The Rab GTPase activating protein TBC-2 regulates endosomal localization of DAF-16 FOXO and lifespan

FOXO transcription factors have been shown to regulate longevity in model organisms and are associated with longevity in humans. To gain insight into how FOXO functions to increase lifespan, we examined the subcellular localization of DAF-16 in C. elegans. We show that DAF-16 is localized to endosomes and that this endosomal localization is increased by the insulin-IGF signaling (IIS) pathway. Endosomal localization of DAF-16 is modulated by endosomal trafficking proteins. Disruption of the Rab GTPase activating protein TBC-2 increases endosomal localization of DAF-16, while inhibition of TBC-2 targets, RAB-5 or RAB-7 GTPases, decreases endosomal localization of DAF-16. Importantly, the amount of DAF-16 that is localized to endosomes has functional consequences as increasing endosomal localization through mutations in tbc-2 reduced the lifespan of long-lived daf-2 IGFR mutants, depleted their fat stores, and DAF-16 target gene expression. Overall, this work identifies endosomal localization as a mechanism regulating DAF-16 FOXO, which is important for its functions in metabolism and aging.

Upon activation, the Insulin/IGF receptor is internalized into endosomes, where it can disassociate from its ligand and recycle back to the plasma membrane, or it can be targeted for lysosomal degradation [9]. The identification of activated IGFR on endosomes suggested that endosomes can serve as a platform for signaling [10]. Subsequently, several components of the IIS pathway have been shown to localize on endosomes. PTEN localizes on PI(3)P positive endosomes through its C2 domain and has been demonstrated to regulate endosome trafficking via dephosphorylation of Rab7 [11,12]. Akt2 localizes to Appl-1 and WDFY-2 positive endosomes to get fully activated and regulate Akt2 specific downstream substrates [13,14]. 14-3-3 proteins can interact with several Akt phosphorylation targets to regulate their subcellular localization and have been found on endosomes [15,16]. However, a role for endosome trafficking in regulation of FOXO transcription factors has not been demonstrated to the best of our knowledge.
Rab5 and Rab7 GTPases localize to early and late endosomes respectively and are critical regulators of trafficking to the lysosome, an organelle important for cargo degradation and metabolic signaling [17,18]. Like many small GTPases, Rabs cycle between a GTP-bound active state and GDP-bound inactive state. This cycling requires guanine nucleotide exchange factors for activation and GTPase Activating Proteins (GAPs) to catalyze GTP hydrolysis and hence Rab inactivation. We previously characterized C. elegans TBC-2 as having in vitro GAP activity towards RAB-5, and some activity towards RAB-7 [19]. Mutations in tbc-2 result in enlarged late endosomes in several tissues including the intestine, an important site of IIS and metabolic regulation [20]. In addition to early to late endosome maturation, TBC-2 regulates phagosome maturation [21], dense core vesicle maturation [22] endosome recycling as an effector of RAB-10 and CED-10/Rac [23,24] and yolk protein trafficking in oocytes and embryos [25]. Yolk protein is prematurely degraded in tbc-2 mutants. As such, tbc-2 mutant larvae hatched in the absence of food had reduced survival during L1 (first larval stage) diapause [25].
Here we explore the subcellular localization of DAF-16 and show that DAF-16 localizes on early and late endosomes in C. elegans intestinal cells. We found that endosome localization of DAF-16 is regulated by nutrient availability and IIS. We show that endosome localization of DAF-16 is increased through mutations in tbc-2, at the expense of nuclear localization. The increased endosomal localization of DAF-16 in tbc-2 mutants decreases lifespan, fat storage and DAF-16 target gene expression in daf-2 IGFR mutant animals. These results demonstrate a role of endosomal localization in the regulation and function of DAF-16 FOXO.

TBC-2 and the RAB-5 and RAB-7 GTPases regulate DAF-16 localization to endosomes in the intestine
We previously reported that tbc-2 is required for survival during L1 diapause [25]. Since daf-16 is also required for survival during L1 diapause [26,27] we sought to test whether TBC-2 might regulate the nuclear versus cytoplasmic localization of DAF-16. Although we determined that loss of tbc-2 did not result in precocious development during L1 diapause as seen daf-16 mutants [25,27], we found that TBC-2 does in fact regulate DAF-16 localization. Unexpectedly, we found that DAF-16a::GFP (zIs356) localized to numerous amorphous vesicles in the intestinal cells of tbc-2(tm2241) deletion mutant animals ( Fig 1B). Furthermore, we found that DAF-16a::GFP localized to cytoplasmic vesicles in the intestine of wild-type animals ( Fig  1A). The percentage of hermaphrodites with DAF-16 vesicles increased during larval development, peaking at L4 and young adults (Figs 1I and S1A). tbc-2(tm2241) animals were about twice as likely to have DAF-16 vesicles than wild type (Figs 1J and S1A). The number of DAF-16 positive vesicles in wild type can range from zero to hundreds (Fig 1I). DAF-16 positive vesicles can be distributed throughout all 20 intestinal cells or be present in high numbers in just a few cells. DAF-16a::GFP localization is not sex specific as we found similar numbers of DAF-16 positive vesicles in males as in hermaphrodites (S1B Fig). Although DAF-16a::GFP nuclear localization is low in normal growth conditions, we find that tbc-2(tm2241) intestinal nuclei appear to have less nuclear DAF-16a::GFP than wild type (Fig 1A and 1B). We quantified the fluorescence intensity of DAF-16a::GFP in nuclei of tbc-2(tm2241) intestinal cells comparing nuclei of cells with vesicles versus nuclei of cells without vesicles (Fig 1K). We found that nuclei of cells with DAF-16 positive vesicles have significantly less nuclear DAF-16a::GFP than cells without vesicles. Thus, DAF-16 localizes to vesicles in wild-type and tbc-2(tm2241) animals, and tbc-2(tm2241) animals have less nuclear DAF-16a::GFP, likely due to sequestration to cytoplasmic vesicles.
To determine if the localization of the GFP tag or the splice variant or expression levels affected DAF-16 vesicular localization we analyzed three other transgenic strains with lower expression levels; GFP::DAF-16a (muIs71), DAF-16a::RFP (lpIs12) and DAF-16f::GFP (lpIs14, with an alternative N-terminus) [28][29][30]. Using the three fluorescent reporters, we detected DAF-16 vesicles in the tbc-2(tm2241) mutant, albeit not in the wild-type background (S2G- S2X Fig). Overexpression of GFP from vha-6 intestinal specific promoter, vhEx1[Pvha-6:: GFP], did not show significant vesicular localization in wild-type animals (S3A Fig). In tbc-2 (tm2241) animals GFP showed some vesicular localization and potential aggregates, but much less than seen with DAF-16a::GFP, indicating that the vesicular localization is due to DAF-16 . DAF-16a::GFP (green) is present on vesicles in both wild-type and tbc-2(tm2241) intestinal cells (A and B) that are positive for autofluorescence (blue) present in the the endolysosomal system (E and F). Corresponding DIC (C and D) and merged (G and H) images are shown. A representative vesicle is shown (arrow head) and the two nuclei of the binucleate cell are marked (n). Note that DAF-16a::GFP is excluded from the large nucleoli (A). (I-J) Grouped bar graphs quantifying the percentage of wild-type (I) and tbc-2(tm2241) (J) animals with 0, 1-10, 11-50 or >50 DAF-16a::GFP (zIs356) positive vesicles at the different larval and not the GFP tag. Of note, we found that GFP expression in the tbc-2 background was visibly stronger than in wild type (S3B-S3E Fig), consistent with vha-6 ranking amongst the top downregulated DAF-16-responsive genes [31] and consistent with TBC-2 facilitating DAF-16 nuclear localization.
To determine if endogenous DAF-16 localizes to vesicles we analyzed daf-16(hq23), a DAF-16::GFP line generated by CRISPR/Cas9 genome editing [32], and found that endogeneously tagged DAF-16 localized to vesicles in both wild type and tbc-2(tm2241) mutants (S2A- S2F  Fig). To determine if the GFP/RFP tag is driving vesicular localization of DAF-16 we analyzed two daf-16 alleles endogenously tagged with evolutionarily distant mNeongreen and mKate2 fluorescent proteins [33,34]. We found that DAF-16::mNG and DAF-16::mK2 both localized to intestinal vesicles in wild-type animals (Fig 2). DAF-16 positive vesicles are marked with arrowheads and are distinct from intestinal autofluorescence in the other fluorescence channels. Furthermore, the percent wild-type and tbc-2(tm2241) animals with endogenous DAF-16::mNG vesicles were comparable to that of the overexpressed DAF-16a::GFP ( Fig 2I). Therefore, DAF-16 localization to vesicles is not due to overexpression and unlikely to be an artifact of the fluorescent tag.

Acute starvation suppresses DAF-16 localization to endosomes
Nuclear localization of DAF-16 is modulated by nutrient availability. As such, starvation promotes DAF-16 cytoplasmic-to-nuclear shuttling [28]. To determine if endosomal DAF-16 can translocate to the nucleus, we tested the effect of acute starvation on DAF-16a::GFP localization to endosomes. We found that starvation strongly suppressed the localization of DAF-16 to endosomes in both wild type and tbc-2(tm2241) mutants (Figs 4A and S4). Upon re-feeding, DAF-16 relocalized to endosomes after 1-2 hours, in both wild type and tbc-2(tm2241) mutants. Thus, DAF-16 localization on endosomal membranes is regulated by nutrient availability.

PLOS GENETICS
TBC-2 regulates endosomal localization of DAF-16 FOXO and lifespan hypomorphic mutant of the insulin/IGF receptor, daf-2(e1370), in which IIS is reduced, particularly at higher temperatures [41]. We compared DAF-16a::GFP localization in three independent daf-2(e1370) strains at 15˚C and shifted overnight to 25˚C to enhance disruption of DAF-2. All three showed a significant reduction in the number of DAF-16 positive vesicles at 25˚C, while the wild-type strain did not (Fig 4C), indicating that DAF-2 promotes DAF-16 localization to endosomes.
DAF-18, the homolog of human tumor suppressor protein PTEN, acts as a negative regulator of the IIS pathway counteracting AGE-1 PI3K signaling by dephosphorylating the 3'phosphate on the PI(3,4,5)P 3 converting it to PI(4,5)P 2 [42]. We analyzed DAF-16a::GFP localization in the daf-18 reference allele, e1375. We generated three independent daf-18 (e1375); zIs356 strains, but only two strains had increased endosome localization of DAF-16. However, combined data from the three strains had a statistically significant increase in the number of animals with DAF-16 endosomes ( Fig 4D). Since daf-18(e1375) is possibly a nonnull allele, we analyzed a deletion allele, daf-18(ok480). We found that five independent daf-18 (ok480); zIs356 strains had more DAF-16 endosomes than wild type that when combined was statistically significant (Fig 4D). Since DAF-16 endosome localization is mildly increased in the background of two distinct daf-18 alleles, it is consistent with increased IIS causing an increase in DAF-16 localization to endosomes.
C. elegans AKT-1 and AKT-2 function upstream of, and can phosphorylate, DAF-16 [43,44]. The SGK-1 Serum Glucorticoid Kinase homolog interacts with AKT kinases and has been shown to regulate DAF-16 nuclear localization [44]. We tested which of these kinases . Arrows mark DAF-16::mNeongreen positive vesicles in the green channel (A) that are distinct from autofluorescence in the red (C) and blue channels shown as a merge (E). Arrows mark DAF-16::mKate2 positive vesicles in the red channel (B) that are distinct from autofluorescence in the green (D) and blue channels shown as a merge (F). For additional context the fluorescent channels were merged with their corresponding DIC image (G and H). Bar graphs displaying the percent wild-type and tbc-2(tm2241) animals with DAF-16::mNG positive vesicles (I). Raw data is available in S1 Data. Fisher's exact test (graphpad.com) was used to determine the statistical difference between conditions. n, total number of animals. ���� P<0.0001. Scale bar (A), 5μm. https://doi.org/10.1371/journal.pgen.1010328.g002

