fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans
- Cellular and Molecular Biology Program, University of Michigan, United States
- Department of Molecular and Integrative Physiology, University of Michigan, United States
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, United States
- Department of Internal Medicine, University of Michigan, United States
Figures
Figure 1 with 2 supplements
Cefmo-4 regulates mTOR and dietary restriction-mediated longevity.
(A) Hydropathy plots of FMO-2 and FMO-4 predicting endoplasmic reticulum (ER) transmembrane (TM) domains based on protein sequence analysis. Analysis was done using Deeploc-2.0. (B) Lifespan analysis of wild-type (WT) worms and fmo-4 knockout (fmo-4 KO) worms on fed and dietary restricted (DR) conditions. (C) Lifespan analysis of WT and fmo-4 KO worms on empty vector (EV) RNAi and rsks-1 RNAi. (D) Lifespan analysis of WT and fmo-4 KO worms on EV and vhl-1 RNAi. (E) Lifespan analysis of WT and fmo-4 KO worms on EV and daf-2 RNAi. (F) Lifespan analysis of WT and fmo-4 KO worms on EV and cyc-1 RNAi. For each lifespan, n = ~120 worms per condition per experiment, and three replicate experiments were performed. Significance was determined at p<0.05 using log-rank analysis and significant interactions between the condition of interest and genotype were determined at p<0.01 using Cox regression analysis. All replicate data can be found in Figure 1—source data 1; Figure 1—source data 2; Figure 1—source data 3; Figure 1—source data 4; Figure 1—source data 5.
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Figure 1—source data 1
DR lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig1-data1-v1.zip
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Figure 1—source data 2
mTOR (rsks-1 RNAi) lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig1-data2-v1.zip
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Figure 1—source data 3
vhl-1 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig1-data3-v1.zip
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Figure 1—source data 4
daf-2 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig1-data4-v1.zip
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Figure 1—source data 5
cyc-1 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig1-data5-v1.zip
Figure 1—figure supplement 1
Full-length alignment of AncFMO5, Human FMO5, and C. elegans FMO-1, FMO-2, and FMO-4.
Alignment was conducted using Clustal Omega and ESPript 3.0 to determine an 88% overlap in catalytic residues between C. elegans FMO-2 and FMO-4.
Figure 1—figure supplement 2
Subcellular localization prediction based on protein sequence of C. elegans FMO-4.
Based on the protein sequence, C. elegans FMO-4 is predicted to be located in the endoplasmic reticulum and the golgi apparatus. Analysis was done using Deeploc-2.0.
Figure 2
fmo-4 is required for the health benefits of fmo-2 overexpression.
(A) Lifespan analysis of wild-type (WT), fmo-2 overexpressing (fmo-2 OE), fmo-4 knockout (fmo-4 KO), and fmo-2 OE;fmo-4 KO (fmo-2OE;4KO) worms on E. coli OP50 (n = ~120 worms per condition, three replicate experiments). Significance was determined at p< 0.05 using log-rank analysis. (B) Survival of WT, fmo-2 OE, fmo-4 KO, and fmo-2OE;4KO worms exposed to 5 mM paraquat at L4 stage (n = ~90 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis. (C) Survival of worms exposed to 37 °C heat shock for 3 hr (hours) at L4 stage (n=100 worms per condition, three replicate experiments). (D) Survival of worms exposed to 0, 1, and 5 µg/mL tunicamycin from the egg until day 1 of adulthood (n = ~60 eggs per condition, three replicate experiments). (E) Healthspan analysis of worm thrashing in a drop of M9 solution for 30 s on day 2 of adulthood (n=10 worms per condition, three replicate experiments). (F) Healthspan analysis of worm thrashing in a drop of M9 solution for 30 s on day 10 of adulthood (n=10 worms per condition, three replicate experiments). For heat stress, tunicamycin stress, and healthspan assessments, * denotes significant change at p<0.05 using unpaired two-tailed t-test or one-way ANOVA. N.S. = not significant. All replicate data can be found in Figure 2—source data 1; Figure 2—source data 2; Figure 2—source data 3; Figure 2—source data 4; Figure 2—source data 5.
