Milestones in the development and implementation of FRET-based sensors of intracellular signals: A biological perspective of the history of FRET.

Fӧrster resonance energy transfer (FRET) has been described for more than a century. FRET has become a mainstay for the study of protein localization in living cells and tissues. It has also become widely used in the fields that comprise cellular signaling. FRET-based probes have been developed to monitor second messenger signals, the phosphorylation state of peptides and proteins, and subsequent cellular responses. Here, we discuss the milestones that led to FRET becoming a widely used tool for the study of biological systems: the theoretical description of FRET, the insight to use FRET as a molecular ruler, and the isolation and genetic modification of green fluorescent protein (GFP). Each of these milestones were critical to the development of a myriad of FRET-based probes and reporters in common use today. FRET-probes offer a unique opportunity to interrogate second messenger signals and subsequent protein phosphorylation - and perhaps the most effective approach for study of cAMP/PKA pathways. As such, FRET probes are widely used in the study of intracellular signaling pathways. Yet, somehow, the potential of FRET-based probes to provide windows through which we can visualize complex cellular signaling systems has not been fully reached. Hence we conclude by discussing the technical challenges to be overcome if FRET-based probes are to live up to their potential for the study of complex signaling networks.