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 was determined at p < 0.01 using Cox regression analysis.

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 5mM 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 hours (hrs) at L4 stage (n = 100 worms per condition, three replicate experiments). (D) Survival of worms exposed to 0, 1, and 5µg/mL tunicamycin from 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 seconds 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 seconds 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.

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 seconds (sec) 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 seconds on day 10 of adulthood (n = 10 worms per condition, three replicate experiments). (D) Survival of worms exposed to 5mM 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 hours (hrs) at L4 stage (n = 100 worms per condition, three replicate experiments). (F) Survival of worms exposed to 0, 1, and 5µg/mL tunicamycin starting from 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 1mg/mL thapsigargin (n = ∼20 worms per condition, three replicate experiments). Quantifications of (G) images in (H-I). For heat stress, tunicamycin stress, thapsigargin stress, and healthspan assessments, * denotes significant change at p < 0.05 using t test. N.S. = not significant.

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 5mM 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 hours (hrs) at L4 stage (n = 100 worms per condition, three replicate experiments). (G) Survival of worms exposed to 0, 1, and 5ug/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 1mg/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 seconds (sec) 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 seconds 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.

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, 300µM carbachol, or 10mM 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 50µM carbachol. (E) Lifespan assessment of WT and fmo-4 knockout (fmo-4 KO) worms exposed to a water control or 50µM carbachol (F) Lifespan assessment of WT and fmo-4 OE worms exposed to a water control or 50µM EDTA. (G) Lifespan assessment of WT and fmo-4 KO worms exposed to a water control or 50µM 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.

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 performed. (D) Survival of WT and fmo-4 OE worms on EV and crt-1 RNAi exposed to 5mM paraquat at L4 stage. (E) Survival of worms on EV and itr-1 RNAi exposed to 5mM paraquat at L4 stage. (F) Survival of worms on EV and mcu-1 RNAi exposed to 5mM 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.

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 5mM 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 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.