Interpreting Heterogeneity in Response of Cells Expressing a Fluorescent Hydrogen Peroxide Biosensor
Autor: | Beijing K. Huang, Kassi T. Stein, Sohail Ali, Hadley D. Sikes |
---|---|
Rok vydání: | 2015 |
Předmět: |
chemistry.chemical_classification
Systems Biophysics Analysis of Variance Reactive oxygen species Escherichia coli Proteins Green Fluorescent Proteins Biophysics Biosensing Techniques Hydrogen Peroxide Peroxide Fluorescence Redox Repressor Proteins chemistry.chemical_compound chemistry Biochemistry Glutaredoxin Humans Hydrogen peroxide Oxidation-Reduction Biosensor Intracellular HeLa Cells Protein Binding |
Zdroj: | Biophysical Journal. 109:2148-2158 |
ISSN: | 0006-3495 |
Popis: | 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. |
Databáze: | OpenAIRE |
Externí odkaz: |