Abstract
FOXO transcription factors play a crucial role in regulating longevity in various organisms, including humans. This article explores the subcellular localization of DAF-16, a C. elegans FOXO transcription factor, to understand how FOXO functions to extend lifespan. DAF-16 is found to be localized to endosomes, and this localization is enhanced by the insulin-IGF signaling (IIS) pathway. Endosomal trafficking proteins modulate this localization. Disruption of TBC-2, a Rab GTPase activating protein, increases endosomal localization of DAF-16. Conversely, inhibiting TBC-2 targets, RAB-5 or RAB-7 GTPases, decreases DAF-16 endosomal localization. The extent of DAF-16 localization to endosomes significantly impacts lifespan, fat storage, and DAF-16 target gene expression. Increased endosomal localization due to mutations in tbc-2 reduces the lifespan of long-lived daf-2 IGFR mutants, depletes their fat stores, and alters DAF-16 target gene expression. These findings highlight the importance of endosomal trafficking in regulating DAF-16 localization and suggest that endosomes are critical sites of FOXO regulation.
Introduction
The insulin/insulin-like growth factor signaling (IIS) pathway is an evolutionarily conserved pathway influencing lifespan, development, metabolism, immunity, and stress responses across species. In C. elegans, IIS-mediated regulation of DAF-16 FOXO was initially identified through genetic characterization of mutants affecting dauer formation and lifespan. Mutations in the insulin-like growth factor receptor (IGFR), DAF-2, can extend lifespan in a DAF-16-dependent manner. Under favorable conditions, the DAF-2 IGFR signals through a conserved PI3K/Akt pathway, leading to phosphorylation and inhibition of nuclear accumulation of DAF-16 FOXO. Disrupting IIS enhances DAF-16 FOXO nuclear entry, inducing stress response genes.
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. 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. Akt2 localizes to Appl-1 and WDFY-2 positive endosomes to get fully activated and regulate Akt2 specific downstream substrates. 14-3-3 proteins can interact with several Akt phosphorylation targets to regulate their subcellular localization and have been found on endosomes. 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. 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. TBC-2 has in vitro GAP activity towards RAB-5, and some activity towards RAB-7. Mutations in tbc-2 result in enlarged late endosomes in several tissues including the intestine, an important site of IIS and metabolic regulation. In addition to early to late endosome maturation, TBC-2 regulates phagosome maturation, dense core vesicle maturation, endosome recycling as an effector of RAB-10 and CED-10/Rac and yolk protein trafficking in oocytes and embryos.
This article explores the subcellular localization of DAF-16 and shows that DAF-16 localizes on early and late endosomes in C. elegans intestinal cells. Endosome localization of DAF-16 is regulated by nutrient availability and IIS. 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. Since daf-16 is also required for survival during L1 diapause, it was tested whether TBC-2 might regulate the nuclear versus cytoplasmic localization of DAF-16. TBC-2 does in fact regulate DAF-16 localization.
DAF-16 FOXO Localizes to Vesicles in Intestinal Cells
DAF-16a::GFP (zIs356) localized to numerous amorphous vesicles in the intestinal cells of tbc-2(tm2241) deletion mutant animals. DAF-16a::GFP localized to cytoplasmic vesicles in the intestine of wild-type animals. The percentage of hermaphrodites with DAF-16 vesicles increased during larval development, peaking at L4 and young adults. tbc-2(tm2241) animals were about twice as likely to have DAF-16 vesicles than wild type. The number of DAF-16 positive vesicles in wild type can range from zero to hundreds. 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 similar numbers of DAF-16 positive vesicles in males as in hermaphrodites were found. Although DAF-16a::GFP nuclear localization is low in normal growth conditions, tbc-2(tm2241) intestinal nuclei appear to have less nuclear DAF-16a::GFP than wild type. Nuclei of cells with DAF-16 positive vesicles have significantly less nuclear DAF-16a::GFP than cells without vesicles.
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Using the three fluorescent reporters, DAF-16 vesicles in the tbc-2(tm2241) mutant were detected, albeit not in the wild-type background. Overexpression of GFP from vha-6 intestinal specific promoter, vhEx1[Pvha-6::GFP], did not show significant vesicular localization in wild-type animals. 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 and not the GFP tag. To determine if endogenous DAF-16 localizes to vesicles daf-16(hq23), a DAF-16::GFP line generated by CRISPR/Cas9 genome editing, was analyzed, and it was found that endogeneously tagged DAF-16 localized to vesicles in both wild type and tbc-2(tm2241) mutants. To determine if the GFP/RFP tag is driving vesicular localization of DAF-16 two daf-16 alleles endogenously tagged with evolutionarily distant mNeongreen and mKate2 fluorescent proteins were analyzed. DAF-16::mNG and DAF-16::mK2 both localized to intestinal vesicles in wild-type animals. 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.
Endogenously Tagged DAF-16::mNeongreen and DAF-16::mKate2 Localize to Vesicles
Representative confocal and differential interference contrast (DIC) images of intestinal cells of daf-16(ot853[daf-16::linker::mNeongreen::3xFlag::AID]) and daf-16(ot821[daf-16::mKate2::3xFlag]) were analyzed. Arrows mark DAF-16::mNeongreen positive vesicles in the green channel that are distinct from autofluorescence in the red and blue channels shown as a merge. Arrows mark DAF-16::mKate2 positive vesicles in the red channel that are distinct from autofluorescence in the green and blue channels shown as a merge. For additional context the fluorescent channels were merged with their corresponding DIC image. Bar graphs displaying the percent wild-type and tbc-2(tm2241) animals with DAF-16::mNG positive vesicles are shown.
RAB-5 and RAB-7 GTPases Promote DAF-16 FOXO Localization to Endosomes
To determine if the DAF-16 vesicles are endosomes, DAF-16a::GFP was co-expressed with RFP::RAB-5, an early endosomal marker, or with mCherry::RAB-7, a late endosomal marker. In wild-type animals, DAF-16a::GFP localizes to a subset of RAB-5 and RAB-7-positive endosomes. Thirty percent of DAF-16a::GFP vesicles were RAB-5 positive while 5% were RAB-7 positive. Furthermore, the genetic requirements for rab-5 and rab-7 for DAF-16 localization were tested by RNAi. rab-5(RNAi) and rab-7(RNAi) knockdown significantly decreased the number of wild-type and tbc-2(tm2241) animals with DAF-16a::GFP vesicles. Together, these data are consistent with DAF-16 localizing to a sub-population of endosomes or endosome-like vesicles in the intestinal cells.
Representative confocal images of the intestinal cells of animals expressing DAF-16a::GFP (green) and either RFP::RAB-5 or mCherry::RAB-7 (magenta) were analyzed. Arrowheads mark examples of vesicles positive for both DAF-16a::GFP and either RFP::RAB-5 or mCherry::RAB-7. Arrows mark examples of DAF-16a::GFP vesicles that are not positive for either RFP::RAB-5 or mCherry::RAB-7. Bar graphs displaying the mean (and SEM) of the percent wild-type and tbc-2(tm2241) animals with DAF-16a::GFP (zIs356) vesicles fed bacteria expressing control empty vector (ev) RNAi (black bars) compared to animals fed rab-5(RNAi) or rab-7(RNAi) (white bars) from three independent experiments.
DAF-16 FOXO Endosome Localization is Regulated by IIS and Nutrient Availability
Nuclear localization of DAF-16 is modulated by nutrient availability. Starvation promotes DAF-16 cytoplasmic-to-nuclear shuttling. The effect of acute starvation on DAF-16a::GFP localization to endosomes was tested to determine if endosomal DAF-16 can translocate to the nucleus. Starvation strongly suppressed the localization of DAF-16 to endosomes in both wild type and tbc-2(tm2241) mutants. Upon re-feeding, DAF-16 relocalized to endosomes after 1â2 hours, in both wild type and tbc-2(tm2241) mutants. To determine if IIS regulates DAF-16 localization to endosomes the effect of disrupting IIS on DAF-16a::GFP localization was analyzed. Firstly, a hypomorphic mutant of the insulin/IGF receptor, daf-2(e1370), in which IIS is reduced, particularly at higher temperatures was analyzed. 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 was compared.
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Bar graphs of the percent wild-type (black) and tbc-2(tm2241) (red) animals with DAF-16a::GFP (zIs356) vesicles in fed animals, animals starved between 4 and 5 hours and starved animals that have been refed for 1 to 2 hours are shown. A diagram of IIS-mediate regulation of DAF-16/FOXO is presented. Grouped bar graphs quantifying the percentage of wild-type and daf-2(e1370) L4 larvae (cumulative data from 3 independent strains) with 0, 1â10, 11â50 or >50 DAF-16a::GFP (zIs356) positive vesicles at 15°C or shifted overnight to 25°C are shown. Bar graphs of percent wild.
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