Data availability
All data supporting the findings of this study are provided in Article and its Supplementary Information. Full versions of all gels and blots are provided in Supplementary Fig. 1. Raw data of all images presented in this manuscript, representative tomograms and proteomic datasets acquired in this study have been deposited in Figshare at https://doi.org/10.6084/m9.figshare.32050848 (ref. 38), https://doi.org/10.6084/m9.figshare.32050956 (ref. 39) and https://doi.org/10.6084/m9.figshare.32050986 (ref. 40), respectively. Source data are provided with this paper.
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Acknowledgements
We thank X. Zhang, X. Niu, Y. Feng, L. Wang, T. Li and X. Li for assisting with sample preparation, CLEM and immunoelectron microscopy imaging; Y. Wan and R. Wang for assistance with flow cytometry; R. Liu and L. Jiao for assistance with microscopy; and all relevant instructors from the Mass Spectrometry Platform, School of Life Sciences, Nankai University, for generous technical support.
Funding
F.Y., Q.C. and Y.L. disclose support for the research of this work from the National Natural Science Foundation of China (grant numbers 32570817, 92357302, 32530029, 92354304 and 32570898), Qingdao Municipal Science and Technology Bureau (grant number 26-1-5-smjk-6-nsh) and the Taishan Scholar Program of Shandong (grant number tsqn202312240). Q.C. discloses support for publication of this work from the National Natural Science Foundation of China (grant number 92357302). L.J., Hanyu Liu, S.Y., Y.Y., H.J., J.L., X.L., Y.D., D.R., J.K., Y.H., Q.M., F.B., S.L., Huadong Liu, W.Z. and J.X. declare no relevant funding.
Author information
Author notes
These authors contributed equally: Fan Yu, Lingyu Jiang
Authors and Affiliations
The State Key Laboratory of Medicinal Chemical Biology and Frontier of Science Center for Cell Response, College of Life Sciences, Nankai University, Tianjin, China
Fan Yu (于凡), Lingyu Jiang (姜凌宇), Hanyu Liu (刘涵宇), Jinming Liu (刘金鸣), Xi Liu (柳曦), Yu Dong (董瑜), Dongfeng Ren (任东峰), Yan Huang (黄燕), Qi Meng (孟琦), Feng Bai (白峰), Yanjun Li (李艳君) & Quan Chen (陈佺)
Qingdao Central Hospital, NHC Key Laboratory of Cardiopulmonary Rehabilitation and Functional Recovery, Industry-Academia-Research Collaborative and Innovation Center for Intelligent Rehabilitation Drug R&D of Shandong Province, Qingdao Key Laboratory of Precision Drug Research for Chronic Disease Rehabilitation, University of Health and Rehabilitation Sciences, Qingdao, China
Fan Yu (于凡), Lingyu Jiang (姜凌宇), Song Yang (杨松), Hongzhen Jin (金洪真), Jie Kang (康洁), Huadong Liu (刘华东), Wei Zhao (赵炜) & Jia Xiao (肖佳)
Aier Academy of Ophthalmology, Central South University, Tianxin District, Changsha, China
Yuan Yuan (袁源) & Jia Xiao (肖佳)
Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Shuoguo Li (李硕果)
Authors
- Fan Yu (于凡)
- Lingyu Jiang (姜凌宇)
- Hanyu Liu (刘涵宇)
- Song Yang (杨松)
- Yuan Yuan (袁源)
- Hongzhen Jin (金洪真)
- Jinming Liu (刘金鸣)
- Xi Liu (柳曦)
- Yu Dong (董瑜)
- Dongfeng Ren (任东峰)
- Jie Kang (康洁)
- Yan Huang (黄燕)
- Qi Meng (孟琦)
- Feng Bai (白峰)
- Yanjun Li (李艳君)
- Shuoguo Li (李硕果)
- Huadong Liu (刘华东)
- Wei Zhao (赵炜)
- Jia Xiao (肖佳)
- Quan Chen (陈佺)
Contributions
F.Y. originally conceived the rationale for this project, planned the experimental direction, interpreted data and wrote the manuscript. L.J. conducted the majority of the experiments and assisted in writing the manuscript. Hanyu Liu constructed most of the plasmids. S.Y. assisted in data collation and figure preparation. Y.Y. established mouse disease models. H.J. completed the model design and drawing. J.L. assisted in processing mouse samples. X.L. constructed some of the plasmids and contributed to the completion of Fig. 5c. Y.D. participated in the completion of Extended Data Fig. 6c,f,g. D.R. was involved in Extended Data Fig. 8h–j. J.K. assisted in cell preparation and collection. Y.H. contributed to Extended Data Fig. 1m. Q.M. and F.B. participated in the work presented in Extended Data Fig. 4a. Y.L., S.L., Huadong Liu and W.Z. provided platform, funding and technical support for this project. J.X. supervised animal experiments. F.Y. and Q.C. conceived and supervised the study and edited the manuscript.
