonal antibody 66E8, which has potential applications in inhibiting cancer metastasis. However, the biophysical mechanisms underlying 66E8-mediated adhesion strengthening are unknown. Here, we use molecular dynamics simulations, site-directed mutagenesis, and single-molecule atomic force microscopy experiments to demonstrate that 66E8 strengthens Ecad binding by stabilizing the primary Ecad adhesive conformation: the strand-swap dimer. By forming electrostatic interactions with Ecad, 66E8 stabilizes the swapped ß-strand and its hydrophobic pocket and impedes Ecad conformational changes, which are necessary for rupture of the strand-swap dimer. Our findings identify fundamental mechanistic principles for strengthening of Ecad binding using monoclonal antibodies.

Light-activated BioID - an optically activated proximity labeling system to study protein-protein interactions.

Proximity labeling with genetically encoded enzymes is widely used to study protein-protein interactions in cells. However, the accuracy of proximity labeling is limited by a lack of control over the enzymatic labeling process. Here, we present a light-activated proximity labeling technology for mapping protein-protein interactions at the cell membrane with high accuracy and precision. Our technology, called light-activated BioID (LAB), fuses the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. We demonstrate, in multiple cell lines, that upon illumination with blue light, CRY2 and CIB1 dimerize, reconstitute split-TurboID and initiate biotinylation. Turning off the light leads to the dissociation of CRY2 and CIB1 and halts biotinylation. We benchmark LAB against the widely used TurboID proximity labeling method by measuring the proteome of E-cadherin, an essential cell-cell adhesion protein. We show that LAB can map E-cadherin-binding partners with higher accuracy and significantly fewer false positives than TurboID.

Engineering the Interactions of Classical Cadherin Cell-Cell Adhesion Proteins.

Classical cadherins are calcium-dependent cell-cell adhesion proteins that play key roles in the formation and maintenance of tissues. Deficiencies in cadherin adhesion are hallmarks of numerous cancers. In this article, we review recent biophysical studies on the regulation of cadherin structure and adhesion. We begin by reviewing distinct cadherin binding conformations, their biophysical properties, and their response to mechanical stimuli. We then describe biophysical guidelines for engineering Abs that can regulate adhesion by either stabilizing or destabilizing cadherin interactions. Finally, we review molecular mechanisms by which cytoplasmic proteins regulate the conformation of cadherin extracellular regions from the inside out.

Strengthening E-cadherin adhesion via antibody mediated binding stabilization.

E-cadherins (Ecads) are a crucial cell-cell adhesion protein with tumor suppression properties. Ecad adhesion can be enhanced by the monoclonal antibody 66E8, which has potential applications in inhibiting cancer metastasis. However, the biophysical mechanisms underlying 66E8 mediated adhesion strengthening are unknown. Here, we use molecular dynamics simulations, site directed mutagenesis and single molecule atomic force microscopy experiments to demonstrate that 66E8 strengthens Ecad binding by stabilizing the primary Ecad adhesive conformation: the strand-swap dimer. By forming electrostatic interactions with Ecad, 66E8 stabilizes the swapped ß-strand and its hydrophobic pocket and impedes Ecad conformational changes, which are necessary for rupture of the strand-swap dimer. Our findings identify fundamental mechanistic principles for strengthening of Ecad binding using monoclonal antibodies.

Characterizing interactions in E-cadherin assemblages.

Cadherin intermolecular interactions are critical for cell-cell adhesion and play essential roles in tissue formation and the maintenance of tissue structures. In this study, we focus on E-cadherin, a classical cadherin that connects epithelial cells, to understand how they interact in cis and trans conformations when attached to the same cell or opposing cells. We employ coevolutionary sequence analysis and molecular dynamics simulations to confirm previously known interaction sites as well as to identify new interaction sites. The sequence coevolutionary results yield a surprising result indicating that there are no strongly favored intermolecular interaction sites, which is unusual and suggests that many interaction sites may be possible, with none being strongly preferred over others. By using molecular dynamics, we test the persistence of these interactions and how they facilitate adhesion. We build several types of cadherin assemblages, with different numbers and combinations of cis and trans interfaces to understand how these conformations act to facilitate adhesion. Our results suggest that, in addition to the established interaction sites on the EC1 and EC2 domains, an additional plausible cis interface at the EC3-EC5 domain exists. Furthermore, we identify specific mutations at cis/trans binding sites that impair adhesion within E-cadherin assemblages.

Characterizing the Biophysical Properties of Adhesive Proteins in Live Cells Using Single-Molecule Atomic Force Microscopy.

Cell adhesion proteins play essential roles in the formation, regeneration, and maintenance of tissue. However, the molecular mechanisms by which cells regulate the conformation and binding properties of adhesion proteins are poorly understood. These biophysical properties can be resolved, with single-molecule resolution, using atomic force microscopy (AFM). Here, we outline how AFM force measurements can be used to study the conformation, cytoskeletal linkage, binding strength, and force-dependent bond lifetimes of adhesion proteins in live cells.

Molecular mechanism for strengthening E-cadherin adhesion using a monoclonal antibody.

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using X-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif: the strand-swap dimer interface. Molecular dynamics simulations and single-molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes: one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped ß-strand and its complementary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.

Cadherins can dimerize via asymmetric interactions.

Cadherins are essential cell-cell adhesion proteins that interact in two distinct conformations: X-dimers and strand-swap dimers. Both X-dimers and strand-swap dimers are thought to exclusively rely on symmetric sets of interactions between key amino acids on both cadherin binding partners. Here, we use single-molecule atomic force microscopy and computer simulations to show that symmetry in cadherin binding is dispensable and that cadherins can also interact in a novel conformation that asymmetrically incorporates key elements of both strand-swap dimers and X-dimers. Our results clarify the biophysical rules for cadherin binding and demonstrate that cadherins interact in a more diverse range of conformations than previously understood.

Inside-out regulation of E-cadherin conformation and adhesion.

Cadherin cell-cell adhesion proteins play key roles in tissue morphogenesis and wound healing. Cadherin ectodomains bind in two conformations, X-dimers and strand-swap dimers, with different adhesive properties. However, the mechanisms by which cells regulate ectodomain conformation are unknown. Cadherin intracellular regions associate with several actin-binding proteins including vinculin, which are believed to tune cell-cell adhesion by remodeling the actin cytoskeleton. Here, we show at the single-molecule level, that vinculin association with the cadherin cytoplasmic region allosterically converts weak X-dimers into strong strand-swap dimers and that this process is mediated by myosin II-dependent changes in cytoskeletal tension. We also show that in epithelial cells, ∼70% of apical cadherins exist as strand-swap dimers while the remaining form X-dimers, providing two cadherin pools with different adhesive properties. Our results demonstrate the inside-out regulation of cadherin conformation and establish a mechanistic role for vinculin in this process.

Robust scan synchronized force-fluorescence imaging.

Simultaneous atomic force microscope (AFM) and sample scanning confocal fluorescence microscope measurements are widely used to obtain mechanistic and structural insights into protein dynamics in live cells. However, the absence of a robust technique to synchronously scan both AFM and confocal microscope piezo stages makes it difficult to visualize force-induced changes in fluorescent protein distribution in cells.  To address this challenge, we have built an integrated AFM-confocal fluorescence microscope platform that implements a synchronous scanning method which eliminates image artifacts from piezo motion ramping, produces accurate pixel binning and enables the collection of a scanned image of a sample while applying force to a single point on the sample. As proof of principle, we use this instrument to monitor the redistribution of fluorescent E-cadherin, an essential transmembrane protein, in live cells, upon application of mechanical force.

Mapping transmembrane binding partners for E-cadherin ectodomains.

We combine proximity labeling and single molecule binding assays to discover transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a "bait" protein with BioID biotin ligase and identify proximal proteins that are biotin tagged on both their extracellular and intracellular regions. We then test direct binding interactions between proximal proteins and the bait, using single molecule atomic force microscope binding assays. Using this approach, we identify binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2ß1, the receptor tyrosine kinase ligand ephrin-B1, and the classical cadherin P-cadherin, all directly interact with E-cadherin ectodomains. Our data shows that combining extracellular and cytoplasmic proximal tagging with a biophysical binding assay increases the precision with which transmembrane ectodomain interactors can be identified.

Pitching single-focus confocal data analysis one photon at a time with Bayesian nonparametrics.

Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.

Single-molecule studies of classical and desmosomal cadherin adhesion.

Classical cadherin and desmosomal cadherin cell-cell adhesion proteins play essential roles in tissue morphogenesis and in maintaining tissue integrity. Deficiencies in cadherin adhesion are hallmarks of diseases like cancers, skin diseases and cardiomyopathies. Structural studies and single molecule biophysical measurements have revealed critical similarities and surprising differences between these key adhesion proteins. This review summarizes our current understanding of the biophysics of classical and desmosomal cadherin adhesion and the molecular basis for their cross-talk. We focus on recent single molecule measurements, highlight key insights into the adhesion of cadherin extracellular regions and their relation to associated diseases, and identify major open questions in this exciting area of research.

An alternative framework for fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS), is a widely used tool routinely exploited for in vivo and in vitro applications. While FCS provides estimates of dynamical quantities, such as diffusion coefficients, it demands high signal to noise ratios and long time traces, typically in the minute range. In principle, the same information can be extracted from microseconds to seconds long time traces; however, an appropriate analysis method is missing. To overcome these limitations, we adapt novel tools inspired by Bayesian non-parametrics, which starts from the direct analysis of the observed photon counts. With this approach, we are able to analyze time traces, which are too short to be analyzed by existing methods, including FCS. Our new analysis extends the capability of single molecule fluorescence confocal microscopy approaches to probe processes several orders of magnitude faster and permits a reduction of photo-toxic effects on living samples induced by long periods of light exposure.

Method for high frequency tracking and sub-nm sample stabilization in single molecule fluorescence microscopy.

While fluorescence microscopes and atomic force microscopes are widely used to visualize, track, and manipulate single biomolecules, the resolution of these methods is limited by sample drift. To minimize drift, active feedback methods have recently been used to stabilize single molecule microscopes on the sub-nanometer scale. However, these methods require high intensity lasers which limits their application in single molecule fluorescence measurements. Furthermore, these feedback methods do not track user-defined regions of the sample, but rather monitor the relative displacement of an unknown point on a fiducial marker, which limits their use in biological force measurements. To overcome these limitations, we have developed a novel method to image, track and stabilize a sample using low laser intensities. We demonstrate the capabilities of our approach by tracking a user-chosen point on a fiducial marker at 8.6 kHz and stabilizing it with sub-nanometer resolution. We further showcase the application of our method in single molecule fluorescence microscopy by imaging and stabilizing individual fluorescently-tagged streptavidin proteins under biologically relevant conditions. We anticipate that our method can be easily used to improve the resolution of a wide range of single molecule fluorescence microscopy and integrated force-fluorescence applications.

E-cadherin binds to desmoglein to facilitate desmosome assembly.

Desmosomes are adhesive junctions composed of two desmosomal cadherins: desmocollin (Dsc) and desmoglein (Dsg). Previous studies demonstrate that E-cadherin (Ecad), an adhesive protein that interacts in both trans (between opposing cells) and cis (on the same cell surface) conformations, facilitates desmosome assembly via an unknown mechanism. Here we use structure-function analysis to resolve the mechanistic roles of Ecad in desmosome formation. Using AFM force measurements, we demonstrate that Ecad interacts with isoform 2 of Dsg via a conserved Leu-175 on the Ecad cis binding interface. Super-resolution imaging reveals that Ecad is enriched in nascent desmosomes, supporting a role for Ecad in early desmosome assembly. Finally, confocal imaging demonstrates that desmosome assembly is initiated at sites of Ecad mediated adhesion, and that Ecad-L175 is required for efficient Dsg2 and desmoplakin recruitment to intercellular contacts. We propose that Ecad trans interactions at nascent cell-cell contacts initiate the recruitment of Dsg through direct cis interactions with Ecad which facilitates desmosome assembly.

Improving estimation of kinetic parameters in dynamic force spectroscopy using cluster analysis.

