STED/AFM as a tool to investigate mechanical and adhesive properties of migrating keratinocytes

E-cadherin is a central junctional molecule of adherens junctions that regulates epithelial integrity. In keratinocytes, wound healing critically depends on dynamic modulation of adhesion and cytoskeletal organization. Here, we applied wound healing assay and the combination of the stimulated emission depletion (STED) microscopy with the atomic force microscopy (AFM) (STED/AFM hybrid technique), to study E-cadherin binding properties during keratinocyte migration. Wound closure occurred within 6 h, an effect associated with E-cadherin accumulation at the leading edge. For location-specific binding measurements with the STED/AFM technique, we overexpressed E-cadherin in mouse keratinocytes and performed single molecule force spectroscopy measurements. Inhibition of actin polymerization abolished E-cadherin binding and further reduced overall cellular stiffness. To study adhesion mechanics during migration, we used the STED/AFM technique and performed single-molecule force spectroscopy at the leading edge of keratinocytes migrating into cell-free areas, generated by removal of two-well inserts. This approach enabled, for the first time, simultaneous measurements of single-molecule binding properties and cellular mechanistic properties in actively migrating keratinocytes. Our results revealed that E-Cad molecules present during migration exhibit binding properties comparable to those of E-Cad in stable AJs. This suggests that these molecules remain functional competent and may be readily available for rapid re-engagement in cell-cell adhesion when required. We introduce a powerful methodology to investigate single molecule binding properties in migrating cells, offering new opportunities to analyze epithelial repair at molecular resolution.