Improved molecular toolkit for cAMP studies in live cells
1 Neurobiology Section, Division of Biological Sciences, Kavli Institute for Brain and Mind, University of California, San Diego, La Jolla, CA 92093, USA
2 INSERM, U839, Université Paris 6, Institut du Fer à Moulin, 17 rue du Fer à Moulin, 75005 Paris, France
BMC Research Notes 2011, 4:241 doi:10.1186/1756-0500-4-241Published: 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.