Cadherin receptors have a well-established role in cell–cell adhesion, cell polarization and differentiation. However, some cadherins also promote cell and tissue movement during embryonic development and tumour progression. In particular, cadherin-11 is upregulated during tumour and inflammatory cell invasion, but the mechanisms underlying cadherin-11 stimulated cell migration are still incompletely understood. Here, we show that cadherin-11 localizes to focal adhesions and promotes adhesion to fibronectin in Xenopus neural crest, a highly migratory embryonic cell population. Transfected cadherin-11 also localizes to focal adhesions in different mammalian cell lines, while endogenous cadherin-11 shows focal adhesion localization in primary human fibroblasts. In focal adhesions, cadherin-11 co-localizes with β1-integrin and paxillin and physically interacts with the fibronectin-binding proteoglycan syndecan-4. Adhesion to fibronectin mediated by cadherin-11/syndecan-4 complexes requires both the extracellular domain of syndecan-4, and the transmembrane and cytoplasmic domains of cadherin-11. These results reveal an unexpected role of a classical cadherin in cell–matrix adhesion during cell migration.

Tissue-embedded cells are often exposed to a complex mixture of extracellular matrix (ECM) molecules, to which they bind with different cell adhesion receptors and affinities. Differential cell adhesion to ECM components is believed to regulate many aspects of tissue function, such as the sorting of specific cell types into different tissue compartments or ECM niches. In turn, aberrant switches in cell adhesion preferences may contribute to cell misplacement, tissue invasion, and metastasis. Methods to determine differential adhesion profiles of single cells are therefore desirable, but established bulk assays usually only test cell population adhesion to a single type of ECM molecule. We have recently demonstrated that atomic force microscopy-based single-cell force spectroscopy (SCFS), performed on bifunctional, microstructured adhesion substrates, provides a useful tool for accurately quantitating differential matrix adhesion of single Chinese hamster ovary cells to laminin and collagen I. Here, we have extended this approach to include additional ECM substrates, such as bifunctional collagen I/collagen IV surfaces, as well as adhesion-passivated control surfaces. We investigate differential single cell adhesion to these substrates and analyze in detail suitable experimental conditions for comparative SCFS, including optimal cell-substrate contact times and the impact of force cycle repetitions on single cell adhesion force statistics. Insight gained through these experiments may help in adapting this technique to other ECM molecules and cell systems, making directly comparative SCFS a versatile tool for comparing receptor-mediated cell adhesion to different matrix molecules in a wide range of biological contexts.

AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest (CNC) cells during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments.