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Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway
Abstract
C. elegans insulin-like signaling regulates metabolism, development, and life span. This signaling pathway negatively regulates the activity of the forkhead transcription factor DAF-16. daf-16 encodes multiple isoforms that are expressed in distinct tissue types and are probable orthologs of human FKHRL1, FKHR, and AFX. We show that human FKHRL1 can partially replace DAF-16, proving the orthology. In mammalian cells, insulin and insulin-like growth factor signaling activate AKT/PKB kinase to negatively regulate the nuclear localization of DAF-16 homologs (reviewed in ). We show that the absence of AKT consensus sites on DAF-16 is sufficient to cause dauer arrest in daf-2(+) animals, proving that daf-16 is the major output of insulin signaling in C. elegans. FKHR, FKRHL1, and AFX may similarly be the major outputs of mammalian insulin signaling. daf-2 insulin signaling, via AKT kinases, negatively regulates DAF-16 by controlling its nuclear localization. Surprisingly, we find that daf-7 TGF-beta signaling also regulates DAF-16 nuclear localization specifically at the time when the animal makes the commitment between diapause and reproductive development. daf-16 function is supported by the combined action of two distinct promoter/enhancer elements, whereas the coding sequences of two major DAF-16 isoforms are interchangeable. Together, these observations suggest that the combined effects of transcriptional and posttranslational regulation of daf-16 transduce insulin-like signals in C. elegans and perhaps more generally.
... The molecular differences between isoforms appear to correlate with their biological functions. daf-16a and daf-16f together regulate longevity, dauer formation, and stress-resistance, with little to no input from daf-16b (Lee et al. 2001;Kwon et al. 2010;Chen et al. 2015). In contrast, daf-16b regulates neuronal morphology and behavior (Christensen et al. 2011;Sun et al. 2019). ...
... With the caveat that we were not able to directly test a daf-16(b) allele, these data suggest that isoforms a and f function together to block 1s pecification and that daf-16b plays at most a minor role in this process. This role is similar to the requirement for those isoforms to regulate dauer formation, lifespan, and stress resistance (Lee et al. 2001;Kwon et al. 2010;Chen et al. 2015). ...
... Taken together, we have found distinct requirements for daf-16 isoforms to block 1˚ and 2˚ cell fate specification during dauer. Promoter swap experiments indicate that differences in expression explain requirements for different isoforms for the regulation of lifespan and dauer formation (Lee et al. 2001;Kwon et al. 2010). daf-16 is required in VPCs to block expression of the 1˚ fate marker used in this study; the focus of daf-16 activity for its role in 2˚ fate is unknown (Karp and Greenwald 2013). ...
FOXO transcription factors regulate development, longevity, and stress-resistance across species. The C. elegans FOXO ortholog, daf-16, has three major isoforms with distinct promoters and N-termini. Different combinations of isoforms regulate different processes. Adverse environments can induce dauer diapause after the second larval molt. During dauer, daf-16 blocks specification of vulval precursor cells, including EGFR/Ras-mediated 1˚ fate specification and LIN-12/Notch-mediated 2˚ fate specification. Using isoform-specific mutants, we find that daf-16a and daf-16f are functionally redundant for the block to the expression of 1˚ fate markers. In contrast, all three isoforms contribute to blocking the expression of 2˚ fate markers.
... The molecular mechanism of TFLP-1 regulating antioxidant and stress resistance in C. elegans has been explored and discussed in this study. The insulin like signaling pathway played an important role in the antioxidant and anti-ageing activities in C. elegans [52,53]. Moreover, daf-16, as a key signaling factor, might be a homologous gene of human FKHRL1, FKHR, and AFX [52]. ...
... The insulin like signaling pathway played an important role in the antioxidant and anti-ageing activities in C. elegans [52,53]. Moreover, daf-16, as a key signaling factor, might be a homologous gene of human FKHRL1, FKHR, and AFX [52]. It was inversely regulated by upstream signaling factors such as daf-2 and age-1, and positively affects downstream signaling factors such as sod-3 and mtl-1, played an important regulatory role in stress resistance [27]. ...
Different methods were used to degrade Tremella fuciformis polysaccharides (TFP) and prepare low molecular weight polysaccharides of Tremella fuciformis (TFLP) to improve their bioavailability. It was found that the TFLP prepared by ultrasonic-assisted H2O2-Vc method showed the highest level of antioxidant activity and stress resistance in C. elegans. The structural characteristics, in vivo antioxidant and stress resistance of TFLP-1 were evaluated after isolation and purification of TFLP, it was found that TFLP-1 was an acid polysaccharide with a molecular weight of 75770 Da, which mainly composed of mannose. Meanwhile, it could regulate the antioxidant activity and stress resistance in C. elegans by upregulating the transcription of fat-5, fat-7, acs-2, glp-1, hsf-1, hsp-1, mtl-1, nhr-49, skn-1 and sod-3 mRNA. The improvement effects were closely related to the significant regulation of galactose metabolism, alpha linolenic acid metabolism, and pantothenate and CoA biosynthesis metabolic pathways. These results provided insights into the high value application of Tremella fuciformis in the food industry and the development of antioxidant related functional foods.
... Cell-or tissuespecific transcriptomic studies suggest that daf-2 is widely expressed [42][43][44] , but it needs confirmation using orthogonal methods as omics data come with a certain level of false identifications. In contrast to the lack of consensus on daf-2 expression, independent studies using fluorescent transgene reporters all agree that daf-16 is ubiquitously expressed in somatic tissues [45][46][47][48] . ...
... We found that DAF-16::GFP was expressed ubiquitously in most or all somatic tissues, such as neurons, intestine, BWM, and hypodermis, and also in the germ cells and oocytes (Fig. 2b). Germline expression of DAF-16::GFP was not detected by earlier transgene reporters [45][46][47][48] . Temporally, ubiquitous expression of DAF-16::GFP from the endogenous locus was detected from the embryonic bean stage to adulthood (Fig. 2c-e and Supplementary Fig. 3). ...
Twenty-nine years following the breakthrough discovery that a single-gene mutation of daf-2 doubles Caenorhabditis elegans lifespan, it remains unclear where this insulin/IGF-1 receptor gene is expressed and where it acts to regulate ageing. Using knock-in fluorescent reporters, we determined that daf-2 and its downstream transcription factor daf-16 are expressed ubiquitously. Using tissue-specific targeted protein degradation, we determined that intracellular DAF-2-to-DAF-16 signaling in the intestine plays a major role in lifespan regulation, while that in the hypodermis, neurons, and germline plays a minor role. Notably, intestine-specific loss of DAF-2 activates DAF-16 in and outside the intestine, causes almost no adverse effects on development and reproduction, and extends lifespan by 94% in a way that partly requires non-intestinal DAF-16. Consistent with intestine supplying nutrients to the entire body, evidence from this and other studies suggests that altered metabolism, particularly down-regulation of protein and RNA synthesis, mediates longevity by reduction of insulin/IGF-1 signaling.
... Early discoveries in the genetics of longevity using C. elegans were founded on prior genetic insight into dauer formation (Riddle et al., 1981). Developing C. elegans proceed through for larval stages (L1-L4), marked by molts before becoming reproductive adults (Cassada and Russell 1975;Felix and Duveau 2012;Frezal and Felix 2015;Lazetic and Fay 2017). At the L2 to L3 transition, C. elegans can enter an alternative L3 developmental stage called dauer, which is a genetic program of developmental diapause, allowing animals to survive food scarcity, overcrowding, or a number of harsh environmental conditions and survive for up to 4 months (Golden and Riddle 1984a;Butcher et al., 2007;Fielenbach and Antebi 2008;Felix and Duveau 2012). ...
... Accumulation of PIP 3 activates PDK-1 (PDPK1, 3phosphoinositide-dependent protein kinase-1), which activates AKT-1/2 (AKT Serine/Threonine Kinase, protein kinase B) via phosphorylation (Paradis et al., 1999). In turn activated AKT-1/2 directly inhibits DAF-16 (FOXO) through phosphorylation and sequestration in the cytosol (Paradis and Ruvkun 1998;Lee et al., 2001;Lin et al., 2001). Mutations within this pathway that decreased ILS lead to activation of DAF-16 and increased longevity (Kenyon et al., 1993;Lin et al., 1997;Ogg et al., 1997). ...
Discoveries made in the nematode Caenorhabditis elegans revealed that aging is under genetic control. Since these transformative initial studies, C. elegans has become a premier model system for aging research. Critically, the genes, pathways, and processes that have fundamental roles in organismal aging are deeply conserved throughout evolution. This conservation has led to a wealth of knowledge regarding both the processes that influence aging and the identification of molecular and cellular hallmarks that play a causative role in the physiological decline of organisms. One key feature of age-associated decline is the failure of mechanisms that maintain proper function of the proteome (proteostasis). Here we highlight components of the proteostatic network that act to maintain the proteome and how this network integrates into major longevity signaling pathways. We focus in depth on the heat shock transcription factor 1 (HSF1), the central regulator of gene expression for proteins that maintain the cytosolic and nuclear proteomes, and a key effector of longevity signals.
... Conversely, in the insulin/IGF pathway, the DAF-2 insulin/IGF receptor transmits the signal through a cascade of phosphorylation events, which culminates in regulation of the FOXO transcription factor DAF-16 ( Fig. 10.2) (Gottlieb & Ruvkun, 1994;Kimura et al., 1997;Ogg et al., 1997;Ogg & Ruvkun, 1998;Paradis & Ruvkun, 1998;Paradis et al., 1999;Lee et al., 2001;Wolkow et al., 2002;Li et al., 2003). Downregulation of insulin signalling and downstream kinases leads to nuclear translocation of unphosphorylated DAF-16, which is required for dauer formation (Lee et al., 2001;Lin et al., 2001;Hertweck et al., 2004). ...
... Conversely, in the insulin/IGF pathway, the DAF-2 insulin/IGF receptor transmits the signal through a cascade of phosphorylation events, which culminates in regulation of the FOXO transcription factor DAF-16 ( Fig. 10.2) (Gottlieb & Ruvkun, 1994;Kimura et al., 1997;Ogg et al., 1997;Ogg & Ruvkun, 1998;Paradis & Ruvkun, 1998;Paradis et al., 1999;Lee et al., 2001;Wolkow et al., 2002;Li et al., 2003). Downregulation of insulin signalling and downstream kinases leads to nuclear translocation of unphosphorylated DAF-16, which is required for dauer formation (Lee et al., 2001;Lin et al., 2001;Hertweck et al., 2004). DAF-16 not only is required for the normal regulation of dauer formation, but also is essential for dauer morphogenesis (Vowels & Thomas, 1992;Ogg et al., 1997;Matyash et al., 2004). ...
... The daf-16 genomic locus encodes several different isoforms with different N-termini transcribed from unique promoters (Fig. 5A) [22][23][24] . These isoforms are expressed in different tissues: daf-16a and daf-16f are expressed in the hypodermis, muscle, neurons, and intestines, while daf-16b is expressed primarily in the neurons, pharynx, and muscles 22,[24][25][26][27] . We tested lines carrying isoform-specific mutations of daf-16: 1) daf-16(mg54) introduced with a premature stop codon to daf-16a, d, f; 2) daf-16(knj36) introduced 7-bp deletion to daf-16b isoform (Fig. 5A). ...
An animal’s ability to sense odors declines during aging, and its olfactory drive is tuned by internal states such as satiety. However, whether internal states modulate an age-dependent decline in odor sensation is unknown. To address this issue, we utilized the nematode Caenorhabditis elegans and compared their chemotaxis abilities toward attractive odorants when aged under different dietary conditions. Feeding with the standard laboratory diet, Escherichia coli attenuated the chemotaxis ability toward diacetyl, isoamyl alcohol, and benzaldehyde when aged. On the other hand, feeding with either the lactic acid bacteria Lactobacillus reuteri or food deprivation selectively maintained the chemotaxis ability toward diacetyl. Our results suggest that ingestion of E. coli causes age-dependent chemotaxis decline. The changes in the chemotaxis behavior are attributed to the different expressions of diacetyl receptor odr-10, and the chemotaxis behavior of aged animals under food deprivation is shown to be dependent on daf-16. Our study demonstrates the molecular mechanism of how diet shapes the trajectory of age-dependent decline in chemosensory behaviors.
... Alternatively, daf-16 may interact with acn-1 by an alternative mechanism. Our experiments have focused primarily on sod-3, a canonical target of DAF-16; however, the expression of DAF-16 target genes is dependent on the specific isoform of DAF-16 (Lee et al., 2001;Lin et al., 2001;Kwon et al., 2010;Chen et al., 2015), the tissue (Kwon et al., 2010;Libina et al., 2003), upstream signaling (Lin et al., 2001;Oh et al., 2005;Robida-Stubbs et al., 2012;Tullet et al., 2014), and other factors that allow DAF-16 to preferentially regulate different target genes (reviewed in (Landis and Murphy, 2010;Tissenbaum, 2018)). Our results do not exclude the possibility that acn-1 functions upstream of daf-16 by mechanisms different from controlling DAF-16 nuclear localization or activation of canonical DAF-16 target genes. ...
The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in C. elegans, Drosophila, and rodents, but its mechanism is not well defined. Here we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril-hypersensitive mutants. We identified a missense mutation that causes a partial loss-of-function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNAi promoted dauer larvae formation, suggesting acn-1 is a daf gene. Captopril-mediated lifespan extension was abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 influence lifespan by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control.
... These kinases, in turn, phosphorylate and sequester FOXO/DAF-16 in the cytoplasm, thereby inactivating it, ensuring normal development and reproduction. Under conditions of low signaling, as in the temperature-sensitive alleles of daf-2, FOXO/DAF-16 is dephosphorylated and it moves into the nucleus to transactivate or repress gene expression leading to extreme longevity and resistance to multiple stresses (Lee et al., 2001;Mukhopadhyay et al., 2006;Ogg et al., 1997). Strong mutations in the IIS receptor also arrest development at dauer diapause, in a DAF-16dependent manner (Gottlieb and Ruvkun, 1994;Larsen et al., 1995). ...
Germ line integrity is critical for progeny fitness. Organisms deploy the DNA damage response (DDR) signaling to protect the germ line from genotoxic stress, facilitating the cell-cycle arrest of germ cells and DNA repair or their apoptosis. Cell-autonomous regulation of germ line quality in response to DNA damage is well-studied; however, how quality is enforced cell non-autonomously on sensing somatic D NA damage is less known. Using Caenorhabditis elegans, we show that DDR disruption, only in the uterus, when insulin-IGF-1 signaling (IIS) is low, arrests oogenesis in the pachytene stage of meiosis I, in a FOXO/DAF-16 transcription factor (TF)-dependent manner. Without FOXO/DAF-16, germ cells of the IIS mutant escape the arrest to produce poor-quality oocytes, showing that the TF imposes strict quality control during low IIS. Activated FOXO/DAF-16 senses DDR perturbations during low IIS to lower ERK/MPK-1 signaling below a threshold to promote germ line arrest. Altogether, we elucidate a new surveillance role of activated FOXO/DAF-16 that ensures optimal germ cell quality and progeny fitness in response to somatic DNA damage.
... To investigate organism-wide DAF-16/FOXO dynamics, we studied the C. elegans L1 arrest, a developmental arrest larvae enter when they encounter high stresses, including starvation, osmotic shock and heat, directly after hatching and that is under control of IIS [20,[22][23][24]28,29] (Fig. 1A). Under unstressed conditions, high insulin-like peptide (ILP) levels activate the insulin receptor DAF-2, causing phosphorylation and cytoplasmic localization of DAF-16/FOXO [30][31][32]. Upon stress, low ILP levels result in DAF-2 inactivation, DAF-16/FOXO dephosphorylation and its subsequent translocation into in the nucleus, where it induces stress response genes [33][34][35] and regulators of cell proliferation [22], ultimately leading to developmental arrest. ...
FOXO transcription factors are highly conserved effectors of insulin and insulin-like growth factor signaling, that are crucial for mounting responses to a broad range of stresses. Key signaling step is the stress-induced translocation of FOXO proteins to the nucleus, where they induce expression of stress response genes. Insulin signaling and FOXO proteins often control responses that impact the entire organism, such as growth or starvation-induced developmental arrest, but how body-wide coordination is achieved is poorly understood. Here, we leverage the small size of the nematode C. elegans, to quantify translocation dynamics of DAF-16, the sole C. elegans FOXO transcription factor, with single-cell resolution, yet in a body-wide manner. Surprisingly, when we exposed individual animals to constant levels of stress that cause larval developmental arrest, DAF-16/FOXO translocated between the nucleus and cytoplasm in stochastic pulses. Even though the occurrence of translocation pulses was random, they nevertheless exhibited striking synchronization between cells throughout the body. DAF-16/FOXO pulse dynamics were strongly linked to body-wide growth, with isolated translocation pulses causing transient reduction of growth and full growth arrest observed only when pulses were of sufficiently high frequency or duration. Finally, we observed translocation pulses of FOXO3A in mammalian cells under nutrient stress. The link between DAF-16/FOXO pulses and growth provides a rationale for their synchrony, as uniform proportions are only maintained when growth and, hence, pulse dynamics are tightly coordinated between all cells. Long-range synchronization of FOXO translocation dynamics might therefore be integral also to growth control in more complex animals.
... DAF-16 in C. elegans is a functional homolog of the FoxO transcription factor in mammalian (Wang et al. 2022). DAF-16 is critical to the resistance and longevity regulated by the IIS pathway (Lee et al. 2001). The IIS pathway, which regulates signal factors and activates a series of genes in the nucleus that mediate oxidative stress, is the most typical life-extending mechanism in C. elegans (Hesp et al. 2015), with the daf-2, age-1, ak-1 and daf-16 genes playing a crucial role (Altintas et al. 2016). ...
Phlorizin, a natural compound, was studied using C. elegans to explore its potential mechanism for extending lifespan. Results showed that phlorizin mitigated the harmful effects of high temperatures and hydrogen peroxide, reduced oxidative stress, increased antioxidant enzyme activity, and reduced MDA levels. Through network pharmacological analysis, it was determined that the AKT1, INSR and SOD2 signaling pathway plays a key role in the anti-aging effects of phlorizin. Experiments with mutants and transgenic C. elegans showed that phlorizin lifespan extension and antioxidant effects are mainly mediated by insulin and SIR-2.1 pathways that regulate DAF-16 and SKN-1 nuclear translocation and its downstream targets SOD-3 and HSP-16.2. These findings were supported by molecular docking experiments. Overall, this study provides insight into the potential use of phlorizin as an anti-aging agent and highlights the importance of the DAF-16/SOD-3 signaling pathway in mediating its action.
... We found that nomilin could not further extend the lifespan in C. elegans insulin-like peptide receptor mutant daf-2(-) (Fig. 1b). Moreover, nomilin supplementation significantly promoted nuclear translocation of DAF-16::GFP, a FOXO transcription factor downstream of DAF-2 IIS 42 (Fig. 1c, d), and daf-16(-) also completely blocked the lifespan extension effect of nomilin (Fig. 1e). To confirm that DAF-16 signalling is involved in nomilin function, the mRNA levels of DAF-16 downstream targets were tested. ...
Citrus fruit has long been considered a healthy food, but its role and detailed mechanism in lifespan extension are not clear. Here, by using the nematode C. elegans, we identified that nomilin, a bitter-taste limoloid that is enriched in citrus, significantly extended the animals’ lifespan, healthspan, and toxin resistance. Further analyses indicate that this ageing inhibiting activity depended on the insulin-like pathway DAF-2/DAF-16 and nuclear hormone receptors NHR-8/DAF-12. Moreover, the human pregnane X receptor (hPXR) was identified as the mammalian counterpart of NHR-8/DAF-12 and X-ray crystallography showed that nomilin directly binds with hPXR. The hPXR mutations that prevented nomilin binding blocked the activity of nomilin both in mammalian cells and in C. elegans. Finally, dietary nomilin supplementation improved healthspan and lifespan in D-galactose- and doxorubicin-induced senescent mice as well as in male senescence accelerated mice prone 8 (SAMP8) mice, and induced a longevity gene signature similar to that of most longevity interventions in the liver of bile-duct-ligation male mice. Taken together, we identified that nomilin may extend lifespan and healthspan in animals via the activation of PXR mediated detoxification functions.
... The key antioxidantrelated genes regulated by Insulin/IGF-1 signaling (IIS pathway), such as DAF-16, DAF-2, sod-3, ctl-2 and hsp-16.2, have been widely concerned because of their important roles in regulating oxidative stress and affecting lifespan [41][42][43]. DAF-16, an ortholog of the FOXO transcription factor, could regulate the downstream target genes, such as sod-3 encoding SOD [44], ctl-2 encoding CAT [43] and hsp-16.2 encoding heat shock protein [28]. ...
The antioxidant effect of probiotics has been widely recognized across the world, which is of great significance in food, medicine, and aquaculture. There are abundant marine microbial resources in the ocean, which provide a new space for humans to explore new probiotics. Previously, we reported on the anti-infective effects of Planococcus maritimu ML1206, a potential marine probiotic. The antioxidant activity of ML1206 in C. elegans was studied in this paper. The study showed that ML1206 could improve the ability of nematodes to resist oxidative stress and effectively prolong their lifespan. The results confirmed that ML1206 could significantly increase the activities of CAT and GSH-PX, and reduce the accumulation of reactive oxygen species (ROS) in nematodes under oxidative stress conditions. In addition, ML1206 promoted DAF-16 transfer to the nucleus and upregulated the expression of sod-3, hsp-16.2, and ctl-2, which are downstream antioxidant-related genes of DAF-16. Furthermore, the expression of the SOD-3::GFP and HSP-16.2::GFP was significantly higher in the transgenic strains fed with ML1206 than that in the control group fed with OP50, with or without stress. In summary, these findings suggest that ML1206 is a novel marine probiotic with an antioxidant function that stimulates nematodes to improve their defense abilities against oxidative stress and prolong the lifespan by regulating the translocation of FOXO/DAF-16. Therefore, ML1206 may be explored as a potential dietary supplement in aquaculture and for anti-aging and antioxidant purposes.
... Freedman, USA, 2007, personal communication). A daf-16::gfp strain exists that displays cytoplasmic GFP expression under normal growth, or nuclear localization when insulin signalling is off (Lee et al., 2001) and during stress (Henderson and Johnson, 2001). These strains may have much potential in terms of biomonitoring. ...
This chapter focuses on transgenic Caenorhabditis elegans developed for ecotoxicology purposes to the end of 2007. This chapter discusses how transgenic Caenorhabditis elegans strains are generated and reviews literature concerning their use in biomonitoring. Developments in the understanding of stress responses is also briefly explored as they have the potential to be translated into novel C. elegans biosensor strains.
... The long-lived and Daf-c phenotypes of daf-2 mutants require the FOXO transcription factor DAF-16 (Ogg et al., 1997;Lin et al., 1997). Activation of the DAF-2/IIRc triggers a cascade of phosphorylation responsible for the retention of DAF-16/FOXO into the cytoplasm (Henderson & Johnson, 2001;Lee et al., 2001;Li et al., 2021;Lin et al., 2001). Down-regulation of this pathway promotes DAF-16/FOXO accumulation into the nucleus where it activates hundreds of genes (Tepper et al., 2013). ...
During aging, preservation of locomotion is generally considered an indicator of sustained good health, in elderlies and in animal models. In Caenorhabditis elegans, mutants of the insulin-IGF-1 receptor DAF2/IIRc represent a paradigm of healthy aging, as their increased lifespan is accompanied by a delay in age-related loss of motility. Here, we investigated the DAF-2/IIRc-dependent relationship between longevity and motility using an auxin-inducible degron to trigger tissue-specific degradation of endogenous DAF-2/IIRc. As previously reported, inactivation of DAF-2/IIRc in neurons or intestine was sufficient to extend the lifespan of worms, whereas depletion in epidermis, germline, or muscle was not. However, neither intestinal nor neuronal depletion of DAF-2/IIRc prevented the age-related loss of motility. In 1-day-old adults, DAF-2/IIRc depletion in neurons reduced motility in a DAF-16/FOXO dependent manner, while muscle depletion had no effect. By contrast, DAF-2 depletion in the muscle of middle-age animals improved their motility independently of DAF-16/FOXO but required UNC-120/SRF. Yet, neuronal or muscle DAF-2/IIRc depletion both preserved the mitochondria network in aging muscle. Overall, these results show that the motility pattern of daf-2 mutants is determined by the sequential and opposing impact of neurons and muscle tissues and can be dissociated from the regulation of the lifespan. This work also provides the characterization of a versatile tool to analyze the tissue-specific contribution of insulin-like signaling in integrated phenotypes at the whole organism level.
... mEAK-7 interacts with catalytic subunits and a peripheral stalk from V-ATPase mEAK-7 is the mammalian homologue of Caenorhabditis elegans EAK-7 (enhancer-of-akt-1-7), which regulates dauer formation in nematodes (Lee et al, 2001;Lin et al, 2001;Alam et al, 2010). Mutations in C. elegans EAK-7 promote diapause and longevity (Alam et al, 2010). ...
V-ATPases are rotary proton pumps that serve as signaling hubs with numerous protein binding partners. CryoEM with exhaustive focused classification allowed detection of endogenous proteins associated with porcine kidney V-ATPase. An extra C subunit was found in ∼3% of complexes, whereas ∼1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 showed that mEAK-7's TLDc domain interacts with V-ATPase's stator, whereas its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike the yeast TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that the mEAK-7:V-ATPase interaction is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. These results show that V-ATPase binding by TLDc domain proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzyme's activity.
... There is a single known insulin/ IGF-like receptor in C. elegans, DAF-2/InsR, which signals through a conserved phosphatidylinositol 3-kinase (PI3K) signaling pathway to antagonize the transcription factor DAF-16/FoxO (Lin et al., 1997;Ogg et al., 1997). When IIS is reduced, such as during starvation, DAF-16 moves to the nucleus and regulates transcription (Henderson and Johnson, 2001;Lee et al., 2001;Lin et al., 2001), promoting starvation resistance (Muñoz and Riddle, 2003;Baugh and Sternberg, 2006;Hibshman et al., 2017). (Roux et al., 2016;Kaplan et al., 2018;Baugh and Hu, 2020;Chen et al., 2022). ...
Starvation resistance is important to disease and fitness, but the genetic basis of its natural variation is unknown. Uncovering the genetic basis of complex, quantitative traits such as starvation resistance is technically challenging. We developed a synthetic-population (re)sequencing approach using molecular inversion probes (MIP-seq) to measure relative fitness during and after larval starvation in C. elegans . We applied this competitive assay to 100 genetically diverse, sequenced, wild strains, revealing natural variation in starvation resistance. We confirmed that the most starvation-resistant strains survive and recover from starvation better than the most starvation-sensitive strains using standard assays. We performed genome-wide association (GWA) with the MIP-seq trait data and identified three quantitative trait loci (QTL) for starvation resistance, and we created near isogenic lines (NILs) to validate the effect of these QTL on the trait. These QTL contain numerous candidate genes including several members of the Insulin/EGF Receptor-L Domain ( irld ) family. We used genome editing to show that four different irld genes have modest effects on starvation resistance. Natural variants of irld-39 and irld-52 affect starvation resistance, and increased resistance of the irld-39; irld-52 double mutant depends on daf-16/FoxO . DAF-16/FoxO is a widely conserved transcriptional effector of insulin/IGF signaling (IIS), and these results suggest that IRLD proteins modify IIS, though they may act through other mechanisms as well. This work demonstrates efficacy of using MIP-seq to dissect a complex trait and it suggests that irld genes are natural modifiers of starvation resistance in C. elegans .
... Conversely, several known defense and stress response modulators, such as the FOXO transcription factor daf-16, failed to exhibit significant changes in gene expression in either of the datasets. Despite the established role of DAF-16 in immune modulation, this was not unexpected as modulation of DAF-16 activity mostly occurs post-translationally, via regulated shuttling between the cytosol and the nucleus (Lee et al., 2001; Figure 2A). ...
Controlling nematode-caused diseases that affect cattle and crops world-wide remains a critical economic issue, owing to the lack of effective sustainable interventions. The interdependence of roundworms and their environmental microbes, including their microbiota, offers an opportunity for developing more targeted anthelminthic strategies. However, paucity of information and a currently narrow understanding of nematode-microbe interactions limited to specific infection contexts has precluded us from exploiting it. With the advent of omics approaches to map host-microbe genetic interactions, particularly in the model roundworm Caenorhabditis elegans, large datasets are now available across multiple models, that enable identification of nematode-microbe-specific pathways. In this work we collected 20 transcriptomic datasets documenting gene expression changes of C. elegans exposed to 20 different commensal and pathogenic microbes, performing gene enrichment analyses followed by functional testing using RNA interference directed toward genes of interest, before contrasting results from transcriptomic meta-analyses and phenomics. Differential expression analyses revealed a broad enrichment in signaling, innate immune response and (lipid) metabolism genes. Amongst signaling gene families, the nematode-divergent and expanded Hedgehog-like signaling (HHLS) pathway featured prominently. Indeed, 24/60 C. elegans Hedgehog-like proteins (HRPs) and 15/27 Patched-related receptors (PTRs) were differentially expressed in at least four microbial contexts, while up to 32/60 HRPs could be differentially expressed in a single context. interestingly, differentially expressed genes followed a microbe-specific pattern, suggestive of an adaptive microbe-specific response. To investigate this further, we knocked-down 96 individual HHLS genes by RNAi, using high-throughput assays to assess their impact on three worm-gut infection models (Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis) and two worm-commensal paradigms (Comamonas sp., and Bacillus subtilis). We notably identified new putative infection response genes whose upregulation was required for normal pathogen resistance (i.e., grl-21 and ptr-18 protective against E. faecalis), as well as commensal-specific host-gene expression changes that are required for normal host stress handling. Importantly, interactions appeared more microbe-specific than shared. Our results thus implicate the Hedgehog-like signaling pathway in the modulation and possibly fine-tuning of nematode-microbe interactions and support the idea that interventions targeting this pathway may provide a new avenue for anthelmintic development.
... Accordingly, the alteration of aging by inhibiting the activity of the insulin/insulin growth factor (IGF) signaling (IIS) cascade (Kenyon et al., 1993) protects model nematodes from proteotoxicity stemming from polyQ (Morley et al., 2002) and AD-causing Ab peptides (Cohen et al., 2006;El-Ami et al., 2014). Upon activation, DAF-2, the sole insulin/IGF receptor of the nematode Caenorhabditis elegans, initiates a signaling cascade that retains DAF-16/FOXO (Lee et al., 2001), SKN-1/Nrf (Tullet et al., 2008), and the heat shock factor 1 (HSF-1) (Chiang et al., 2012) in the cytosol. This prevents these transcription factors from regulating their target genes. ...
The protein homeostasis (proteostasis) network (PN) encompasses mechanisms that maintain proteome integrity by controlling various biological functions. Loss of proteostasis leads to toxic protein aggregation (proteotoxicity), which underlies the manifestation of neurodegeneration. How the PN responds to dissimilar proteotoxic challenges and how these responses are regulated at the organismal level are largely unknown. Here, we report that, while torsin chaperones protect from the toxicity of neurodegeneration-causing polyglutamine stretches, they exacerbate the toxicity of the Alzheimer’s disease-causing Aβ peptide in neurons and muscles. These opposing effects are accompanied by differential modulations of gene expression, including that of three neuropeptides that are involved in tailoring the organismal response to dissimilar proteotoxic insults. This mechanism is regulated by insulin/IGF signaling and the transcription factor SKN-1/NRF. Our work delineates a mechanism by which the PN orchestrates differential responses to dissimilar proteotoxic challenges and points at potential targets for therapeutic interventions.
... In well-fed animals, insulin/ IGF-1Àlike peptides are secreted by neuroendocrine cells and bind to DAF-2, the C. elegans homolog of the insulin/IGF-1 receptor (Hu, 2007;Kimura et al., 1997). Ligand activation of DAF-2 further transduces the nutrient signal by activating downstream kinases that eventually phosphorylate and sequester the DAF-16/FOXO transcription factor in the cytoplasm (Lee et al., 2001;Lin, 1997;Lin et al., 2001;Ogg et al., 1997). Reduced activity of the insulin/IGF-1 signaling pathway due to nutrient stress or mutations in upstream genes allows DAF-16 to enter the nucleus and promote the expression of target genes necessary for dauer transformation and arrest. ...
Molting is a widespread feature in the development of many invertebrates, including nematodes and arthropods. In Caenorhabditis elegans, the highly conserved protein kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (NEKLs) promote molting through their involvement in the uptake and intracellular trafficking of epidermal cargos. We found that the relative requirements for NEKL-2 and NEKL-3 differed at different life-cycle stages and under different environmental conditions. Most notably, the transition from the second to the third larval stage (L2→L3 molt) required a higher level of NEKL function than during several other life stages or when animals had experienced starvation at the L1 stage. Specifically, larvae that entered the pre-dauer L2d stage could escape molting defects when transiting to the (non-dauer) L3 stage. Consistent with this, mutations that promote entry into L2d suppressed nekl-associated molting defects, whereas mutations that inhibit L2d entry reduced starvation-mediated suppression. We further showed that loss or reduction of NEKL functions led to defects in the transcription of cyclically expressed molting genes, many of which are under the control of systemic steroid hormone regulation. Moreover, the timing and severity of these transcriptional defects correlated closely with the strength of nekl alleles and with their stage of arrest. Interestingly, transit through L2d rescued nekl-associated expression defects in suppressed worms, providing an example of how life-cycle decisions can impact subsequent developmental events. Given that NEKLs are implicated in the uptake of sterols by the epidermis, we propose that loss of NEKLs leads to a physiological reduction in steroid-hormone signaling and consequent defects in the transcription of genes required for molting.
