Specific and regulated adhesion is required for cellular interactions with substrates, other cells, or tissues. Although most of the proteins involved in adhesion have been identified, the mechanisms by which cells regulate adhesion are not well understood. One reason for this is that commonly used methods do not provide quantitative and systematic information on mechanisms that regulate cell adhesion. Others and we have implemented atomic force microscopy-based single-cell force spectroscopy (SCFS) assays to examine the mechanisms of cell adhesion down to the contribution of single adhesive bonds.
We applied SCFS to examine the adhesion strength of integrin α2β1-expressing cells to nanopatterned collagen type I matrices over time. Interestingly, within 2 min of contact to these matrices cells convert to an adhesion enhanced state. Using the high force resolution offered by SCFS we showed that the enhanced adhesion state was the result of cooperative binding of α2β1 integrins.
Mechanisms that regulate cell adhesion can also be studied using SCFS. This was demonstrated in a study on the contribution of galectins-3 to the adhesion of epithelial cells to collagen type I. Galectin-3 depleted cells had an increased probability of entering the enhanced adhesion state. Adhesion enhancement was specific to integrin α2β1, as it was not observed when cells adhered to ECM substrates by other integrins. It was proposed that galectin-3 influences integrin α2β1-mediated adhesion complex formation by altering receptor clustering.
To control adhesion, cells regulate adhesion receptors. One regulatory process is crosstalk, where the binding of one type of receptor influences the activity of another type. We developed a SCFS assay to identify such crosstalks and gain insight into their mechanisms is described. With this assay the interactions of integrin receptors being specifically attached to one ligand are characterized in dependence of another ligand-bond receptor pair. With this assay a crosstalk between collagen-binding integrin α1β1 and fibronectin-binding integrin α5β1 was identified in HeLa cells. This crosstalk was directional from integrin α1β1 to integrin α5β1 and appeared to regulate integrin α5β1 by inducing its endocytosis.
These studies demonstrate the power of SCFS to elucidate the mechanism involved in cell adhesion at the molecular level.