Physical biology of GPCR signalling dynamics inferred from fluorescence spectroscopy and imaging

The physical biology of G protein-coupled receptor (GPCR) signalling can be inferred from imaging of single molecules and single living cells. In this opinion paper, we highlight recent developments in technologies to study GPCR signalling in vitro and in cyto. We start from mobility and localisation characteristics of single receptors in membranes. Subsequently, we discuss the kinetics of shifts in receptor-conformation equilibrium due to allosteric binding events and G protein activation. We continue with recent insights into downstream signalling and the role of delayed negative feedback to suppress GPCR signalling. Finally, we discuss new strategies to reveal how the multiplex signalling responses of cells to ligand mixtures, mediated by their entire receptor arsenal, can be disentangled, using single-cell data.