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Real-time monitoring of cyclic nucleotide signaling in neurons using genetically encoded FRET probes

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Brain Cell Biology

Abstract

Signaling cascades involving cyclic nucleotides play key roles in signal transduction in virtually all cell types. Elucidation of the spatiotemporal regulation of cyclic nucleotide signaling requires methods for tracking the dynamics of cyclic nucleotides and the activities of their regulators and effectors in the native biological context. Here we review a series of genetically encoded FRET-based probes for real-time monitoring of cyclic nucleotide signaling with a particular focus on their implementation in neurons. Current data indicate that neurons have a very active metabolism in cyclic nucleotide signaling, which is tightly regulated through a variety of homeostatic regulations.

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References

  • Adams, S. R. et al. (1991) Fluorescence ratio imaging of cyclic AMP in single cells. Nature, 349, 694–697

    Article  PubMed  CAS  Google Scholar 

  • Allen, M. D. & Zhang, J. (2006) Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem. Biophys. Res. Commun., 348, 716–721

    Article  PubMed  CAS  Google Scholar 

  • Bacskai, B. J. et al. (1993) Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons. Science, 260, 222–226

    Article  PubMed  CAS  Google Scholar 

  • DiPilato, L. M., Cheng, X. & Zhang, J. (2004) Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc. Natl. Acad. Sci. USA, 101, 16513–16518

    Article  PubMed  CAS  Google Scholar 

  • Domingo, B. et al. (2007). Imaging FRET standards by steady-state fluorescence and lifetime methods. Microsc. Res. Tech. 70, 1010–1021

    Google Scholar 

  • Dunn, T. A. et al. (2006) Imaging of cAMP levels and protein kinase a activity reveals that retinal waves drive oscillations in second-messenger cascades. J. Neurosci., 26, 12807–12815

    Article  PubMed  CAS  Google Scholar 

  • Gervasi, N. et al. (2007) Dynamics of PKA signaling at the membrane, in the cytosol and in the nucleus of neurons in mouse brain slices. J. Neurosci., 27, 2744–2750

    Article  PubMed  CAS  Google Scholar 

  • Gibbs, C. S. et al. (1992) Systematic mutational analysis of cAMP-dependent protein kinase identifies unregulated catalytic subunits and defines regions important for the recognition of the regulatory subunit. J. Biol. Chem., 267, 4806–4814

    PubMed  CAS  Google Scholar 

  • Goaillard, J.-M., Vincent, P. & Fischmeister, R. (2001) Simultaneous measurements of intracellular cAMP and L-type Ca2+ current in single frog ventricular myocytes. J. Physiol., 530, 79–91

    Article  PubMed  CAS  Google Scholar 

  • Gorbunova, Y. V. & Spitzer, N. C. (2002) Dynamic interactions of cyclic AMP transients and spontaneous Ca(2+) spikes. Nature, 418, 93–96

    Article  PubMed  CAS  Google Scholar 

  • Gordon, G. W. et al. (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J., 74, 2702–2713

    Article  PubMed  CAS  Google Scholar 

  • Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem., 260, 3440–3450

    PubMed  CAS  Google Scholar 

  • Gu, Y. et al. (2004) Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing. J. Microsc., 215, 162–173

    Article  PubMed  CAS  Google Scholar 

  • Heim, N. et al. (2007) Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor. Nat. Methods, 4, 127–129

    Article  PubMed  CAS  Google Scholar 

  • Hempel, C. M. et al. (1996) Spatio-temporal dynamics of cAMP signals in an intact neural circuit. Nature, 384, 166–169

    Article  PubMed  CAS  Google Scholar 

  • Hepp, R. et al. (2007) Phosphodiesterase type 2 and the homeostasis of cyclic GMP in living thalamic neurons. J. Neurochem., 102, 1875–1886

    Article  PubMed  CAS  Google Scholar 

  • Honda, A. et al. (2001) Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc. Natl. Acad. Sci. USA, 98, 2437–2442

    Article  PubMed  CAS  Google Scholar 

  • Honda, A. et al. (2005) Cygnets: in vivo characterization of novel cGMP indicators and in vivo imaging of intracellular cGMP. Methods Mol. Biol., 307, 27–43

    PubMed  CAS  Google Scholar 

  • Hoppe, A., Christensen, K. & Swanson, J. A. (2002) Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys. J., 83, 3652–3664

    Article  PubMed  CAS  Google Scholar 

  • Houslay, M. D. & Milligan, G. (1997) Tailoring cAMP-signalling responses through isoform multiplicity. Trends Biochem. Sci., 22, 217–224

    Article  PubMed  CAS  Google Scholar 

  • Kaupp, U. B. & Seifert, R. (2001) Molecular diversity of pacemaker ion channels. Annu. Rev. Physiol, 63, 235–257

    Article  PubMed  CAS  Google Scholar 

  • Kemp, B. E. (1980) Phosphorylation of acyl and dansyl derivatives of the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly by the cAMP-dependent protein kinase. J. Biol. Chem., 255, 2914–2918

    PubMed  CAS  Google Scholar 

  • Lissandron, V. et al. (2005) Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction. J. Mol. Biol., 354, 546–555

    Article  PubMed  CAS  Google Scholar 

  • Lissandron, V. et al. (2007) Transgenic fruit-flies expressing a FRET-based sensor for in vivo imaging of cAMP dynamics. Cell. Signal., 19, 2296–2303

    Article  PubMed  CAS  Google Scholar 

  • Lohse, M. J. et al. (2008) Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors. Trends Pharmacol. Sci., 29, 159–165