... When nutritional resources are abundant, insulin-like ligands signaling through DAF-2 leads to DAF-16 phosphorylation and its consequent sequestration in the cytoplasm (Henderson and Johnson, 2001;Lee et al., 2001;Lin et al., 2001). Conversely, when food is scarce, DAF-2 signaling decreases, leading to DAF-16 translocation to the nucleus and subsequent gene expression (Ogg et al., 1997;Honda and Honda, 1999;Henderson and Johnson, 2001;Garsin et al., 2003;Libina et al., 2003). ...
Food produces powerful reinforcement that can lead to overconsumption and likely contributes to the obesity epidemic. The present studies examined molecular mechanisms mediating food-induced reinforcement in the model system C. elegans. After a 1-h training session during which food (bacteria) is paired with the odorant butanone, odor preference for butanone robustly increased. Glucose mimicked this effect of bacteria. Glucose-induced odor preference was enhanced similarly by prior food withdrawal or blocking glucose metabolism in the presence of food. Food- and glucose-induced odor preference was mimicked by serotonin signaling through the serotonin type-4 (5-HT4) receptor. Dopamine (thought to act primarily through a D1-like receptor) facilitated, whereas the D2 agonist bromocriptine blocked, food- and glucose-induced odor preference. Furthermore, prior food withdrawal similarly influenced reward produced by serotonin, dopamine, or food, implying post-synaptic enhancement of sensitivity to serotonin and dopamine. These results suggest that glucose metabolism plays a key role in mediating both food-induced reinforcement and enhancement of that reinforcement by prior food withdrawal and implicate serotonergic signaling through 5-HT4 receptor in the re-enforcing properties of food.
... The C. elegans wild-type reference strains N2/Bristol (Stiernagle, 2006), CB4270 tra-1 (e2200) (Zarkower et al., 1994), CB3844 fem-3 (e2006) IV (Hodgkin, 1986), RA7 (rdEx1) (Mathies et al., 2004), and TJ356 daf-16 (zIs356) IV (Lee et al., 2001;Lin et al., 2001) were obtained from the Caenorhabditis Genetics Center-CGC (University of Minnesota, Minneapolis, MN, United States), and are summarized in Table 1. All worm strains were maintained at 20°C, except of the CB4270 tra-1 mutant (e2200), which was maintained at 15°C. ...
Broccoli-derived isothiocyanate sulforaphane inhibits inflammation and cancer. Sulforaphane may support healthy aging, but the underlying detailed mechanisms are unclear. We used the C. elegans nematode model to address this question. Wild-type and 4 mutant C. elegans worm strains were fed in the presence or absence of sulforaphane and E. coli food bacteria transfected with RNA interference gene constructs. Kaplan–Meier survival analysis, live imaging of mobility and pharyngeal pumping, fluorescence microscopy, RT–qPCR, and Western blotting were performed. In the wild type, sulforaphane prolonged lifespan and increased mobility and food intake because of sulforaphane-induced upregulation of the sex-determination transcription factor TRA-1, which is the ortholog of the human GLI mediator of sonic hedgehog signaling. In turn, the tra-1 target gene daf-16 , which is the ortholog of human FOXO and the major mediator of insulin/IGF-1 and aging signaling, was induced. By contrast, sulforaphane did not prolong lifespan and healthspan when tra-1 or daf-16 was inhibited by RNA interference or when worms with a loss-of-function mutation of the tra-1 or daf-16 genes were used. Conversely, the average lifespan of C. elegans with hyperactive TRA-1 increased by 8.9%, but this longer survival was abolished by RNAi-mediated inhibition of daf-16 . Our data suggest the involvement of sulforaphane in regulating healthy aging and prolonging lifespan by inducing the expression and nuclear translocation of TRA-1/GLI and its downstream target DAF-16/FOXO.
... DAF-2 activates AGE-1, and the downstream kinases AKT-1, AKT-2 and SGK-1 [31][32][33][34]. Activation of these kinases results in the phosphorylation of the transcription factor DAF-16 which is retained in the cytoplasm [35][36][37]. Upon inhibition of the IIS cascade, DAF-16 is activated [37][38][39] and triggers the expression of a plethora of genes involved in the regulation of lifespan [40]. The nematode C. elegans is one of the premier model organisms on ageing research due to its short life cycle and lifespan, its small size and transparency, the ease of its laboratory maintenance, and genetic manipulation. ...
The mitochondrial prohibitin (PHB) complex, composed of PHB-1 and PHB-2, is an evolutionarily conserved context-dependent modulator of longevity. This extremely intriguing phenotype has been linked to alterations in mitochondrial function and lipid metabolism. The true biochemical function of the mitochondrial PHB complex remains elusive, but it has been shown to affect membrane lipid composition. Recent work, using large-scale biochemical approaches, has highlighted a broad effect of PHB on the C. elegans metabolic network. Collectively, the biochemical data support the notion that PHB modulates, at least partially, worm longevity through the moderation of fat utilisation and energy production via the mitochondrial respiratory chain. Herein, we review, in a systematic manner, recent biochemical insights into the impact of PHB on the C. elegans metabolome.
... Similarly, salt aversive learning was impaired by the absence of DAF-16 (Nagashima et al. 2019;Tomioka et al. 2006). This is curious as DAF-16 activity is normally suppressed by insulin signalling (Lee et al. 2001), but activation of the insulin pathway appears to be required for salt aversive learning , suggesting that this pathway affects this behavioural paradigm via a different downstream target. DAF-16 normally localises in the cytoplasm but, upon activation, translocates into nuclei to regulate gene transcription ( Fig. 1) (Henderson & Johnson 2001). ...
Learning is an essential biological process for survival since it facilitates behavioural plasticity in response to environmental changes. This process is mediated by a wide variety of genes, mostly expressed in the nervous system. Many studies have extensively explored the molecular and cellular mechanisms underlying learning and memory. This review will focus on the advances gained through the study of the nematode Caenorhabditis elegans. C. elegans provides an excellent system to study learning because of its genetic tractability, in addition to its invariant, compact nervous system (~300 neurons) that is well‐characterised at the structural level. Importantly, despite its compact nature, the nematode nervous system possesses a high level of conservation with mammalian systems. These features allow the study of genes within specific sensory‐, inter‐ and motor neurons, facilitating the interrogation of signalling pathways that mediate learning via defined neural circuits. This review will detail how learning and memory can be studied in C. elegans through behavioural paradigms that target distinct sensory modalities. We will also summarise recent studies describing mechanisms through which key molecular and cellular pathways are proposed to affect associative and non‐associative forms of learning.
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... (39) Several reports have indicated that DAF-7 and DAF-12 positively regulate DAF-16 function. (40,41) Based on those findings, one might infer that S. thermophilus-mediated daf-16 upregulation is related to the activation of DAF-7/TGF-β pathway, but not of IIS. ...
Streptococcus thermophilus bacteria, which are widely used as fermented starter for dairy production, exert various beneficial health effects. Nevertheless, even though pro-longevity effects of various probiotics have been reported, no report has described Streptococcus thermophilus effects on longevity. This study was conducted to evaluate Streptococcus thermophilus effects on lifespan extension and to elucidate the Streptococcus thermophilus-mediated longevity mechanism using Caenorhabditis elegans worms as a model animal. They were fed standard food (Escherichia coli OP50) or Streptococcus thermophilus from the young adult stage. Feeding with Streptococcus thermophilus, compared to Escherichia coli OP50, to Caenorhabditis elegans extend the lifespan, reduced lipofuscin accumulation, and maintain vigorous locomotion. Feeding with Streptococcus thermophilus did not alter the worm growth curve or the offspring number, indicating that the Streptococcus thermophilus-mediated lifespan extension is not attributable to caloric restriction. The qRT-PCR data showed that Streptococcus thermophilus increased the expression of daf-16 and some of its downstream antioxidant genes. Furthermore, the pro-longevity effects of Streptococcus thermophilus were decreased in loss-of-function mutant of daf-16. Results show that Streptococcus thermophilus extends the lifespan of Caenorhabditis elegans through DAF-16-mediated antioxidant pathway activation.
... These signals are released and then received in multiple tissues where insulin signaling blocks the activity of the downstream DAF-16/FOXO transcription factor (Kimura et al. 1997;Ogg et al. 1997;Lin et al. 1997;Pierce et al. 2001). In adverse environments, DAF-16 is active and regulates the expression of genes that promote dauer formation (Lin et al. 2001;Lee et al. 2001). ...
Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16 /FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp . Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29 . lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development lin-41 opposes adult cell fate by inhibiting the translation of lin-29 , which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41 , but does not require lin-29 . Taken together, this work demonstrates a novel role for daf-16 /FOXO as a heterochronic gene that promotes expression of lin-41 /TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.
... Thus, knocking down the activity of DAF-2 by either mutation or RNA interference (RNAi) hyper-activates HSF-1, DAF-16 and SKN-1, creating long-lived, youthful and stress-resistant worms. These longevity and stress resistance effects of daf-2 knockdown are dependent upon each of the aforementioned transcription factors [12,15,16]. Similarly to worms, reduced IGF-1 signaling was shown to extend the lifespan of mice [9], and mutations in components of the same pathway correlate with extreme longevity of humans [7,17], indicating that the aging-regulating roles of the IIS are conserved from worms to mammals. ...
Lowering the activity of the Insulin/IGF-1 Signaling (IIS) cascade results in elevated stress resistance, enhanced protein homeostasis (proteostasis) and extended lifespan of worms, flies and mice. In the nematode Caenorhabditis elegans (C. elegans), the longevity phenotype that stems from IIS reduction is entirely dependent upon the activities of a subset of transcription factors including the Forkhead factor DAF-16/FOXO (DAF-16), Heat Shock Factor-1 (HSF-1), SKiNhead/Nrf (SKN-1) and ParaQuat Methylviologen responsive (PQM-1). While DAF-16 determines lifespan exclusively during early adulthood and governs proteostasis in early adulthood and midlife, HSF-1 executes these functions foremost during development. Despite the central roles of SKN-1 as a regulator of lifespan and proteostasis, the temporal requirements of this transcription factor were unknown. Here we employed conditional knockdown techniques and discovered that in C. elegans, SKN-1 is primarily important for longevity and proteostasis during late larval development through early adulthood. Our findings indicate that events that occur during late larval developmental through early adulthood affect lifespan and proteostasis and suggest that subsequent to HSF-1, SKN-1 sets the conditions, partially overlapping temporally with DAF-16, that enable IIS reduction to promote longevity and proteostasis. Our findings raise the intriguing possibility that HSF-1, SKN-1 and DAF-16 function in a coordinated and sequential manner to promote healthy aging.
