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The insulin/IGF signaling cascade modulates SUMOylation to regulate aging and proteostasis in Caenorhabditis elegans

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Cite this article as: eLife 2018;7:e38635 doi: 10.7554/eLife.38635

Abstract

Although aging-regulating pathways were discovered a few decades ago, it is not entirely clear how their activities are orchestrated, to govern lifespan and proteostasis at the organismal level. Here, we utilized the nematode Caenorhabditis elegans to examine whether the alteration of aging, by reducing the activity of the Insulin/IGF signaling (IIS) cascade, affects protein SUMOylation. We found that IIS activity promotes the SUMOylation of the germline protein, CAR-1, thereby shortening lifespan and impairing proteostasis. In contrast, the expression of mutated CAR-1, that cannot be SUMOylated at residue 185, extends lifespan and enhances proteostasis. A mechanistic analysis indicated that CAR-1 mediates its aging-altering functions, at least partially, through the notch-like receptor glp-1. Our findings unveil a novel regulatory axis in which SUMOylation is utilized to integrate the aging-controlling functions of the IIS and of the germline and provide new insights into the roles of SUMOylation in the regulation of organismal aging.

https://doi.org/10.7554/eLife.38635.001

eLife digest

Aging may seem inescapable, but there are many factors, from diet to genetic mutations, that can affect this process. In fact, scientists have started to uncover the mechanisms that control and influence this slow decline. For example, in the small worm Caenorhabditis elegans, removing the germs cells – which give rise to eggs – extends the lifespan. Similarly, interfering with the activity of the Insulin/IGF-1 signaling (IIS) pathway leads to a longer life for the animals. However, it is unclear whether these two mechanisms work together, or if they operate in parallel.

To explore this, Moll, Roitenberg et al. first looked at how the IIS pathway regulates a type of protein modification known as SUMOylation in C. elegans. Reducing the activity of the IIS pathway slowed down aging in the worms. It also decreased the levels of SUMOylation of certain proteins, including CAR-1, which is found in the structures that produce germ cells. Further experiments showed that stopping the SUMOylation of CAR-1 extended the lifespan of the animals. In fact, replacing the protein with a mutated version of CAR-1 that cannot accept the SUMO element makes the worms live longer and resist a toxic protein that causes Alzheimer’s disease in humans. These results therefore show that, in C. elegans, the IIS pathway and a mechanism that involves CAR-1 in germ cells work together to determine the pace of aging. Further studies are now needed to dissect how the IIS pathway influences SUMOylation, and whether the findings hold true in mammals.

https://doi.org/10.7554/eLife.38635.002

Introduction

The view that aging is solely driven by stochastic events has changed as mounting evidences showed that certain, apparently independent, genetic and metabolic modulations, slow aging and extend lifespans of various organisms. Dietary restriction (DR), reduced activity of the insulin/IGF signaling pathway (IIS) or of the mitochondrial electron transport chain (ETC), and removal of germ cells (Kenyon, 2005), all slow the pace of aging. Among these, most prominent is IIS reduction, which extends lifespan and elevates stress resistance of worms (Kenyon et al., 1993), mice (Holzenberger et al., 2003), and presumably humans (Suh et al., 2008). In the nematode Caenorhabditis elegans (C. elegans), the sole insulin/IGF receptor, DAF-2, initiates a signaling cascade that negatively regulates the activity of at least three transcription factors by modulating phosphorylation. Direct phosphorylation of DAF-16/FOXO (Lee et al., 2001) and of SKN-1/NRF (Tullet et al., 2008) prevents these factors from entering the nucleus and from regulating their target genes. Similarly, the IIS inhibits the phosphorylation of DDL-1, which retains the Heat Shock Factor 1 (HSF-1) in the cytosol (Chiang et al., 2012). Thus, IIS reduction by daf-2 RNA interference (RNAi) or by mutation, hyperactivates its downstream transcription factors, creating long-lived worms (Kenyon, 2005). IIS reduction also elevates resistance to a variety of stresses including heat (Lithgow et al., 1995), ultraviolet (UV) radiation (Murakami and Johnson, 1996), and pathogenic bacteria (Singh and Aballay, 2006). In addition, IIS reduction protects worms and mice from toxic aggregation (proteotoxicity) of various neurodegeneration-causing proteins (reviewed in Carvalhal Marques et al., 2015). Finally, IIS reduction also modulates reproduction and egg-laying patterns, as knocking down daf-2 by RNAi, reduces the worm’s brood size but extends the reproduction period (Dillin et al., 2002). Although it was shown that the IIS is locally neutralized in germ cells (Narbonne et al., 2015) and that DAF-2 responds to food availability by modulating oogenesis through RAS-ERK signaling (Lopez et al., 2013), whether changes in post-translational modifications are involved in the IIS-mediated control of reproduction, is only partially understood. Moreover, despite the evidences that the ablation of germ cells extends lifespan (Hsin and Kenyon, 1999) and promotes proteostasis in C. elegans (Shemesh et al., 2013), it is unclear how the aging-regulating mechanisms downstream of the IIS and those that are activated by the reproduction system are linked, and whether post-translational modifications play roles in the orchestration of these mechanisms.

SUMOylation is a post-translational modification involving a reversible covalent attachment of a small ubiquitin-like modifier (SUMO) to specific lysine residues of proteins (Melchior, 2000). While mammals ubiquitously express three forms of SUMO (SUMO-1, 2, and 3), C. elegans expresses only one SUMO-encoding gene, smo-1 that encodes a polypeptide of 91 amino acids with a predicted molecular weight of 10.2 kDa (Choudhury and Li, 1997). SUMOylation controls various biological processes and plays important roles in development and survival (Johnson, 2004). Among other functions, smo-1 is critically needed for germline development and fertility of the nematode (Broday, 2017).

Here, we examined whether IIS activity controls SUMOylation of C. elegans’ proteins and if this post-translational modification plays roles in aging-associated functions of this pathway. To address this, we compared global SUMOylation patterns of proteins that were extracted from untreated and from daf-2 RNAi-treated animals, and found that among other modulations, IIS reduction lowers the SUMOylation rate of the protein CAR-1 (Cytokinesis/Apoptosis/RNA-binding protein 1) but has no effect on the expression level of car-1. CAR-1 is an RNA-binding protein, which acts in association with the RNA helicase, CGH-1 in the germline (Audhya et al., 2005). The knockdown of car-1 increases the levels of GLP-1 during late oogenesis (Noble et al., 2008), thereby leading to germ cell death and to defective embryonic cytokinesis (Boag et al., 2005; Squirrell et al., 2006). We show that knocking down car-1 shortens lifespan and enhances proteotoxicity in model worms. On the contrary, the expression of a mutant CAR-1, which cannot be SUMOylated on lysine residue 185 (K185), extends lifespan and promotes proteostasis. These effects are conferred, at least partially, through the GLP-1 axis in a DAF-16-dependent manner, but probably also through an additional, DAF-16-independent pathway. Interestingly, we found that GLP-1 positively controls the expression of car-1 establishing a regulatory circuit. Our findings unveil a novel link between the reproductive system and the IIS, demonstrating that one downstream arm of this pathway regulates certain aspects of aging through the SUMOylation of CAR-1.

Results

IIS reduction results in differential protein SUMOylation in C. elegans

In order to test whether IIS reduction affects global protein SUMOylation in C. elegans, we employed three worm strains: wild-type animals (strain N2) and two conditionally sterile nematode strains: CF512 and CF1903, all exhibit natural IIS activity. CF512 animals become sterile when exposed to 25°C during development, as they cannot produce sperm. CF1903 animals harbor a temperature-sensitive glp-1 mutant that renders them sterile upon exposure to 25°C during development (Arantes-Oliveira et al., 2002). Using these conditionally sterile worm strains, we could compare protein SUMOylation in adult tissues with no background from developing embryos. Eggs of all worm strains were extracted from animals that were grown in 15°C, and placed on plates that were seeded with either control bacteria, harboring the empty RNAi vector (EV), or with daf-2 RNAi expressing bacteria. The plates were incubated at 25°C for 48 hr, transferred to 20°C for additional 24 hr and the worms were harvested at day 1 of adulthood. As expected, wild-type worms were fertile while CF512 and CF1903 worms were sterile (Figure 1—figure supplement 1). Global protein SUMOylation patterns were determined by western blot (WB) analysis, using an anti SUMO antibody. Our results indicated that IIS reduction modulates the patterns of SUMOylation in all three worm strains (Figure 1A), as the SUMOylation levels of several proteins were increased (arrowheads) and of others were decreased (arrows) upon treatment with daf-2 RNAi. Differences in SUMOylation patterns between strains suggest strain-specific protein SUMOylation.

Figure 1 with 2 supplements see all
The knockdown of daf-2 modulates the SUMOylation of CAR-1 in C.elegans. 

(A) Global protein SUMOylation patterns in homogenates of daf-2 RNAi-treated and untreated wild-type (N2), CF512 and CF1903 worms were compared by western blot using an anti-SUMO antibody. In all three worm strains, several proteins exhibit enhanced levels of SUMOylation upon the knockdown of daf-2 (arrowheads) and others show decreased levels (arrows). (B) Schematic illustration of pulldown procedure to isolate covalently SUMOylated proteins from NX25 animals that express His-Flag-smo-1 in a smo-1 knockout background. Covalently SUMOylated proteins were pulled down and identified by mass spectrometry. (C) Nematodes expressing GFP-tagged CAR-1 (strain WH346) were treated with daf-2 RNAi or left untreated (EV), harvested at day 1 of adulthood and GFP-CAR-1 was immune-precipitated by a GFP antibody and blotted using a SUMO antibody. daf-2 RNAi treatment reduced the level of SUMOylated GFP-CAR-1 that migrated as two bands. One was migrating as a protein of ~ 60 kDa and the other as a protein of ~ 250 kDa. (D) Reblotting the membrane with a GFP antibody showed that daf-2 RNAi treatment had no effect on the amounts of the precipitated GFP-CAR-1 protein (this blot serves as a loading control for C). (E) WH346 worms were either grown on control bacteria (EV) or on daf-2 RNAi bacteria, harvested at day 1 of adulthood and total GFP-CAR-1 amounts in the worm homogenates were analyzed by a western blot. No difference in the total levels of GFP-CAR-1 was observed. (F) Comparison of CAR-1-GFP signals in three independent experiments as in E. G. CF512 worms that were either treated with daf-2 RNAi or left untreated (EV) express similar levels of car-1 as measured by quantitative real-time PCR.

https://doi.org/10.7554/eLife.38635.003

To identify proteins which are differentially SUMOylated upon IIS reduction, we used worms that were deprived of the endogenous smo-1 gene and express a dually tagged smo-1 transgene instead (His-Flag-smo-1, strain NX25 [Pferdehirt and Meyer, 2013]). NX25 worms were treated from hatching to day 1 of adulthood with daf-2 RNAi or left untreated (EV), harvested and SUMOylated proteins were pulled-down by tandem-purification procedure (Figure 1B and Figure 1—figure supplement 2). Sediment proteins were analyzed by quantitative Mass Spectrometry (Supplementary file 1, full data set can be accessed at http://www.ebi.ac.uk/pride project ID: PXD010011). Our analysis showed that, among other affected proteins, the SUMOylation of CAR-1 is approximately threefold lower in daf-2 RNAi-treated worms compared to the levels observed in untreated NX25 animals.

IIS reduction lessens the SUMOylation of CAR-1

To further examine whether the IIS governs the rate of CAR-1 SUMOylation, we utilized worms that express CAR-1 fused to the green fluorescent protein (GFP) under the regulation of the pie-1 promoter (strain WH346, GFP-CAR-1 [Squirrell et al., 2006]). These nematodes were used due to the high efficiency and specificity of GFP pulldown. The animals were developed on EV or daf-2 RNAi bacteria, harvested at day 1 of adulthood and GFP-CAR-1 was immuno-precipitated using a GFP antibody, and blotted by an anti SUMO antibody. The intensities of two bands were remarkably higher in homogenates of untreated worms (EV) compared to homogenates of daf-2 RNAi-treated worms (Figure 1C, arrows). One band migrated as a ~ 60 kDa protein, the size corresponding to the mono SUMOylated GFP-CAR-1. The other band migrated as a protein of approximately 250 kDa, suggesting that SUMOylated GFP-CAR-1 is a component of a highly stable protein complex, perhaps with the RNA helicase CGH-1 (Boag et al., 2005). This complex appears to be less abundant, or less SUMOylated, in worms that exhibit low IIS activity. To compare the total amounts of GFP-CAR-1 in this pulldown experiment, we re-exposed the blot to an anti GFP antibody and found no difference in the quantities of GFP-CAR-1 molecules which migrated as a protein of approximately 50 kDa (Figure 1D).

We next tested whether IIS reduction destabilizes the GFP-CAR-1 protein. WH346 worms were cultured from hatching on EV or daf-2 RNAi bacteria, homogenized at day 1 of adulthood and WB analysis using an anti-GFP antibody was utilized to compare the relative levels of GFP-CAR-1. Our results showed similar amounts of GFP-CAR-1 in daf-2 RNAi-treated and untreated worms (Figure 1E). Quantification of the GFP-CAR-1 signals in three independent repeats of this WB experiment confirmed that IIS reduction does not significantly changes the levels of this chimeric protein (Figure 1F). Finally, we tested the possibility that the lower level of SUMOylated CAR-1, observed in daf-2 RNAi-treated worms (Figure 1C), stems from the regulation of car-1 expression by the IIS. To address this, we employed CF512 worms, quantitative real-time PCR (qPCR) and car-1-specific primers and found no significant difference in the expression levels of car-1 in daf-2 RNAi-treated and untreated worms (Figure 1G).

Taken together, our observations indicate that the IIS modulates the SUMOylation of a sub-population of CAR-1 molecules and show that this signaling pathway affects neither the level of car-1 expression nor the amounts of CAR-1 protein within the worm population.

The roles of CAR-1 in the regulation of lifespan

Previous observations regarding the role of CAR-1 as a negative regulator of glp-1 expression in germ cells (Noble et al., 2008), the well-documented effects of glp-1 on aging (Arantes-Oliveira et al., 2002), and our findings of IIS-mediated SUMOylation of CAR-1 (Figure 1), have led us to speculate that CAR-1 is involved in the regulation of lifespan. To test this hypothesis, we compared the lifespans of wild-type worms and of nematodes that are car-1 null. To obtain nematodes that lack car-1, we used worms that carry only one copy of the gene (strain WH377) and selected for progeny that lack both copies of car-1 (car-1 knockout worms are sterile). Lifespans of car-1 knockout worms were found to be significantly shorter than these of wild-type animals (strain N2) (Figure 2A, Supplementary file 2, mean lifespans (LS) of 14.81 ± 0.41 and 17.56 ± 0.52 days, respectively, p<0.001). A parallel experiment, using CF512 worms and RNAi towards car-1 or daf-16, showed similar lifespan shortening by car-1 RNAi (Figure 2—figure supplement 1A, and Supplementary file 3, mean LS of 14.87 ± 0.48 (car-1 RNAi) and 18.01 ± 0.63 (EV) days, p<0.001). Nevertheless, the car-1 RNAi-mediated lifespan shortening effect was less prominent than that of daf-16 RNAi (Figure 2—figure supplement 1A, mean LS of 12.19 ± 0.44 days).

Figure 2 with 4 supplements see all
car-1 regulates lifespan through the germline of C.elegans.

