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. 2019 Mar 12;8(3):236.
doi: 10.3390/cells8030236.

ATG-18 and EPG-6 Are Both Required for Autophagy but Differentially Contribute to Lifespan Control in Caenorhabditis elegans

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Free PMC article

ATG-18 and EPG-6 Are Both Required for Autophagy but Differentially Contribute to Lifespan Control in Caenorhabditis elegans

Zsuzsanna Takacs et al. Cells. .
Free PMC article

Abstract

During macroautophagy, the human WIPI (WD-repeat protein interacting with phosphoinositides) proteins (WIPI1⁻4) function as phosphatidylinositol 3-phosphate effectors at the nascent autophagosome. Likewise, the two WIPI homologues in Caenorhabditis elegans, ATG-18 and EPG-6, play important roles in autophagy, whereby ATG-18 is considered to act upstream of EPG-6 at the onset of autophagy. Due to its essential role in autophagy, ATG-18 was found to be also essential for lifespan extension in Caenorhabditis elegans; however, this has not yet been addressed with regard to EPG-6. Here, we wished to address this point and generated mutant strains that expressed the autophagy marker GFP::LGG-1 (GFP-LC3 in mammals) and harbored functional deletions of either atg-18 (atg18(gk378)), epg-6 (epg-6(bp242)) or both (atg-18(gk378);epg-6(bp242)). Using quantitative fluorescence microscopy, Western blotting, and lifespan assessments, we provide evidence that in the absence of either ATG-18 or EPG-6 autophagy was impaired, and while atg-18 mutant animals showed a short-lived phenotype, lifespan was significantly increased in epg-6 mutant animals. We speculate that the long-lived phenotype of epg-6 mutant animals points towards an autophagy-independent function of EPG-6 in lifespan control that warrants further mechanistic investigations in future studies.

