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Mitochondria-associated condensates maintain mitochondrial homeostasis and promote lifespan

Nature Aging (2025)Cite this article

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Abstract

Membraneless organelles assembled by liquid–liquid phase separation interact with diverse membranous organelles to regulate distinct cellular processes. It remains unknown how membraneless organelles are engaged in mitochondrial homeostasis. Here we demonstrate that mitochondria-associated translation organelles (MATOs) mediate local synthesis of proteins required for structural and functional maintenance of mitochondria. In Caenorhabditis elegans, the RNA-binding protein LARP-1 (La-related protein 1) orchestrates coalescence of translation machinery and multiple RNA-binding proteins via liquid–liquid phase separation into MATOs that associate with mitochondria in a translocase of the outer membrane complex-dependent manner. LARP-1 deficiency markedly reduces mitochondrial protein levels, impairing cristae organization and ATP production. Specifically, we show that the membrane-shaping MICOS subunit IMMT-1(MIC60) and the ATP synthase β subunit ATP-2, both being important for cristae organization, are synthesized in LARP-1 MATOs. During aging and starvation, LARP-1 MATOs dissociate from mitochondria; however, mitochondrion-persistent LARP-1 MATOs protect mitochondrial health and greatly extend lifespan. These findings suggest an important mitochondrion-regulating mechanism in aging and stress.

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Fig. 1: larp-1 mutations impair mitochondrial structure and functions.
Fig. 2: Loss of larp-1 affects mitochondrial cristae by down-regulating proteins important for cristae organization.
Fig. 3: LARP-1 is engaged in translation on mitochondria and interacts with translation machinery and additional RBPs.
Fig. 4: LARP-1 undergoes LLPS to form condensates.
Fig. 5: LARP-1 orchestrates the coalescence of translation machinery, RBPs, and mRNAs on the mitochondrial surface.
Fig. 6: The TOM complex is required for mitochondrial association of LARP-1 membraneless organelles.
Fig. 7: Mitochondria-associated LARP-1 membraneless organelles are essential for mitochondrial homeostasis.
Fig. 8: Mitochondria-anchored LARP-1 MATOs promote mitochondrial functions and animal lifespan.

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Data availability

All data are available in the main text and the Supplementary Information. The RNA analysis data generated in this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus under the accession number GSE300049. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the Proteomics Identifications Database (PRIDE) partner repository with the dataset identifiers PXD065160 and PXD065187. Numerical source data underlying all graphical representations and statistical descriptions and uncropped gels and blots are provided with the paper. All other data supporting the findings of this study are available from the authors on reasonable request.

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Acknowledgements

This research was supported by grants 92354303 and 91954204 from the National Science Foundation of China, 2021YFA1300302 from the National Basic Research Program of China, and 202001BB050077 from Yunnan Province Science and Technology Department. C.Y. is supported by the Program of Yunnan Province Leading Talents in Science and Technology (202105AB160003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank I. Hanson for proofreading the manuscript and the Caenorhabditis Genetics Center for C. elegans strains used in this study.

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  1. These authors contributed equally: Yan Bai, Tengfei Ma.

Authors and Affiliations

  1. State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan and Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China

    Yan Bai, Tengfei Ma, Shan Zhao, Shalan Li, Xin Wang, Jingyang Li, Wenhao Sun, Yang Yang, Fenglian Liu, Qian Shan, Zizhen Qin, Nan Liu, Jie Zhang, Fei Tian, Mei Duan, Shunkai Chen, Fan Lai, Qingfeng Chen, Xuna Wu & Chonglin Yang

  2. Southwest United Graduate School, Kunming, China

    Fan Lai & Chonglin Yang

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Contributions

C.Y. supervised the study. Y.B. and T.M. performed most of the experiments and interpreted the data with C.Y. S.Z. screened for the mutants. X. Wang performed TEM and analysis. S.L. and X. Wu performed proteomic analysis. J.L. and F. Lai helped perform data analysis. W.S., Y.Y., F. Liu, Q.S., Z.Q., N.L., J.Z., F.T., M.D., S.C. and Q.C. contributed to the experiments and materials. Y.B., T.M. and C.Y. prepared the manuscript with discussion between all authors.

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Correspondence to Chonglin Yang.

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Extended data

Extended Data Fig. 1 Characterization of the larp-1 gene.

a, Schematic representation of the C. elegans larp-1 gene (top), the encoded protein and human LARP1 (bottom). Filled boxes and lines indicate exons and introns in the gene. The yq421 and yq565 mutations are indicated. In C. elegans (C. e) LARP-1 and human (H. s) LARP1 proteins, the LAM (La motif), RRM (RNA recognition motif), and DM15 domains are indicated in different colors. The similarity of each domain between the two proteins is indicated. b, Relative mRNA levels in indicated larp-1 mutants, n = 4 independent experiments for each genotype. c, LARP-1 abundance determined with LC-MS in indicated larp-1 mutants. n = 5 (N2), 4 (larp-1(yq421)) and 4 (larp-1(yq565)) independent experiments. d-e Expression of the larp-1 gene in different tissues. Images (d) of LARP-1::GFP driven by the larp-1 promoter (Plarp-1larp-1::gfp) are shown for the indicated tissues. Boxed regions are magnified (3 ×) in bottom left in merged images. Quantifications of LARP-1::GFP and TOMM-20 intensities along the line in insets of merged images in (d) are shown in (e). f,g, Representative images (f) and quantification (g) of the rescue of defective mitochondria in the hypodermis of larp-1(yq565) mutants by transgenic Plarp-1larp-1::gfp. h,i, Representative images (h) and quantification (i) of the rescue of defective mitochondria in the hypodermis of larp-1(yq421) mutants by human LARP1 (hLARP1) under the control of the larp-1 promoter (Plarp-1hLARP1::gfp). ≥120 animals were analyzed for each genotype. n = 5 independent experiments. Scale bars, 5 μm. For statistical analyses, P values were determined using one-way ANOVA (b-c) and two-way ANOVA (g, i). Individual data points (mean ± s.e.m.) and exact P values are shown. ***, P < 0.001.

Source data

Extended Data Fig. 2 Proteomic analysis of mitochondrial proteins in larp-1 mutants and TEM analysis of mitochondrial structure in MICOS mutants and animals with RNAi of ATP subunits.

a, Schematic summary of affinity-based purification of mitochondria and LC-MS analysis. b, Western blotting of IMMT-1 in mitochondria immunoprecipitated from indicated strains. The yq640 strain expresses TOMM-20::RFP::3xHA. c, Graphic summary of unchanged (Other-Mito, light orange) and down-regulated (Sig. Down-Mito, deep blue) mitochondrial proteins in larp-1 mutants. Ensembl-Mito is the set of C. elegans proteins in the ENSEMBL database annotated with the GO Cellular Component term “mitochondrion”. Mass-Mito is the subset of Ensembl-Mito proteins identified by MS in both N2 and larp-1 mutants. d, TEM images (left) and quantification (right) of abnormal mitochondria in mutants of MICOS components. Arrowheads indicate inner membrane stacks. ≥116 mitochondria were analyzed in n = 5 (hypodermis) and 4 (pharyngeal muscle) (immt-1(yq160)), n = 3 (hypodermis) and 3 (pharyngeal muscle) (chch-3(tm2336)), n = 4 (hypodermis) and 3 (pharyngeal muscle) (F54A3.5(yq206)), n = 6 (hypodermis) and 3 (pharyngeal muscle) (W04C9.2(yq663)), n = 4 (hypodermis) and 3 (pharyngeal muscle) (moma-1(yq664)) animals. e, TEM images (left) and quantification (right) of abnormal mitochondria in animals treated with RNAi against larp-1 and genes encoding the indicated subunits of ATP synthase and respiratory complex 1. Arrowheads indicate inner membrane stacks. ≥265 mitochondria were analyzed in n = n = 4 (Ctrl), 4 (atp-2), 5 (atp-3), 6 (atp-5), 4 (larp-1), 5 (ndub-7), 4 (nuo-4), 5 (nuo-6) RNAi animals. P values were determined using the one-way ANOVA (e). Individual data points (mean ± s.e.m.) and exact P values are shown. ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, not significant (P > 0.05).

Source data

Extended Data Fig. 3 Amelioration of defects in larp-1 mutants by over-expressing IMMT-1::GFP and other components of MICOS, ATP synthase and respiratory complex 1.

a-f, Amelioration of ATP levels, n = 4 independent experiments (a), body bends, n = 10 animals (b), number of progeny, n = 5 independent experiments (c), developmental time, n = 5 independent experiments (d) embryonic lethality, n = 5 independent experiments (e) and adult lifespan, n = 4 independent experiments (f) of larp-1(yq421) mutants by over-expressed IMMT-1::GFP. g, Representative images (left) and quantification (right) of rescue of defective mitochondria in the hypodermis of larp-1(yq421) mutants by W04C9.2::GFP, MOMA-1::GFP, and ATP-2::GFP under the control of the semo-1 promoter. ≥200 animals were analyzed for each genotype. n = 8 independent experiments. h, TEM images (left) and quantification (right) of mitochondria with inner membrane stacks in larp-1(yq421) mutants over-expressing W04C9.2::GFP, MOMA-1::GFP, and ATP-2::GFP. ≥149 mitochondrial were analyzed for each genotype. n = 4 or 5 independent experiments. i, Representative images (left) and quantification (right) of rescue of defective mitochondria in the hypodermis of larp-1(yq421) mutants by CHCH-3::GFP, F54A3.5::GFP, and NUO-4::GFP under the control of the semo-1 promoter. ≥240 animals were analyzed for each genotype. n = 8 independent experiments. Scale bars, 5 μm (g, i), 1 μm (h). For statistical analyses, P values were determined using the two-tailed unpaired Student’s t test (a-f) and two-way ANOVA (g-i). Individual data points (mean ± s.e.m.) and exact P values are shown. ***, P < 0.001.

Source data

Extended Data Fig. 4 Proximity labeling-based identification of mRNAs and proteins associated with LARP-1.

a, Schematic depiction of proximity labeling and identification of mRNAs and proteins associated with LARP-1. Proximity labeling was conducted with LARP-1::APEX2 animals, and LARP-1::GFP-expressing animals were used as control. b, Graphic summary of data reproducibility (mRNAs) derived from the control (LARP-1::GFP) and LARP-1::APEX2 samples. 3 repeats were performed for each group. c, Graphic summary of LARP-1::APEX2-enriched mRNAs encoding proteins of different intracellular organelles. d. Comparison of LARP-1::APEX2-enriched mRNAs encoding mitochondrial proteins with down-regulated mitochondrial protein identified by LC-MS in larp-1 mutants. e-g, qPCR analysis of the levels of mRNAs encoding the indicated protein subunits of MICOS complex (e), respiratory complex I (f), and ATP synthase complex (g)in N2, larp-1(yq421) and larp-1(yq565) animals. n = 3 independent experiments. h, Western-blotting analysis of LARP-1::APEX2-enriched proteins with Streptavidin-HRP antibody. i, Representative heatmaps of RNA-binding proteins enriched by LARP-1::APEX2. Triplicate samples were analyzed for each group. Data are graphed as mean ± s.e.m. P values were determined using one-way ANOVA (e-g). NS, not significant (P > 0.05). Exact P values are shown in SourceData ED Fig 4 (e-g).

