Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Oct;8(10):831-8.
doi: 10.1038/nchembio.1059.

Ceramide Targets Autophagosomes to Mitochondria and Induces Lethal Mitophagy

Affiliations
Free PMC article

Ceramide Targets Autophagosomes to Mitochondria and Induces Lethal Mitophagy

R David Sentelle et al. Nat Chem Biol. .
Free PMC article

Erratum in

  • Nat Chem Biol. 2012 Dec;8(12):1008

Abstract

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C(18)-pyridinium ceramide treatment or endogenous C(18)-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C(18)-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 β-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1- and C(18)-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

Figures

Figure 1
Figure 1
Exogenous C18-Pyr-Cer (C18PC) induces autophagic cell death. (a) C18-Pyr-Cer. (b) Effects of C18-Pyr-Cer on the lipidation of LC3B (LC3B-II) were examined by western blot in the absence or presence of a pan caspase inhibitor Z-VAD compared to vehicle-treated controls (lanes 1-2 and 3-4, respectively). Full blots can be found in Supplementary Figure 13. (c) Formation of double-membrane autophagosomal vesicles in the absence or presence of C18-Pyr-Cer was visualized by TEM (left and right panels, respectively). Higher magnification of TEM visualization is shown in lower panels. Scale bars represent 10 microns (top) and 500 nm (bottom). (d) Effects of siRNA-mediated knockdown of Atg3 or Atg7 on cell death in the absence or presence of C18-Pyr-Cer were determined compared to controls transfected with Scr siRNAs using trypan blue exclusion assay. (e) Roles of C18-Pyr-Cer in the regulation of lethal autophagy were assessed after treatment of MEFs isolated from wt (ATG5+/+) versus ATG5-/- k/o mice with C18-Pyr-Cer. (f) Effects of C18-Pyr-Cer on caspase-dependent or –independent cell death were determined in MEFs isolated from Bax-/-/Bak-/- (dko) or caspase3-/-/7-/- (dko) mice compared to cells isolated from wt (Bax+/+/Bak+/+) or caspase3+/-/7+/- mice, used as controls. Data shown are an average of at least three experiments ± s.d. (*P <0.05).
Figure 2
Figure 2
C18-Pyr-Cer mediates targeting of autophagolysosomes to mitochondria and the inhibition of mitochondrial function. (a) Targeting of autophagolysosomes to mitochondria in the absence or presence of C18-Pyr-Cer (C18PC) at 2 h in UM-SCC22A cells was examined by visualizing the co-localization of MTG and LTR using live cell imaging and confocal microscopy. Scale bars represent 10 microns. (b) Effects of C18-dihydro-Cer-14-piperidine (Dihydro-C18PC, left panel) on OCR were measured using the SeaHorse compared to C18-Pyr-Cer (C18PC) and vehichle-treated controls (right panel). Data shown are an average of at least two independent experiments performed in duplicates ± s.d. (*P <0.05).
Figure 3
Figure 3
Induction of endogenous C18-ceramide by CerS1 expression mediates lethal mitophagy. (a) Effects of induction of wt-CerS1 versus its catalytically inactive mutant, which cannot generate C18-ceramide, expression (containing V5 tags) on the formation of LC3B-II were determined by western blot (first panel, +/-tet, respectively). Successful induction of wt- and mutant-CerS1 expression was confirmed using the anti-V5 antibody. Beta-actin was used as a loading control. Full blots can be found in Supplementary Figure 13. (b) Generation of endogenous C18- and C18:1-ceramides in response to wt-CerS1 compared to the mutant-CerS1 induction was measured by LC/MS/MS. (c) Effects of wt-CerS1/C18-ceramide induction (+tet) on GFP or LC3B-GFP lipidation were visualized using confocal microscopy compared to non-induced controls (-tet). Scale bars represent 10 microns. (d) Roles of wt-CerS1/C18-ceramide versus the catalytically inactive mutant-CerS1 induction (+tet) in the regulation of mitochondrial function was assessed by measuring oxygen consumption rate using the SeaHorse, compared to non-induced controls (-tet). (e) Targeting mitochondria with autophagolysosomes in the absence or presence of wt-CerS1/C18-ceramide (-/+ tet, repectively) was visualized by co-localization of MTG and LTR using confocal microscopy. (f) Effects of wt and mutant CerS1 (-/+ tet) on ATP generation. Scale bars represent 10 microns. (g) Effects of wt-CerS1 (-/+ tet) on ATP generation in the absence or presence of shRNA-mediated knockdown of LC3B were measured. Data shown are an average of at least three experiments ± s.d. (*P <0.05).
