The protein L-isoaspartyl-O-methyltransferase functions in the Caenorhabditis elegans stress response

Abstract

The efficient use of nutrients is important in development and aging. In this study, we asked if the protein repair methyltransferase has a related or additional role in energy metabolism and stress response in the nematode Caenorhabditis elegans. Worms lacking the pcm-1 gene encoding this enzyme exhibit reduced longevity as SDS-isolated dauer larvae and as arrested L1 larvae under starvation stress, while overexpression leads to increased adult longevity. These findings led us to question whether pcm-1 deficient C. elegans may have inappropriate metabolic responses to stress. We assayed dauer and dauer-like larvae for starvation survival and observed a two-fold reduction of median survival time for pcm-1 mutants compared to N2 wild-type worms. Under these conditions, pcm-1 deficient dauer larvae had reduced fat stores, suggesting that PCM-1 may have a role in the initiation of the correct metabolic responses to stress starvation. We show expression of the pcm-1 gene in neurons, body wall and reproductive tissues. Upon heat shock and dauer formation-inducing conditions, we observe additional pcm-1 expression in body wall muscle nuclei and actomyosin filaments and in hypodermal cells. These results suggest that this enzyme may be important in stress response pathways, including proper decision making for energy storage.

Fig. 1

Dauer and Dauer-like Larvae Survival. Non-SDS isolated dauer and dauer-like larvae were aged in M9 at 25 °C and survival was assayed at various time points. Error bars indicate standard deviation from three replicate samples of 25–30 animals from the total population and asterisks indicate p-values less than 0.05 when comparing N2 and pcm-1 survival at the specified time point. Similar results were obtained in two replicate experiments at 25 °C.

Fig. 2

Dauer Larvae Recovery. Starved dauer larvae were placed on OP50-seeded, NGM plates at various ages and incubated at 20 °C for two days. Recovery was defined as the ability to exit dauer arrest and is expressed as a percentage of the number of animals originally scored as alive. Error bars indicate the standard deviation obtained from three replicate measurements of the total population.

Fig. 3

Dauer and Dauer-like Larvae Fat Accumulation. N2 and pcm-1 dauer and dauer-like larvae after various times of arrest were stained with Sudan Black and observed at 40 power magnification. Representative images are shown for three independent experiments analyzing worms at day 1 (2 experiments), day 5, day 6, day 10, day 13, day 16, day 20, and day 25. In each case, we observe a progressive decrease in Sudan Black staining in larvae from day 1 of dauer arrest; pcm-1 mutant larvae also appear to have an decrease in initial fat stores in comparison to N2 larvae.

Fig. 4

PCM-1 Expression Pattern. Two constructs were prepared for observing the expression pattern of PCM-1: a pcm-1 promoter GFP fusion, and a PCM-1::GFP fusion. (A) pcm-1 genomic DNA is in the anti-sense direction (a). The PCM-1::GFP fusion was created to included genomic regions that previously rescued pcm-1 dauer formation defects (Banfield et al., 2008) (b). The Ppcm-1::GFP fusion was made by fusing 3 kb upstream of the pcm-1 start site with GFP containing a nuclear localization signal (NLS), which is shown in red (c). Blue represents coding regions while gray represents non-coding regions. (B–D) GFP, DIC II, and merged images of an adult animal containing PCM-1::GFP transgene shows GFP positive areas in unidentified head neurons. PCM-1::GFP expression is also seen in the distal tip (dt), vulva (vul) and spermatheca (spm) (E–G) of adult nematodes as well as in the root ganglia (rg) and nerve cords (vc) (H–J) (A). Transgenic animals were analyzed for GFP expression at 40 power magnification.

Fig. 5

PCM-1 is Expressed in Muscle Tissue after Heat Shock Treatment. Transgenic L4 and young adult animals were incubated at 34 °C for 3 hours before analysis. PCM-1::GFP expression is observed in a striated pattern in body wall muscles of the head of treated animals but not in untreated animals (A–B). Similar striated muscle expression is also seen throughout the midbody of treated animals (C–D). Expression of PCM-1 can also be seen in the nuclei of the body wall muscles. One of the muscles from panel D is enlarged for detail in panel E. Arrows in panels B, C, and D indicate GFP positive muscles. Pictures were taken with identical exposure times and microscope settings.

Fig. 6

PCM-1 Dauer Larvae Expression. PCM-1::GFP animals were allowed to lay eggs on pheromone plates. GFP positive L4 (panels A–C) and dauer larvae (panels D–F) progeny were compared. The expression pattern of PCM-1::GFP is similar in dauer larvae as in L4, with the exception that dauer larvae show more ubiquitous expression including hypodermal cells (as indicated by an arrow in panel E). Pictures were taken with identical exposure times and microscope settings.