    PubMed  CAS  Google Scholar 

  • Mongillo, M. et al. (2004) Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ. Res., 95, 67–75

    Article  PubMed  CAS  Google Scholar 

  • Namiki, S. et al. (2005) NO signalling decodes frequency of neuronal activity and generates synapse-specific plasticity in mouse cerebellum. J. Physiol., 566, 849–863

    Article  PubMed  CAS  Google Scholar 

  • Nausch, L.W. et al. (2008) Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors. Proc. Natl. Acad. Sci. USA, 105, 365–370

    Article  PubMed  CAS  Google Scholar 

  • Nikolaev, V. O. et al. (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J. Biol. Chem., 279, 37215–37218

    Article  PubMed  CAS  Google Scholar 

  • Nikolaev, V. O. et al. (2006a) Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching {beta}1-adrenergic but locally confined {beta}2-adrenergic receptor-mediated signaling. Circ. Res., 99, 1084–1091

    Article  PubMed  CAS  Google Scholar 

  • Nikolaev, V. O., Gambaryan, S. & Lohse, M. J. (2006b) Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat. Methods, 3, 23–25

    Article  PubMed  CAS  Google Scholar 

  • Ponsioen, B. et al. (2004) Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep., 5, 1176–1180

    Article  PubMed  CAS  Google Scholar 

  • Rich, T. C. & Karpen, J. W. (2002) Review article: cyclic AMP sensors in living cells: what signals can they actually measure? Ann. Biomed. Eng., 30, 1088–1099

    Article  PubMed  Google Scholar 

  • Rich, T. C. et al. (2001a). In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J. Gen. Physiol. 118, 63–78

    Google Scholar 

  • Rich, T. C. et al. (2001b). A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell. Proc. Natl. Acad. Sci. USA 98, 13049–13054

    Google Scholar 

  • Russwurm, M. et al. (2007) Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach. Biochem. J., 407, 69–77

    Article  PubMed  CAS  Google Scholar 

  • Sato, M., Hida, N., and Umezawa, Y. (2005). Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells. Proc. Natl. Acad. Sci. USA 102, 14515–14520

    Google Scholar 

  • Sato, M. et al. (2000) Fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins. Anal. Chem., 72, 5918–5924

    Article  PubMed  CAS  Google Scholar 

  • Saucerman, J. J. et al. (2006) Systems analysis of PKA-mediated phosphorylation gradients in live cardiac myocytes. Proc. Natl. Acad. Sci. USA, 19, 2650–2658

    Google Scholar 

  • Shafer, O. T. et al. (2008) Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of drosophila revealed by real-time cyclic AMP imaging. Neuron, 58, 161–163

    Article  CAS  Google Scholar 

  • Smith, C. M. et al. (1999) The catalytic subunit of cAMP-dependent protein kinase: prototype for an extended network of communication. Prog. Biophys. Mol. Biol., 71, 313–341

    Article  PubMed  CAS  Google Scholar 

  • Tsien, R. Y. & Harootunian, A. T. (1990) Practical design criteria for a dynamic ratio imaging system. Cell Calcium, 11, 93–109

    Article  PubMed  CAS  Google Scholar 

  • Vincent, P. & Brusciano, D. (2001) Cyclic AMP imaging in neurones in brain slice preparations. J. Neurosci. Methods, 108, 189–198

    Article  PubMed  CAS  Google Scholar 

  • Vincent, P. et al. (2006) Live imaging of neural structure and function by fibred fluorescence microscopy. EMBO Rep., 7, 1154–1161

    Article  PubMed  CAS  Google Scholar 

  • Violin, J. D. et al. (2008) β2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics. J. Biol. Chem., 283, 2949–2961

    Article  PubMed  CAS  Google Scholar 

  • Willoughby, D. & Cooper, D. M. (2008) Live-cell imaging of cAMP dynamics. Nat. Methods, 5, 29–36

    Article  PubMed  CAS  Google Scholar 

  • Zaccolo, M. et al. (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat. Cell Biol., 2, 25–29

    Article  PubMed  CAS  Google Scholar 

  • Zacharias, D. A. et al. (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science, 296, 913–916

    Article  PubMed  CAS  Google Scholar 

  • Zetterqvist, O. et al. (1976) The minimum substrate of cyclic AMP-stimulated protein kinase, as studied by synthetic peptides representing the phosphorylatable site of pyruvate kinase (type L) of rat liver. Biochem. Biophys. Res. Commun., 70, 696–703

    Article  PubMed  CAS  Google Scholar 

  • Zhang, J. et al. (2001) Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc. Natl. Acad. Sci. USA, 98, 14997–15002

    Article  PubMed  CAS  Google Scholar 

  • Zhang, J. et al. (2005) Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes. Nature, 437, 569–573

    Article  PubMed  CAS  Google Scholar 

  • Zimmermann, T. et al. (2002) Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett., 531, 245–249

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank Professor Ron Harris-Warrick for critically reading this manuscript. This work was supported by CNRS, UPMC, “Fondation pour la Recherche Médicale” and “Fondation pour la Recherche sur le Cerveau” (to P. V.); and by NIH (DK073368 and CA122673), the American Heart Association, the Young Clinical Scientist Award Program of the Flight Attendant Medical Research Institute, and 3M (to J. Z.).

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Correspondence to Jin Zhang.

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Vincent, P., Gervasi, N. & Zhang, J. Real-time monitoring of cyclic nucleotide signaling in neurons using genetically encoded FRET probes. Brain Cell Bio 36, 3–17 (2008). https://doi.org/10.1007/s11068-008-9035-6

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  • DOI: https://doi.org/10.1007/s11068-008-9035-6

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