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. 2012 Jun;52(11-12):2254-65.
doi: 10.1016/j.freeradbiomed.2012.04.004. Epub 2012 Apr 17.

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes

Michèle Dardalhon  1 , Chitranshu Kumar, Ismail Iraqui, Laurence Vernis, Guy Kienda, Agata Banach-Latapy, Tiantian He, Roland Chanet, Gérard Faye, Caryn E Outten, Meng-Er Huang
Affiliations

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes

Michèle Dardalhon et al. Free Radic Biol Med. 2012 Jun.

Abstract

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H₂O₂ bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.

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Figures

Figure 1
Figure 1
Localization of nucleus-rxYFP. (A) Wild-type yeast cells expressing nucleus-rxYFP and stained by DAPI were analyzed by fluorescence microscopy. Differential interference contrast (DIC) image and overlay image of YFP and DAPI signal (merge) are also shown. (B) NAB2-mRFP strain transformed with a nucleus-rxYFP expressing plasmid. (C) Wild-type yeast cells expressing nucleus-rxYFP and Nup49-mCherry were also analyzed for localization of YFP relative to the nuclear marker. (D) Wild-type yeast cells expressing cytosol-rxYFP and stained by DAPI were analyzed by DIC and fluorescence microscopy. (E) Nuclei or cytosol from intact cells were prepared (Materials and Methods) and subjected to Western blot analysis using antibodies directed against Pgk1 (cytosol marker), Nop1 (nucleus marker) and rxYFP. In the right panel, isolated nuclei (40 μg of protein) from cells expressing nucleus-rxYFP (n) or cytosol-rxYFP (c) was prepared and analyzed. The same blot was cut and blotted for indicated antibody. In the right panel, cytosol portion (40 μg of protein) from cells expressing nucleus-rxYFP (n) or cytosol-rxYFP (c) was prepared and analyzed. The same blot was cut and blotted for indicated antibody.
Figure 2
Figure 2
Nucleus-rxYFP senses the nuclear GSH/GSSG redox status and responds to oxidant and reductant treatment. Wild-type yeast cells expressing cytosol- or nucleus-rxYFP were treated with 180 μM 4-DPS or 50 mM DTT for 20 min and collected for redox Western blot analysis. Reduced (red) and oxidized (ox) forms of rxYFP were quantified using an Odyssey Infrared Imaging System. Two representative blots are shown. The reported values are the mean ± SD of at least three independent experiments.
Figure 3
Figure 3
rxYFP redox response in various mutant cells (W303 background) affecting GSH or TRX redox systems and effects of deletions of thioredoxin genes on the growth of trr1 mutants. (A) Representative redox Western blots of the samples are shown. Percentage of oxidized rxYFP was quantified using an Odyssey Infrared Imaging System. The reported values are the mean ± SD for 3–7 independent experiments. *P < 0.05, **P < 0.01. (B) Tetrads from diploids heterozygous for trr1, trx1 and trx2 were dissected and analyzed for the presence of selection or auxotrophic markers (trr1::kanMX4, trx1::HIS3, and trx2::URA3). Circles indicate trr1 single mutants; triangles indicate trr1 trx1 mutants; squares indicate trr1 trx2 mutants; diamonds indicate trr1 trx1 trx2 mutants; hexagons indicate trx1 trx2 mutants.
Figure 4
Figure 4
Dynamic subcellular redox changes of gsh1 cells during GSH depletion. The gsh1 cells expressing the rxYFP sensors were inoculated into SC–Leu medium containing 500 μM GSH for overnight growth. These preculture cells were diluted to an appropriate density in SC–Leu with or without 500 μM GSH. During the 48 h time course, cultures were diluted when necessary to maintain exponential growth. (A) Cell density (OD600) of gsh1 cells was monitored at 0, 8, 24, 32 or 48 h after onset of GSH depletion. (B) and (C) Time course of intracellular GSH (B) and GSSG (C) concentrations during GSH depletion. Samples were removed 0, 8, 24, 32 and 48 h after onset of GSH depletion and prepared for GSH and GSSG quantification. (D) Redox Western blot of the samples removed from two parallel cultures, with or without GSH, at 0, 8, 24, 32 and 48 h after onset of GSH depletion. Percentage of oxidized forms of rxYFP was quantified using an Odyssey Infrared Imaging System. In A–D, the reported values are the means ± SD for two independent experiments.
Figure 5
Figure 5
Subcellular redox changes in response to H2O2 bursts. Wild-type strains expressing the rxYFP sensors at mid-exponential phase were treated with 0.4 mM H2O2 (A) or 3.0 mM H2O2 (B). Redox Western blots were performed on samples removed before and 5, 15, 30, 60, 120, 240 min after addition of H2O2 (left). Percentage of oxidized forms of rxYFP was quantified using an Odyssey Infrared Imaging System (right). (C) Time course of total intracellular GSH concentration in response to 0.4 mM H2O2 and 3.0 mM H2O2 bursts. Samples were removed before and 5, 15, 30, 60, 120, 240 min after addition of H2O2 and prepared for GSH quantification. The reported values are the mean ± SD of at least two independent experiments.
Figure 6
Figure 6
Effect of GSH depletion on mitochondrial and nuclear genome stability. The strains (S288c background) were inoculated into SC medium containing 500 μM GSH for overnight growth before dilution to an appropriate density in SC without GSH. (A) Effect of GSH depletion on respiratory incompetency. The gsh1 cells were removed at 0, 8, 24, and 48 h after onset of GSH depletion and plated onto YPD plates to form colonies. The frequency of petite mutations was calculated as described in Materials and Methods. The reported values are the means ± SD for 2–5 independent experiments. (B) Effect of GSH depletion on nuclear DNA mutation. The gsh1 cells that underwent 24 h GSH depletion were either inoculated in SC without GSH or supplemented with indicated concentrations of GSH for another 24 h. Cells removed from these defined culture conditions were plated onto YPD and selection plates containing canavanine to determine the total number of viable cells and the number of mutants, respectively. The reported mutation frequency is the median frequency of at least five independent cultures. (C) Effect of GSH depletion on nuclear DNA mutation in tsa1 gsh1 mutants. The tsa1 gsh1 mutants that underwent 24 h GSH depletion were either inoculated into SC medium containing 500 μM GSH or without GSH for another 24 h. Canr mutation frequencies were calculated as above and is the median frequency of 21 independent cultures. The error bars indicate 95% interval confidence based on order statistics with the formula available at http//www.math.unb.ca/~knight/utility/Medlnt95.htm. Statistical significance was evaluated by the Mann-Whitney test using programs available at http://faculty.vassar.edu/lowry/vshome.htm.
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References

    1. Halliwell B, Gutteridge JMC, editors. Free Radicals in Biology and Medicine. 4. Oxford: Oxford University Press; 2007.
    1. Herrero E, Ros J, Belli G, Cabiscol E. Redox control and oxidative stress in yeast cells. Biochim Biophys Acta. 2008;1780:1217–1235. - PubMed
    1. Toledano MB, Kumar C, Le Moan N, Spector D, Tacnet F. The system biology of thiol redox system in Escherichia coli and yeast: differential functions in oxidative stress, iron metabolism and DNA synthesis. FEBS Lett. 2007;581:3598–3607. - PubMed
    1. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med. 2001;30:1191–1212. - PubMed
    1. Ohtake Y, Yabuuchi S. Molecular cloning of the gamma-glutamylcysteine synthetase gene of Saccharomyces cerevisiae. Yeast. 1991;7:953–961. - PubMed

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