Investigating G protein-coupled receptor endocytosis and trafficking by TIR-FM.

G protein-coupled receptors (GPCRs) represent the largest and most versatile family of signaling receptors. Their actions are highly regulated, both under physiological conditions and in response to clinically relevant drugs. A key element in this regulation is control of the number of functional receptors at the cell surface. Major processes that mediate this regulation are vesicular endocytosis and exocytosis of receptors. These trafficking events involve a concerted series of steps, some of which occur on a rapid timescale similar to that of functional signaling itself. Here, we describe and discuss an optical imaging approach, based on evanescent field or total internal reflection-fluorescence microscopy (TIR-FM), to investigate receptor endocytosis and recycling at the level of discrete membrane fission and fusion events. TIR-FM facilitates the study of receptor trafficking events near the plasma membrane with much greater spatial and temporal resolution than afforded by traditional methods. TIR-FM has already provided new insight to GPCR regulation, and we believe that this method has great potential for addressing a variety of questions in GPCR biology.