DOI: https://doi.org/10.1039/D3RA01390A
Development of green fluorescent protein-based cAMP indicators with expanded dynamic range and various working ranges
DOI:
https://doi.org/10.51094/jxiv.108Keywords:
biosensor, fluorescent protein, protein engineering, cyclic AMP, live-cell imaging, molecular evolutionAbstract
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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Submitted: 2022-06-30 07:04:18 UTC
Published: 2022-07-04 04:31:17 UTC
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Copyright (c) 2022
Sohei Hiasa
Takeru Fujimori
Saki Aiki
Hiroshi Ueda
Takashi Tsuboi
Tetsuya Kitaguchi
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