iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP

Abstract

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F₀-F₁ ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

Keywords: ATP; fluorescent sensor; neuronal metabolism.

Fig. 1.

Design and characterization of iATPSnFR2 in vitro and in fibroblasts. (A) Artistic rendering of iATPSnFR2.HaloTag made with PyMol. The N-terminal fragment (residues 1 to 109) of the epsilon subunit of ATPase (orange, based on 2E5Y.PDB) is fused to cpSFGFP (green). The C terminus of cpSFGFP is fused to the residual ATP-binding helix (residues 110 to 129) of the epsilon subunit (pink), which itself is fused to HaloTag (cyan). “Linkers” are shown as sticks in yellow. ATP is shown as sticks in black. HaloTag fluorophore is shown as sticks in red. (B) iATPSnFR2.HaloTag-JFX650 affinity variants titrated with ATP. Gray, S29W.A95K (4 µM); orange, A95K (16 µM), yellow, A95A.A119L (530 µM). (C–E) Average ratio of green (iATPSnFR2) to red (HaloTag-JFX650) fluorescence in fibroblasts transfected with three affinity variants of iATPSnFR2-HaloTag. Gray, S29W.A95K (high affinity); orange, A95K (medium affinity); yellow, A95A.A119L (low affinity). Cells were incubated for 21 min in buffer containing 10 mM glucose and then switched to buffer containing 10 mM 2-deoxyglucose. (C) Rho° cells (n = 23, 32, 18 cells, respectively). (D) Parental LMTK cell line (n = 37, 42, 49 cells). (E) Parental cell line treated with both 2-deoxyglucose and oligomycin (n = 2, 30, 51 cells).

Fig. 2.

Response of iATPSnFR2 in the matrix of axonal mitochondria to glycolytic and mitochondrial ATP synthase inhibitors. iATPSnFR2.A95A.A119L.HaloTag targeted to mitochondria with 4x-COX8 signal sequence was imaged for HaloTag-JF635 in 5 mM glucose. iATPSnFR2.A95A.A119L was imaged with perfusion of buffers containing 5 mM glucose, 5 mM glucose + 10 µM KA, 1.25 mM lactate + 1.25 mM pyruvate, and finally 1.25 mM lactate + 1.25 mM pyruvate + 10 µM oligomycin. (Scale bar: 10 µm.) Data are represented as the mean of 4 cells ± SE. Images at the top align with treatments and trace below. JF635 channel (red image) remained stable throughout the experiment.

Fig. 3.

Depletion of cytosolic ATP in nerve terminals during AP firing. (Bottom Left) Ratio of green to red fluorescence of axonal terminals in cultured neurons expressing iATPSnFR2.HaloTag-JFX650 during a burst of AP firing [6 s at 50 Hz (gray bar)] in either 5 mM glucose (dashed lines) or 5 mM glucose + 10 µM KA (solid lines). Low affinity variant A95A.A119L in yellow (n = 12) and medium affinity variant A95K in orange (n = 7). Identical experiments carried out using cpSFGFP control (green) showed no change for the same stimulus for either condition (n = 6). (Top) Images at the completion of the experiment in both green and red channels; (scale bar: 10 µm.) (Bottom Right) Maximum change in ratio during the stimulation period in the absence of KA (open circles) or presence of 10 µM KA (filled circles). ***P < 0.001, *P < 0.05, ^n.s.P > 0.05 paired t test.

Fig. 4.

Spontaneous depletion of ATP in boutons detected by iATPSnFR2. iATPSnFR2 was targeted to boutons by fusing it to the C-terminal of synaptophysin. (A) Average ratio of green to red fluorescence of iATPSnFR2-HaloTag-JFX650 normalized to pretreatment baseline. Yellow, low affinity variant A95A.A119L (n = 7); orange, medium affinity variant A95K (n = 6), ± SE. Treatment with 5 mM glucose + 10 µM KA + 0.3 µM TTX initiated at t = 0. (B) Variation in ATP across individual boutons (n = 577) and across individual neurons pre- and post-ATP depletion by KA of the 7 cells shown in A with the low affinity variant. (C) Representative raw traces from individual boutons expressing A95A.A119L variant. (D) Normalization of representative traces illustrates that individual boutons have unique half-times (t_½) to ATP depletion and unique rates of ATP consumption. (E) Cross-correlation of single-bouton depletion rates vs. t_½ values shows that there is in general an inverse relationship between these parameters, with some cells occupying regions of the parameter space. (F) Boutons from only two cells (cells 4 and 7 in B) shown for clarity.