Forward genetics in C. elegans reveals genetic adaptations to polyunsaturated fatty acid deficiency
- Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden
- Department of Molecular and Clinical Medicine/Wallenberg Laboratory, Institute of Medicine, University of Gothenburg, Sweden
Figures
Figure 1
C.elegans fatty acid synthesis pathway and FAT-2 desaturase.
(A) Simplified pathway of fatty acid synthesis and desaturation in C. elegans. Boxes indicate the name of the enzymes, with the FAT-2 desaturase being indicated in a red box. Fatty acids whose synthesis is dependent on FAT-2 are indicated in red. Fatty acid abbreviations are as follow: palmitic acid (PA), palmitoleic acid (POA), vaccenic acid (VA), stearic acid (SA), oleic acid (OA), linoleic acid (LA), alpha-linolenic acid (ALA), gamma-linolenic acid (GLA), stearidonic acid (STA), dihomo-gamma-linolenic acid (DGLA), eicosatetraenoic acid (ETA), arachidonic acid (AA), and eicosapentaenoic acid (EPA). (B) AlphaFold2 predicted the FAT-2 structure with the serine at position 101 indicated with a red arrow. (C) Same structure as in B, zoomed in and angled to show that the S101 position that is mutated to phenylalanine in the fat-2(wa17) allele lies in a loop connecting two alpha helices.
Figure 2
Characterization and rescue of fat-2(wa17).
(A) Introduction of the wild-type fat-2(+) allele on an extrachromosomal array rescues the fat-2(wa17) growth defect. n=20 for each genotype. (B–C) Fluorescence recovery after photobleaching (FRAP) curve and Thalf value show that fat-2(wa17) has rigid membranes similar to paqr-2(tm3410) control. n=8 for each genotype. (D–I, K–M) The lengths of fat-2(wa17) worms grown from L1 stage for 72 hr in the indicated conditions; horizontal dashed lines indicate the approximate lengths of the synchronized L1s at the start of the experiments. n=20 for each genotype/condition. (J) FRAP Thalf values show that NP-40 rescues fat-2(wa17) rigid membranes similarly to paqr-2(tm3410). From left to right, n=6, 8, 7, 7, 8. Error bars show the standard error of the mean. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control (ordinary one-way ANOVA with Tukey multiple comparisons test).
Figure 3 with 1 supplement
Lipidomic analysis of fat-2(wa17) mutant.
(A) Saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) levels in phosphatidylcholines (PCs) of fat-2(wa17) grown in various conditions. Note that cultivation on 2 mM LA boosts PUFA levels. Linoleic acid (LA) to NGM worms were grown on 2 mM LA before being transferred to NGM 6 hr prior to harvesting. (B) Heatmap of phosphatidylcholine (PC) species in fat-2(wa17) in all conditions. (C) Levels of individual FA species in PCs for all conditions. The inset shows that levels of 20:5 FA are increased by providing fat-2(wa17) with linoleic acid. n=4 populations for each genotype/condition. For A and C (inset), *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control and using one-way ANOVA followed by a Dunnett’s multiple comparison test.
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Figure 3—source data 1
Data from the targeted lipidomics analysis, related to Figures 3 and 7, Figure 3—figure supplement 1, Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/104181/elife-104181-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Lipidomics analysis of phosphatidylethanolamines (PEs) in fat-2(wa17) in various cultivation conditions.
(A) Saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) levels in phosphatidylethanolamine (PEs) of fat-2(wa17) grown in various conditions. Cultivation on 2 mM linoleic acid (LA) boosts PUFA levels. LA to NGM worms were grown on 2 mM LA before being transferred to NGM 6 hr prior to harvesting. (B) Heatmap of PE species in fat-2(wa17) in all conditions. (C) Levels of individual FA species in PEs for all conditions. Inset shows that the levels of C20:5 are increased by providing fat-2(wa17) with LA. n=4 populations for each genotype/condition. For A and C (inset), *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control and using one-way ANOVA followed by a Dunnett’s multiple comparison test.
Figure 4
Membrane fluidizing mutations partially rescue fat-2(wa17).
(A–D) Fluidizing paqr-2(tm3410) suppressor mutations only slightly rescue fat-2(wa17) growth. Dashed horizontal lines indicate the approximate length of L1s at the start of the experiments; length was measured 72 hr post-synchronization. n=20 for each genotype. Error bars show the standard error of the mean. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control (ordinary one-way ANOVA with Tukey multiple comparisons test). (E–G) Oil Red O staining of day 1 adults shows that the high lipid abundance in fat-2(wa17) is not suppressed by paqr-2(tm3410) fluidizing mutations.
