Quantitative real-time imaging of glutathione

Abstract

Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe-designated as RealThiol (RT)-that can quantitatively monitor the real-time glutathione dynamics in living cells. Using RT, we observe enhanced antioxidant capability of activated neurons and dynamic glutathione changes during ferroptosis. RT is thus a versatile tool that can be used for both confocal microscopy and flow cytometry based high-throughput quantification of glutathione levels in single cells. We envision that this new glutathione probe will enable opportunities to study glutathione dynamics and transportation and expand our understanding of the physiological and pathological roles of glutathione in living cells.

Figure 1. Characterization of reversible reaction-based glutathione probe RT.

(a) The reversible Michael addition reaction between RT and GSH. The function of each moiety in RT is highlighted (QY: quantum yield). (b) Ultraviolet–visible (unshaded) and fluorescence (shaded) spectra of RT (green) and RT-GSH (blue), the GSH adduct. (c) Linear relationship between F₄₀₅/F₄₈₈ and GSH concentrations. F₄₀₅ and F₄₈₈ are the fluorescence intensities for RT-GSH and RT, respectively. (d) Dynamic ranges of Grx1-roGFP2 and RT probe. Fluorescence ratio changes on treatment with different concentrations of H₂O₂ in HeLa cells is measured, R_60s/R_0s is calculated as the ratio R value 60 s after H₂O₂ treatment divided by the R value before treatment. Each point is the mean value of >8 cells analysed from 2 independent experiments. Error bars represent s.e.m.

Figure 2. Reaction selectivity of RT towards GSH under different conditions.

(a) Fluorescence ratio after RT probe equilibrated in vitro with physiological relevant concentrations of different compounds. In particular, small-molecule thiols are removed in lysate for testing RT reactivity towards proteins in vitro. Refer to method for details. (b) Relative ratio of RT and RT-GSH adduct under different pH and viscosity conditions. Blue symbols are intentionally lowered by 0.2 units for clarity. (c) Responses of RT with GSH and cysteine. Fluorescence spectra of RT reaction with increasing concentrations of GSH and cysteine (0.1–50 mM) at λ_ex=488 nm and λ_ex=405 nm; maximum fluorescence signals and the corresponding ratio changes as a function of thiol concentrations are illustrated. Physiological concentrations of both thiols are shaded. (d) Flow chart of RT selectivity profiling against intracellular proteins in living cells. (e) Fluorescence signals (λ_ex=405 nm, λ_em=478 nm) of GPC traces of the lysate from RT stained HeLa cells. Each data point represents the mean value of three independent experiments. Error bars represent s.e.m. and are too small to show.

Figure 3. Kinetics of RT reaction with GSH.

(a) RT reacts rapidly with GSH in both forward and reverse reactions. All data were recorded at 37 °C. The kinetic traces for reverse reactions were shifted on x axis for clarity. (b) Catalytic effect of GST on the reaction rate between GSH and RT. Data points represent the fluorescence signal from newly generated RT-GSH conjugate. No significant differences can be observed with or without GST. One representative set of data out of three independent experiments are shown.

Figure 4. RT-based imaging and quantification of glutathione in living cells.

(a) Confocal and ratiometric images of HeLa cells stained with RT (refer to Supplementary Fig. 4 for details on generating ratiometric images). Scale bar, 20 μm. (b) Quantitative analysis of GSH level fluctuations in HeLa cells on consecutive treatment with H₂O₂ (500 μM) and GSH ester (100 μM). Each data point represents the mean value of 46 cells analysed from one representative time-lapsed imaging experiment. Error bars represent s.e.m. (c) Representative images of dynamic changes of GSH levels in HeLa cells on consecutive treatments with H₂O₂ and GSH ester. Scale bar, 20 μm. (d) Lysate-based GSH levels measured using LCMS. HeLa cells were treated with H₂O₂ only (orange), H₂O₂ followed by GSH ester (moss), GSH ester only (turquoise) or PBS (pink). The cells were lysed and the amounts of GSH were quantified using LCMS. Each data point represents the mean value of two independent experiments. Error bars represent s.d.

