Skip to main content
SpringerLink
Log in

Live Imaging of cAMP Signaling in D. discoideum Based on a Bioluminescent Indicator, Nano-lantern (cAMP)

  • Protocol
  • First Online:
cAMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2483))

  • 881 Accesses

Abstract

Bioluminescence imaging of cellular function is a promising strategy. It has advantages over fluorescence imaging such as high sensitivity, no phototoxicity or no autofluorescence, and compatibility to deep-tissue imaging or optogenetics. However, functional imaging of cellular signaling by bioluminescence is not so easy due to the limited availability of bright bioluminescent indicators.

Here we describe a detailed strategy to detect cellular cAMP dynamics by using Nano-lantern (cAMP1.6), one of the brightest bioluminescent indicator for cAMP . Both induced and spontaneous cAMP signaling in social amoeba, with a large and small signal change, respectively, were imaged by this method.

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 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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

References

  1. Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA et al (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2(1):25–29. https://doi.org/10.1038/71345

    Article  CAS  PubMed  Google Scholar 

  2. Kim N, Shin S, Bae SW (2021) cAMP biosensors based on genetically encoded fluorescent/luminescent proteins. Biosensors 11(2):39. https://doi.org/10.3390/bios11020039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Jiang LI, Collins J, Davis R, Lin K-M, DeCamp D, Roach T et al (2007) Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway. J Biol Chem 282(14):10576–10584. https://doi.org/10.1074/jbc.M609695200

    Article  CAS  PubMed  Google Scholar 

  4. Fan F, Binkowski BF, Butler BL, Stecha PF, Lewis MK, Wood KV (2008) Novel genetically encoded biosensors using firefly luciferase. ACS Chem Biol 3(6):346–351. https://doi.org/10.1021/cb8000414

    Article  CAS  PubMed  Google Scholar 

  5. Binkowski BF, Butler BL, Stecha PF, Eggers CT, Otto P, Zimmerman K et al (2011) A luminescent biosensor with increased dynamic range for intracellular cAMP. ACS Chem Biol 6(11):1193–1197. https://doi.org/10.1021/cb200248h

    Article  CAS  PubMed  Google Scholar 

  6. Saito K, Chang YF, Horikawa K, Hatsugai N, Higuchi Y, Hashida M et al (2012) Luminescent proteins for high-speed single-cell and whole-body imaging. Nat Commun 3(1):1262. https://doi.org/10.1038/ncomms2248

    Article  CAS  PubMed  Google Scholar 

  7. Loening AM, Fenn TD, Wu AM, Gambhir SS (2006) Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Eng Des Sel 19(9):391–400. https://doi.org/10.1093/protein/gzl023

    Article  CAS  PubMed  Google Scholar 

  8. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20(1):87–90. https://doi.org/10.1038/nbt0102-87

    Article  CAS  PubMed  Google Scholar 

  9. Takai A, Nakano M, Saito K, Haruno R, Watanabe TM, Ohyanagi T et al (2015) Expanded palette of nano-lanterns for real-time multicolor luminescence imaging. Proc Natl Acad Sci 112(14):4352. https://doi.org/10.1073/pnas.1418468112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fey P, Kowal AS, Gaudet P, Pilcher KE, Chisholm RL (2007) Protocols for growth and development of Dictyostelium discoideum. Nat Protoc 2(6):1307–1316. https://doi.org/10.1038/nprot.2007.178

    Article  CAS  PubMed  Google Scholar 

  11. Gaudet P, Pilcher KE, Fey P, Chisholm RL (2007) Transformation of Dictyostelium discoideum with plasmid DNA. Nat Protoc 2(6):1317–1324. https://doi.org/10.1038/nprot.2007.179

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are grateful to Kenji Osabe for proofreading the manuscript. This work was supported by JST SENTAN KEISOKU program (16812798 to T.N.), Grant-in-Aid for Scientific Research on Innovative Areas “Singularity Biology (No.8007)” (18H05408 to T.N., 18H05415 to K.H.), MEXT, Japan, and the Research Program of “Five-star Alliance” in “NJRC Mater. & Dev.” (T.N., K.H).

Author information

Authors and Affiliations

  1. Department of Optical Imaging, Advanced Research Promotion Center, Tokushima University, Tokushima City, Tokushima, Japan

    Kazuki Horikawa

  2. Department of Biomolecular Science and Engineering, SANKEN (The Institute of Scientific and Industrial Research), Osaka University, Ibaraki, Osaka, Japan

    Takeharu Nagai

Authors
  1. Kazuki Horikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takeharu Nagai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takeharu Nagai .

Editor information

Editors and Affiliations

  1. Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, UK

    Manuela Zaccolo

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Horikawa, K., Nagai, T. (2022). Live Imaging of cAMP Signaling in D. discoideum Based on a Bioluminescent Indicator, Nano-lantern (cAMP). In: Zaccolo, M. (eds) cAMP Signaling. Methods in Molecular Biology, vol 2483. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2245-2_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2245-2_14

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2244-5

  • Online ISBN: 978-1-0716-2245-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation