LIN-3 is expressed in epithelial cells of adults. To examine LIN-3 expression, we obtained transgenic syIs107 nematodes that express GFP protein with a nuclear localization signal from a genomic lin-3 promoter. (A) L2 animal showing expression (arrows) from lin-3 promoter in the pharynx. Bar, 100 μm. (B) L3 animal showing expression from the lin-3 promoter in the anchor cell along the ventral mid-body (the dorsal and ventral cuticle is outlined in white). L4 animals later show vulF cell expression in this same region of the body. Bar, 50 μm. While not detected in larvae, there is epithelial expression from the lin-3 promoter in adults starting at L4+24 h in (C) the intestine and (D) the hypodermis. Bar, 100 μm.

A GFP reporter for polyubiquitination and proteasomal degradation. (A) Schematic representation of the Ub-GFP and Ub^G76V-GFP chimeras. The amino-acid sequences for ubiquitin are in yellow, whereas the sequences for GFP are in green. The Ub-GFP chimera is co-translationally cleaved, releasing a stable GFP protein. In contrast, the Ub^G76V-GFP chimera, which contains a mutation in the terminal residue of ubiquitin, cannot be cleaved. The resulting protein is a substrate for polyubiquitination (indicated by the circles labelled with ‘u') and degradation. (B) Co-expression of Ub^G76V-GFP (green) and mRFP (red) in C. elegans epithelia using the col-19 promoter. Ub^G76V-GFP fluorescence is visible 24 h after L4 stage, but gone 48 h after L4. (C) Relative levels of Ub^G76V-GFP (green line) and mRFP (red line) fluorescence quantified (AU indicates arbitrary units) from micrographs at different time points after L4 stage. Black line indicates the relative ratio of Ub^G76V-GFP to actin from western blot analysis. Error bars indicate s.e.m. (D) GFP and actin detected by western blot at specific times (in hours) after L4. ‘Load' indicates that equivalent amounts of protein were loaded into each lane, except for the ‘36 h' and ‘48 h' lanes, in which twice the amount was loaded, so as to maximize Ub^G76V-GFP detection. For the anti-GFP blot, the multiple bands represent the Ub^G76V-GFP chimeric protein with different numbers of additional ubiquitins attached. (E) Co-expression of Ub^G76V-GFP (green, top panels) and mRFP (red, bottom panels) proteins in C. elegans at 48 h after L4 stage. ‘Control RNAi' indicates wild-type animals raised on bacteria containing an empty RNAi vector. N2 animals raised on pas-5(RNAi) or pbs-3(RNAi) bacteria, both of which knockdown proteasome subunit genes, showed stable GFP. Mutants for rpn-10(ok1865), which lack the RPN-10 proteasome regulatory subunit, also contained stable GFP. Bar, 50 μm.

SKR-5 is upregulated by EGF/EOR-1 signalling and is required for Ub^G76V-GFP turnover. (A) Gene structure of skr-5. Boxes indicate exons. Brackets indicate the location of individual motifs within the protein. The arrow indicates the start of transcription. The red line indicates the sequences deleted by the ok3068 deletion. (B) Alignment of SKR-5 protein with yeast Skp1, C. elegans SKR-1, and human Skp1a (black indicates conserved identity, grey indicates conserved similarity). The red arrow indicates the breakpoint of the ok3068 deletion within SKR-5; sequences C-terminal to the arrow are absent in the mutants. (C–F) Fluorescence from adult (L4+48 h) animals expressing a P_skr−5∷GFP transgene in the indicated genotype. Expression is observed in intestinal epithelia and hypodermis, as well as in vulval muscle. (G) Quantification of average whole animal fluorescence from P_skr−5∷GFP in animals of the indicated genotype. Error bars indicate s.e.m. ^***P<0.001, ^**P<0.01 by ANOVA/Dunnetts post hoc comparison with wild type. Bar, 100 μm. (H, I) Co-expression of Ub^G76V-GFP (green, top panels) and mRFP (red, bottom panels) proteins in C. elegans at either 24 or 48 h after L4 stage for (H) skr-5(ok3068) single mutants or (I) let-23(sa62) skr-5(ok3068) double mutants. Bar, 50 μm.

A hypothetical model for the role of EGF signalling and the UFD complex in ageing animals. (A) Larvae maintain protein homoeostasis primarily via chaperones. In larvae, the primary function of the UPS is likely a combination of protein synthesis quality control (e.g. ERAD) and targeted destruction of specific regulatory proteins (e.g. cyclins). Small HSP proteins (brown trapezoid) prevent proteins from unfolding and keep unfolded or misfolded proteins from aggregating. (B) EGF switches adults towards maintaining protein homoeostasis via the UPS. As animals grow older, the number of unfolded and oxidized proteins increases, perhaps in response to a buildup in ROS over time. The EGF/Ras/MAPK pathway becomes activated, triggering a change in gene expression by the EOR-1 and EOR-2 transcription factors. Small HSP proteins are downregulated, resulting in a greater likelyhood that proteins will become unfolded and aggregate. In contrast, SKR-5 and other UPS-associated genes are upregulated, which target oxidized proteins (red elipse) for ubiquitination. In addition, the UFD complex recognizes multiple oligoubiquitinated proteins and adds additional ubiquitins. The resulting polyubiquitinated proteins (red elipse with yellow ubiquitins attached) are substrates for the 26S proteasome, which degrades them. Eventually, even this maintenance system becomes overwhelmed by the misfolded protein load in very old animals, or perhaps damaged itself, contributing to the animal's final decline.

