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Reduced noise level of pm-Epac2-camps-GFP/mCherry compared to pm-Epac2-camps-CFP/YFP. (A) Normalized excitation (solid lines) and emission (dashed lines) of GFP, mCherry, CFP and YFP. Emission spectra of CFP and YFP largely overlap, making FRET measurement difficult. The large separation of GFP and mCherry emission spectra significantly reduces crosstalk between acceptor and donor. (B) ΔR/R0 was computed as (R-R0)/R0 where R is donor:acceptor ratio and R0 the mean value of this ratio before forskolin stimulation. ΔR/R0 of both pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry increase after 10 μM forskolin application. The lag between the pm-Epac2-camps-GFP/mCherry and pm-Epac2-camps-CFP/YFP signals falls under the precision of the perfusion system and is unlikely to reflect a difference between the sensors. No correction factor was applied. ≥ 15 cells were scored for each condition. (C) Acceptor and donor emission of pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry before and after 1000 frame acquisitions (excitation with a 442 nm laser line). Photobleaching is less pronounced for pm-Epac2-camps-GFP/mCherry. (D) Fluorescence intensity of acceptors and donors of pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry after 1000 frames, expressed as a fraction of the fluorescence intensity of the first frame. ** p < 0.01, *** p < 0.001. (E) Noise levels for pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry are equivalent for the first 50 frames. After 950 frames, only the noise level for pm-Epac2-camps-CFP/YFP has increased. Data are normalized to the value at the beginning of the recording as in B (ΔR/R0). (F) The standard deviation of ΔR/R0 over 40 frames, indicating the noise level, shows a progressive increase for pm-Epac2-camps-CFP/YFP but not for pm-Epac2-camps-GFP/mCherry. * p < 0.05 from frame 819 to 1000. C-F, 5 cells per condition. Error bars, sem. D, Kruskal-Wallis test. F, Mann-Whitney U test.
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Figure 2: Reduced noise level of pm-Epac2-camps-GFP/mCherry compared to pm-Epac2-camps-CFP/YFP. (A) Normalized excitation (solid lines) and emission (dashed lines) of GFP, mCherry, CFP and YFP. Emission spectra of CFP and YFP largely overlap, making FRET measurement difficult. The large separation of GFP and mCherry emission spectra significantly reduces crosstalk between acceptor and donor. (B) ΔR/R0 was computed as (R-R0)/R0 where R is donor:acceptor ratio and R0 the mean value of this ratio before forskolin stimulation. ΔR/R0 of both pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry increase after 10 μM forskolin application. The lag between the pm-Epac2-camps-GFP/mCherry and pm-Epac2-camps-CFP/YFP signals falls under the precision of the perfusion system and is unlikely to reflect a difference between the sensors. No correction factor was applied. ≥ 15 cells were scored for each condition. (C) Acceptor and donor emission of pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry before and after 1000 frame acquisitions (excitation with a 442 nm laser line). Photobleaching is less pronounced for pm-Epac2-camps-GFP/mCherry. (D) Fluorescence intensity of acceptors and donors of pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry after 1000 frames, expressed as a fraction of the fluorescence intensity of the first frame. ** p < 0.01, *** p < 0.001. (E) Noise levels for pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry are equivalent for the first 50 frames. After 950 frames, only the noise level for pm-Epac2-camps-CFP/YFP has increased. Data are normalized to the value at the beginning of the recording as in B (ΔR/R0). (F) The standard deviation of ΔR/R0 over 40 frames, indicating the noise level, shows a progressive increase for pm-Epac2-camps-CFP/YFP but not for pm-Epac2-camps-GFP/mCherry. * p < 0.05 from frame 819 to 1000. C-F, 5 cells per condition. Error bars, sem. D, Kruskal-Wallis test. F, Mann-Whitney U test.

Mentions: In addition to the improvement of PACα to manipulate cAMP, we modified the FRET pair of the existing Epac2-camps cAMP sensor to improve the stability of its signal-to-noise ratio over longer periods of time. We replaced the CFP/YFP pair by GFP/mCherry, with the same linkers as in the original probe [10]. mCherry and GFP have a low photobleaching rate [16] and constitute an efficient FRET pair [12] with smaller overlap between the emission spectra of the acceptor and the donor (Figure 2A). In addition, we included a mutation in the cAMP binding domain (K405E) that has been shown to reduce the Kd of the probe from 900 nM to 300 nM [11]. To demonstrate the improvement of the sensor, mRNA coding for membrane-targeted Epac2-camps including either CFP/YFP (pm-Epac2-camps-CFP/YFP) or GFP/mCherry (pm-Epac2-camps-GFP/mCherry) was transcribed and expressed in Xenopus laevis neural tube cultures as described for mCherry-PACα. Cultures were continuously superfused and three images per minute were acquired with 442 nm laser excitation and filters adapted for each FRET pair (CFP: 460 nm-500 nm; YFP: 525 nm-580 nm; GFP: 480 nm-550 nm; mCherry: 590 nm-700 nm). 10 μM forskolin induced a similar FRET ratio increase for pm-Epac2-camps-CFP/YFP and pm-Epac2-camps-GFP/mCherry, revealing an appropriate FRET response to cAMP elevation for both probes (Figure 2B). To assess the resistance to photobleaching, cells were continuously excited at 442 nm. CFP and YFP intensity decreased rapidly whereas GFP and mCherry showed reduced photobleaching (Figure 2C and 2D). Although 442 nm is not at the peak of excitation of GFP, a forskolin-induced increase of the GFP:mCherry ratio can be detected using this excitation wavelength (Figure 2B). Exciting GFP with a 442 nm laser line has the advantage of minimizing the direct excitation of the acceptor (mCherry) that perturbs FRET measurements when longer excitation wavelengths are used. To assess the noise level, we monitored the standard deviation of CFP:YFP and GFP:mCherry FRET ratio changes over 40 frames during the 1000 frames imaging period. The standard deviation of pm-Epac2-camps-GFP/mCherry was stable throughout the measurement period, while the standard deviation of pm-Epac2-camps-CFP/YFP increased over time (Figure 2E and 2F). The progressive increase of the noise level over time may be due to the increased relative contribution of the noise to the fluorescence measurement of each channel: the fluorescence intensity decreases because of photobleaching, but the noise level is not affected. Consequently, the noise level of the donor:acceptor ratio is higher after photobleaching.

Improved molecular toolkit for cAMP studies in live cells

Hong KP, Spitzer NC, Nicol X - BMC Res Notes (2011)

Bottom Line: We have improved the suitability of cAMP manipulating and monitoring tools for live cell imaging.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.

Affiliation: Neurobiology Section, Division of Biological Sciences, Kavli Institute for Brain and Mind, University of California, San Diego, La Jolla, CA 92093, USA. xavier.nicol@inserm.fr.

ABSTRACT

Background: 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.

Findings: 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.

Conclusions: 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.

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