Classical genetic analysis is invaluable for understanding the genetic interactions underlying specific phenotypes, but requires laborious and subjective experiments to characterize polygenic and quantitative traits. Contrarily, transcriptomic analysis enables the simultaneous and objective identification of multiple genes whose expression changes are associated with specific phenotypes. Here, we conducted transcriptomic analysis of genes crucial for longevity using datasets with daf‐2 /insulin/IGF‐1 receptor mutant Caenorhabditis elegans . Our analysis unraveled multiple epistatic relationships at the transcriptomic level, in addition to verifying genetically established interactions. Our combinatorial analysis also revealed transcriptomic changes associated with longevity conferred by daf‐2 mutations. In particular, we demonstrated that the extent of lifespan changes caused by various mutant alleles of the longevity transcription factor daf‐16 / FOXO matched their effects on transcriptomic changes in daf‐2 mutants. We identified specific aging‐regulating signaling pathways and subsets of structural and functional RNA elements altered by different genes in daf‐2 mutants. Lastly, we elucidated the functional cooperation between several longevity regulators, based on the combination of transcriptomic and molecular genetic analysis. These data suggest that different biological processes coordinately exert their effects on longevity in biological networks. Together our work demonstrates the utility of transcriptomic dissection analysis for identifying important genetic interactions for physiological processes, including aging and longevity.
Stem cells are pivotal players in the intricate dance of embryonic development, tissue maintenance, and regeneration. Their behavior is delicately balanced between maintaining their pluripotency and differentiating as needed. Disruptions in this balance can lead to a spectrum of diseases, underscoring the importance of unraveling the complex molecular mechanisms that govern stem cell fate. Forkhead box O (FOXO) proteins, a family of transcription factors, are at the heart of this intricate regulation, influencing a myriad of cellular processes such as survival, metabolism, and DNA repair. Their multifaceted role in steering the destiny of stem cells is evident, as they wield influence over self-renewal, quiescence, and lineage-specific differentiation in both embryonic and adult stem cells. This review delves into the structural and regulatory intricacies of FOXO transcription factors, shedding light on their pivotal roles in shaping the fate of stem cells. By providing insights into the specific functions of FOXO in determining stem cell fate, this review aims to pave the way for targeted interventions that could modulate stem cell behavior and potentially revolutionize the treatment and prevention of diseases.
In C. elegans mechanisms by which peripheral organs relay internal state information to the nervous system remain unknown, although strong evidence suggests that such signals do exist. Here we report the discovery of a peptide of the ancestral insulin superfamily called INS-7 that functions as an enteroendocrine peptide and is secreted from specialized cells of the intestine. INS-7 secretion increases during fasting, and acts as a bona fide gut-to-brain homeostatic signal that attenuates neuronally induced fat loss during food shortage. INS-7 functions as an antagonist at the canonical DAF-2 receptor in the nervous system, and phylogenetic analysis suggests that INS-7 bears greater resemblance to members of the broad insulin/relaxin superfamily than to conventional mammalian insulin and IGF peptides. The discovery of an endogenous insulin antagonist secreted by specialized intestinal cell with enteroendocrine functions suggests that much remains to be learned about the intestine and its role in directing neuronal functions.
FOXO family of proteins are transcription factors involved in many physiological and pathological processes including cellular homeostasis, stem cell maintenance, cancer, metabolic and cardiovascular diseases. Genetic evidence has been accumulating to suggest a prominent role of FOXOs in lifespan regulation in animal systems from hydra, C elegans, Drosophila and mice. Together with the observation that FOXO3 is the second most replicated gene associated with extreme human longevity suggests that pharmacological targeting of FOXO proteins can be a promising approach to treat cancer and other age-related diseases and extend life and health span. However, due to the broad range of cellular functions of the FOXO family members FOXO1, 3, 4 and 6, isoform-specific targeting of FOXOs might lead to greater benefit and cause less side effects. Therefore, a deeper understanding of the common and specific features of these proteins as well as their redundant and specific functions in our cells represents the basis of specific targeting strategies. In this review, we provide an overview on the evolution, structure, function and disease-relevance of each of the FOXO family members.
Caenorhabditis elegans can enter a diapause stage called “dauer” when it senses that the environment is not suitable for development. This implies a detour from the typical developmental trajectory and requires a tight control of the developmental clock and a massive tissue remodeling. In the last decades, core components of the signaling pathways that govern the dauer development decision have been identified, but the tissues where they function for the acquisition of dauer-specific traits are still under intense study. Growing evidence demonstrates that these pathways engage in complex cross-talk and feedback loops. In this review, we summarize the current knowledge regarding the transcriptional regulation of the dauer program and the relevant tissues for its achievement. A better understanding of this process will provide insight on how developmental plasticity is achieved and how development decisions are under a robust regulation to ensure an all-or-nothing response. Furthermore, this developmental decision can also serve as a simplified model for relevant developmental disorders.
Abbreviations: AID Auxin Induced Degron DA dafachronic acid Daf-c dauer formation constitutive Daf-d dauer formation defective DTC Distal Tip Cells ECM modified extracellular matrix GPCRs G protein-coupled receptors IIS insulin/IGF-1 signaling ILPs insulin-like peptides LBD Ligand Binding Domain PDL4 Post Dauer L4 TGF-β transforming growth factor beta WT wild-type
Patulin is one of the mycotoxins that exists in abundance in fruits and derivative products and is easily exposed in daily life, leading to various toxicities such as genotoxicity, teratogenicity, immunotoxicity, and carcinogenicity in the human body, while the efficient removal or degradation measures are still in urgent demand. In this work, Saccharomyces cerevisiae, a natural yeast with both patulin degradation and intestine damage protection abilities, was first applied to prevent and decrease the hazard after patulin intake. In vitro, Saccharomyces cerevisiae KD (S. cerevisiae KD) could efficiently degrade patulin at high concentrations. In a Canenorhabditis elegans (C. elegans) model fed on S. cerevisiae KD, locomotion, oxidative stress, patulin residual, intestine damage, and gene expression were investigated after exposure to 50 μg mL-1 patulin. The results demonstrated that S. cerevisiae KD could efficiently degrade patulin, as well as weaken the oxidative stress and intestinal damage caused by patulin. Moreover, S. cerevisiae KD could regulate the gene expression levels of daf-2 and daf-16 through the IGF-1 signaling pathway to control the ROS level and glutathione (GSH) content, thus decreasing intestinal damage. In summary, this work uncovers the outstanding characteristic of an edible probiotic S. cerevisiae KD in patulin degradation and biotoxicity alleviation and provides enlightenment toward solving the hazards caused by the accumulation of patulin.
Cellular fates are determined by genes interacting across large, complex biological networks. A critical question is how to identify causal relationships spanning distinct signaling pathways and underlying organismal phenotypes. Here, we address this question by constructing a Boolean model of a well-studied developmental network and analyzing information flows through the system. Depending on environmental signals Caenorhabditis elegans develop normally to sexual maturity or enter a reproductively delayed, developmentally quiescent ‘dauer’ state, progressing to maturity when the environment changes. The developmental network that starts with environmental signal and ends in the dauer/no dauer fate involves genes across 4 signaling pathways including cyclic GMP, Insulin/IGF-1, TGF- β and steroid hormone synthesis. We identified three stable motifs leading to normal development, each composed of genes interacting across the Insulin/IGF-1, TGF- β and steroid hormone synthesis pathways. Three genes known to influence dauer fate, daf-2 , daf-7 and hsf-1 , acted as driver nodes in the system. Using causal logic analysis, we identified a five gene cyclic subgraph integrating the information flow from environmental signal to dauer fate. Perturbation analysis showed that a multifactorial insulin profile determined the stable motifs the system entered and interacted with daf-12 as the switchpoint driving the dauer/no dauer fate. Our results show that complex organismal systems can be distilled into abstract representations that permit full characterization of the causal relationships driving developmental fates. Analyzing organismal systems from this perspective of logic and function has important implications for studies examining the evolution and conservation of signaling pathways.
Polysaccharide is one of the main active ingredients in Lonicera japonica Thunb. (L. japonica). In this study, we examined the anti-aging activities of L. japonica polysaccharides (LJPs) and further explored the mechanisms. Polysaccharides from L. japonica including the crude LJP (CLJP) and the purified fraction (LJP-2-1) were characterized. The molecular weights of CLJP and LJP-2-1 were 1450 kDa and 1280 kDa, respectively. Meanwhile, CLJP was mainly composed of galacturonic acid (23.57 %), galactose (23.45 %) and arabinose (23.45 %). LJP-2-1 was mainly composed of galacturonic acid (51.25 %) and arabinose (22.89 %). In Caenorhabditis elegans (C. elegans), LJPs maximally prolonged mean lifespan by 13.97 %, promoted fitness with increased motility by 40.92 % and pharyngeal pumping by 25.72 %, and decreased lipofuscin accumulation by 38.9 % with intact body length and fecundity. Moreover, CLJP extended the mean lifespan of nematodes under oxidative and heat stress by 16.76 % and 14.05 % respectively by activating stress-related genes and the antioxidant system. Further, CLJP required DAF-16 to prolong the lifespan of nematodes. CLJP upregulated the expression of daf-16 and its targeted downstream genes, including sod-3, gst-4 and hsp-16.2. Moreover, nuclear accumulation of DAF-16 was promoted upon CLJP treatment. Together, our data uncover the role of LJPs in extending lifespan and healthspan through DAF-16.
C. elegans insulin/insulin-like growth factor 1 signaling (IIS) affects diverse physiological processes through the DAF-16/FOXO transcription factor. Despite its presence in all somatic cells, DAF-16's physiological effects, such as modulation of dauer formation, synapse maturation, axon regeneration, and adult longevity, exhibit prevalent tissue-specificity as well as tissue crosstalk. This implies that tissue-specific DAF-16 transcriptional programs contribute to the functional diversity of IIS. To further examine this possibility, we sought to identify tissue-specific and direct transcriptional targets of DAF-16 in muscle cells. Following FACS-sorting to enrich mature muscle cells from young adult animals, we compared the muscle transcriptomes under high and low IIS signaling conditions, with and without DAF-16. We further analyzed and compared the DAF-16 docking sites in muscle and intestine cells from published datasets. These analyses revealed 14 potential muscle-specific DAF-16 transcriptional targets, among which we validated two that are strongly and specifically activated by DAF-16 in muscles: a secreted protein C54F6.5 and a calcium-binding protein CEX-1/Calexcitin. Both genes exhibit DAF-16-independent non-muscle expression, explaining their low rank or absence from the current DAF-16 target lists generated by multiple independent whole-animal microarray or mRNA-sequencing analyses. These results support the notion of tissue-specific DAF-16 transcriptional programs and highlight the importance of verifying FOXO targets in a cell-type-specific manner.
The high substrate selectivity of the ubiquitin/proteasome system is mediated by a large group of E3 ubiquitin ligases. The ubiquitin ligase CHIP regulates the degradation of chaperone-controlled and chaperone-independent proteins. To understand how CHIP mediates substrate selection and processing, we performed a structure-function analysis of CHIP and addressed its physiological role in Caenorhabditis elegans and human cells. The conserved function of CHIP in chaperone-assisted degradation requires dimer formation to mediate proteotoxic stress resistance and to prevent protein aggregation. The CHIP monomer, however, promotes the turnover of the membrane-bound insulin receptor and longevity. The dimer-monomer transition is regulated by CHIP autoubiquitylation and chaperone binding, which provides a feedback loop that controls CHIP activity in response to cellular stress. Because CHIP also binds other E3 ligases, such as Parkin, the molecular switch mechanism described here could be a general concept for the regulation of substrate selectivity and ubiquitylation by combining different E3s.
