Real-time monitoring of redox changes in the mammalian endoplasmic reticulum

Van Lith, M., Tiwari, S., Pediani, J. , Milligan, G. and Bulleid, N. J. (2011) Real-time monitoring of redox changes in the mammalian endoplasmic reticulum. Journal of Cell Science, 124(14), pp. 2349-2356. (doi: 10.1242/jcs.085530)

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Redox-sensitive GFPs with engineered disulphide bonds have been used previously to monitor redox status in the cytosol and mitochondria of living cells. The usefulness of these redox probes depends on the reduction potential of the disulphide, with low values suiting the cytosol and mitochondrion, and higher values suiting the more oxidising environment of the endoplasmic reticulum (ER). Here, we targeted a modified redox-sensitive GFP (roGFP1-iL), with a relatively high reduction potential, to the ER of mammalian cells. We showed that the disulphide is partially oxidised, allowing roGFP1-iL to monitor changes in ER redox status. When cells were treated with puromycin, the redox balance became more reducing, suggesting that the release of nascent chains from ribosomes alters the ER redox balance. In addition, downregulating Ero1. prevented normal rapid recovery from dithiothreitol (DTT), whereas downregulating peroxiredoxin IV had no such effect. This result illustrates the contribution of the Ero1. oxidative pathway to ER redox balance. This first report of the use of roGFP to study the ER of mammalian cells demonstrates that roGFP1-iL can be used to monitor real-time changes to the redox status in individual living cells.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Pediani, Dr John and Van Lith, Dr Marcel and Bulleid, Professor Neil and Milligan, Professor Graeme
Authors: Van Lith, M., Tiwari, S., Pediani, J., Milligan, G., and Bulleid, N. J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience
Journal Name:Journal of Cell Science
Publisher:Company of Biologists
ISSN (Online):1477-9137
Published Online:21 June 2011
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
528621Regulating the redox conditions within the mammalian endoplasmic reticulumNeil BulleidWellcome Trust (WELLCOME)088053/Z/08/AInstitute of Molecular Cell and Systems Biology
510631The organisational structure of class A GPCRs: implications for function and drug designGraeme MilliganMedical Research Council (MRC)G0900050Institute of Neuroscience and Psychology