Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging

PLoS One. 2014 Jun 24;9(6):e100252. doi: 10.1371/journal.pone.0100252. eCollection 2014.

Abstract

Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Calcium / metabolism
  • Cell Line
  • Chlorocebus aethiops
  • Cyclic AMP / metabolism*
  • Fluorescent Dyes
  • Gene Expression
  • Gene Order
  • HeLa Cells
  • Humans
  • Luminescent Proteins / genetics*
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence
  • Molecular Imaging* / methods
  • Plasmids / genetics

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • Cyclic AMP
  • Calcium

Grants and funding

This research was supported by the Waseda University Organization for University Research Initiatives, a Grant-in aid for Scientific Research to T.K. (grant number 25350970) and the Supporting Project to Form the Strategic Research Platforms for a Private University from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.