doi: 10.1038/s41422-020-00416-2.
Epub 2020 Sep 28.
COPII mitigates ER stress by promoting formation of ER whorls
Affiliations
- PMID: 32989223
- PMCID: PMC8026990
- DOI: 10.1038/s41422-020-00416-2
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COPII mitigates ER stress by promoting formation of ER whorls
Cell Res.
2021 Feb.
Abstract
Cells mitigate ER stress through the unfolded protein response (UPR). Here, we report formation of ER whorls as an effector mechanism of the ER stress response. We found that strong ER stress induces formation of ER whorls, which contain ER-resident proteins such as the Sec61 complex and PKR-like ER kinase (PERK). ER whorl formation is dependent on PERK kinase activity and is mediated by COPII machinery, which facilitates ER membrane budding to form tubular-vesicular ER whorl precursors. ER whorl precursors then go through Sec22b-mediated fusion to form ER whorls. We further show that ER whorls contribute to ER stress-induced translational inhibition by possibly modulating PERK activity and by sequestering translocons in a ribosome-free environment. We propose that formation of ER whorls reflects a new type of ER stress response that controls inhibition of protein translation.
Conflict of interest statement
The authors declare no competing interests.
Figures
a NRK cells were treated with DMSO or 0.6 μM Tg for 6 h, and then stained with ER-Tracker Red and visualized by confocal microscopy. Scale bar, 10 μm. b Cells from a were quantified for % cells with ER rings (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. c Cells from a were quantified for the number of ER rings per cell (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. d TEM images of a NRK cell treated with Tg for 6 h. Scale bar, 2 μm. The right panel shows an enlarged image of the region of interest outlined in the left panel. e Cross-sectional FIB-SEM image analysis of ER whorls. Representative images (left) show individual cross-sections of the structure. Scale bar, 100 nm. Side view (right) of a 3D reconstruction from the aligned cross-sections showing the sphere-like structure. Different colors represent different layers. f B cells were purified from B6 splenocytes using MACS beads conjugated with anti-CD19 antibody and resuspended in culture medium. 2 × 106 cells were cultured in the absence or presence of LPS (10 μg/mL) for 48 h, and then stained with ER-Tracker Red and visualized by Airyscan microscopy. Scale bar, 1 μm. Regions outlined with white dashed lines are magnified in the insets. g Cells from f were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. h Representative TEM micrographs of cells from f. Scale bar, 2 μm. The boxed region is magnified on the right. i INS-1 cells were treated with BSA or PA for 6 h, and then stained with ER-Tracker Red and visualized by 3D-SIM microscopy (three-dimensional structured illumination microscopy). Scale bar, 5 μm. The region outlined with white dashed lines is magnified in the insets. j Cells from i were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. k Representative TEM micrographs of cells from i. Scale bar, 1 μm. Regions outlined with white dashed lines are magnified on the right. l GFP-Sec61β-expressing NRK cells were treated with Tg for 0 or 6 h and then observed by confocal microscopy. Scale bar, 5 μm. The region outlined with white dashed lines is magnified in the inset. m Immunostaining of endogenous Sec61β in NRK cells treated with Tg for 0 or 6 h. Scale bar, 5 μm. The region outlined with white dashed lines is magnified in the inset. n RFP-Sec61β-expressing NRK cells transfected with the sheet ER marker Climp-63-GFP were treated with Tg for 0 or 6 h and then observed by confocal microscopy. Regions of interest are boxed and magnified on the right. Scale bar, 5 μm. o NRK cells stably expressing GFP-Sec61β were treated with Tg for 6 h, and then Tg was withdrawn. Representative confocal images of the cells are shown at the indicated times after Tg withdrawal. Scale bar, 5 μm. P values were calculated using a two-tailed, unpaired t-test (b, c, g, j).
