Development of green fluorescent protein-based cAMP indicators for covering a wide range of cAMP concentrations

Abstract

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 values (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 a dynamic range of over 3-fold. Green Falcans showed a high specificity for cAMP over its structural analogues. When we expressed Green Falcans in HeLa cells, these indicators were applicable for visualization of cAMP dynamics in the low concentration range compared to the previously developed cAMP indicators, and visualized distinct kinetics of cAMP in various pathways with high spatiotemporal resolution in living cells. 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 a new avenue for understanding hierarchal and cooperative interactions with other molecules in various cAMP signaling pathways by multi-color imaging.