A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

Area of Science:

• Molecular Biology
• Neuroscience
• Biochemistry

Background:

• Cyclic adenosine monophosphate (cAMP) is crucial for cellular functions, including neural plasticity.
• Understanding cAMP's spatiotemporal dynamics in vivo is limited by current indicator sensitivity and brightness.

Purpose of the Study:

• To develop a novel, highly sensitive genetically encoded fluorescent indicator for intracellular cAMP.
• To characterize the indicator's performance and demonstrate its utility in live organisms.

Main Methods:

• Development of a circularly permuted GFP (cpGFP)-based indicator, G-Flamp1.
• Biochemical characterization including affinity (K_d) and response kinetics.
• Structural analysis of the cAMP-bound indicator.
• In vivo imaging in fruit flies and mice.

Main Results:

• G-Flamp1 exhibits a large fluorescence increase (up to 1100% ΔF/F₀) with good brightness and fast kinetics.
• The indicator has an appropriate cAMP affinity (K_d = 2.17 μM).
• Crystal structure revealed a unique linker conformation potentially acting as a fluorescence switch.
• G-Flamp1 successfully monitored endogenous cAMP signals in relevant brain regions of fruit flies and mice.

Conclusions:

• G-Flamp1 is a sensitive and effective fluorescent indicator for intracellular cAMP.
• This tool enables sensitive monitoring of cAMP dynamics in vivo, advancing the study of neural plasticity and related functions.