Rewiring of the ubiquitinated proteome determines ageing in C. elegans

Abstract

Ageing is driven by a loss of cellular integrity¹. Given the major role of ubiquitin modifications in cell function², here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome³, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament⁴ and the EPS-8 modulator of RAC signalling⁵. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

Fig. 1. Rewiring of the Ub-proteome with age.

a, Scheme of ubiquitin proteomics by di-glycine (diGly) peptide enrichment in wild-type (WT) and long-lived mutant worms. b, Heat maps representing log₂-transformed fold changes in Ub-peptide levels at different days (D) of adulthood compared with the corresponding day-1 adult strain. For each strain, only Ub-peptides significantly changed in at least one age are shown. c, Number of significantly downregulated and upregulated Ub-peptides compared with the respective day-1 adult strain. d, Percentage of downregulated and upregulated Ub-peptides among the total number of significantly changed Ub-peptides per condition. e, The log₂-transformed fold changes of differentially abundant Ub-peptides and their corresponding total protein levels comparing day-15 and day-5 wild-type worms. f, The log₂-transformed fold changes of differentially abundant Ub-peptides in day-15 wild-type worms and comparison with age-matched eat-2 and daf-2 mutants. In b–f, n = 4; two-sided t-test, false discovery rate (FDR) < 0.05 was considered significant. g–i, Immunoblot of Ub-proteins in wild-type (g), eat-2(ad1116) (h) and daf-2(e1370) (i) worms at different days of adulthood. α-tubulin was used as a loading control. The images are representative of four independent experiments. j, Immunoblot of Ub-proteins in wild-type worms treated with 13.7 μg ml⁻¹ PR-619 (broad-spectrum DUB inhibitor) or vehicle control (dimethyl sulfoxide, DMSO) for 4 h before lysis. Representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Fig. 2. Age-related deubiquitination impairs targeted degradation of longevity regulators.

a, Ten Ub-proteins increase after rpn-6 RNAi treatment in young wild-type adults (rpn-6 RNAi/Vector RNAi) and become less ubiquitinated but more abundant with age (day 15/day 5). b, Volcano plot of proteins containing Lys48-linked polyUb in day-5 adult wild-type worms (n = 3, FDR < 0.05). The −log₁₀(P value) of a two-sided t-test is plotted against the log₂-transformed fold change values from immunoprecipitation (IP) with an antibody against Lys48-linked polyUb compared with a control anti-Flag antibody. Red dots indicate age-dysregulated proteasome targets. c, d, Knockdown of either ifb-2 (c) or eps-8 (d) after development extends lifespan (P < 0.0001). e, Western blot analysis with an antibody against IFB-2 of wild-type worms at different days of adulthood. α-tubulin is the loading control. Representative of four independent experiments. f, Western blot analysis with an antibody against EPS-8 of wild-type worms. Representative of three independent experiments. g, Western blot analysis with an antibody against IFB-2 of wild-type and IFB-2(K255R/K341R) (Ub-less) mutant worms at day 2 of adulthood. Representative of three independent experiments. h, Ubiquitin-less IFB-2 mutant worms have a shorter lifespan than wild-type worms (P < 0.0001). i, Western blot analysis with an antibody against EPS-8 of worms expressing endogenous wild-type EPS-8::HA or EPS-8(K524R/K583R/K621R::HA) (Ub-less) at day 1 of adulthood. Representative of three independent experiments. j, EPS-8 (Ub-less) mutants are short-lived (P < 0.0001). In lifespan experiments, P values were determined by two-sided log-rank test; n = 96 worms per condition. Lifespan data are representative of at least two independent experiments. Supplementary Table 11 contains replicate data of independent lifespan experiments. For gel source data, see Supplementary Fig. 1.

