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

Abstract

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 F0F1-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.Given widespread roles in energy homeostasis and cell signaling 3-5 , several methods have been used to detect ATP using small-molecule chemical approaches 6 , firefly luciferase 7,8 , ion channel-expressing "sniffer" cells 9-11 , voltammetry 12 , and different types of microelectrodes 13,14 . However, these methods lack spatial resolution, cannot be easily used in tissue slices or in vivo, respond to off-target ligands, need unwieldy photon-counting cameras, are imprecise and/or unavoidably damage tissue during probe placement. Importantly, none of these methods can be genetically targeted to specific cells, sub-cellular compartments or whole organisms, and they all lack cellular-scale spatial resolution. Although luciferase is genetically encoded 15 , it requires the exogenous substrate luciferin, addition of which complicates use in tissue slices and in vivo. It also saturates at nanomolar ATP, far lower than concentrations expected during ATP signaling (~1 µM to ~1 mM). Additionally, luciferase's bioluminescent output yields low photon fluxes and rules out cellular-resolution imaging. To address these issues, genetically-encoded fluorescent ATP sensors have been developed, including Perceval and PercevalHR 16,17 , ATeam 18 , and QUEEN 19 . These sensors are valuable, but they have limitations. They either respond to ADP/ATP ratio instead of ATP concentration or are susceptible to optical overlap with cellular sources of auto-fluorescence, and all are incompatible with single-wavelength fluorescence imaging, the workhorse of functional fluorescence microscopy 20 .Our goals were to: 1) develop an ATP sensor that could be used in routine fluorescence imaging, e.g. at GFP's 488 nm excitation and ~525 nm emission and 2) deploy such a sensor on the cell surface and within cells to image ATP. QUEEN is an excitation ratio sensor, ATeam is a fluorescence resonance energy transfer (FRET) sensor, and the Perceval sensors are fluorescence lifetime indicators -all of these methods require customized equipment, substantially slow down imaging rates, complicate use with other reagents, and typically have lower signal-to-noise ratio (SNR)...

High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions

“…For example, dyes are available to analyze lysosomal labeling (e.g., LysoTracker), mitochondrial calcium signaling (e.g., Fura-2, Fluo-3), and/or other oxidative stress parameters (e.g., CellROX) 67 . Similarly, genetically encoded sensors exist to monitor changes in pH, ATP, redox cofactors (e.g., NADH, NADPH), and oxidative stress [68][69][70][71][72] , and these could be applied to this workflow. Indeed, the Cytation5 imaging system permits the use of various filter cube sets allowing visualization of many different fluorescent channels.…”

G‐protein‐coupled receptor‐based sensors for imaging neurochemicals with high sensitivity and specificity

“…These sensors were originally designed by combining a ligand‐binding protein as the ‘sensing scaffold’ with a circular‐permutated fluorescent protein (cpFP) (Baird et al ) as the ‘reporting module’. Previously, prokaryotic periplasmic‐binding proteins (PBPs) were used as the scaffold for detecting several neurochemicals, including glutamate (iGluSnFR), γ‐aminobutyric acid (iGABASnFR), and adenosine triphosphate (iATPSnFR), suggesting that this strategy may be applicable for sensor engineering (Marvin et al ; Marvin et al ; Lobas et al ). However, this design strategy is not feasible in cases which a suitable PBP is not available for the molecule of interest.…”

Accurate quantification of astrocyte and neurotransmitter fluorescence dynamics for single-cell and population-level physiology

“…Recording astrocytic dynamics to decoding their disparate roles in neural circuits has centered on expression of genetically encoded probes to carry out intracellular calcium (Ca 2+ ) imaging using GCaMP variants 3 . In addition, many groups study astrocytic function by performing extracellular glutamate imaging using GluSnFR 2 , and several more recently developed genetically encoded fluorescent probes for neurotransmitters such as GABA 11 , norepinephrine (NE) 12 , ATP 13 , and dopamine 14 are poised to further expand our understanding of astrocytic circuit biology.…”