TBC-2 is required for lifespan extension and increased fat storage of daf-2 (e1370) mutants
To determine if the increased endosomal localization of DAF-16 seen in tbc-2 mutants affects DAF-16 activity, we tested if TBC-2 regulates adult lifespan. We found that the tbc-2(sv41) and tbc-2(tm2241) deletion alleles had similar lifespans as compared to wild type (Fig 5A and 5B). As expected, daf-2(e1370) animals lived significantly longer than wild type. We found that both tbc-2 alleles significantly shortened the lifespan of daf-2(e1370) animals (Fig 5A and 5B). Thus, TBC-2 is not required for normal lifespan, but is partly required for the extended lifespan of daf-2(e1370) mutants.
To assess TBC-2's contribution to other daf-2 mutant phenotypes, we tested whether TBC-2 is required for the increased fat storage of daf-2(e1370) mutants. Consistent with previous findings we found that daf-2(e1370) had significantly more fat than wild-type animals as determined by Nile Red and Oil Red O staining of fixed L4 larvae [50,51] (Fig 5C-5F). We found that tbc-2 (tm2241) larvae had less fat than wild type with Nile Red staining, but not with Oil Red O staining (Fig 5D and 5F). This difference might reflect the lower sensitivity of Oil Red O for quantifying lipid abundance as compared to Nile Red [52]. Interestingly, we found that tbc-2(tm2241) significantly suppressed the daf-2(e1370) increased lipid staining by both Nile Red and Oil Red O ( Fig  5C-5F). Thus, TBC-2 is required for the increased fat storage of daf-2(e1370) mutants.