Figure 5 with 1 supplement
A forward genetic screen reveals that fat-2(wa17) is suppressed by mutations in the HIF-1 pathway.
(A) Overview of the forward genetics screen strategy to isolate fat-2(wa17) suppressors. (B) Identity and position of the fat-2(wa17) suppressors as well as the positions of functional domains. Novel mutations are marked by a black triangle with the corresponding allele name and mutation effect; the red triangle in FAT-2 indicates the original wa17 allele. Gene names in red represent loss- or reduction-of-function mutations; gene names in green represent gain-of-function mutations. (C) Proposed pathway of fat-2(wa17) suppression by mutations in the HIF-1 pathway. Reduction of EGL-9 constitutively activates HIF-1, and HIF-1 activation inhibits FTN-2. The loss of FTN-2 increases the levels of Fe2+, thus boosting FAT-2 desaturase activity. Gain-of-function mutations are labeled in green, loss- or reduction-of-function mutations are labeled in red. (D) Length of all fat-2(wa17) suppressors measured 72 hr after the L1 stage. (E) Representative images of fat-2(wa17) suppressors after 72 h of growth. (F–H) Null alleles of egl-9 and hif-1 do not rescue fat-2(wa17), but the null allele of ftn-2 does, confirming that ftn-2(et67) and ftn-2(et68) are loss-of-function alleles. Lengths were measured 72 hr after L1 synchronization. (I) ftn-2(et68) rescue of fat-2(RNAi) worms, confirming that the suppressors are not wa17 specific. The horizontal dashed line indicates the approximate length of L1s at the start of each experiment. n=20 for each genotype/condition. Error bars show the standard error of the mean. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control (ordinary one-way ANOVA with Tukey multiple comparisons test).
Figure 5—figure supplement 1
fat-2(wa17) and fat-2(syb7458) with suppressors.
(A) Confirmation of hif-1(et69) suppression of fat-2(wa17) by CRISPR-Cas9. (B) The hif-1(et69) allele acts best as a fat-2(wa17) suppressor when in a heterozygous state. (C) ftn-2(et68) does not act as a suppressor for the fat-2(syb7458) allele. (D) ftn-2(et68) still suppresses fat-2(wa17) in a hif-1 null background. (E) hif-1(et69) suppresses fat-2(wa17) in a ftn-2 null background. (A–E) All length measurements were taken 72 hr after L1 synchronization. The horizontal dashed line represents the approximate size of L1s at the start of each experiment. n=20 for each genotype/conditions. Error bars show the standard error of the mean. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control (ordinary one-way ANOVA with Tukey multiple comparisons test).
Figure 6 with 1 supplement
fat-2(wa17) suppressors belong in the HIF-1 pathway and influence HIF-1 levels.
(A) Western blot confirming that hif-1::3xFLAG levels in fat-2(wa17) are increased by egl-9(et60), but not by ftn-2(et68). Hypoxia treatment increases HIF-1 levels in wild-type (WT) and fat-2(wa17), confirming successful protein tagging. (B) Quantification of Western blot in A showing relative intensity of the HIF-1 signal normalized to that of tubulin. (C) mRNA expression of FTN-2, confirming that hif-1(et69) reduces FTN-2 levels. n = the mean of 3 independent normalized replicates for each genotype. *p<0.05 (unpaired t-test). (D) Western blot confirming that fat-2::HA levels in fat-2(wa17) are greatly reduced but increased in suppressor strains. (E) Quantification of Western blot in D showing relative intensity of the FAT-2 signal normalized to that of tubulin.
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Figure 6—source data 1
PDF file containing original western blots for Figure 6, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104181/elife-104181-fig6-data1-v1.zip
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Figure 6—source data 2
Original files for western blot analysis displayed in Figure 6.
- https://cdn.elifesciences.org/articles/104181/elife-104181-fig6-data2-v1.zip
Figure 6—figure supplement 1
Suppressors influence HIF-1 and FAT-2 levels.
(A) Western blot showing that hif-1::3xFLAG levels in fat-2(wa17) are increased by egl-9(et60). (B) Quantification of Western blot in A showing normalized relative intensity of the HIF-1 signal to that of tubulin. (C) Western blot showing that fat-2::HA levels are reduced in fat-2(wa17) but increased by suppressors. (D) Quantification of Western blot in C showing normalized relative intensity of HIF-1 signal to that of tubulin.
