Monitoring the dynamics and mobility of membrane proteins tagged with green fluorescent protein.

Publisher Summary This chapter discusses the methods that can be used to study intracellular membrane dynamics using green fluorescent protein (GFP)-tagged membrane proteins expressed in living cells. The use of GFP chimeras overcomes many of the limitations inherent to the studies of fixed specimens labeled with antibodies. Not only can GFP chimeras be visualized in the unperturbed environment of a living cell, virtually any protein can be tagged with GFP, producing fusion proteins that are intrinsically fluorescent and photostable and that usually retain parent molecule targeting and function. The chapter discusses approaches for optimizing GFP chimera brightness and expression levels within cells, so that intracellular membranes expressing GFP constructs can be readily resolved. The chapter also discusses the techniques for acquiring time-lapse images and methods to analyze them. Approaches for quantitating the number of GFP molecules being imaged in a cell have been described, so that the kinetic steps of membrane transport can be analyzed. The chapter describes the methods of photobleaching that can be used to measure the diffusional mobility of GFP chimeras and the extent of their movement between membrane-bound compartments.