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.
13 12Ecad, are a weaker adhesive structure and serve as an intermediate during the formation 13 and rupture of strand swap dimers 5, 6, 7 . Using cell-free, single molecule experiments, we 14 previously showed that X-dimers and strand-swap dimers can be distinguished based on 15 their distinctly different response to mechanical force. When a strand-swap dimer is pulled, 16 its lifetime decreases with increasing force, resulting in the formation of a slip bond 8, 9 . In 17 contrast, an X-dimer responds to pulling force by forming a catch bond, where bond 18 lifetime initially increases up to a threshold force, and then subsequently decreases 8, 10 . 19We also showed that wild type Ecad ectodomains in solution can interconvert between X-20 dimer and strand-swap dimer conformations 9 . However, the biophysical mechanisms by 21 which Ecad conformations (and adhesion) are regulated on the cell surface are unknown.Ecad adhesion 21, 22, 23 . Here, we directly map the allosteric effects of cytoplasmic proteins 15 and demonstrate, at the single molecule level, that vinculin association with the Ecad 16 cytoplasmic region switches X-dimers to strand-swap dimers, thereby regulating 17 ectodomain structure and adhesion. 18 19 RESULTS 20 Single molecule Atomic Force Microscope (AFM) measurements of specific Ecad 21 trans adhesion. We measured interactions between recombinant Ecad extracellular 22regions immobilized on AFM cantilevers and Ecad endogenously expressed on the apical 2A). In contrast, in the Ecad-KO cells, event rates using AFM tips with/without Ecad were 17 similar to each other and were comparable to the nonspecific event rates measured with 18 the other cell lines (Figure 2A). Taken together, this confirmed that the measured specific 19 events corresponded to Ecad-Ecad binding interactions. 20Ecad conformation and cytoskeletal-linkage in each cell line were determined from 21 previously reported 'signatures' in the measured force curves. Since force measurements 22 have shown that pulling on a cell surface protein that is not linked to the cytoskeleton 23 results in the formation of a membrane-tether 26, 27 (Figure 3A), we used membrane-tethers 16 4.
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