DAF-16 localizes to endosomes
We were surprised to find that DAF-16 FOXO localizes to a subset of RAB-5 and RAB-7 endosomes in wild-type animals. Many studies have used DAF-16 cytoplasmic versus nuclear localization to assess IIS activity under various conditions. We assume that DAF-16 positive endosomes were not discovered earlier as nuclear translocation can be assessed at low magnification where DAF-16 positive endosomes are not apparent, and DAF-16 positive endosomes are not present in every cell or every animal. Furthermore, we first took notice of DAF-16 positive endosomes in the tbc-2 mutant background where they are more prominent. These are likely not an artefactual consequence of overexpression, as we see these vesicles in the endogenously tagged daf-16::GFP and we do not see similar vesicles in a GFP overexpression strain. DAF-16::mNG and DAF-16::mK2 also localize to vesicles indicating that membrane localization is unlikely to be an artifact of the GFP tag. Additionally, DAF-16-vesicles are regulated by IIS.
Many components of the IIS pathway localize to endosomes in mammalian cells including active insulin receptor and downstream signaling components such as PI3K, Akt, PTEN and 14-3-3 proteins [9,11,15,57-61]. In the case of Akt and PTEN, both have demonstrated roles in regulation of endosome trafficking independent of IIS [12, [62][63][64]. On the other hand, the PI3Ks are Rab5 effectors and Rab5 has been shown to promote Akt activity on endosomes [60,61,[65][66][67]. While endosomal localization of FOXO proteins has not been reported to the best of our knowledge, knockdown of Rab5 in mouse liver results in a strong increase in phosphorylated FOXO1 [68]. This is contrary to the finding that Rab5 promotes Akt phosphorylation [61,66], which could be a consequence of indirect regulation or suggest tissue-specific regulation. The fact that TBC-2 is a RAB-5 GAP is consistent with increased RAB-5 activity promoting DAF-16 localization on endosomes. This is further supported by the fact that rab-5 and rab-7 RNAi knockdown reduces the number of animals with DAF-16 vesicles in both wild type and tbc-2 mutants. Given the importance of RAB-5 for endosome trafficking, it is difficult to parse whether RAB-5 is promoting a platform for DAF-16 localization or if it also has a role in IIS.
We demonstrated that DAF-16 localized to a subset of RAB-5 and RAB-7 positive endosomes. Since RAB-5 and RAB-7 promote trafficking to the lysosome and promote receptor tyrosine kinase degradation, it is possible that DAF-16-positive endosomes are signaling endosomes, in which case we would expect other upstream signaling components might be present. Consistent with that hypothesis, knockdown of IIS reduces the number of animals with DAF-16 vesicles. However, when analyzing DAF-16 localization in daf-2(e1370) mutants at 15˚C vs. 25˚C, we find that while there is a reduction in the number of DAF-16 endosomes, these endosomes are noticeably fainter at 25˚C. This suggests that there are not necessarily less endosomes being generated, but rather less DAF-16 on the vesicles which may be inconsistent with these being signaling endosomes derived from DAF-2 internalization at the plasma membrane. On the other hand, the fact that Akt and 14-3-3 can localize to endosomes in mammalian cells [15,59,61] and that AKT-1 and FTT-2 promote DAF-16 localization to endosomes, suggests that these proteins might recruit DAF-16 onto endosomes rather than DAF-16 interacting directly with membranes. Future studies should test whether DAF-2 IGFR and downstream IIS components actively recruit DAF-16 to endosomes or whether IIS has a passive role. IIS inhibition of nuclear DAF-16 could result in increased DAF-16 in the cytoplasm where it can bind endosomes.
If these are not signaling endosomes, then what are the DAF-16 endosomes? Since RAB-5 and RAB-7 are also regulators of autophagy, so it is possible that these endosomes contribute to degradation of inactive excess DAF-16. There is precedent for selective autophagy in the degradation of the GATA4 transcription factor [69]. Alternatively, these endosomes could serve as a reservoir of inactive DAF-16 that can be quickly mobilized if environmental stress is encountered. For example, we found that acute starvation is a potent regulator of DAF-16 endosome localization, even in the tbc-2 mutant background.
We find it interesting that there is such variability within the population, and amongst the intestinal cells in a given animal, as to whether there will be DAF-16 positive endosomes or not. It suggests that each intestinal cell autonomously senses changes in IIS, or possibly other nutrient and stress sensing pathways, to regulate DAF-16 localization. Then, why is endosomal DAF-16 more prominent in tbc-2 mutants? One explaination would be that the expansion of endosomal membranes in a tbc-2 mutant create more storage space for inactive DAF-16. Another would be that IIS or other pathways are more active in tbc-2 mutants, or some combination of the two. The fact that loss of IIS does not eliminate DAF-16 localization from endosomes suggests that additional signaling pathways could regulate DAF-16 endosome localization. GLP-1/Notch signaling in the germline regulates longevity in a DAF-16-dependent manner as well as DAF-16 nuclear translocation [70], and IIS post-translationally regulates GLP-1 signaling [71], thus it would be interesting to determine if DAF-16 localization to endosomes are regulated by GLP-1/Notch signaling and if TBC-2 regulates GLP-1 to DAF-16 target gene expression [72]. Additionally, AMPK, JNK and LET-363/mTor signaling regulate DAF-16 and could regulate DAF-16 localization to endosomes or be subject to regulation by TBC-2, particularly mTor which localizes to lysosomes [70,[73][74][75][76][77].

