Visualizing dynamic activities of signaling enzymes using genetically encodable FRET-based biosensors from designs to...

Living cells respond to various environmental cues and process them into a series of spatially and temporally regulated signaling events, which can be tracked in real time with an expanding repertoire of genetically encodable FRET-based biosensors. A series of these biosensors, designed to track dynamic activities of signaling enzymes such as protein kinases and small GTPases, have yielded invaluable information regarding the spatiotemporal regulation of these enzymes, shedding light on the orchestration of signaling pathways within the native cellular context. In this chapter, we first review the generalizable modular designs of FRET-based biosensors, followed by a detailed discussion about biosensors for reporting protein kinase activities and GTPase activation. Two general designs, uni- and bimolecular reporters, will be discussed with an analysis of their strengths and limitations. Finally, an example of using both uni- and bimolecular kinase activity reporters to visualize PKA activity in living cells will be presented to provide practical tips for using these biosensors to explore specific biological systems.