Fig. 3. Increased IFB-2 levels induce age-related intestinal alterations.

a, Intestinal-specific knockdown (KD) of ifb-2 extends lifespan. P value determined by two-sided log-rank test; n = 96 worms per condition. Lifespan data are representative of two independent experiments. Supplementary Table 11 contains replicate data of independent experiments. b, Filter trap analysis with an antibody against IFB-2 of wild-type worms at different ages. Representative of eight independent experiments. c, Filter trap analysis with an antibody against IFB-2 of wild-type and IFB-2 (Ub-less) mutant worms. Representative of four independent experiments. d, e, Filter trap experiments with an antibody against GFP of worms expressing IFC-1::GFP under the ifc-1 promoter (d) or IFP-1::GFP under the ifp-1 promoter (e). Representative of two independent experiments. f, Quantification of bacterial colonization. Fluorescence of mCherry-expressing E. coli within the intestine relative to day 1 (D1) Vector RNAi. Data are mean ± s.e.m. D1 Vector RNAi, n = 56 worms from 3 independent experiments; D1 ifb-2 RNAi, n = 35; D5 Vector RNAi, n = 53; D5 ifb-2 RNAi, n = 55; D10 Vector RNAi, n = 45; D10 ifb-2 RNAi, n = 39. g, Bacterial colonization relative to day-10 adult wild-type worms. Data are mean ± s.e.m. Wild-type, n = 50 worms from 3 independent experiments; Ub-less IFB-2, n = 47. In f and g, P values were determined by two-sided t-test. NS, not significant. In all experiments, RNAi was initiated at day 1 of adulthood. Source data

Fig. 4. Increased EPS-8 levels shorten lifespan through RAC hyperactivation.

a, b, Muscle-specific (a) and neuronal-specific (b) knockdown of eps-8 after development extends lifespan. c, Knockdown of mig-2 after development extends longevity and rescues the short lifespan induced by ubiquitin-less EPS-8. P values in a–c were determined by two-sided log-rank test; n = 96 worms per condition. Supplementary Table 11 contains replicate data of independent experiments. d, Staining of filamentous actin with phalloidin. eps-8 RNAi prevents age-associated destabilization of actin filaments. Scale bar, 20 μm. Representative of two independent experiments. e, Knockdown of mig-2 rescues the disruption of actin filaments in day-3 adult worms induced by ubiquitin-less EPS-8. Scale bar, 20 μm. Representative of two independent experiments. f, Thrashing movements per 30 s (n = 45 worms per condition from three independent experiments). Knockdown of mig-2 suppresses motility deficits induced by ubiquitin-less EPS-8 in young adult worms. Data are mean ± s.e.m. P values in f determined by two-sided t-test. In all experiments, RNAi was initiated at day 1 of adulthood. Source data

Extended Data Fig. 1. Differences in the levels of Ub-peptides often do not directly correlate with a change in the total amounts of the protein.

a, Principal component analysis indicates high reproducibility in proteomics of Ub-peptides among the four biological replicates for each experimental condition (wild type, eat-2(ad1116), daf-2(e1370) at day 1, 5, 10 and 15 of adulthood). b–d, Heat maps representing log₂-transformed fold changes of all the differentially abundant Ub-peptides and their corresponding total protein levels at the indicated day and genetic background (n = 4, two-sided t-test, FDR <0.05 was considered significant). In each heat map, only significantly changed Ub-peptides are shown. Supplementary Table 3 contains complete list of all identified Ub-peptides and levels of the respective protein. e, Integrated proteomics analysis of log₂-transformed fold changes in the levels of Ub-peptides and corresponding proteins comparing day-15 and day-5 wild-type worms (n = 4, two-sided t-test, FDR < 0.05). f, The total protein levels of myosin MYO-2 and paramyosin UNC-15 remain similar during ageing, but they contain multiple downregulated and upregulated Ub-sites. EPS-8 protein becomes more abundant with age, and most of its Ub-sites are significantly downregulated (n = 4, two-sided t-test, FDR < 0.05 was considered significant). Because EPS-8 has many different isoforms, we show the ubiquitination sites numbered according to the leading isoform identified in our proteomics data (EPS-8 protein isoform g). g, Among the 1,813 downregulated ubiquitin modifications in aged wild-type worms (WT d15/WT d5), age-matched eat-2 (eat-2 d15/WT d15) and daf-2 mutants (daf-2 d15/WT d15) exhibited increased ubiquitination for 952 and 336 peptides, respectively. Among the 350 upregulated ubiquitin modifications in aged wild-type worms, age-matched eat-2 and daf-2 worms exhibited decreased ubiquitination for 234 and 251 peptides, respectively.

