Mitochondria–lysosome contacts regulate mitochondrial fission via RAB7 GTP hydrolysis

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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Figure 1: Mitochondria and lysosomes form stable membrane contact sites.
Figure 2: RAB7 GTP hydrolysis promotes mitochondria–lysosome contact untethering.
Figure 3: Mitochondrial recruitment of TBC1D15, a RAB7 GAP, by FIS1 drives RAB7 GTP hydrolysis to promote mitochondria–lysosome contact untethering.
Figure 4: Mitochondria–lysosome contacts mark sites of mitochondrial fission regulated by RAB7 GTP hydrolysis.

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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).

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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.

Corresponding author

Correspondence to Dimitri Krainc.

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The authors declare no competing financial interests.

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Extended data figures and tables

Extended Data Figure 1 Correlative light electron microscopy and 3D structured illumination microscopy of mitochondria–lysosome contacts.

a–c, Representative electron microscopy images of mitochondria (M) and lysosome (L) contacts (yellow arrows) in untreated HeLa cells (insets shown on right) (n = 55 examples from 20 cells). d, e, Representative correlative light electron microscopy and confocal images of HeLa cells (from n = 14 images from 6 cells) incubated with LysoTracker Red to label lysosomes or late endosomes (red arrows) that contain electron-dense lumen with irregular content and/or multilamellar membrane sheets (d, see insets on right), and form a stable membrane contact site with mitochondria (e, yellow arrows; see inset on right), while simultaneously forming contact sites with the endoplasmic reticulum (e, purple arrows). Early endosomes lacking electron-dense lumen are LysoTracker-negative (d, blue arrows). f, Representative structured illumination microscopy (N-SIM) images of mitochondria–lysosome contacts (yellow arrows) in fixed HeLa cells stained for endogenous LAMP1 (lysosomes) or TOM20 (mitochondria) and imaged in Z-stacks showing contacts extending more than 200 nm in the Z-plane (n = 210 examples from 26 cells). Scale bars, 200 nm (ad); 100 nm (ad, insets on right; e, left, middle); 50 nm (e, right); 500 nm (f).

Extended Data Figure 2 Characterizing mitochondria–lysosome contacts in living cells.

a–d, Representative images of mitochondria–lysosome contacts (lasting more than 10 s) in living HeLa cells expressing LAMP1–mGFP (lysosomes) and mApple–TOM20 (mitochondria) (n = 23 cells). a, Examples of small LAMP1 vesicles (vesicle diameter <0.5 μm) contacting mitochondria. b, Examples of larger LAMP1 vesicles (vesicle diameter >1 μm) contacting mitochondria. c, Examples of a single LAMP1 vesicles contacting multiple mitochondria. d, Examples of multiple LAMP1 vesicles contacting a single mitochondrion. e, Representative images of contacts (yellow arrows) in fixed HeLa cells stained for endogenous LAMP1 (green) and TOM20 (red) (n = 341 examples from 25 cells). f, g, Representative images of living HeLa cells (n = 23 cells) expressing LAMP1–mGFP (lysosomes) and mApple–TOM20 (outer mitochondrial membrane) with corresponding linescans showing a mitochondria-lysosome contact at close proximity (f), distinct from lysosomal engulfment of mitochondrial TOM20 (g). All scale bars, 0.5 μm.

Extended Data Figure 3 Structured illumination microscopy and FRET imaging of mitochondria–lysosome contacts in living cells.

ac, Representative N-SIM images (a, b) of mitochondria–lysosome contacts (yellow arrows) in living HeLa cells (n = 43 examples from 10 cells) expressing LAMP1–mGFP (lysosomes) and mApple–TOM20 (mitochondria) and quantification of duration of mitochondria–lysosome contacts from N-SIM time-lapse images (c). d, Model of newly generated FRET pairs targeted to the outer mitochondrial membrane (TOM20–Venus) and the lysosomal membrane (LAMP1–mTurquoise2). e, Representative time-lapse images of a living HeLa cell (n = 200 cells) expressing FRET pairs (TOM20–Venus, LAMP1–mTurquoise2) and RAB7a(Q67L)–mRuby3 demonstrating preferentially increased SE-FRET signal over 60 s at the interface between mitochondria and lysosomes (white arrows). f, Quantification of normalized SE-FRET intensity per cell in conditions expressing wild-type RAB7a or RAB7a(Q67L) (n = 200 cells per condition) showing an approximately twofold increase in cells expressing RAB7a(Q67L). Data are means ± s.e.m. ***P < 0.0001, unpaired two-tailed t-test (f). Scale bars, 4 μm (a); 1 μm (b, e).

Extended Data Figure 4 Mitochondria–lysosome contacts are distinct from mitochondria-derived vesicles and mitophagy.

ac, Representative images (a) of living HeLa cells expressing LAMP1–RFP (lysosomes), mito–BFP (mitochondrial matrix) and SMAC–EGFP (mitochondrial intermembrane space), and corresponding linescans (b, c) showing that mitochondrial intermembrane space and matrix proteins do not undergo bulk transfer into lysosomes at contacts (yellow arrows) (n = 57 events from 12 cells). d, e, Representative images (d) in a living HeLa cell expressing mApple–TOM20 (mitochondrial outer membrane), mito–BFP (mitochondrial matrix) and LAMP1–mGFP (lysosomes) and linescan (e, corresponding to top panel in d) showing that mitochondria that form contacts with lysosomes (yellow arrows) are positive for mitochondrial matrix protein mito-BFP and are not TOM20-positive MDVs (n = 104 events from 23 cells). f, Representative linescan in a living HeLa cell expressing mEmerald–TOM20 (mitochondrial outer membrane), DsRed2–Mito (mitochondrial matrix) and mBFP2–Lys (lysosomes) showing that mitochondria that form contacts with lysosomes are positive for mitochondrial matrix protein DsRed2–mito and are not TOM20-positive MDVs (n = 94 events from 16 cells). gi, Representative images (g) in a living HeLa cell expressing mApple–TOM20 (outer mitochondrial membrane), LAMP1–mGFP (lysosomal membrane) and fluid-phase marker dextran blue pulse-chased into the lysosomal lumen, and corresponding linescans (h, i) showing that lysosomal luminal contents (blue) do not undergo bulk transfer into mitochondria at contacts (yellow arrows) (n = 66 events from 18 cells). j, Representative images in a living HeLa cell expressing LAMP1–RFP (lysosomes), mito–BFP (mitochondrial matrix) and EGFP–LC3 (autophagosome) showing that mitochondria that form contacts with lysosomes (yellow arrows) are not engulfed by autophagosomes (not undergoing mitophagy) (n = 142 events from 17 cells). k, Autophagosome biogenesis proteins (ULK1–GFP, mCherry–ATG5, mEmerald–ATG12, GFP–DFCP1 and EGFP–LC3) do not mark sites of mitochondria–lysosome contacts in living cells (number of events analysed in n = 14 cells (ULK1), n = 17 cells (ATG5, ATG12, LC3) or n = 13 cells (DFCP1), top; quantification, bottom). Mitochondria (M) and lysosomes (L) are indicated in linescans. Data are means ± s.e.m. Scale bars, 0.5 μm (a); 1 μm (d, g, j).

Extended Data Figure 5 FIS1 recruits TBC1D15 to mitochondria.

ae, Representative images and quantification of localization of HA–TBC1D15 to mitochondria (stained with endogenous TOM20) in fixed HeLa cells showing that mitochondrial localization is not disrupted by TBC1D15 GAP mutants (D397A or R400K) but is disrupted by mutating the FIS1-binding site of TBC1D15 (Δ231–240) (n = 293 cells, WT; n = 228 cells, D397A; n = 181 cells, R400K; n = 379 cells, Δ231–240). Δ231–240 versus WT (*P = 0.0178), D397A (*P = 0.0131), and R400K (*P = 0.0112), ANOVA with Tukey’s post-hoc test. f, Quantification showing that localization of YFP–TBC1D15 to mitochondria is greatly decreased by the Flag–FIS1(LA) mutant (which cannot bind TBC1D15) as compared to wild-type Flag–FIS1 (n = 290 cells, FIS1; n = 281 cells, FIS1(LA)). ***P < 0.0001, unpaired two-tailed t-test. g, Examples of HA–TBC1D15 GAP mutants (D397A and R400K) or FIS1-binding mutant (Δ231–240) inducing enlarged lysosomes (white arrows) (LAMP1–mGFP) not observed in cells expressing wild-type HA–TBC1D15 (n = 293 cells, WT; n = 228 cells, D397A; n = 181 cells, R400K; n = 379 cells, Δ231–240). Data are means ± s.e.m. Scale bars, 10 μm (ad, g); 1 μm (ad, insets).

Extended Data Figure 6 Recruitment of TBC1D15 by FIS1 to mitochondria promotes mitochondria–lysosome contact untethering.

a, b, Representative time-lapse images of stable mitochondria–lysosome contacts (yellow arrows) for over 100 s before untethering (white arrow) in living HeLa cells expressing mApple–TOM20 (mitochondria), LAMP1–mGFP (lysosome) and the RAB7 GAP mutant TBC1D15(D397A) (n = 38 events from 10 cells). c, TBC domain mutants TBC1D15(D397A) and TBC1D15(R400K), which lack GAP activity, do not alter the percentage of lysosomes in contacts (n = 12 cells per condition), as compared to wild-type TBC1D15 (N.S., not significant). d, e, TBC1D15−/− HCT116 cells have increased duration (d, n = 18 events from 6 cells, WT; n = 16 events from 7 cells, TBC1D15−/−) but no change in the number of mitochondria–lysosome contacts (e, n = 15 cells, WT; n = 14 cells, TBC1D15−/−) compared to wild-type HCT116 cells (*P < 0.0491, N.S., not significant). f, Expression of the Flag–FIS1(LA) mutant (unable to bind TBC1D15) increases the percentage of lysosomes in mitochondria–lysosome contacts compared to wild-type FIS1 in living HeLa cells (n = 18 cells, FIS1; n = 16 cells, FIS1(LA); *P < 0.0117). g, h, FIS1−/− HCT116 cells have an increased duration (g, n = 18 events from 6 cells, WT; n = 14 events from 6 cells, FIS1−/−) and number of mitochondria–lysosome contacts (h, n = 15 cells, WT; n = 13 cells, FIS1−/−) compared to wild-type HCT116 cells (*P < 0.0442, ***P < 0.0001). i, j, Localization of HA–TBC1D15 (i, n = 293 cells) and Flag–FIS1 (j, n = 272 cells) to mitochondria in fixed HeLa cells is not restricted to mitochondria–lysosome contacts. Data are means ± s.e.m. ANOVA with Tukey’s post-hoc test (c), unpaired two-tailed t test (dh). Scale bars, 0.5 μm (a); 1 μm (b, i (insets), j (insets)); 10 μm (i, j).

Extended Data Figure 7 Mitochondrial fission sites are marked by mitochondria–lysosome contacts in multiple cell types.

a, b, Representative time-lapse images of lysosomes contacting mitochondria at site of mitochondrial fission (yellow arrow, top) before mitochondrial fission (white arrows, middle) in living HeLa cells expressing mGFP–LAMP1 (lysosomes) and mApple–TOM20 (mitochondria) with corresponding linescans (right) showing lysosomes at the site of fission (yellow arrow; linescan) after mitochondrial division into two daughter mitochondria (grey arrows, linescan) (n = 62 events from 23 cells). c, Electron microscopy image of mitochondria (M) in contact (<30 nm) with a lysosome (L; yellow arrows) at site of mitochondrial constriction in untreated HeLa cells (from n = 20 cells imaged). d–g, Lysosomes (yellow arrows in eg; mGFP–LAMP1) mark sites of mitochondrial fission (white arrows in eg; mApple–TOM20) at similar rates (d) in living H4 neuroglioma, HEK293 and HCT116 cells as in HeLa cells by time-lapse confocal imaging (n = 49 events from 10 cells, HeLa; n = 36 events from 13 cells, H4; n = 18 events from 9 cells, HEK293; n = 9 events from 6 cells, HCT116). Data are means ± s.e.m. N.S., not significant, ANOVA with Tukey’s post-hoc test. Scale bars, 1 μm (a, b, eg, insets); 200 nm (c); 2.5 μm (eg).