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Figure 2—source data 1
fmo-2 OE;4KO lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig2-data1-v1.zip
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Figure 2—source data 2
fmo-2 OE;4KO paraquat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig2-data2-v1.zip
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Figure 2—source data 3
fmo-2 OE;4KO heat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig2-data3-v1.zip
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Figure 2—source data 4
fmo-2 OE;4KO tunicamycin stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig2-data4-v1.zip
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Figure 2—source data 5
fmo-2 OE;4KO healthspan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig2-data5-v1.zip
Figure 3 with 3 supplements
Overexpressing fmo-4 is sufficient for lifespan extension, paraquat stress resistance, and improved healthspan.
(A) Lifespan analysis of wild-type (WT), fmo-4 overexpressing (fmo-4 OE), and fmo-4 knockout (fmo-4 KO) worms on E. coli OP50 (n = ~120 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis. (B) Healthspan analysis of worms thrashing in a drop of M9 solution for 30 s (seconds) on day 2 of adulthood (n=10 worms per condition, three replicate experiments). (C) Healthspan analysis of worms thrashing in a drop of M9 solution for 30 s on day 10 of adulthood (n=10 worms per condition, three replicate experiments). (D) Survival of worms exposed to 5 mM paraquat starting from L4 stage (n = ~90 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis. (E) Survival of worms exposed to 37 °C for 3 hr (hours) at L4 stage (n=100 worms per condition, three replicate experiments). (F) Survival of worms exposed to 0, 1, and 5 µg/mL tunicamycin starting from the egg until day 1 of adulthood (n = ~60 eggs per condition, three replicate experiments). (G) Brightfield images of WT and ubiquitous fmo-4 OE worms exposed to DMSO or 1 mg/mL thapsigargin (n = ~20 worms per condition, three replicate experiments). Quantifications of (G) images in (H, I). (J) Development (Dev) time in hours (hrs) of WT and fmo-4 OE worms grown on DMSO control or 1 mg/mL thapsigargin (Thaps) from L2 stage (n=~10 worms per condition, three replicate experiments). * denotes significant change at p<0.05 using t-test. N.S. = not significant. All replicate data can be found in Figure 3—source data 1; Figure 3—source data 2; Figure 3—source data 3; Figure 3—source data 4; Figure 3—source data 5; Figure 3—source data 6; Figure 3—source data 7.
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Figure 3—source data 1
mo-4 OE ubuitous lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-data1-v1.zip
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Figure 3—source data 2
fmo-4 OE ubiquitous healthspan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-data2-v1.zip
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Figure 3—source data 3
fmo-4 OE ubuitous paraquat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-data3-v1.zip
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Figure 3—source data 4
fmo-4 OEubuitous heat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-data4-v1.zip
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Figure 3—source data 5
fmo-4 OE ubuitous tunicamycin stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-data5-v1.zip
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Figure 3—source data 6
fmo-4 OE ubuitous thapsigargin stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-data6-v1.zip
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Figure 3—source data 7
fmo-4 OE ubuitous thapsigargin development replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-data7-v1.zip
Figure 3—figure supplement 1
Development time of experimental worm strains compared to wild-type.
Development (Dev) time in hours (hrs) of wild-type (WT), fmo-2 overexpressing (OE), ubiquitous fmo-4 OE (fmo-4 OE ubiq), hypodermal-specific fmo-4 OE (fmo-4 OE Hyp), fmo-2 knockout (KO), fmo-4 KO, fmo-2 OE;fmo-4 KO (fmo-2OE;4KO), fmo-2 OE;fmo-4 OE (fmo-2OE;4 OE), and fmo-4 OE;fmo-2 KO (fmo-4OE;2KO) worms. (n = ~10 worms per condition, three replicate experiments). * denotes significant change in development time compared to WT. p<0.05 using unpaired two-tailed t-test. NS = not significant. All replicate data can be found in Figure 3—figure supplement 1—source data 1.
