Single-molecule imaging technique to study the dynamic regulation of GPCR function at the plasma membrane

The lateral diffusion of a G-protein-coupled receptor (GPCR) in the plasma membrane determines its interaction capabilities with downstream signaling molecules and critically modulates its function. Mechanisms that control GPCR mobility, like compartmentalization, enable a cell to fine-tune its response through local changes in the rate, duration, and extent of signaling. These processes are known to be highly dynamic and tightly regulated in time and space, usually not completely synchronized in time. Therefore, bulk studies such as protein biochemistry or conventional confocal microscopy will only yield information on the average properties of the interactions and are compromised by poor time resolution. Single-particle tracking (SPT) in living cells is a key approach to directly monitor the function of a GPCR within its natural environment and to obtain unprecedented detailed information about receptor mobility, binding kinetics, aggregation states, and domain formation. This review provides a detailed description on how to perform single GPCR tracking experiments.