Dataset Information

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

ABSTRACT: 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.

SUBMITTER: van Lith M

PROVIDER: S-EPMC3124370 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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

van Lith Marcel M Tiwari Shweta S Pediani John J Milligan Graeme G Bulleid Neil J NJ

Journal of cell science 20110621 Pt 14

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 ...[more]

PMID: 21693587

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Project description:BACKGROUND:Bacterial biofilms are communities of surface-associated microorganisms living in cellular clusters or micro-colonies, encapsulated in a complex matrix composed of an extracellular polymeric substance, separated by open water channels that act as a circulatory system that enable better diffusion of nutrients and easier removal of metabolic waste products. The monitoring of biofilms can provide important information on fundamental biofilm-related processes. That information can shed light on the bacterial processes and enable scientists to find ways of preventing future bacterial infections. Various approaches in use for biofilm analysis are based on microscopic, spectrochemical, electrochemical, and piezoelectrical methods. All these methods provide significant progress in understanding the bio-process related to biofilm formation and eradication, nevertheless, the development of novel approaches for the real-time monitoring of biochemical, in particular metabolic activity, of bacterial species during the formation, life and eradication of biofilms is of great potential importance. RESULTS:Here, detection and monitoring of the metabolic activity of bacterial biofilms in high-ionic-strength solutions were enabled as a result of novel surface modification by an active redox system, composed of 9,10-dihydroxyanthracene/9,10-anthraquinone, on the oxide layer of the SiNW, yielding a chemically-gated FET array. With the use of enzymatic reactions of oxidases, metabolites can be converted to H2O2 and monitored by the nanosensors. Here, the successful detection of glucose metabolites in high-ionic-strength solutions, such as bacterial media, without pre-processing of small volume samples under different conditions and treatments, has been demonstrated. The biofilms were treated with antibiotics differing in their mechanisms of action and were compared to untreated biofilms. Further examination of biofilms under antibiotic treatment with SiNW-FET devices could shed light on the bioprocess that occurs within the biofilm. Moreover, finding proper treatment that eliminates the biofilm could be examined by the novel nanosensor as a monitoring tool. CONCLUSIONS:To summarize, the combination of redox-reactive SiNW-FET devices with micro-fluidic techniques enables the performance of rapid, automated, and real-time metabolite detection with the use of minimal sample size, noninvasively and label-free. This novel platform can be used as an extremely sensitive tool for detection and establishing medical solutions for bacterial-biofilm eradication and for finding a proper treatment to eliminate biofilm contaminations. Moreover, the sensing system can be used as a research tool for further understanding of the metabolic processes that occur within the bacterial biofilm population.

Dataset Information

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

Publications

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

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