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

28 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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Color
image analysis
Imaging techniques
color
Luminance
cells
Energy Transfer
Cell Surface Receptors
Second Messenger Systems
Colforsin
Chromophores
calcium
HeLa Cells
forskolin
Cyclic AMP
Cytosol
Energy transfer
second messengers
cyclic AMP
cell nucleus

ASJC Scopus subject areas

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

Cite this

Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging. / Odaka, Haruki; Arai, Satoshi; Inoue, Takafumi; Kitaguchi, Tetsuya.

In: PLoS One, Vol. 9, No. 6, e100252, 24.06.2014.

Research output: Contribution to journalArticle

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