a NRK cells were transfected with nonspecific (NS) or PERK siRNA. Cells were treated with Tg for 6 h, and then stained with ER-Tracker Red and visualized by confocal microscopy. Scale bar, 5 μm. b Cells from a were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. c PERK knockdown efficiency in cells from a was determined by western blot. d NRK cells were transfected with NS, ATF6, or IRE1a siRNA. Cells were treated with Tg for 6 h, and then stained with ER-Tracker Red and visualized by confocal microscopy. Scale bar, 5 μm. e Cells from d were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. f Knockdown efficiency in cells from d was determined by western blot. g A PERK KO cell line was generated by CRISPR-Cas9. WT and PERK KO cells were treated with Tg for 6 h, and then stained with ER-Tracker Red and visualized by confocal microscopy. Scale bar, 10 μm. h Cells from g were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. i Representative TEM micrographs of cells from g. Scale bar, 2 μm. Regions outlined with white dashed lines are magnified on the right. j PERK KO cells transfected with GFP, GFP-PERK, or GFP-PERK-Mut (K622A) were treated with Tg for 6 h and then observed by Opera Phenix microscopy with 60× confocal mode. Scale bar, 5 μm. k Cells from j were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. l NRK cells were treated with Tg (0.6 μM), Tm (2.5 μg/mL), CPA (20 μM), DTT (1 mM) or DTT (2 mM) for the indicated times and analyzed by western blot. P values were calculated using a two-tailed, unpaired t-test (b, e, h, k).
a Representative TEM micrographs of NRK cells treated with Tg for the indicated times. Regions of interest (outlined with white dashed lines) are magnified at the bottom. Red arrows in the lower 1 h panel indicate vesicles. Scale bar, 2 μm. b A TEM micrograph from a at 1 h. Scale bar, 200 nm. c A representative TEM micrograph of an NRK cell treated with Tg for 1 h. The block is processed to 300-nm-thick sections for observation. Scale bar, 100 nm. d NRK cells stably expressing GFP-Sec61β were treated with Tg and time-lapse images were acquired by Opera Phenix microscopy with 60× confocal mode. Regions of interest are magnified at the bottom. Scale bar, 5 μm. e NRK cells stably expressing GFP-Sec61β were treated with Tg for 2.5 h and visualized by GI-SIM at 100 nm resolution. Scale bar, 2 μm. The region of interest is magnified at the bottom. f CLEM imaging of a GFP-Sec61β-expressing NRK cell treated with Tg for 2 h. Left, the confocal image; middle, the TEM image; right, the enlarged images of the regions of interest outlined in the middle panel. 1, enlarged area of the GFP-Sec61β-condensed area; 2, enlarged area outside of the GFP-Sec61β-condensed area. Scale bar, 5 μm. g TEM image showing the DAB staining pattern in NRK cells stably expressing APEX2-GFP-Sec61β after treatment with Tg for 0 or 1 h. Scale bar, 2 μm.
a Sar1a-GFP-expressing NRK cells transfected with RFP-Sec61β were treated with Tg for 2.5 or 6 h and then images were acquired by Opera Phenix microscopy with 60× confocal mode. Scale bar, 5 μm. The regions outlined with white dashed lines are magnified on the right (Merge). b RFP-Sec61β-expressing NRK cells were transfected with Sar1a-GFP (WT) or Sar1a[H79G]-GFP (H79G E80V). Cells were treated with Tg for 0 or 6 h, and then imaged with a confocal microscope. Scale bar, 5 µm. c NRK cells stably expressing Sar1a-GFP and NRK cells were treated with decreasing concentrations of Tg for 6 h, and then stained with ER-Tracker Red and visualized by confocal microscopy. Cells were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. d NRK cells were transfected with Sar1a-GFP (Sar1a WT), Δ25-Sar1a-GFP, Sar1a[8,9AA]-GFP, or Sar1a[F5D]-GFP. Cells were treated with Tg (1 nM) for 6 h, and then stained with ER-Tracker Red and visualized by confocal microscopy. Scale bar, 10 μm. e Cells from d were quantified for ER whorls (n = 3 independent experiments; >50 cells were assessed per independent experiment). Data represent means ± SE. P values were calculated using a two-tailed, unpaired t-test. f Permeabilized NRK cells treated with Tg for 0 or 1 h were employed in an in vitro COPII budding assay. The resulting vesicle fractions and permeabilized cell input were separated by SDS-PAGE and visualized by immunoblotting.
