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MIROs and DRP1 drive mitochondrial-derived vesicle biogenesis and promote quality control

Nature Cell Biology volume 23, pages 1271–1286 (2021)Cite this article

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Abstract

Mitochondrial-derived vesicles (MDVs) are implicated in diverse physiological processes—for example, mitochondrial quality control—and are linked to various neurodegenerative diseases. However, their specific cargo composition and complex molecular biogenesis are still unknown. Here we report the proteome and lipidome of steady-state TOMM20+ MDVs. We identified 107 high-confidence MDV cargoes, which include all β-barrel proteins and the TOM import complex. MDV cargoes are delivered as fully assembled complexes to lysosomes, thus representing a selective mitochondrial quality control mechanism for multi-subunit complexes, including the TOM machinery. Moreover, we define key biogenesis steps of phosphatidic acid-enriched MDVs starting with the MIRO1/2-dependent formation of thin membrane protrusions pulled along microtubule filaments, followed by MID49/MID51/MFF-dependent recruitment of the dynamin family GTPase DRP1 and finally DRP1-dependent scission. In summary, we define the function of MDVs in mitochondrial quality control and present a mechanistic model for global GTPase-driven MDV biogenesis.

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Fig. 1: Biochemical purification of TOMM20+ MDVs.
Fig. 2: The proteome of TOMM20+ MDVs.
Fig. 3: TOMM20+ MDVs contain assembled import and β-barrel protein complexes.
Fig. 4: MIRO1/2-dependent membrane protrusions precede TOM+-MDV biogenesis.
Fig. 5: TOM+-MDV biogenesis requires microtubules.
Fig. 6: The lipidome of TOM+ MDVs.
Fig. 7: TOM+-MDV formation depends on DRP1.
Fig. 8: TOM+-MDV scission depends on the recruitment of DRP1 by MID49, MID51 or MFF.

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Data availability

The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD023077. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank B. Kornmann, M. Frohman and S. Jakobs for sharing constructs and giving advice regarding super-resolution microscopy. This work was supported by a Feodor Lynen postdoctoral research fellowship awarded to T.K. by the Alexander von Humboldt Foundation and CIHR grant no. MOP#133549 to H.M.M. H.M.M is supported by a Canada Research Chair in Mitochondrial Cell Biology. M.J.A. was supported by a postdoctoral fellowship from the Healthy Brains Healthy Lives initiative. T.L. is supported by the Max–Planck Society and grants from the Deutsche Forschungsgemeinschaft (CRC1218, project number 269925409).

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Authors and Affiliations

  1. Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada

    Tim König, Mari J. Aaltonen, Michiel Krols, Thomas Stroh & Heidi M. McBride

  2. Max Planck Institute for Biology of Ageing, Cologne, Germany

    Hendrik Nolte, Takashi Tatsuta & Thomas Langer

  3. Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

    Thomas Langer

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Contributions

T.K. and H.M.M. designed the study and wrote the manuscript. T.K., H.N., M.J.A., T.T., M.K. and T.S. planned and performed the experiments. T.L. contributed to the manuscript and fruitful discussions.

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Correspondence to Heidi M. McBride.

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Extended data

Extended Data Fig. 1 Mitochondrial integrity and pre-clearing efficiency.

a) Representative confocal images of TOMM20+-MDVs in COS-7 cells. MDVs are positive for TOMM20 and negative for PDH. Scale bar = 5 µm. b) Representative confocal images of TOMM20+-MDVs in COS-7 cells expressing human TOMM20-3xHA and MAPL-h319L-Halo. Scale bar = 5 µm. The close-up demonstrates the unchanged cargo specificity of TOMM20+-MDVs in these cells. Orange circles = MDVs only positive for TOMM20. Scale bar = 1 µm. c) SDS-PAGE analysis of mitochondrial integrity after homogenization of cells by protease protection. Mitochondria were either untreated, swollen, or swollen and solubilized by Triton X-100 (TX100) in the presence of Proteinase K. OMM = outer mitochondrial membrane, IMS = intermembrane space. d) Quantification of the intermembrane space protein YME1L after Proteinase K treatment as a readout for the integrity of the outer mitochondrial membrane. Three experiments with single values (circles), averages (bars) and standard deviations (error bars) are shown (n = 3 biologically independent experiments). e-g) SDS-PAGE analysis (e) and quantifications (f, g) of pre-clearing efficiency through Halo link resin. Three experiments with single values (circles), averages (bars) and standard deviations (error bars) are shown (two-tailed unpaired Student’s t-test: ** = p ≤ 0.01, *** = p ≤ 0.001, n = 3 biologically independent experiments). Unprocessed blots and numerical source data including exact p values and test statistics are available in source data.

