Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor

<div><p>Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens.</p></div

ecATeam3.10 detects ATP hydrolysis by ectonucleotidases.

<p>Live Neuro2A cells expressing ecAT3.10 were imaged under non-perfusion, static bath conditions. (A) The addition of 10 μM ATP did not elicit a response (black) unless the cells were pre-treated with the ectonucleotidase inhibitor ARL67156 at 100 μM (red). Apyrase addition degrades extracellular ATP confirming a reversible ATP-specific sensor response. (B) Ectonucleotidase inhibition by ARL67156 pre-treatment also potentiated responses when 30 μM ATP was added. Vehicle, black. ARL67156, red. (A) and (B), n = 6, 2 independent investigators. (C) ARL67156 does not directly affect the ATeam3.10 sensor response to ATP.</p

ecAT3.10 detects nucleotide-stimulated release of ATP from cells.

<p>(A) A biphasic response was apparent on average when 100 μM ATP was added at time = 30 minutes to the static bath of ecAT3.10-expressing Neuro2A cells. The decrease in signal at time = 45 minutes is due to ARL67156-sensitive ectonucleotidase activity, and the subsequent increase in signal after time = 55 minutes reports a secondary release of endogenous ATP from cells. (B) Pretreatment with 100 μM ARL67156 abrogates the transient decrease in ectonucleotidase activity. (C) Addition of 100 μM ADP also stimulate a release of ATP from cells, which was potentiated by pretreatment with ARL67156 (D). Apyrase addition degrades extracellular ATP confirming a reversible ATP-specific sensor response. Solid lines show average responses from ecAT3.10, and dashed traces show average responses from the negative control ecATYEMK sensor (n = 3).</p

ADP-stimulated release of ATP release from Neuro2A cells may be mediated in part by purinergic pathways.

<p>(A) Average time course of ecAT3.10 ratio signal (baseline normalized, n = 6 experiments) from Neuro2A cells that were imaged under static bath conditions. 30 μM ADP was added to stimulate release of ATP. Pretreatment with 100 μM ARL67156 (red) potentiated the response in comparison to vehicle treatment (black). Treatment with 3.3 μM suramin (green) attenuated extracellular ATP levels compared to vehicle (black), and treatment with suramin in the presence of ARL67156 (blue) attenuated the release of ATP compared to ARL67156 alone (red). Apyrase addition degrades extracellular ATP confirming a reversible ATP-specific sensor response. (B) Summary of cell averaged peak responses for individual experiments. Peak responses: vehicle, 1.06 ± 0.01; ARL67156, 1.13 ± 0.01; suramin, 1.027 ± 0.002; ARL67156 plus suramin, 1.048 ± 0.006, mean ± sem. t-test, *p = 0.004, **p = 0.02, ***p = 0.0004. n = 6, 2 independent investigators.</p

ecATeam3.10 detects ADP-stimulated release of endogenous ATP from live Neuro2A cells.

<p>(A) ecAT3.10-expressing Neuro2A cells were imaged under continuous perfusion conditions to demonstrate that ecAT3.10 has greater sensitivity to ATP compared to ADP. (B) Under static bath conditions, paired measurements by ecAT3.10 real-time imaging and endpoint luciferase assays demonstrate that ADP stimulates ATP release. In (A) and (B) grey traces are individual cells, and the bold trace is the cell mean. Arrows in (B) and (C) indicate the addition of ADP and apyrase, sequentially. In (C), time points represent samples from the bath solution taken during the ecAT3.10 imaging experiment shown in (B).</p

Surface-localized ecAT3.10 responds to the addition of extracellular ATP.

<p>High magnification confocal microscopy demonstrates that the ecAT3.10 ratio signal response to extracellular ATP occurs exclusively at the membrane. (A) Representative confocal fluorescence intensity images in the CFP and FRET channels show strong membrane localized signal and some intracellular puncta. Scale bar is 20 μm. (B) Pixel-by-pixel FRET/CFP ratio images before (left) and after addition of 100 μM ATP (right) illustrates a response at the membrane only. (C-E) Line profile analysis for the two example cells shown in (A). A 5-pixel width line region of interest was drawn through the membrane and peak areas of intracellular fluorescence. (C) The fluorescence intensity line profiles exhibit peaks at the membrane and also from intracellular locations that are likely ER/Golgi in origin. The line profiles from the ratio images shown in (B) before (D) and after (E) the addition of extracellular ATP clearly show that the ecAT3.10 ratio signal increases at the membrane and not from the intracellular sites. (F) After 3 minutes, 100 μM ATP was added. The time course shows that the membrane-localized ratio signals increase, but the signals from intracellular regions do not change (mean ± sem for three experiments with n = 32 cells, n = 60 cells, and n = 46 cells).</p