Variable-Angle Total Internal Reflection Fluorescence Microscopy: Exploring Integrin-mediated Adhesion

Abstract

Cell adhesion plays a global role in cell communication and regulation. During adhesion, cells interact with each other and with their environment, through a broad range of specific and nonspecific interactions. The specific cell binding is regulated by lock-and-key mechanisms, related to the presence of transmembrane proteins, such as integrins. These integrins can specifically recognize ligands and are the keystone of focal adhesion complexes (FA). Cell adhesion also results from the synergy between long-range and short-range cell-substrate nonspecific interactions, mediated for example, by cellular glycocalyx. Therefore, our team recently proposed a real-time imaging technique to probe simultaneously the specific and the nonspecific aspects of adhesion process by measuring distances from the substrate to the plasma membrane, and to quantify adhesion strength at the single cell level. This technique, called variable-angle total internal reflection fluorescence microscopy (va-TIRFM), allows us to map the membrane-substrate separation distance, with a nanometric axial resolution. Va-TIRFM can also be used to extract the profile of potential energy related to membrane/substrate interactions, which reflects an energy of adhesion. Recently we used va-TIRFM to study live glioblastoma metastatic cells in adhesion on fibronectin. By blocking the adhesion signaling pathways using very well-known alpha-5 beta-1 and alpha-v beta-3 integrin antagonists, we showed a change in FA organization from local points to adhesion plaques, which can take up to 50 percent of cell surface, followed by glycocalyx remodeling. Surprisingly these modifications were accompanied by a decrease of membrane/substrate distance and energy of adhesion.