Combinatorial Live Cell Homo- and Hetero-FRET Microscopy of Membrane Proteins

Abstract

The structure-function-activity relationships of transmembrane receptors are often mediated not only by ligand-induced signaling but also homo- and hetero-philic binding interactions. Understanding the molecular basis for these interactions is therefore critical for elucidating receptor function. A powerful means of addressing these phenomena is to apply combinatorial microscopies that allow one to probe not only location but also orientation, association, and dynamics. By applying a coupled confocal-total internal reflection fluorescence (TIRF) microscopy imaging scheme, we are examining the distribution, association, and ligand accessibility of two families of transmembrane receptors: carcinoembryonic-antigen-related cell- adhesion molecule 1 (CEACAM1), and fibroblast growth factor receptor 1 (FGFR1), thought to associate with FGF21 co-receptor Klotho-beta (KLB). By using this coupled imaging platform, we can address differences in receptor behaviour, dynamics and structure on the free cell apical surface as well as in the cell itself by confocal microscopy and at the cell-substrate interface by TIRF microscopy. The use of homo- and hetero- Forster Resonance Energy Transfer (FRET) analysis provides us with a powerful means of examining real-time association kinetics of these systems and the effect of soluble ligands on receptor association.