Mitochondrial function is challenged by toxic by-products of metabolism as well as by pathogen attack1,2. Caenorhabditis elegans normally responds to mitochondrial dysfunction with activation of mitochondrial-repair, drug-detoxification and pathogen-response pathways1,2,3,4,5,6,7. Here, from a genome-wide RNA interference (RNAi) screen, we identified 45 C. elegans genes that are required to upregulate detoxification, pathogen-response and mitochondrial-repair pathways after inhibition of mitochondrial function by drug-induced or genetic disruption. Animals defective in ceramide biosynthesis are deficient in mitochondrial surveillance, and addition of particular ceramides can rescue the surveillance defects. Ceramide can also rescue the mitochondrial surveillance defects of other gene inactivations, mapping these gene activities upstream of ceramide. Inhibition of the mevalonate pathway, either by RNAi or statin drugs, also disrupts mitochondrial surveillance. Growth of C. elegans with a significant fraction of bacterial species from their natural habitat causes mitochondrial dysfunction. Other bacterial species inhibit C. elegans defence responses to a mitochondrial toxin, revealing bacterial countermeasures to animal defence.
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We thank C. Haynes (Memorial Sloan Kettering Cancer Center), F. Ausubel (Massachusetts General Hospital) and the Caenorhabditis Genetics Center for providing strains; and the V. Mootha laboratory (Massachusetts General Hospital) for providing C2C12 cells. We thank J. Larkins-Ford, C. Phillips, S. Garcia and M. Pellegrino for technical support, as well as M. A. Félix for bacterial strains and for guidance in the collection of microbial strains from C. elegans natural habitats. Y.L. is supported by the Helen Hay Whitney research fellowship; B.S.S. is supported by the Charles King Trust postdoctoral fellowship. The work is supported by a grant from the National Institutes of Health, awarded to G.R. (NIH AG043184-16).
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Mitochondrial dysfunction activates homeostatic, detoxification and pathogen responses.
a, Drug-induced food-avoidance phenotypes on live or dead bacteria. Dead bacteria were obtained by heating bacteria at 90 °C for 30 min. Avoidance behaviour was also observed when antimycin was added to dead bacteria, showing that the drug acts directly on C. elegans and is not transformed by the bacteria. b, Quantification of the food-avoidance phenotypes of Extended Data Fig. 1a (n = 4). Error bars represent s.d. c, A dose response of hsp-6p::gfp induction with the addition of antimycin.
For the detailed experimental procedure, see Methods.
a, A graph showing the fold change in C. elegans sptl-1 transcript level compared to control (n = 3). Error bars represent s.d. b, hsp-6p::gfp worms raised on control or sptl-1(RNAi) (n = 40). c, Body wall muscle animals expressing a mitochondrially localized GFP reporter. The animals were subjected to sptl-1(RNAi) for 36 h and transferred onto the second RNAi (control, fzo-1, fis-1 or drp-1). The hyperfusion of mitochondria observed under sptl-1(RNAi) is dependent on the fusion machinery as disruption of mitochondrial fusion by inactivating fzo-1 or fis-1 partially restored the tubular structure. By contrast, inhibition of the gene drp-1 that governs fission led to mitochondrial hyperfusion. The images were taken 36 h after they were placed on the second RNAi. d, A graph showing the percentage of worms which avoid the spg-7(RNAi) bacteria lawn 48 h after they were initially placed on the plates (n = 4). Error bars represent s.d. e, A graph showing the percentage of worms that avoid the bacteria lawn 8 h after the addition of antimycin (n = 4). Error bars represent s.d. f, Wild-type N2 or sptl-1(ok1693) mutant animals raised in the presence or absence of 0.8 μM antimycin. Photos were taken 4 days after the synchronized L1 worms were placed on the plates. g, hsp-6p::gfp animals were raised on sptl-1(RNAi) for 36 h and transferred onto a subset of wild microbes. The images were taken after 2 days.
a, Genotyping of the sphingolipid metabolism pathway mutant alleles. b, c, Fold difference in hsp-6 transcript levels in wild type or sphingolipid metabolism pathway mutants (b) and hyl-1 or lagr-1 mutants (c) (n = 3). Error bars represent s.d. d, hsp-6p::gfp worms raised on sptl-1(RNAi) in the presence of increasing amounts of ceramide. e, hsp-6p::gfp in the presence or absence of ceramide.
a, The percentage of worms that avoid the bacterial lawn 8 h after the addition of antimycin. Animals were pre-treated with control RNAi, control RNAi with ceramide, sptl-1(RNAi) or sptl-1(RNAi) with ceramide (n = 4). Error bars represent s.d. b, Time course experiment for the induction of hsp-6p::gfp with antimycin. c, Dissected young adults after 4 h antimycin treatment were stained with anti-COX-IV antibody (green) and anti-ceramide antibody (red). d, Nomarski (top) and fluorescent (bottom) images of intestinal cells in atfs-1;hsp-16p::atfs-1Δ1-32.myc::gfp transgenic animals. e, hsp-6p::gfp worms raised on indicated RNAi in the presence or absence of ceramide.
a, hsp-6p::gfp animals raised on control or hmgs-1(RNAi) in the presence of antimycin. b, Immunoblotting of GFP expressed by hsp-6p::gfp animals, with or without antimycin. c, Antimycin-induced food avoidance in control or hmgs-1(RNAi) animals. d, Quantification of food avoidance (n = 4). Error bars represent s.d. e, Body wall muscle of control or hmgs-1(RNAi) animals expressing a mitochondrially localized GFP reporter. f, hsp-6p::gfp animals raised on hmgs-1(RNAi), or hmgs-1(RNAi) with addition of mevalonate exposed to antimycin. g, hsp-6p::gfp animals treated with antimycin after pre-treatment with simvastatin or mevastatin. h, Mitochondrial immunostaining in HEK293T cells. i, ATP levels in C2C12 myotubes after treating with simvastatin or mevastatin (n = 3). Error bars represent s.d.
a, Diagram of the mevalonate pathway for the biosynthesis of cholesterol, ubiquinone and haem A, and protein N-glycosylation and prenylation. b, hsp-6p::gfp animals treated with antimycin after pre-treatment with increasing concentration of simvastatin. c, hsp-6p::gfp animals with mock, 80 μg ml−1 simvastatin or 80 μg ml−1 mevastatin treatment. d, hsp-6p::gfp animals raised on control RNAi, hmgs-1(RNAi), or hmgs-1(RNAi) with the addition of geranylgeranyl pyrophosphate. The animals were then treated with antimycin to induce mitochondrial damage. Statin toxicity has been proposed to be caused by the inhibition of Rab prenylation24. Geranylgeranyl pyrophosphate (GGPP), a precursor of protein prenylation rescued the statin side effect in cell culture24. GGPP also partially rescued the deficiency of mitochondrial surveillance and activated hsp-6p::gfp in antimycin-treated hmgs-1(RNAi) animals.
The cells were treated with DMSO, 10 μM simvastatin or 10 μM mevastatin for 2 days.
Animals were filmed after 48 hours of growth on control RNAi. (MOV 1725 kb)
Animals were filmed after 48 hours of growth on sptl-1(RNAi) (MOV 2107 kb)
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Liu, Y., Samuel, B., Breen, P. et al. Caenorhabditis elegans pathways that surveil and defend mitochondria. Nature 508, 406–410 (2014). https://doi.org/10.1038/nature13204
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