(A) The knockout of car-1 (strain WH377) shortens lifespan compared to wild-type worms (N2) (mean LS of 14.81 ± 0.41 and 17.56 ± 0.52 days, p<0.001). (B) daf-2 mutant (e1370) worms were treated with car-1, daf-16 RNAi or left untreated (EV) and lifespans were followed. car-1 RNAi as well as daf-16 RNAi, shortened lifespan compared to control worms, however, the lifespan-shortening effect of daf-16 RNAi was more prominent than that of car-1 RNAi (mean LS of 17.56 ± 0.56, 40.02 ± 1.40 and 50.52 ± 1.38 days, respectively (p<0.004, EV vs. car-1 RNAi)). (C) The lifespans of car-1 RNAi-treated and untreated daf-16 mutant worms (strain CF1038) were indistinguishable (mean LS of 15.74 ± 0.47 and 15.37 ± 0.48 days, p=0.3). (D–E) Worms that express CAR-1 K185R (EHC118) live approximately 53% longer than their wild-type counterparts (mean LS of 22.51 ± 0.60 and 14.70 ± 0.59 days respectively, p<0.001), as shown by a representative experiment (D) and a summary of three independent experiments (E). (F) Worms that express CAR-1 K257R (EHC121) and wild-type animals have nearly identical lifespans (16.32 ± 0.54 and 16.84 ± 0.56 days, respectively, p=0.25). (G) The longevity of CAR-1 K185R expressing worms is DAF-16-dependent (mean LS of 18.32 ± 0.47 (daf-16 RNAi) and 21.73 ± 0.61 (EV) days, p<0.001). However, daf-16 RNAi-treated CAR-1 K185R and wild-type worms had nearly identical lifespans (16.90 ± 0.63 and 16.67 ± 0.52 respectively, p=0.42). (H) daf-16 RNAi shortens the lifespans of CAR-1 K257R expressing worms (mean LS of 14.95 ± 0.47 days) compared to untreated wild-type animals (18.09 ± 0.41 days, p<0.001).

https://doi.org/10.7554/eLife.38635.006

Next, we asked whether CAR-1 is needed for the full longevity phenotype of nematodes that carry a weak daf-2 allele (e1370, strain CB1370). The worms were either grown from hatching on daf-16, car-1 RNAi, or left untreated (EV) and their lifespans were recorded. The knockdown of car-1 significantly shortened the lifespan of daf-2 mutant worms, compared to untreated animals (Figure 2B, Supplementary file 2, mean LS of 40.02 ± 1.40 (car-1 RNAi) and 50.52 ± 1.38 (EV) days, respectively, p<0.001). This effect was less prominent than that of daf-16 knockdown (mean LS of 17.56 ± 0.56 days). A similar trend was seen when an additional daf-2 mutant worm strain (e1368) was used (Figure 2—figure supplement 1B and Supplementary file 2). Yet, the lifespan reduction that we observed among untreated and car-1 RNAi-treated daf-2 (e1370) mutant worms, which was similar to the difference observed among wild-type and car-1 knockout animals (Figure 2A), questioned the notion that CAR-1 is involved in IIS-mediated regulation of lifespan. Thus, these results suggest that the SUMOylation of CAR-1 by the IIS may be involved in other functions of this signaling pathway.

To further characterize the roles of car-1 as a regulator of lifespan, we tested whether the knockdown of car-1 affects the lifespan of daf-16 mutant animals (strain CF1038), and found that car-1 RNAi had no effect on the lifespans of these worms (Figure 2C and Supplementary file 2). This observation implies that the lifespan regulatory functions of car-1 are DAF-16 dependent. However, since DAF-16 is involved in several longevity-controlling mechanisms (Hsin and Kenyon, 1999), we further tested whether CAR-1 is mechanistically linked to IIS.

Since IIS reduction lowers CAR-1 SUMOylation levels (Figure 1C), we sought to test whether the SUMOylation state of CAR-1 affects lifespan. To directly address this, we used the computational tool GPS-SUMO (Zhao et al., 2014), for identifying SUMOylation consensus motifs in the sequence of CAR-1. Two lysine residues, K185 and K257 were found to be located within predicted SUMOylation motifs. Among the two, K185 is more likely to serve as a SUMOylation site (Figure 2—figure supplement 2). If SUMOylation of K185 or K257 reduces CAR-1 activity and shortens lifespan, it was expected that the overexpression of a SUMOylation-resistant CAR-1 mutant would extend lifespan. To test this hypothesis, we created worms that over-express mutated car-1 genes in which either K185 or K257 were substituted with arginine (CAR-1 K185R (EHC118) and CAR-1 K257R (EHC121), both strains also express the endogenous, wild-type car-1). These mutations prevent potential SUMOylation but maintain the hydrophobicity of the protein. To control for the effect of car-1 over-expression on lifespan, we also created worms that over-express the wild-type car-1 gene (strain EHC117). The three exogenous car-1 genes were fused to an N-terminal double HA tag, and their expression levels were controlled by the car-1 promoter. To compare the levels of SUMOylated CAR-1 in these worm strains, we homogenized young adult worms of the three strains and subjected equal amounts of protein to IP. Using an HA antibody, we pulled down CAR-1, separated total proteins of each worm strain and blotted SUMOylated CAR-1 by a SUMO antibody. Our results (Figure 2—figure supplement 3) show that worms that express the WT CAR-1 contain much higher levels of SUMOylated CAR-1 compared to their counterparts that overexpress either CAR-1 K185R or CAR-1 K257R, indicating that these are SUMOylation sites. Our results also show that CAR-1 is SUMOylated on more than one site, as the substitution of either K185 or of K257 with arginine, did not abolish SUMOylation of the protein.

Performing lifespan assays we found that the over-expression of CAR-1 K185R significantly extended the worms’ lifespans compared to those of wild-type animals (Figure 2D, Supplementary file 2, mean LS of 22.51 ± 0.60 and 14.70 ± 0.59, p<0.001). Three independent repeats confirmed the significance of this phenotype (Figure 2E, Figure 2—figure supplement 4, A and B, and Supplementary file 2). In contrast, no lifespan extension was observed in worms that overexpress CAR-1 K257R (Figure 2F and Supplementary file 3) or wild-type CAR-1 (Figure 2—figure supplement 4C and Supplementary file 3), indicating that the SUMOylation of CAR-1 on lysine 185, but not on lysine 257, plays a role in lifespan determination.

To examine whether the lifespan-extending mechanism that is activated by CAR-1 K185R is DAF-16-dependent, we utilized CAR-1 K185R worms (of a second clone). The worms were either treated with daf-16 RNAi or left untreated (EV), and their lifespans were monitored. Surprisingly, our results (Figure 2G) show that daf-16 RNAi-treated CAR-1 K185R worms and wild-type (N2) animals, exhibited indistinguishable lifespans (see also Supplementary file 2,3). In contrast, daf-16 RNAi-treated CAR-1 K257R worms (Figure 2H) and animals that over-express the wild-type CAR-1 and fed with daf-16 RNAi bacteria (Figure 2—figure supplement 4C and Supplementary file 3) had shorter lifespans compared to their wild-type (N2) counterparts. These results suggest that CAR-1 also regulates lifespan by a DAF-16-independent mechanism.

Taken together, our observations indicate that CAR-1 is needed for wild-type worms to live their natural lifespan and for daf-2 mutant animals to exhibit their full longevity phenotype. They also indicate that SUMOylation of K185 plays a role in the regulation of lifespan. Interestingly, DAF-16 is needed for CAR-1 K185R to extend lifespan; however, the knockdown of daf-16 reduces the lifespans of CAR-1 K185R-expressing animals to be similar to these of wild-type worms, but not shorter as expected.

The mechanisms of CAR-1-mediated lifespan regulation

One possible explanation to the lifespan shortening effect of car-1 RNAi, and the longevity conferred by CAR-1 K185R, suggests that CAR-1 modulates lifespan by negatively regulating the activity of GLP-1. Accordingly, knocking down car-1 by RNAi is expected to hyperactivate GLP-1 and shorten lifespan, whereas the expression of the SUMOylation-resistant, hyperactive CAR-1 K185R, is expected to lower the activity of GLP-1, thereby extending lifespan. To scrutinize this hypothesis, we utilized CF1903 worms. If the lifespan shortening effect of car-1 RNAi is mediated by hyper-activating GLP-1, it was expected that the knockdown of car-1 would not shorten the long lifespans of these worms, which lack functional GLP-1. CF1903 worms were either grown throughout life on control bacteria (EV), or treated with RNAi towards daf-16 or car-1, and lifespans were recorded (in this experiment the worms were developed at 25°C and transferred to 20°C at day 1 of adulthood). While daf-16 RNAi-treated animals had shorter lifespans (Figure 3A, Supplementary file 2, mean LS of 10.71 ± 0.33 days, p<0.001), untreated and car-1 RNAi-treated worms had very similar lifespans (mean LS of 18.13 ± 0.85 (EV) and 17.76 ± 0.72 (car-1 RNAi) days, respectively, p=0.37).

Figure 3 with 4 supplements see all
The knockdown of car-1 modulates the activity of glp-1.

(A) Untreated (EV) and car-1 RNAi-treated, long-lived glp-1 mutant worms (strain CF1903) show no difference in lifespans (mean LS of 17.76 ± 0.72 and 18.13 ± 0.85 days, respectively p=0.37). In contrast, daf-16 RNAi reduced the lifespan of these animals (10.71 ± 0.0.33 days, p<0.001). (B) car-1 RNAi treatment has no significant effect on the lifespans of kri-1 mutant worms (mean LS of 16.59 ± 0.43 (EV) and 15.96 ± 0.39 (car-1 RNAi), p=0.14). In contrast, daf-16 RNAi shortened the lifespans of these animals (mean LS of 13.37 ± 0.26 days (daf-16 RNAi), p<0.001). (C) DAPI stained image of gonads of worms of the indicated genotypes. Bar = 50 mm. (D) The over expression of CAR-1 (strain EHC117) or of the mutated K185R CAR-1 (EHC118) resulted in significantly reduced number of germ cells in the worms’ gonads. The expression of K185R CAR-1 (in EHC118 animals) reduces the number of germ cells by ~ 22% compared to the number that was observed in worms that express the wild-type CAR-1 (EHC117) (p<0.01). (E) Quantification of germline apoptosis by acridine orange staining. car-1 RNAi elevates the average number of apoptotic cells in the gonads of wild-type worms by ~ 2.5-fold. The average numbers of apoptotic cells in the gonads of untreated N2 and of EHC117 worms are nearly identical (3.36 and 3.48, respectively). In contrast, the expression of K185R CAR-1 elevates the average number of apoptotic cells by ~ 80% compared to untreated N2 and EHC117 (p<0.0001, bars represent ± SEM). (F–H) Worms expressing CAR-1 K185R (EHC118) have reduced number of progeny compared to N2 animals (F). The average total number of progeny of EHC118 animals was 105 while control animals had an average of 271 offspring (G). No significant difference in the brood size of wild-type (N2) and EHC117 worms (H). (I) The knockdown of car-1 increases the expression levels of sygl-1 compared to the levels detected in untreated daf-2 (e1370) but decreases the levels of lst-1. (J) The knockdown of glp-1 (in CF1903 worms that were exposed to 25°C) lowers the expression levels of car-1. No such effect was observed in wild-type animals.

https://doi.org/10.7554/eLife.38635.011

These results show that car-1 RNAi does not affect lifespan in the absence of functional GLP-1, and support the theme that CAR-1 modulates lifespan by controlling the activity of GLP-1.

To further assess the hypothesis that CAR-1 affects lifespan through the GLP-1 axis, we utilized worms that carry a mutant kri-1 gene (strain CF2052 (ok1251)). kri-1 is essential for the mediation of longevity by germ cell ablation but not by IIS reduction (Berman and Kenyon, 2006). Thus, if car-1 affects lifespan through the modulation of GLP-1 activity, car-1 RNAi is expected not to affect the lifespan of kri-1 mutant worms. CF2052 nematodes were treated with daf-16 or car-1 RNAi and lifespans were recorded. Although daf-16 RNAi-treated CF2052 animals exhibited a short mean lifespan (13.37 ± 0.26 days, p<0.001), untreated and car-1 RNAi-treated worms had similar mean lifespans of 16.59 ± 0.43 and 15.96 ± 0.39 days, respectively (Figure 3B, p=0.14, and Supplementary file 2). Together these observations support the theme that car-1 governs lifespan, at least partially, through a glp-1-controlled mechanism.

The possible involvement of car-1 in the regulation of lifespan through an additional, daf-16-independent mechanism has led us to ask whether the knockdown of car-1 is linked to the aging-regulating pathway downstream of the transforming growth factor β (TGF-β). This pathway converges with the IIS at the nuclear hormone receptor DAF-12, whose activation is regulated by the cytochrome P450 enzyme, DAF-9 (Gerisch et al., 2001). To test this, we followed the lifespans of daf-9 (strain CF2531) and of daf-12 (strain AA86) mutant worms that were either treated with daf-16, car-1 RNAi or left untreated, and found that the lifespans of both worm strains were shortened by daf-16 RNAi as well as by the knockdown of car-1 (Figure 3—figure supplement 1, A and B, Supplementary file 4). The similar rates of lifespan shortening that resulted from the knockdown of car-1 in wild-type worms (Figure 2A), daf-9 and daf-12 mutant worms, strongly suggest that car-1 RNAi shortens lifespan by a daf-9 and daf-12-independent mechanism.

The roles of CAR-1 in GLP-1-mediated functions

Beside its roles in lifespan determination, GLP-1 is also involved in germ cells proliferation and reproduction (Austin and Kimble, 1987). Thus, we asked whether the modulation of car-1 expression and activity modifies the amount of germ cells. To test this, we compared the numbers of germ cells in gonads of four groups of worms: (i) untreated, wild-type worms (strain N2), (ii) car-1 RNAi-treated wild-type animals, (iii) worms that over-express the natural car-1 (EHC117), and (iv) nematodes that over-express the CAR-1 K185R mutant (EHC118). Nuclei were stained with DAPI and germ cells were counted. We found that the knockdown of car-1 by RNAi had no significant effect on the number of germ cells as untreated and car-1 RNAi-treated animals had similar numbers of germ cells (Figure 3, C and D; average of 724 ± 45.25 and 719 ± 32.36 cells, respectively). In contrast, worms that over-express the natural CAR-1 (EHC117) had significantly less germ cells compared to untreated or car-1 RNAi-treated worms (414.2 ± 6.3 cells, p<0.001). The over-expression of CAR-1 K185R resulted in further reduction in the number of germ cells (average of 324.8 ± 21.37 germ cells/gonad, p<0.01 compared to EHC117).

The observation that the knockdown of car-1 shows no effect on the number of germ cells may emanate from an efficient SUMOylation-mediated inactivation of CAR-1 in wild-type worms. Thus, CAR-1 is less active and its knockdown has a small effect on the number of germ cells. In contrast, the over-expression of CAR-1 (in EHC117 worms), may exceeds the capacity of the SUMOylation mechanism and thus, hyper-activates CAR-1, which in turn lowers the activity of GLP-1, thereby reducing the number of germ cells. According to this explanation, the over-expression of the hyper-active CAR-1 K185R (EHC118) further suppresses the activity of GLP-1, resulting in an even lower number of germ cells.

The knockdown of car-1 by RNAi was reported to enhance physiological apoptosis in hermaphrodites (Boag et al., 2005). Thus, we examined how knocking down car-1, or over expressing wild-type or the mutant CAR-1 K185R, affect the rate of apoptosis in the gonads. Our results (Figure 3E) indicate that, as shown previously (Boag et al., 2005), the knockdown of car-1 elevates the number of apoptotic nuclei in the gonad by approximately 2.5 fold. A similar increase in the rate of apoptosis was seen in EHC118 worms, but not in EHC117 animals. These observations raise the question of how the effects of CAR-1 and its SUMOylation on the number of germ cells and of apoptotic nuclei, affect reproduction.

To address this we tested how the expression of wild-type or of CAR-1 K185R affects brood size by comparing the egg laying capabilities of N2, EHC117 and EHC118 worms. The animals were grown from hatching on control bacteria. At L4 larval stage, 12 animals of each strain were transferred onto new plates, one animal per plate. The worms were transferred onto new plates in 12 hr intervals and viable progeny were counted 48 hr thereafter. We found that EHC118 animals lay fewer eggs than control worms (Figure 3, F and G). While in total, N2 worms had an average of 271.1 progeny, each CAR-1 K185R worm had an average of 104.8 living offspring (Figure 3G). A small but not significant difference was observed among EHC117 and N2 worms, as on average EHC117 worms had 171.4 viable offspring and N2 worms had 201.9 (Figure 3H).

Our observations indicate that SUMOylation of CAR-1 on residue 185 is involved in the modulation of reproduction and suggest that this phenotype may be associated with the effects of IIS reduction on egg laying (Dillin et al., 2002). Thus, we examined how the knockdown of car-1 affects the egg-laying pattern of worms that exhibit impaired IIS. To test this, we employed daf-2 (e1370) mutant and daf-16 mutant (mu86, strain CF1038) worms. e1370 worms were treated with either car-1 RNAi, daf-16 RNAi, or were left untreated (EV), while CF1038 animals were treated with car-1 RNAi or fed on control bacteria (EV). Egg-laying patterns were followed as described above. Our results (Figure 3—figure supplement 2, A and B) confirmed that untreated e1370 worms had a much longer reproductive period compared to wild-type animals. However, the total number of progeny per untreated daf-2 mutant worm was on average 95.7, much lower than that of wild-type nematodes (Figure 3G). Both phenotypes were largely rescued by the knockdown of daf-16, which shifted egg laying to early adulthood (Figure 3—figure supplement 2A) and restored the total number of eggs to the average of 255.4 (Figure 3—figure supplement 2B).

Surprisingly, RNAi-mediated knockdown of car-1 resulted in nearly complete sterility of both daf-2 and daf-16 mutant worms (Figure 3—figure supplement 2, A and B). These observations show that worms that have impaired IIS activity are much more sensitive to the knockdown of car-1 than wild-type animals, implying that this gene is involved in the control of reproduction by the IIS. Yet, this reduction in brood size may be partially due to additive effects of IIS reduction and knocking down car-1.

We also tested whether knocking down car-1 influences the egg-laying patterns of kri-1 mutant (CF2052) nematodes. As shown previously (Dillin et al., 2002), untreated wild-type worms laid the highest number of eggs between day 1 and 1.5 of adulthood (Figure 3F). In contrast, N2 worms that were treated with car-1 RNAi laid the highest number of eggs at the beginning of their reproductive stage (day 0 to 0.5), and the number of eggs declined thereafter (Figure 3—figure supplement 2, C and D). While untreated N2 worms laid on average 244 eggs in total, their car-1 RNAi-treated counterparts laid only 133 eggs (a reduction of ~ 45% (Figure 3—figure supplement 2D)). The reduced reproduction was surprising, as the knockdown of car-1 by RNAi had no effect on the number of germ cells (Figure 3, C and D). Nevertheless, this reduction, which is consistent with a previous report (Boag et al., 2005), may be explained by the increase in the number of apoptotic cells in the gonads of these animals (Figure 3E). kri-1 mutant worms (CF2052) laid fewer eggs than N2 animals, but car-1 RNAi treatment further reduced the number of progeny of both strains. While untreated kri-1 mutant worms laid an average of 116 eggs, car-1 RNAi-treated worms of the same strain laid merely 10 eggs (Figure 3—figure supplement 2, C and D).

Unexpectedly, the knockdown of car-1 affects neither egg-laying patterns, nor brood size of daf-9 and daf-12 mutant worms (Figure 3—figure supplement 2, E and F). These observations show that the egg-laying modulation that resulted from the knockdown of car-1 by RNAi is dependent on the presence of functional daf-9 and daf-12, linking CAR-1 also with the DAF-9/DAF-12 pathway.

Altogether, despite the similarity in the number of germ cells in car-1 RNAi-treated and untreated N2 worms, the reduction in brood size of wild-type worms by car-1 RNAi, strongly suggests that CAR-1 regulates reproduction by GLP-1-dependent and GLP-1-independent mechanisms. In addition, our observation that the knockdown of car-1 leads to nearly complete sterility of worms carrying weak kri-1 or daf-2 alleles or nonfunctional daf-16, suggests that CAR-1 is also a component of the reproduction-regulating mechanism downstream of the IIS.

The levels of car-1 modulate the transcriptional activity of the GLP-1 pathway

To directly test whether car-1 affects the transcriptional activity of the GLP-1 pathway, we asked how the knockdown of car-1 affects the expression levels of the glp-1-target genes sygl-1 and lst-1 (Shin et al., 2017). First, we used N2 and CF1903 to confirm the regulatory roles of GLP-1 on the transcription of these genes. Worms of both strains were either grown at 15 or 25°C (to inactivate GLP-1 in CF1903 animals) and the expression levels of sygl-1 and lst-1 were determined by qPCR. While an increase in the expression levels of both genes was observed in N2 worms upon exposure to 25°C (a significant increase for sygl-1 and a non-significant trend for lst-1), the inactivation of GLP-1 in CF1903 worms, by exposing them to 25°C during development, resulted in a significant reduction in the expression of both, sygl-1 and lst-1 (Figure 3—figure supplement 3, A and B). These results indicate that GLP-1 positively regulates the expression of these two genes.

We next utilized daf-2 (e1370) mutant animals to test how the knockdown of car-1 affects the expression of sygl-1 and lst-1 in these nematodes. The worms were grown from hatching on EV or on car-1 RNAi bacteria, harvested at day 1 of adulthood and gene expression levels were compared by qPCR. If CAR-1 is a negative regulator of GLP-1 that is negatively controlled by IIS-mediated SUMOylation, it is expected that CAR-1 is hyperactive in daf-2 mutant worms. Accordingly, the knockdown of car-1 by RNAi is predicted to activate GLP-1 and elevate the expression of sygl-1 and lst-1. Indeed, we observed significantly elevated levels of sygl-1 in e1370 animals (Figure 3I). A non-significant elevation in the expression of sygl-1 was also seen in car-1 RNAi-treated N2 worms (Figure 3—figure supplement 4). This lack of significance may be explained by the low activity of CAR-1 in these worms, due to its SUMOylation. These results indicate that CAR-1 negatively controls the activity of GLP-1 as a transcriptional regulator of sygl-1.

Unexpectedly, the knockdown of car-1 reduced the expression level of lst-1 in e1370 worms (Figure 3I) showing that CAR-1 could play opposing roles on the expression levels of GLP-1 target genes. This observation is consistent with the finding that in some cases, transcriptional co-factors affect the expression of some target genes but not of others (Volovik et al., 2014b), and show the complex regulatory relations between car-1 and glp-1.

Since the IIS controls the expression levels of some of its components (Alic et al., 2011), we asked whether GLP-1 controls the expression of car-1. Using qPCR, CF1903, and wild-type worms, we found that CF1903 animals that were developed at 25°C and thus, lack functional GLP-1, have reduced car-1 levels compared to their counterparts that were grown at 15°C. No significant difference in the expression of car-1 was observed in wild-type worms (Figure 3J). This reduction of approximately 65% in the levels of car-1, shows that GLP-1 positively regulates the expression of car-1, and raises the question of whether CAR-1 also plays roles in another feature of the GLP-1-controlled mechanism, the maintenance of proteostasis.

CAR-1 is involved in maintaining proteostasis

The known regulatory roles of glp-1 on proteostasis (Shemesh et al., 2013) has led us to examine whether CAR-1 also controls proteotoxicity. To address this, we utilized worms that express the Alzheimer's disease associated, human Aβ3-42 peptide (McColl et al., 2009), in their body wall muscles (strain CL2006, Aβ worms) (Link, 1995). The expression of Aβ causes progressive paralysis within the worm population, a phenotype that can be alleviated by the knockdown of daf-2 (Cohen et al., 2006). Eggs of Aβ worms were placed on plates seeded with daf-2 or car-1 RNAi bacteria, or left untreated (EV). Rates of paralysis were followed up until day 12 of adulthood. While the knockdown of daf-2 protected the worms from Aβ-mediated toxicity, animals that were treated with car-1 RNAi exhibited higher rate of paralysis than untreated worms (Figure 4A). Five independent repeats confirmed the significance of this phenotype (Figure 4B).

Figure 4 with 1 supplement see all
CAR-1 modulates proteostasis in C.elegans.

(A–B) Worms expressing Aβ3-42 in their body wall muscles were treated with car-1, daf-2 RNAi or left untreated (EV). While daf-2 RNAi protected from paralysis, car-1 RNAi significantly increased paralysis (N = 5). (C–D) The dilutions of car-1 RNAi (orange) or of daf-2 RNAi (red) bacteria with control bacteria do not significantly change the effects of these treatments on Aβ-mediated paralysis. Concurrent knockdown of daf-2 and car-1 by RNAi only partially protects Aβ worms from paralysis (C, blue). The increased rate of paralysis after car-1 RNAi treatment and the reduced paralysis after car-1 and daf-2 knockdown were significant compared to the level seen in untreated worms (EV) (N = 4, p<0.04) (D). (E–F) The expression of CAR-1 K185R in Aβ worms (strain EHC124) protects the animals from paralysis (E). Three independent experiments confirmed the significance of this observation (F) (p<0.004).

https://doi.org/10.7554/eLife.38635.016

We next examined whether CAR-1 is needed for daf-2 RNAi-conferred protection from proteotoxicity, by performing paralysis assays using Aβ worms that were grown on different mixtures of RNAi bacterial strains. First, we checked if the dilution of bacteria expressing either car-1 or daf-2 RNAi with control bacteria (EV) reduces the effects of these treatments on paralysis. Worms that were solely treated with car-1 RNAi and their counterparts that were fed with a mixture of car-1 RNAi and EV bacteria exhibited very similar rates of paralysis over time. Likewise, similar protection from paralysis was seen in worms that were exclusively fed with daf-2 RNAi bacteria and those which were fed with a mixture of daf-2 RNAi and EV bacteria (Figure 4, C and D). These results show that the dilution of car-1 and of daf-2 RNAi bacteria with another bacterial strain does not significantly changes the effects of these treatments on proteostasis.

We next examined whether the mixture of daf-2 and car-1 RNAi treatments prevents IIS reduction from promoting its full counter-proteotoxic effect, and found that concurrent knockdown of these genes resulted in an enhanced rate of paralysis compared to the rate observed in animals that were treated solely with daf-2 RNAi (Figure 4, C and D, blue, p<0.04). These findings imply that CAR-1 is needed for proteostasis-maintenance, downstream of the IIS however, the reduced paralysis rates that we observed in worms that were concomitantly treated with daf-2 and car-1 RNAi imply that the knockdown of daf-2 protects from proteotoxicity by additional, CAR-1-independent mechanisms. Yet, it is also possible that knocking down car-1, may inflict damage by an IIS-independent mechanism. According to this notion, the observed cumulative paralytic effect results from both, daf-2 RNAi-mediated protection and car-1 RNAi-promoted damage. To directly address this hypothesis, we expressed CAR-1 K185R in Aβ worms (strain EHC124). If SUMOylation on K185 reduces the activity of CAR-1, it was expected that the expression of the SUMOylation-resistant CAR-1 K185R mutant would protect the animals from Aβ toxicity. Indeed, we found that Aβ worms expressing CAR-1 K185R are largely (Figure 4E) and significantly (Figure 4F, from day 7 p<0.004) protected from proteotoxicity. The protective effect of CAR-1 K185R is DAF-16-dependent, since no protection was seen when EHC124 worms were treated with daf-16 RNAi (Figure 4—figure supplement 1A). These daf-16 RNAi-treated worm population exhibited similar rates of paralysis to these of CL2006 worms that were treated with the same RNAi (Cohen et al., 2006; Cohen et al., 2010). The observation that Aβ worms expressing CAR-1 K257R (strain EHC125) and Aβ worms (CL2006) exhibit indistinguishable rates of paralysis (Figure 4—figure supplement 1B) indicates that the counter-proteotoxic effect that is conferred by CAR-1, is suppressed by the SUMOylation of lysine 185. Finally, the more efficient protection from Aβ proteotoxicity that is conferred by daf-2 RNAi (Figure 4C), supports the idea that the protective mechanisms that are activated by IIS reduction and by CAR-1 K185R only partially overlap.

We further examined whether CAR-1 modulates proteotoxicity by employing worms that express fluorescently tagged, poly-glutamine stretches of 67 repeats in their neurons (polyQ67-YFP, strain AM716). Expanded glutamine stretches cause various human neurodegenerative maladies, including Huntington's disease (Bates, 2003), and lead to impaired neuronal activity in worms (Vilchez et al., 2012). AM716 worms were grown on EV, daf-2 or car-1 RNAi bacteria and placed in a drop of liquid at days 1 and 4 of adulthood. To measure proteotoxicity, the number of body bends per 30 s were counted (Volovik et al., 2014a). As anticipated, knocking down daf-2 protected from proteotoxicity at both day 1 (p<0.001) and day 4 (p<0.001) of adulthood. In contrast, car-1 RNAi treatment decreased the number of body bends at day 4 of adulthood (p<0.001) but not at day 1 (Figure 5—figure supplement 1). These results confirm the roles of CAR-1 as a modulator of age-onset proteotoxicity.

CAR-1 maintains proteostasis through the germline

Our results suggest that CAR-1 modulates proteostasis, at least partially, by negatively regulating glp-1 activity. To examine this possibility, we used worms that express polyQ35-YFP in their body wall muscles (strain AM140), and thus exhibit a motility defect (Morley et al., 2002), and harbor a temperature-sensitive glp-1 mutant (strain ABZ21). These animals are protected from polyQ-mediated paralysis when grown at 25°C (Shemesh et al., 2013). The rates of paralysis of polyQ35-YFP and of ABZ21 worms that were developed at 25°C and were either grown on EV, daf-2, or car-1 RNAi bacteria, were compared. While car-1 RNAi-treated polyQ35-YFP worms (that express a functional GLP-1) showed an increased rate of paralysis compared to untreated animals, daf-2 RNAi provided nearly complete protection from paralysis (Figure 5, A and B). In contrast, both untreated and car-1 RNAi-treated ABZ21 worms were protected from paralysis. This set of experiments shows that knocking down car-1 has no deleterious effect on proteostasis when glp-1 is inactive (p<0.05), indicating that CAR-1 controls proteostasis through GLP-1.

Figure 5 with 3 supplements see all
CAR-1 modulates proteostasis through the GLP-1 axis.