Corresponding authors
Correspondence to Fan Yu (于凡), Jia Xiao (肖佳) or Quan Chen (陈佺).
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Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature thanks Emily Sontag who co-reviewed with Chloe Langridge; Wen-Xing Ding, Luca Scorrano and the other, anonymous, reviewer for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Various stresses induce SIMPA.
a, Schematic of SIMPA induction across multiple stress models, including H2O2 stimulation and heat shock (HS). b, Confocal images of stress-induced SIMPA formation. Representative immunofluorescence of SIMPA (MitoTracker Red, red) and mitochondria (TOM20, green) under H2O2 (30 μM) and heat shock (42 °C) conditions. c, Schematic of H2O2 dose-dependent analysis. U-2OS cells were treated with different H2O2 concentrations for 60 min, with MitoTracker Red staining during the final 30 min. d, Morphological changes of SIMPA within mitochondria under H2O2 stress. Representative confocal images showing the size and number of SIMPA (MitoTracker Red, red) in mitochondria (TOM20, green). e, MitoTracker dose-dependent SIMPA formation in HeLa cells. Immunofluorescence of HeLa cells stained with 50-400 nM MitoTracker Red for 30 min and recovered for 24 h, showing SIMPA size and density. f, Experimental workflow for AIE-mediated photo stress induction. U-2OS cells were treated with AIE (0-8 μM) for 1 h, followed by 10 min light stimulation, incubated for 4 h, and then stained with MitoTracker Red for 30 min. g, Confocal microscopy of AIE-induced SIMPA formation. Representative images depicting the dose-dependent formation of SIMPA (MitoTracker Red, red) after photostimulation of AIE (green). h, Quantification of AIE dose-dependent SIMPA-positive cells (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). i, Protocol for inhibiting the mitochondrial respiratory chain. U-2OS cells were treated with Rotenone (10 μM), Antimycin A (5 μM), NaN3 (1 μM), or Oligomycin (2 μM) for 12 h, followed by MitoTracker Red staining (30 min). j, SIMPA formation in response to respiratory chain inhibitors. Confocal images showing the size and quantity of MitoTracker Red-positive aggregates (SIMPA, red) in treated cells. k, Statistical analysis of inhibitor-induced SIMPA incidence (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). l, U-2OS cells were transfected with varying concentrations of ΔOTC–GFP plasmid for 24 h. To maintain constant total DNA amount, the lower-concentration groups were supplemented with empty PE–GFP vector. After 18 h, cells were treated with MG132 for 6 h. Mitochondria were labelled with MitoTracker Red during the last 30 min. m, Representative fluorescence micrographs illustrating the colocalization of SIMPA (MitoTracker Red, red) and ΔOTC-GFP aggregates (green) under different plasmid concentrations. n, Percentage of U-2OS cells with SIMPA following transfection with different amounts of ΔOTC-GFP plasmid (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). Scale bar, 10 μm.