Figure 5. Neuron activity characterization and RT-based neuron imaging.

(a) Representative traces of membrane potentials (top panels) in response to somatic current injections (bottom panels) from a human ESC-derived neuron show action potentials, which were blocked by a voltage-gated sodium channel blocker, TTX (0.5 μM). Action potentials were detected in all 6 recorded neurons. The resting membrane potential, input resistance and membrane capacitance were −64.3±1.5 mV, 1.30±0.14 GΩ and 33.9±8.1 pF (n=6), respectively. TTX was applied to three recorded neurons and blocked action potentials in all three neurons. (b) Representative traces of membrane currents recorded at a holding potential of −70 mV from a human ESC-derived neuron show spontaneous EPSCs in the presence or absence of a GABA_A receptor antagonist SR95531 (10 μM), which were blocked by glutamate receptor antagonists, NBQX (10 μM) and CPP (10 μM). Spontaneous EPSC frequency was increased with SR95531 treatment in all 6 recorded neurons (P=0.0313, n=6, Wilcoxon matched-pairs signed rank test (two-sided)). NBQX and CPP were applied to 2 recorded neurons and blocked spontaneous EPSCs in both neurons. (c) Quantification of the real-time GSH levels in neurons treated with H₂O₂ in d (refer to Supplementary Fig. 2 for calibration curve). Each data point represents the mean value of 11 (grey), 16 (pink), 16 (blue) and 11 (turquoise) cells analysed from one representative time-lapsed experiment. Error bars represent s.e.m. (d) Time-lapsed ratiometric GSH imaging of untreated control (resting condition), bicuculline/4-AP treated (activation condition) and bicuculline/4-AP/APV treated (activation with paired inhibition of NMDAR channels) neurons on treatment with exogenous H₂O₂ (100 μM). The neural cell bodies are pointed out with arrows in the DIC images. Scale bar, 10 μm.

Figure 6. Comparison of GSH quantifications using RT probe and lysate-based measurements.

(a) A BSO concentration-dependent decrease of GSH levels measured using FACS. HeLa cells treated with a series of concentrations (0.9–500 μM) of BSO for 72 h were harvested and analysed using RT. R=F_405 nm/F_488 nm; R_min and R_max are the corresponding R values with 0 and saturating GSH concentrations, respectively. Each data point represents the mean value of 17,000–28,000 cells analysed from three independent experiments. Error bars represent s.d. (b) Correlation of GSH levels in BSO-treated HeLa cells measured using the RT-based flow cytometry method in a and a lysate-based biochemical assay. Each point in the lysate measurements represents the mean value of a total of nine replicates from three independent experiments. Error bars represent s.d.

Figure 7. GSH quantification using RT-based FACS in ferroptotic HT1080 cells and representative images.

(a) Histogram of ferroptotic HT1080 cells measured using RT on a flow cytometer. Cells were treated with erastin (10 μM) for 0 (pink), 6 (blue) and 24 h (turquoise). (b) Statistical analysis of ferroptotic HT1080 cells measured with FACS and LCMS. For FACS, each data point represents the mean value of >13,000 cells analysed from two independent experiments. Standard parametric unpaired t-test was used to analyse the data with P<0.0001 between groups. For LCMS, each data point represents the mean value of two independent experiments. Error bars represent s.d. (c) Representative images of HT1080 cells treated with 10 μM erastin. Scale bar, 10 μm. Despite significant morphology changes, GSH level did not change significantly over 3 min time span.

Figure 8. GSH probe prototypes.

(a) Chemical structures of TQG-CN and TQG-CN-GSH and its confocal images in HeLa cells. (b) Chemical structures of RT-NH₂ and RT-NH₂-GSH and its confocal images in HeLa cells. Scale bar 10 μm. It should be noted that neither TQG-CN nor RT-NH₂ distributes in the nucleus, which is a hallmark of protein binding for fluorescent probes.