(A–B) Worms expressing polyQ35-YFP in their body wall muscles were crossed with CF1903 animals carrying a ts mutant glp-1. PolyQ35-YFP and polyQ35-YFP/glp-1 worms were exposed during development to 25˚C and either left untreated (EV) or treated with daf-2 or car-1 RNAi and subjected to paralysis assay. While daf-2 RNAi protected worms of both strains from paralysis, car-1 RNAi enhanced paralysis of polyQ35-YFP worms but not of polyQ35-YFP/glp-1 animals (A). Three independent experiments confirmed the significance of these phenotypes (B). (C–D) Aβ worms were either left untreated or fed with daf-2, car-1 or cgh-1 RNAi bacteria and rates of paralysis were followed. While daf-2 RNAi protected the worms from proteotoxicity, the knockdown of cgh-1 and car-1 enhanced paralysis compared to control animals (C). Three independent experiments confirmed the significance of this phenotype (D, p<0.035).

https://doi.org/10.7554/eLife.38635.018

Since the CAR-1-associated helicase CGH-1, which is expressed in meiotic germ cells, modulates lifespan (Figure 5—figure supplement 2, Supplementary file 5 and (Seo et al., 2015)), we tested whether this helicase also controls proteostasis. To address this, we let Aβ worms develop on EV bacteria or treated them with RNAi towards daf-2, car-1, or cgh-1, followed their rates of paralysis and found that the knockdown of cgh-1 as well as of car-1, significantly (p<0.035) increase the rates of paralysis compared to untreated worms (Figure 5, C and D). Similar results were obtained when worms that harbor a metastable perlecan (unc-52 (ts)), that misfolds and causes paralysis of worms that are grown at 25°C (strain HE250, [Shemesh et al., 2013]), were treated with either car-1 or cgh-1 RNAi (Figure 5—figure supplement 3, A and B). These results further support the view that CAR-1 and CGH-1 promote proteostasis by modulating germ cell activity, plausibly by negatively regulating GLP-1.

The role of CAR-1 in stress resistance

The roles of CAR-1 in lifespan determination and proteostasis maintenance have led us to ask whether it also controls stress resistance. To examine whether car-1 influences heat stress resistance we used two worm strains, N2 and CF512 (the exposure of CF512 worms to 25˚C during development does not activate the heat shock response [Volovik et al., 2012]). The worms were treated from hatching with car-1 RNAi or left untreated, and exposed at day 1 of adulthood to 35°C for 11 hr. We observed no significant effect of car-1 RNAi on resistance to heat (Figure 6, A and B). Similarly, car-1 RNAi did not abolish the elevated heat resistance of daf-2 mutant worms (Figure 6C). Surprisingly, the expression of CAR-1 K185R elevated the survival after heat shock compared to control worms (N2, p<0.04), however a trend but not a significant effect was observed when the natural car-1 was over-expressed (Figure 6D).

The roles of car-1 in stress resistance.

(A–C) The survival rates of heat-stressed CF512 worms (A) N2 animals (B) that were exposed to 35°C for 11 hr, and of daf-2(e1370) mutant worms (strain CB1370) (C) that were exposed for 19 hr to 35°C, were not significantly affected by car-1 RNAi. In contrast, daf-16 RNAi significantly reduced the survival rates of heat-stressed daf-2 mutant animals (p<0.001). (D) The expression of CAR-1 K185R (EHC118) significantly elevates survival compared to the wild-type animals (average survival of 67.39% and 43.51% respectively, N = 3, p<0.04). A trend but no significant effect was observed in worms that over-express the natural CAR-1 protein (EHC117). (E) car-1 RNAi-treated CF512 worms are more resistant to the pathogenic bacteria Pseudomonas aeruginosa than control worms (mean survival of 6.77 ± 0.21 and 5.88 ± 0.15 days, p<0.001). daf-2 RNAi prolonged and daf-16 RNAi reduced the survival of worms that were grown throughout adulthood with P. aeruginosa (mean survival rates of 11.96 ± 0.42 and 4.28 ± 0.11 days, respectively, p<0.001 for both treatments). (F) The over expression of wild-type CAR-1 (strain EHC117) or of CAR-1 K185R (strain EHC118) shortens survival of worms that were grown during adulthood on P. aeruginosa (mean survival of 6.05 ± 0.17, 5.43 ± 0.14 and 5.66 ± 0.17 days for N2, EHC117 (p<0.005) and EHC118 (p=0.1029), respectively). (G) The survival of CF512 worms that were exposed to sub-lethal dose of UV radiation was significantly increased by daf-2 and car-1 RNAi treatments compared to control animals (EV); (mean survival of 10.45 ± 0.22, 9.23 ± 0.22 and 7.97 ± 0.21 days, respectively, p<0.001). (H) In agreement, the over-expression of wild-type CAR-1 (strain EHC117) or of CAR-1 K185R (strain EHC118) shortens survival of animals that were exposed to UV radiation (mean survival of 12.78 ± 0.33, 8.89 ± 0.19 and 10.87 ± 0.27 days for N2, EHC117 (p<0.001) and EHC118 (p<0.001), respectively).

https://doi.org/10.7554/eLife.38635.022

We next used CF512 worms to analyze resistance to pathogenic bacteria. The worms were treated throughout development with daf-2, daf-16, or car-1 RNAi or left untreated (EV). At day 1 of adulthood, the nematodes were transferred onto plates seeded with the pathogenic bacteria Pseudomonas aeruginosa. As expected (Singh and Aballay, 2006), the knockdown of daf-2 extended, whereas daf-16 RNAi shortened the mean survival of the worms compared to control animals (mean survival of 11.96 ± 0.42, 4.28 ± 0.11 and 5.88 ± 0.15 days, respectively, p<0.001). Interestingly, car-1 RNAi had a small but significant protective effect from pathogenic bacteria (mean survival of 6.77 ± 0.21 days, p<0.001, Figure 6E, Supplementary file 6). To further test this effect, we conducted the reciprocal experiment asking whether the over-expression of the wild-type CAR-1 (strain EHC117) or the K185R CAR-1 (strain EHC118), shortens the survival rates of worms that were cultured on P. aeruginosa. Our results (Figure 6F) show a small but significant lifespan shortening effect that stemmed from the over-expression of the wild-type (p<0.01) and a non-significant trend in worms that express the K185R CAR-1. These results confirm that CAR-1 is deleterious when the worms are exposed to these pathogenic bacteria.

We also assessed whether CAR-1 is involved in protection from UV radiation by following the survival of CF512 worms that were treated with RNAi as above, and exposed to a sub-lethal dose of UV. The knockdown of daf-2 protected the worms from UV and the survival rate of the worms treated with car-1 RNAi was also increased compared to control animals (mean survival rates of 10.45 ± 0.22 (daf-2 RNAi), 9.23 ± 0.22 (car-1 RNAi) and 7.97 ± 0.21 (EV) days, respectively, p<0.001) (Figure 6G, Supplementary file 6). Similarly, the over-expression of the wild-type CAR-1 or of the CAR-1 K185R mutant significantly shortened the lifespans of worms that were exposed to UV radiation (mean survivals of 12.78 ± 0.33 (EV), 8.89 ± 0.19 (wt CAR-1) and 10.87 ± 0.27 (CAR-1 K185R) days, p<0.001) (Figure 6H and Supplementary file 6).

Together, these results show that CAR-1 plays minor roles in resistance to heat as well as in survival after exposure to pathogenic bacteria and UV radiation.

The data obtained in this work culminate to suggest the following model (Figure 7): besides regulating the cellular localization of its downstream transcription factors (7-I), the IIS also governs aging-associated functions by SUMOylating CAR-1 on lysine 185. This post-translational modification inhibits CAR-1’s function (7-II), thereby activating GLP-1 (7-III). Accordingly, the expression of the hyper-active, SUMOylation-resistant CAR-1 K185R, efficiently represses GLP-1 thereby, mimicking one aspect of IIS reduction, and promotes proteostasis. Interestingly, GLP-1 regulates the expression of car-1 to create a regulatory circuit (7-IV). Importantly, CAR-1 may also affects lifespan, proteostasis, stress resistance, and reproduction by a DAF-16-independent mechanism (7 V).

A model The IIS negatively regulates its downstream transcription factors DAF-16, SKN-1 and HSF-1 by inhibiting their entrance into the nucleus (I).

Thus, knocking down daf-2 hyper-activates these transcription factors resulting in longevity, proteostasis, stress resistance and modulated reproduction profile. The IIS also governs aging by SUMOylating CAR-1 on lysine 185 to mitigate its regulatory function (II), on GLP-1 (III). The RNA-helicase CGH-1 acts in cooperation with CAR-1 to regulate glp-1. IIS reduction hyper-activates CAR-1 by lowering the level of its SUMOylation on K185. This modulates the activity of GLP-1 to mediate longevity and enhance proteostasis in a DAF-16-dependent manner. Our results also indicate that GLP-1 positively controls the expression of car-1 (IV). CAR-1 also appears to affect lifespan, proteostasis, stress resistance and reproduction by a DAF-16-independent mechanism (V).

https://doi.org/10.7554/eLife.38635.023

Discussion

Several observations suggest a mechanistic link between the IIS and the germline. First, knocking down daf-2 extends lifespan solely during reproductive adulthood (Dillin et al., 2002). This finding is particularly interesting as other functions of the IIS are regulated at other stages of the nematode’s lifecycle; egg laying is controlled during development (Dillin et al., 2002) and proteostasis is governed by the IIS during early and late adulthood (Cohen et al., 2010). Moreover, the key roles of DAF-16 in the longevity phenotype that results from both, IIS reduction (Kenyon et al., 1993) and loss of germ cells (Berman and Kenyon, 2006), further support the idea that the IIS and germline are inter-related. Indeed, DAF-2 signaling was reported to govern oogenesis through the RAS-ERK pathway according to food availability (Lopez et al., 2013) and the phosphatase DAF-18/PTEN locally antagonizes IIS activity in the germline (Narbonne et al., 2015). In addition, germline signaling synergizes the longevity mechanisms downstream of the IIS and of the target of rapamycin (TOR) pathway (Chen et al., 2013). Nevertheless, whether the IIS and the germline coordinate aging and proteostasis in an orchestrated manner and if so, how this coordination is achieved, remained largely unexplored. Here, we show that IIS reduction lessens the SUMOylation of CAR-1, a germline protein that is involved in mRNA processing and negatively regulates the levels of glp-1 (Noble et al., 2008). Our data suggest that by SUMOylating CAR-1, the IIS suppresses its activity, thereby activating glp-1 to shorten lifespan and impair proteostasis. This notion is supported by several observations. First, the knockdown of car-1 shortens the lifespans of CF512 and of daf-2 mutant worms (Figure 2B and Figure 2—figure supplement 1, A–B), but not of animals that lack functional glp-1 or kri-1 (Figure 3, A and B). On the other hand, the knockdown of car-1 exhibits similar shortening effects on the lifespans of CF512 and daf-2 mutant animals, questioning the role of car-1 in the lifespan-controlling functions of the IIS. Nevertheless, the expression of CAR-1 K185R which lacks a putative SUMOylation site, extends lifespan (Figure 2, D and E) and protects model worms from proteotoxicity (Figure 4, E and F) (it is important to note that it is not clear whether this mutated CAR-1 restores the natural functions of the protein or activate another lifespan extending mechanism). In addition, the simultaneous knockdown of daf-2 and car-1 prevents IIS reduction from conferring its full protective effect on Aβ worms (Figure 4, C and D). These results argue that CAR-1 is needed for IIS reduction to fully protect the worm from proteotoxicity. Finally, using daf-2 mutant worms and qPCR we found that the knockdown of car-1 by RNAi elevates the expression of sygl-1 and lessens the levels of lst-1 (Figure 3I), which are regulated by GLP-1 (Figure 3—figure supplement 3, A and B). These results indicate that CAR-1 is a co-regulator of GLP-1 activity, however, while in the case of sygl-1 the knockdown of car-1 activates GLP-1-mediated transcription, car-1 RNAi treatment reduces the expression of lst-1. These results show that CAR-1 can function as either negative or positive regulator of GLP-1 and show that the relations between CAR-1 and GLP-1 require further elucidation.

CAR-1 and SUMOylation coordinate signaling of the IIS and the germline but probably affect lifespan through an additional mechanism

Post-translational modifications are known to have various biological functions, including the regulation of aging. Phosphorylation regulates the activities of DAF-16, SKN-1, and HSF-1 downstream of the IIS (Lee et al., 2001; Tullet et al., 2008; Chiang et al., 2012) and SUMOylation controls the localization of the IGF-1 receptor and its signaling activity in mammalian tissues (Sehat et al., 2010). SUMOylation is also critical for aging-associated modulation of mevalonate biosynthesis (Sapir et al., 2014), a metabolite that has been implicated in the development of clinical conditions (Mokarram et al., 2017). In this study, we unveiled a novel role of SUMOylation in the regulation of aging, serving as a functional switch of CAR-1, which is governed by the IIS. This raises the question of how the IIS controls the SUMOylation state of CAR-1. One possible explanation stems from the correlation between AKT function, the stability of SUMO, and the SUMO-conjugating enzyme UBC-9 in mammals (Lin et al., 2016). According to this theme, reduced IIS lowers AKT activity, resulting in SMO-1 destabilization and in reduction of UBC-9 activity. This cascade of events may lower the rate of global protein SUMOylation. This possibility appears less likely as our results (Figure 1A) indicate that while daf-2 RNAi leads to lower SUMOylation of some proteins others proteins exhibit increased SUMOylation upon IIS reduction. Alternatively, the expression of specific genes that encode for proteins involved in CAR-1 SUMOylation may be positively regulated by the IIS, which lowers the expression of these genes and reduces the rate of CAR-1 SUMOylation. Future research is needed to clarify this issue.

Although SUMOylation appears to be a pivotal post-translational modification that influences aging, a simultaneous knockdown of car-1 and daf-2 only partially protects worms from proteotoxicity and only partially shortens lifespans of daf-2 mutant worms. On one hand, these observations suggest that IIS reduction also protects from proteotoxicity and extends lifespan by additional, CAR-1-independent mechanisms. However, on the other hand, the knockdown of daf-16 in CAR-1 K185R-expressing worms did not shorten lifespan below those of wild-type nematodes (Figure 2G), suggesting that CAR-1 also governs lifespan by an additional, DAF-16-independent mechanism (the lack of functional daf-16 shorten the lifespan of N2 worms by approximately 30% [Kenyon et al., 1993]). Nevertheless, the knockdown of car-1 has not further shortened the lifespan of daf-16 mutant animals (Figure 2C). The lack of additive effect may be a result of the very short lifespan of daf-16 mutant worms, which does not allow the knockdown of car-1 to further shorten lifespan or perhaps by the limited efficiency of RNAi-mediated knockdown of car-1.

Opposing effects of car-1 on stress resistance and proteostasis

An additional interesting aspect of this study is the differential effects of car-1 on distinct environmental insults. While the knockdown of car-1 has no effect on heat stress resistance (Figure 6, A-C), the expression of CAR-1 K185R, mildly but significantly elevates the survival of heat-stressed worms (Figure 6D). In contrast, the knockdown of car-1 has a small but reproducible protective effect on resistance to UV radiation (Figure 6G) and to pathogenic bacteria (Figure 6E). In agreement, the over expression of either the wild-type CAR-1 or the mutated CAR-1 K185R is deleterious to worms that were exposed to these insults (Figure 6, F and H). The observed protection from UV radiation, conferred by car-1 RNAi, is consistent with a previous report that CAR-1 and CGH-1 negatively regulate DNA-damage-mediated apoptosis (Tomazella et al., 2012). Nevertheless, despite its protective effect when the worm is exposed to stress conditions, the knockdown of car-1 shortens lifespan (Figure 2B). These results support the theme that the ability to resist stresses such as heat (Maman et al., 2013; Volovik et al., 2014b) and oxidation (Van Raamsdonk and Hekimi, 2012) are not necessarily coupled with lifespan determination. They also coincide with the reports that IIS-regulated factors may be involved in the regulation of certain stress resistance mechanisms but not of others. For instance, the transcription factor SMK-1 is needed for the worm to resist UV radiation and pathogenic bacteria but is dispensable for coping with heat (Wolff et al., 2006). Similarly, we recently reported that the knockdown of caveolin-1 extends lifespan and provides partial protection from pathogenic bacteria, but has no role in heat stress resistance (Roitenberg et al., 2018).

The car-1 RNAi-mediated protection from certain stresses appears to be contradictive to the observation that knocking down this gene exposes the animal to proteotoxicity. However, it has been already shown that abolishing the nematode’s ability to resist heat by knocking down neuronal components, provides the worm with partial protection from proteotoxicity (Prahlad and Morimoto, 2011; Volovik et al., 2014b). Our findings show that manipulating the activity of a germline protein can also confer opposing effects on stress resistance and proteostasis, and raise the question of how CAR-1 promotes these opposing effects. One possible explanation suggests that CAR-1 may differentially affect the expression levels of different GLP-1-controlled genes. Such differential effects of transcriptional co-regulators have been reported. For instance, the knockdown of the DAF-16 transcriptional co-factor nhl-1, lowers the expression level of sod-3 and of sip-1, but has no effect on the expression of mtl-1, which are all known target genes of DAF-16 (Volovik et al., 2014b). The opposing effects of car-1 RNAi on the expression levels of sygl-1 and lst-1 propose a similar mechanism of differential effects on the expression of distinct genes. How this mechanism functions and what cellular components are involved in the mediation of proteostasis by the SUMOylation-resistant CAR-1, are questions that require further elucidation.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain
background
(Caenorhabditis elegans)
N2Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/N2
Strain, strain
background
(Caenorhabditis elegans)
CF512Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/CF512
Strain, strain
background
(Caenorhabditis elegans)
CF1903Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/CF1903
Strain
(Caenorhabditis elegans)
CB1370(e1370)Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/CB1370
Strain, strain
background
(Caenorhabditis elegans)
e1368Dr. Andrew Dillin,
University of California,
Berkeley, USA
Strain, strain
background
(Caenorhabditis elegans)
CF1038Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/CF1038
Strain, strain
background
(Caenorhabditis elegans)
CF2052Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/CF2052
Strain, strain
background
(Caenorhabditis elegans)
AA86Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/AA86
Strain, strain
background
(Caenorhabditis elegans)
CF2531Caenorhabditis
Genetic Center (CGC)
Strain, strain
background
(Caenorhabditis elegans)
ABZ21otherDr. Anat Ben-Zvi,
Ben Gurion University,
Israel
Strain, strain
background
(Caenorhabditis elegans)
CL2006Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/CL2006
Strain, strain
background
(Caenorhabditis elegans)
WH377Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/WH377
Strain, strain
background
(Caenorhabditis elegans)
WH346Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/WH346
Strain, strain
background
(Caenorhabditis elegans)
NX25otherDr. Limor Broday,
Tel-aviv University,
Israel
Strain, strain
background
(Caenorhabditis elegans)
AM140Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/AM140
Strain, strain
background
(Caenorhabditis elegans)
AM716otherDr. Richard I Morimoto,
Northwestern University,
IL, USA
Strain, strain
background
(Caenorhabditis elegans)
HE250Caenorhabditis
Genetic Center (CGC)
https://cgc.umn.edu/strain/HE250
Strain, strain
background
(Caenorhabditis elegans)
EHC117This paperN/ApUC18-car-1p::2HAcar-1
injected to N2 worms
Strain, strain
background
(Caenorhabditis elegans)
EHC118This paperN/ApUC18-car-1p::2 HA car-1
K185R injected to N2
worms
Strain, strain
background
(Caenorhabditis elegans)
EHC121This paperN/ApUC18-car-1p::2 HA car-1
K257R injected to N2
worms
Strain, strain
background
(Caenorhabditis elegans)
EHC124This paperN/ApUC18-car-1p::2 HA car-1
K185R injected to CL2006
worms
Strain, strain
background
(Caenorhabditis elegans)
EHC125This paperN/ApUC18-car-1p::2 HA car-1
K257R injected to CL2006
worms
Genetic reagentempty vector (EV) (pAD12)DOI: 10.1126/
science.1074240
ID_addgene: 34832
Genetic reagentdaf-2 RNAi (pAD48)DOI: 10.1126/
science.1074240
ID_addgene: 34834
Genetic reagentdaf-16 RNAi (pAD43)DOI: 10.1126/
science.1074240
ID_addgene: 34833
Genetic reagentcar-1 RNAividal RNAi libraryProduct code:
3320_Cel_ORF_RNAi
Genetic reagentcgh-1 RNAividal RNAi libraryProduct code:
3320_Cel_ORF_RNAi
Antibodyanti-GFP antibody
(Rabbit monoclonal)
Cell Signalingca#2956(1:1000)
Antibodyanti-SUMO-1
antibody
(Rabbit polyclonal)
Milliporeca#09–409(1:2000)
Antibodyanti-HA.11
epitope tag
(Mouse monoclonal)
BioLegendca#901501(1:2000)
Antibodyanti-FLAG M2,
Clone M2
(Mouse monoclonal)
Sigmaca#F1804(1:1000)
Antibodyanti-actin antibody
(Mouse monoclonal)
Sigmaca#A5441(1:5000)
Commercial
assay or kit
HisPurTM Ni-NTA
Resin
Thermo Fisher Scientificca#88221
Commercial
assay or kit
Red ANTI-FLAG
M2 Affinity Gel
Sigmaca#F2426
Commercial
assay or kit
GFP-Trap_AChromotekcode#gta-100
Commercial
assay or kit
Pierce Crosslink
Immunoprecipitation
Kit
Thermo Fisher Scientificca#26147
Commercial
assay or kit
NucleoSpin RNA kitMACHEREY-NAGELca#740955.50
Commercial
assay or kit
iScript cDNA
Synthesis Kit
Bioradca#170–8891
Commercial
assay or kit
EvaGreen supermixBioradca#172–5204
Commercial
assay or kit
BCA kitThermo Fisher Scientificca#23225
Sequence-based
reagent
qPCR act-1
forward primer (5'-->3')
This paper, IDTN/AGAG CAC GGT ATC GTC ACC AA
Sequence-based
reagent
qPCR act-1
reverse primer (5'-->3')
This paper, IDTN/ATGT GAT GCC AGA TCT TCT CCA T
Sequence-based
reagent
qPCR cdc-42
forward primer (5'-->3')
This paper, IDTN/ACTG CTG GAC AGG AAG ATT ACG
Sequence-based
reagent
qPCR cdc-42
reverse primer (5'-->3')
This paper, IDTN/ACTC GGA CAT TCT CGA ATG AAG
Sequence-based
reagent
qPCR car-1
forward primer (5'-->3')
This paper, IDTN/AAGG AGA GAG AAA CGA ATC AG
Sequence-based
reagent
qPCR car-1
reverse primer (5'-->3')
This paper, IDTN/ATTG TAA CCT CCA TAT CCG C
Sequence-based
reagent
qPCR sygl-1
forward primer (5'-->3')
This paper, IDTN/AAGG CAA AGG AAT CAA GC
Sequence-based
reagent
qPCR sygl-1
reverse primer (5'-->3')
This paper, IDTN/ATTA CGA TAC TTC AGG TTG G
Sequence-based
reagent
qPCR lst-1
forward primer (5'-->3')
This paper, IDTN/ACCA CGC TTG TTA TTT TCG
Sequence-based
reagent
qPCR lst-l-1
reverse primer (5'-->3')
This paper, IDTN/AAGT TGT TTC TTC TTG GAG G
Software, algorithmMass SpectrometryThe PRIDE PRoteomics
IDEntifications (PRIDE)
database
ID_pride archive: PXD010011
Software, algorithmComputational
tool GPS-SUMO
DOI: 10.1093/nar/gku383
Software, algorithmImageJNIHhttps://imagej.nih.gov/ij/

Worm and RNAi strains

N2 (wild-type, Bristol), CB1370 (daf-2(e1370) mutant worms), CL2006 (unc-54p::human Aβ3-42), CF512 (fer-15(b26)II; fem-1(hc17)IV), CF1903 (glp-1(e2141) III.), AM140 (Punc54::Q35::YFP), HE250 (unc-52(e669su250) II.), WH377 (car-1(tm1753) I/hT2), WH346 (unc-119(ed3) III. ojIs34 [GFP::car-1+unc-119(+)], CF2052 kri-1(ok1251) (I), AA86 daf-12(rh61rh411) X., CF2531 daf-9(rh50) X., CF1038 (daf-16(mu86)I) were obtained from the Caenorhabditis Genetic Center (CGC, Minneapolis, MN). Worms that over-express the K185R or K257R mutated car-1 (strains EHC118 and EHC121 respectively) or WT CAR-1 tagged to 2xHA tag (strain EHC117) were generated by injecting a plasmid that carries the gene downstream of the natural car-1 promoter region (1074 bp upstream of the ORF) into N2 worms. rol-6 driven by the unc-54 promoter or gfp driven by the elt-2 promoter were used as selection markers. Worms expressing in their body wall muscle and CAR-1 K185R or CAR-1 K257R were generated by injecting the same plasmids into CL2006 worms (strains EHC124 and EHC125, respectively). AM716 (rmIs284[pF25B3.3::Q67::YFP]) worms were obtained from Dr. Richard I Morimoto (Northwestern, IL). ABZ21 animals (Punc54::Q35::YFPxglp-1 (CF1903)) were a gift of Dr. Anat Ben-Zvi (Ben-Gurion, Israel). NX25 (smo-1 (ok359);tvEx25[psmo-1::His-FLAG-SMO-1; rol-6]) were obtained from Dr. Limor Broday (TAU, Israel). CF512 (fer-15(b26)II; fem-1 (hc17)IV), CF1903 (glp-1(e2144) III.) nematodes are heat-sensitive sterile and were thus, grown at 15°C. To avoid egg lying, these worms were developed at 25°C and transferred at day 1°C to 20°C until harvested. To achieve sterility, ABZ21 worms were grown at 25°C until harvesting. Other strains were synchronized and grown on the indicated RNAi bacteria at 20°C until day 1 of adulthood. To reduce gene expression, we used bacterial strains expressing dsRNA: empty vector (pAD12), daf-2 (pAD48), daf-16 (pAD43). car-1 and cgh-1 dsRNA-expressing bacteria were obtained from the Vidal RNAi library. RNAi bacteria were grown at 37°C in LB with 100 μg/ml ampicillin and then seeded on NG-ampicillin plates with the addition of 100 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG ~ 1 mM final concentration).

Purification of SUMOylated proteins

To isolate SUMOylated proteins, 300,000 NX25 worms were grown on EV or daf-2 RNAi bacteria until day 1 of adulthood, collected and froze in liquid nitrogen (Figure 1, B, C and D). The worms were then homogenized and equilibration buffer (1XPBS, 8M UREA, 5 mM NEM and protease inhibitors) was added prior to centrifugation (10 min, 9,391 g). Protein concentrations were measured and equalized by Bradford reagent. First, a His-tag purification using HisPur Ni-NTA Resin (Thermo Scientific, #88221) was performed. Lysates were incubated with the resin for 50 min at RT and washed (1XPBS (pH7.4), 8M UREA, 250 mM Imidazole). SUMOylated proteins were eluted using buffer 1 (1XPBS, 2M UREA, 250 mM Imidazole) followed by elution buffer 2 (1XPBS, 1M UREA, 250 mM Imidazole). Aliquots of the samples were blotted by WB for validation. The remaining eluted samples were used for Flag-tag purification with the Red ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich, #F2426). Samples were diluted with RIPA buffer (50 mM Tris HCl pH7.5, 150 mM NaCl, 5 mM EDTA, 1% TritionX-100, 0.1% SDS, 1X Protease Inhibitor (Calbiochem set III #539134, 1 mM BetaME)) and incubated over night at 4°C with the Anti-Flag beads. The beads were washed with RIPA buffer followed by elution (100 mM Glycine pH3.5, 150 mM NaCl).

To detect the SUMOylation state of CAR-1 (Figure 1, C and D), WH346 worms were treated as described above. The homogenized worms were dissolved in RIPA buffer and the GFP immunoprecipitation was performed using GFP-Trap_A (#gta-100, Chromotek, Germany) according to the manufacturer’s instructions. The beads were incubated with the lysates over night at 4°C and the trapped proteins were eluted and analyzed by WB.

To test the SUMOylation state of the CAR-1 (WT), CAR-1 K185R and CAR-1 K257R mutants (strains EHC117, EHC118 and EHC121, respectively) 120,000 worms were harvested as described above. CAR-1 was purified by performing an immunoprecipitation using the anti-HA.11 Epitope Tag antibody and the Pierce Crosslink Immunoprecipitation Kit. The crosslinked beads and the worm lysates were incubated over night at 4°C, bound proteins were eluted and analyzed by WB.

RNA isolation and quantitative real-time PCR

Total RNA was isolated from synchronized worm populations using QIAzol reagent (QIAGEN, Hilden Germany #79306) and NucleoSpin RNA kit (MACHEREY-NAGEL, #740955.50). cDNA was synthesized using iScript cDNA Synthesis Kit (Biorad, #170–8891). Quantitative real-time PCR reactions were performed with EvaGreen supermix (Biorad, #172–5204). Quantities were normalized to levels of act-1 and of cdc-42 cDNA.

SDS-PAGE and western blot analysis

To blot SUMOylated proteins (Figure 1A), N2, CF512, and CF1903 worms that were grown at 15°C, were bleached to obtain synchronized eggs. The eggs were placed on plates that were seeded with control bacteria (EV) or daf-2 RNAi bacteria and incubated for 48 hr at 25°C (to sterilize the CF512 and CF1903 worms). The worms were transferred thereafter to 20°C for additional 24 hr. For the experiment displayed at Figure 1E, the worms were hatched and grown at 20°C (strain WH346). At day 1 of adulthood, the worms were washed twice with M9, and homogenized using a bullet grinder (full speed, 10 s, three times). The worm homogenates were spun for 3 min at 850 g (3000 rpm in a benchtop Qiagen centrifuge) to sediment debris. The post debris supernatants were collected, protein amounts were measured by a BCA kit (Thermo Fisher #23225), supplemented with loading buffer (10% glycerol, 125 mM Tris base, 1% SDS) and heated at 95°C for 10 min. For each treatment, equal protein quantities were loaded and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a PVDF membrane (Millipore, Billerica MA) and probed with the indicated antibody: GFP antibody (Cell Signaling, Danvers, MA cat #2956), anti-SUMO-1 antibody (Millipore, #09–409), anti-HA.11 antibody (BioLegend, San Diego, CA, #901501) or anti-actin antibody (Simga, #A5441). HRP-conjugated secondary antibody and a luminescent image analyzer (ChemiDoc XRS + BioRad) were used to detect protein signals.