Extended Data Fig. 2 MitoTracker treatment induces SIMPA.
a, U-2OS cells were stained with MitoTracker Red for 30 min, or stained with MitoTracker Red for 30 min, and then incubated after MitoTracker washout for 24 h. b, Schematic of SIMPA induction by different types of MitoTracker. U-2OS cells were stained with MitoTracker DeepRed for 30 min, transferred to normal medium for 24 h, then stained with MitoTracker Red for 30 min. c, Representative images of SIMPA induced by Mitotracker DeepRed. Confocal images depict SIMPA (red) induced by MitoTracker DeepRed (purple) within mitochondrial networks (TOM20, green). d, Representative images of SIMPA induced by Mitotracker Green, showing MitoTracker Green (green) co-localized with resulting SIMPA (indicated by MitoTracker Red, red). e, U-2OS cells were stained with MitoTracker Red (red) for 30 min, 1 d, or 2 d, respectively, and subsequently stained with PK Mito (green) for 30 min. Representative fluorescent images at different MitoTracker staining times. f, U-2OS cells were stained with PK Mito (green) for 30 min, 1 d, or 2 d, respectively, and then stained with MitoTracker Red (red) for 30 min. Representative fluorescence images of different PK Mito staining durations. g, Protocol for MitoTracker washout versus continuous staining to examine SIMPA size and quantity changes. U-2OS cells were stained with MitoTracker Red for 30 min, washed, and incubated for 24 h, or continuously stained for 24 h. h, Confocal imaging of SIMPA size and quantity changes with varying MitoTracker staining time. U-2OS cells were stained with MitoTracker Red for 30 min, then either washed or left unwashed and cultured for 24 h. Scale bar, 10 μm.
Extended Data Fig. 3 Isolation and analysis of SIMPA components.
a, Time-lapse structured-illumination microscopy (SIM) of SIMPA biogenesis. U-2OS cells were imaged at 30-s intervals, capturing formation and growth of MitoTracker Red-labelled SIMPA (red) and mitochondrial cristae changes (PK Mito, green). See Supplementary Video 4. b, Isolation and flow cytometry analysis of mitochondrial fractions. Mitochondria were enriched using Dounce homogenization and differential centrifugation, followed by fluorescence-activated cell sorting (FACS) of MitoTracker Red-stained cells at 24 h post-stimulation. Scatter plots show separation of SIMPA-positive (high PE intensity) and SIMPA-negative (low PE intensity) fractions. c, Proteomics-based clustering of SIMPA and non-SIMPA mitochondrial components. Bubble plot depicts differentially enriched proteins in SIMPA fractions (high PE intensity) versus non-SIMPA controls (low PE intensity), identified by label-free quantitative proteomics. d,e, Immunofluorescence localization of SIMPA with mtDNA and mtDNA-binding proteins. d, Representative fluorescence images and co-localization analysis of ATP6 (mtDNA-binding protein, green) with SIMPA aggregates (MitoTracker, red). e, Representative fluorescence images and the corresponding co-localization analysis of mitochondrial dsDNA (green) with SIMPA aggregates (MitoTracker, red). Scale bar, 10 μm.
Extended Data Fig. 4 GRP75 is critical for SIMPA aggregation, and SIMPA can be cleared.
a, Representative confocal images of SIMPA (MitoTracker Red, red) and heat shock proteins (HSP60, HSPA8, HSPA1L, GRP75; green). b, Pearson’s correlation coefficients for HSP60, HSPA8, HSPA1L, and GRP75 with MitoTracker Red signals (n = 30 biologically independent cells; mean ± s.d.). c, WB validation of GRP75 knockdown efficiency. d, Representative images of U-2OS cells transfected with siGRP75 or siControl, stained with MitoTracker Red, and immunostained for GRP75 (cyan) and HSP60 (green). e, Statistical analysis of SIMPA-positive cells after GRP75 knockdown (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). f, Statistical analysis of the proportion of viable cells among all cells in MitoTracker-stained samples at day 0 (control) and day 1 (1 d), with or without knockdown of GRP75 (n = 3 biologically independent experiments; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). g, Protocol for SIMPA formation and clearance. U-2OS cells were stained with MitoTracker Red for 30 min, then incubated for 0-4 days to monitor dynamics. h, Confocal imaging of SIMPA density over a 4-day time course. Representative immunofluorescence of MitoTracker Red-labeled SIMPA (red) and mitochondrial morphology (TOM20, green) at Day 0, 1, 2, and 4. i, Quantification of SIMPA-positive cells (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). j, Protocol for H2O2-induced SIMPA formation and recovery over four days. U-2OS cells were treated with 100 μM H2O2 for one hour, then incubated for 0 to 4 days. k, Immunofluorescence imaging of H2O2-induced SIMPA changes. Representative images of SIMPA (red) and mitochondria (TOM20, green) at indicated times. l, Statistical analysis of SIMPA incidence during H2O2 stress recovery (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). Scale bar, 10 μm.
Extended Data Fig. 5 GRP75 and MAM are involved in the SIMPA clearance process.
a, Long-term dynamics of SIMPA in GRP75-overexpressing cells. Confocal images of GRP75-Tg (green) and WT cells stained for SIMPA (red) over 4 days. b, Percentage of SIMPA-positive GRP75-Tg and WT cells at 0-4 days (n = 3 biologically independent experiments; mean ± s.d.). c, WB analysis of key ISR and mtUPR pathway proteins during SIMPA induction and recovery (days 1-6). For gel source data, see Supplementary Fig. 1. d, MAM-SIMPA interactions in GRP75-knockdown cells. Confocal images of MAM-GFP (green) and SIMPA (red) in siControl and siGRP75 cells after Mitotracker staining at 0 and 1 day. Meanwhile, immunostaining of the GRP75 protein was performed (cyan). e, Statistical analysis of i, MAM structure incidence following GRP75 knockdown (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). f, Co-IP analysis of GRP75 interactions with VDAC1/ ITPR3 during SIMPA turnover. Ctrl, control; R3, Recovery Day 3; R6, Recovery Day 6; IgG, negative control. g, Proximity ligation assay (PLA) showing pairwise interactions between GRP75, VDAC1, and ITPR3 in control (Day 0) and SIMPA-recovery (Day 2) cells, indicated by green dots. h,i, WB validation of VDAC1 and ITPR3 knockdown efficiency. j, Representative images of SIMPA (MitoTracker Red, red) and MAM (green) in siControl, siVDAC1, and siITPR3 cells over 4 days in VDAC1/ITPR3-knockdown split-MAM-GFP cells. k, Quantification of SIMPA-positive cells (n = 3 biologically independent experiments; mean ± s.d.). Scale bar, 10 μm.
Extended Data Fig. 6 Construction of GRP75 mutants and their effects on SIMPA.
a, Design of synonymous mutants for the entire GRP75 protein sequence based on siRNA. Plasmid maps of GRP75 truncation and localization variant constructs, including localization-deficient and localization-variant mutants; substrate binding domain (SBD) deficient mutants; ATPase-deficient mutants. b, Flowchart for functional and localization validation of GRP75 variants. GRP75 was knocked down in split-MAM-GFP U-2OS cells, followed by transfection with plasmids of GRP75 mutants. MitoTracker staining was performed for 30 min, followed by 24 h incubation and immunofluorescence analysis. c, Representative immunofluorescence image showing functional recovery of SIMPA (MitoTracker Red, red) and MAM (split-GFP, green) in split-MAM-GFP U-2OS cells after GRP75 knockdown and transfection with GRP75 WT and localization-deficient/modified variants. d, Statistical analysis of SIMPA functional recovery (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). e, Statistical analysis of MAM functional recovery (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). f, Representative immunofluorescence image showing functional recovery of SIMPA (MitoTracker Red, red) and MAM (split-GFP, green) in split-MAM-GFP U-2OS cells after knockdown and transfection with GRP75 WT and ATPase-deficient mutants. g, Representative immunofluorescence image showing functional recovery of SIMPA (MitoTracker Red, red) and MAM (split-GFP, green) in split-MAM-GFP U-2OS cells after knockdown and transfection with GRP75 WT and its SBD deficient mutants. h,i, Statistical analysis of SIMPA functional recovery (n = 30 biologically independent cells; ordinary one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.d.; ****P < 0.0001). j,k, Statistical analysis of MAM functional recovery (n = 30 biologically independent cells; ordinary one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.d.; ****P < 0.0001). Scale bar, 10 μm.