Dynamic Force Spectroscopy (DFS) is a widely used technique to characterize the dissociation kinetics and interaction energy landscape of receptor-ligand complexes with single-molecule resolution. In an Atomic Force Microscope (AFM)-based DFS experiment, receptor-ligand complexes, sandwiched between an AFM tip and substrate, are ruptured at different stress rates by varying the speed at which the AFM-tip and substrate are pulled away from each other. The rupture events are grouped according to their pulling speeds, and the mean force and loading rate of each group are calculated. These data are subsequently fit to established models, and energy landscape parameters such as the intrinsic off-rate (koff) and the width of the potential energy barrier (xß) are extracted. However, due to large uncertainties in determining mean forces and loading rates of the groups, errors in the estimated koff and xß can be substantial. Here, we demonstrate that the accuracy of fitted parameters in a DFS experiment can be dramatically improved by sorting rupture events into groups using cluster analysis instead of sorting them according to their pulling speeds. We test different clustering algorithms including Gaussian mixture, logistic regression, and K-means clustering, under conditions that closely mimic DFS experiments. Using Monte Carlo simulations, we benchmark the performance of these clustering algorithms over a wide range of koff and xß, under different levels of thermal noise, and as a function of both the number of unbinding events and the number of pulling speeds. Our results demonstrate that cluster analysis, particularly K-means clustering, is very effective in improving the accuracy of parameter estimation, particularly when the number of unbinding events are limited and not well separated into distinct groups. Cluster analysis is easy to implement, and our performance benchmarks serve as a guide in choosing an appropriate method for DFS data analysis.

Minimizing open-loop piezoactuator nonlinearity artifacts in atomic force microscope measurements.

Atomic force microscopes (AFMs) are widely used to study molecular interactions with piconewton force sensitivity. In an AFM, interaction forces are measured by reflecting a laser beam off a cantilever onto a position sensitive detector and monitoring cantilever deflection. Precise measurements of interaction forces rely on accurately determining the optical lever sensitivity, i.e., the relationship between cantilever deflection and changes in detector voltage. The optical lever sensitivity is measured by pressing the cantilever against a hard substrate using a piezoactuator and recording the resulting change in detector voltage. However, nonlinearities in the motion of commonly used open-loop piezo actuators introduce significant errors in measured optical lever sensitivities. Here, the authors systematically characterize the effect of piezo actuator hysteresis and creep on errors in optical lever sensitivity and identify measurement conditions that minimize these errors.

Biophysical basis of cadherin mediated cell-cell adhesion.

Classical cadherin transmembrane cell-cell adhesion proteins play essential roles in tissue morphogenesis and in mediating tissue integrity. Cadherin ectodomains from opposing cells interact to form load-bearing trans dimers that mechanically couple cells. Cell-cell adhesion is believed to be strengthened by cis clustering of cadherins on the same cell surface. This review summarizes biophysical studies of the structure, interaction kinetics and biomechanics of classical cadherin ectodomains. We first discuss the structure and equilibrium binding kinetics of classical cadherin trans and cis dimers. We then discuss how mechanical stimuli alters the kinetics of cadherin interaction and tunes adhesion. Finally, we highlight open questions on the role of mechanical forces in influencing cadherin structure, function and organization on the cell surface.

Molecular determinants of cadherin ideal bond formation: Conformation-dependent unbinding on a multidimensional landscape.

Classical cadherin cell-cell adhesion proteins are essential for the formation and maintenance of tissue structures; their primary function is to physically couple neighboring cells and withstand mechanical force. Cadherins from opposing cells bind in two distinct trans conformations: strand-swap dimers and X-dimers. As cadherins convert between these conformations, they form ideal bonds (i.e., adhesive interactions that are insensitive to force). However, the biophysical mechanism for ideal bond formation is unknown. Here, we integrate single-molecule force measurements with coarse-grained and atomistic simulations to resolve the mechanistic basis for cadherin ideal bond formation. Using simulations, we predict the energy landscape for cadherin adhesion, the transition pathways for interconversion between X-dimers and strand-swap dimers, and the cadherin structures that form ideal bonds. Based on these predictions, we engineer cadherin mutants that promote or inhibit ideal bond formation and measure their force-dependent kinetics using single-molecule force-clamp measurements with an atomic force microscope. Our data establish that cadherins adopt an intermediate conformation as they shuttle between X-dimers and strand-swap dimers; pulling on this conformation induces a torsional motion perpendicular to the pulling direction that unbinds the proteins and forms force-independent ideal bonds. Torsional motion is blocked when cadherins associate laterally in a cis orientation, suggesting that ideal bonds may play a role in mechanically regulating cadherin clustering on cell surfaces.