Red fluorescent redox-sensitive biosensor Grx1-roCherry

Abstract

Redox-sensitive fluorescent proteins (roFPs) are a powerful tool for imaging intracellular redox changes. The structure of these proteins contains a pair of cysteines capable of forming a disulfide upon oxidation that affects the protein conformation and spectral characteristics. To date, a palette of such biosensors covers the spectral range from blue to red. However, most of the roFPs suffer from either poor brightness or high pH-dependency, or both. Moreover, there is no roRFP with the redox potential close to that of 2GSH/GSSG redox pair. In the present work, we describe Grx1-roCherry, the first red roFP with canonical FP topology and fluorescent excitation/emission spectra of typical RFP. Grx1-roCherry, with a midpoint redox potential of - 311 mV, is characterized by high brightness and increased pH stability (pKa 6.7). We successfully used Grx1-roCherry in combination with other biosensors in a multiparameter imaging mode to demonstrate redox changes in cells under various metabolic perturbations, including hypoxia/reoxygenation. In particular, using simultaneous expression of Grx1-roCherry and its green analog in various compartments of living cells, we demonstrated that local H₂O₂ production leads to compartment-specific and cell-type-specific changes in the 2GSH/GSSG ratio. Finally, we demonstrate the utility of Grx1-roCherry for in vivo redox imaging.

Keywords: 2GSH/GSSG; Biosensor; Grx1-roCherry; Multiparameter imaging; Redox-sensitive fluorescent protein.

Fig. 1

Redox-sensitive biosensor Grx1-roCherry. (A) Diagram of Grx1-roCherry structure. Grx1-roCherry consists of a mutated fluorescent protein mCherry, a human glutaredoxin 1 and a polypeptide linker. The diagram shows the pair of redox-active cysteine residues and other mutations in the biosensor structure. (B) Images of Grx1-roCherry in transiently transfected HeLa Kyoto cells exposed to 150 μM of H₂O₂ after 40 s of imaging. Numbers indicate timing in seconds. Scale bar, 40 µm. Lookup table indicates intensity of red fluorescence. (C) Timing of H₂O₂ induced fluorescence change (F₅₈₉) in HeLa Kyoto cells expressing Grx1-roCherry (red line) or roCherry without Grx1 (black line). Signal values of Grx1-roCherry and roCherry were normalized to the initial value. Signals were averaged from 67 cells for Grx1-roCherry and 101 cells for roCherry in 3 experiments. Error bars indicate standard error of mean. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 2

Characterization of Grx1-roCherry biosensor. (A) Excitation and emission spectra of purified Grx1-roCherry. (B) Emission spectrum of 40 nM Grx1-roCherry in PBS (pH 7.4) in reduced form (with 1 mM GSH, 2 mM NADPH, 0.5–1.5 U/ml glutathione reductase) and oxidized form (with 1 mM GSSG). Excitation at 577 nm. (C) Changes in Grx1-roCherry fluorescence intensity in the presence of different GSSG and H₂O₂ concentrations as a function of time. All lines were normalized to values obtained from an intact protein sample incubated at the same conditions to eliminate component of atmosphere oxygen-driven oxidation. The moment of GSSG or H₂O₂ addition is marked with an arrow. The total volume of the sample is 1 ml with protein concentration of 40 nM (PBS, pH = 7.4). (D) pH dependency of purified reduced Grx1-roCherry. (E) Amplitude of signal change of reduced Grx1-roCherry (F₅₈₉) and Grx1-roGFP2 (F₄₀₅/F₄₈₈) responding to various oxidants. Incubation time was 3 min for samples 1–5 and 10 min for samples 6–7. Error bars indicate standard error of mean from 3 experiments.

