Letter | Published:

Mitochondria–lysosome contacts regulate mitochondrial fission via RAB7 GTP hydrolysis

Nature volume 554, pages 382386 (15 February 2018) | Download Citation

Abstract

Both mitochondria and lysosomes are essential for maintaining cellular homeostasis, and dysfunction of both organelles has been observed in multiple diseases1,2,3,4. Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network, which drives cellular metabolism5. Lysosomes similarly undergo constant dynamic regulation by the RAB7 GTPase1, which cycles from an active GTP-bound state into an inactive GDP-bound state upon GTP hydrolysis. Here we have identified the formation and regulation of mitochondria–lysosome membrane contact sites using electron microscopy, structured illumination microscopy and high spatial and temporal resolution confocal live cell imaging. Mitochondria–lysosome contacts formed dynamically in healthy untreated cells and were distinct from damaged mitochondria that were targeted into lysosomes for degradation6,7. Contact formation was promoted by active GTP-bound lysosomal RAB7, and contact untethering was mediated by recruitment of the RAB7 GTPase-activating protein TBC1D15 to mitochondria by FIS1 to drive RAB7 GTP hydrolysis and thereby release contacts. Functionally, lysosomal contacts mark sites of mitochondrial fission, allowing regulation of mitochondrial networks by lysosomes, whereas conversely, mitochondrial contacts regulate lysosomal RAB7 hydrolysis via TBC1D15. Mitochondria–lysosome contacts thus allow bidirectional regulation of mitochondrial and lysosomal dynamics, and may explain the dysfunction observed in both organelles in various human diseases.

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Acknowledgements

We thank K. Trajkovic and all members of the Krainc laboratory for advice, F. Korobova for electron microscopy assistance and J. Z. Rappoport and D. Kirchenbuechler for N-SIM assistance. All imaging work was performed at the Northwestern University Center for Advanced Microscopy, supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. Structured illumination microscopy was performed on a Nikon N-SIM system, purchased with the support of NIH 1S10OD016342-01. The spinning disk confocal system was acquired through a NCRR shared instrumentation grant awarded to V. Gelfand (S10 RR031680-01). TBC1D15 and FIS1 constructs were gifts from N. Ishihara. HCT116 wild-type and knockout cells were gifts from R. Youle. This work was supported by NIH/NINDS grants to Y.C.W. (T32 NS041234 and F32 NS101778) and D.K. (R01 NS076054).

Author information

Affiliations

  1. Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA

    • Yvette C. Wong
    • , Daniel Ysselstein
    •  & Dimitri Krainc

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Contributions

Y.C.W. and D.K. designed the overall study, analysed data and wrote the manuscript. Y.C.W. performed cell culture, electron microscopy, correlative light electron microscopy, structured illumination microscopy, confocal live cell imaging and immunofluorescence. D.Y. designed, performed and analysed FRET experiments.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Dimitri Krainc.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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    Life Sciences Reporting Summary

Videos

  1. 1.

    Live imaging of mitochondria-lysosome contact

    Confocal microscopy imaging of a stable mitochondria-lysosome contact which remains in contact for over 1 min (60 sec) in a living Hela cell expressing Lamp1-mGFP (lysosome; green) and mApple-TOM20 (mitochondria; red). Video was acquired at 1 frame/2 seconds for 60 sec and played back at 5 frames/second (10x speed). Video corresponds to Fig. 1d. Scale bar, 1 μm.

  2. 2.

    Live imaging of mitochondria-lysosome contact formation

    Time-lapse confocal image of a lysosome approaching mitochondria, forming a stable contact for 24 sec, and subsequently leaving mitochondria, in a living Hela cell expressing Lamp1-mGFP (lysosome; green) and mApple-TOM20 (mitochondria; red). Video was acquired at 1 frame/2 seconds for 34 sec and played back at 5 frames/second (10x speed). Video corresponds to Fig. 2a. Scale bar, 0.5 μm.

  3. 3.

    Live imaging of increased duration of mitochondria-lysosome contact with Rab7 Q67L GTP hydrolysis mutant

    Time-lapse confocal image of a lysosome (left) in cytosol approaching mitochondria to form a stable contact of increased duration for > 2 min (152 sec) before leaving mitochondria in a living Hela cell expressing constitutively active mutant Rab7Q67L-GFP unable to undergo GTP hydrolysis (lysosome; green) and mApple-TOM20 (mitochondria; red). Video was acquired at 1 frame/2 seconds for 178 sec and played back at 5 frames/second (10x speed). Video corresponds to Fig. 2c. Scale bar, 0.5 μm.

  4. 4.

    Live imaging of increased duration of mitochondria-lysosome contact with TBC1D15 D397A (TBC domain mutant) lacking GAP activity

    Time-lapse confocal image of a stable mitochondria-lysosome contact which remains in contact for increased duration of > 5 min (326 sec) in a living Hela cell expressing Lamp1-mGFP (lysosome; green), mApple-TOM20 (mitochondria; red) and TBC1D15/Rab7-GAP D397A (TBC domain mutant) lacking GAP activity. Video was acquired at 1 frame/2 seconds for 326 sec and played back at 10 frames/second (20x speed). Video corresponds to Fig. 3b. Scale bar, 0.5 μm.

  5. 5.

    Live imaging of mitochondria-lysosome contact at site of mitochondrial fission

    Confocal microscopy imaging of a mitochondria-lysosome contact at the site of mitochondrial fission in a living Hela cells expressing Lamp1-mGFP (lysosome; green) and mApple-TOM20 (mitochondria; red). Video was acquired at 1 frame/2 seconds for 12 sec and played back at 4 frames/second (8x speed). Video corresponds to Fig. 4a. Scale bar, 0.5 μm.

  6. 6.

    Live imaging of mitochondria-lysosome contact at site of mitochondrial fission

    Time-lapse confocal image of a mitochondria-lysosome contact at the site of mitochondrial fission in a living Hela cells expressing Lamp1-mGFP (lysosome; green) and mApple-TOM20 (mitochondria; red). Video was acquired at 1 frame/2 seconds for 20 sec and played back at 4 frames/second (8x speed). Video corresponds to Fig. 4b. Scale bar, 0.5 μm.

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DOI

https://doi.org/10.1038/nature25486

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