Extended Data Figure 8 Mitochondria–lysosome contacts mark sites of mitochondrial fission upon induction of mitochondrial fragmentation.

ad, Lysosomes (yellow arrows; mGFP–LAMP1) mark sites of mitochondrial fission (white arrows; mApple–TOM20) at similar rates (d) in untreated living HeLa cells as in cells treated for up to 20 min with actinomycin D (a), STS (b) or CCCP (c) (n = 49 events from 10 cells, control; n = 29 events from 14 cells, actinomycin D; n = 36 events from 10 cells, STS; n = 49 events from 14 cells, CCCP). Data are means ± s.e.m. N.S., not significant, ANOVA with Tukey’s post-hoc test. Scale bars, 5 μm (a-c); 1 μm (ac, insets).

Extended Data Figure 9 Mitochondrial fission sites marked by lysosomes are positive for DRP1 and endoplasmic reticulum tubules.

a, Representative time-lapse images of a lysosome (mBFP2–Lys) contacting mitochondria (mEmerald–TOM20) at the site of mitochondrial division (yellow arrow) before fission (white arrows) in a living HeLa cell showing mCherry–DRP1 oligomerization at the site of mitochondrial division (n = 41 events from 11 cells). b, c, Representative image (b, inset time-lapse images shown in c) of a lysosome (mBFP2–Lys) contacting mitochondria (mEmerald–TOM20) at the site of mitochondrial division (yellow arrow) before fission (white arrows) in a living HeLa cell showing an endoplasmic reticulum tubule (mCherry–ER) at the site of mitochondrial division (n = 54 events from 16 cells). Scale bars, 1 μm (a, c); 5 μm (b).

Extended Data Figure 10 Regulation of mitochondrial network dynamics by RAB7 GTP hydrolysis.

a, Examples of mitochondria not undergoing fission for more than 120 s in living HeLa cells expressing mApple–TOM20 (mitochondria) and RAB7(Q67L)–GFP (n = 13 cells). b, c, Examples of mitochondria undergoing fission (white arrows) after 36 s in living HeLa cells expressing mApple–TOM20 (mitochondria) and wild-type TBC1D15 (n = 13 cells). d, e, Examples of mitochondria not undergoing fission for more than 240 s in living HeLa cells expressing mApple–TOM20 (mitochondria) and GAP mutants TBC1D15(D397A) (d) or TBC1D15(R400K) (e) (n = 13 cells per condition). fi, The percentage of mitochondrial fission sites marked by lysosomes (mGFP–LAMP1; f, h) or endoplasmic reticulum (mCherry–ER; g, i) is not disrupted by the RAB7(Q67L) GTP-hydrolysis-deficient mutant (f, g; n = 12 events from 15 cells) or by TBC1D15 GAP mutants (D397A or R400K) (h, i; n = 22 events from 10 cells, WT; n = 17 events from 19 cells, D397A; n = 27 events from 22 cells, R400K). jl, Examples of RAB7(Q67L) and HA–TBC1D15 GAP mutants (D397A and R400K) inducing elongated mitochondria (j, yellow arrows; >10 μm length) compared to control cells, and quantification of RAB7(Q67L) (k; *P = 0.0321) and HA-TBC1D15 GAP mutants (D397A and R400K) (l; *P = 0.0297, **P = 0.0051) leading to decreased percentages of cells with normal mitochondrial networks (no elongated mitochondria >10 μm length or hyperfused or tethered networks) (n = 47 cells, RAB7; n = 72 cells, RAB7(Q67L); n = 88 cells, TBC1D15 WT; n = 168 cells, TBC1D15(D397A); n = 132 cells, TBC1D15(R400K)). Data are means ± s.e.m. N.S., not significant; ANOVA with Tukey’s post-hoc test (h, i, l), unpaired two-tailed t-test (f, g, k). Scale bars, 0.5 μm (a); 1 μm (be); 10 μm (j).

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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. (MOV 185 kb)

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. (MOV 109 kb)

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. (MOV 994 kb)

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. (MOV 1372 kb)

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. (MOV 27 kb)

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. (MOV 52 kb)

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Wong, Y., Ysselstein, D. & Krainc, D. Mitochondria–lysosome contacts regulate mitochondrial fission via RAB7 GTP hydrolysis. Nature 554, 382–386 (2018). https://doi.org/10.1038/nature25486

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