title: 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.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Beijing K
authors.initials: BK
authors.lastname: Huang
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Sohail
authors.initials: S
authors.lastname: Ali
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Kassi T
authors.initials: KT
authors.lastname: Stein
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts. Electronic address: sikes@mit.edu.
authors.forename: Hadley D
authors.initials: HD
authors.lastname: Sikes
mesh_terms: D000704:Analysis of Variance; D015374:Biosensing Techniques / Q000379:methods* / Q000592:standards; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C499299:oxyR protein, E coli; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide