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MISO regulates mitochondrial dynamics and mtDNA homeostasis by establishing membrane subdomains

Nature Cell Biology volume 28, pages 255–267 (2026)Cite this article

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Abstract

Mitochondrial dynamics and mtDNA homeostasis have been linked to specialized mitochondrial subdomains known as small MTFP1-enriched mitochondria (SMEM), though the underlying molecular mechanisms remain unclear. Here we identified MISO (mitochondrial inner membrane subdomain organizer), a conserved protein that regulates both mitochondrial dynamics and SMEM formation in Drosophila and mammalian cells. MISO inhibits fusion by recruiting MTFP1 and promotes fission through FIS1–DRP1. Furthermore, MISO drives SMEM biogenesis and facilitates their peripheral fission that promotes lysosomal degradation of mtDNA. Genetic ablation of MISO abolishes SMEM generation, confirming that MISO is both necessary and sufficient for SMEM formation. Inner mitochondrial membrane stresses, including mtDNA damages, OXPHOS dysfunction and cristae disruption, stabilize the otherwise short-lived MISO protein, thereby triggering SMEM assembly. This process depends on the C-terminal domain of MISO, likely mediated by oligomerization. Together, our findings reveal a molecular pathway through which inner mitochondrial membrane stresses modulate mitochondrial dynamics and mtDNA homeostasis via MISO-orchestrated SMEM organization.

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Fig. 1: Drosophila MISO/CG30159 forms mitochondrial subdomains and regulates mitochondrial dynamics.
Fig. 2: Mammalian MISO/C3orf33 functions as a regulator of mitochondrial morphology.
Fig. 3: MISO-enriched subdomains exert dual regulatory control over mitochondrial dynamics.
Fig. 4: MISO constitutes SMEM that regulate mtDNA homeostasis.
Fig. 5: IMM stress leads to SMEM formation through the stabilization of the MISO protein.
Fig. 6: MISO promotes inter-membrane interactions to drive SMEM formation.

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Data availability

All data generated or analysed during this study are included in the article or Supplementary Information. Raw data are available in the source data file. Exact genotypes used for each experiment are listed in Supplementary Table 6. Detailed parameters for image acquisition and processing are provided in Supplementary Table 7. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Protein mass spectrometry data have been deposited in the ProteomeXchange PRIDE database under accession number PXD057461. Source data are provided with this paper.

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Acknowledgements

We thank N. Perrimon and R. Binari at the Harvard Medical School for supporting the initial genetic screen. We also thank the Cryo-EM Center at the University of Science and Technology of China and the Harvard Medical School for assistance with TEM. Funding for this work was from the National Natural Science Foundation of China (nos. 32470754 and 32070750), USTC Cultivation Fund of Innovative Research Team (WK2030000074) and Opening Foundation of National Engineering Research Center of Genetic Medicine, China (NERCGM-OF-20250201) to L.H.; the K.C. Wong Education Foundation, the National Natural Science Foundation of China (no. 32430049, 82370596), National Key R&D Program of China (no. 2021YFA08049000), Science and Technology Project in Guangzhou (no. 202102070001) and the Guangdong Basic and Applied Basic Research Foundation (no. 2023B1515120089) to Q.Z.; and the National Natural Science Foundation of China (nos. 82370576 and 32170772) to J. L.

Author information

Authors and Affiliations

  1. The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Yue Zhang, Yuchen Xia, Xinhui Wang, Yueqin Xia, Shang Wu & Li He

  2. Shantou Clinical Medical College of Jinan University (Shantou Central Hospital), College of Life Science and Technology, Jinan University, Guangzhou, China

    Jianshuang Li & Qinghua Zhou

  3. State Key Laboratory of Bioactive Molecules and Druggability Assessment, Guangdong Basic Research Center of Excellence for Natural Bioactive Molecules and Discovery of Innovative Drugs, College of Life Science and Technology, Jinan University, Guangzhou, China

    Jianshuang Li & Qinghua Zhou

  4. Life Science Institute, Jinzhou Medical University, Jinzhou, China

    Xuan Guo

  5. National Engineering Research Center of Genetic Medicine, Guangzhou, China

    Qinghua Zhou

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Contributions

Conceptualization and experimental design were carried out by L.H., Y.Z. and Q.Z. Experiments, data collection and analysis were carried out by Y.Z., Yuchen Xia, X.W., Yueqin Xia, S.W., J.L., X.G., Q.Z. and L.H. All authors read and agreed to the published version of the paper.

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Correspondence to Xuan Guo, Qinghua Zhou or Li He.

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Nature Cell Biology thanks Martin van der Laan, Zhiyin Song and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 CG30159 is expressed in Drosophila intestinal stem cells (ISCs) and forms mitochondrial subdomains in cultured insect cells.

a. Expression pattern of CG30159-Gal4 in fly intestine visualized with myr-tdTomato. DAPI staining indicates the cell nuclei throughout the figure. Scale bars: 500 μm. b. CG30159 is expressed in ISCs identified by Delta staining in regions R1, R2, R4 and R5 of the intestine. Arrowheads denote ISCs. Scale bars: 20 μm. c. CG30159-Gal4 driven tdTomato co-staining with Dl-LacZ. Arrowheads denote Dl+ stem cells. Scale bars: main panel 20 μm, magnified insets 10 μm. d. CG30159-Gal4 driven tdTomato co-staining with Su(H)Gbe-LacZ. The white arrowheads point to Su(H)Gbe+ enteroblasts. Scale bars: main panel 20 μm, magnified insets 10 μm. e. Representative images of CG30159 immunofluorescence staining in esg+ stem cells. Arrowheads denote membrane subdomains. Scale bar: 1 μm. f. Representative images of immunofluorescence staining for Flag-tagged CG30159 in S2 cells. Scale bars: main panels 5 μm, magnified insets 2 μm. g. qPCR analysis of CG30159 knockdown efficiency in adult flies. n = three biological replicates. Data are presented as mean ± S D. (g): two-tailed unpaired Student’s t-test.

Extended Data Fig. 2 Structural and truncation analysis of MISO.

a. AlphaFold structure prediction of CG30159 (dMISO) and its human orthologue C3orf33 (hMISO). The prediction confidence scores (0-100) are visually represented using a colour scale. The alignment of the structures of the two is displayed on the right, with aligned regions superimposed and highlighted in colour. b. Schematic representation of full-length MISO and corresponding truncation mutants. Transmembrane domains are highlighted in yellow. c. Representative images of U2OS cells expressing Flag-tagged full-length MISO and corresponding truncation mutants. Scale bars: 10 μm. d,e. Representative expansion microscopy images (d) and corresponding line profiles (e) showing the localization of MISO-N-Flag and TOMM20 in U2OS cells. Scale bars: 2 μm (post-expansion). f,g. Representative expansion microscopy images (f) and corresponding line profiles (g) showing the localization of MISO-ΔC-Flag and TOMM20 in U2OS cells. Scale bars: 5 μm (post-expansion).

Extended Data Fig. 3 MISO regulates mitochondrial morphology both in vitro and in vivo.

a. Sequence analysis of MISO alleles in wild-type (WT) and CRISPR/Cas9-generated MISO knockout (MISO-KO) U2OS cells. b. PCR-based genotyping of MISO locus in WT and MISO-KO U2OS cells. c-d. Representative TEM images (c) and corresponding quantification (d) of mitochondrial morphology in WT and MISO-KO U2OS cells. Scale bars: main panels 2 μm, magnified insets 500 nm. n = 44, 57 for MISO-WT and MISO-KO from three biologicalal replicates. e. Representative images illustrating the methodology for the quantitative analysis of mitochondrial morphology using Fiji. Scale bar: 2 µm. f-g. Representative images (f) and corresponding quantification (g) of mitochondrial morphology in WT and MISO-KO U2OS cells expressing an empty vector (pLenti), MISO isoform 1-Flag (MISO iso1-Flag) or MISO isoform 2-Flag (MISO iso2-Flag). Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. h. Representative images show that both MISO isoform 1 and isoform 2 can establish mitochondrial subdomains. Scale bars: main panels 10 μm, magnified insets 2 μm. i. Representative images and line profiles showing the localization of MISO isoform 1 and TOMM20 in U2OS cells. Scale bars: main panel 10 μm, magnified insets 1 μm. j. Representative images and line profiles showing the localization of MISO isoform 2 and TOMM20 in U2OS cells. Scale bars: main panel 10 μm, magnified insets 1 μm. k. qPCR analysis of MISO knockdown efficiency in various cell lines treated with the indicated shRNAs. NT: non-targeted. n = three biological replicates. l. Representative images and corresponding quantification of mitochondrial morphology in U2OS cells treated with the indicated shRNAs. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. m. Representative images and corresponding quantification of mitochondrial morphology in PLC/PRF/5 cells treated with the indicated shRNAs. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. n. Representative images and corresponding quantification of mitochondrial morphology in HuH-7 cells treated with the indicated shRNAs. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. o. Schematic of mMISO/E130311K13Rik flox allele and KO allele generated by cre recombination. p. PCR-based genotyping of mMISO locus in mMISOflox/flox and mMISO−/− mice. q. Representative images and corresponding quantification of mitochondrial morphology in mMISOflox/flox and mMISO−/− primary mouse hepatocytes. Scale bars: main panels 10 μm, magnified insets 5 μm. n = Three biological replicates. r. Representative images and corresponding quantification of mitochondrial morphology in mMISOflox/flox and mMISO−/− mouse embryonic fibroblasts (MEFs). Scale bars: main panels 20 μm, magnified insets 5 μm. n = three biological replicates. s-t. Representative TEM images (s) and corresponding quantification (t) of mitochondrial morphology in liver cells from mMISOflox/flox and mMISO−/− mice. Scale bars: main panels 2 μm, magnified insets 500 nm. n = 32, 34 for mMISOflox/flox and mMISO−/− from three biological replicates. u. Representative images and corresponding quantification of mitochondrial morphology in WT and MISO-KO U2OS cells treated with CCCP. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. v. Representative images and corresponding quantification of mitochondrial morphology in WT and MISO-KO U2OS cells expressing MFF-Flag. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. w. Quantification of mitochondrial parameters from Fig. 2 (l). n = three biological replicates. x. Immunoblots showing DRP1 and FIS1 knockdown efficiency in U2OS cells. Data are presented as mean ± SD. (d) and (t): two-tailed non-parametric Mann-Whitney U-test; (g) and (w): two-tailed nested t-test; (k), (m) and (n): ordinary one-way ANOVA with Tukey’s multiple comparisons test; (l), (q) and (r): two-way ANOVA with Tukey’s multiple comparisons test.

Extended Data Fig. 4 MISO forms inner mitochondrial membrane subdomains across a range of expression levels.

a. Representative images showing the mitochondrial localization of MISO–Flag expressed in different cell types. Mitochondria were marked with HSP60. Scale bars: main panels 10 μm, magnified insets 2 μm. b. Immunoblot analysis of lysates from MISO-WT and MISO-KO expressing MISO–Flag cells by anti-MISO antibody. Arrowheads indicate the expected band, and asterisks denote non-specific bands. c. Representative images of MISO localization in U2OS cells expressing untagged MISO, detected with an anti-MISO antibody. Scale bars: main panel 10 μm, magnified insets 5 μm. d. A diagram of mMISO gene structure with 3×Flag tag knock-in element. e. Immunoblot analysis of endogenous mMISO expression across various mouse tissues. HSP60 and GAPDH were used as loading controls. f. A Scheme of the lentiviral cassette for the inducible expression of MISO–Flag. g. Western blot analysis of MISO–Flag expression in U2OS cells following induction with 10 ng/ml doxycycline. h. Representative images of MISO–Flag fluorescence intensity in mitochondrial filaments and subdomains across a range of expression levels. Scale bars: main panels 10 μm, magnified insets 2 μm. i. Representative images and corresponding colocalization analysis demonstrating MISO localization to the inner mitochondrial membrane. Scale basr: 1 μm. j. Cell fractionation assay of MISO–Flag stable expressing cells. WCL: whole cell lysate; Cyto: cytoplasm; Mito: mitochondria. k. Protease protection assay of mitochondria isolated from U2OS cells stably expressing MISO–Flag. l. Mitochondrial protein extraction was performed using sodium carbonate treatment. Total mitochondria (T) were separated into supernatant (S) and pellet (P) fractions.

Extended Data Fig. 5 PHBs are enriched in MISO-induced subdomains but are dispensable for subdomain formation.

a-b. Representative images (a) and corresponding colocalization quantification (b) of MISO with the indicated mitochondrial proteins in U2OS cells. Colocalization was assessed using Pearson’s correlation coefficient. Scale bars: 2 μm. Number of analysed mitochondrial subdomains: n = 20 (SLC25A4-Myc), 17 (SLC25A5-Myc), 21 (SLC25A6-Myc), 21 (PHB1-Myc), 21 (PHB2-Myc), 17 (TIMM23), 19 (VDAC1-HA), 19 (VDAC2-HA), 21 (COX5B), 16 (TOMM20), 22 (TOMM40), 20 (TOMM70), 20 (SDHB), 22 (STOML2), 18 (ATAD3A-Myc), and 19 (ATAD3B-Myc). Three biological replicates. c. Representative images demonstrating the colocalization of MISO–Flag (green) with endogenous PHB2 (magenta). Scale bars: main panel 10 μm, magnified insets 5 μm. d. Immunoblots of the indicated proteins from anti-Flag immunoprecipitation of HEK293T cells overexpressing either an empty vector (pcDNA3.1), Flag-MISO or MISO–Flag. e. Representative images of mitochondrial subdomains in U2OS cells expressing MISO–Flag and treated with the indicated shRNAs. Scale bars: main panels 10 μm, magnified insets 2 μm. f. Immunoblots showing the knockdown efficiency of PHB1 and PHB2 in U2OS cells.

Extended Data Fig. 6 MISO-enriched subdomains drive mitochondrial fission while suppressing fusion.

a. Representative images illustrating the methodology for quantitative analysis of mitochondrial length and subdomain number using Fiji. Scale bar: 2 µm. b. Representative images showing the localization of PHB2–mNeonGreen in live U2OS cells, with or without MISO–Flag expression. Scale bars: 10 µm. c. Representative time-lapse images showing subdomain movement toward the mitochondrial periphery in cells co-expressing MISO–Flag, PHB2–mNeonGreen, and Mito–RFP. Scale bar: 2 µm. d. Representative time-lapse images showing the dissociation of subdomain from the mitochondrial periphery. Scale bar: 2 µm. e. Representative images of mitochondrial fusion in HeLa cells following PEG-mediated fusion assays. Scale bars: 10 μm. f. Representative images showing the absence of OPA1 within MISO-enriched mitochondrial subdomains in U2OS cells. Scale bars: 2 µm. g. Representative images showing the presence of MFN1 and MFN2 within MISO-enriched mitochondrial subdomains in U2OS cells. Scale bars: 2 µm. h-i. Representative images (h) and corresponding quantification (i) of mitochondrial morphology in U2OS cells overexpressing MISO–Flag, with or without co-expression of MFN1-mCherry or MFN2-mCherry. Scale bars: main panels 20 μm, magnified insets 5 μm. n = three biological replicates. j-k. Representative images (j) and corresponding quantification (k) of mitochondrial morphology in WT and MISO-KO U2OS cells treated with the indicated shRNAs. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. l. Immunoblots showing the knockdown efficiency of MFN1, MFN2, and OPA1 in U2OS cells. m. Representative images of MISO–Flag localization relative to various cellular markers in U2OS cells. Scale bars: main panels 10 μm, magnified insets 2 μm. n. Colocalization analysis with quantification of Mander’s coefficient between MISO–Flag and various cellular markers. n = 26, 27, 26, 28, 30, 28 cells for Mito, ER, Golgi, Lyso, Perox and LD from three biological replicates. Data are presented as mean ± SD. (i): two-way ANOVA with Tukey’s multiple comparisons test; (k): two-tailed nested t-test. (n): ordinary one-way ANOVA with Sidak’s multiple comparisons test.

Extended Data Fig. 7 MISO is involved in the recruitment of mitochondrial nucleoids and modulates the level and activity of OXPHOS complexes.

a. Representative images of co-staining for the Mito–GFP and mtDNA/mtRNA (labelled via FASTKD2). Scale bars: main panels 10 μm, magnified insets 2 μm. b. Representative images showing the spatial association between mtDNA and MISO-enriched subdomains in U2OS cells with or without EB treatment. Scale bars: main panels 10 μm, magnified insets 2 μm. c. Quantification of DNA enrichment in subdomains from (b). n = 21, 21cells for Vehicle and EB (3 d), respectively. Three biological replicates. d. Representative expansion microscopy images showing the association between MISO and TFAM in U2OS cells. Scale bars: main panel 20 μm (post-expansion), magnified insets 5 μm (post-expansion). e. Representative expansion microscopy images depicting distinct types of mitochondrial subdomains associated with TFAM in U2OS cells. Scale bars: 5 μm (post-expansion). f. Representative time-lapse images of mtDNA (labelled with PicoGreen) and PHB1-mCherry (marking MISO) in U2OS cells. Arrowheads indicate the subdomain. Scale bar: 2 μm. 1g. Representative images showing the spatial association among MISO-enriched subdomains, mtDNA and replicating DNA (labelled with EdU) in U2OS cells. Scale bars: main panels 10 μm, magnified insets 2 μm. h. Representative images showing the spatial association among MISO-enriched subdomains, mtDNA and SSBP1 in U2OS cells. Scale bars: main panels 10 μm, magnified insets 2 μm. i. Immunoprecipitation with anti-Flag validates the interaction between MISO and SSBP1. j. Representative images of mitochondrial subdomains in U2OS cells expressing MISO–Flag with SSBP1 knockdown. k. Immunoblots showing the knockdown efficiency of SSBP1 in U2OS cells. l. Representative images of U2OS cells expressing PHB2–mNeonGreen and MISO–Flag incubated with MitoSOX or TMRM. MitoSOX or TMRM intensities were represented by calibrated colour scale. Scale bars, 2 μm. m. The gating strategy for determining TMRM fluorescence intensity. n. Flow analysis and quantification of TMRM fluorescence intensity in WT and MISO-KO U2OS cells. n = three biological replicates. o. Seahorse analysis of the mitochondrial respiratory capacity in WT and MISO-KO U2OS cells. n = six biological replicates. p. Flow cytometry analysis and quantification of TMRM fluorescence intensity in U2OS cells expressing an empty vector (pLenti) or MISO–Flag. n = three biological replicates. q. Seahorse analysis of the mitochondrial respiratory capacity in U2OS cells expressing an empty vector (pLenti) or MISO–Flag. r-s. Immunoblots (r) and corresponding quantifications (s) of indicated proteins in MISO-WT, MISO-KO and MISO-OE U2OS cells. n = three biological replicates. t-u. Immunoblots (t) and corresponding quantifications (u) of mitochondrial OXPHOS complexes in liver tissues from mMISOflox/flox and mMISO−/− mice. Each lane represents an individual mouse. n = four biological replicates. v-w. Blue-native PAGE-based in-gel activity assays (v) and corresponding quantification (w) of mitochondrial respiratory complexes I, II, IV, and V in WT and MISO-KO U2OS cells. n = three biological replicates. Data are presented as mean ± SD. (c): two-tailed non-parametric Mann-Whitney U-test; (n), (o), (p), (q), (u) and (w): two-tailed unpaired Student’s t-test; (s): ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Extended Data Fig. 8 MISO controls mtDNA homeostasis, and MTFP1 acts as a key effector in MISO-driven mitochondrial fission.

a. Representative image MISO-enriched subdomains with loss of TOMM20. Scale bar: 1 μm. b. A line profiles of MISO-enriched subdomain with loss of TOMM20. c. qPCR analysis of mtDNA levels in mMISOflox/flox and mMISO−/− Hepatocytes. n = twelve biological replicates. d. qPCR analysis of mtDNA levels in mMISOflox/flox and mMISO−/− MEFs. n = thirteen biological replicates. e. qPCR analysis of mtDNA levels in tissues from wild-type and mMISO knockout (KO) mice. n =eight biological replicates for Liver, Kidney and eBAT; n =seven biological replicates for Brain; n =ten biological replicates for iBAT. f. Representative images depicting mitochondrial subdomains in U2OS cells expressing MISO–Flag with MTFP1 knockdown. Scale bars: main panels 10 μm, magnified insets 2 μm. g. Immunoblots showing the knockdown efficiency of two distinct shRNA constructs targeting MTFP1 in U2OS cells. h. Representative images of mitochondrial morphology in U2OS cells expressing pLenti or MISO–Flag and treated with the indicated shRNAs. Scale bars: main panels 10 μm, magnified insets 5 μm. i. Quantification of mitochondrial parameters from (h). n = three biological replicates. j-k. Representative images (j) and corresponding quantification (k) of mitochondrial morphology in WT and MISO-KO U2OS cells expressing an empty vector (pcDNA3.1) or MTFP1-Myc. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. l. Quantification of mitochondrial parameters from (j). n = three biological replicates. m-n. Representative immunoblots (m) and corresponding quantifications (n) of indicated proteins from WT and MISO-KO U2OS cells incubated in the presence or absence of EB. n = three biological replicates. Data are presented as mean ± SD. (c), (d), (e) and (k): two-tailed unpaired Student’s t-test; (i) and (l): two-tailed nested t-test; (n): two-way ANOVA with Tukey’s multiple comparisons test.

Extended Data Fig. 9 IMM stress stabilizes MISO to promote SMEM formation.

a. CHX chase assay assessing MISO protein stability in U2OS cells treated in the presence or absence of EB. b. Quantification of indicated protein levels from (a). n = three biological replicates. c. Representative images of the Drosophila larval fat body harbouring GFP-marked somatic clones of the indicated genotypes, immunostained for HA-tagged endogenous dMISO. Scale bars: 50 μm. d. Quantification of relative MISO fluorescence intensity from (c). n = 20, 25, 22, 23, 25, 20, 20, 20, 21, 23, 28 cell clones for Luc-i, NP15.6-i, ND-49-i, ND-23-i, SdhC-i, UQCR-Q-i, RFeSP-i, COX6C-i, COX5A-i, ATPsyn-beta-i-1 and ATPsyn-beta-i-2, respectively. Three biological replicates. e. Representative confocal images of the Drosophila larval fat body harbouring GFP-marked somatic clones of dMISO knockdown. Scale bars: 50 μm. f. Quantification of relative MISO fluorescence intensity from (e). n = 20, 20 cell clones for Luc-i and dMISO-i from three biological replicates. g-h. Immunoblots (g) and corresponding quantifications (h) of MISO protein levels in U2OS cells treated for 24 h with the indicated compounds (1 µM Rotenone, 5 mM NaN3, 2 µM Oligomycin and 2 µM CCCP), except for EB, which was applied at 50 ng/mL for 3 days. Relatively low dosages of the compounds were used to avoid triggering significant mitophagy. n = three biological replicates. i-j. Representative images (i) and corresponding quantifications (j) of PHB2-enriched microdomains (white arrows) in WT and MISO-KO U2OS cells treated with the indicated compounds as described in (g-h). Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. k-l. Representative images (k) and corresponding quantification (l) of mitochondrial phenotype in WT and MISO-KO U2OS cells expressing mCherry-Parkin, treated with DMSO or 10 μM CCCP for 12 h. Scale bars: main panels 20 μm, magnified insets 5 μm. n = three biological replicates. m-n. Representative images (m) and corresponding quantifications (n) of PHB2-enriched microdomains (white arrows) in WT and MISO-KO U2OS cells treated with indicated shRNAs. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. o. Immunoblots showing the knockdown efficiency of MIC60 and ATP5A in U2OS cells. p. CHX chase assay assessing MISO protein stability in U2OS cells stably expressing MISO and treated with the indicated shRNAs. q. Quantification of indicated protein levels from (p). n = three biological replicates. r. qPCR analysis of MISO mRNA levels in U2OS cells transduced with the indicated shRNAs. n = three biological replicates. Data are presented as mean ± SD. (b) and (f): two-tailed unpaired Student’s t-test; (d): ordinary one-way ANOVA with Dunnett’s multiple comparisons; (h), (j), (n), (q) and (r): ordinary one-way ANOVA with Tukey’s multiple comparisons test; (l): two-way ANOVA with Tukey’s multiple comparisons test.

Extended Data Fig. 10 SMEM arise from collapsed and condensed inner mitochondrial membranes through MISO oligomerization.

a. Representative TEM images of mitochondria in U2OS cells expressing APEX2-MISO. Scale bar: 500 nm. b. Representative images of U2OS cells expressing PHB1-mCherry and MISO–Flag, incubated with nonyl acridine orange (NAO). White arrows indicate subdomains. Scale bars: main panels 10 μm, magnified insets 2 μm. c-d. Representative immunoblots (c) and corresponding quantification (d) of proteins involved in the maintenance of mitochondrial cristae structure and morphology from U2OS cells expressing an empty vector (pLenti) or MISO–Flag. n = three biological replicates. e. Crosslinking analysis of the oligomerization of two MISO isoforms in U2OS cells using disuccinimidyl glutarate (DSG). Arrowheads point to the MISO dimers. Asterisks denote non-specific bands. f. Crosslinking analysis of MISO oligomerization using DSG in U2OS cells expressing MISO–Flag and treated with the indicated shRNAs. Red arrowheads point to the MISO dimers and trimers. g. Crosslinking analysis of oligomerization of MISO and corresponding truncation variants in U2OS cells. h-i. Representative images (h) and corresponding quantification (i) of mitochondrial morphology in HEK293T cells expressing OMM-GFP or OMM-MISO. Scale bars: main panels 10 μm, magnified insets 2 μm. n = three biological replicates. j-k. Representative images (j) and corresponding quantification (k) of mitochondrial morphology in U2OS cells expressing OMM-MISO truncation mutants. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. l. Representative images showing the localization of IMM-MISO and IMM-MISO-M in U2OS cells. m-n. Representative images (m) and corresponding quantification (n) of mitochondrial morphology in U2OS cells expressing IMM-MISO truncation mutants. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. Data are presented as mean ± SD. (d) and (i): two-tailed unpaired Student’s t-test; (k) and (n): ordinary one-way with Tukey’s multiple comparisons.

Supplementary information

Reporting Summary (download PDF )

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Supplementary Tables 1–7.

Supplementary Video 1 (download MP4 )

Translocation of SMEM to the mitochondrial periphery. Timelapse imaging of U2OS cells stably expressing PHB2–mNeonGreen, MISO-3×Flag and mito–RFP. Scale bar, 2 μm.

Supplementary Video 2 (download MP4 )

Representative live imaging of type I and type II fission events relative to SMEM. Timelapse imaging of U2OS cells stably expressing PHB2–mNeonGreen, MISO-3×Flag and mito–RFP. Type I fission: fission event occurring within a 0–1 μm range from the SMEM spot. Type II fission: fission event occurring within a 1–2 μm range from the SMEM spot. Scale bar, 2 μm.

Supplementary Video 3 (download MP4 )

Detachment of SMEM from mitochondria through peripheral fission. Timelapse imaging of U2OS cells stably expressing PHB2–mNeonGreen, MISO-3×Flag and mito–RFP. Scale bar, 2 μm.

Supplementary Video 4 (download MP4 )

Representative live imaging of type I, II and III mitochondrial fusion events. Timelapse imaging of U2OS cells stably expressing PHB2–mNeonGreen, MISO-3×Flag and mito–RFP. Type I fusion: fusion between two mitochondrial termini without SMEM. Type II fusion: fusion between two mitochondrial termini with SMEM present on one terminus. Type III fusion: fusion between two mitochondrial termini with SMEM present on both termini. Scale bar, 2 μm.

Supplementary Video 5 (download MP4 )

Live imaging of mtDNA and SMEM. U2OS cells stably expressing PHB1-mCherry and MISO-3×Flag were stained with PicoGreen to visualize mtDNA. Scale bar, 2 μm.

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Unprocessed western blots and gels for main figures and extended data figures.

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Statistical source data for main figures and extended data figures.

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Zhang, Y., Xia, Y., Wang, X. et al. MISO regulates mitochondrial dynamics and mtDNA homeostasis by establishing membrane subdomains. Nat Cell Biol 28, 255–267 (2026). https://doi.org/10.1038/s41556-025-01829-0

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