Extended Data Fig. 7 ER stress pathway involves in SIMPA clearance.
a, Confocal images and localization analysis show co-localization of p-IRE1α with SIMPA under stress. Representative immunofluorescence of U-2OS cells and its corresponding colocalization analysis depict the relative positional relationship between phosphorylated IRE1α (p - IRE1α, green) and SIMPA (MitoTracker Red, red) at 2 days post-SIMPA formation. b,c, Impact of XBP1 knockdown on SIMPA during long-term recovery. b, Confocal images of SIMPA (red) and mitochondria (HSP60, green) in siXBP1#1/#2 and siControl cells at 3 days post-SIMPA formation (Day 3) and 5 days post-SIMPA formation (Day 5). c, Percentage of cells with residual SIMPA foci (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). d, Co-IP of GRP75 with ER UPR signaling components. Western blots detect interactions between GRP75 and p-PERK or p-IRE1α at 0 (Ctrl), 3 (R3), and 6 (R6) days post-SIMPA formation. For gel source data, see Supplementary Fig. 1. e, Statistical analysis of relative p-IRE1α and p-PERK levels around GRP75 (n = 3 biologically independent experiments; mean ± s.d). f,g, WB analysis of the phosphorylation levels of ER UPR proteins following the knockdown of key MAM formation components. f, Relative expression levels of p-IRE1α/IRE1α and p-eIF2α/eIF2α in VDAC1 knockdown cells at 4 days post-recovery. g, Similar analysis in ITPR3 knockdown cells. β-actin, loading control. h, Representative confocal images of U-2OS cells under SIMPA (MitoTracker Red, red) induction and prolonged recovery (0-5 days), with inhibition by two ER UPR pathway-specific inhibitors added on Recovery Day 2. i, Percentage of cells with SIMPA from day 0 to 5 post-induction, with or without ER UPR inhibitors (n = 3 biologically independent experiments; mean ± s.d). Scale bar, 10 μm.
Extended Data Fig. 8 ER-associated degradation pathway involves in SIMPA clearance.
a, Expression levels of ER-associated degradation (ERAD) markers (VCP1, NPLOC4, UFD1) at 0 to 6 days post-MitoTracker treatment. For gel source data, see Supplementary Fig. 1. b, Statistical chart of relative abundance changes of the aforementioned ERAD protein detected by Western blot experiments (n = 3 biologically independent experiments; mean ± s.d.). c,d, Pharmacological modulation of ER UPR affects SIMPA formation. c, Confocal images of U-2OS cells treated with Azoramide (0-1.25 μM) for 24 h post-MitoTracker staining, showing SIMPA (MitoTracker, red) and mitochondrial morphology (HSP60, green). d, Quantification of SIMPA-positive cells (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). e, Confocal images of U-2OS cells treated with 0.75 μM Azoramide 24 h post MitoTracker staining with/without XBP1 knockdown. f, Statistical graph of the proportion of cells with SIMPA (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). g, Time-lapse imaging of mitochondrial outer membrane Ca2+ (OMM-NEMO, green) at SIMPA forming sites in U-2OS cells stained with MitoTracker (MitoT, Red) and PK Mito DeepRed (blue). Images were acquired every 5 s. See Supplementary Video 5. h, EdU assay showing cell proliferation rate changes during SIMPA formation and recovery. U-2OS cells were collected on Day 0-6 post 30-min MitoTracker staining, and EdU staining was performed 2 h before flow cytometry analysis (n = 3 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). i, PI live-cell staining to show the proportion of apoptotic cells during the formation and recovery of SIMPA. U-2OS cells were collected on Day 0-6 post 30-min MitoTracker staining, and PI staining was performed 10 min before flow cytometry analysis (n = 3 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). j, PI staining was used to detect the proportions of G1, G2/M, and S phase in the cell cycle during the process of SIMPA. U-2OS cells were collected on Day 0-6 post 30-min MitoTracker staining, then fixed (70% ethanol) and permeabilized, and PI staining was performed 10 min before flow cytometry analysis (n = 3 biologically independent samples; mean ± s.d.). Scale bar, 10 μm.
