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.

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.

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.

Different roles of cadherins in the assembly and structural integrity of the desmosome complex.

Adhesion between cells is established by the formation of specialized intercellular junctional complexes, such as desmosomes. Desmosomes contain isoforms of two members of the cadherin superfamily of cell adhesion proteins, desmocollins (Dsc) and desmogleins (Dsg), but their combinatorial roles in desmosome assembly are not understood. To uncouple desmosome assembly from other cell-cell adhesion complexes, we used micro-patterned substrates of Dsc2aFc and/or Dsg2Fc and collagen IV; we show that Dsc2aFc, but not Dsg2Fc, was necessary and sufficient to recruit desmosome-specific desmoplakin into desmosome puncta and produce strong adhesive binding. Single-molecule force spectroscopy showed that monomeric Dsc2a, but not Dsg2, formed Ca(2+)-dependent homophilic bonds, and that Dsg2 formed Ca(2+)-independent heterophilic bonds with Dsc2a. A W2A mutation in Dsc2a inhibited Ca(2+)-dependent homophilic binding, similar to classical cadherins, and Dsc2aW2A, but not Dsg2W2A, was excluded from desmosomes in MDCK cells. These results indicate that Dsc2a, but not Dsg2, is required for desmosome assembly through homophilic Ca(2+)- and W2-dependent binding, and that Dsg2 might be involved later in regulating a switch to Ca(2+)-independent adhesion in mature desmosomes.

Ideal, catch, and slip bonds in cadherin adhesion.

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.

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.

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.

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.