Source data

Extended Data Fig. 5 Rescue of larp-1 mutants by transgenic LARP::GFP and FRAP analysis of LARP-1::GFP.

a-b, Images (a) and quantification (b) of the rescue of defective mitochondria in the hypodermis in larp-1(yq421) mutants by transgenic LARP-1::GFP driven by the col-19 promoter (Pcol-19larp-1::gfp). ≥169 animals were analyzed for each genotype. n = 5 independent experiments. c-d, Images (c) and quantification (d) of the rescue of defective mitochondria in the hypodermis and muscle in larp-1(yq421) mutants by transgenic TOMM-20(1-70)::LARP-1::GFP driven by the larp-1 promoter (Plarp-1tomm-20(1-70)::larp-1::gfp). ≥172 animals were analyzed for each genotype. n = 5 independent experiments. e-f, Images (e) and quantification (f) of the rescue of defective mitochondria in the hypodermis in larp-1(yq421) mutants by transgenic SCAV-3::LARP-1::GFP driven by the larp-1 promoter (Plarp-1scav-3::larp-1::gfp). ≥134 animals were analyzed for each genotype. n = 5 independent experiments. g-h, Left: Time-course images of the recovery of LARP-1::GFP signal in FRAP assays on mitochondria (labeled with TOMM-20::RFP) in the hypodermis (g) and oocyte (h). Photo-bleached regions are framed with dashed lines. Right: Quantification of the recovery of LARP-1::GFP signals. Scale bars, 5 μm. For statistical analyses, P values were determined using two-way ANOVA (b, d, f). Individual data points (mean ± s.e.m.) and exact P values are shown. ***, P < 0.001. NS, not significant (P > 0.05).

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Extended Data Fig. 6 Characterization of LLPS of LARP-1 with translation machinery proteins and RBPs.

a, Graphic summary of the IDRs in LARP-1, predicted with the Database of Disordered Protein Predictions (D2P2) software. b, Graphic depiction of in vitro LLPS assays. c, Western blotting of LARP-1::GFP in sedimentation assays. S, supernatant, P, pellet. 3 independent experiments with similar results were performed. d, Graphic summary of the correlation of LARP-1::GFP LLPS with NaCl concentration. e, Coomassie-blue staining of the indicated translation proteins and RBPs fused with His6 and Strep tags, as well as mCh or BFP for use in in vitro LLPS assays. ≥ 3 independent experiments with similar results were performed. f, Individual mCh-fused translation machinery proteins (3 μM) as indicated were incubated with GFP or LARP-1::GFP for 1 min or 30 min. Images are shown for mCh and GFP fluorescence. g-h, Time-course images (g) and western blotting (h) of LLPS of LARP-1::GFP with His6-fused PAB-1::mCh, IFE-4::BFP, EIF-4A3::BFP, and RPL-38::BFP. For western blotting, sedimentation was performed and proteins in the supernatant (S) and pellet (P) were detected with antibodies against GFP, mCh and His6. 3 independent experiments with similar results were performed. i-j, Individual mCh-fused RBPs (3 μM) as indicated were incubated with GFP or LARP-1::GFP for 1 min or 30 min. Images are shown for mCh and GFP fluorescence. Scale bars, 5 μm (f-g, i-j).

Extended Data Fig. 7 Analysis of LLPS of RBPs with translation machinery proteins.

a, Individual mCh-fused RBPs (3 μM) as indicated were incubated with BFP or BFP-fused translation machinery proteins for 30 min. Images are shown for mCh and BFP fluorescence. Arrows indicate liquid droplets. Bars, 5 μm. b, Overexpression of RBPs failed to rescue the mitochondrial defects in larp-1 mutants. Representative images and quantifications are shown on the left and right, respectively. Bars, 5 μm. ≥181 animals were analyzed for each genotype. n = 6 independent experiments. For statistical analyses, P values were determined using one-way ANOVA (b). Individual data points (mean ± s.e.m.) and exact P values are shown. NS, not significant (P > 0.05).

