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Live-cell imaging of cAMP dynamics

Nature Methods volume 5, pages 29–36 (2008)Cite this article

Abstract

Spatial and temporal compartmentalization of cAMP (and its target proteins) is central to the ability of this second messenger to govern cellular activity over timescales ranging from milliseconds to several hours. Recent years have witnessed a burgeoning of methodologies that enable researchers to directly monitor rapid subcellular cAMP dynamics, which are unobtainable by traditional cAMP assays. In this review, we examine cAMP biosensors that are currently available for measuring cAMP at the single-cell level, compare their various operating principles and discuss their applications.

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Figure 1: Localization of the currently available cAMP sensors.
Figure 2: Apparent dynamics of the cAMP signal depend on the cAMP sensor used.
Figure 3: Problems associated with saturation of sensors, as illustrated by cAMP sensor signals during agonist-evoked transient and sustained rises in cAMP.

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References

  1. Buxton, I.L. & Brunton, L.L. Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J. Biol. Chem. 258, 10233–10239 (1983).

    CAS  PubMed  Google Scholar 

  2. Bootman, M.D. & Berridge, M.J. The elemental principles of calcium signaling. Cell 83, 675–678 (1995).

    Article  CAS  Google Scholar 

  3. Petersen, O.H. et al. Calcium signalling: past, present and future. Cell Calcium 38, 161–169 (2005).

    Article  CAS  Google Scholar 

  4. Butcher, R.W. et al. Adenosine 3′,5′-monophosphate in biological materials. II. The measurement of adenosine 3′,5′-monophosphate in tissues and the role of the cyclic nucleotide in the lipolytic response of fat to epinephrine. J. Biol. Chem. 240, 4515–4523 (1965).

    CAS  PubMed  Google Scholar 

  5. Gilman, A.G. A protein binding assay for adenosine 3′:5′-cyclic monophosphate. Proc. Natl. Acad. Sci. USA 67, 305–312 (1970).

    Article  CAS  Google Scholar 

  6. Brooker, G. et al. Gammaflow: a completely automated radioimmunoassay system. Science 194, 270–276 (1976).

    Article  CAS  Google Scholar 

  7. Evans, T. et al. Muscarinic cholinergic receptors of two cell lines that regulate cyclic AMP metabolism by different molecular mechanisms. Mol. Pharmacol. 26, 395–404 (1984).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Zaccolo, M. & Pozzan, T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295, 1711–1715 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Fagan, K.A. et al. Adenovirus encoded cyclic nucleotide–gated channels: a new methodology for monitoring cAMP in living cells. FEBS Lett. 500, 85–90 (2001).

    Article  CAS  Google Scholar 

  13. DiPilato, L.M. et al. 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 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Xu, X. et al. Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. Proc. Natl. Acad. Sci. USA 104, 10264–10269 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Gorbunova, Y.V. & Spitzer, N.C. Dynamic interactions of cyclic AMP transients and spontaneous Ca2+ spikes. Nature 418, 93–96 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Mongillo, M. et al. 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 (2004).

    Article  CAS  Google Scholar 

  22. Dyachok, O. et al. Oscillations of cyclic AMP in hormone-stimulated insulin-secreting β-cells. Nature 439, 349–352 (2006).

    Article  CAS  Google Scholar 

  23. Terrin, A. et al. PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases. J. Cell Biol. 175, 441–451 (2006).

    Article  CAS  Google Scholar 

  24. Warrier, S. et al. β-Adrenergic and muscarinic receptor–induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor. Am. J. Physiol. Cell Physiol. 289, C455–C461 (2005).

    Article  CAS  Google Scholar 

  25. Prinz, A. et al. Novel, isotype-specific sensors for protein kinase A subunit interaction based on bioluminescence resonance energy transfer (BRET). Cell. Signal. 18, 1616–1625 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Nikolaev, V.O. et al. Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis. J. Biol. Chem. 280, 1716–1719 (2005).

    Article  CAS  Google Scholar 

  28. Jiang, L.I. et al. Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway. J. Biol. Chem. 282, 10576–10584 (2007).

    Article  CAS  Google Scholar 

  29. Kaupp, U.B. & Seifert, R. Cyclic nucleotide–gated ion channels. Physiol. Rev. 82, 769–824 (2002).

    Article  CAS  Google Scholar 

  30. Rochais, F. et al. Negative feedback exerted by cAMP-dependent protein kinase and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels. J. Biol. Chem. 279, 52095–52105 (2004).

    Article  CAS  Google Scholar 

  31. Willoughby, D. et al. An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics. EMBO J. 25, 2051–2061 (2006).

    Article  CAS  Google Scholar 

  32. Rich, T.C. et al. Cyclic nucleotide–gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. J. Gen. Physiol. 116, 147–161 (2000).

    Article  CAS  Google Scholar 

  33. Heine, M. et al. 5-HT-receptor–induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel. Pflugers Arch. 443, 418–426 (2002).

    Article  CAS  Google Scholar 

  34. Nikolaev, V.O. et al. Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β1-adrenergic but locally confined β2-adrenergic receptor-mediated signaling. Circ. Res. 99, 1084–1091 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  36. Bauman, A.L. et al. Dynamic regulation of cAMP synthesis through anchored PKA-adenylyl cyclase V/VI complexes. Mol. Cell 23, 925–931 (2006).

    Article  CAS  Google Scholar 

  37. Rudolf, R. et al. Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle. J. Cell Biol. 173, 187–193 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Landa, L.R., Jr. et al. Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 β-cell line. J. Biol. Chem. 280, 31294–31302 (2005).

    Article  CAS  Google Scholar 

  40. Willoughby, D. & Cooper, D.M.F. Ca2+ stimulation of adenylyl cyclase generates dynamic oscillations in cyclic AMP. J. Cell Sci. 119, 828–836 (2006).

    Article  CAS  Google Scholar 

  41. Liu, M. et al. Calcium-calmodulin modulation of the olfactory cyclic nucleotide–gated cation channel. Science 266, 1348–1354 (1994).

    Article  CAS  Google Scholar 

  42. Trudeau, M.C. & Zagotta, W.N. Calcium/calmodulin modulation of olfactory and rod cyclic nucleotide–gated ion channels. J. Biol. Chem. 278, 18705–18708 (2003).

    Article  CAS  Google Scholar 

  43. Reinscheid, R.K. et al. High-throughput real-time monitoring of Gs-coupled receptor activation in intact cells using cyclic nucleotide–gated channels. Eur. J. Pharmacol. 478, 27–34 (2003).

    Article  CAS  Google Scholar 

  44. Fagan, K.A. et al. Adenovirus-mediated expression of an olfactory cyclic nucleotide–gated channel regulates the endogenous Ca2+-inhibitable adenylyl cyclase in C6-2B glioma cells. J. Biol. Chem. 274, 12445–12453 (1999).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK

    Debbie Willoughby & Dermot M F Cooper

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  1. Debbie Willoughby
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  2. Dermot M F Cooper
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Correspondence to Dermot M F Cooper.

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Competing interests

D.M.F.C. is one of the authors on an issued patent relating to cyclic nucleotide–gated channels.

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Willoughby, D., Cooper, D. Live-cell imaging of cAMP dynamics. Nat Methods 5, 29–36 (2008). https://doi.org/10.1038/nmeth1135

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