Extended Data Fig. 2. Loss of ubiquitination in aged wild-type worms is not associated with lower expression or half-life of protein ubiquitin itself.

a, Immunoblot of Ub-proteins in wild-type worms at the indicated days of adulthood. The images are representative of four independent experiments. b, Immunoblot of Ub-proteins in eat-2(ad1116) mutant worms. Representative of four independent experiments. c, Immunoblot of Ub-proteins in daf-2(e1370) mutant worms. Representative of three independent experiments. d, qPCR analysis of ubiquitin (ubq-1), ubiquitin-ribosomal fusion (ubq-2, ubl-1) and ubiquitin-stress response (usp-14) genes comparing day-1 and day-15 wild-type adult worms. Graph represents the relative expression to day 1 adult wild-type worms (mean ± s.e.m., n = 9). P values: ubq-1 (P = 0.0117), ubq-2 (P = 0.0042), ubl-1 (P = 0.0598), usp-14 (P = 0.5326). e, qPCR analysis comparing day-1 and day-15 eat-2 mutant worms. Graph represents the relative expression to day 1 adult eat-2 mutant worms (mean ± s.e.m., n = 9). P values: ubq-1 (P = 0.0668), ubq-2 (P = 0.0144), ubl-1 (P < 0.0001), usp-14 (P = 0.0786). f, qPCR analysis comparing day 1 and day 15 daf-2 mutant worms. Graph represents the relative expression to day 1 adult daf-2 mutant worms (mean ± s.e.m., day 1 (n = 9); day 15 (n = 8)). P values: ubq-1 (P = 0.0057), ubq-2 (P = 0.5710), ubl-1 (P = 0.7593), usp-14 (P = 0.2199). g, qPCR analysis comparing day 15 wild-type worms with age-matched long-lived mutant worms. Graph represents the relative expression to wild-type worms (mean ± s.e.m., wild-type (n = 9); eat-2 (n = 9); daf-2 (n = 8)). Wild-type worms express higher levels of ubq-1 gene at day 15 of adulthood (wild-type versus eat-2: P = 0.0341; wild-type versus daf-2: P < 0.0001). h, The log₂-transformed label-free quantification (LFQ) values of protein ubiquitin (UBQ-1 ;UBQ-2) from global proteomics analysis of wild-type, eat-2(ad1116) and daf-2(e1370) worms at the indicated ages (n = 4, mean ± s.e.m.). The total amounts of protein ubiquitin itself slightly increase in wild-type worm with age (wild-type d1 versus wild-type d15: P < 0.0001). Old wild-type and aged-matched daf-2 mutant worms have similar levels of total protein ubiquitin (wild-type d15 versus daf-2 d15: P = 0.2669). Label-free proteomics of total protein levels (Supplementary Table 2) was performed in input samples separated from the same lysates used for analysis of the Ub-modified proteome before the enrichment with an anti-diGly antibody. In d–h, P values were determined by two-sided t-test. i, Western blot of free ubiquitin (Ub) in worms treated with 54.5 μg ml⁻¹ cycloheximide (CHX) to block ubiquitin synthesis. Worms were lysed at the indicated time after CHX treatment. The images are representative of three independent experiments. j, Quantification of the western blots presented in the previous figure. Graph represents the percentage of free Ub levels relative to time point 0 h of CHX treatment (mean ± s.e.m., n = 3 independent experiments). For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 3. Single knockdown of specific age-dysregulated DUBs ameliorates the global decline in Ub-protein levels of aged wild-type worms.