Figure 4
Figure 4
Mitochondrial localization of CerS1-generated C18-ceramide induces targeting of mitochondria by LC3B-II-containing autophagosomes. (a) Subcellular localization of ceramide, generated by wt-CerS1 induction (+tet), in mitochondria were visualized by colocalization of ceramides and mitochondria using anti-ceramide and anti-Tom-20 antibodies, respectively, using confocal microscopy. (b-c) Mitochondrial targeting of LC3B-II and C18-ceramide, generated by induction of wt-CerS1 (b) versus the catalytically inactive mutant CerS1 (c) expression (+tet) was visualized by co-localization of ceramide (green), LC3B-CFP (blue) and MitoTracker Red (MTR) using anti-ceramide antibody and confocal microscopy. Non-induced cells (-tet) were used as controls. Scale bars represent 10 microns.
Figure 5
Figure 5
C18-ceramide interacts with LC3B via lipid-protein association. (a) Binding of wt-, Ile35Ala-, Phe52Ala- or Gly120Ala-LC3B-FLAG proteins with endogenous C18-ceramide, generated in response to wt-CerS1 induction (upper panel) or exogenous C18-Pyr-Cer (lower panel) were measured using LC/MS/MS after pull-down using anti-FLAG antibody-conjugated beads. Data shown are an average of at least three experiments ± s.d. (*P <0.05). (b) Effects of CerS1/C18-ceramide induction on the lipidation of wt-, Ile35Ala-, Phe52Ala- or Gly120Ala-LC3B-FLAG proteins were examined by western blott. Non-induced cells (-tet) were used as controls. Beta-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.
Figure 6
Figure 6
C18-ceramide-LC3B-II interaction on mitochondrial membranes is regulated downstream of Drp1-mediated mitochondrial fission. (a-b) Effects of Gly120Ala and Phe52Ala conversions, which perturbed C18-ceramide-binding of LC3B, on targeting mitochondria by LC3B-containing autophagosomes (visualized by confocal microscopy using anti-FLAG and anti-Tom-20 antibodies; a) or mitochondrial function (measurement of oxygen consumption OCR; b). (c) Roles of siRNA-mediated knockdown of p62, NIX and Drp1 in the alteration of OCR in the absence or presence of CerS1/C18-ceramide induction (-/+Tet) were measured using the SeaHorse. Data shown are an average of at least three experiments ± s.d. (*P <0.05). (d) Effects of siRNA-mediated knockdown of Drp1 on the localization of ceramide within mitochondrial membranes were visualized using the co-localization of anti-ceramide (red) and anti-Tom20 (green) antibodies under confocal microscopy, in the absence or presence of CerS1/C18-ceramide induction (-/+ Tet) compared to Scr-siRNA-transfected controls (right and left, upper and lower panels, respectively). Scale bars represent 10 microns in (a) and (d).
Figure 7
Figure 7
C18-ceramide-LC3B interaction induces lethal mitophagy and subsequent tumor suppression in vivo. (a-b) Roles of shRNA-mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function (a), or UM-SCC-22A xenograft-derived tumor growth in the absence/presence of wt-CerS1/C18-ceramide induction (-/+ tet) were determined using the SeaHorse, or measurement of tumor volumes in the flanks of SCID mice (n=6/group), respectively. Data shown are an average of at least three experiments ± s.d. (*P <0.05). (c) Mechanism by which C18-ceramide induces lethal autophagy involves, at least in part, the lipidation of LC3B, forming LC3B-II, and interaction of LC3B-II and ceramide on mitochondrial membranes upon Drp1-mediated mitochondrial fission, which targets autophagolysosomes to mitochondria for lethal mitophagy.

Similar articles

See all similar articles

Cited by 142 articles

See all "Cited by" articles

References

    1. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature. 2008;451:1069–1075. - PMC - PubMed
    1. Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol. 2010;12:814–822. - PMC - PubMed
    1. Tanida I, Ueno T, Kominami E. Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autophagosomal membranes. J Biol Chem. 2004;279:47704–10. - PubMed
    1. Rabinowitz JD, White E. Autophagy and metabolism. Science. 2010;330:1344–1348. - PMC - PubMed
    1. Vara D, et al. Anti-tumoral action of cannabinoids on hepatocellular carcinoma: role of AMPK-dependent activation of autophagy. Cell Death Differ. 2011;18:1099–1111. - PMC - PubMed

Publication types

Feedback