Lifespan and paralysis assays

Synchronized worm eggs were placed on master NG-Ampicillin plates seeded with the indicated RNAi bacterial strain and supplemented with 100 mM IPTG. The eggs were incubated at 20°C until transferred onto small NG- Ampicillin plates, 12 animals per plate (CF1903 and CF512 were incubated throughout development at 25°C, to induce sterility). Adult worms were transferred onto freshly seeded plates every 3 days. Worms that failed to move their noses when tapped twice with a platinum wire were scored as dead. Dead worms were scored daily. Lifespan analyses were conducted at 20°C.

Heat, UV and innate immunity stress assays

For all stress assays synchronized eggs were placed on NG plates seeded with the RNAi bacteria (as indicated). For heat-stress assays, 120 day one adult animals were transferred onto fresh plates (12 animals per plate) spotted with RNAi bacteria and exposed to 35°C (N2, EHC117, EHC118, EHC121 and CF512 worms for 11 hr and CB1370 worms for 19 hr) and survival rates were recorded. To assess resistance to ultra-violet (UV) radiation, day 1 adult CF512 worms were exposed to sub-lethal UV dose (800 j/cm2). Survival rates were scored daily. To evaluate resistance to pathogenic bacteria (innate immunity), eggs of CF512 worms were placed on plates seeded with the indicated RNAi bacteria, grown to day 1 of adulthood, and transferred onto plates seeded with P. aeruginosa. Survival rates were followed daily.

Germ cells number quantification

20–24 hr post L4 worms were dissected in egg buffer (0.025 mM Hepes pH 7.4, 118 mM NaCl, 48 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.1% Tween 20), transferred to superfrost plus slide and freeze cracked. Gonads were fixed in −20°C MeOH for 1 min, and 4% PFA for 30 min. Slides were washed twice in PBST (PBS with 0.1% Tween 20) for 5 min, and incubated in PBST with 0.5 µg/ml DAPI for 10’. Finally, the slides were washed for 10 min in PBST, again in 10 mM Tris 7.5% and 0.1% Tween 20 for 5 min, and sealed with Vectashield (Vector Laboratories, # H-1000). Imaging was done with Olympus IX81 inverted fluorescent microscope, and 3D images we collected and deconvolved with AutoQuant X3. Germ cells nuclei were manually counted from the mitotic tip to the end of pachytene.

Quantitative analysis of germ-cell apoptosis

Germ cell corpses were scored in 20 hr post-L4 adult hermaphrodites using acridine orange (AO), as described in Melchior (2000). A minimum of 23 gonads were scored for each genotype. Statistical analyses were performed using the two-tailed Mann–Whitney test (95% C.I.)

Statistical analyses

Statistical significance of the results was performed using the Student T-test, two-tailed distribution and two-sample equal variance. The analyses were done using at least three independent biological repeats of each experiment, as indicated. Statistical information of lifespan experiments is presented in Supplementary files 26 as mean LS ± SEM.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
    SUMO modification of Akt regulates global SUMOylation and substrate SUMOylation specificity through Akt phosphorylation of Ubc9 and SUMO1
    1. CH Lin
    2. SY Liu
    3. EH Lee
    (2016)
    Oncogene, 35, 10.1038/onc.2015.115, 25867063.
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
    SUMO--nonclassical ubiquitin
    1. F Melchior
    (2000)
    Annual Review of Cell and Developmental Biology 16:591–626.
    https://doi.org/10.1146/annurev.cellbio.16.1.591
  30. 30
  31. 31
  32. 32
    A genetic pathway conferring life extension and resistance to UV stress in Caenorhabditis elegans
    1. S Murakami
    2. TE Johnson
    (1996)
    Genetics 143:1207–1218.
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54

Decision letter

  1. Inna Slutsky
    Reviewing Editor; Tel Aviv University, Israel
  2. K VijayRaghavan
    Senior Editor; National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "The Insulin/IGF Signaling Cascade Modulates SUMOylation to Regulate Aging and Proteostasis in C. elegans" for consideration by eLife. Your article has been reviewed by K VijayRaghavan as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Veena Prahlad (Reviewer #1); Andrew Dillin (Reviewer #2). A further reviewer remains anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The manuscript "The Insulin/IGF Signaling Cascade Modulates SUMOylation to Regulate Aging and Proteostasis in C. elegans" by Moll et al., presents a very detailed description of one of the molecular mechanisms downstream of IIS-mediated proteotoxicity and lifespan extension. The Cohen group have done an extraordinary amount of work highlighting the molecular mechanism of CAR-1, which is described as a protein downstream of the IIS cascade and upstream of GLP-1 in regulation of this highly studied, but still poorly understood, paradigm of lifespan extension. The experimental methods are elegant and there is a lot of data presented, but the style of the data presentation and writing invite a lot of questions and potential experiments, all of which are listed below. Most of the comments can be addressed textually, and many of the experiments are perhaps not necessary should the writing be made more concise and informative. Again, a majority of the experimental suggestions and questions came up due to the presentation style, which had a major lack of descriptive reasoning behind the experiments, the methods by which the experiments were performed, and conclusions drawn from the data. Either the text needs to be cleaned up considerably to remove the confusions drawn out below, or perhaps the experiments suggested need to actually be performed.

Essential revisions:

1) Is the increased sumoylation found in CF512 (Figure 1A) due to differences that result in growth at 25 °C? Were the N2 worms also grown at 25 °C for this assay to control for this difference? The Materials and methods do not make it clear – it looks like it was performed at different temperatures. Several studies have shown that 25 °C can cause dramatic differences in proteostasis, so this needs to be re-evaluated or made clearer in the text. The authors are also urged to check other reproduction-deficient mutants, such as glp-1 and glp-4, or via chemical sterilization, such as FUDR, to see if these major differences are due to the lack of germline, or specific to CF512. If it is specific to CF512 and the reasoning behind why there are such major differences in sumoylation cannot be rectified, this begs the question of whether this is an appropriate model to use. Another question that comes up is whether CAR-1 protein itself is differentially sumoylated in CF512 background.

2) Figure 1C-D are confusing. How do we know that 1C is showing SUMOylated-GFP-CAR-1 and not other protein that gets pulled down with an anti-SUMO antibody? Is this actually a double-IP where it is pulled down with SUMO antibodies, then GFP antibodies? This is especially confusing if probing with GFP-antibodies cannot visualize the larger, SUMOylated versions of the proteins – does SUMOylation prevent the GFP antibody from binding? Please clear up the experimental details and the conclusions drawn from the data.

3) Since car-1 RNAi/KO decreases lifespan of wild-type and daf-2 mutants to a similar extent, is this just an additive phenotype? (Figure 2A-B)

4) A single copy rescue experiment of the CAR-1 K185R should be performed in the car-1 KO to ensure that this CAR-1 still maintains the normal (albeit hyperactive) functions of the protein (e.g. rescues sterility) and isn't a completely mutant form of the protein with brand new functions.

5) The lifespan decrease of car-1 RNAi on daf-9 mutants seems much milder than in wild-type or other mutant worms. Does this suggest daf-9 and car-1 is partially overlapping/interdependent? (Compare Figure 3—figure supplement 1A and B and Figure 2—figure supplement 1A).

6) If CAR-1 drives lifespan extension by inhibiting GLP-1, why does a CAR-1 (WT) overexpression, which has a dramatic effect on number of germ cells have no effect on lifespan, and the very modest change in germ cells of CAR-1 (WT) overexpression versus CAR-1 K185R overexpression (this is a very modest decline compared to the dramatic decline versus wild-type) have such a profound effect on lifespan? (Compare Figure 3C-D to Figure 2—figure supplement 4A/C).

7) What was the purpose of measuring the effect of car-1 RNAi on egg laying of kri-1 mutant worms? It seems unsurprising that car-1 RNAi and kri-1 mutants, both of which decrease egg laying, have an additive phenotype. Moreover, the initial car-1 mutant experiments expressed that ¬car-1 mutant animals are sterile, so why would it be surprising that car-1 RNAi decreases egg-laying? Perhaps it would be less confusing if this section is removed, as it doesn't seem to add any additional benefits to the story. Alternatively, it can be moved into supplements as a control for the overexpression data (Figure 3E-F).

8) Is it surprising that EHC117 worms have no significant decrease in egg-laying when they have such a dramatic decline in germ cells? This should be textually addressed. This sentence can be added in the text where the differences in car-1 RNAi gonad versus egg-laying is compared – subsection “The roles of CAR-1 in GLP-1-mediated functions” (Compare Figure 3C-D to Figure 3I).

9) How do we know that the decreased egg-laying of CB1370 and car-1 RNAi are not just additive since both decrease egg-laying in otherwise WT animals? (Figure 3—figure supplement 2Figure)

10) Can you show that car-1 RNAi on its own does not affect sygl-1 activity? Why is this only done in the daf-2 mutant and not in WT animals? The authors should either include these controls, or explicitly state in the text why these experiments are not significant. The reviewers think that showing that overexpression of CAR-1 (WT or K185R) can decrease sygl-1 transcripts shows more direct evidence that CAR-1 negatively regulates transcriptional activity of GLP-1 (Figure 3J). Another suggestion is to stain for GLP-1 instead of measuring sygl-1 levels. K185R mutants should have decreased GLP-1 levels and car-1 RNAi should have decreased GLP-1 levels based on the model.

11) If car-1's effects are through GLP-1, car-1 RNAi should lead to an increase in GLP-1 protein. This should yield glp-1 gain of function phenotypes (e.g. persistent mitosis, increase in mitotic cells and fewer meiotic cells). Conversely, CAR-1 K185R should yield larger meiotic germ line cells causing a premature entry into meiosis (e.g. Pepper et al., 2003; Maine and Kimble, 1993). The absence of the phenotype in car-1 RNAi animals could suggests alternative mechanisms.

12) To further support the hypothesis that CAR-1 K185R that cannot be SUMOylated acts to increase longevity by decreasing GLP-1 would be to examine whether animals harboring CAR-1 K185R suppress glp-1 gain of function phenotypes (e.g. Pepper et al., 2003; Maine and Kimble, 1993), by sequestering glp-1 mRNA and decreasing its translation into protein.

13) Is the daf-16 CAR-1 K185 overexpression similar to daf-16 RNAi? This seems like an extremely important control to show that the two are not simply an additive effect if daf-16 RNAi has higher proteotoxicity. (Figure 4—figure supplement 1A). It would also be stronger argument that CAR-1 activity is falling within the daf-2 cascade if daf-2 RNAi/mutants do not have an additive effect with CAR-1 K185.

14) Why are all the paralysis experiments done with car-1 RNAi, which is an indirect way to show that car-1 is affecting proteoxicity through GLP-1? All of these experiments should be done with the CAR-1 K185 to show directly that hyperactive CAR-1 can actually protect against proteoxicity. Minimally, the text should explain why these experiments are done with RNAi to increase proteotoxicity (Figure 5 and Figure 5—figure supplement 1, Figure 5—figure supplement 3A-B).

15) For Figure 6A-C, why was CF512 worms used? Were these grown at 15, 20, or 25 C? Growth at 25 C can mildly stress worms and can affect HSR or thermotolerance. The actual growth conditions should be specified, and experiments should be repeated if worms were developed at 25 C. Moreover, the thermotolerance and Pseudomonas data seems to suggest that hsf-1 may be activated. It is recommended that the role of HSF-1 in these paradigms be tested.

16) For Figure 6D, if car-1 RNAi normally decreases lifespan, then the mild increase in lifespan is actually pretty substantial if normalized for this fact. Maybe something on this should be added in the text.

17) The experiments in Figure 6 really spark the curiosity of whether all of these phenotypes are truly due to car-1 or through non-specific pleotropic effects of car-1 RNAi. All of these experiments should be performed with the CAR-1 K185 strain to directly test whether these phenotypes are truly due to CAR-1. Moreover, would CAR-1 overexpression make worms more sensitive to pathogenic bacteria or UV (opposite of car-1 RNAi data), despite extending lifespan? Seems counter to the entire argument of the paper. These issues need to be addressed.

18) The article conflates fundamentally distinct concepts/roles, and in two cases that impact the overall conclusions. One, the role of Car1 in reproduction vs its role in longevity are considered interchangeably. The authors use effects on fertility to draw conclusions about longevity functions and vice versa and it is incorrect and confusing. Two, the function of GLP1 protein in germline development is substituted with the glp1 temperature sensitive mutant phenotype that is a surrogate for longevity brought on by germline loss. They use some results to directly implicate GLP1 protein function (e.g. sygl-1 expression) and others to deduce roles in germline eliminated longevity pathway (kri1, daf12 tests). This is also misleading and makes the article difficult to follow.

Car1's genetic interaction with Glp1 protein is published so if the premise is that it acts with/through Glp1 to modulate germline events that impact longevity, the experiments conducted here (Figure 3) do not test it or offer evidence for or against it. That requires examining the effect of CAR1 modulation on GLP1 (and the very well-characterized Notch pathway for translation control in the germline) within the germline and the fertility and lifespan consequences thereof. Similarly, if the hypothesis is that the Car1-Glp1 relationship operates in the context of germline-less longevity paradigm, the evidence provided is insufficient to infer any conclusions. The daf16/kri1/daf12/daf36 pathway is activated upon germline loss and solely impacts somatic Daf16. The relationships of these proteins within the germline are different (indeed, even in germline development these relationships change based on developmental state and/or environmental conditions). The effects of Car1 manipulations on reproduction of kri1 or daf12 mutants do not reveal their relationship in determining longevity, especially of sterile glp1 mutants. The germline-less longevity pathway has been shown by numerous studies to be genetically parallel to IIS and the authors overlook this concept while concluding that Car1 integrates 'aging controlling functions of IIS and germline'.

19) An alternative hypothesis that has not been addressed and would be consistent with much of the data such as the observed effects of overexpressing both wild-type CAR-1 and CAR-1 K185R on the number of germ line nuclei, is the role of CAR-1 on germline apoptosis (Boag et al., 2005). Programmed cell death mutations in C. elegans affect stress resistance, albeit reports show that the mechanisms are rather complex (e.g. Judy et al., 2013). It would strengthen the manuscript if this were directly examined, but this point needs to be addressed, at the very least in the Discussion section.

https://doi.org/10.7554/eLife.38635.034

Author response

Essential revisions:

1) Is the increased sumoylation found in CF512 (Figure 1A) due to differences that result in growth at 25 °C? Were the N2 worms also grown at 25 °C for this assay to control for this difference? The Materials and methods do not make it clear – it looks like it was performed at different temperatures. Several studies have shown that 25 °C can cause dramatic differences in proteostasis, so this needs to be re-evaluated or made clearer in the text. The authors are also urged to check other reproduction-deficient mutants, such as glp-1 and glp-4, or via chemical sterilization, such as FUDR, to see if these major differences are due to the lack of germline, or specific to CF512. If it is specific to CF512 and the reasoning behind why there are such major differences in sumoylation cannot be rectified, this begs the question of whether this is an appropriate model to use. Another question that comes up is whether CAR-1 protein itself is differentially sumoylated in CF512 background.