A direct role for TBC-2 in regulating daf-2(e1370) IGFR mutant phenotypes
Our finding that tbc-2 was required for the extended lifespan and increased fat storage of daf-2 (e1370) mutants suggests that TBC-2 might have a more specific role related to IIS. However, TBC-2 is not the first endosomal regulator required for lifespan extension of daf-2(e1370) mutants. C. elegans BEC-1, a homolog of human Beclin1, is a regulator of autophagy and endosome trafficking, and bec-1 mutants accumulate large late endosomes in the intestinal cells [78][79][80]. Mutations in bec-1 suppress the increased lifespan of daf-2(e1370), and were reported to be required for the increased fat storage [78]. Similarly, other autophagy regulators, atg-7 and atg-12, have been shown to be required for daf-2 longevity [81]. An RNAi screen identified regulators of endosome to lysosome trafficking, including RAB-7, and components of the ESCRT and HOPS complexes as being required for the lifespan extension phenotypes of daf-2 IGFR mutants [82]. However, the mechanisms by which they regulate lifespan are not known. In daf-2(e1370) mutants, there is an increase in autophagy and lysosome function, both of which are required for the extended lifespan [78,[83][84][85], consistent with increased stress resistance contributing to increased longevity. Since the human homologs of TBC-2 are implicated in autophagy [86][87][88][89], and tbc-2 mutants accumulate autophagy protein LGG-1 (LC3/Atg8) in enlarged endosomes [19], it is possible that TBC-2 also regulates autophagy and thus daf-2(-) longevity. However, our findings that TBC-2 regulates DAF-16 nuclear vs. endosome localization and that TBC-2 is required for the DAF-16 target gene expression in daf-2 mutants demonstrate that TBC-2 has a more direct role in IIS, as opposed to being required for downstream cellular responses. It will be interesting to test if TBC-2 regulates DAF-16-independent mechanisms of longevity as well as to determine if other endosome and autophagyregulating genes can regulate DAF-16 localization to endosomes.
In conclusion, we demonstrate that the DAF-16 FOXO transcription factor localizes to endosomes. This endosomal localization of DAF-16 FOXO is regulated by both IIS and the TBC-2 Rab GAP. TBC-2 promotes the nuclear localization of DAF-16 FOXO, and this localization has functional effects on both longevity and metabolism through modulation of DAF-16 target gene expression. Our data suggest that endosomes serve as an important location for DAF-16 FOXO transcription factor regulation, and suggest that endomembranes may function as a site of transcription factor regulation.

C. elegans genetics and strain construction
C. elegans strains were cultured as described in Wormbook (www.wormbook.org). The C. elegans N2 Bristol strain was the wild-type parent strain and HB101 E. coli strain was used as a food source. Both were obtained from the Caenorhabditis Genetic Center (CGC) as were many of the strains used in this study (S1 Table). New strains were constructed using standard methods and the presence of mutations were confirmed by PCR and DNA sequencing.

RNAi experiments
C. elegans RNAi feeding experiments were conducted essentially as described in [90]. RNAi feeding clones were obtained from the Ahringer RNAi library and confirmed by sequencing (S1 Table) [91,92]. The L4440 empty RNAi feeding vector transformed into HT115(DE3) was used as a negative control [93].