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Extended Data Fig. 8 Characterization of the colocalization of LARP-1 membraneless organelles with nascent mRNAs and neopeptides of IMMT-1, ATP-2, and RBPs on the mitochondrial surface.

a, Schematic depiction of transgenic expression of 12×SunTag- and 12×MS2-fused immt-1 or atp-2 and the recognition of 12×SunTag in the neopeptides by scFv::mKate2 and MS2 in the nascent mRNAs by MCB::BFP. b-c, Images of foci containing ATP-2neo (recognized by scFv::mKate2, red) and MS2-fused atp-2 nascent mRNA (recognized by MCP-BFP, purple) on the surface of mitochondria in the hypodermis of N2 and larp-1(yq421) animals expressing Psemo-1SunTag::atp-2-MS2, Psemo-1scFv::mKate2, and Psemo-1MCP::bfp. Mitochondria are labeled with TOMM-20::GFP. Boxed regions are magnified in the adjacent “Zoom 2 ×” images. Arrowheads indicate representative foci on mitochondria. ≥150 mitochondria in each animal were analyzed, n = 10 independent animals. d, Colocalization of LARP-1::GFP with foci containing ATP-2neo (scFv::mKate2, red) and MS2-fused atp-2 nascent mRNA (MCP-BFP, purple) in the hypodermis of N2 animals. Boxed regions are magnified in the adjacent Zoom (2 ×) images. e, Quantification of the fluorescence of LARP-1::GFP (green), ATP-2neo (scFv::mKate2, blue) and atp-2 mRNA (MCP::BFP, purple) in the foci along the purple line in the merged image in (d). f, Representative images showing colocalization within foci of LARP-1::GFP and IMMT-1 (scFv::BFP, purple) with the indicated RBPs in the hypodermis of N2 animals. Individual RBPs fused with mKate2 were expressed under the control of the semo-1 promoter with Plarp-1larp-1::gfp, Psemo-1SunTag::immt-1-MS2, Psemo-1scFv::bfp. Scale bars, 5 μm (b, d, f). For statistical analyses, P values were determined using the two-tailed unpaired Student’s t test. Individual data points (mean ± s.e.m.) and exact P values are shown. ***, P < 0.001.

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Extended Data Fig. 9 The effect of RNAi of translation machinery proteins and RBPs on mitochondrial association of LARP-1 translation-mediating condensates.

a-d, Representative images (a, c) and quantification (b, d) of foci containing LARP-1::GFP-IMMTneo (indicated with scFv::BFP, purple) on mitochondria (labeled with MDH-2::mKate2, red) in the hypodermis of N2 animals treated with RNAi against the indicated translation machinery proteins (a, b) and RBPs (c, d). Scale bars, 5 μm (a, c). ≥30 animals for each group were analyzed, n = 3 independent experiments. For statistical analyses, P values were determined using one-way ANOVA (b and d). Individual data points (mean ± s.e.m.) and exact P values are shown. ***, P < 0.001.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. 457 significantly down-regulated mitochondrial proteins in LC/MS analysis of mitochondria purified by affinity-based approaches. Supplementary Table 2. 653 mRNAs enriched by LARP-1::APEX2-mediated proximity labeling of RNAs. Supplementary Table 3. Mitochondrial protein-encoding mRNAs enriched in LARP-1::APEX2-mediated proximity labeling of RNAs. Supplementary Table 4. Translation-related proteins identified by LARP-1::APEX2-mediated proximity labeling of proteins. Supplementary Table 5. RNA binding proteins enriched by LARP-1::APEX2-mediated proximity labeling of proteins. Supplementary Table 6. The 9 LARP-1-interacting RNA-binding proteins which caused mitochondrial fragmentation when subjected to RNAi knockdown in C. elegans. Supplementary Table 7. List of outer mitochondrial membrane proteins screened by RNAi. Supplementary Table 8. C. elegans strains used in this study. Supplementary Table 9. Expression vectors. Supplementary Table 10. Oligonucleotides used for CRISPR/Cas9-mediated gene editing. Supplementary Table 11. Oligonucleotides used for qRT-PCR. Supplementary Table 12. Antibodies used in this study. Supplementary Table 13. Summary of lifespan experiments.

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Bai, Y., Ma, T., Zhao, S. et al. Mitochondria-associated condensates maintain mitochondrial homeostasis and promote lifespan. Nat Aging (2025). https://doi.org/10.1038/s43587-025-00942-x

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