Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function

Abstract

Hydrogen peroxide (H₂O₂) is a key redox intermediate generated within cells. Existing probes for H₂O₂ have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H₂O₂ probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H₂O₂ diffusion from the mitochondrial matrix, to find a functional output of H₂O₂ gradients in polarized cells, and to prove the existence of H₂O₂ gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H₂O₂ imaging in various biological contexts.

Keywords: D-amino acid oxidase; H(2)O(2); H(2)O(2) gradients; HyPer7; cell migration; chemogenetics; genetically encoded probes; hydrogen peroxide; mitochondria; redox signaling.

Figure 1.

Design of the ultrasensitive H₂O₂ indicator. (A) Neighbor joining-based phylogenetic tree of the OxyR-RD domain sequence based on the multiple sequence alignment derived from Supplementary Figure 1. The tested 11 OxyR regulatory domain sequences are highlighted in bold, and the regulatory domain sequence of HyPer7 (from Neisseria meningitidis) is highlighted in red. (B) cpYFP from HyPer probe was integrated into 11 selected OxyR-RD protein domains. The most sensitive version was further improved by mutagenesis.

Figure 2.

In vitro characterization of HyPer7. (A) Fluorescence excitation and emission spectra of HyPer7 in oxidized and reduced state. (B) pH-dependency of HyPer7 and HyPer3 in the physiological pH range. F500 and F400 are fluorescence intensities excited at 500 nm and 400 nm, respectively. (C) HyPer7 responds much faster than HyPerRed to the addition of exogenous H₂O₂ added to Ea926.hy human endothelial cells. Curves represent simultaneous responses to H₂O₂ treatment (50 μM) of Ea926.hy cells co-expressing HyPer7 (black curve) and HyPerRed (red curve). The speed of HyPer7 response is principally limited by the imaging system used. (D) The response of HyPer7 of the indicated concentrations to increasing concentrations of H₂O₂. The y-axis represents the HyPer7 ratio, normalized to the ratio prior to the addition of oxidant. Each point represents the mean of three repeats, the error bars denote the standard deviation. The inset depicts the lower nM range for HyPer7 at a concentration of 1 μΜ. (E) Selectivity of HyPer7. Reduced HyPer7 was incubated with the respective substances. XOX = xanthine oxidase; X = xanthine (50 μΜ); cat = catalase (100 nM); tBOOH = tert-butyl hydroperoxide; CumOOH = cumene hydroperoxide; benzOOH = benzoyl hydroperoxide; 4H2N = 4-hydroperoxy 2-nonenal. Catalase was added to ONOO⁻ sample to remove any H₂O₂. The y-axis represents the HyPer7 ratio, normalized to the ratio obtained with the addition of an equal volume of vehicle (100 mM phosphate buffer, pH 7.4 + 0.1 mM DTPA or acetone for the organic and lipid hydroperoxides). Each bar represents the mean of three replicates and the error bars denote the standard deviation. The dotted vertical lines separate two different concentrations of the organic hydroperoxides and H₂O₂. The horizontal black line separates the conditions where HyPer7 reacts to the oxidant (gray bars) from those where it does not, i.e. normalized ratio >1 (white bars).

Figure 3.

Mitochondrial H₂O₂ production: diffusion and topology. (A) HyPer7 F500/F400 ratio in cytosol (NES, nuclear exclusion signal) and mitochondrial matrix (mito) of transiently transfected K562 cells exposed to different concentrations of H₂O₂. Data are the results of flow cytometry analysis. Each bar represents the mean of N replicates and the error bars denote the standard deviation (at [H₂O₂] ≤ 0.2 μM N=5, at [H₂O₂] > 0.2 μM N=3; *,** P < 0.05 versus control using unpaired t-test) (B) Images of HyPer7 and HyPerRed in different compartments of HeLa-Kyoto cells expressing mito-DAO before and after the addition of D-Ala. HyPer7 is targeted to the mitochondrial matrix (mito, scale bar 20 μm), mitochondrial intermembrane space (IMS, scale bar 30 μm), cytosol (NES, nuclear export signal, scale bar 15 μm), nucleus (NLS, nuclear localization signal, scale bar 10 μm). Cells on the lowest panel were preincubated with auranofin (Aur). (C, D) H₂O₂ dynamics in various compartments of mito-DAO expressing cells in the absence (C) or in the presence (D) of auranofin detected with HyPer7 and HyPerRed. Traces represent mean and standard deviation from at least 10 cells in each of 3 biological replicates. (E, F) Flow cytometry histograms displaying mito-HyPer (E) and mito-HyPer7 (F) ratio signals in K562 cells exposed to rotenone. (G) HyPer7 ratio in cytosol (NES, nuclear exclusion signal) and mitochondrial matrix (mito) of transiently transfected K562 cells exposed to different concentrations of rotenone. Data are the results of flow cytometry analysis. Each bar represents the mean of four replicates and the error bars denote the standard deviation (* P < 0.05 versus control using unpaired t-test).

Figure 4.

H₂O₂ gradients in cell polarization. NIH-3T3 cells transiently expressing HyPer7-LifeAct were imaged during spontaneous migration. (A) Images of a migrating cell at different time points. Upper and middle rows: fluorescent images excited by 405 nm and 488 nm. Lower row: ratiometric images. Addition of PEG-catalase (PEG-Cat) destroys the gradient and leads to retraction of the protrusions. Lines 1 and 2 were used to build kymographs. Scale bar shows 10 μm. (B) Kymographs of the HyPer7-LifeAct ratio along lines 1 and 2 representing the leading edge and the side of the cell from images shown in panel A, respectively. Notice that a more extensive gradient makes the protrusion more stable. (C, D) Correlation plots between the protrusion lifetime and either HyPer-LifeAct ratio gradient (C) or the mean HyPer-LifeAct ratio in the cell demonstrates positive correlation between protrusion stability and the intensity of the H₂O₂ gradient from the protrusion to the cell body (correlation coefficient 0.37), but not the mean H₂O₂ levels (correlation coefficient −0.06).

Figure 5.

Monitoring H₂O₂ production with HyPer7 in zebrafish larvae after wounding. Zebrafish embryos were injected with HyPer7 or HyPer7-C121S mRNAs at the one-cell stage and tail fin amputation assay was performed on 48 hpf larvae. Images were taken before amputation and time lapse imaging was performed up to 60 min post-amputation (mpa). HyPer7 and HyPer7-C121S ratios were quantified at the amputation plane and normalized to the mean fluorescence of the uncut tail for each larvae. (A) Representative images of HyPer7 and HyPer7-C121S expressing larvae before and after amputation. Scale bar is 100µm. (B) Time course of HyPer7 (black lines) or HyPer7-C121S (gray lines) in the wounding site of the larvae tail fin (values ± SEM, N=2) experiments, n≥4 embryos/time point; **, P<0,01; ***, P<0,001). Uncut tails - dashed line.