Development of green fluorescent protein-based cAMP indicators with expanded dynamic range and various working ranges

Sohei Hiasa; Takeru Fujimori; Saki Aiki; Hiroshi Ueda; Takashi Tsuboi; Kitaguchi, Tetsuya

doi:10.51094/jxiv.108

##article.authors##

Sohei Hiasa School of Life Science and Technology, Department of Life Science and Technology, Tokyo Institute of Technology
Takeru Fujimori School of Life Science and Technology, Department of Life Science and Technology, Tokyo Institute of Technology
Saki Aiki Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo
Hiroshi Ueda Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology
Takashi Tsuboi Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo
Kitaguchi, Tetsuya Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology

DOI:

https://doi.org/10.51094/jxiv.108

キーワード:

biosensor、 fluorescent protein、 protein engineering、 cyclic AMP、 live-cell imaging、 molecular evolution

抄録

There is a wide range in the concentration of intracellular cyclic adenosine 3’, 5’-monophosphate (cAMP), which mediates specific effects as a second messenger in pathways affecting many physiological processes. Here, we developed green fluorescent cAMP indicators, named Green Falcan (Green fluorescent protein-based indicator visualizing cAMP dynamics) with various EC₅₀ (0.3, 1, 3, 10 µM) for covering the wide range of intracellular cAMP concentrations. The fluorescence intensity of Green Falcans increased in a cAMP dose-dependent manner, with the dynamic range of over 3-fold. Green Falcans showed a high specificity for cAMP over its structural analogues. When we expressed Green Falcans to HeLa cells, these indicators visualized dynamics and distinct kinetics of cAMP in various pathways in living cells with high spatiotemporal resolution. Furthermore, we demonstrated that Green Falcans are applicable to dual-color imaging with R-GECO, a red fluorescent Ca²⁺indicator, in the cytoplasm and the nucleus. This study shows that Green Falcans open up new avenue for understanding hierarchal and cooperative interaction with other molecules in various cAMP signaling pathways by multi-color imaging.

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