Microscopy
DAF-16 vesicular localization was analyzed in the intestinal cells of hermaphrodites. Hermaphrodite worms at the L4 stage were imaged alive at room temperature unless it is stated otherwise. Animals were picked and mounted onto 4% agarose pads, animals were anesthetized with levamisole.
Differential interference contrast (DIC) and fluorescent imaging were performed with an Axio Imager A1 compound microscope with a 100×1.3 NA Plan-Neofluar oil-immersion objective lens (Zeiss) and images were captured by using an Axio Cam MRm camera and AxioVision software (Zeiss). Confocal microscopy was performed on an Axio Observer Z1 LSM780 laser scanning confocal microscope with a 63×1.4 NA Plan-Apochromat oil-immersion objective lens (Zeiss) in a multi-track mode using an argon multiline laser (405 nm excitation for autofluoresence, 488 nm excitation for GFP and a 561/ 594 nm excitation for mCherry/RFP). Images were captured with a 32 channel GaAsP detector and ZEN2010 image software. Raw data was analyzed using Fiji (ImageJ) or Zen 2010 Lit programs, and images were modified by using Fiji (ImageJ).
To compare DAF-16 (zIs356) nuclear intensity in cells with or without DAF-16 vesicles, animals at L4 stage were imaged using an LSM780 scanning laser microscope. To ensure consistency, only the anterior most intestine cells were imaged. First, the nucleus of intestinal cells were focused under bright field and without changing the position, GFP, autofluoresence and bright field signals were imaged. Each animal was imaged using the same confocal settings. After data collection, each nucleus was categorized as a nucleus with adjacent DAF-16-positive vesicles or a nucleus without any DAF-16 positive vesicles. Total GFP intensity inside the nucleus was measured using Fiji (Image J) software. The nucleus is circled using DIC/Bright Field and autofluorescence channels as reference. Since intestinal cells have two nuclei per cells, if there are two nuclei within the focus, their GFP intensity is averaged for statistical analysis. Prism 8 (GraphPad) were used to graph the data and determine statistical analysis using an unpaired t-test.

Starvation-refeeding and temperature shift experiments
For starvation and refeeding experiments animals were synchronized at the L1 stage and grown on NGM plates with HB101 E. coli till the L4 stage. Then animals were collected washed 3 times for 5 mins with M9 buffer to remove bacteria in their gut. After the third wash animals were plated to regular NGM plates with or without HB101 E. coli, and incubated for 4-5 hours before scoring. Animals are scored for the presence of DAF-16::GFP-positive vesicles the intestine using an A1 Zeiss microscope. After 4h-5h of starvation, animals were harvested with M9 buffer from the starved plates and washed once with M9 buffer and plated to NGM plates with HB101 E. coli and incubated for 1-2 hours at 20˚C before scoring.

Life span analysis
Replicate strains were maintained for several generations prior to beginning the lifespan assays which were conducted at 20˚C. For each strain 25 young adult hermaphrodites were picked to two NGM plates without FUDR seeded with HB101 E. coli for three independent replicates totaling 150 animals. Strains were coded and scored blindly to reduce bias. Animals were transferred to fresh NGM plates to avoid contamination and getting crowded out by their progeny. Animals were scored every 2-3 days and were considered dead when they stop exhibiting spontaneous movement and fail to move in response to 1) a gentle touch of the tail, 2) a gentle touch of the head, and 3) gently lifting the head. Animals that die of unnatural causes (internal hatching of embryos, bursting, or crawling off the plate) are omitted. Graphs and statistics were done using Graphpad Prism. None of the strains used in the lifespan assays carry the fln-2(ot611) mutation found in a N2 male stock strain and found to extend median lifespan [94,95].

Fat staining
L4 animals were fixed for staining with Nile Red (Invitrogen) or Oil Red O (Sigma-Aldrich) as previously described [52]. Imaging and analysis was done as previously described [96]. Graphs and statistics were done using Graphpad Prism.

Quantitative real-time RT-PCR
C. elegans RNA was isolated from young adults maintained at 15˚C using TRIZOL reagent (Invitrogen). 1 ug of RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). Quantitative real-time PCR was performed using 1 μl of the cDNA preparation with SYBR-Green Reagents and a Vii7 qPCR analyzer (Applied Biosystems). Each DAF-16 target gene was amplified using PCR primers as described in [97] and compared to act-3 (S1 Table).

Statistical analysis
Statistical analysis was carried out using GraphPad Prism software. For the analysis of two groups, a student t test was performed using two-tailed distribution for analysis involving two groups of samples. Fishers' exact test was used for comparing groups of four. For each analysis, P<0.05 was considered as significant. Raw data is available in S1 Data. The difference was determined to be not significant (ns) in an unpaired t test. (TIF) S1 Table. List of key resources used in this study including bacterial strains, C. elegans strains, RNAi-feeding clones, and oligonucleotides. (DOCX) S1 Data. Raw numbers and statistical analyses summarized in the bar graphs.