Improved molecular toolkit for cAMP studies in live cells

Kwan Pyo Hong,Nicholas C Spitzer,Xavier Nicol

doi:10.1186/1756-0500-4-241

Kwan Pyo Hong, Nicholas C Spitzer + Show 1 more

Open Access

https://doi.org/10.1186/1756-0500-4-241

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Abstract

BackgroundcAMP 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.FindingsWe 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.ConclusionsThe 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.

Highlights

CAMP is a ubiquitous second messenger involved in a wide spectrum of cellular processes including gene transcription, cell proliferation, and axonal pathfinding
Background 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 [1], cell adhesion and migration [2], and axonal growth and pathfinding [3]. 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

Summary

Introduction

CAMP is a ubiquitous second messenger involved in a wide spectrum of cellular processes including gene transcription, cell proliferation, and axonal pathfinding. 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. MCherry excitation did not affect circus movements and mCherry-negative cells did not exhibit plasma membrane movement arrest following stimulation by either wavelength (Figure 1B and 1C and see Additional file 1).

Results

Conclusion