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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.<\/jats:p>","DOI":"10.1083\/jcb.201211155","type":"journal-article","created":{"date-parts":[[2013,4,15]],"date-time":"2013-04-15T15:57:13Z","timestamp":1366041433000},"page":"337-349","update-policy":"https:\/\/doi.org\/10.1083\/jcb.crossmarkpolicy","source":"Crossref","is-referenced-by-count":100,"title":["Lifetime imaging of a fluorescent protein sensor reveals surprising stability of ER thiol redox"],"prefix":"10.1083","volume":"201","author":[{"given":"Edward","family":"Avezov","sequence":"first","affiliation":[{"name":"University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge CB2 0QQ, England, UK 1"}]},{"given":"Benedict C.S.","family":"Cross","sequence":"additional","affiliation":[{"name":"University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge CB2 0QQ, England, UK 1"}]},{"given":"Gabriele S.","family":"Kaminski Schierle","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, England, UK 2"}]},{"given":"Mikael","family":"Winters","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, England, UK 2"}]},{"given":"Heather P.","family":"Harding","sequence":"additional","affiliation":[{"name":"University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge CB2 0QQ, England, UK 1"}]},{"given":"Eduardo Pinho","family":"Melo","sequence":"additional","affiliation":[{"name":"University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge CB2 0QQ, England, UK 1"},{"name":"Centre for Molecular and Structural Biomedicine, Universidade do Algrave, 8005-139 Faro, Portugal 3"}]},{"given":"Clemens F.","family":"Kaminski","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, England, UK 2"}]},{"given":"David","family":"Ron","sequence":"additional","affiliation":[{"name":"University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge CB2 0QQ, England, UK 1"}]}],"member":"291","published-online":{"date-parts":[[2013,4,15]]},"reference":[{"key":"2023072307173389600_bib1","doi-asserted-by":"publisher","first-page":"223","DOI":"10.1126\/science.181.4096.223","article-title":"Principles that govern the folding of protein chains","volume":"181","author":"Anfinsen","year":"1973","journal-title":"Science."},{"key":"2023072307173389600_bib2","doi-asserted-by":"publisher","first-page":"2977","DOI":"10.1038\/emboj.2008.202","article-title":"A novel disulphide switch mechanism in Ero1alpha balances ER oxidation in human cells","volume":"27","author":"Appenzeller-Herzog","year":"2008","journal-title":"EMBO J."},{"key":"2023072307173389600_bib3","doi-asserted-by":"publisher","first-page":"3318","DOI":"10.1038\/emboj.2010.203","article-title":"Disulphide production by Ero1\u03b1-PDI relay is rapid and effectively regulated","volume":"29","author":"Appenzeller-Herzog","year":"2010","journal-title":"EMBO J."},{"key":"2023072307173389600_bib4","doi-asserted-by":"publisher","first-page":"7193","DOI":"10.1021\/jm300713s","article-title":"Discovery of 7-methyl-5-(1-[3-(trifluoromethyl)phenyl]acetyl-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)","volume":"55","author":"Axten","year":"2012","journal-title":"J. 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