Interpreting Heterogeneity in Response of Cells Expressing a Fluorescent Hydrogen Peroxide Biosensor

Abstract

Fluorescent, genetically encoded sensors of hydrogen peroxide have enabled visualization of perturbations to the intracellular level of this signaling molecule with subcellular and temporal resolution. Ratiometric sensors hold the additional promise of meaningful quantification of intracellular hydrogen peroxide levels as a function of time, a longstanding goal in the field of redox signaling. To date, studies that have connected the magnitudes of observed ratios with peroxide concentrations have either examined suspensions of cells or small numbers of adherent cells (∼10). In this work, we examined the response of all cells in several microscopic fields of view to an identical perturbation and observed a striking degree of heterogeneity of fluorescence ratios from individual cells. The expression level of the probe and phase within the cell cycle were each examined as potential contributors to the observed heterogeneity. Higher ratiometric responses correlated with greater expression levels of the probe and phase in the cell cycle were also shown to influence the magnitude of response. To aid in the interpretation of experimental observations, we incorporated the reaction of the reduced probe with peroxide and the reactions of the oxidized probe with glutathione and glutaredoxin into a larger kinetic model of peroxide metabolism. The predictions of the kinetic model suggest possible explanations for the experimental observations. This work highlights the importance of a systems-level approach to understanding the output of genetically encoded sensors that function via redox reactions involving thiol and disulfide groups.

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer

“…In fact, the character of this dependence indicates that in H 2 O 2 -exposed cells, HyPer reduction cannot be considered as a reaction of pseudo-first order, and its rate is dependent on the level of reduced HyPer-interacting enzymes, which may be depleted after H 2 O 2 exposure. For instance, recent theoretical considerations have shown that depletion of the reduced form of glutaredoxin can affect HyPer oxidation after a bolus exposure of HeLa cells to 20 μM of H 2 O 2 [21]. In addition, we suggest that the level of reduced thioredoxins can also be lowered in the H 2 O 2 -exposed cells due to the massive reduction of oxidized peroxiredoxins [22].…”

Modulating and Measuring Intracellular H2O2 Using Genetically Encoded Tools to Study Its Toxicity to Human Cells

“…We observed that HyPer’s ratio increased to its peak value for that H 2 O 2 concentration within the first 2 min post bolus addition, followed by a gradual decrease back to the baseline (Supplementary Figure S1). Because the kinetics of the fluorescence signal are determined by the slow conformational change of oxidized HyPer and its reduction by a limited pool of glutaredoxin, a delay exists between the cytosolic H 2 O 2 changes and the resulting signal output of HyPer . Therefore, we assumed that the peak of each HyPer kinetic curve corresponds to the maximum cytosolic H 2 O 2 concentration during bolus addition, with a time lag due to the conformational change of the sensor.…”

AQP8 is a crucial H2O2 transporter in insulin-producing RINm5F cells

“…This could be explained by the high H 2 O 2 generation in the ER during insulin biosynthesis that oxidizes HyPer by leaving the ER lumen. Generally, the maximal Hyper ratio after H 2 O 2 perifusion depends on the basal oxidation status of the HyPer protein that correlates with the oxidation level of the observed compartment [ 58 ]. AQP8 overexpression shows a faster HyPer response compared to mock cells at the cytosolic face of the ER membrane indicating a faster H 2 O 2 influx through the plasma membrane.…”