We agree with the referees that a clarification of methods and text were needed here. To clarify this issue, we first reproduced the experiment that is depicted as Figure 1A and modified the text to better explain the rationale. In the new experiment, we added a third worm strain that lack functional glp-1 when exposed to 25ᵒC during development (strain CF1903). Similarly, to CF512 worms, the exposure of CF1903 worms to 25ᵒC renders the animals sterile. The experiment was conducted according to a revised protocol in which all worm strains were cultured in identical temperatures. All three parental (F0) worm populations (N2, CF512, and CF1903) were grown at 15ᵒC to ensure fertility, eggs were extracted and placed on plates that were seeded with either control bacteria (EV) or on daf-2 RNAi bacteria. All plates were incubated for 48 hours at 25ᵒC and transferred to 20ᵒC for additional 24 hours, until all worms completed their development and reached day 1 of adulthood. As expected, CF512 and CF1903 worm populations, but not N2 animals, were sterile as displayed in Figure 1—figure supplement 1 of the revised manuscript. The worms were homogenized and total proteins were separated on a 10% PAA gel and blotted with a SUMO antibody.

We have modified the text (subsection “IIS reduction results in differential protein SUMOylation in C. elegans”) and the Materials and methods section to accurately explain how the experiment was performed. We thank the reviewers for this important comment.

2) Figure 1C-D are confusing. How do we know that 1C is showing SUMOylated-GFP-CAR-1 and not other protein that gets pulled down with an anti-SUMO antibody? Is this actually a double-IP where it is pulled down with SUMO antibodies, then GFP antibodies? This is especially confusing if probing with GFP-antibodies cannot visualize the larger, SUMOylated versions of the proteins – does SUMOylation prevent the GFP antibody from binding? Please clear up the experimental details and the conclusions drawn from the data.

We agree with the referees, revised the text and updated the Figure (1C) to better explain the experimental procedure and prevent confusion (please see subsection “IIS reduction lessens the SUMOylation of CAR-1”). In brief, WH346 worms were developed from hatching on either EV or daf-2 RNAi bacteria. At day 1 of adulthood, the worms (~300,000 per treatment) were homogenized and GFP-CAR-1 was pulled down by a GFP antibody. The sediment proteins were separated on a gel and blotted with a SUMO antibody. The large number of worms was needed due to the relative weak signal of SUMOylated proteins. In Figure 1D we display the same pulldown experiment as in 1C after reblotting it with a GFP antibody. Thus, Figure 1D serves as a loading control for Figure 1C. Since the signal of GFP antibody is much stronger than that of the SUMO antibody, the short exposure of the blot was not sufficient to detect SUMOylated proteins. To better analyze the total amounts of GFP-CAR-1 in these worms, we added Figure 1E, which displays another experiment in which we used a smaller number of worms (~4000 per treatment) and whole worm homogenates were loaded onto the gel. This result supports the conclusion that the knockdown of daf-2 does not affect the levels of GFP-CAR-1. In addition, we labelled Figure 1C-E to indicate what is displayed in each panel, modified the text to clearly explain these experiments and changed the figure legend accordingly.

3) Since car-1 RNAi/KO decreases lifespan of wild-type and daf-2 mutants to a similar extent, is this just an additive phenotype? (Figure 2A-B.)

This is a valid point. We agree with the referees, tested how the knockdown of car-1 by RNAi affects the lifespan of an additional daf-2 mutant strain (e1368, Figure 2—figure supplement 1B), and modified the text to explain this issue. We added the sentence: “Yet, the lifespan reduction that we observed among untreated and car-1 RNAi-treated daf-2 (e1370) mutant worms, which was similar to the difference observed among wild-type and car-1 knockout animals (Figure 2A), questioned the notion that CAR-1 is involved in IIS-mediated regulation of lifespan” to subsection “The roles of CAR-1 in the regulation of lifespan”, to reflect this issue. We also suggest that various longevity mechanisms are regulated by the insulin/IGF signaling cascade, and thus, the SUMOylation of CAR-1 is only one such IIS-controlled mechanism. Accordingly, the effect of car-1 RNAi on the lifespan of daf-2 mutant worms is partial but may be related to the activity of this pathway. Please also see the Discussion section of the revised manuscript.

We thank the referees for highlighting this important issue.

4) A single copy rescue experiment of the CAR-1 K185R should be performed in the car-1 KO to ensure that this CAR-1 still maintains the normal (albeit hyperactive) functions of the protein (e.g. rescues sterility) and isn't a completely mutant form of the protein with brand new functions.

This is an important point that we tried addressing by injecting car-1 heterozygous worms (strain WH377) with the CAR-1 K185 construct and searched for fertile worms that lack the endogenous car-1 and express the CAR-1 K185R protein. Unfortunately, we could not identify such worms. This was plausibly due to the impaired fertility of worms that over-express the mutated car-1 K185R (Figure 3G). Although we could not solve this issue experimentally within the time devoted for revision, we mentioned this important issue in the Discussion section of the revised manuscript: “(it is important to note that it is not clear whether this mutated CAR-1 restores the natural functions of the protein or activate another lifespan extending mechanism)”.

5) The lifespan decrease of car-1 RNAi on daf-9 mutants seems much milder than in wild-type or other mutant worms. Does this suggest daf-9 and car-1 is partially overlapping/interdependent? (Compare Figure 3—figure supplement 1A and B and Figure 2—figure supplement 1A).

We kindly disagree with the reviewers here, as the average differences in mean lifespans are similar. Each lifespan experiment was conducted three independent times. When daf-2 (e1370) mutant worms were fed with car-1 RNAi the average shortening in mean lifespan following car-1 RNAi treatment was 12.53% and car-1 KO animals exhibited 15.32% lifespan shortening compared to their wild-type counterparts (see Supplementary file 2). Similarly, the knockdown of car-1 by RNAi shortened the mean lifespan of daf-9 mutant worms (CF2531) in 10.47% and of daf-12 mutant animals (strain AA86) in 12.31% (see Supplementary file 4 for details). These differences are not significant and thus, we think that the shortening in mean lifespans are comparable and do not suggest an overlap in the pathways’ activities.

To better clarify this, we modified the text (please see subsection “The mechanisms of CAR-1-mediated lifespan regulation”).

6) If CAR-1 drives lifespan extension by inhibiting GLP-1, why does a CAR-1 (WT) overexpression, which has a dramatic effect on number of germ cells have no effect on lifespan, and the very modest change in germ cells of CAR-1 (WT) overexpression versus CAR-1 K185R overexpression (this is a very modest decline compared to the dramatic decline versus wild-type) have such a profound effect on lifespan? (Compare Figure 3C-D to Figure 2—figure supplement 4A/C).

We totally agree that this is an important issue, which requires clarification. It is likely that the over-expression of wild-type CAR-1 has only a little effect on lifespan, as the IIS promotes an efficient SUMOylation of the exogenous wtCAR-1 molecules and prevents them from affecting lifespan (Figure 2—figure supplement 4C). This may be similar to the case of DAF-16, which is efficiently phosphorylated by the IIS-regulated kinases and thus, daf-16 RNAi treatment has a small effect on the lifespan of wild-type worms. Since the CAR-1 K185R protein is SUMOylation-resistant, this mutant is not (or less) affected by the IIS and thus, the over-expression of this construct efficiently suppresses the activity of GLP-1 and extend lifespan (Figure 2D and E). Nevertheless, as noted accurately by the referees, the remarkable reducing effect of over-expressing wtCAR-1 on the number of germ cells is interesting and surprising (Figure 3D). This observation suggests that germ cells are more sensitive to perturbations in the levels of CAR-1 than lifespan. Accordingly, it is possible that although most exogenous CAR-1 molecules are SUMOylated by the IIS, a residual amount of non-SUMOylated CAR-1 molecules, could be sufficient to reduce the number of germ cells but not to extend lifespan. Importantly, the over-expression of the SUMOylation-resistant CAR-1 K185R further reduces the number of germ cells (Figure 3D). We further discussed this in subsection “The roles of CAR-1 in GLP-1-mediated functions” of the revised manuscript.

To further address the question why the over-expression of the wtCAR-1 reduces the number of germ cells (Figure 3D) but have a limited effect on the number of progeny (Figure 3H) we conducted an additional experiment. One possible explanation to this puzzling observation suggests that the over-expression of wtCAR-1 does not affect the number of apoptotic nuclei while expressing the mutated CAR-1 K185R does. If this is correct, the enhanced apoptosis may explain the different effects of the brood sizes. We tested this by comparing the number of apoptotic nuclei and found that indeed, the knockdown of car-1 by RNAi, or the over-expression of the SUMOylation resistant CAR-1 K185R, but not of the wtCAR-1, elevate the number of apoptotic nuclei (Figure 3E). This new experiment is described in subsection “The roles of CAR-1 in GLP-1-mediated functions” of the revised manuscript.

7) What was the purpose of measuring the effect of car-1 RNAi on egg laying of kri-1 mutant worms? It seems unsurprising that car-1 RNAi and kri-1 mutants, both of which decrease egg laying, have an additive phenotype. Moreover, the initial car-1 mutant experiments expressed that ¬car-1 mutant animals are sterile, so why would it be surprising that car-1 RNAi decreases egg-laying? Perhaps it would be less confusing if this section is removed, as it doesn't seem to add any additional benefits to the story. Alternatively, it can be moved into supplements as a control for the overexpression data (Figure 3E-F).

The rationale behind this experiment was to examine whether the knockdown of kri-1 and car-1 have an additive effect. Yet, we agree with the referees that this rationale is not easy to follow and thus, we moved these results to the supplemental section as suggested. This experiment is now depicted as Figure 3—figure supplement 2C and D of the revised manuscript.

8) Is it surprising that EHC117 worms have no significant decrease in egg-laying when they have such a dramatic decline in germ cells? This should be textually addressed. This sentence can be added in the text where the differences in car-1 RNAi gonad versus egg-laying is compared – subsection “The roles of CAR-1 in GLP-1-mediated functions” (Compare Figure 3C-D to Figure 3I).

We thank the reviewers for this important suggestion and followed their guidance. As explained above, in the revised manuscript we also compared the number of apoptotic nuclei in the gonads of wild-type, EHC117 and EHC118 worms and found that the expression of the SUMOylation resistant CAR-1 K185R mutant, but not of the wtCAR-1 protein, elevates the numbers of apoptotic nuclei. This apparent contradiction between the number of germ cells and the brood size of worms that over-express the wtCAR-1 as well as the new experiment are described in subsection “The roles of CAR-1 in GLP-1-mediated functions” of the revised manuscript.

9) How do we know that the decreased egg-laying of CB1370 and car-1 RNAi are not just additive since both decrease egg-laying in otherwise WT animals? (Figure 3—figure supplement 2).

We agree that these effects could be additive and modified the text to reflect this issue. The sentence: “Yet, this reduction in brood size may be partially due to additive effects of IIS reduction and knocking down car-1” was added to subsection “The roles of CAR-1 in GLP-1-mediated functions”.

10) Can you show that car-1 RNAi on its own does not affect sygl-1 activity? Why is this only done in the daf-2 mutant and not in WT animals? The authors should either include these controls, or explicitly state in the text why these experiments are not significant. The reviewers think that showing that overexpression of CAR-1 (WT or K185R) can decrease sygl-1 transcripts shows more direct evidence that CAR-1 negatively regulates transcriptional activity of GLP-1 (Figure 3J). Another suggestion is to stain for GLP-1 instead of measuring sygl-1 levels. K185R mutants should have decreased GLP-1 levels and car-1 RNAi should have decreased GLP-1 levels based on the model.

This is an important point. We used here daf-2 mutant worms as we expect that in these animals CAR-1 is much less SUMOylated (due to the low IIS activity), thereby should be more active. Thus, the effect of car-1 RNAi is expected to be more prominent than in wild-type animals in which CAR-1 activity is suppressed by SUMOylation. However, we agree with the referee that a control experiment showing the effect of car-1 RNAi on the levels of sygl-1 in wild-type worms, is critical here. Accordingly, we used qPCR using N2 worms and sygl-1 primers (Figure 3—figure supplement 4 of the revised manuscript). The results of five independent experiments show that, as expected, the knockdown of car-1 by RNAi elevates the levels of sygl-1 in N2 worms, however, this effect is not significant. This observation suggests that in wild-type worms CAR-1 is less active than in daf-2 mutant animals and thus, the effect of knocking down this gene on the expression of sygl-1 is smaller than in daf-2 mutant animals. This experiment is delineated in subsection “The levels of car-1 modulate the transcriptional activity of the GLP-1 pathway”.

The reviewers think that showing that overexpression of CAR-1 (WT or K185R) can decrease sygl-1 transcripts shows more direct evidence that CAR-1 negatively regulates transcriptional activity of GLP-1 (Figure 3J). Another suggestion is to stain for GLP-1 instead of measuring sygl-1 levels. K185R mutants should have decreased GLP-1 levels and car-1 RNAi should have decreased GLP-1 levels based on the model.

We addressed this important issue by measuring the expression levels of an additional GLP-1 target gene, lst-1. While the exposure of CF1903 worms to 25ᵒC remarkably reduced the expression levels of lst-1 (Figure 3—figure supplement 3B), this gene showed reduced expression upon the knockdown of car-1. This shows that the relations between CAR-1 and GLP-1 are complex and probably depends on additional factors. We modified the text to discuss this important issue and thank the referees for their scientific guidance. Please see subsection “The levels of car-1 modulate the transcriptional activity of the GLP-1 pathway” and the Discussion section.

11) If car-1's effects are through GLP-1, car-1 RNAi should lead to an increase in GLP-1 protein. This should yield glp-1 gain of function phenotypes (e.g. persistent mitosis, increase in mitotic cells and fewer meiotic cells). Conversely, CAR-1 K185R should yield larger meiotic germ line cells causing a premature entry into meiosis (e.g. Pepper et al., 2003; Maine and Kimble, 1993). The absence of the phenotype in car-1 RNAi animals could suggests alternative mechanisms.

We took two measures to expand our analysis of the relations between CAR-1 and GLP-1. First, we compared the number of apoptotic nuclei in the gonads of wild type, EHC117 and EHC118 worms. In addition, we followed the expression levels of an additional GLP-1 target gene, lst-1. The comparison of apoptotic nuclei in the gonads of EHC117, EHC118 and wild-type animals is now displayed as Figure 3E and discussed in subsection “The roles of CAR-1 in GLP-1-mediated functions”. The complex relations of CAR-1 and GLP-1 are demonstrated by the opposing effects of car-1 RNAi on the expression levels of sygl-1 and lst-1 and explained in subsection “The levels of car-1 modulate the transcriptional activity of the GLP-1 pathway” and the Discussion section.

12) To further support the hypothesis that CAR-1 K185R that cannot be SUMOylated acts to increase longevity by decreasing GLP-1 would be to examine whether animals harboring CAR-1 K185R suppress glp-1 gain of function phenotypes (e.g. Pepper et al., 2003; Maine and Kimble, 1993), by sequestering glp-1 mRNA and decreasing its translation into protein.

This is an important comment. We tried to adopt a functional approach and injected GC833 worms ((glp-1(ar202) III), that develop tumor due to the activation of GLP-1 (Pepper et al., Genetics 2003)) with the CAR-1 K185R plasmid. However, this rendered the animals sterile and we could not isolate rescued animals.

Thus, we modified the text to explain the complex relations between CAR-1 and GLP-1 (Discussion section).

13) Is the daf-16 CAR-1 K185 overexpression similar to daf-16 RNAi? This seems like an extremely important control to show that the two are not simply an additive effect if daf-16 RNAi has higher proteotoxicity. (Figure 4—figure supplement 1A). It would also be stronger argument that CAR-1 activity is falling within the daf-2 cascade if daf-2 RNAi/mutants do not have an additive effect with CAR-1 K185.

