Studying Integrin-Mediated Cell Adhesion at the Single-Molecule Level Using AFM Force Spectroscopy

The establishment of cell adhesion involves specific recognition events between individual cell-surface receptors and molecules of the cellular environment. However, characterizing single-molecule adhesion events in the context of a living cell presents an experimental challenge. The atomic force microscope (AFM) operated in force spectroscopy mode provides an ultrasensitive method to investigate cell adhesion forces at the level of single receptor-ligand bonds. With a living cell attached to the AFM cantilever, the number of cell-substrate interactions can be controlled and limited to the formation of single receptor-ligand bonds. From force-distance (F-D) curves recorded during cell detachment, the strength of single receptor-ligand bonds can be determined. Furthermore, by varying the rate of force application during bond rupture, a dynamic force spectrum (DFS) can be generated from which additional parameters that describe the energy landscape of the interaction, such as dissociation rate and energy barrier width, can be obtained. Using the example of α 2 β 1 integrin–mediated adhesion to type I collagen, we provide a detailed description of how dynamic AFM single-cell force spectroscopy (SCFS) adhesion measurements can be performed with single-molecule sensitivity, and how specific energy landscape parameters of the integrin-collagen bond can be extracted from the DFS.