a RFP-Sec61β-expressing NRK cells were transfected with NS or Sec13 siRNA. Cells were treated with Tg for 6 h. Scale bar, 20 μm. Regions outlined with white dashed lines are magnified on the right. b Cells from a were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. c Sec13 knockdown efficiency in cells from a was determined by western blot. d RFP-Sec61β-expressing NRK cells were transfected with NS or Sec24a siRNA. Cells were treated with Tg for 6 h. Scale bar, 10 μm. Regions outlined with white dashed lines are magnified on the right. e Cells from d were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. f Sec24a knockdown efficiency in cells from d was determined by western blot. g Representative TEM micrographs of cells from a and d. Scale bar, 1 μm. Regions outlined with white dashed lines are magnified. h RFP-Sec61β-expressing NRK cells transfected with GFP-Sec22b were treated with Tg for 0 or 6 h and then observed by confocal microscopy. Scale bar, 5 μm. i RFP-Sec61β-expressing NRK cells were transfected with NS or Sec22b siRNA. Cells were treated with Tg for 6 h. Scale bar, 5 μm. j Cells from i were quantified for ER whorls (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. k Sec22b knockdown efficiency in cells from i was determined by western blot. l Representative TEM micrographs of cells from i. Scale bar, 2 μm. Regions outlined with white dashed lines are magnified. P values were calculated using a two-tailed, unpaired t-test (b, e, j).
a NRK cells were transfected with NS or Sec13 siRNA. Cells were treated with Tg for 0 or 6 h and analyzed by western blot using an antibody against BiP. b NRK cells were transfected with NS or Sec13 siRNA, and treated with Tg for the indicated times. Total RNA was subsequently extracted for semiquantitative RT-PCR analysis of XBP1 mRNA species (Xbp1 S: spliced XBP1 mRNA band; Xbp1 U: unspliced XBP1 mRNA band). c Flag-ATF6-expressing NRK cells were transfected with NS or Sec13 siRNA. Cells were treated with Tg for 0 or 6 h and analyzed by western blot using an antibody against Flag. d NRK cells were transfected with NS or Sec13 siRNA. Cells were treated with Tg for the indicated times and analyzed by western blot using an antibody against phospho-PERK (Thr980) and phospho-eIF2α (Ser51). e RFP-Sec61β-expressing NRK cells transfected with GFP-PERK were treated with Tg for 0 or 6 h and then observed by confocal microscopy. Scale bar, 5 μm. f Immunostaining of endogenous PERK and RFP in RFP-Sec61β-expressing NRK cells treated with Tg for 6 h. Scale bar, 5 μm. Regions outlined with white dashed lines are magnified. g NRK cells treated with Tg for 0 or 6 h were homogenized and the lysates were subjected to 1000× g centrifugation to discard the nuclei. The supernatant was ultracentrifuged in OptiPrep density gradient medium. The distribution of PERK in the fractions was monitored by western blot. 1 is the top fraction. h In vitro protein kinase assays. Membranes were collected from Fraction 4 of NRK cells treated with Tg for 6 h as described in g and mixed with ATP and the peptides eIF2α p(45–56) or mutant eIF2α p(45–56, S51A). Kinase reactions were resolved on SDS-PAGE and visualized by immunoblotting using an antibody against phospho-eIF2α (Ser51).
a All three translocon components (GFP-Sec61α, GFP-Sec61β, GFP-Sec61γ) localize on ER whorls in cells treated with Tg for 6 h. Regions of interest are outlined with white dashed lines and magnified in the insets. Scale bar, 5 μm. b RFP-Sec61β-expressing NRK cells were treated with Tg for 6 h and visualized by 2D-confocal microscopy. Scale bar, 5 μm. c Cells from b were analyzed for IOD (integrated optical density) of ER whorls and the rest of the ER by Image-Pro plus (n = 3 independent experiments; >100 cells were assessed per independent experiment). Data represent means ± SE. d TEM images showing the DAB staining pattern in NRK cells stably expressing APEX2-GFP-Sec61β after treatment with Tg for 0 or 6 h. Scale bar, 5 μm. e Representative images of APEX2-GFP-Sec61β-labeled structures in cells from d. Scale bar, 500 nm. f NRK cells were transfected with NS or Sec13 siRNAi. Cells were treated with Tg for the indicated times and incubated with puromycin for 30 min. Membrane proteins and cytosolic proteins were separately detected by immunoblotting using an anti-puromycin antibody. g–j Average density of ribosome footprints on all coding genes aligned by their start codons. The analyses were done in NRK cells treated with NS (g), PERK siRNA (h), Sec13 siRNA (i), or Sec22b siRNA (j), before (0 h) and after (6 h) Tg treatment. The ribosome footprints were allocated to each codon according to their P-sites, and the count of footprints on each codon was normalized by the average count per codon after the first 30 codons. k A model of ER whorl formation and function.
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