Fluorescence imaging detection of nanodomain redox signaling events at organellar contacts

David M Booth; Péter Várnai; Suresh K Joseph; György Hajnóczky

doi:10.1016/j.xpro.2021.101119

Fluorescence imaging detection of nanodomain redox signaling events at organellar contacts

STAR Protoc. 2022 Jan 20;3(1):101119. doi: 10.1016/j.xpro.2021.101119. eCollection 2022 Mar 18.

Authors

David M Booth¹, Péter Várnai², Suresh K Joseph¹, György Hajnóczky¹

Affiliations

¹ MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Jefferson Alumni Hall 1020 Locust Street, Philadelphia, PA 19107, USA.
² Department of Physiology, Semmelweis University, Faculty of Medicine, 1444 Budapest, Hungary.

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).

Keywords: Cell Biology; Microscopy; Molecular/Chemical Probes.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Hep G2 Cells
Humans
Kinetics
Optical Imaging / methods*
Organelles / metabolism*
Oxidation-Reduction
Signal Transduction*

Abstract

Publication types

MeSH terms

Grants and funding