Real-Time Redox Measurements during Endoplasmic Reticulum Stress Reveal Interlinked Protein Folding Functions

Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
Cell (Impact Factor: 32.24). 12/2008; 135(5):933-47. DOI: 10.1016/j.cell.2008.10.011
Source: PubMed


Disruption of protein folding in the endoplasmic reticulum (ER) causes unfolded proteins to accumulate, triggering the unfolded protein response (UPR). UPR outputs in turn decrease ER unfolded proteins to close a negative feedback loop. However, because it is infeasible to directly measure the concentration of unfolded proteins in vivo, cells are generically described as experiencing "ER stress" whenever the UPR is active. Because ER redox potential is optimized for oxidative protein folding, we reasoned that measureable redox changes should accompany unfolded protein accumulation. To test this concept, we employed fluorescent protein reporters to dynamically measure ER redox status and UPR activity in single cells. Using these tools, we show that diverse stressors, both experimental and physiological, compromise ER protein oxidation when UPR-imposed homeostatic control is lost. Using genetic analysis we uncovered redox heterogeneities in isogenic cell populations, and revealed functional interlinks between ER protein folding, modification, and quality control systems.

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    • "To assess whether oxidative signaling is involved in regulation of nuclear actin filaments induced by MMS, we generated a construct containing the redox-sensing fluorophore roGFP2 fused with 3×NLS (roGFP2-NLS) (Lohman and Remington, 2008). roGFP2 is variant of Green Fluorescent Protein (GFP) engineered to introduce 2 cysteines to the interior of the GFP beta barrel, and it has been extensively used to measure oxidation changes in live cells (Merksamer et al., 2008; Al-Mehdi et al., 2012). The roGFP2 excitation spectrum contains 2 peaks, at 488 nm and at 405 nm. "
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    ABSTRACT: Actin filaments assemble inside the nucleus in response to multiple cellular perturbations, including heat shock, protein misfolding, integrin engagement, and serum stimulation. We find that DNA damage also generates nuclear actin filaments-detectable by phalloidin and live-cell actin probes-with three characteristic morphologies: (i) long, nucleoplasmic filaments; (ii) short, nucleolus-associated filaments; and (iii) dense, nucleoplasmic clusters. This DNA damage-induced nuclear actin assembly requires two biologically and physically linked nucleation factors: formin-2 and Spire-1/Spire-2. Formin-2 accumulates in the nucleus after DNA damage, and depletion of either formin-2 or actin's nuclear import factor, importin-9, increases the number of DNA double-strand breaks (DSBs), linking nuclear actin filaments to efficient DSB clearance. Nuclear actin filaments are also required for nuclear oxidation induced by acute genotoxic stress. Our results reveal a previously unknown role for nuclear actin filaments in DNA repair and identify the molecular mechanisms creating these nuclear filaments.
    eLife Sciences 08/2015; 4. DOI:10.7554/eLife.07735 · 9.32 Impact Factor
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    • "We first estimated ER red/ox conditions in a Drosophila stable S2R+ cell line that conditionally express an ER-localized roGFP variant (eroGFP) (fig. 3A; [26]). The excitation peak of the reporter is dependent on the red/ox state of the ER lumen, and decreases in the ratio of signal obtained at 400 nm (oxidized species) as compared with that derived at 490 nm (reduced species) suggests a reducing environment exists at the ER lumen, which could hamper protein folding. "
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    ABSTRACT: The function and capacity of the endoplasmic reticulum (ER) is determined by multiple processes ranging from the local regulation of peptide translation, translocation, and folding, to global changes in lipid composition. ER homeostasis thus requires complex interactions amongst numerous cellular components. However, describing the networks that maintain ER function during changes in cell behavior and environmental fluctuations has, to date, proven difficult. Here we perform a systems-level analysis of ER homeostasis, and find that although signaling networks that regulate ER function have a largely modular architecture, the TORC1-SREBP signaling axis is a central node that integrates signals emanating from different sub-networks. TORC1-SREBP promotes ER homeostasis by regulating phospholipid biosynthesis and driving changes in ER morphology. In particular, our network model shows TORC1-SREBP serves to integrate signals promoting growth and G1-S progression in order to maintain ER function during cell proliferation.
    PLoS ONE 07/2014; 9(7):e101164. DOI:10.1371/journal.pone.0101164 · 3.23 Impact Factor
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    • "The roGFPs conveniently exhibit two distinct excitation peaks responding reciprocally to redox changes, thereby enabling ratiometric analyses. The excitation ratio from these two wavelengths (400 and 490 nm for roGFP2) is much less dependent upon probe expression level and varying fluorescence output due to photobleaching, thus simplifying comparison between samples [6] [7]. The fluorescence ratio indicates the extent of probe oxidation, and can be used for quantification of glutathione redox potential after additional calibration by exposing cells to strong reducing and oxidizing agents at the conclusion of an experiment [4] [6]. "
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    ABSTRACT: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.
    Biochemical and Biophysical Research Communications 09/2013; 439(4). DOI:10.1016/j.bbrc.2013.09.011 · 2.30 Impact Factor
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