Source data

Extended Data Fig. 2 Validation of MDV hits by super-resolution microscopy.

a) SDS-PAGE analysis of the immuno-isolation of TOMM20+-MDVs validating the specific enrichment of a subset of mitochondrial proteins. Mitochondrial proteins LONP1, LRPPRC, and cytosolic free-Ubiquitin serve as negative markers. IN = input, E = elution, OMM = outer mitochondrial membrane, IMM = inner mitochondrial membrane. b) SDS-PAGE analysis demonstrating the enrichment of TOMM20+-MDVs cargoes in MDVs. 5 µg of isolated mitochondria (MITO) and MDVs were loaded. LONP1 and LRPPRC serve as negative markers. c-d) Super-resolution microscopy of selected endogenous MDV hits from the outer mitochondrial membrane (c) and the inner mitochondrial membrane (d) in COS-7 cells. TOMM20 serves as positive MDV marker and PDH/MRPL12 as MDV-negative mitochondrial marker. Orange circles = double positive MDVs, cyan circles = MDVs only positive for TOMM20, scale bar = 1 µm. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 Blue-native PAGE analysis of oligomeric protein complexes.

a) Blue-native PAGE analysis of digitonin-solubilized (1.5%, 6 g digitonin/g protein) COS-7 mitochondria and biochemically purified TOMM20+-MDVs. Comparison of mitochondria with MDVs reveals assembly of MDV-enriched respiratory chain subunits into oligomeric complexes comparable to their mitochondrial assemblies. b) SDS-Page analysis to confirm the siRNA-mediated knockdown of USP30 in COS-7 cells after 72 hours. * = unspecific cross reaction. c) SDS-PAGE analysis of biochemically purified TOMM20+-MDVs to address the ubiquitylation state of TOM complex subunits in MDVs in comparison to mitochondria (MITO). IN = input, E = elution. d) SDS-PAGE analysis of PARK2 CRISPR/Cas9 KO COS-7 cell lines. * = unspecific cross reaction. e-f) Quantification of TOM+-MDVs (e) in non-targeting (NT) and FUNDC1-silenced COS-7 cells after 72 hours verified by SDS-PAGE (f). Counted MDVs are positive for TOMM40 and negative for PDH and MRPL12. Single values and means of three experiments are depicted in orange, cyan and grey. Means were used to calculate averages (horizontal bars), standard deviations (error bars), and p-values (two-tailed unpaired Student’s t-test: n = 3 biologically independent experiments). Unprocessed blots and numerical source data including exact p values and test statistics are available in source data.

Source data

Extended Data Fig. 4 SNX9 and STX17 are not required for biogenesis of TOM+-MDVs.

a) Confocal images of TOMM20+-MDVs in COS-7 cells stably expressing hTOMM20-EGFP (green) used for live cell microscopy. Mitochondria are stained with MitoTracker Deep Red FM (magenta). Scale bar = 5 µm. b-e) Quantifications and SDS-PAGE analysis of TOM+-MDVs in SNX9 (b, d) and STX17 (c, e) CRISPR/Cas9 KO and COS-7 control cells. Counted MDVs are positive for TOMM40 and negative for PDH and MRPL12. Single values and means of three experiments are depicted in orange, cyan and grey. Means were used to calculate averages (horizontal bars), standard deviations (error bars), and p-values (two-tailed unpaired Student’s t-test: n = 3 biologically independent experiments). * = unspecific cross reaction. f) Super-resolution microscopy of ectopically-expressed 3xFLAG-hMIRO1 and 3xFLAG-hMIRO2 in COS-7 cells. TOMM20 serves as positive MDV marker and PDH/MRPL12 as MDV-negative mitochondrial marker. Orange circles = double positive MDVs. Scale bar = 1 µm. g-i) SDS-PAGE analysis to confirm the siRNA-mediated knockdown of MIRO1/2 (g), CENP-F (h) and ARMCX1/3 (i) in COS-7 cells after 72 hours. Unprocessed blots and numerical source data including exact p values and test statistics are available in source data.

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Extended Data Fig. 5 PTPIP51, YME1L and MCU are TOM+-MDV cargoes.

a-f) Quantification of TOM+-MDVs in non-targeting (NT), PTPIP51- (a), YME1L- (b), and MCU-silenced (c) COS-7 cells after 72 hours verified by SDS-PAGE (d-f). Counted MDVs are positive for TOMM40 and negative for PDH and MRPL12. Single values and means of three experiments are depicted in orange, cyan and grey. Means were used to calculate averages (horizontal bars), standard deviations (error bars), and p-values (two-tailed unpaired Student’s t-test: n = 3 biologically independent experiments). g-h) Quantifications of TOM+-MDVs in non-targeting (NT) and MIRO1/MIRO2-silenced COS-7 cells after 72 hours grown (g), or COS-7 cells treated with 15 µM Nocodazole for 2 hours (h) under galactose conditions (5 days). Counted MDVs are positive for TOMM40 and negative for PDH and MRPL12. Single values and means of three experiments are depicted in orange, cyan and grey. Means were used to calculate averages (horizontal bars), standard deviations (error bars), and p-values (two-tailed unpaired Student’s t-test: *** = p ≤ 0.001, n = 3 biologically independent experiments). i) Representative images of double-positive TOM+/PDH+-MDVs and PDH+-MDVs in COS-7 cells grown under galactose conditions (5 days). Scale bar = 10 µm / 2 µm (close-up). Unprocessed blots and numerical source data including exact p values and test statistics are available in source data.