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Figure 3—figure supplement 1—source data 1
Replicates of development time of experimental worm strains compared to wild-type.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
fmo-4 acts in the same pathway as fmo-2.
(A) Lifespan analysis of wild-type (WT), fmo-2 overexpressing (fmo-2 OE), fmo-4 overexpressing (fmo-4 OE), and fmo-2 OE;fmo-4 OE (fmo-2OE;4 OE) worms starting from egg (n = ~120 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis. (B) Survival of worms exposed to 5 mM paraquat starting from L4 stage (n = ~90 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis. (C) Survival of worms exposed to 37 °C heat for 3 hr (hours) at L4 stage (n=100 worms per condition, three replicate experiments). (D) Survival of worms exposed to 0, 1, and 5 ug/mL tunicamycin starting from egg until day 1 of adulthood (n = ~60 eggs per condition, three replicate experiments). (E) Brightfield images of WT, fmo-2 OE, fmo-4 OE, and fmo-2OE;4 OE worms exposed to DMSO or 1 mg/mL thapsigargin (n = ~20 worms per condition, three replicate experiments). Quantification of (E) in (F–I). (J) For heat stress, tunicamycin stress, and thapsigargin stress, * denotes significant change at p<0.05 using unpaired two-tailed t-test or one-way ANOVA. N.S. = not significant. All replicate data can be found in Figure 3—figure supplement 2—source data 1; Figure 3—figure supplement 2—source data 2; Figure 3—figure supplement 2—source data 3; Figure 3—figure supplement 2—source data 4; Figure 3—figure supplement 2—source data 5.
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Figure 3—figure supplement 2—source data 1
fmo-2 OE;4OE lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source data 2
fmo-2 OE;4OE paraquat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp2-data2-v1.zip
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Figure 3—figure supplement 2—source data 3
fmo-2 OE;4OE heat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp2-data3-v1.zip
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Figure 3—figure supplement 2—source data 4
fmo-2 OE;4OE tunicamycin stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp2-data4-v1.zip
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Figure 3—figure supplement 2—source data 5
fmo-2 OE;4OE thapsigargin stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp2-data5-v1.zip
Figure 3—figure supplement 3
Knocking out fmo-2 does not affect fmo-4 overexpression.
(A) Lifespan analysis of wild-type (WT), fmo-4 overexpressing (fmo-4 OE), fmo-2 knockout (fmo-2 KO), and fmo-4 OE;fmo-2 KO (fmo-4OE;2KO) worms starting from egg (n = ~120 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis. (B) Survival of worms exposed to 5 mM paraquat starting at L4 stage (n = ~90 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis. (C) Survival of worms exposed to 37 °C heat for 3 hr (hours) from L4 stage (n=100 worms per condition, three replicate experiments). (D) Survival of worms exposed to 0, 1, and 5 ug/mL tunicamycin starting from egg until day 1 of adulthood (n = ~60 eggs per condition, three replicate experiments). (E) Brightfield images of WT, fmo-4 OE, fmo-2 KO, and fmo-4OE;2KO worms exposed to DMSO or 1 mg/mL thapsigargin (n = ~20 worms per condition, three replicate experiments). Quantification of (E) in (F–I). For heat stress, tunicamycin stress, and thapsigargin stress, * denotes significant change at p<0.05 using unpaired two-tailed t test or one-way ANOVA. N.S. = not significant. All replicate data can be found in Figure 3—figure supplement 3—source data 1; Figure 3—figure supplement 3—source data 2; Figure 3—figure supplement 3—source data 3; Figure 3—figure supplement 3—source data 4; Figure 3—figure supplement 3—source data 5.
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Figure 3—figure supplement 3—source data 1
fmo-4 OE;2KO lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp3-data1-v1.zip
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Figure 3—figure supplement 3—source data 2
fmo-4 OE;2KO paraquat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp3-data2-v1.zip
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Figure 3—figure supplement 3—source data 3
fmo-4 OE;2KO heat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp3-data3-v1.zip
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Figure 3—figure supplement 3—source data 4
fmo-4 OE;2KO tunicamycin stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp3-data4-v1.zip
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Figure 3—figure supplement 3—source data 5
fmo-4 OE;2KO thapsigargin stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig3-figsupp3-data5-v1.zip
Figure 4
Overexpressing fmo-4 in the hypodermis is sufficient for lifespan extension and paraquat stress resistance.
(A) Complete cell atlas of C. elegans aging cluster map highlighting regions of fmo-4 gene expression. (B) Image of the ubiquitous fmo-4 overexpressing (fmo-4 OE) worm by the eft-3 promoter showing expression throughout its body. (C) Image of the hypodermal-specific fmo-4 OE worm by the dpy-7 promoter showing expression in the hypodermis. (D) Lifespan analysis of wild-type (WT), fmo-4 OE, hypodermal-specific fmo-4 OE (fmo-4 OEHyp), and fmo-4 knockout (fmo-4 KO) worms (n = ~120 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis. (E) Survival of worms exposed to 5 mM paraquat starting at L4 stage (n = ~90 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis. (F) Survival of worms exposed to 37 °C heat for 3 hr (hours) at L4 stage (n=100 worms per condition, three replicate experiments). (G) Survival of worms exposed to 0, 1, and 5 ug/mL tunicamycin starting at egg until day 1 of adulthood (n = ~60 eggs per condition, three replicate experiments). (H) Brightfield images of WT and fmo-4 OEHyp worms exposed to DMSO or 1 mg/mL thapsigargin (n = ~20 worms per condition, three replicate experiments). Quantification of (H) in (I–J). (K) Healthspan analysis of worms thrashing in a drop of M9 solution for 30 s (seconds) on day 2 of adulthood (n=10 worms per condition, three replicate experiments). (L) Healthspan analysis of worms thrashing in a drop of M9 solution for 30 s on day 10 of adulthood (n=10 worms per condition, three replicate experiments). For heat stress, tunicamycin stress, thapsigargin stress, and healthspan assessments, * denotes significant change at p<0.05 using t-test. N.S. = not significant. All replicate data can be found in Figure 4—source data 1; Figure 4—source data 2; Figure 4—source data 3; Figure 4—source data 4; Figure 4—source data 5; Figure 4—source data 6.
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Figure 4—source data 1
fmo-4 OE hypodermal lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig4-data1-v1.zip
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Figure 4—source data 2
fmo-4 OE hypodermal paraquat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig4-data2-v1.zip
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Figure 4—source data 3
fmo-4 OE hypodermal heat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig4-data3-v1.zip
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Figure 4—source data 4
fmo-4 OE hypodermal tunicamycin stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig4-data4-v1.zip
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Figure 4—source data 5
fmo-4 OE hypodermal thapsigargin stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig4-data5-v1.zip
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Figure 4—source data 6
fmo-4 OE hypodermal healthspan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig4-data6-v1.zip
Figure 5 with 1 supplement
fmo-4 OE transcriptomics reveals a link to calcium regulation.