Fig. 3

Redox equilibria of Grx1-roCherry (A) and Grx1-roGFP2 (B) by titration against DTT. The total concentration of DTT_red and DTT_ox is 1 mM. Probes were incubated for 3 h at room temperature. All buffers shared a pH value of 7.4. (C) Redox titration of Grx1-roCherry (F₅₈₉) and Grx1-roGFP2 (F₄₀₅/F₄₈₈) coexpressed in cytoplasm of HeLa Kyoto cells. The dynamics of both biosensors were imaged in real time. Sensors reached the maximum level of response range within a few seconds after addition of H₂O₂. Error bars indicate standard error of mean from 3 experiments. (D) The dynamics of Grx1-roCherry (F₅₈₉ signal) and Grx1-roGFP2 (F₄₈₈ signal) in response to exogenous addition of 100 μM 2-AAPA (inhibitor of GR). Grx1-roCherry and Grx1-roGFP2 were coexpressed in HeLa Kyoto cells in cytoplasm and mitochondria, respectively. The functional activity of the biosensors was examined by addition of 150 μM H₂O₂. Signal values of Grx1-roCherry and Grx1-roGFP2 were normalized to the initial values. Signals were averaged from 32 cells in 2 experiments. Error bars indicate standard error of mean. (E) Fluorescence dynamics in HeLa Kyoto cells expressing Grx1-roCherry in the cytoplasm (F₅₈₉ signal, red line) and Grx1-roGFP2 in mitochondria (F₄₈₈ signal, green line) exposed to external H₂O₂. Cells were pretreated with either 50 μM DMF for 24 h or DMSO as the negative control. Signal values of Grx1-roCherry and Grx1-roGFP2 were normalized to the initial value. Signals were averaged from 103 cells in 3 experiments with DMF; from 119 cells in 3 experiments with DMSO. Error bars indicate standard error of mean. (F) Experiment described in (E) with localization of Grx1-roCherry in mitochondria (F₅₈₉ signal, red line) and Grx1-roGFP2 in cytoplasm (F₄₈₈ signal, green line). Signal values of Grx1-roCherry and Grx1-roGFP2 were normalized to the initial value. Signals were averaged from 30 cells in 2 experiments with DMF; from 34 cells in 2 experiments with DMSO. Error bars indicate standard error of mean. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 4

(A) HeLa Kyoto cells coexpressing Grx1-roCherry in cytoplasm (F₅₈₉ signal, red line) and Grx1-roGFP2 in mitochondria (F₄₀₅/F₄₈₈ signal, green line) were exposed to hypoxia, with subsequent reoxygenation. Signal values of Grx1-roCherry and Grx1-roGFP2 were normalized to the initial value. Signals were averaged from 28 cells in 2 experiments. Error bars indicate standard error of mean. (B) HeLa Kyoto cells coexpressing Grx1-roCherry and SoNar in cytoplasm were analyzed by flow cytometry after incubation with 30 mM DCA for 48 h (shown in blue) in comparison with untreated cells (shown in red). For each graph 10,000 cells were analyzed. (C) Experiment described in (B) with HEK293 cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 5

Dynamics of glutathione redox state in cytosol (red line) and mitochondrial matrix (green line) caused by local generation of Н₂O₂ by DAO in neurons and HeLa Kyoto cells. Neurons of mixed mouse primary embryonic cortex cell culture and HeLa Kyoto cells were cotransfected by cytosolic Grx1-roCherry (F₅₈₉ signal) and mitochondrial Grx1-roGFP2 (F₄₈₈ signal). In the same cells, DAO was localized to nuclei (A, B) or mitochondria (С-F). The arrows in all graphs indicate the start of inducible H₂O₂-generation in nuclei or mitochondria caused by the addition of 2 mM D-norvaline to the cell culture medium. To evaluate the effect of TrxR inhibition, HeLa Kyoto cells coexpressing Grx1-roCherry in cytoplasm, Grx1-roGFP2 and DAO in mitochondria were preincubated overnight with 2.5 μM auranofin (E) and DMSO (control probe) (F), followed by 2 mM D-norvaline treatment. In all graphs, error bars indicate standard error of mean. Signal values of Grx1-roCherry and Grx1-roGFP2 were normalized to the initial value. Signals in each series were averaged in 3 experiments from at least 11 neurons and 40 HeLa Kyoto cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Fig. 6

Grx1-roCherry in tissues of zebrafish D. rerio. (A) Image of zebrafish larva at 48 h stage injected with mRNA encoding Grx1-roCherry. White ROI indicates area in which fluorescent signal was detected in (B). Scale bar 100 µM. Lookup table indicates intensity of red fluorescence. (B) Dynamics of Grx1-roCherry signal in the noted area (A) of fish tissue in response to oxidation caused by the exogenous addition of 50 mM H₂O₂.