Limits...
A Mutation in Caenorhabditis elegans NDUF-7 Activates the Mitochondrial Stress Response and Prolongs Lifespan via ROS and CED-4.

Rauthan M, Ranji P, Abukar R, Pilon M - G3 (Bethesda) (2015)

Bottom Line: Statins, i.e., cholesterol-lowering drugs that inhibit the rate-limiting enzyme in the mevalonate pathway, HMG-CoA reductase, are lethal to Caenorhabditis elegans even though this animal lacks the branch of the mevalonate pathway that leads to cholesterol synthesis.To better understand the effects of statins that are not related to cholesterol, we have adopted the strategy of isolating statin-resistant C. elegans mutants.We conclude that the nduf-7(et19) mutant allele causes an increase in reactive oxygen species that activate ATFS-1, hence UPR(mt)-mediated statin resistance, and extends life span via CED-4.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, S-405 30, Sweden.

No MeSH data available.


Characterization of atfs-1(et15) suppressors. (A) Overview of the screening strategy: atfs-1(et15);zcIs9(Phsp60::GFP) worms were mutagenized for 4 hr by incubation in the presence of 0.5% ethyl methane sulfonate, and approximately 27,000 mutagenized haploid genomes were screened among the F2 progeny, leading to the isolation of 14 GFP-negative mutants. (B) Images of hsp-60::GFP expression in control worms, atfs-1(et15) worms, and the atfs-1(et15) suppressors et27 and et29. (C) Quantification of the fluorescence levels for representative atfs-1(et15) suppressor mutants. Note that the et29 allele retains some hsp-60::GFP expression. (D) Length of 10 atfs-1(et15) suppressors 96 hr after being deposited as L1s on culture plates containing 0.5 mM fluvastatin; the atfs-1(et15) is also included as a control. Note that the et29 allele retains a partial resistance to fluvastatin. (E) Molecular definition of the atfs-1(et15) suppressors. The main structural features of the ATFS-1 protein are depicted, and the nature and position of the alleles used in this study are indicated. For each allele, the atfs-1 coding regions were PCR-amplified and both strands were sequenced to define the mutation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4528320&req=5

fig5: Characterization of atfs-1(et15) suppressors. (A) Overview of the screening strategy: atfs-1(et15);zcIs9(Phsp60::GFP) worms were mutagenized for 4 hr by incubation in the presence of 0.5% ethyl methane sulfonate, and approximately 27,000 mutagenized haploid genomes were screened among the F2 progeny, leading to the isolation of 14 GFP-negative mutants. (B) Images of hsp-60::GFP expression in control worms, atfs-1(et15) worms, and the atfs-1(et15) suppressors et27 and et29. (C) Quantification of the fluorescence levels for representative atfs-1(et15) suppressor mutants. Note that the et29 allele retains some hsp-60::GFP expression. (D) Length of 10 atfs-1(et15) suppressors 96 hr after being deposited as L1s on culture plates containing 0.5 mM fluvastatin; the atfs-1(et15) is also included as a control. Note that the et29 allele retains a partial resistance to fluvastatin. (E) Molecular definition of the atfs-1(et15) suppressors. The main structural features of the ATFS-1 protein are depicted, and the nature and position of the alleles used in this study are indicated. For each allele, the atfs-1 coding regions were PCR-amplified and both strands were sequenced to define the mutation.

Mentions: The genetic pathway linking nduf-7(et19) and the UPRmt reporter hsp-60::GFP likely involves no additional components between atfs-1 and its target hsp-60 promoter; a forward genetic screen to identify suppressors of the previously identified atfs-1(gof) allele et15(Rauthan et al. 2013) resulted in the isolation of 14 intragenic loss-of-function alleles of atfs-1 itself (Figure 5, A–C). Ten of these mutants were characterized in some detail. All were growth-inhibited by 0.5 mM fluvastatin, to which the atfs-1(et15) allele is resistant, and all harbored mutations within the coding region of atfs-1 (Figure 5, D and E). There are at least three possible explanations for having isolated only atfs-1 intragenic suppressors in this screen: (1) atfs-1 may act directly on the hsp-60 promoter; (2) atfs-1 may act together with essential genes that cause lethality when mutated; and (3) atfs-1 may act together with any of several redundant genes. In any case, it may be difficult to further investigate the pathway between atfs-1 and its target promoter using a forward genetics approach. We therefore focused our effort on better understanding how the nduf-7(et19) mutation leads to atfs-1 activation.


