Imaging Sub-plasma Membrane cAMP Dynamics with Fluorescent Translocation Reporters

  • Anders TengholmEmail author
  • Olof Idevall-Hagren
Part of the Methods in Molecular Biology book series (MIMB, volume 1294)


Imaging cAMP dynamics in single cells and tissues can provide important insights into the regulation of a variety of cellular processes. In recent years, a large number of tools for cAMP measurements have been developed. While most cAMP reporters are designed to undergo changes in fluorescence resonance energy transfer (FRET), there are alternative techniques with advantages for certain applications. Here, we describe protocols for cAMP measurements in the sub-plasma membrane space based on the detection of the cAMP-induced translocation of engineered fluorescent protein-tagged subunits of protein kinase A between the cytoplasm and the plasma membrane. Total internal reflection fluorescence (TIRF) imaging of the changes in reporter localization yields robust signal changes and has contributed to the discovery of cAMP oscillations in the sub-plasma membrane space of insulin-secreting β-cells stimulated with glucose and gluco-incretin hormones. We also demonstrate how the technique can be combined with measurements of the cytosolic Ca2+ concentration or with recordings of the subcellular localization of the cAMP effector protein Epac2. The translocation reporter approach provides a valuable complement to other methods for imaging sub-membrane cAMP dynamics in various types of cells.


cAMP oscillations Plasma membrane Protein kinase A Translocation Total internal reflection fluorescence 



We thank Drs Hongyan Shuai and Geng Tian for their help with the preparation of Figs. 3 and Fig. 4. The authors’ work is supported by grants from the European Foundation for the Study of Diabetes, the family Ernfors Foundation, the Novo Nordisk Foundation, the Swedish Diabetes Association, and the Swedish Research Council (67X-14643, 67P-21262, 325-2012-6778, 524-2013-298).


  1. 1.
    Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4:517–529CrossRefPubMedGoogle Scholar
  2. 2.
    Willoughby D, Cooper DM (2008) Live-cell imaging of cAMP dynamics. Nat Methods 5:29–36CrossRefPubMedGoogle Scholar
  3. 3.
    Berrera M, Dodoni G, Monterisi S et al (2008) A toolkit for real-time detection of cAMP: insights into compartmentalized signaling. Handb Exp Pharmacol 186:285–298CrossRefPubMedGoogle Scholar
  4. 4.
    Adams SR, Harootunian AT, Buechler YJ et al (1991) Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349:694–697CrossRefPubMedGoogle Scholar
  5. 5.
    Zaccolo M, De Giorgi F, Cho CY et al (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2:25–29CrossRefPubMedGoogle Scholar
  6. 6.
    Zaccolo M, Pozzan T (2002) Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295:1711–1715CrossRefPubMedGoogle Scholar
  7. 7.
    Nikolaev VO, Bunemann M, Hein L et al (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218CrossRefPubMedGoogle Scholar
  8. 8.
    Nikolaev VO, Bunemann M, Schmitteckert E et al (2006) Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2-adrenergic receptor-mediated signaling. Circ Res 99:1084–1091CrossRefPubMedGoogle Scholar
  9. 9.
    Ponsioen B, Zhao J, Riedl J et al (2004) Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep 5:1176–1180CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    DiPilato LM, 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 U S A 101:16513–16518CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Piston DW, Kremers GJ (2007) Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem Sci 32:407–414CrossRefPubMedGoogle Scholar
  12. 12.
    Vogel SS, Thaler C, Koushik SV (2006) Fanciful FRET. Sci STKE 2006:re2. doi: 10.1126/stke.3312006re3312002 PubMedGoogle Scholar
  13. 13.
    Rich TC, Fagan KA, Nakata H et al (2000) Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. J Gen Physiol 116:147–161CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Dyachok O, Isakov Y, Sågetorp J et al (2006) Oscillations of cyclic AMP in hormone-stimulated insulin-secreting β-cells. Nature 439:349–352CrossRefPubMedGoogle Scholar
  15. 15.
    Scott JD, Dessauer CW, Tasken K (2013) Creating order from chaos: cellular regulation by kinase anchoring. Annu Rev Pharmacol Toxicol 53:187–210CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Steyer JA, Almers W (2001) A real-time view of life within 100 nm of the plasma membrane. Nat Rev Mol Cell Biol 2:268–275CrossRefPubMedGoogle Scholar
  17. 17.
    Dyachok O, Idevall-Hagren O, Sågetorp J et al (2008) Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion. Cell Metab 8:26–37CrossRefPubMedGoogle Scholar
  18. 18.
    Tian G, Sandler S, Gylfe E et al (2011) Glucose- and hormone-induced cAMP oscillations in α- and β-cells within intact pancreatic islets. Diabetes 60:1535–1543CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Tian G, Sågetorp J, Xu Y et al (2012) Role of phosphodiesterases in the shaping of sub-plasma membrane cAMP oscillations and pulsatile insulin secretion. J Cell Sci 125:5084–5095CrossRefPubMedGoogle Scholar
  20. 20.
    Li J, Shuai HY, Gylfe E et al (2013) Oscillations of sub-membrane ATP in glucose-stimulated beta cells depend on negative feedback from Ca2+. Diabetologia 56:1577–1586CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Hansen C, Howlin J, Tengholm A et al (2009) Wnt-5a-induced phosphorylation of DARPP-32 inhibits breast cancer cell migration in a CREB-dependent manner. J Biol Chem 284:27533–27543CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Malmersjö S, Liste I, Dyachok O et al (2010) Ca2+ and cAMP signaling in human embryonic stem cell-derived dopamine neurons. Stem Cells Dev 19:1355–1364CrossRefPubMedGoogle Scholar
  23. 23.
    Idevall-Hagren O, Barg S, Gylfe E et al (2010) cAMP mediators of pulsatile insulin secretion from glucose-stimulated single β-cells. J Biol Chem 285:23007–23018CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Idevall Hagren O, Jakobsson I, Xu Y et al (2013) Spatial control of Epac2 activity by cAMP and Ca2+-mediated activation of Ras. Sci Signal 6:ra29. doi: 10.1126/scisignal.2003932 CrossRefPubMedGoogle Scholar
  25. 25.
    Miyazaki J, Araki K, Yamato E et al (1990) Establishment of a pancreatic β cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127:126–132CrossRefPubMedGoogle Scholar
  26. 26.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedGoogle Scholar
  27. 27.
    Zhao Y, Araki S, Wu J et al (2011) An expanded palette of genetically encoded Ca2+ indicators. Science 333:1888–1891CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Akerboom J, Carreras Calderon N, Tian L et al (2013) Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neurosci 6:2. doi: 10.3389/fnmol.2013.00002 CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Medical Cell Biology, Biomedical CentreUppsala UniversityUppsalaSweden

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