EGF signalling regulates longevity. (A) Survival curves (number of live animals assayed each day after L4 stage) for the indicated mutants at 20°C, P_log−rank<0.0001, and N=180–447 animals per genotype. (B) RNA from sample pairs of let-23(sa62) and eor-1(cs28) mutants were compared with Agilent oligonucleotide microarrays to identify genes that are upregulated by EGF signalling via EOR-1 activity. Regulated genes were identified by hierarchical clustering and SAM. Relative enrichment in let-23(sa62) versus eor-1(cs28) for mRNAs from select genes is shown, with EGF/EOR-1-upregulated genes in red, downregulated genes in green, and unregulated genes (controls) in grey. (C–F) Oxidative-stress-induced gcs-1 expression was examined by introducing a P_gcs−1∷GFP transgene into (C) wild type, (E) lin-3(e1417), or (F) let-23(sy1) animals and observing their fluorescence. For comparison, (D) wild-type animals carrying the transgene were induced by 1 h exposure to sodium arsenite (5 mM). Constitutive expression is observed in the pharynx of all animals (brackets), whereas expression induced by stress is observed in the intestine (arrowheads). Error bars indicate s.e.m. Bar, 100 μm.

EGF signalling regulates the aggregation of Poly(Q)₄₀∷YFP. Fluorescence from (A–C) wild type, (D–F) let-23(sa62) mutants, and (G–I) eor-1(cs28) mutants containing a transgene that expresses Poly(Q)₄₀∷YFP in epithelial cells from (A, D, G) L4 stage, (B, E, H) 24 h post-L4, and (C, F, I) 48 h post-L4. Bar, 50 μm. (J) Per cent of animals containing aggregates of Poly(Q)₄₀∷YFP at the indicated time after hatching. N=1236–1563 animals per genotype.

Ub^G76V-GFP turnover in adults is mediated by the UFD complex and the EGF/Ras/MAPK-signalling pathway. (A, B) Co-expression of Ub^G76V-GFP (green, top panels) and mRFP (red, bottom panels) proteins in C. elegans at either (A) 24 h or (B) 48 h after L4 stage for the indicated genotypes. (C) Relative levels of Ub^G76V-GFP and mRFP fluorescence are quantified as a ratio (AU indicates arbitrary units) at either 24 h (solid bars) or 48 h (stippled bars) after L4 stage for the indicated genotypes. Hypomorphic mutants for let-60, mek-2, and eor-2 show a modest change in the ratio; it is unclear whether this change is due to residual gene activity in the mutants or indicates that these factors do not exclusively mediate this effect. ^***P<0.0001, ^**P<0.001, ^*P<0.01 by two-way ANOVA/Bonferoni's post hoc comparisons between the 24 and 48-h time points for each genotype. For the statistical effect of genotype on the time-dependent outcome, P<0.0001 for all mutants compared with wild type. Error bars indicate s.e.m., N=15–25 animals per trial. (D) Ub^G76V-GFP and actin were detected by western blot at either 24 or 48 h after L4 for the indicated genotypes. Equivalent amounts of protein were loaded into each lane. Bar, 100 μm.

Increased polyubiquitination in adults requires EGF signalling and the UFD complex. (A) Ubiquitin or actin were detected by western blot at either 24 or 48 h post-L4 as indicated. Equivalent amounts of total protein were loaded into each lane. All lanes are from the same gel. In wild-type animals, polyubiquitinated proteins (75–250 kDa) begin to accumulate 48 h after L4 (60% increase compared with animals at 24 h post-L4). Mutations that reduce UFD complex activity or EGF/Ras/MAPK signalling reduce such accumulation. (B) Relative levels of polyubiquitinated proteins (area of interest included 75–250 kDa) and actin were quantified as a ratio (arbitrary units) at either 24 or 48 h (as indicated) after L4 stage for the given genotypes, and normalized to the wild-type sample at 48 h from the same western blot. The mean normalized ratios from six blots are indicated. ^**P<0.01, ^*P<0.05 by ANOVA/Dunnetts post hoc comparison with wild type. Error bars indicate s.e.m. (C, D) Survival curves (number of live animals assayed each day after hatching) for animals of the indicated genotype at 20°C. P_log−rank<0.0001, N=281–447 animals per genotype. (E, F) Oxidative-stress-induced gcs-1 expression was examined by introducing a P_gcs−1∷GFP transgene into (E) wild type and (F) hecd-1 mutants. Constitutive expression is observed in the pharynx of all animals (brackets), whereas expression induced by stress is observed in the intestine (arrowheads). Bar, 100 μm.