Lifetime imaging of a fluorescent protein sensor reveals surprising stability of ER thiol redox
| Autor: | Edward Avezov, David Ron, Clemens F. Kaminski, Benedict C. S. Cross, Eduardo P. Melo, Mikael U. Winters, Gabriele S. Kaminski Schierle, Heather P. Harding |
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| Přispěvatelé: | Avezov, Edward [0000-0002-2894-0585], Kaminski Schierle, Gabriele [0000-0002-1843-2202], Harding, Heather [0000-0002-7359-7974], Kaminski, Clemens [0000-0002-5194-0962], Ron, David [0000-0002-3014-5636], Apollo - University of Cambridge Repository |
| Rok vydání: | 2013 |
| Předmět: |
Green Fluorescent Proteins
chemistry.chemical_element Biosensing Techniques Biology Calcium Endoplasmic Reticulum Redox Cell Line Tools Mice 03 medical and health sciences 0302 clinical medicine In vivo Animals Humans Sulfhydryl Compounds Protein disulfide-isomerase Research Articles 030304 developmental biology 0303 health sciences Protein Stability Endoplasmic reticulum Cell Biology Endoplasmic Reticulum Stress Fluorescence Cell biology Microscopy Fluorescence chemistry Unfolded Protein Response Unfolded protein response Protein folding Oxidation-Reduction 030217 neurology & neurosurgery |
| Zdroj: | The Journal of Cell Biology; Vol 201 The Journal of Cell Biology |
| ISSN: | 1540-8140 0021-9525 |
| Popis: | Fluorescent lifetime imaging of an ER-tuned redox-responsive probe revealed an unanticipated stability of ER thiol redox to fluctuations in unfolded protein load, in contrast with sensitivity to lumenal calcium. 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. |
| Databáze: | OpenAIRE |
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