Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging

Kazuki Harada,Motoki Ito,Ayumu Konno,Xiaowen Wang,Mika Tanaka,Hirokazu Hirai,Devina Wongso,Takashi Tsuboi,Hajime Hirase,Tetsuya Kitaguchi

doi:10.1038/s41598-017-07820-6

Abstract

cAMP is a common second messenger that is involved in various physiological processes. To expand the colour palette of available cAMP indicators, we developed a red cAMP indicator named “Pink Flamindo” (Pink Fluorescent cAMP indicator). The fluorescence intensity of Pink Flamindo increases 4.2-fold in the presence of a saturating dose of cAMP, with excitation and emission peaks at 567 nm and 590 nm, respectively. Live-cell imaging revealed that Pink Flamindo is effective for monitoring the spatio-temporal dynamics of intracellular cAMP generated by photoactivated adenylyl cyclase in response to blue light, and in dual-colour imaging studies using a green Ca2+ indicator (G-GECO). Furthermore, we successfully monitored the elevation of cAMP levels in vivo in cerebral cortical astrocytes by two-photon imaging. We propose that Pink Flamindo will facilitate future in vivo, optogenetic studies of cell signalling and cAMP dynamics.

Highlights

Cyclic adenosine monophosphate is an important second messenger that mediates hormone secretion, cell migration and memory formation[1,2,3]
Since Cyclic adenosine monophosphate (cAMP) functions in concert with various other intracellular signalling molecules, expansion of the colour palette of genetically-encoded fluorescent protein (FP)-based cAMP indicators is a key requirement for studies aimed at delineating the interplay and/or hierarchy between cAMP and these molecules
The green FP-based cAMP indicator, Flamindo[2], was originally constructed by fusing the green FP variant, Citrine, and the cAMP binding domain of the mouse exchange protein that is directly activated by cAMP 1 (Epac[1], NP_ 01171281, 199–358 aa) to two linker peptides[9]

Summary

Introduction

Cyclic adenosine monophosphate (cAMP) is an important second messenger that mediates hormone secretion, cell migration and memory formation[1,2,3]. It is difficult to combine optogenetic strategies with blue light-excited fluorescent indicators, such as Flamindo, as many optogenetic tools (including channel rhodopsins and photoactivated adenylyl cyclases (PACs)) are themselves activated by blue light[10, 11]. To overcome these drawbacks, we developed a red FP-based cAMP indicator named Pink Flamindo (Pink Fluorescent cAMP indicator).

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Methods

Results

Conclusion