Abstract
Understanding intracellular redox chemistry requires new tools for the site-specific visualization of intracellular oxidation. We have developed a spatially-resolved intracellular sensor of hydrogen peroxide, HyPer-Tau, for time-resolved imaging in live cells. This sensor consists of a hydrogen peroxide-sensing protein tethered to microtubules. We demonstrate the use of the HyPer-Tau sensor for three applications; dose-dependent response of human cells to exogenous hydrogen peroxide, a model immune response of mouse macrophages to stimulation by bacterial toxin, and a spatially-resolved response to localized delivery of hydrogen peroxide. These results demonstrate that HyPer-Tau can be used as an effective tool for tracking changes in spatially localized intracellular hydrogen peroxide and for future applications in redox signaling.
Highlights
Understanding intracellular redox chemistry requires new tools for the site-specific visualization of intracellular oxidation
Hydrogen peroxide (H2O2) is an essential extracellular and intracellular signaling molecule that reacts with protein cysteine thiols to confer reversible post-translational modifications[1,2,3,4]
HyPer consists of yellow fluorescent protein (YFP) inserted into a bacterial hydrogen peroxide-sensing protein (OxyR)[22] developed by Belousov et al for the detection of H2O2 in cells[18]
Summary
Understanding intracellular redox chemistry requires new tools for the site-specific visualization of intracellular oxidation. We have developed a spatially-resolved intracellular sensor of hydrogen peroxide, HyPer-Tau, for time-resolved imaging in live cells. This sensor consists of a hydrogen peroxide-sensing protein tethered to microtubules. We demonstrate the use of the HyPer-Tau sensor for three applications; dose-dependent response of human cells to exogenous hydrogen peroxide, a model immune response of mouse macrophages to stimulation by bacterial toxin, and a spatiallyresolved response to localized delivery of hydrogen peroxide These results demonstrate that HyPerTau can be used as an effective tool for tracking changes in spatially localized intracellular hydrogen peroxide and for future applications in redox signaling. We develop a new ratiometric fusion protein sensor, HyPer-Tau, for spatially resolving intracellular and extracellular H2O2 gradients by tethering to a microtubule-binding protein, Tau
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