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Figure 6—figure supplement 1—source data 1
PDF file containing original western blots for Figure 6—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104181/elife-104181-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/104181/elife-104181-fig6-figsupp1-data2-v1.zip
Figure 7 with 1 supplement
Lipidomic analysis of fat-2(wa17) suppressors reveals that polyunsaturated fatty acid (PUFA) levels are increased.
(A) Levels of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and PUFAs in phosphatidylcholines (PCs) measured in fat-2(wa17) suppressors confirm that the suppressors increase PUFA levels in fat-2(wa17). Worms were homozygous for all indicated genotypes, but note that the hif-1(et69) allele suppresses fat-2(wa17) best in a heterozygous state. (B) Heat map analysis of PC species in suppressor mutants. (C) Levels of individual FA species in PCs in fat-2(wa17) suppressors, the insert shows that levels of C20:5 are significantly increased in all double mutant strains. n=4 populations for each genotype. For A and C (inset), *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control and using one-way ANOVA followed by a Dunnett’s multiple comparison test. Note that the N2 and fat-2(wa17) samples are the same as in Figure 3.
Figure 7—figure supplement 1
Lipidomics of phosphatidylethanolamines (PEs) in fat-2(wa17) suppressors.
(A) Levels of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) in PEs measured in fat-2(wa17) suppressors confirm that the suppressors increase PUFA levels in fat-2(wa17). (B) Heat map analysis of PE species in suppressor mutants. (C) Levels of individual FA species in PEs in fat-2(wa17) suppressors, the insert shows that levels of 20:5 are increased in all double mutant strains. n=4 populations for each genotype. For A and C (inset), *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control and using one-way ANOVA followed by a Dunnett’s multiple comparison test.
Figure 8
ftn-2(et68) rescues fat-2(wa17)’s stress responses.
(A) Representative image of a fluorescence recovery after photobleaching (FRAP) experiment, showing pGLO-1::GFP-CAAX-positive intestinal membranes. The rectangle indicates the bleached area. (B–C) Thalf values and FRAP curves show that ftn-2(et68);fat-2(wa17) has less rigid membranes than fat-2(wa17). From left to right, n=7, 9, 10. (D–E) Representative images and quantification of ftn-2(et68) rescue of fat-2(wa17) mitochondrial stress with a hsp-60::gfp reporter. atfs-1(et15) serves as a control for high mitochondrial UPR activation. n=20 for each genotype. (F–G) Representative images and quantification of DAF-16::GFP localization showing that the DAF-16 stress response is constitutively active in the fat-2(wa17) mutant but normalized by ftn-2(et68). Chi-squared test shows that fat-2(wa17) is significantly different from wild-type (WT). n=100 for each genotype. (H–I) Representative images and quantification of mild ER stress in fat-2(wa17) that is slightly rescued by ftn-2(et68) using a hsp-4::gfp reporter. n=20 for each genotype. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control (ordinary one-way ANOVA with Tukey multiple comparisons test).
Figure 9 with 1 supplement
Exogenous treatments that mimic fat-2(wa17) suppressors partially rescue fat-2(wa17).
(A–E) Length assay of fat-2(wa17) cultivated with different treatments for 72 hr after L1 stage synchronization. The horizontal dashed line represents the approximate length of L1 worms at the start of each experiment. n=20 for each genotype/condition. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control (ordinary one-way ANOVA with Tukey multiple comparisons test).
Figure 9—figure supplement 1
Exogenous treatment of fat-2(wa17) to mimic suppressors.
(A–E) Length of fat-2(wa17) treated with various diets for 72 hr after L1 synchronization, attempting to mimic effects of egl-9, ftn-2, and hif-1 suppressors. Horizontal dashed line represents the approximate size of worms at the start of each experiment. n=20 for each genotype/condition. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences compared to the fat-2(wa17) control (ordinary one-way ANOVA with Tukey multiple comparisons test).
Additional files
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Supplementary file 1
Primers for genotyping.
- https://cdn.elifesciences.org/articles/104181/elife-104181-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/104181/elife-104181-mdarchecklist1-v1.docx
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Forward genetics in C. elegans reveals genetic adaptations to polyunsaturated fatty acid deficiency
eLife 13:RP104181.
https://doi.org/10.7554/eLife.104181.4