a, Heat map representing all the differentially abundant E3 ubiquitin ligases in old (day 15) wild-type worms compared with young (day 5) wild-type worms (n = 4, two-sided t-test, FDR < 0.05 was considered significant). The levels of differentially abundant E3s in old wild-type worms were also compared with age-matched long-lived eat-2 and daf-2 mutants. b, Heat map representing all the differentially abundant DUBs in old wild-type worms compared with young wild-type worms (n = 4, two-sided t-test, FDR < 0.05 was considered significant). The levels of differentially abundant DUBs in old wild-type worms were also compared with age-matched long-lived eat-2 and daf-2 mutants. c, Immunoblot of Ub-proteins and α-tubulin in day 10 wild-type worms upon knockdown of prp-8, eif-3.F, cyk-3 and F07A11.4 after development. The images are representative of three independent experiments. d, Immunoblot of Ub-proteins in day-10 wild-type worms upon knockdown of usp-5 and otub-3 after development. Representative of three independent experiments. e, Immunoblot of Ub-proteins in day-10 wild-type worms upon knockdown of math-33 and H34C03.2 after development. Representative of four independent experiments. f, Immunoblot of Ub-proteins in day-10 wild-type worms upon knockdown of cyld-1 and csn-6 after development. Representative of four independent experiments. g, Immunoblot of Ub-proteins in day-10 wild-type worms upon knockdown of usp-50, csn-5 and usp-48 after development. Representative of four independent experiments. In c–g, RNAi was initiated at day 1 of adulthood. h, Wild-type worms treated with 13.7 μg ml⁻¹ of the broad-spectrum DUB inhibitor PR-619 at day 9 of adulthood for 24 h (P = 0.0162) or from day 9 of adulthood until the end of the experiment (P < 0.0001) live longer compared with untreated wild-type worms. P values: two-sided log-rank test, n = 96 worms per condition. Supplementary Table 11 contains statistics and replicate data of independent lifespan experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 4. Ageing does not increase the mRNA levels of age-dysregulated proteasome targets.

a, Heat map representing log₂-transformed fold changes of all the differentially abundant proteins comparing rpn-6 RNAi-treated wild-type worms with Vector RNAi-treated wild-type worms at day 5 of adulthood (n = 3, two-sided t-test, FDR < 0.05 was considered significant). Loss of proteasome activity results in widespread changes in the individual protein levels of young adults, including 297 downregulated proteins and 509 upregulated proteins. See Supplementary Table 8 for complete list of all the detected proteins. b, The mRNA levels of age-dysregulated proteasome targets do not increase with age. In fact, ifb-2 (P = 0.0026) and eps-8 (P = 0.0194) mRNA levels decrease in aged worms. Graph represents the relative expression to young (day 5) adult worms (mean ± s.e.m., n = 3). c, Loss of rpn-6 does not upregulate the mRNA levels of age-dysregulated proteasome targets in young worms (day 5), with the exception of the deubiquitinase usp-5 (P = 0.0019) that could indicate a compensatory mechanism to ameliorate ubiquitin deficits triggered by dysregulated proteasome activity. Graph represents the relative expression to Vector RNAi control (mean ± s.e.m., n = 3). RNAi treatment was initiated at day 1 of adulthood. In b, c, P values were determined by two-sided t-test. d, Volcano plot of proteins containing Lys63-linked polyUb chains in wild-type worms at day 5 of adulthood (n = 3, FDR < 0.05). −log₁₀(P value) of a two-sided t-test is plotted against the log₂-transformed fold change of LFQ values from immunoprecipitation experiments using an antibody against Lys63-linked polyUb compared with anti-Flag antibody. Red dots indicate age-dysregulated proteasome targets. e, Knockdown of either hsp-43 (P = 0.0010) or usp-5 (P = 0.0146) during adulthood shortens lifespan, indicating that these genes are essential for adult viability. Knockdown of either ddi-1 (P = 0.8277) or lec-1 (P = 0.7034) does not significantly affect lifespan. In contrast, single knockdown of rpl-4 (P = 0.0283), M01G12.9 (P < 0.0001), C46C2.2 (P = 0.0196) and F54D1.6 (P = 0.0400) extends lifespan. In each lifespan experiment, RNAi was initiated at day 1 of adulthood. P values were determined by two-sided log-rank test; n = 96 worms per condition. Lifespan data are representative of two independent experiments. Supplementary Table 11 contains statistics and replicate data of independent lifespan experiments. f, Western blot analysis with an antibody against GFP of integrated transgenic worms expressing ifb-2p::ifb-2a::CFP. α-tubulin is the loading control. Representative of two independent experiments. g, Western blot with an antibody against GFP of worms expressing endogenous IFB-2 tagged with GFP. Representative of two independent experiments. h, Western blot analysis of total IFB-2 levels with an anti-IFB-2 antibody in whole wild-type C. elegans extracts without mild centrifugation to collect supernatant. Representative of three independent experiments. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 5. The age-associated decline in ubiquitination levels occurs across tissues.

a, Bioinformatics classification of proteins that exhibit differences in their Ub-peptides in aged wild-type worms (day 15) according to the tissues where these proteins are expressed. b, Immunoblot of Ub-proteins in lysates of whole worms or isolated germlines, intestines and heads from wild-type worms at the indicated days of adulthood. Representative of two independent experiments. c, Western blot analysis with an antibody against EPS-8 of isolated intestines and heads from wild-type worms at the indicated days. Representative of two independent experiments. d, Western blot analysis with an antibody against IFB-2 of isolated intestines and heads from wild-type worms at the indicated days. Representative of two independent experiments. e, Western blot with antibodies to EPS-8 and IFB-2 after intestinal-specific knockdown (KD) of rpn-6. RNAi rescued in the intestine of RNAi-deficient worms (rde-1(ne219); nhx-2p::rde-1 strain). f, Western blot with antibodies to EPS-8 and IFB-2 after neuronal-specific KD of rpn-6. RNAi rescued in the neurons of RNAi-deficient worms (sid-1(pk3321); unc-119p::sid-1 strain). g, Western blot with antibodies to EPS-8 and IFB-2 after muscle-specific KD of rpn-6. RNAi rescued in the muscle of RNAi-deficient worms (rde-1(ne300); myo-3p::rde-1 strain). h, Western blot with antibodies to EPS-8 and IFB-2 after epidermal-specific KD of rpn-6. RNAi rescued in the epidermis of RNAi-deficient worms (rde-1(ne219); lin-26p::rde-1 strain). In e–h, RNAi treatment was initiated at day 1 of adulthood. Worms were analysed at day 5 of adulthood. Representative of two independent experiments. i, Immunoblot of Ub-proteins in wild-type and unc-13(e51) mutant worms at the indicated days of adulthood. Representative of three independent experiments. j, Western blot with an antibody against EPS-8 in wild-type and unc-13(e51) mutant worms at day 10 of adulthood. Representative of three independent experiments. k, Western blot with an antibody against IFB-2 in wild-type and unc-13(e51) mutant worms at day 10 of adulthood. Representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 6. Increased levels of IFB-2 and EPS-8 shorten adult lifespan.

a, Western blot with an antibody against IFB-2 of whole lysates from wild-type worms and IFB-2 K255R/K341R (Ub-less) mutant worms at day 2 of adulthood. The images are representative of two independent experiments. b, Non-integrated transgenic DVG197 and DVG198 strains overexpressing ifb-2 under sur-5 promoter exhibit a short lifespan phenotype compared with the control strain (P < 0.0001). P values were determined by two-sided log-rank test; n = 96 worms per condition. Lifespan data are representative of two independent experiments. c, Percentage of hatched eggs (mean ± s.e.m., n = 10 worms scored per condition from 2 independent experiments). EPS-8(K524R/K583R/K621R) (Ub-less) mutants do not exhibit embryonic lethality (P = 0.7806). d, Percentage of L1 larvae that developed into adults (mean ± s.e.m., n = 10 worms scored per condition from 2 independent experiments). Ubiquitin-less EPS-8 mutants do not exhibit developmental arrest (P = 0.0507). In c, d, P values were determined by two-sided t-test. e, Ubiquitin-less EPS-8 mutant worms live shorter than with worms that express wild-type EPS-8 (EPS-8 (WT) + Vector RNAi versus EPS-8 (Ub-less) + Vector RNAi, P = 0.0030). Knockdown of eps-8 after development suppresses the deleterious effects on lifespan induced by Ub-less EPS-8 mutations (EPS-8 (WT) + eps-8 RNAi versus EPS-8 (Ub-less) + eps-8 RNAi, P = 0.3394). P values determined by two-sided log-rank test, n = 96 worms per condition. Supplementary Table 11 contains statistics and replicate data of independent lifespan experiments. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 7. Ageing induces mislocalization and aggregation of IFB-2 and other intestinal intermediate filaments.

