Revealing the unbinding mechanics of hyaluronan·receptor interactions on live cells

The extracellular matrix polysaccharide hyaluronan (HA) and its receptors are important mediators of cell adhesion and migration, and tissue mechanics. While mechanical forces are clearly important in these processes, there is a lack of methods to study the effect of mechanical forces on individual bonds of HA with its receptors directly on the surface of cells. Here, we present an assay based on atomic force microscopy to probe the frequency of bond formation (along with the receptor surface density) and the mechanical resistance of HA·receptor bonds to a force ramp on live cells. We demonstrate the method using a lymphoma cell line with stably transfected CD44 and one‐end anchored HA chains. We validate that HA·CD44 unbinding forces on cells are high compared to their relatively low binding affinity, and that bond rupture is dominated by a single energy barrier. The new live cell single HA chain force spectroscopy assay can be used to reveal the interaction mechanics with CD44 or other receptors in distinct interaction geometries, and adapted for other glycosaminoglycans and their receptors.