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

Haruki Odaka, Satoshi Arai, Takafumi Inoue, Tetsuya Kitaguchi

Research output: Contribution to journalArticle

35 Citations (Scopus)

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 Ca 2+ 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.

Original languageEnglish
Article numbere100252
JournalPloS one
Volume9
Issue number6
DOIs
Publication statusPublished - 2014 Jun 24

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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