a, Muscle-specific knockdown of ifb-2 does not affect lifespan (P = 0.3755). b, Neuronal-specific knockdown of ifb-2 does not affect lifespan (P = 0.6381). In each lifespan experiment, RNAi was initiated during adulthood, P values determined by two-sided log-rank test, n = 96 worms per condition. Supplementary Table 11 contains statistics and replicate data of independent lifespan experiments. c, Representative images of transgenic worms expressing IFB-2::CFP under ifb-2 promoter at different days of adulthood. On the left, IFB-2::CFP is presented in greyscale to show more visibly the IFB-2 aggregates. Merge images present IFB-2::CFP (blue) + DIC (differential interference contrast). Scale bar, 200 μm. Representative of three independent experiments. d, Images of worms expressing endogenous IFB-2 tagged with GFP at different days of adulthood. Scale bar, 100 μm. Representative of two independent experiments. e, Filter trap analysis with an antibody against GFP of worms expressing endogenous IFB-2 tagged with GFP. Representative of three independent experiments. f, Images of worms expressing endogenous IFC-2 tagged with YFP or the integrated transgenes ifc-1p::IFC-1::GFP and ifp-1p::IFP-1::GFP. Scale bar, 100 μm. Representative of two independent experiments. g, Filter trap analysis with an antibody against GFP of C. elegans expressing IFC-1::GFP under ifc-1 promoter. Representative of two independent experiments. h, Filter trap with an antibody to GFP of C. elegans expressing endogenous intestinal intermediate filament IFC-2 tagged with YFP. Representative of three independent experiments. i, Filter trap with an antibody to GFP of C. elegans expressing IFP-1::GFP under ifp-1 promoter. Representative of two independent experiments. j, Images of worms expressing the intestinal filament organizer IFO-1::YFP under ifo-1 promoter. Scale bar, 100 μm. Representative of three independent experiments. k, Filter trap with an antibody to GFP of worms expressing IFO-1::YFP under ifo-1 promoter. Representative of four independent experiments.

Extended Data Fig. 8. Downregulation of increased IFB-2 levels ameliorates loss of intestinal integrity and bacterial colonization.

a, ifb-2 RNAi ameliorates age-related changes in the intracellular distribution and aggregation of the intestinal intermediate filaments IFC-1, IFC-2 and IFP-1 as well as the intestinal filament organizer IFO-1. We examined worms expressing endogenous IFC-2 tagged with YFP or the integrated transgenes ifc-1p::IFC-1::GFP, ifp-1p::IFP-1::GFP, and ifo-1p::IFO-1::GFP. ifb-2 RNAi was initiated during adulthood. Scale bar, 25 μm. Representative of two independent experiments. b, Filter trap analysis with an antibody to GFP of C. elegans expressing endogenous intestinal intermediate filament IFC-2 tagged with YFP. ifb-2 RNAi was initiated during adulthood. Representative of four independent experiments. c, Filter trap analysis with an antibody to GFP of worms expressing IFO-1::YFP under ifo-1 promoter. ifb-2 RNAi was initiated during adulthood. Representative of three independent experiments. d, Images of bacterial colonization in the intestine of wild-type C. elegans at different days of adulthood. RNAi was initiated during adulthood. Scale bar, 200 μm. Representative of three independent experiments. e, Images of bacterial colonization in the intestine of wild-type and IFB-2(K255R/K341R) (Ub-less) mutant worms at day 10 of adulthood. Scale bar, 200 μm. Representative of three independent experiments. Quantifications of bacterial colonization are shown in Fig. 3f, g.

Extended Data Fig. 9. Age-related hyperactivation of RAC by increased EPS-8 levels increases JNK phosphorylation.