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Extended Data Fig. 6 Phospholipid composition of TOM+-MDVs, the outer mitochondrial membrane and mitochondria.

a-b) Schematic representation (A) and SDS-PAGE analysis (B) of the purification of mitochondria by Percoll-density-gradient centrifugation. c-d) Comparison of phospholipid composition, ceramides (CER) and sphingomyelin (SM) (d) between the outer mitochondrial membrane (OMM) and Percoll-purified mitochondria (MITO). Three experiments with single values (circles), averages (bars) and standard deviations (error bars) are shown. Inlay: close-up for phosphatidic acid (PA) and phosphatidylglycerol (PG). Two-tailed unpaired Student’s t-test (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, n = 3 biologically independent experiments). PC = phosphatidylcholine, PE = phosphatidylethanolamine, PI = phosphatidylinositol, PS = phosphatidylserine, CL = cardiolipin. e-f) Comparison of acyl chain length (e) and level of saturation (f) of phospholipids between the OMM, TOM+-MDVs (MDV) and Percoll-purified mitochondria (MITO). Three experiments with single values (circles), averages (bars) and standard deviations (error bars) are shown. (n = 3 biologically independent experiments) Unprocessed blots and numerical source data including exact p values and test statistics are available in source data.

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Extended Data Fig. 7 Biogenesis of PDH-containing MDVs depends on DRP1.

a-c) SDS-PAGE analysis of DNM2 and DRP1 KO COS-7 cell lines (a), DRP1 KO COS-7 cells stably expressing human DRP1 (b) and DRP1 KO HeLa cells (c). * = unspecific cross reaction. d) Quantification of TOM+-MDVs in HeLa WT and two DRP1 KO clones. Counted MDVs are positive for TOMM40 and negative for PDH and MRPL12. Single values and means of three experiments are depicted in orange, cyan and grey. Means were used to calculate averages (horizontal bars), standard deviations (error bars), and p-values (two-tailed unpaired Student’s t-test: ** = p ≤ 0.01, n = 3 biologically independent experiments). e-g) Quantifications of TOM+-MDVs (e), double-positive TOM+/PDH+-MDVs (f) as well as classical PDH+-MDVs (g) in WT, DRP1 KO and DRP1 KO COS-7 cells rescued with human DRP1 grown under galactose conditions (5 days). Counted MDVs are negative for PDH and MRPL12 (e), MRPL12 (f), or TOMM40 and MRPL12 (g). Single values and means of three experiments are depicted in orange, cyan and grey. Means were used to calculate averages (horizontal bars), standard deviations (error bars), and p-values (two-tailed unpaired Student’s t-test: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, n = 3 biologically independent experiments). Unprocessed blots and numerical source data including exact p values and test statistics are available in source data.

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Extended Data Fig. 8 Mitochondrial morphology changes do not correlate with TOM+-MDV formation.

a) SDS-PAGE analysis of isolated mitochondria from WT and different combinations of MID49, MID51 and MFF double KO COS-7 cells. * = unspecific cross reaction. b-d) Quantification of mitochondrial morphology changes in COS-7 cells treated with 20 µM CCCP for 30 minutes (b), 10 µg / ml cycloheximide (CHX) for 2 hours (c) or 30 µM Forskolin (FORS.) for 2 hours (d). Three experiments with single values (orange, cyan & grey circles), averages (bars) and standard deviations (error bars) are shown. Two-tailed unpaired Student’s t-test (*** = p ≤ 0.001, n = 3 biologically independent experiments). e-f) Quantification of mitochondrial morphology changes (e) in DRP1 KO COS-7 cells stably expressing human DRP1 mutants and SDS-PAGE analysis of the expression levels (f). Three experiments with single values (orange, cyan & grey circles), averages (bars) and standard deviations (error bars) are shown (n = 3 biologically independent experiments). Unprocessed blots and numerical source data including exact p values and test statistics are available in source data.

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König, T., Nolte, H., Aaltonen, M.J. et al. MIROs and DRP1 drive mitochondrial-derived vesicle biogenesis and promote quality control. Nat Cell Biol 23, 1271–1286 (2021). https://doi.org/10.1038/s41556-021-00798-4

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