A Mutation in Caenorhabditis elegans NDUF-7 Activates the Mitochondrial Stress Response and Prolongs Lifespan via ROS and CED-4.

Rauthan M, Ranji P, Abukar R, Pilon M - G3 (Bethesda) (2015)

Characterization of atfs-1(et15) suppressors. (A) Overview of the screening strategy: atfs-1(et15);zcIs9(Phsp60::GFP) worms were mutagenized for 4 hr by incubation in the presence of 0.5% ethyl methane sulfonate, and approximately 27,000 mutagenized haploid genomes were screened among the F2 progeny, leading to the isolation of 14 GFP-negative mutants. (B) Images of hsp-60::GFP expression in control worms, atfs-1(et15) worms, and the atfs-1(et15) suppressors et27 and et29. (C) Quantification of the fluorescence levels for representative atfs-1(et15) suppressor mutants. Note that the et29 allele retains some hsp-60::GFP expression. (D) Length of 10 atfs-1(et15) suppressors 96 hr after being deposited as L1s on culture plates containing 0.5 mM fluvastatin; the atfs-1(et15) is also included as a control. Note that the et29 allele retains a partial resistance to fluvastatin. (E) Molecular definition of the atfs-1(et15) suppressors. The main structural features of the ATFS-1 protein are depicted, and the nature and position of the alleles used in this study are indicated. For each allele, the atfs-1 coding regions were PCR-amplified and both strands were sequenced to define the mutation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4528320&req=5

fig5: Characterization of atfs-1(et15) suppressors. (A) Overview of the screening strategy: atfs-1(et15);zcIs9(Phsp60::GFP) worms were mutagenized for 4 hr by incubation in the presence of 0.5% ethyl methane sulfonate, and approximately 27,000 mutagenized haploid genomes were screened among the F2 progeny, leading to the isolation of 14 GFP-negative mutants. (B) Images of hsp-60::GFP expression in control worms, atfs-1(et15) worms, and the atfs-1(et15) suppressors et27 and et29. (C) Quantification of the fluorescence levels for representative atfs-1(et15) suppressor mutants. Note that the et29 allele retains some hsp-60::GFP expression. (D) Length of 10 atfs-1(et15) suppressors 96 hr after being deposited as L1s on culture plates containing 0.5 mM fluvastatin; the atfs-1(et15) is also included as a control. Note that the et29 allele retains a partial resistance to fluvastatin. (E) Molecular definition of the atfs-1(et15) suppressors. The main structural features of the ATFS-1 protein are depicted, and the nature and position of the alleles used in this study are indicated. For each allele, the atfs-1 coding regions were PCR-amplified and both strands were sequenced to define the mutation.
Mentions: The genetic pathway linking nduf-7(et19) and the UPRmt reporter hsp-60::GFP likely involves no additional components between atfs-1 and its target hsp-60 promoter; a forward genetic screen to identify suppressors of the previously identified atfs-1(gof) allele et15(Rauthan et al. 2013) resulted in the isolation of 14 intragenic loss-of-function alleles of atfs-1 itself (Figure 5, A–C). Ten of these mutants were characterized in some detail. All were growth-inhibited by 0.5 mM fluvastatin, to which the atfs-1(et15) allele is resistant, and all harbored mutations within the coding region of atfs-1 (Figure 5, D and E). There are at least three possible explanations for having isolated only atfs-1 intragenic suppressors in this screen: (1) atfs-1 may act directly on the hsp-60 promoter; (2) atfs-1 may act together with essential genes that cause lethality when mutated; and (3) atfs-1 may act together with any of several redundant genes. In any case, it may be difficult to further investigate the pathway between atfs-1 and its target promoter using a forward genetics approach. We therefore focused our effort on better understanding how the nduf-7(et19) mutation leads to atfs-1 activation.

Bottom Line: Statins, i.e., cholesterol-lowering drugs that inhibit the rate-limiting enzyme in the mevalonate pathway, HMG-CoA reductase, are lethal to Caenorhabditis elegans even though this animal lacks the branch of the mevalonate pathway that leads to cholesterol synthesis.To better understand the effects of statins that are not related to cholesterol, we have adopted the strategy of isolating statin-resistant C. elegans mutants.We conclude that the nduf-7(et19) mutant allele causes an increase in reactive oxygen species that activate ATFS-1, hence UPR(mt)-mediated statin resistance, and extends life span via CED-4.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, S-405 30, Sweden.

No MeSH data available.