In Vivo Detection of Reactive Oxygen Species and Redox Status in Caenorhabditis elegans

Abstract

Significance: Due to its large families of redox-active enzymes, genetic amenability, and complete transparency, the nematode Caenorhabditis elegans has the potential to become an important model for the in vivo study of redox biology.

Recent advances: The recent development of several genetically encoded ratiometric reactive oxygen species (ROS) and redox sensors has revolutionized the quantification and precise localization of ROS and redox signals in living organisms. Only few exploratory studies have applied these sensors in C. elegans and undoubtedly much remains to be discovered in this model. As a follow-up to our recent findings that the C. elegans somatic gonad uses superoxide and hydrogen peroxide (H2O2) signals to communicate with the germline, we here analyze the patterns of H2O2 inside the C. elegans germline.

Critical issues: Despite the advantages of genetically encoded ROS and redox sensors over classic chemical sensors, still several general as well as C. elegans-specific issues need to be addressed. The major concerns for the application of these sensors in C. elegans are (i) decreased vitality of some reporter strains, (ii) interference of autofluorescent compartments with the sensor signal, and (iii) the use of immobilization methods that do not influence the worm's redox physiology.

Future directions: We propose that several of the current issues may be solved by designing reporter strains carrying single copies of codon-optimized sensors. Preferably, these sensors should have their emission wavelengths in the red region, where autofluorescence is absent. Worm analysis could be optimized using four-dimensional ratiometric fluorescence microscopy of worms immobilized in microfluidic chips. Antioxid. Redox Signal. 25, 577-592.

FIG. 1.

Expression of roGFP2-Orp1 in the Caenorhabditis elegans germline. Schematic overview of the cytosolic (A) and the mitochondrial (B) roGFP2-Orp1 gene construct. Ratiometric image of cytosol (C) and mitochondrial (D) roGFP-Orp1 in the gonad of intact living animals. Both images show the gradual increase in H₂O₂ levels from the distal (upper) part toward the proximal (lower) part of the gonad. H₂O₂, hydrogen peroxide; MLS, mitochondrial localization sequence; roGFP, reduction–oxidation-sensitive green fluorescent protein

FIG. 2.

Determining the experimental dynamic range of roGFP2-Orp1 in the C. elegans germline. Ratiometric image of the gonad arm in intact living animals under reducing (A, B) and oxidizing (C, D) conditions of worms expressing roGFP-Orp1 in the cytosol (A, C) and in the mitochondria (B, D). Reducing conditions were obtained by exposing the worms to 5 mM DTT immediately before imaging; oxidizing conditions were obtained with 5 mM H₂O₂ immediately before imaging. DTT, dithiothreitol.

FIG. 3.

To increase mitochondrial H₂O₂ production, worms were exposed to several concentrations of PQ for 24 h before imaging. Following this treatment, ratiometric images of the cytosolic (A) and the mitochondrial (B) roGFP2-Orp1 probe in the gonad of living animals showed that H₂O₂ levels were increased in both the cytosol and inside mitochondria compared to the control condition. PQ, paraquat.

FIG. 4.

To analyze the effect of mitochondrial stress on H₂O₂ levels, worms were exposed to RNAi against the mitochondrial germline-specific protein ASB-1 for 24 h before imaging. Depletion of ASB-1 caused a clear increase in cytosolic H₂O₂ levels (A). Undiluted asb-1 RNAi arrested import of the mitochondrially targeted roGFP2-Orp1 (not shown), while with 1/10 diluted asb-1 RNAi, roGFP2-Orp1 was still targeted to the mitochondria, but no changes in H₂O₂ levels could be observed (B). RNAi, RNA interference.

FIG. 5.

The C. elegans germline shows a H₂O₂ gradient. (A) Schematic overview of the Caenorhabditis elegans germline, showing the regions where relative H₂O₂ levels were quantified. Regions 1–3 correspond to measurements in the distal germline, 4–6 to the proximal germline, 7 to one-cell embryos, and 8 to two-cell embryos. To calculate and plot relative H₂O₂ levels in the cytosol (B) and mitochondria (C), ratiometric images of individual gonads were generated, after which the roGFP-Orp1 ratiometric signal was measured in these eight different regions. In control conditions, both the cytosolic and mitochondrial H₂O₂ levels show a gradual increase from the distal toward the proximal region of the gonad, followed by a decrease in the two-cell embryo. These spatial differences in H₂O₂ levels became more pronounced when animals were exposed to paraquat (upper panels), and, specifically for cytosolic H₂O₂ levels, also when worms were treated with asb-1 RNAi (lower panels).

FIG. 6.

Density effects in microplate-based fluorimetry of large worm populations. (A) Nonlinear correlation of green fluorescence (530 nm) after violet (405 nm) and blue (490 nm) excitation of the sensors roGFP2-Orp1, Grx1-roGFP2, and HyPer. At least three independent replicate populations of each worm strain were diluted. Each symbol represents a single dilution of a given replicate experiment. The symbols consist of vertical and horizontal standard deviations calculated as the fluctuation of 13 consecutive fluorescence measurements over a 20-min time period. (B) Fluorescence ratio plotted against sensor concentration (estimated by fluorescence intensity following violet excitation in varying population densities of unstressed worms expressing the roGFP2-Orp1, Grx1-roGFP2, or HyPer sensor). At low sensor concentrations, the fluorescence ratio tends to show an incline. Fluorescence ratios were calculated as the 405/490 nm signal for roGFP2-Orp1 and Grx1-roGFP2, and as the 490/405 nm signal for HyPer. HyPer, hydrogen peroxide sensor.