Figure 1.
Aggregation of DCAP-1 protein in response to heat-shock.
(A) Representative confocal images of somatic tissues in 1-day adult worms expressing the transcriptional fusion Pdcap-1::gfp (BRF154) or the translational fusion dcap-1::gfp in N2 (BRF155 and BRF261) and germline-deficient glp-1(e2141) worms (BRF219), normally grown at 25°C (-HS) or transiently subjected to heat-shock (+HS, 35°C for 3 h). Arrows point to DCAP-1::GFP granules in body wall muscles that are highly induced upon stress. Asterisks denote the intestinal autofluorescence. Scale bar: 25 µm. (B) Quantification of data in (A). Values on Y axis show the number of granules per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). (C) Endogenous dcap-1(mRNA) levels in 1-day adults N2, grown at 25°C before (-HS) or after heat shock (+HS, 35°C for 3 h) were measured by quantitative RT-PCR and normalized to endogenous ama-1(mRNA) levels. Error bars represent the standard deviation of the means of five independent experiments (p-value = 0.3466, calculated by Student's t-test). (D) Western blot analysis of DCAP-1::GFP protein expression in 1-day adult BRF155 transgenic animals, before (-HS) or after heat shock (+HS), using anti-GFP or anti-Actin (as a loading control) antibodies. Band intensity of DCAP-1::GFP normalized to actin gives a value of 0.83, showing similar protein levels in the two conditions.
Figure 2.
Accumulation of DCAP-1-containing granules under heat-shock is rapid, reversible and sensitive to cgh-1(RNAi).
(A) Representative confocal images of 1-day adult worms expressing dcap-1::gfp (BRF155 and BRF261 strains) normally grown at 25°C (-HS) or transiently subjected to heat-shock (+HS, 35°C for 3 h),following recovery for 3 h at 20°C (Recovery). (B) Quantification of data in (A). (C) Representative confocal images of 1-day adult worms expressing dcap-1::gfp (BRF155) fed from eggs with Control(RNAi) or cgh-1(RNAi) bacteria and grown at 25°C (-HS) or subjected to heat-shock at 35°C for 3 h (+HS). In all cases arrows point to DCAP-1::GFP granules in body wall muscles. (D). Quantification of data in (C). Values on Y axis show the number of granules per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). Scale bar: 25 µm.
Figure 3.
(A) Representative confocal images of 1-day and 5-day adults, grown at 25°C and expressing the transcriptional fusion Pdcap-1::gfp reporter (BRF154) or the translational fusions dcap-1::gfp (BRF155 and BRF261). Arrows point to age-induced DCAP-1::GFP granules. Scale bar: 25 µm. (B) Quantification of data in (A). Values on Y axis show the number of granules per head (mean±SD, see Materials and Methods and Table S4). (C) Endogenous dcap-1(mRNA) levels in 1-day and 7-day N2 adults grown at 25°C before (-HS) or after heat shock (+HS, 35°C for 3 h) were measured by quantitative RT-PCR and normalized to endogenous ama-1(mRNA) levels. Error bars represent the standard deviation of the means of six independent experiments (p-value = 0.9697, calculated by Student's t-test). (D) Western blot analysis of DCAP-1::GFP protein expression in 1-day and 5-day adult BRF155 transgenic animals, grown at 25°C, using anti-GFP or anti-β-Actin (as a loading control) antibodies. Band intensity of DCAP-1::GFP normalized to actin gives a value of 0.95, showing similar protein levels in the two conditions.
Figure 4.
Disruption of mRNA degradation, but not of translation factors, triggers PBs formation that lack ife-2/eIF4E.
(A) Representative confocal images of 1-day adults expressing dcap-1::gfp in N2 (BRF155 and BRF261) fed with ife-2(RNAi), eIF2a(RNAi), rsks-1(RNAi), eIF2Bγ(RNAi) or xrn-1(RNAi) bacteria. (B) Quantification of data in (A). Values on Y axis show the number of granules per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). (C) Endogenous eIF2Bγ(mRNA) levels in 1-day adults BRF155 worms grown at 25°C and fed with eIF2Bγ(RNAi) from eggs, were measured by quantitative RT-PCR and normalized to ama-1(mRNA) levels. Error bars represent the standard deviation of the means of three independent experiments (***p-value<0.0001, in unpaired t-test). (D) Representative confocal images of 1-day adults expressing dcap-1::gfp in N2 (BRF155) or ife-2(ok306) (BRF220) background. (E) Representative confocal images of 1-day adults co-expressing dcap-1::rfp and ife-2::gfp (BRF313), and treated with xrn-1(RNAi) to monitor the lack of co-localization of the two proteins. In all cases, targeted RNAi was initiated from eggs at 25°C in parallel with Control(RNAi) to assess the localization of DCAP-1::GFP into PBs. Asterisks show the intestinal autofluorescence. Scale bar: 25 µm.
Figure 5.
