A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP

Adenosine 5’ triphosphate (ATP) is a universal intracellular energy source 1 and an evolutionarily ancient 2 extracellular signal 3–5 . Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F 0 F 1 -ATPase from Bacillus PS3 . On the cell surface and within the cytosol, iATPSnFR 1.0 responded to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. iATPSnFRs represent promising new reagents for imaging ATP dynamics.