title: A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range.
abstract: FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP) have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs). Through a conformational change in Epac1, alterations in cellular cAMP levels lead to a change in FRET that is most commonly detected by either Fluorescence Lifetime Imaging (FLIM) or by Sensitized Emission (SE), e.g., by simple ratio-imaging. We recently reported a range of different Epac-based cAMP sensors with high dynamic range and signal-to-noise ratio. We showed that constructs with cyan FP as donor are optimal for readout by SE, whereas other constructs with green FP donors appeared much more suited for FLIM detection. In this study, we present a new cAMP sensor, termed (T)Epac(VV), which employs mTurquoise as donor. Spectrally very similar to CFP, mTurquoise has about doubled quantum efficiency and unlike CFP, its fluorescence decay is strictly single-exponential. We show that (T)Epac(VV) appears optimal for detection both by FLIM and SE, that it has outstanding FRET span and signal-to-noise ratio, and improved photostability. Hence, (T)Epac(VV) should become the cAMP sensor of choice for new experiments, both for FLIM and ratiometric detection.
authors.affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Klarenbeek
authors.forename: Joachim
authors.initials: J
authors.lastname: Goedhart
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hink
authors.forename: Theodorus W J
authors.initials: TW
authors.lastname: Gadella
authors.forename: Kees
authors.initials: K
authors.lastname: Jalink
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000737:chemistry*; D004867:Equipment Design; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D012815:Signal Processing, Computer-Assisted
chemical_list: D005456:Fluorescent Dyes; D000242:Cyclic AMP