Force Spectroscopy of the Leukocyte Function-Associated Antigen-1/Intercellular Adhesion Molecule-1 Interaction

Abstract

Interactions between leukocyte function-associated antigen-1 (LFA-1) with its cognate ligand, intercellular adhesion molecule-1 (ICAM-1) play a crucial role in leukocyte adhesion. Because the cell and its adhesive components are subject to external perturbation from the surrounding flow of blood, it is important to understand the binding properties of the LFA-1/ICAM-1 interaction in both steady state and in the presence of an external pulling force. Here we report on atomic force microscopy (AFM) measurements of the unbinding of LFA-1 from ICAM-1. The single molecule measurements revealed the energy landscape corresponding to the dissociation of the LFA-1/ICAM-1 complex and provided the basis for defining the energetic determinants of the complex at equilibrium and under the influence of an external force. The AFM force measurements were performed in an experimental system consisting of an LFA-1-expressing T cell hybridoma, 3A9, attached to the end of the AFM cantilever and an apposing surface expressing ICAM-1. In measurements covering three orders of magnitude change in force loading rate, the LFA-1/ICAM-1 force spectrum (i.e., unbinding force versus loading rate) revealed a fast and a slow loading regime that characterized a steep inner activation barrier and a wide outer activation barrier, respectively. The addition of Mg(2+), a cofactor that stabilizes the LFA-1/ICAM-1 interaction, elevated the unbinding force of the complex in the slow loading regime. In contrast, the presence of EDTA suppressed the inner barrier of the LFA-1/ICAM-1 complex. These results suggest that the equilibrium dissociation constant of the LFA-1/ICAM-1 interaction is regulated by the energetics of the outer activation barrier of the complex, while the ability of the complex to resist a pulling force is determined by the divalent cation-dependent inner activation barrier.

The Role of Glycocalyx in Nanocarrier-Cell Adhesion Investigated Using a Thermodynamic Model and Monte Carlo Simulations

“…Fitting the above expression to the force spectroscopy data of Moy et al 19 gives k 0 and k (see Appendix A3).…”

“…19,33 This experiment is repeated a number of times to give a rupture force distribution at a given loading rate.…”

Revealing Early Steps of α2β1Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

“…Furthermore, the effect of inhibitors or augmenters of adhesion can be monitored directly at the single-molecule level. Atomic force microscope (AFM)-based SCFS offers the possibility to measure molecular forces with piconewton accuracy and has been adapted to measure cell-substrate (Zhang et al, 2002;Puech et al, 2005) Abbreviations used: AFM, atomic force microscopy; CHO, Chinese hamster ovary; DFS, dynamic force spectroscopy; SCFS, single-cell force spectroscopy. …”

Recent progress in AFM molecular recognition studies

“…Using such force spectroscopy experiments, a variety of ligand-receptor forces have been measured at the singlemolecule level including those associated with avidin/ streptavidin [8,9] antibodies [16,17,22], DNA [13,23,24], carbohydrates [15], lectins [14,25], cadherins [26,27], integrins [28,29], selectins [30], and bacterial adhesins [12]. Knowledge of these forces contributes to refine our understanding of the molecular basis of molecular recognition events such as those mediating cell adhesion and immunological processes, and offers promising prospects in nanobiotechnology for the rapid detection of bio-analytes.…”