- Technical Note
- Open Access
Improved molecular toolkit for cAMP studies in live cells
© Nicol et al; licensee BioMed Central Ltd. 2011
Received: 6 April 2011
Accepted: 20 July 2011
Published: 20 July 2011
cAMP is a ubiquitous second messenger involved in a wide spectrum of cellular processes including gene transcription, cell proliferation, and axonal pathfinding. Precise spatiotemporal manipulation and monitoring in live cells are crucial for investigation of cAMP-dependent pathways, but existing tools have several limitations.
We have improved the suitability of cAMP manipulating and monitoring tools for live cell imaging. We attached a red fluorescent tag to photoactivated adenylyl cyclase (PACα) that enables reliable visualization of this optogenetic tool for cAMP manipulation in target cells independently of its photoactivation. We show that replacement of CFP/YFP FRET pair with GFP/mCherry in the Epac2-camps FRET probe reduces photobleaching and stabilizes the noise level during imaging experiments.
The modifications of PACα and Epac2-camps enhance these tools for in vitro cAMP studies in cultured living cells and in vivo studies in live animals in a wide range of experiments, and particularly for long term time-lapse imaging.
cAMP is a major cellular second messenger that activates and integrates multiple intracellular signaling pathways and modulates a large range of cellular processes, including gene transcription , cell adhesion and migration , and axonal growth and pathfinding . cAMP studies rely on methods to manipulate and monitor cAMP concentrations in live cells. Existing tools have been very useful in identifying cAMP-dependent cellular processes, but have some limitations when it comes to understanding cAMP dynamics and localization in living cells. Forskolin and 3-isobutyl-1-methylxanthine (IBMX) are powerful pharmacological compounds enabling the generation of sustained elevations of cAMP. Forskolin directly stimulates most transmembrane adenylyl cyclases  and IBMX inhibits cAMP hydrolysis by phosphodiesterases. Recently, the use of photoactivated adenylyl cyclase alpha (PACα) from the flagellate Euglena gracilis, synthesizing cAMP in response to blue light, has allowed precise spatiotemporal manipulation of cAMP . It has been attached to GFP for visualization in live cells . However, the excitation wavelength of this visible reporter overlaps with the excitation spectrum of PACα, making it difficult to use this fusion construct for independent PACα excitation and reporter imaging.
Monitoring cAMP in live cells has been made possible by the use of FRET probes [7–10]. Epac2-camps is a cAMP indicator that is widely used to monitor cAMP  and has been recently improved with a mutation increasing its affinity for cAMP . However, fast photobleaching of the commonly used CFP/YFP FRET pair limits its use in live cell imaging experiments over extended periods of time because the signal-to-noise ratio decreases progressively. The GFP/mCherry FRET pair has been successfully used for cAMP sensors , but its photostability and signal-to-noise ratio have not been assessed.
Independent excitation of PACα and mCherry in living cells and live animals
Additional file 2:Photoactivation of PACα induces X. laevis embryo twitching. mCherry-PACα-injected embryos twitch when illuminated with blue light to excite PACα, but not when exposed to green light to excite mCherry. (AVI 5 MB)
To verify that this construct can be used in vivo, Xenopus embryos injected with mCherry-PACα mRNA were illuminated with green and blue light in alternation using a fluorescence dissecting microscope with GFP and Texas-red filter cubes. Blue light illumination (excitation filter: BP 470/40) induced embryos to twitch, whereas they remain completely immobile under green light illumination (excitation filter: BP 560/40) (Figure 1D and 1E and see Additional file 2). Uninjected embryos did not exhibit light-induced twitching. Excitation of mCherry did not affect embryos' behavior, confirming the spectral compatibility of mCherry and PACα.
Reduced photobleaching of Epac2-camps using the GFP/mCherry FRET pair
In summary, we have generated an improved toolkit for cAMP studies. mCherry-PACα allows spatiotemporal control of cAMP in living cells after identification of cells expressing it. Localized illumination of a cell is likely to increase cAMP concentration locally. Further development of PACα may include its targeting to subcellular compartments to go beyond the limit of precision of optical stimulation and achieve cAMP manipulation bearing closer resemblance to physiological signals. It would be useful to ensure for each experimental condition that the cyclase activity of PACα in the dark does not affect intracellular signaling [5, 17]. To limit cAMP synthesis without light exposure, we used a mutated PACα (R330A) that has a limited cyclase activity in the dark (G. Nagel, personal communication). This was enough to avoid perturbation of circus cells movement by the cyclase activity of PACα in the dark. In case an extremely low dark activity is needed, bPAC, a bacterial light-sensitive adenylyl cyclase, could be used at the cost of less stringent temporal control of cAMP signaling .
pm-Epac2-camps-GFP/mCherry has greater photostability than pm-Epac2-camps-CFP/YFP and a lower noise level after extended periods of imaging. However GFP and mCherry make a less effective FRET pair than CFP and YFP, and its use may be beneficial only for FRET experiments requiring an extended period of imaging. Versions of CFP and YFP (mTurquoise and Venus respectively) with increased photostability are now available and make an efficient FRET pair for cAMP sensors . Testing its noise level stability would allow comparison of the behaviour of GFP/mCherry and mTurquoise/Venus as FRET pairs for prolonged experiments. The sensor described here has the advantage over the mTurquoise/Venus probe  of sensitivity to lower concentrations of cAMP, because it is an Epac2-based sensor including a mutation that reduces its Kd . It would be useful to compare it to the Epac1-based sensor using mTurquoise/Venus, with the higher Kd for cAMP [9, 10, 18].
The use of both tools in the same cell is not yet possible due to the overlap of excitation wavelengths, but further improvements may include the shift of PACα excitation towards the UV to avoid wavelength conflict with the FRET probe excitation, in combination with the switch of the mCherry tag to a longer wavelength fluorescent protein such as mKate to avoid the overlap of emission between the PACα tag and the FRET acceptor.
We thank Dr M. Roe for the gift of pm-Epac2-camps, and Dr G. Nagel for the gift of PACα. We are grateful to members of our lab for thoughtful discussion. This work was supported by a Fondation pour la Recherche Médicale fellowship and a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme to X.N., and by NIH NS15918 to N.C.S.
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