Classical cadherin cell-cell adhesion proteins play key morphogenetic roles during development and are essential for maintaining tissue integrity in multicellular organisms. Classical cadherins bind in two distinct conformations, X-dimer and strand-swap dimer; during cellular rearrangements, these adhesive states are exposed to mechanical stress. However, the molecular mechanisms by which cadherins resist tensile force and the pathway by which they convert between different conformations are unclear. Here, we use single molecule force measurements with an atomic force microscope (AFM) to show that E-cadherin, a prototypical classical cadherin, forms three types of adhesive bonds: catch bonds, which become longer lived in the presence of tensile force; slip bonds, which become shorter lived when pulled; and ideal bonds that are insensitive to mechanical stress. We show that X-dimers form catch bonds, whereas strand-swap dimers form slip bonds. Our data suggests that ideal bonds are formed as X-dimers convert to strand-swap binding. Catch, slip, and ideal bonds allow cadherins to withstand tensile force and tune the mechanical properties of adhesive junctions.single molecule biomechanics | force clamp | trans dimers | protein conformation | structure-function relationship

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.

Single-molecule localization microscopy has the ability to measure spatial proximity between individual molecules with tens of nanometers precision. Extracting meaningful biological results, however, requires fully characterizing the distribution of molecular behaviors, which in turn, necessitates analyzing large numbers of individual measurements. Making large numbers of replicate measurements in a single imaging session has been made possible in recent years by large area detectors that afford an ultrawide field-of-view as well as fast frame rates. A remaining barrier to ultrawide-field imaging is that optical aberrations become pronounced when imaging far away from the central optical axis, which can compromise the precision and accuracy of point-spreadfunction (PSF) fitting across the field-of-view. Here, we present a computational phase retrieval routine based on vectorial PSF models to account for the spatially-variant aberrations in two color channels of a 3D singlemolecule localization microscope. By computationally correcting the aberrations during data post-processing, we are able to localize emitters in an ultrawide filed-of-view with improved precision and accuracy compared to approaches based on analytical PSF models. The use of a spatially-variant PSF model enables accurate emitter localization in x, y and z over the entire field-of-view, so that the reconstructed super-resolution images and singlemolecule trajectories accurately reproduce the relative spatial arrangement among all localized emitters.

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.