Skip to main content
SpringerLink
Log in

Simultaneous Assessment of cAMP Signaling Events in Different Cellular Compartments Using FRET-Based Reporters

  • Protocol
  • First Online:
cAMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1294))

Abstract

Several aspects of the cAMP signaling cascade, including the levels of the messenger itself and the activity of its main effector protein kinase A (PKA), can be measured in living cells, thanks to genetically encoded probes based on fluorescence resonance energy transfer (FRET). While these biosensors enable the assessment of cAMP or PKA activity with great spatial and temporal resolution, concomitant events triggered by the same stimuli at the same or other cellular compartments are not easily assessed. In this chapter we present a simple approach that allows the simultaneous measurement of cAMP and its actions in subcellular compartments of neighboring cells. As proof of principle, we compare cAMP signals and PKA activity in the cytosol of neighboring HEK cells. We propose that this flexible and powerful method can significantly improve the direct comparison of cAMP signals and their action in specific cellular domains.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Lefkimmiatis K, Zaccolo M (2014) cAMP signaling in subcellular compartments. Pharmacol Ther 143:295–304. doi:10.1016/j.pharmthera.2014.03.008

    Article  CAS  PubMed  Google Scholar 

  2. Lim CJ, Kain KH, Tkachenko E et al (2008) Integrin-mediated protein kinase A activation at the leading edge of migrating cells. Mol Biol Cell 19:4930–4941

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Burdyga A, Conant A, Haynes L et al (2013) cAMP inhibits migration, ruffling and paxillin accumulation in focal adhesions of pancreatic ductal adenocarcinoma cells: effects of PKA and EPAC. Biochim Biophys Acta 1833:2664–2672

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Zimmerman NP, Roy I, Hauser AD et al (2013) Cyclic AMP regulates the migration and invasion potential of human pancreatic cancer cells. Mol Carcinog 54:203–215. doi: 10.1002/mc.22091

  5. Insel PA, Zhang L, Murray F et al (2012) Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger. Acta Physiol (Oxf) 204:277–287

    Article  CAS  Google Scholar 

  6. Beavo JA, Brunton LL (2002) Cyclic nucleotide research—still expanding after half a century. Nat Rev Mol Cell Biol 3:710–718

    Article  CAS  PubMed  Google Scholar 

  7. Hayes JS, Brunton LL, Mayer SE (1980) Selective activation of particulate cAMP-dependent protein kinase by isoproterenol and prostaglandin E1. J Biol Chem 255:5113–5119

    CAS  PubMed  Google Scholar 

  8. Hayes JS, Brunton LL, Brown JH et al (1979) Hormonally specific expression of cardiac protein kinase activity. Proc Natl Acad Sci U S A 76:1570–1574

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Buxton IL, Brunton LL (1983) Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem 258:10233–10239

    CAS  PubMed  Google Scholar 

  10. Zaccolo M, De Giorgi F, Cho CY et al (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2:25–29

    Article  CAS  PubMed  Google Scholar 

  11. 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 5:1176–1180

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. 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 101:16513–16518

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Nikolaev VO, Bünemann M, Hein L et al (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218

    Article  CAS  PubMed  Google Scholar 

  14. Zhang J, Ma Y, Taylor SS, Tsien RY (2001) Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A 98:14997–15002

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Zhang J, Hupfeld CJ, Taylor SS et al (2005) Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes. Nature 437:569–573

    Article  CAS  PubMed  Google Scholar 

  16. Depry C, Allen MD, Zhang J (2011) Visualization of PKA activity in plasma membrane microdomains. Mol Biosyst 7:52–58

    Article  CAS  PubMed  Google Scholar 

  17. Klarenbeek J, Jalink K (2014) Detecting cAMP with an EPAC-based FRET sensor in single living cells. Methods Mol Biol 1071:49–58

    Article  CAS  PubMed  Google Scholar 

  18. Klarenbeek JB, Goedhart J, Hink MA et al (2011) A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range. PLoS One 6:e19170

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Van der Krogt GNM, Ogink J, Ponsioen B, Jalink K (2008) A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example. PLoS One 3:e1916

    Article  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  21. Lefkimmiatis K, Leronni D, Hofer AM (2013) The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics. J Cell Biol 202:453–462

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Terrin A, Di Benedetto G, Pertegato V et al (2006) PGE(1) stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases. J Cell Biol 175:441–451

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Stangherlin A, Koschinski A, Terrin A et al (2014) Analysis of compartmentalized cAMP: a method to compare signals from differently targeted FRET reporters. Methods Mol Biol 1071:59–71

    Article  PubMed  Google Scholar 

  24. Miranda JG, Weaver AL, Qin Y et al (2012) New alternately colored FRET sensors for simultaneous monitoring of Zn2+ in multiple cellular locations. PLoS One 7:e49371

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Sample V, DiPilato LM, Yang JH et al (2012) Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP. Nat Chem Biol 8:375–382

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Shaner NC, Campbell RE, Steinbach PA et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572

    Article  CAS  PubMed  Google Scholar 

  27. Hofer AM, Curci S, Doble MA et al (2000) Intercellular communication mediated by the extracellular calcium-sensing receptor. Nat Cell Biol 2:392–398

    Article  CAS  PubMed  Google Scholar 

  28. Lefkimmiatis K, Srikanthan M, Maiellaro I et al (2009) Store-operated cyclic AMP signalling mediated by STIM1. Nat Cell Biol 11:433–442

    Article  CAS  PubMed  Google Scholar 

  29. Lochner A, Moolman JA (2006) The many faces of H89. Cardiovasc Drug Rev 24(3–4):261–274

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Physiology, Anatomy and Genetics, Burdon Sanderson Cardiac Science Centre, BHF Centre of Research Excellence, University of Oxford, Sherrington Building, Parks Road, Oxford, OX1 3PT, UK

    Alex Burdyga & Konstantinos Lefkimmiatis

Authors
  1. Alex Burdyga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Konstantinos Lefkimmiatis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Konstantinos Lefkimmiatis .

Editor information

Editors and Affiliations

  1. Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, United Kingdom

    Manuela Zaccolo

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Burdyga, A., Lefkimmiatis, K. (2015). Simultaneous Assessment of cAMP Signaling Events in Different Cellular Compartments Using FRET-Based Reporters. In: Zaccolo, M. (eds) cAMP Signaling. Methods in Molecular Biology, vol 1294. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2537-7_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2537-7_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2536-0

  • Online ISBN: 978-1-4939-2537-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation