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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    • "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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    • "However, the effect of the resulting stress on ER thiol redox remains poorly characterized. In budding yeast, an ER-localized roGFP2 had been reported to undergo reduction on exposure to tunicamycin, an inhibitor of N-linked glycosylation that causes unfolded protein stress (Merksamer et al., 2008). However, given the reducing midpoint potential of roGFP2, 0.272 V (Hanson et al., 2004), it is suited to detect catastrophic deviations in redox state, but cannot track physiological variations around the organelles' midpoint lifetime, as expected (Fig. S2 A). "
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    ABSTRACT: Interfering with disulfide bond formation impedes protein folding and promotes endoplasmic reticulum (ER) stress. Due to limitations in measurement techniques, the relationships of altered thiol redox and ER stress have been difficult to assess. We report that fluorescent lifetime measurements circumvented the crippling dimness of an ER-tuned fluorescent redox-responsive probe (roGFPiE), faithfully tracking the activity of the major ER-localized protein disulfide isomerase, PDI. 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.
    The Journal of Cell Biology 04/2013; 201(2):337-49. DOI:10.1083/jcb.201211155 · 9.83 Impact Factor
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