Figure 5. Reduced insulin/IGF-1-like signaling protects against age-dependent protein insolubility and aggregation.

(A) Sypro Ruby staining revealed a decrease in overall age-dependent protein insolubility in gon-2(−)/gonad-less animals treated with daf-2 RNAi compared to control RNAi (1.6-fold compared to a 3.6-fold increase with age, quantified in the red outlined areas). daf-2 RNAi treatment prevented the insolubility of multiple proteins that appear with age in the total staining of insoluble proteins in control animals. (B) Western blot detection of specific candidates showed a slight delay in insolubility or the absence of insolubility in animals treated with daf-2 RNAi. Quantification of the Western blots: KIN-19: daf-2 RNAi, 2-fold increase with age; control RNAi, 3.4-fold. PAR-5: daf-2 RNAi, 1.8-fold increase with age; control RNAi, 2.1-fold. (C) Decreased insolubility with reduced insulin/IGF-1 signaling is not correlated with a decrease in total protein levels of these proteins. Interestingly, daf-2 RNAi treatment prevented the shift in size of DAF-21 in older animals. (D–E) The strong mutation daf-2(e1370) prevented KIN-19::tagRFP aggregation in the pharynx. (D) daf-2(e1370); Pkin-19::kin-19::tagrfp animals had significantly fewer KIN-19::tagRFP puncta in their anterior pharyngeal bulbs than did wild-type animals expressing Pkin-19::kin-19::tagrfp (Day 6: p<0.0001, Day 12: p<0.0001, Yates' Chi-square test). No further increase in the number of puncta was observed after Day 12 in the daf-2(e1370) background, suggesting that reduced insulin/IGF-1-like signaling somehow caps the process of KIN-19::tagRFP puncta formation. The number of animals is indicated in the bars. (E) KIN-19::tagRFP puncta remained mostly soluble in a daf-2 mutant background. FRAP analysis of a KIN-19::tagRFP puncta in the anterior pharyngeal bulb of daf-2(e1370); Pkin-19::kin-19::tagrfp animal, Day 38 (Laser setting: 10% in 0.8 µm²). As with this example, most KIN-19::tagRFP puncta present in a daf-2 mutant background uniformly lost fluorescence in the whole punta when bleached in a restricted area (Table 1). These results suggested that KIN-19::tagRFP does not aggregate in these puncta. Scale bar: 2 µm.