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. 2018 Apr:14:679-685.
doi: 10.1016/j.redox.2017.11.015. Epub 2017 Nov 21.

Local redox environment beneath biological membranes probed by palmitoylated-roGFP

Yuta Hatori  1 , Sachiye Inouye  2 , Reiko Akagi  2 , Toshio Seyama  2
Affiliations

Local redox environment beneath biological membranes probed by palmitoylated-roGFP

Yuta Hatori et al. Redox Biol. 2018 Apr.

Abstract

Production of reactive oxygen species (ROS) and consequent glutathione oxidation are associated with various physiological processes and diseases, including cell differentiation, senescence, and inflammation. GFP-based redox sensors provide a straight-forward approach to monitor ROS levels and glutathione oxidation within a living cell at the subcellular resolution. We utilized palmitoylated versions of cytosolic glutathione and hydrogen peroxide sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively) and demonstrated a unique redox environment near biological membranes. In HeLa cells, cytosolic glutathione was practically completely reduced (EGSH/GSSG = - 333mV) and hydrogen peroxide level was under the detectable range. In contrast, the cytoplasmic milieu near membranes of intracellular vesicles exhibited significant glutathione oxidation (EGSH/GSSG > - 256mV) and relatively high H2O2 production, which was not observed for the plasma membrane. These vesicles colocalized with internalized EGFR, suggesting that H2O2 production and glutathione oxidation are characteristics of cytoplasmic surfaces of the endocytosed vesicles. The results visually illustrate local redox heterogeneity within the cytosol for the first time.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Membrane targeting of glutathione- and H2O2- specific redox sensors. A, Schematic representation of palmitoylated redox sensors, Grx1-roGFP2Palm and roGFP2-Orp1Palm. Grx1-roGFP2 and roGFP2-Orp1 were fused with the first 29 amino acid residues of human guanine nucleotide exchange factors (GEF) which contains S-palmitoylation motif (Cys23/25/26). Palmitoylated proteins can associate with biological membranes from the cytoplasmic side. B, Grx1-roGFP2Palm and roGFP2-Orp1Palm were transfected into HeLa cells and protein localization was analyzed using confocal microscopy. For comparison, the data for the original cytosolic constructs are shown. Scale bars, 10 µm. C, Localizations of Grx1-roGFP2Palm and roGFP2-Orp1Palm were assessed by immunostaining for organelle markers; EGFR at the plasma membrane and endocytic vesicles, ABCD3 at peroxisome, phosphodiesterase (PDE) at endoplasmic reticulum, TUFM in mitochondria, LAMP1 at lysosome. Scale bars, 10 µm. Co-localizations at the plasma membrane (PM; open arrow heads) and vesicular membranes (VM; filled arrow heads) are indicated.
Fig. 2
Fig. 2
Membrane association of Grx1-roGFP2Palm and roGFP2-Orp1Palm assessed by fluorescence recovery after photobleach (FRAP) experiments. A, Confocal images of HeLa cells expressing fluorescent redox sensors. Bleaching laser was shot on 10 µm × 10 µm area (dashed square) and images were taken after indicated time periods. As non-diffusible controls, cells were fixed with 3.7% paraformaldehyde prior to FRAP (Grx1-roGFP2 Fixed). B, FRAP kinetics for cells shown in A.
Fig. 3
Fig. 3
Palmitoylation did not affect reactivity of Grx1-roGFP2 and roGFP2-Orp1. Grx1-roGFP2Palm and roGFP2-Orp1Palm were transfected into HeLa cells. Redox reactivity was tested by sequential treatment with 100 µM H2O2 (oxidant, Time = 10 min) and 5 mM DTT (reductant, Time = 40 min in A and 35 min in B) during live cell imaging. I405 and I488 Images were taken by confocal microscopy and the ratio RI405/I488 was obtained for each cell. Time-dependent changes of RI405/I488 values for Grx1-roGFP2Palm (A) and roGFP2-Orp1Palm (B) are shown. PM, plasma membrane. VM, vesicular membrane.
Fig. 4
Fig. 4
Redox imaging demonstrated that the cytoplasmic surfaces the plasma membrane and vesicular membrane are characterized by different levels of glutathione oxidation and H2O2 production. A, Grx1-roGFP2Palm was transfected into HeLa cells and I405/I488 time-lapse imaging was performed by live-cell confocal microscopy. R value (I405/I488) represents the degree of sensor oxidation and shown in a false-color scale. Time is indicated in each image (h:m:s). Scale bar, 10 µm. Signals from the plasma membrane (PM; open arrow heads) and vesicular membranes (VM; filled arrow heads) are indicated. Time-lapse movie is available as Supplementary Data 5. B and D, Grx1-roGFP2Palm and roGFP2-Orp1Palm were transfected into HeLa cells and steady-state I405/I488 images were taken (z-sections). X-Y image in the right corresponds to the focal plane indicated by *. Signals from the plasma membrane (PM; open arrow heads) and vesicular membranes (VM; filled arrow heads) are indicated. C and E, Average RI405/I488 values from multiple cells (n = 5). PM, plasma membrane. VM, vesicular membrane.
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