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

Mark A Lobas,Mira T Kronschläger,Loren L Looger,Jun Nagai,Philip M Borden,Baljit S Khakh,Jonathan S Marvin,Rongkun Tao

doi:10.1038/s41467-019-08441-5

Abstract

Adenosine 5′ triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. 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 F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds 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. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.

Highlights

Adenosine 5′ triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species
After unsuccessful attempts to modify ATP-gated P2X receptors[33] and the bacterial regulatory protein GlnK1 to suit our needs, we turned to microbial F0F1-ATP synthase epsilon subunits[34], which have been adapted to create the fluorescence resonance energy transfer (FRET) sensor ATeam[22] and the excitation ratio sensor QUEEN22,23, upon which our sensors are based
We used the bright, stable circularly permuted superfolder GFP36 (cpSFGFP) scaffold, rational design, and library screening to direct the selection of ATP sensors with desired characteristics

Summary

Introduction

Adenosine 5′ triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Luciferase’s bioluminescent output yields low photon fluxes and limits cellular-scale resolution imaging Despite these potential limitations, several pioneering studies have successfully used genetically targeted luciferase-based probes to image ATP dynamics and this remains a useful approach in the purinergic signaling and intracellular ATP homeostasis fields[16,17,18,19]. FRET, which would require excitation at 442 nm for imaging YFP emission: 442 nm lasers are not standard on confocal microscopes and CFP in the existing sensors has one of the lowest quantum yields of the available GFP-based proteins This would translate to inefficient energy transfer from the donor’s excited state to the acceptor by dipole–dipole coupling. We report a GFP-based genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP

Objectives

Methods

Results

Conclusion