Mechanical forces have a major influence on cell migration and are predicted to significantly impact cancer metastasis, yet this idea is currently poorly defined. In this study we have asked if changes in traction stress and migratory properties correlate with the metastatic progression of tumor cells. For this purpose, four murine breast cancer cell lines derived from the same primary tumor, but possessing increasing metastatic capacity, were tested for adhesion strength, traction stress, focal adhesion organization and for differential migration rates in two-dimensional and three-dimensional environments. Using traction force microscopy (TFM), we were surprised to find an inverse relationship between traction stress and metastatic capacity, such that force production decreased as the metastatic capacity increased. Consistent with this observation, adhesion strength exhibited an identical profile to the traction data. A count of adhesions indicated a general reduction in the number as metastatic capacity increased but no difference in the maturation as determined by the ratio of nascent to mature adhesions. These changes correlated well with a reduction in active beta-1 integrin with increasing metastatic ability. Finally, in two dimensions, wound healing, migration and persistence were relatively low in the entire panel, maintaining a downward trend with increasing metastatic capacity. Why metastatic cells would migrate so poorly prompted us to ask if the loss of adhesive parameters in the most metastatic cells indicated a switch to a less adhesive mode of migration that would only be detected in a three-dimensional environment. Indeed, in three-dimensional migration assays, the most metastatic cells now showed the greatest linear speed. We conclude that traction stress, adhesion strength and rate of migration do indeed change as tumor cells progress in metastatic capacity and do so in a dimension-sensitive manner.

Summary Cadherin and nectin are distinct transmembrane proteins of adherens junctions. Their ectodomains mediate adhesion while their cytosolic regions couple the adhesive contact to the cytoskeleton. Both these proteins are essential for adherens junction formation and maintenance. However, some basic aspects of these proteins, such as their organization in adherence junctions, have remained open. Therefore, using super-resolution microscopy and live-imaging, we focused on the subjunctional distribution of these proteins. We showed that cadherin and nectin in the junctions of A431 cells and human keratinocytes are located in separate clusters. The size of each cluster is independent of that of the adjacent clusters and can significantly fluctuate over time. Several nectin and cadherin clusters that constitute an individual adherens junction are united by the same actin filament bundle. Surprisingly, interactions between each cluster and F-actin are not uniform since neither vinculin nor LIM domain actin-binding proteins match the boundaries of cadherin or nectin clusters. Thus, the adherens junction is not a uniform structure but a mosaic of different adhesive units with very diverse modes of interaction with the cytoskeleton. We propose that such a mosaic architecture of adherence junctions is important for the fast regulation of their dynamics.

SUMMARY Type II cadherins are cell-cell adhesion proteins critical for tissue patterning and neuronal targeting but whose molecular binding code remains poorly understood. Here, we delineate binding preferences for type II cadherin cell-adhesive regions, revealing extensive heterophilic interactions between specific pairs, in addition to homophilic interactions. Three distinct specificity groups emerge from our analysis with members that share highly similar heterophilic binding patterns and favor binding to one another. Structures of adhesive fragments from each specificity group confirm near-identical dimer topology conserved throughout the family, allowing interface residues whose conservation corresponds to specificity preferences to be identified. We show that targeted mutation of these residues converts binding preferences between specificity groups in biophysical and co-culture assays. Our results provide a detailed understanding of the type II cadherin interaction map and a basis for defining their role in tissue patterning and for the emerging importance of their heterophilic interactions in neural connectivity.

Adherens junctions (AJs) play a fundamental role in tissue integrity; however, the organization and dynamics of the key AJ transmembrane protein, E-cadherin, both inside and outside of AJs, remain controversial. Here we have studied the distribution and motility of E-cadherin in punctate AJs (pAJs) of A431 cells. Using single-molecule localization microscopy, we show that pAJs in these cells reach more than 1 μm in length and consist of several cadherin clusters with crystal-like density interspersed within sparser cadherin regions. Notably, extrajunctional cadherin appears to be monomeric, and its density is almost four orders of magnitude less than observed in the pAJ regions. Two alternative strategies of tracking cadherin motion within individual junctions show that pAJs undergo actin-dependent rapid-on the order of seconds-internal reorganizations, during which dense clusters disassemble and their cadherins are immediately reused for new clusters. Our results thus modify the classical view of AJs by depicting them as mosaics of cadherin clusters, the short lifetimes of which enable stable overall morphology combined with rapid internal rearrangements.

Epithelial apicobasal polarity in Drosophila depends on the basolateral protein Scribble (Scrib), but the role of mammalian Scrib is unclear. Choi et al. now report that mammalian Scrib also mediates epithelial apicobasal polarity but this function is obscured by compensatory activity of other LAP proteins, Erbin and Lano.