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Fluorescence imaging detection of nanodomain redox signaling events at organellar contacts.

ABSTRACT: This protocol describes how to visualize, detect, and analyze redox signals (oxidative bursts) at the ER-mitochondrial interface. It uses drug-inducible crosslinking to target the genetically encoded glutathione redox sensor Grx1roGFP2 to organellar contact sites to measure local redox changes associated with transient depolarizations of the mitochondrial membrane potential (flickers). The strategy allows imaging of the oxidized to reduced glutathione ratio (GSSG:GSH) in subcellular regions below the diffraction limit with good temporal resolution and minimum phototoxicity. Moreover, the strategy also applies to diverse parameters including pH, H₂O₂, and Ca²⁺. For complete details on the use and execution of this profile, please refer to Booth et al. (2016) and Booth et al. (2021).

SUBMITTER: Booth DM

PROVIDER: S-EPMC8783204 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Fluorescence imaging detection of nanodomain redox signaling events at organellar contacts.

Booth David M DM Várnai Péter P Joseph Suresh K SK Hajnóczky György G

STAR protocols 20220120 1

This protocol describes how to visualize, detect, and analyze redox signals (oxidative bursts) at the ER-mitochondrial interface. It uses drug-inducible crosslinking to target the genetically encoded glutathione redox sensor Grx1roGFP2 to organellar contact sites to measure local redox changes associated with transient depolarizations of the mitochondrial membrane potential (flickers). The strategy allows imaging of the oxidized to reduced glutathione ratio (GSSG:GSH) in subcellular regions belo ...[more]

PMID: 35098166

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