Extended Data Fig. 9 SIMPA is present in multiple liver injury disease models.
a, Liver sections from mice immunofluorescently labelled with Grp75 (SIMPA marker, red) at 4, 12, and 24 h post-alcohol administration (20% ethanol, 5 g/kg, intragastric gavage). b,The proportion of SIMPA positive cells in all statistically analysed cells (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). c, The number of GRP75-positive aggregates (SIMPA) per cell (n = 30 biologically independent cells; Ordinary one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.d.). d-f, Acute carbon tetrachloride (CCl4)-induced liver injury. d, Confocal imaging of Grp75-labelled SIMPA in hepatocytes. Representative images of Grp75-positive SIMPA (red) at 6 and 24 h post intraperitoneal injection of 1 mL/kg CCl4. e, Proportion of SIMPA-positive cells (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). f, Average Grp75-positive aggregate (SIMPA) number per cell (n = 30 biologically independent cells; ordinary one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.d.). g-i, Thioflavin T staining of protein aggregates in three liver injury models. Representative immunofluorescence images of mouse liver sections stained with Thioflavin T (cyan), Tom20 (green), and Grp75 (red, SIMPA) in the ALD model at 0 h and 24 h (g), and in the APAP (h) and CCl4 (i) models at 0 h and 12 h. j, Whole-organ fluorescence imaging after AAV-mediated Grp75 (GFP-Grp75, green) or control (GFP-NC, green) overexpression. k, Representative confocal images of liver sections from Grp75-Tg and WT mice 12 h after alcohol administration. l, Statistical analysis of the proportion of SIMPA-positive cells (n = 10 biologically independent samples; Welch’s ANOVA with Dunnett’s T3 multiple comparison test; mean ± s.d.). m, The number of SIMPA dots per cell (n = 30 biologically independent cells; ordinary one-way ANOVA with Dunnett’s multiple comparison test; mean ± s.d.). n, Representative images of Grp75-labeled SIMPA (red) in AAV-Grp75-Tg (green) livers at 0, 4, and 12 h post-ethanol gavage (5 g/kg), showing differences in SIMPA size and quantity between exogenous high and low expression of AAV-Grp75. o, Long-term survival curves of Sham, APAP, APAP + AAV-Grp75, and APAP + AAV-NC group (n = 15 animals per group). Scale bar, 10 μm.
Extended Data Fig. 10 Promoting the SIMPA pathway ameliorates disease phenotype.