(A) Gene ontology (GO) analysis of significantly regulated pathways unique to fmo-4 overexpression (fmo-4 OE). (B) Fluorescence images of fmo-4p::mCherry reporter worms exposed to a water control, dietary restriction (DR) control, 300 µM carbachol, or 10 mM EDTA, which is quantified in (C) (n = ~20 worms per condition, three replicate experiments). (D) Lifespan assessment of wild-type (WT) and fmo-4 OE worms exposed to a water control or 50 µM carbachol. (E) Lifespan assessment of WT and fmo-4 knockout (fmo-4 KO) worms exposed to a water control or 50 µM carbachol (F) Lifespan assessment of WT and fmo-4 OE worms exposed to a water control or 50 µM EDTA. (G) Lifespan assessment of WT and fmo-4 KO worms exposed to a water control or 50 µM EDTA. For all lifespan analyses, n = ~120 worms per condition, three replicate experiments performed. Significance was determined at p<0.05 using log-rank analysis and significant interactions between the condition of interest and genotype was determined at p<0.01 using Cox regression analysis. For imaging experiments, * denotes significant change at p<0.05 using unpaired two-tailed t-test. N.S. = not significant. All replicate data can be found in Figure 5—source data 1; Figure 5—source data 2; Figure 5—source data 3.
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Figure 5—source data 1
Carbachol and EDTA imaging replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig5-data1-v1.zip
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Figure 5—source data 2
Quantification of Carbachol and EDTA imaging replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig5-data2-v1.zip
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Figure 5—source data 3
Carbachol and EDTA lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig5-data3-v1.zip
Figure 5—figure supplement 1
Carbachol and EthyleneDiamineTetraAcetic acid (EDTA) alter GCaMP7f fluorescence intesity.
(A) Fluorescence images of neuronal GCaMP7f calcium indicator worms on water control, 300 µM carbachol, or 10 mM EDTA. Fluorescence intensity is quantified in (B) (n = ~20 worms per condition, three replicate experiments). (C) Fluorescence images of the neuronal GCaMP7f calcium indicator worms on 300 µM carbachol assessed at multiple time points including 0 min (minutes), 0.5 min, 5 min, 10 min, 15 min, and 20 min. Fluorescence intensity is quantified in (D) (n = ~20 worms per condition, three replicate experiments). (E) Fluorescence images of the neuronal GCaMP7f calcium indicator worms on 10 mM EDTA assessed at multiple time points including 0 min (minute), 0.5 min, 5 min, 10 min, and 20 min. Fluorescence intensity is quantified in (F) (n = ~20 worms per condition, three replicate experiments). * denotes significant change in fluorescence compared to control. p<0.05 using an unpaired two-tailed t-test. N.S.=not significant. All replicate data can be found in Figure 5—figure supplement 1—source data 1; Figure 5—figure supplement 1—source data 2.
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Figure 5—figure supplement 1—source data 1
GCaMP7f on Carbachol imaging replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
GCaMP7f on EDTA imaging replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig5-figsupp1-data2-v1.zip
Figure 6 with 1 supplement
fmo-4 interacts with calcium signaling genes to promote longevity and paraquat resistance.
(A) Survival analysis of wild-type (WT) and fmo-4 overexpressing (fmo-4 OE) worms on empty vector (EV) and crt-1 RNAi. (B) Survival analysis of worms on EV and itr-1 RNAi. (C) Survival analysis of worms on EV and mcu-1 RNAi. For all lifespan analyses, n = ~120 worms per condition, three replicate experiments were performed. (D) Survival of WT and fmo-4 OE worms on EV and crt-1 RNAi exposed to 5 mM paraquat at L4 stage. (E) Survival of worms on EV and itr-1 RNAi exposed to 5 mM paraquat at L4 stage. (F) Survival of worms on EV and mcu-1 RNAi exposed to 5 mM paraquat at L4 stage. For all paraquat survival assays, n = ~90 worms per condition, three replicate experiments performed. Significance was determined at p<0.05 using log-rank analysis and significant interactions between the condition of interest and genotype were determined at p <0.01 using Cox regression analysis. All replicate data can be found in Figure 6—source data 1; Figure 6—source data 2; Figure 6—source data 3; Figure 6—source data 4.
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Figure 6—source data 1
fmo-4 OE worms on crt-1 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig6-data1-v1.zip
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Figure 6—source data 2
fmo-4 OE worms on itr-1 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig6-data2-v1.zip
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Figure 6—source data 3
fmo-4 OE worms on mcu-1 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig6-data3-v1.zip
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Figure 6—source data 4
Paraquat stress replicates of fmo-4 OE worms on crt-1, itr-1 and mcu-1 RNAi.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig6-data4-v1.zip
Figure 6—figure supplement 1
Lifespan and paraquat survival assessments of fmo-4 KO worms on crt-1, itr-1, and mcu-1 RNAi.
(A) Lifespan assessment of wild-type (WT) and fmo-4 knockout (fmo-4 KO) worms on empty vector (EV) and crt-1 RNAi. (B) Lifespan assessment of WT and fmo-4 KO worms on EV and itr-1 RNAi. (C) Lifespan assessment of WT and fmo-4 KO worms on EV and mcu-1 RNAi. For all lifespan assays, n = ~120 worms per condition, three replicate experiments performed. (D) Survival of WT and fmo-4 KO worms on EV and crt-1 RNAi exposed to 5 mM paraquat at L4 stage. (E) Survival of WT and fmo-4 KO worms on EV and itr-1 RNAi exposed to 5 mM paraquat at L4 stage. (F) Survival of WT and fmo-4 KO worms on EV and mcu-1 RNAi exposed to 5 mM paraquat at L4 stage. (G) Survival of WT and fmo-4 overexpressing (OE) worms on EV and vdac-1 RNAi exposed to 5 mM paraquat at L4 stage. (H) Survival of WT and fmo-4 KO worms on EV and vdac-1 RNAi exposed to 5 mM paraquat at L4 stage. For all paraquat survival assays, n = ~90 worms per condition, three replicate experiments performed. Significance was determined at p<0.05 using log-rank analysis and significant interactions between the condition of interest and genotype was determined at p<0.01 using Cox regression analysis. All replicate data can be found in Figure 6—figure supplement 1—source data 1; Figure 6—figure supplement 1—source data 2; Figure 6—figure supplement 1—source data 3; Figure 6—figure supplement 1—source data 4.
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Figure 6—figure supplement 1—source data 1
fmo-4 KO worms on crt-1 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
fmo-4 KO worms on itr-1 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig6-figsupp1-data2-v1.zip
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Figure 6—figure supplement 1—source data 3
fmo-4 KO worms on mcu-1 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig6-figsupp1-data3-v1.zip
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Figure 6—figure supplement 1—source data 4
Paraqaut stress assays of fmo-4 KO worms on crt-1, itr-1, mcu-1, and vdac-1 RNAi replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig6-figsupp1-data4-v1.zip
Figure 7 with 1 supplement
atf-6 KD regulates fmo-4-mediated longevity and paraquat stress resistance.
(A) Fluorescence imaging of the fmo-4p::mCherry reporter worms on empty vector (EV) and atf-6 RNAi. (B) Quantification of (A) (n = ~20 worms per condition, three replicate experiments). (C) Lifespan analysis of wild-type (WT) and fmo-4 knockout (fmo-4 KO) worms on EV and atf-6 RNAi (n = ~120 worms per condition, three replicate experiments). (D) Survival of WT and fmo-4 KO worms on EV and atf-6 RNAi exposed to 5 mM paraquat at L4 stage (n = ~90 worms per condition, three replicate experiments). Significance was determined at p<0.05 using log-rank analysis and significant interactions between the condition of interest and genotype was determined at p<0.01 using Cox regression analysis. (E) Working model showing that fmo-4 acts downstream of multiple longevity promoters including dietary restriction, reduction in rsks-1, and reduction in atf-6. Reduced expression of atf-6 induces fmo-4 expression, which regulates calcium regulation from the endoplasmic reticulum (ER) to the mitochondria to promote lifespan extension and paraquat stress resistance. For imaging experiments, * denotes significant change at p<0.05 using unpaired two-tailed t test. N.S. = not significant. All replicate data can be found in Figure 7—source data 1; Figure 7—source data 2; Figure 7—source data 3; Figure 7—source data 4.
