title: Lifetime imaging of a fluorescent protein sensor reveals surprising stability of ER thiol redox.
abstract: Interfering with disulfide bond formation impedes protein folding and promotes endoplasmic reticulum (ER) stress. Due to limitations in measurement techniques, the relationships of altered thiol redox and ER stress have been difficult to assess. We report that fluorescent lifetime measurements circumvented the crippling dimness of an ER-tuned fluorescent redox-responsive probe (roGFPiE), faithfully tracking the activity of the major ER-localized protein disulfide isomerase, PDI. In vivo lifetime imaging by time-correlated single-photon counting (TCSPC) recorded subtle changes in ER redox poise induced by exposure of mammalian cells to a reducing environment but revealed an unanticipated stability of redox to fluctuations in unfolded protein load. By contrast, TCSPC of roGFPiE uncovered a hitherto unsuspected reductive shift in the mammalian ER upon loss of luminal calcium, whether induced by pharmacological inhibition of calcium reuptake into the ER or by physiological activation of release channels. These findings recommend fluorescent lifetime imaging as a sensitive method to track ER redox homeostasis in mammalian cells.
authors.affiliation: University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge CB2 0QQ, England, UK.
authors.forename: Edward
authors.initials: E
authors.lastname: Avezov
authors.forename: Benedict C S
authors.initials: BC
authors.lastname: Cross
authors.forename: Gabriele S
authors.initials: GS
authors.lastname: Kaminski Schierle
authors.forename: Mikael
authors.initials: M
authors.lastname: Winters
authors.forename: Heather P
authors.initials: HP
authors.lastname: Harding
authors.forename: Eduardo Pinho
authors.initials: EP
authors.lastname: Melo
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
authors.forename: David
authors.initials: D
authors.lastname: Ron
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D059865:Endoplasmic Reticulum Stress; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D010084:Oxidation-Reduction; D055550:Protein Stability; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D056811:Unfolded Protein Response
chemical_list: D013438:Sulfhydryl Compounds; D049452:Green Fluorescent Proteins; D002118:Calcium