We thank the reviewers for this important comment. We modified the text to add a comparison of the rates of paralysis in daf-16 RNAi-treated CL2006 worms in published articles (Cohen et al., 2006, Cohen et al., 2010) to the rate seen in EHC124 animals that were grown on daf-16 RNAi bacteria (Figure 4—figure supplement 1A). The similar rates of paralysis suggest that the protective effect of CAR-1 K185R is daf-16 dependent. This is described in subsection “CAR-1 is involved in maintaining proteostasis”.

14) Why are all the paralysis experiments done with car-1 RNAi, which is an indirect way to show that car-1 is affecting proteoxicity through GLP-1? All of these experiments should be done with the CAR-1 K185 to show directly that hyperactive CAR-1 can actually protect against proteoxicity. Minimally, the text should explain why these experiments are done with RNAi to increase proteotoxicity (Figure 5 and Figure 5—figure supplement 1, Figure 5—figure supplement 3A-B).

In Figure 4 we tested whether CAR-1 is a modulator of proteotoxicity. To address this we used: (i) car-1 RNAi (Figure 4A and B), (ii) mixes of car-1 RNAi with other RNAi bacterial strains (Figure 4C and D) and (iii) Aβ worms which express the CAR-1 K185R proteins (Figure 4E and F). We also tested whether the protection that is conferred by CAR-1 K185R is daf-16 dependent and whether the CAR-1 K257R affects Aβ toxicity (Figure 4—figure supplement 1A and B). The results of all of these experiments culminate to show that the knockdown of car-1 expose the worms to Aβ proteotoxicity, the over-expression of the hyper-active CAR-1 K185R, but not of the CAR-1 K257R, protects the animals from Aβ toxicity in a daf-16 dependent manner.

The rationale behind the experiments that we display in Figure 5A and B, Figure 5—figure supplement 1 and Figure 5—figure supplement 3 is different. Here we addressed two questions. First, we asked whether CAR-1 also modulates proteotoxicity of an additional disease-linked, aggregation-prone protein, polyQ-YFP. We found that similarly to the effect seen in Aβ worms, the knockdown of car-1 exposes the worms to proteotoxicity in a glp-1 dependent manner. Secondly, we asked whether the RNA helicase CGH-1, is also a modulator of proteotoxicity. Our results (Figure 5C-D and Figure 5—figure supplement 3) show that it indeed modulates proteotoxicity.

We modified the text as suggested, to better explain the experiments of Figure 5 and Figure 5—figure supplement 3 (Results section).

15) For Figure 6A-C, why was CF512 worms used? Were these grown at 15, 20, or 25 C? Growth at 25 C can mildly stress worms and can affect HSR or thermotolerance. The actual growth conditions should be specified, and experiments should be repeated if worms were developed at 25 C. Moreover, the thermotolerance and Pseudomonas data seems to suggest that hsf-1 may be activated. It is recommended that the role of HSF-1 in these paradigms be tested.

We agree with the referees and repeated the heat tolerance experiment that is presented as Figure 6A using N2 worms. Our new results (Figure 6B) show that similarly to the observations that we obtained using CF512 worms, the knockdown of car-1 by RNAi has no significant effect on heat tolerance of wild-type worms. In addition, in our previous study (Volovik et al., 2012) we have shown no HSF-1 activation in CF512 animals that were developed in 25ᵒC and transferred to 20ᵒC at day 1 of adulthood (as judged by the lack of induction of the HSF-1-target gene hsp-16.2). To clarify this issue we cited our 2012 Aging Cell paper and to describe the new experiment (Results section).

16) For Figure 6D, if car-1 RNAi normally decreases lifespan, then the mild increase in lifespan is actually pretty substantial if normalized for this fact. Maybe something on this should be added in the text.

We agree with the referees, conducted additional experiments and modified the text to better explain the slight increase in resistance to Pseudomonas aeruginosa. First, we examined whether the over-expression of wtCAR-1 (strain EHC117) and/or of CAR-1 K185R (strain EHC118) affect the survival of worms that were exposed to Pseudomonas aeruginosa. We found that the over-expression of car-1 (wild-type or mutant) slightly shortens the worms’ survival. These results (Figure 6F) support the notion that CAR-1 activity is deleterious when the animals are exposed to pathogenic bacteria. We also expanded the textual explanation to better discuss this important issue (please see Results section).

17) The experiments in Figure 6 really spark the curiosity of whether all of these phenotypes are truly due to car-1 or through non-specific pleotropic effects of car-1 RNAi. All of these experiments should be performed with the CAR-1 K185 strain to directly test whether these phenotypes are truly due to CAR-1. Moreover, would CAR-1 overexpression make worms more sensitive to pathogenic bacteria or UV (opposite of car-1 RNAi data), despite extending lifespan? Seems counter to the entire argument of the paper. These issues need to be addressed.

We thank the referees for highlighting this important issue and conducted the suggested experiment. We compared the survival of N2, EH117, and EHC118 worms (over-expressing the wild-type or K185R car-1 mutant respectively) that were exposed to heat (Figure 6D). We also tested whether EHC118 worms are more sensitive to pathogenic bacteria (Figure 6F) and to sub-lethal dose of UV radiation (Figure 6H). Our results show that, as predicted by the referees, the over-expression of the CAR-1 K185R acts in opposite to the knockdown of car-1 by RNAi as it lowers survival of stressed animals.

These new experiments are described in the Results section of the revised manuscript.

It is important to note that lifespan, stress resistance and proteostasis were shown by multiple studies to be separable. For instance, the ability to respond to heat stress comes at the expense of the worms capacity to cope with proteotoxicity (Prahlad and Morimoto, 2011). Similarly, we have shown (Maman et al., 2013) that the knockdown of gtr-1 elevates heat sensitivity but has no effect on lifespan. Thus, in the light of these insights, the opposing effects of car-1 RNAi on lifespan and stress resistance are not surprising.

We further discussed the relations between lifespan and stress resistance in the revised Discussion section.

18) The article conflates fundamentally distinct concepts/roles, and in two cases that impact the overall conclusions. One, the role of Car1 in reproduction vs its role in longevity are considered interchangeably. The authors use effects on fertility to draw conclusions about longevity functions and vice versa and it is incorrect and confusing. Two, the function of GLP1 protein in germline development is substituted with the glp1 temperature sensitive mutant phenotype that is a surrogate for longevity brought on by germline loss. They use some results to directly implicate GLP1 protein function (eg., sygl-1 expression) and others to deduce roles in germline eliminated longevity pathway (kri1, daf12 tests). This is also misleading and makes the article difficult to follow.

We see the point here and took several measures to clarify the manuscript and avoid confusion.

First, since theoretically it possible that, in addition to the reported link between CAR-1 and GLP-1 (Noble et al., 2008), CAR-1 influences additional cellular pathways, we sought to directly test if the knockdown of car-1 affects the transcriptional activity of the GLP-1-controlled pathway. To address this, we tested whether the knockdown of car-1 by RNAi affects the expression of sygl-1 and lst-1, both well-defined targets of the GLP-1 pathway. Our results (Figure 3I) provide a direct evidence to the negative regulation of CAR-1 on the transcriptional activity of GLP-1 on sygl-1 but not on lst-1. This highlights the complex relations of CAR-1 and GLP-1 that are now explained in the Results section and the Discussion section.

Secondly, to further examine the roles of CAR-1 of GLP-1 mediated functions, we tested how CAR-1 germ cell proliferation (Figure 3, C and D) and egg laying (Figure 3, F-H). In addition, since GLP-1 regulates the activity of DAF-16 through a well-defined set of components, including KRI-1 (Berman and Kenyon, 2006), we tested whether car-1 RNAi affects egg laying patterns and lifespan of worms that lack functional kri-1. We found that it reduces the brood size of kri-1 mutant worms (Figure 3—figure supplement 2C and D) but does not affect the lifespan of these animals (Figure 3B).

Finally, in the revised manuscript we tested how GLP-1 affects the expression level of car-1 (Figure 3J) and found that the expression of car-1 is lower in worms that lack functional GLP-1 compared to their counterparts the harbor functional GLP-1.

Together, these results describe the complex relations of CAR-1 and of GLP-1. The text has been modified to further discuss these relations (Results section).

Car1's genetic interaction with Glp1 protein is published so if the premise is that it acts with/through Glp1 to modulate germline events that impact longevity, the experiments conducted here (Figure 3) do not test it or offer evidence for or against it. That requires examining the effect of CAR1 modulation on GLP1 (and the very well-characterized Notch pathway for translation control in the germline) within the germline and the fertility and lifespan consequences thereof.

Our results show that the knockdown of car-1 shortens lifespan of wild-type and daf-2 mutant worms (Figure 2A-B) but not of worms that lack functional GLP-1 (Figure 3A). This shows that the effect of CAR-1 on lifespan is GLP-1 dependent. Moreover, the knockdown of car-1 modulates the transcription of GLP-1-target genes (sygl-1 and lst-1), showing that CAR-1 regulates at least some of the activities of GLP-1.

We also tested how the knockdown and over-expression of car-1 affect the number of germ cells, reproduction profile, and number of apoptotic nuclei (Figures 3C-E and G-J).

We hope that the modified text (Results section) as well as the wealth of new results further clarify these issues raised here by the referees.

Similarly, if the hypothesis is that the Car1-Glp1 relationship operates in the context of germline-less longevity paradigm, the evidence provided is insufficient to infer any conclusions.

Our hypothesis suggests that since CAR-1 regulates the activity of GLP-1, as the CAR-1-GLP-1 relationship should have no effect on lifespan of worms that lack functional GLP-1 (Figure 3A) or functional DAF-16 (Figure 2C). In addition, the expression of CAR-1 K185R extends lifespan. These results clearly show that CAR-1 is a lifespan regulator, probably through the GLP-1 controlled mechanism.

The daf16/kri1/daf12/daf36 pathway is activated upon germline loss and solely impacts somatic Daf16. The relationships of these proteins within the germline are different (indeed, even in germline development these relationships change based on developmental state and/or environmental conditions). The effects of Car1 manipulations on reproduction of kri1 or daf12 mutants do not reveal their relationship in determining longevity, especially of sterile glp1 mutants. The germline-less longevity pathway has been shown by numerous studies to be genetically parallel to IIS and the authors overlook this concept while concluding that Car1 integrates 'aging controlling functions of IIS and germline'.

The key conclusion of this work is that indeed, the aging-regulating pathways, not necessarily longevity, but stress resistance and proteostasis, downstream of the IIS and the germ cells are inter-related through the SUMOylation of CAR-1. Our data challenge the conclusion of previous studies that claimed that these two pathways are independent. Nevertheless, we expanded the Discussion section to better discuss this paradigm.

19) An alternative hypothesis that has not been addressed and would be consistent with much of the data such as the observed effects of overexpressing both wild-type CAR-1 and CAR-1 K185R on the number of germ line nuclei, is the role of CAR-1 on germline apoptosis (Boag et al., 2005). Programmed cell death mutations in C. elegans affect stress resistance, albeit reports show that the mechanisms are rather complex (e.g. Judy et al., 2013). It would strengthen the manuscript if this were directly examined, but this point needs to be addressed, at the very least in the Discussion section.

We followed the referee’s guidance and tested the proposed hypothesis by comparing the number of apoptotic nuclei in the gonads of untreated and car-1 RNAi-treated wild-type worms, as well as in EHC117 and EHC118 animals. Our results (Figure 3E of the revised manuscript) show that the knockdown of car-1 by RNAi, and the over-expression of the SUMOylation resistant CAR-1 K185R elevate the numbers of apoptotic nuclei in the gonad. No such effect was seen when the wtCAR-1 was over-expressed. These results provide a possible explanation to the lack of significant difference in the brood sizes of N2 and EHC117 worms (Figure 3H). We thank the referee for this important comment and agree that this mechanism is complex. Accordingly, we expanded the Discussion section to explain the roles of CAR-1 in controlling germ cells apoptosis.

https://doi.org/10.7554/eLife.38635.035

Article and author information

Author details

  1. Lorna Moll

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Conceptualization, Data curation, Validation, Investigation, Methodology, Writing—review and editing
    Contributed equally with
    Noa Roitenberg
    Competing interests
    No competing interests declared
  2. Noa Roitenberg

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Conceptualization, Data curation, Investigation, Methodology, Writing—review and editing
    Contributed equally with
    Lorna Moll
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2181-3313
  3. Michal Bejerano-Sagie

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Conceptualization, Validation, Investigation, Writing—original draft
    Competing interests
    No competing interests declared
  4. Hana Boocholez

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Investigation, Performed stress resistance assays including heat, UV and survival on pathogenic bacteria as well as lifespan assays, Involved in data analysis and interpretation
    Competing interests
    No competing interests declared
  5. Filipa Carvalhal Marques

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Investigation, Performed paralysis assays and created transgenic worms, Assisted with data interpretation
    Competing interests
    No competing interests declared
  6. Yuli Volovik

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Investigation, Performed paralysis assays
    Competing interests
    No competing interests declared
  7. Tayir Elami

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Investigation, Performed UV stress assays and paralysis assays
    Competing interests
    No competing interests declared
  8. Atif Ahmed Siddiqui

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Methodology, Created transgenic worms for the project
    Competing interests
    No competing interests declared
  9. Danielle Grushko

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Investigation, Performed qPCR experiments
    Competing interests
    No competing interests declared
  10. Adi Biram

    Departments of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
    Contribution
    Investigation, Conducted Western blot experiments
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6169-9861
  11. Bar Lampert

    Departments of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
    Contribution
    Investigation, Performed germ cell analyses in worm gonads
    Competing interests
    No competing interests declared
  12. Hana Achache

    Departments of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
    Contribution
    Methodology, Compared the number of apoptotic nuclei in worm gonads
    Competing interests
    No competing interests declared
  13. Tommer Ravid

    Departments of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
    Contribution
    Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  14. Yonatan B Tzur

    Departments of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
    Contribution
    Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  15. Ehud Cohen

    Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University School of Medicine, Jerusalem, Israel
    Contribution
    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Methodology, Writing—original draft, Project administration
    For correspondence
    ehudc@ekmd.huji.ac.il
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5552-7086

Funding

Israel Science Foundation (EC 981/16)

  • Hana Boocholez
  • Danielle Grushko
  • Ehud Cohen

Israel Science Foundation (YBT 2090/15)

  • Bar Lampert
  • Yonatan B Tzur

Israel Science Foundation (YBT 1283/15)

  • Bar Lampert
  • Yonatan B Tzur

European Research Council (EC 281010)

  • Lorna Moll
  • Noa Roitenberg
  • Michal Bejerano-Sagie
  • Filipa Carvalhal Marques
  • Yuli Volovik
  • Tayir Elami
  • Danielle Grushko
  • Ehud Cohen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This study was supported by the European Research Council (ERC) (EC#281010) and the Israel Science Foundation (ISF) EC#981/16 and YBT#1283/15 and 2090/15.

Senior Editor

  1. K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

Reviewing Editor

  1. Inna Slutsky, Tel Aviv University, Israel

Publication history

  1. Received: May 24, 2018
  2. Accepted: November 6, 2018
  3. Accepted Manuscript published: November 7, 2018 (version 1)
  4. Version of Record published: December 3, 2018 (version 2)

Copyright

© 2018, Moll et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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