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Figure 7—source data 1
atf-6 RNAi imaging replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig7-data1-v1.zip
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Figure 7—source data 2
Quantification of atf-6 RNAi imaging replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig7-data2-v1.zip
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Figure 7—source data 3
fmo-4 KO worms on atf-6 RNAi lifespan replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig7-data3-v1.zip
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Figure 7—source data 4
fmo-4 KO worms on atf-6 RNAi paraquat stress replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig7-data4-v1.zip
Figure 7—figure supplement 1
fmo-4 gene expression is not induced by the other branches of UPRER.
(A) Fluorescence images of the fmo-4p::mCherry reporter worms on empty vector (EV) RNAi, atf-4 RNAi, pek-1 RNAi, ire-1 RNAi, and xbp-1 RNAi, quantified in (B) (n = ~20 worms per condition, three replicate experiments). * denotes significant change in fluorescence compared to EV RNAi control. p<0.05 using unpaired two-tailed t test. N.S. =not significant. All replicate data can be found in Figure 7—figure supplement 1—source data 1; Figure 7—figure supplement 1—source data 2.
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Figure 7—figure supplement 1—source data 1
Fluorescent images of reporter worms on atf-4, pek-1, ire-1, and xbp-1 RNAi replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig7-figsupp1-data1-v1.zip
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Figure 7—figure supplement 1—source data 2
Quantification of fluorescent images of reporter worms on atf-4, pek-1, ire-1, and xbp-1 RNAi replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-fig7-figsupp1-data2-v1.zip
Additional files
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Supplementary file 1
qPCR validation of worm strains.
All replicate data can be found in Supplementary file 7.
- https://cdn.elifesciences.org/articles/99971/elife-99971-supp1-v1.xlsx
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Supplementary file 2
RNA-sequencing calcium-related transcripts from DAVID analysis.
- https://cdn.elifesciences.org/articles/99971/elife-99971-supp2-v1.xlsx
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Supplementary file 3
List of RNAi used.
- https://cdn.elifesciences.org/articles/99971/elife-99971-supp3-v1.xlsx
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Supplementary file 4
List of worm strains used in this paper.
- https://cdn.elifesciences.org/articles/99971/elife-99971-supp4-v1.xlsx
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Supplementary file 5
List of genotyping primers used to validate worm strains.
- https://cdn.elifesciences.org/articles/99971/elife-99971-supp5-v1.xlsx
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Supplementary file 6
List of qPCR primers used to validate worm strains.
- https://cdn.elifesciences.org/articles/99971/elife-99971-supp6-v1.xlsx
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Supplementary file 7
qPCR validation of worm strains replicates.
- https://cdn.elifesciences.org/articles/99971/elife-99971-supp7-v1.zip
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MDAR checklist
- https://cdn.elifesciences.org/articles/99971/elife-99971-mdarchecklist1-v1.docx
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Source data 1
RNA-sequencing results showing the significantly upregulated and significantly downregulated transcripts when fmo-4 is overexpressed in C. elegans.
- https://cdn.elifesciences.org/articles/99971/elife-99971-data1-v1.xlsx
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Source data 2
DAVID analysis shows that calcium ion binding transcripts are regulated when fmo-4 is overexpressed in C. elegans.
- https://cdn.elifesciences.org/articles/99971/elife-99971-data2-v1.xlsx
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Source data 3
Log-rank and Cox regression analyses of all lifespans and stress resistance assays which were run in RStudio.
- https://cdn.elifesciences.org/articles/99971/elife-99971-data3-v1.zip
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fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans
eLife 13:RP99971.
https://doi.org/10.7554/eLife.99971.3
