(598c) Interpreting Heterogeneity in the Response of Cells Expressing a Fluorescent Biosensor to Stimulation with Hydrogen Peroxide

Fluorescent, genetically encoded sensors of hydrogen peroxide have enabled visualization of perturbations to the intracellular level of this signaling molecule with sub-cellular 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). 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 investigated as potential contributors to the observed heterogeneity and found to be important. 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 reversible redox reactions involving thiol and disulfide groups.