Hyaluronan binding to CD44 and LYVE-1—Methods to probe and mechanistic insight into the mechanics of key interactions for immune cell trafficking

The extracellular matrix polysaccharide hyaluronan (HA) and its cell surface receptors CD44 and LYVE-1 play central roles in immune cell adhesion and migration across blood and lymphatic vessels, and better mechanistic insight of these processes could enable new therapeutic interventions. HA•receptor interactions occur under mechanical stress imposed by fluid flow or cell contractility, yet their unbinding mechanics under force remain poorly characterised in the cellular context. Moreover, HA interacts with CD44 vs. LYVE-1 through ‘sticking’ vs. ‘sliding’ interactions, respectively, but the physical origins of such distinctive mechanics remain unclear. This thesis develops a force spectroscopy method to reveal HA•receptor mechanics on live cells, and a reductionist computational model to explain HA•receptor mechanics in terms of activation barriers that are overcome by tensile force exerted by the flexible HA chain.
In Chapter 2, a new assay was developed to probe individual HA•receptor interactions directly on live cells. First applied to HA•CD44 interactions on stably transfected lymphoma cells, it confirmed that the sticking interaction previously seen with purified, surface-anchored CD44 ectodomains is qualitatively preserved for full-length CD44 embedded in the plasma membrane of live cells, albeit quantitatively distinct owing to variations in receptor glycosylation and/or the receptor host organism, and the cell membrane compliance. The assay also enabled estimation of receptor densities on the cell surface. These findings highlight how the cellular context shapes receptor unbinding mechanics. Chapter 3 extended this approach to HA•LYVE-1 interactions on stably transfected lymphocytes, uncovering that detection of its distinct sliding behaviour is constrained by receptor density and cell mechanics, highlighting density as a key regulator of HA adhesion. In Chapter 4, a reductionist computational approach to study the unbinding mechanics of HA•receptor interactions was developed that integrates HA chain mechanics via the worm-like chain model and force-modulated unbinding across an activation barrier through the Bell-Evans model. Comparison with experimental data revealed that LYVE-1 movement along the HA chain occurs remarkably fast, with a zero-force rate of approximately one step per 0.1 ms, offering new mechanistic insight into HA•LYVE-1 interactions.
These studies expand prior work with purified receptors to live cell experiments and theoretical models. The new experimental and computational tools can now be extended to other biopolymer•receptor systems and advance our mechanistic understanding of HA•receptor adhesion.