Visualizing Dynamic Activities of Signaling Enzymes Using Genetically Encodable Fret-Based Biosensors

Abstract

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.

GFP-Based Biosensors

“…PKA and ERK areenzymes that transfer the γ-phosphate of ATP to a number of protein substrates thereby affecting a conformational change. Kinase induced conformational changes are important because they are involved in the control a number of critical cellular processes that include glycogen synthesis, hormonal response, and ion transport [70]. A number of signaling cascades that involve kinases require a means of dynamic control and spatial compartmentalization of the kinase activity; a requirement highlights the need for a mechanism to continuously track kinase activity in different compartments and signaling microdomains in vivo.…”

“…Genetically encodable FRET GFP biosensors have proven to be useful in characterizing the dynamic phosphorylation dependent regulation of small GTPases [70]. Ras GTPases play essential roles in regulating cell growth, cell differentiation, cell migration, and lipid vesicle trafficking.…”

“…These sensors are constructed so that the substrate protein of the kinase of interest is flanked with a fluorescent protein pair in such a way that the conformational change imparted by phosphorylation translates into a change in the FRET signal (Figure7) [70]. These biosensors can be localized to particular sites of interest with the aid of appropriate targeting signal sequences, allowing the imaging of site-specific kinase activity.G-protein coupled receptors, when used in a biosensor, provide a mechanism for transducingdrug mediated effects on PKA activity into a light signal.…”

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

“…Several groups have described the use of fluorescence lifetime imaging as an effective approach for measuring FRET in vivo [42][43][44] . The ratiometric approach offers a few key advantages over fluorescence lifetime-based approaches.…”

Intravital FRET: comprehending life at single-molecule level. Focus on “A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy”

“…A large variety of highly elaborate FRET constructs have been developed in the past decades (1,8,10). The key properties necessary for effective FRET quantification in vivo are high FRET efficiency to yield a large dynamic range, specificity, low cross talk with other cellular processes and fast response time, since intravital experiments aim at monitoring the highly dynamic motion of cells in living organisms.…”