Edible insects, a novel source of protein, are gaining interest for their health promoting attributes. In this study, the in vivo and in vitro antioxidant effect of tropical banded crickets (Gryllodes sigillatus) peptides was evaluated. Antioxidant activity by 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity, oxygen radical antioxidant capacity (ORAC) and caco-2 cellular antioxidant acidity, were measured in hydrolyzed followed by simulated gastrointestinal digested (SGD) peptides. In vivo analysis was conducted using Caenorhabditis elegans as a model. In vitro analysis showed cricket peptides had greater (p< 0.05) antioxidant activity than the unhydrolyzed protein (control). In C. elegans, the lifespan of nematodes fed SGD peptides increased under chronic and acute oxidative stress conditions. Reactive oxygen species (ROS) levels of nematodes fed SGD peptides under paraquat induced oxidative stress were lower (p<0.05) than that of the control group. Further studies using polymerase chain reaction (PCR) indicated that the increased resistance to oxidative stress in C. elegans fed SGD peptides could be due to the increased expression of the stress-related gene gst-4. Taken together, these results indicate that tropical banded cricket peptides could be used as a functional food and nutraceutical to combat oxidative stress.
Mitochondrial function is closely related to the body's oxidative stress level and lifespan. Our previous research demonstrated that the combination of blueberry extracts (BE) and apple peel extracts (APE) could synergistically promote the oxidative stress and lifespan of Caenorhabditis elegans (C. elegans). The purpose of this study was to determine whether the improvement of the oxidative stress and lifespan is involved in the regulation of mitochondrial function, and to explore its potential mechanisms. A model of nematode mitochondrial dysfunction was established using RNAi technology. The results showed that supplementation with BE plus APE could prolong the lifespan of RNAi(mev-1) C. elegans, and improve mitochondrial function, while there was no significant effect on RNAi(isp-1) C. elegans. In addition, treatment with BE plus APE could down-regulate the overexpression of reactive oxygen species (ROS) in clk-1(e2519) mutants. Meanwhile, administration with BE plus APE abolished the extension of the lifespan of clk-1(e2519) mutants, and inhibited the expression of clk-1 downstream genes, including mev-1, isp-1, nuo-2, cco-1, cyc-1 and atp-3, thereby regulating the function of the mitochondrial respiratory chain and improving its oxidative stress ability to prolong the lifespan of nematodes. In short, the combination of BE and APE could improve the oxidative stress and extend the lifespan of nematodes via regulating mitochondrial function. This study provided a theoretical basis for exploring the role of mitochondrial function in regulating the body's oxidative stress level and aging process.
SIN3/HDAC is a multi-protein complex that acts as a regulatory unit and functions as a co-repressor/co-activator and a general transcription factor. SIN3 acts as a scaffold in the complex, binding directly to HDAC1/2 and other proteins and plays crucial roles in regulating apoptosis, differentiation, cell proliferation, development, and cell cycle. However, its exact mechanism of action remains elusive. Using the Caenorhabditis elegans (C. elegans) model, we can surpass the challenges posed by the functional redundancy of SIN3 isoforms. In this regard, we have previously demonstrated the role of SIN-3 in uncoupling autophagy and longevity in C. elegans. In order to understand the mechanism of action of SIN3 in these processes, we carried out a comparative analysis of the SIN3 protein interactome from model organisms of different phyla. We identified conserved, expanded, and contracted gene classes. The C. elegans SIN-3 interactome -revealed the presence of well-known proteins, such as DAF-16, SIR-2.1, SGK-1, and AKT-1/2, involved in autophagy, apoptosis, and longevity. Overall, our analyses propose potential mechanisms by which SIN3 participates in multiple biological processes and their conservation across species and identifies candidate genes for further experimental analysis.
Mutations in the well-known tumor suppressor PTEN are observed in many cancers. PTEN is a dual-specificity phosphatase that harbors lipid and protein-phosphatase activities. The Caenorhabditis elegans PTEN ortholog is daf-18, which has pleiotropic effects on dauer formation, aging, starvation resistance, and development. Function of three daf-18 point-mutants, G174E, D137A and C169S, had previously been investigated using high-copy transgenes in a daf-18 null background. These alleles were generated based on their mammalian counterparts and were treated as though they specifically disrupt lipid or protein-phosphatase activity, or both, respectively. Here, we investigated these alleles using genome editing of endogenous daf-18. We assayed three traits relevant to L1 starvation resistance, and we show that each point mutant is essentially as starvation-sensitive as a daf-18 null mutant. Furthermore, we show that G174E and D137A do not complement each other, suggesting overlapping effects on lipid and protein-phosphatase activity. We also show that each allele has strong effects on nucleocytoplasmic localization of DAF-16/FoxO and dauer formation, both of which are regulated by PI3K signaling, similar to a daf-18 null allele. In addition, each allele also disrupts M-cell quiescence during L1 starvation, though D137A has a weaker effect than the other alleles, including the null. Our results confirm that daf-18/PTEN is important for promoting starvation resistance and developmental arrest and that it is a potent regulator of PI3K signaling, and they highlight challenges of using genetic analysis to link specific DAF-18/PTEN enzymatic activities to particular phenotypes.
BACKGROUND
Artemisia selengensis Turcz. (AST) is a common edible and medicinal herb possessing extensive biological activities and various health‐promoting functions. However, the anti‐aging effects of AST have been neglected. This work evaluated the effects of AST leaf extract (ASTE) on stress tolerance and longevity in Caenorhabditis elegans.
RESULTS
ASTE treatment enhanced stress resistance and significantly extended the lifespan of C. elegans. Moreover, ASTE prolonged the healthspan by increasing body bending and pharyngeal pumping rates, and by reducing the intestinal lipofuscin level and accumulation of intracellular reactive oxygen species (ROS). Caffeoylquinic acids in ASTE, especially dicaffeoylquinic acids, were the major components responsible for these benefits. The mechanism underlying the anti‐aging effect of ASTE occurred by activating insulin/insulin‐like growth factor, SIR‐2.1 signaling and mitochondrial dysfunction pathways, which in turn induced the activity of the transcription factors DAF‐16/FOXO and SKN‐1/Nrf2.
CONCLUSION
These findings provide direct evidence for the anti‐aging effects of AST and reveal its potential on promoting healthy aging. © 2022 Society of Chemical Industry.
Germline integrity is critical for progeny fitness. Organisms deploy the DNA damage response (DDR) signalling to protect germline from genotoxic stress, facilitating cell-cycle arrest of germ cells and DNA repair or their apoptosis. Cell-autonomous regulation of germline quality is well-studied; however, how quality is enforced cell non-autonomously on sensing somatic DNA damage is less known. Using Caenorhabditis elegans, we show that DDR disruption, only in the uterus, when insulin-IGF-1 signalling (IIS) is low, arrests germline development and induces sterility in a FOXO/DAF-16 transcription factor (TF)-dependent manner. Without FOXO/DAF-16, germ cells of the IIS mutant escape arrest to produce poor quality oocytes, showing that the TF imposes strict quality control during low IIS. In response to low IIS in neurons, FOXO/DAF-16 works cell autonomously as well as non-autonomously to facilitate the arrest. Activated FOXO/DAF-16 promotes transcription of checkpoint and DDR genes, protecting germline integrity. However, on reducing DDR during low IIS, the TF decreases ERK/MPK-1 signaling below a threshold, and transcriptionally downregulates genes involved in spermatogenesis-to-oogenesis switch as well as cdk-1/Cyclin B to promote germline arrest. Altogether, our study reveals how cell non-autonomous function of FOXO/DAF-16 promotes germline quality and progeny fitness in response to somatic DNA damage.
The aggregation of β-amyloid (Aβ) has the neurotoxicity, which is thought to play critical role in the pathogenesis of Alzheimer's disease (AD). Inhibiting Aβ deposition and neurotoxicity has been considered as an important strategy for AD treatment. 3,6'-Disinapoyl sucrose (DISS), one of the oligosaccharide esters derived from traditional Chinese medicine Polygalae Radix, possesses antioxidative activity, neuroprotective effect and anti-depressive activity. This study was to explore whether DISS could attenuate the pathological changes of Aβ1-42 transgenic Caenorhabditis elegans (C. elegans). The results showed that DISS (5 and 50 μM) treatment significantly prolonged the life span, increased the number of egg-laying, reduced paralysis rate, decreased the levels of lipofuscin and ROS and attenuated Aβ deposition in Aβ1-42 transgenic C. elegans. Gene analysis showed that DISS could up-regulate the mRNA expression of sod-3, gst-4, daf-16, bec-1 and lgg-1, while down-regulate the mRNA expression of daf-2 and daf-15 in Aβ1-42 transgenic C. elegans. These results suggested that DISS has the protective effect against Aβ1-42 -induced pathological damages and prolongs the life span of C. elegans, which may be related to the reduction of Aβ deposition and neurotoxicity by regulating expression of genes related to antioxidation and autophagy.
Acrylamide (AC), a proved toxin is mainly used in industrial fields and proved to possess various toxicities. In recent years, AC has been found in starch-containing foods due to Maillard reaction in a high-temperature process. Therefore, how to mitigate the toxic effect of AC is a research spot. Blumea laciniata is a widely used folk medicine in Asia and the extract from B. laciniata (EBL) exhibited a strong protection on cells against oxidative stress. In this work, we used EBL to protect Hep G2 cells and Caenorhabditis elegans against AC toxicity. As the results turned out, EBL increased cell viability under AC stress and notably reduced the cell apoptosis through decreasing the high level of ROS. Moreover, EBL extended the survival time of C. elegans, while EBL failed to prolong the survival time of mutants that were in Insulin signaling pathway. Besides, the expressions of antioxidant enzymes were activated after the worms were treated with EBL and daf-16 gene was activated. Our results indicated that EBL exhibited a protective effect against AC induced toxicity in Hep G2 cells and C. elegans via Insulin/IGF-1 signaling pathway. These outcomes may provide a promising natural drug to alleviate the toxic effect of AC.
Chemical agents released into the environment can induce oxidative stress in organisms, which is detrimental for health. Although environmental exposures typically include multiple chemicals, organismal studies on oxidative stress derived from chemical agents commonly study exposures to individual compounds. In this work, we explore how chemical mixtures drive the oxidative stress response under various conditions in the nematode C. elegans, by quantitatively assessing levels of gst-4 expression. Our results indicate that naphthoquinone mixtures drive responses differently than individual components, and that altering environmental conditions, such as increased heat and reduced food availability, result in dramatically different oxidative stress responses mounted by C. elegans. When exposed to heat, the oxidative stress response is diminished. Notably, when exposed to limited food, the oxidative stress response specific to juglone is significantly heightened, while identified antagonistic interactions between some naphthoquinone components in mixtures are abolished. This implies that organismal responses to xenobiotics is confounded by environment and stressor interactions. Given the high number of variables under study, and their potential combinations, a simplex centroid design was used to capture such non-trivial response over the design space. This makes the case for the adoption of Design of Experiments approaches as they can greatly expand the experimental space probed in noisy biological readouts, and in combinatorial experiments. Our results also reveal gaps in our current knowledge of the organismal oxidative stress response, which can be addressed by employing sophisticated design of experiments approaches to identify significant interactions.
During ageing, preservation of locomotion is generally considered an indicator of sustained good health, in elderlies and in animal models. In C. elegans , mutants of the insulin-IGF-1 receptor DAF-2/IIRc represent a paradigm of healthy ageing, as their increased lifespan is accompanied by a delay in age-related loss of motility. However, these animals are less mobile than wild-type animals in early adulthood. Here we investigated the DAF-2/IIRc- dependent relationship between longevity and motility using an auxin-inducible degron to trigger tissue-specific degradation of endogenous DAF-2/IIRc. As previously reported, inactivation of DAF-2/IIRc in neurons or intestine was sufficient to extend the lifespan of worms, whereas depletion in epidermis, germline or muscle was not. However, neither intestinal nor neuronal depletion of DAF-2/IIRc prevented the age-related loss of motility. In 1-day-old adults, DAF-2/IIRc depletion in neurons reduced motility in a DAF-16/FOXO dependent manner, while muscle depletion had no effect. By contrast, DAF-2 depletion in the muscle of middleage animals improved their motility independently of DAF-16/FOXO but required UNC-120/SRF.
Yet, neuronal or muscle DAF-2/IIRc depletion both preserved the mitochondria network in ageing muscle. Overall, these results show that the motility pattern of daf-2 mutants is determined by the sequential and opposing impact of neurons and muscle tissues and can be dissociated from the regulation of the lifespan. This work also provides the characterization of a versatile tool to analyze the tissue-specific contribution of insulin-like signaling in integrated phenotypes at the whole organism level.
FoxO transcription factors (TFs) control metabolism, development, and aging in diverse species. Mouse models implicate FoxO dysregulation in the pathogenesis of age-related disease, including type 2 diabetes, cancer, osteoporosis, and cardiovascular disease. In the nematode C. elegans, the FoxO ortholog DAF-16 extends life span more than 2-fold in response to reduced signaling from the insulin-like growth factor receptor (IGFR) ortholog DAF-2. However, DAF-16/FoxO regulates numerous target genes, posing a significant challenge to understanding the mechanistic details of life span extension. Here, we have developed a logical framework to prioritize functional testing of DAF-16/FoxO target genes highly associated with longevity. Multiple DAF-16/FoxO isoforms are expressed in C. elegans, and we dissected the contributions of individual isoforms to life span control using novel isoform-specific daf-16/FoxO mutants. Whole-transcriptome profiling (RNA-seq) revealed the sets of target genes regulated by each DAF-16/FoxO isoform. An integrative analysis of the life span phenotypes and gene expression profiles of daf-16/FoxO isoform-specific mutants yielded an experimentally tractable list of high-priority genes likely to influence life span. We then screened these genes using a novel method and discovered critical longevity targets of DAF-16/FoxO. These findings suggest that a small subset of DAF-16/FoxO target genes may play a disproportionate role in life span control. Furthermore, most of the genes we identified are conserved in humans and may be involved in human age-related disease. Our approach integrating genetics, phenotypic analysis, and gene expression profiling may be generally useful in dissecting the mechanisms by which transcription factors influence complex processes like aging.
In mammals, insulin signalling regulates glucose transport together with the expression and activity of various metabolic enzymes. In the nematode Caenorhabditis elegans, a related pathway regulates metabolism, development and longevity. Wild-type animals enter the developmentally arrested dauer stage in response to high levels of a secreted pheromone, accumulating large amounts of fat in their intestines and hypodermis. Mutants in DAF-2 (a homologue of the mammalian insulin receptor) and AGE-1 (a homologue of the catalytic subunit of mammalian phosphatidylinositol 3-OH kinase) arrest development at the dauer stage. Moreover, animals bearing weak or temperature-sensitive mutations in daf-2 and age-1 can develop reproductively, but nevertheless show increased energy storage and longevity. Here we show that null mutations in daf-16 suppress the effects of mutations in daf-2 or age-1; lack of daf-16 bypasses the need for this insulin receptor-like signalling pathway. The principal role of DAF-2/AGE-1 signalling is thus to antagonize DAF-16. daf-16 is widely expressed and encodes three members of the Fork head family of transcription factors. The DAF-2 pathway acts synergistically with the pathway activated by a nematode TGF-β-type signal, DAF-7, suggesting that DAF-16 cooperates with nematode SMAD proteins in regulating the transcription of key metabolic and developmental control genes. The probable human ortholognes of DAF-16, FKHR and AFX, may also act downstream of insulin signalling and cooperate with TGF-β effectors in mediating metabolic regulation. These genes may be dysregulated in diabetes.
In Caenorhabditis elegans, an insulin-like signaling pathway to phosphatidylinositol 3-kinase (PI 3-kinase) and AKT negatively regulates the activity
of DAF-16, a Forkhead transcription factor. We show that in mammalian cells, C.elegans DAF-16 is a direct target of AKT and that AKT phosphorylation generates 14-3-3 binding sites and regulates the nuclear/cytoplasmic
distribution of DAF-16 as previously shown for its mammalian homologs FKHR and FKHRL1. In vitro, interaction of AKT- phosphorylated DAF-16 with 14-3-3 prevents DAF-16 binding to its target site in the insulin-like growth
factor binding protein-1 gene, the insulin response element. In HepG2 cells, insulin signaling to PI 3-kinase/AKT inhibits
the ability of a GAL4 DNA binding domain/DAF-16 fusion protein to activate transcription via the insulin-like growth factor
binding protein-1-insulin response element, but not the GAL4 DNA binding site, which suggests that insulin inhibits the interaction
of DAF-16 with its cognate DNA site. Elimination of the DAF-16/1433 association by mutation of the AKT/14-3-3 sites in DAF-16,
prevents 14-3-3 inhibition of DAF-16 DNA binding and insulin inhibition of DAF-16 function. Similarly, inhibition of the DAF-16/14-3-3
association by exposure of cells to the PI 3-kinase inhibitor LY294002, enhances DAF-16 DNA binding and transcription activity.
Surprisingly constitutively nuclear DAF-16 mutants that lack AKT/14-3-3 binding sites also show enhanced DNA binding and transcription
activity in response to LY294002, pointing to a 14-3-3-independent mode of regulation. Thus, our results demonstrate at least
two mechanisms, one 14-3-3-dependent and the other 14-3-3-independent, whereby PI 3-kinase signaling regulates DAF-16 DNA
binding and transcription function.
Dauer larva formation in Caenorhabditis elegans is controlled by chemosensory cells that respond to environmental cues. Genetic interactions among mutations in 23 genes that affect dauer larva formation were investigated. Mutations in seven genes that cause constitutive dauer formation, and mutations in 16 genes that either block dauer formation or result in the formation of abnormal dauers, were analyzed. Double mutants between dauer-constitutive and dauer-defective mutations were constructed and characterized for their capacity to form dauer larvae. Many of the genes could be interpreted to lie in a simple linear epistasis pathway. Three genes, daf-16, daf-18 and daf-20, may affect downstream steps in a branched part of the pathway. Three other genes, daf-2, daf-3 and daf-5, displayed partial or complex epistasis interactions that were difficult to interpret as part of a simple linear pathway. Dauer-defective mutations in nine genes cause structurally defective chemosensory cilia, thereby blocking chemosensation. Mutations in all nine of these genes appear to fall at a single step in the epistasis pathway. Dauer-constitutive mutations in one gene, daf-11, were strongly suppressed for dauer formation by mutations in the nine cilium-structure genes. Mutations in the other six dauer-constitutive genes caused dauer formation despite the absence of functional chemosensory endings. These results suggest that daf-11 is directly involved in chemosensory transduction essential for dauer formation, while the other Daf-c genes play roles downstream of the chemosensory step.
We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single-stranded oligonucleotide was co-injected with the double-stranded DNA.
In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences.
A C. elegans neurosecretory signaling system regulates whether animals enter the reproductive life cycle or arrest development at the
long-lived dauer diapause stage. daf-2, a key gene in the genetic pathway that mediates this endocrine signaling, encodes an insulin receptor family member. Decreases
in DAF-2 signaling induce metabolic and developmental changes, as in mammalian metabolic control by the insulin receptor.
Decreased DAF-2 signaling also causes an increase in life-span. Life-span regulation by insulin-like metabolic control is
analogous to mammalian longevity enhancement induced by caloric restriction, suggesting a general link between metabolism,
diapause, and longevity.
The wild-type Caenorhabditis elegans nematode ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced
activity of the genedaf-2, a homolog of the insulin and insulin-like growth factor receptors, age more slowly than normal and live more than twice as
long. These mutants are active and fully fertile and have normal metabolic rates. The life-span extension caused by daf-2mutations requires the activity of the gene daf-16. daf-16appears to play a unique role in life-span regulation and encodes a member of the hepatocyte nuclear factor 3 (HNF-3)/forkhead
family of transcriptional regulators. In humans, insulin down-regulates the expression of certain genes by antagonizing the
activity of HNF-3, raising the possibility that aspects of this regulatory system have been conserved.
Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In this study, we demonstrate that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.
The activity of the DAF-2 insulin-like receptor is required for Caenorhabditis elegans reproductive growth and normal adult life span. Informatic analysis identified 37 C. elegans genes predicted to encode insulin-like peptides. Many of these genes are divergent insulin superfamily members, and many are clustered, indicating recent diversification of the family. The ins genes are primarily expressed in neurons, including sensory neurons, a subset of which are required for reproductive development. Structural predictions and likely C-peptide cleavage sites typical of mammalian insulins suggest that ins-1 is most closely related to insulin. Overexpression of ins-1, or expression of human insulin under the control of ins-1 regulatory sequences, causes partially penetrant arrest at the dauer stage and enhances dauer arrest in weak daf-2 mutants, suggesting that INS-1 and human insulin antagonize DAF-2 insulin-like signaling. A deletion of the ins-1 coding region does not enhance or suppress dauer arrest, indicating a functional redundancy among the 37 ins genes. Of five other ins genes tested, the only other one bearing a predicted C peptide also antagonizes daf-2 signaling, whereas four ins genes without a C peptide do not, indicating functional diversity within the ins family.
We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.
Following the discovery that the proto-oncogene protein kinase B (PKB) functions as a downstream element
in signalling from phosphoinositide 3′-kinase (PI3-kinase) (Burgering & Coffer, 1995), PKB has been shown
to mediate a diverse array of PI3-kinase dependent cellular responses. Most recently PKB-dependent
phosphorylation of 3 members of the family of Forkhead transcription factors has been demonstrated to
play a role in PI3-kinase dependent effects on transcription. This review focuses on this newly discovered
function of PKB in conveying the diversity of PI3-kinase dependent cellular responses.
Alveolar rhabdomyosarcomas are associated with unique chromosomal translocations t(2;13) and t(1;13), which arise from fusion of the genes for the paired box proteins PAX3 and PAX7, respectively, to the FKHR (forkhead in rhabdomyosarcoma) gene on chromosome 13q14. Here we report the identification and characterization of three novel human forkhead genes with similarity to FKHR. The three genes (HGMW-approved symbols FKHRP1, FKHRL1, and FKHRL1P1) map to chromosomal regions 5q35.2–q35.3, 6q21, and 17p11, respectively. Based on amino acid sequence comparisons of their forkhead domains, FKHRL1, FKHRL1P1, and FKHRP1 share 86, 84, and 68% identity, respectively, with FKHR. While FKHR and FKHRL1 are expressed in every human adult tissue examined, FKHRP1 mRNA expression could not be detected, and FKHRL1P1 expression was present only at low levels. FKHR and FKHRL1 share a similar genomic organization, each having a very large intron 1 (FKHR ∼130 kb and FKHRL1 >90 kb), which bisects their respective forkhead domains at identical positions, as well as a second intron just downstream of each stop codon. FKHRP1 and FKHRL1P1 lack introns and contain stop codons that prevent them from yielding full-length proteins. Thus, while FKHR and FKHRL1 represent functional genes, FKHRP1 and FKHRL1P1 probably are processed pseudogenes. These results suggest that these four genes represent an FKHR-like gene subfamily within the larger human forkhead gene family.