a, Intestinal-specific knockdown of eps-8 after development does not affect lifespan (P = 0.1486). b, Epidermal-specific knockdown of eps-8 during adulthood does not affect lifespan (P = 0.3149). c, Single knockdown of RAC orthologues mig-2 (P < 0.0001) and rac-2 (P = 0.0008) during adulthood extends lifespan in wild-type worms. Knockdown of RAC orthologue ced-10 does not affect lifespan (P = 0.3227). d, Intestinal-specific knockdown of either mig-2 (P = 0.7340) or rac-2 (P = 0.6021) during adulthood does not affect lifespan. e, Muscle-specific knockdown of either mig-2 (P < 0.0001) or rac-2 (P = 0.0003) during adulthood extends lifespan. f, Neuronal-specific knockdown of either mig-2 (P < 0.0001) or rac-2 (P < 0.0001) during adulthood extends lifespan. g, Western blot analysis with antibodies to phosphorylated JNK (P-JNK), total JNK and α-tubulin of wild-type worms at different days of adulthood. Representative of three independent experiments. h, Western blot analysis with antibodies to P-JNK, total JNK and α-tubulin of wild-type worms at day 10 of adulthood. eps-8 RNAi was initiated during adulthood. Representative of two independent experiments. i, Knockdown of kgb-1 after development extends longevity (EPS-8 (WT) Vector RNAi versus EPS-8 (WT) kgb-1 RNAi, P < 0.0001) and rescues the short lifespan induced by ubiquitin-less EPS-8 mutant variant (EPS-8 (WT) Vector RNAi versus EPS-8 (Ub-less) Vector RNAi (P < 0.0001); EPS-8 (WT) kgb-1 RNAi versus EPS-8 (Ub-less) kgb-1 RNAi (P = 0.8061)). In each lifespan experiment, RNAi was initiated at day 1 of adulthood. P values were determined by two-sided log-rank test, n = 96 worms per condition. Supplementary Table 11 contains statistics and replicate data of independent lifespan experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 10. Lowering hyperactivated RAC signalling prevents alterations in actin networks induced by increased EPS-8 levels.

a, Myosin heavy chain tagged to GFP indicates destabilization of myosin filaments in muscle cells during ageing, whereas knockdown of eps-8 during adulthood maintains organization of the myosin network. Scale bar, 20 μm. Representative of three independent experiments. b, Data are mean ± s.e.m. thrashing movements over a 30-s period on day 1 (n = 30 worms per condition, three independent experiments), day 3 (n = 30 worms per condition, three independent experiments) and day 10 (n = 45 worms per condition, three independent experiments) of adulthood. Knockdown of eps-8 after development ameliorates the age-associated decline in motility (day 1 vector RNAi versus day 1 eps-8 RNAi, P = 0.2127; day 3 Vector RNAi versus day 3 eps-8 RNAi, P < 0.0001; day 10 Vector RNAi versus day 10 eps-8 RNAi, P < 0.0001). P values were determined by two-sided t-test. c, Images of transgenic worms expressing LifeAct::mRuby in the epidermis. Scale bar, 20 μm. Representative of two independent experiments. d, Images of transgenic worms expressing LifeAct::mRuby in the intestine. Scale bar, 100 μm. Representative of two independent experiments. e, Filter trap analysis with an antibody to β-actin in wild-type worms. Knockdown of eps-8 during adulthood reduces the amounts of actin aggregates in aged worms. Representative of six independent experiments. f, Filter trap analysis with an antibody to β-actin. Muscle-specific knockdown of eps-8 during adulthood reduces the amounts of actin aggregates in aged worms. Representative of three independent experiments. g, Neuronal-specific knockdown of eps-8 results in decreased actin aggregates during ageing. Representative of three independent experiments. h, Epidermal-specific knockdown of eps-8 does not decrease age-related actin aggregation. Representative of three independent experiments. i, Intestinal-specific knockdown of eps-8 does not decrease age-related actin aggregation. Representative of three independent experiments. j, Knockdown of either mig-2 or rac-2 during adulthood ameliorates the age-associated destabilization of actin filaments in muscle cells. RNAi treatment was initiated at day 1 of adulthood. Scale bar, 20 μm. Representative of two independent experiments. k, Knockdown of either mig-2 or rac-2 during adulthood ameliorates the age-associated destabilization of myosin filaments in muscle cells. Scale bar, 20 μm. Representative of two independent experiments. l, Filter trap with an antibody to β-actin in worms expressing endogenous wild-type EPS-8 or mutant Ub-less EPS-8 at day 3 of adulthood. Representative of three independent experiments. In all the experiments, RNAi was initiated at day 1 of adulthood. Source data