a, Representative H&E images of livers at 6 and 24 h post Sham/APAP treatment in Grp75-overexpressing (AAV-Grp75) and control (AAV-NC) mice. Scale bar, 100 μm. b, Quantification of area of necrosis in APAP model (n = 5 biologically independent animals; two-tailed unpaired t test; mean ± s.d.). c,d, Serum ALT (c) and AST (d) levels in each group of the APAP model (n = 5 biologically independent animals; two-tailed unpaired t test; mean ± s.d.). e,f, WB analysis of liver tissue from a, showing Cyp2E1 and Grp75 expression in each group at 6 h (e) and 24 h (f) post-APAP treatment. g, Representative H&E images of livers at 4, 12, and 24 h post Sham/ALD treatment in Grp75-overexpressing (AAV-Grp75) and control (AAV-NC) mice. Scale bar, 100 μm. h, Histopathological scoring of each group via NAFLD Activity Score (NAS) in the ALD model. Quantification of steatosis, inflammation, and ballooning (0-8 scale. Scored by 4 independent hepatologists and each point represents the average score) (n = 3 biologically independent samples; two-tailed unpaired t test; mean ± s.d.). i,j, Serum ALT (i) and AST (j) levels in each group of the ALD model (two-tailed unpaired t test; mean ± s.d.). k,l, WB analysis of liver tissue from g, showing Cyp2E1 and Grp75 expression in each group at 4 h (k) and 24 h (l) post-ALD treatment. m, Representative H&E images of livers at 12 and 24 h post Sham/CCl4 treatment in Grp75-overexpressing (AAV-Grp75) and control (AAV-NC) mice. Scale bar, 100 μm. n, Quantification of area of necrosis in the CCl4 model (n = 5 biologically independent samples; two-tailed unpaired t test; mean ± s.d.). o,p, Serum ALT (o) and AST (p) levels in each group of the CCl4 model (n = 5 biologically independent animals; two-tailed unpaired t test; mean ± s.d.). q,r, WB analysis of liver tissue from m, showing Cyp2E1 and Grp75 in each group at 12 h (q) and 24 h (r) post-CCl4 treatment. s, Immunofluorescence of SIMPA (Grp75, red) in mouse liver sections with or without Azoramide at 0, 6, and 24 h post APAP treatment. Tom20 is used to label mitochondria. Scale bar, 10 μm. t, H&E staining of liver sections with/without Azoramide at 0, 6, and 24 h post-APAP treatment. Scale bar, 100 μm. For gel source data, see Supplementary Fig. 1.
Supplementary information
Supplementary information (download PDF )
This file contains all complete, uncropped versions of every gel and blot image shown in the Figs. and Extended Data Figs. of the main text, and Supplementary Table 1.
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Supplementary Video 1 (download MP4 )
Formation of SIMPA condensates. The video shows SIMPA (red) formation in relation to the OMM (green) and the mitochondrial matrix (blue). It directly corresponds to and supports the timelapse sequence presented in Figure 1g of the main text.
Supplementary Video 2 (download MP4 )
Fusion of SIMPA condensates. This video illustrates the fusion of SIMPA condensates (red) within mitochondria, with the OMM shown in green and the matrix in blue. It is directly related to and supports the dynamic process displayed in the timelapse sequence of Fig. 1h in the main text.
Supplementary Video 3 (download MP4 )
FRAP analysis of SIMPA. The video presents FRAP of SIMPA (red). It corresponds directly to and corroborates the dynamic process shown in the timelapse sequence of Fig. 1i in the main text.
Supplementary Video 4 (download MP4 )
Remodelling of the mitochondrial inner membrane during SIMPA formation. This video captures changes in mitochondrial cristae (PK Mito, green) during the formation of SIMPA condensates (red). It is directly related to and supports the timelapse sequence shown in Extended Data Fig. 3a of the main text.
Supplementary Video 5 (download MP4 )
Ca2+ release during SIMPA formation. The video shows Ca2+ release (OMM-NEMO, green) at sites of SIMPA formation in U-2 OS cells stained with MitoTracker (red) and PK Mito DeepRed (blue). It directly corresponds to and reinforces the dynamic process presented in the timelapse sequence of Extended Data Fig. 8g in the main text
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Yu, F., Jiang, L., Liu, H. et al. GRP75-dependent SIMPA mediates inter-organellar quality control. Nature (2026). https://doi.org/10.1038/s41586-026-10733-y
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DOI: https://doi.org/10.1038/s41586-026-10733-y