Fluorodeoxyuridine (FUdR), an inhibitor of DNA synthesis, was examined for its ability to prevent a synchronous population of C. elegans from reproducing without otherwise interfering with the organism's post-maturational development and aging. When a synchronized population was exposed to 400 μM FUdR just as the population reached sexual maturity, the FUdR induced complete sterility within five hours by preventing eggs from hatching. Any larvae that hatched from eggs made before the FUdR was added remained small in the presence of FUdR and were easily removed by filtration or sedimentation. FUdR-sterilized adults showed no morphological abnormalities. Age-associated changes seen in controls also occurred in FUdR-treated worms, including atrophy of the gonads, increased pigmentation, sluggishness and increased transparency. Life span was not shortened by FUdR treatment.
Our observations suggest that treatment with FUdR under carefully controlled conditions is a reasonable way to maintain synchronously aging populations of C. elegans.
Three environmental cues influence both the entry into and exit from the developmentally arrested dispersal stage called the dauer larva: a dauer-inducing pheromone, food, and temperature. The pheromone, which is a measure of population density, induces dauer larva formation at the second (L2) molt and inhibits recovery in a dose-dependent manner. Food acts competitively to reduce the frequency of dauer larva formation and to enhance recovery. The pheromone causes a specific extension of the second larval stage, coupled with a transient decrease in the growth rate of the L2. Second-stage larvae grown in the presence of added pheromone are morphologically distinguishable from L2 larvae grown without pheromone. We have named the pre-dauer L2 larva the L2d. Commitment to dauer larva formation can occur at the L2d molt. When L2d larvae are shifted out of pheromone to a lawn of E. coli just before the L2d molt, a few worms complete development into dauer larvae. In contrast, worms are essentially committed to the non-dauer life cycle by the first larval molt if the L1 larvae are not grown in appropriately high levels of pheromone. In the presence of pheromone, the percentage of dauer larva formation is enhanced at higher temperatures within the normal growth range. Temperature down-shifts induce dauer larva recovery. Temperature-shift experiments show that the enhancement of dauer larva formation requires exposure to the higher temperature around the L1 molt. Two sensory mutants defective in thermotaxis are altered in their sensitivity to the dauer-inducing pheromone, but their pheromone response retains temperature dependence. Response of dauer larvae to environmental cues is highly age dependent, with older dauer larvae exhibiting an increased tendency to recover.
Under conditions of high population density and low food, Caenorhabditis elegans forms an alternative third larval stage, called the dauer stage, which is resistant to desiccation and harsh environments. Genetic analysis of some dauer constitutive (Daf-c) and dauer defective (Daf-d) mutants has revealed a complex pathway that is likely to function in particular neurons and/or responding tissues. Here we analyze the genetic interactions between three genes which comprise a branch of the dauer formation pathway that acts in parallel to or downstream of the other branches of the pathway, the Daf-c genes daf-2 and daf-23 and the Daf-d gene daf-16. Unlike mutations in other Daf-c genes, mutations in both daf-2 and daf-23 cause non-conditional arrest at the dauer stage. Our epistasis analysis suggests that daf-2 and daf-23 are functioning at a similar point in the dauer pathway. First, mutations in daf-2 and daf-23 are epistatic to mutations in the same set of Daf-d genes. Second, daf-2 and daf-23 mutants are suppressed by mutations in daf-16. Mutations in daf-16 do not suppress any of the other Daf-c mutants as efficiently as they suppress daf-2 and daf-23 mutants. Third, double mutants between either daf-2 or daf-23 and several other daf-d mutants exhibit an unusual interaction. Based on these results, we present a model for the function of daf-2, daf-23 and daf-16 in dauer formation.
It has long been postulated that protein tyrosine phosphatases may act as tumor suppressors because of their ability to counteract the oncogenic actions of protein tyrosine kinases. Here we report the cloning and characterization of a novel human protein tyrosine phosphatase, TEP1. TEP1 contains the protein tyrosine phosphatase signature motif, and we show that it possesses an intrinsic protein tyrosine phosphatase activity. TEP1 also shares extensive homology with tensin, a cytoskeletal protein localized to focal adhesions, and with auxilin, a protein involved in synaptic vesicle transport. Immunofluorescence studies show that TEP1 is a cytoplasmic protein. The abundance of TEP1 transcription is altered in many transformed cells. In the transforming growth factor beta-sensitive cells, TEP1 expression is rapidly down-regulated by transforming growth factor beta, a cytokine shown to be involved in regulating cell adhesion and cell motility. We have also mapped the gene encoding TEP1 to chromosome 10q23, a locus that is frequently deleted in a variety of human cancers. TEP1 protein is identical to the protein encoded by the candidate tumor suppressor gene PTEN/MMAC1. Our functional studies of the TEP1 protein suggest that its tumor suppressor function may associate with its intrinsic protein tyrosine phosphatase activity and its cytoplasmic localization.
Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
A fusion of the sur-5 protein to the green fluorescent protein containing a nuclear localization signal is demonstrated as a marker for genetic mosaic analysis in the nematode Caenorhabditis elegans. Because of an extensive accumulation of bright fluorescence in many nuclei, normal growth plates, each containing hundreds of worms, can be rapidly screened with a dissecting microscope for rare mosaic individuals. As the marker can also be used to detect transgenic worms, the construction of strains for mosaic analyses can be minimized. In the course of examining rare mosaic animals, an unexpected pattern of fluorescence was noticed for hyp6, a syncytial component of the hypodermis, which indicated that the marker may serve as a means of assessing cellular fusions during development. Immunofluorescent staining of adherens junctions confirmed a postembryonic fusion of hyp6 with hyp7, the major syncytium of the hypodermis.
A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. We show here that two C. elegansAkt/PKB homologs, akt-1 and akt-2,transduce insulin receptor-like signals that inhibit dauer arrest and that AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1,revealed by a genetic screen, as well as increased dosage of wild-typeakt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 andakt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation.
Keywords
• Insulin signaling
• dauer
• Fork head transcription factor
• life span
The precise arrangement of molecules within the thick filament, as well as the mechanisms by which this arrangement is specified, remains unclear. In this article, we have exploited a unique genetic interaction between one isoform of myosin heavy chain (MHC) and paramyosin in Caenorhabditis elegans to probe the molecular interaction between MHC and paramyosin in vivo. Using chimeric myosin constructs, we have defined a 322-residue region of the MHC A rod critical for suppression of the structural and motility defects associated with the unc-15(e73) allele. Chimeric constructs lacking this region of MHC A either fail to suppress, or act as dominant enhancers of, the e73 phenotype. Although the 322-residue region is required for suppression activity, our data suggest that sequences along the length of the rod also play a role in the isoform-specific interaction between MHC A and paramyosin. Our genetic and cell biological analyses of construct behavior suggest that the 322-residue region of MHC A is important for thick filament stability. We present a model in which this region mediates an avid interaction between MHC A and paramyosin in parallel arrangement in formation of the filament arms.
An insulinlike signaling pathway controlsCaenorhabditis elegans aging, metabolism, and development. Mutations in the daf-2 insulin receptor–like gene or the downstream age-1 phosphoinositide 3-kinase gene extend adult life-span by two- to threefold. To identify tissues where this pathway regulates
aging and metabolism, we restored daf-2pathway signaling to only neurons, muscle, or intestine. Insulinlike signaling in neurons alone was sufficient to specify
wild-type life-span, but muscle or intestinal signaling was not. However, restoring daf-2 pathway signaling to muscle rescued metabolic defects, thus decoupling regulation of life-span and metabolism. These findings
point to the nervous system as a central regulator of animal longevity.
Following the discovery that the proto-oncogene protein kinase B (PKB) functions as a downstream element in signalling from phosphoinositide 3'-kinase (PI3-kinase) (Burgering & Coffer, 1995), PKB has been shown to mediate a diverse array of PI3-kinase dependent cellular responses. Most recently PKB-dependent phosphorylation of 3 members of the family of Forkhead transcription factors has been demonstrated to play a role in PI3-kinase dependent effects on transcription. This review focuses on this newly discovered function of PKB in conveying the diversity of PI3-kinase dependent cellular responses.
Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
The lifespan of Caenorhabditis elegans is regulated by the insulin/insulin-like growth factor (IGF)-1 receptor homolog DAF-2, which signals through a conserved phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Mutants in this pathway remain youthful and active much longer than normal animals and can live more than twice as long. This lifespan extension requires DAF-16, a forkhead/winged-helix transcription factor. DAF-16 is thought to be the main target of the DAF-2 pathway. Insulin/IGF-1 signaling is thought to lead to phosphorylation of DAF-16 by AKT activity, which in turn shortens lifespan. Here, we show that the DAF-2 pathway prevents DAF-16 accumulation in nuclei. Disrupting Akt-consensus phosphorylation sites in DAF-16 causes nuclear accumulation in wild-type animals, but, surprisingly, has little effect on lifespan. Thus the DAF-2 pathway must have additional outputs. Lifespan in C. elegans can be extended by perturbing sensory neurons or germ cells. In both cases, lifespan extension requires DAF-16. We find that both sensory neurons and germline activity regulate DAF-16 accumulation in nuclei, but the nuclear localization patterns are different. Together these findings reveal unexpected complexity in the DAF-16-dependent pathways that regulate aging.
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Phosphatidylinositol 3-kinase signaling inhibits DAF-16 DNA binding and function via 14-3-3-dependent and 14-3-3- independent pathways
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Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor
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predauer stage Figure 3c. The L2d stage is an extension
predauer stage Figure 3c. The L2d stage is an extension
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The Forkhead transcription factor DAF-16 transduces longed feeding, and darker, apparently due to storage Brief Communication 1957 insulin-like metabolic and longevity signals in C. elegans
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Brief Communication 1957
insulin-like metabolic and longevity signals in C. elegans.
Nature 1997, 389:994-999.
insulin-like metabolic and longevity signals in C. elegans
insulin-like metabolic and longevity signals in C. elegans.
Nature 1997, 389:994-999.
The Forkhead transcription factor DAF-16 transduces longed feeding, and darker, apparently due to storage Brief Communication
- S Ogg
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Ogg S, Paradis S, Gottlieb S, Patterson G, Lee L, Tissenbaum H,
et al.: The Forkhead transcription factor DAF-16 transduces
longed feeding, and darker, apparently due to storage
Brief Communication 1957
The Forkhead transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans
- Ogg
daf-2, daf-16 and daf-23
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Phosphatidylinositol 3-kinase signaling inhibits DAF-16 DNA binding and function via 14-3-3-dependent and 14-3-3-independent pathways
- Cahill
The C. elegans dauer larva
- Golden
Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor
- Brunet