PB components are important for normal lifespan and stress response.
(A) Survival curves of N2, dcap-1 and dcap-2 mutants at 20°C. (B) Survival of adults dcap-1 and dcap-2 mutants in heat-shock (1-day adults at 35°C for 6 h), oxidative stress (1-day adults in 5 mM sodium arsenite for 48 h) and UV-irradiation (5-day adults at 0.2 J/cm2), compared to N2. (C) Survival curves of N2 worms treated post-developmentally with dcap-1/-2(RNAi), patr-1(RNAi) or xrn-1(RNAi) at 20°C. (D) Lifespan of daf-2, daf-2; dcap-1 and daf-2; dcap-2 mutants at 20°C and (E) survival of 1-day adults after heat-shock (35°C for 8 h). (F) Survival of 1-day adult glp-1 germline-deficient mutants that overexpress dcap-1::gfp after heat-shock (at 35°C for 6 h) or oxidative stress (5 mM sodium arsenite for 48 h). In lifespan assays the p-values were determined using the log-rank test (see Tables S5 and S6) and in stress assays the error bars show the SD in unpaired t-tests (see Materials and Methods). ** indicates very significant (p-value 0.001 to 0.01); *** indicates extremely significant (p<0.001).
Figure 6.
Aggregation of TIAR-1 and TIAR-2 proteins in response to stress but not to age.
(A) The domain structure of TIAR proteins, designed using the Prosite MyDomains (http://prosite.expasy.org/mydomains/). The graphic was created using the Exon-Intron Graphic Maker (http://wormweb.org/exonintron). RRM: RNA-recognition motif, GLY_R: Glycine rich domain, GLN_R: Glutamine rich domain. (B) Representative confocal images of 1-day adults of the indicated transgenic strains grown at 25°C (Control 1-d ad), upon heat-shock (HS, 35°C for 3 h), oxidative stress (SA, 15 mM sodium arsenite for 3 h) or compared to 6-day adults under normal conditions. Arrows point to formed granules and quantification of the granule number is shown in the lower panel for each strain. Values on Y axis show the number of granules per head (BRF211), per 100 µM length of excretory cell (BRF255) or per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). Asterisks show the intestinal auto-fluorescence. Scale bar: 25 µm. (C–D) Expression levels of tiar-1 or tiar-2 genes in (C) the indicated transgenic strains expressing gfp::tiar-1 and gfp::tiar-2 by their own promoter (BRF211 and BRF255, respectively) or the muscle specific Pmyo-3::gfp::tiar-2 (BRF310) and (D) N2 worms after heat shock (35°C for 3 h). Quantification of each mRNA level, relative to ama-1(mRNA) levels, in 1-day adults and the mean ± SD of biological triplicates are shown (p>0.05 in Student's t-tests).
Figure 7.
Rapid formation and dissociation of heat-induced SGs.
Representative confocal images of 1-day adults expressing gfp::tiar-1 and gfp::tiar-2 by their own promoter (BRF211 and BRF255, respectively) or the muscle specific Pmyo-3::gfp::tiar-2 (BRF310) under normal growth conditions (-HS) and upon heat-shock (+HS, 35°C for 45 min to 2.5 h), following recovery at 20°C for 2 h. Arrows point to formed granules and quantification of the granule number is shown in the lower panel for each strain. Values on Y axis show the number of granules per head (BRF211), per 100 µM length of excretory cell (BRF255) or per 2500 µm2 per worm (mean±SD, see Materials and Methods and Table S4). Asterisks show the intestinal auto-fluorescence. Scale bar: 25 µm.
Figure 8.
Effects of tiar genes deletion on development, fertility, lifespan and stress survival.
(A) Developmental rate and brood size of tiar-1, tiar-2, tiar-3 and tiar-1;tiar-2 mutant worms, compared to N2. (B) Survival curves of the above mutants or of N2 worms treated post-developmentally with tiar-1(RNAi), tiar-2(RNAi) or tiar-3(RNAi). (C) Survival of adults of the above mutants in oxidative stress (1-day adults at 5 mM sodium arsenite for 48 h), heat-shock (1-day adults at 35°C for 6 h), UV-irradiation (5-day adults at 0.2 J/cm2) or osmotic stress (1-day adults at 400 mM NaCl for 24 h, compared to N2. (D) Survival of tiar-1 mutants expressing the gfp::tiar-1 rescuing transgene in oxidative stress (1-day adults at 5 mM sodium arsenite for 24 h) compared to N2 or tiar-1 animals carrying only the rol-6(su1006) roller marker. In lifespan assays the p-values were determined using the log-rank test (see Tables S5 and S6) and in stress assays the error bars show the SD in unpaired t-tests (see Materials and Methods). ns indicates not significant (p>0.05); * indicates significant (p-value 0.01 to 0.05); ** indicates very significant (p-value 0.001 to 0.01); *** indicates extremely significant (p<0.001).