Keywords: ATG-18; EPG-6; GFP::LGG-1; WIPI3; WIPI4; autophagy; lifespan.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Viability and brood size decreased in the absence of functional autophagy related 18 (ATG-18). (A) Light microscopy images of Caenorhabditis elegans wild-type (N2), atg-18(gk378), epg-6(bp242), and atg-18(gk378);epg-6(bp242) strains were taken using a 1.2× objective and representative images are shown, n ≥ 9. (B) Single hermaphrodites were transferred to nematode growth medium (NGM) plates and the total number of eggs during the reproductive period per hermaphrodite was counted (n > 4). (C) The number of eggs hatching and reaching the larval stage 4 (L4)/adult stage was counted, and the percentages, based on the total egg number, was calculated. Mean values (+STDEV) of the number of eggs (B) and the brood size (C) are shown, and significant differences compared to wild-type indicated ** p < 0.01, *** p < 0.001, one-way ANOVA.
Figure 2
Figure 2
GFP::LGG-1 puncta formation decreased in early embryos, but enlarged puncta accumulate in late-stage embryos of atg-18 and epg-6 mutant animals. GFP::LGG-1 puncta formation was analyzed in wild-type, atg-18(gk378), and epg-6(bp242) embryos expressing the adIS2122 transgene. The scheme in (A) indicates the developmental stage where GFP::LGG-1 puncta assessments were conducted, and the images in (B) show early- and late-stage embryos as examples. Fluorescence microscopy images were quantified using the stress granule counter plugin of ImageJ. The average number (C) and the average size (D) of GFP::LGG-1 puncta were calculated and representative images are shown (E,F). Scale bar: 20 μm. Significances were calculated using the Kruskal–Wallis test, ns: not significant, ** p < 0.01, *** p < 0.001. For more information, see Data S1.
Figure 3
Figure 3
GFP::LGG-1 puncta accumulated in L1 larvae. GFP::LGG-1 puncta formation was analysed in the head and seam cells of wild-type, atg-18(gk378), and epg-6(bp242) L1 larvae expressing the adIS2122 transgene. The scheme in (A) indicates the developmental larval stage (L1) where GFP::LGG-1 puncta assessments were conducted, and the images in (B) show head and mid-body regions, the latter with seam cells, as examples. Fluorescence microscopy images were quantified using the stress granule counter plugin of ImageJ. The average number (C) and size (D) of GFP::LGG-1 puncta were calculated. Representative images are shown (E). Scale bar: 20 μm. Significances calculated using the Kruskal–Wallis test, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. For more information, see Data S1.
Figure 4
Figure 4
GFP::LGG-1 puncta accumulated in L2/3 larvae in the head but decreased in numbers in seam cells. GFP::LGG-1 puncta formation was analysed in the head and seam cells of wild-type, atg-18(gk378), and epg-6(bp242) L2/3 larvae expressing the adIS2122 transgene. The scheme in (A) indicates the developmental larval stage (L2/3) where GFP::LGG-1 puncta assessments were conducted, and the images in (B) show head and mid-body regions, the latter with seam cells, as examples. Fluorescence microscopy images were quantified using the stress granule counter plugin of ImageJ. The average number (C) and size (D) of GFP::LGG-1 puncta were calculated. Representative images are shown (E). Scale bar: 20 μm. Significances calculated using the Kruskal–Wallis test, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. For more information, see Data S1.
Figure 5
Figure 5
GFP::LGG-1 puncta accumulated in L4 larvae in the head but decreased in numbers in seam cells. GFP::LGG-1 puncta formation was analysed in the head and seam cells of wild-type, atg-18(gk378), and epg-6(bp242) L4 larvae expressing the adIS2122 transgene. The scheme in (A) indicates the developmental larval stage (L4) where GFP::LGG-1 puncta assessments were conducted, and the images in (B) show head and mid-body regions, the latter with seam cells, as examples. Fluorescence microscopy images were quantified using the stress granule counter plugin of ImageJ. The average number (C) and size (D) of GFP::LGG-1 puncta were calculated. Representative images are shown (E). Scale bar: 20 μm. Significances calculated using the Kruskal–Wallis test, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. For more information, see Data S1.
Figure 6
Figure 6
Western blot analysis of starved L1 animals revealed a block in autophagic flux upon loss of ATG-18 or EPG-6 function. The scheme indicates that GFP::LGG-1 localizes both to the outside and the inside of the autophagosome, hence become degraded after the fusion of the autophagosome with the lysosome (A, left). Cytosolic GFP-LGG-1 as well as membrane-bound GFP::LGG-1-PE and cleaved GFP is detected by Western blotting using GFP::LGG-1 expressing strains along with wild-type N2 control (A, right). Cleaved GFP, which can also be visualized by Western blotting, accumulates upon lysosomal inhibition due to a decelerated degradation using NH4Cl (A, right). L1 animals of wild-type (Control), atg-18(gk378), and epg-6(bp242) expressing the adIS2122 transgene were starved overnight in the presence (+) or absence (-) of NH4Cl, and proteins were extracted for anti-GFP Western blotting (B). A representative short exposure is shown and GFP::LGG-1 and GFP::LGG-1-PE migrations are indicated (B, upper panel). Likewise, a longer exposure is shown and the migration of cleaved GFP indicated (B, lower panel). Protein extracts from equal numbers of L1 larvae were used for anti-GFP western blotting and the appearance of cleaved GFP quantified, n = 3 (C). Significances calculated using two-tailed Student’s t-test, * p < 0.05.
Figure 7
Figure 7
Survival of L1 larvae upon starvation decreased in the absence of ATG-18 and EPG-6. The overall experimental set-up for the survival analysis upon starvation conditions (A). L1 larvae hatched in nutrient-free medium and were incubated in this medium for the indicated time. The larvae were then tested for recovery on bacteria-covered plates, while their movement was recorded. Subsequently, the percentage of animals that recovered from starvation in the presence of food was calculated (B). The percentages of animals that showed movement upon starvation of 1, 2 or 3 days (C, left panel) is shown, as well as the percentages of animals recovering and surviving from the indicated starvation periods in the presence of food: e.g., 1 (+2) stands for 1 day of starvation followed by 2 days of feeding (C, right panel). Likewise, over a period of 1–29 days, the percentages of surviving animals were recorded where atg-18 and/or epg-6 mutant L1 larvae showed a decreased tolerance to starvation (D).
Figure 8
Figure 8
Lifespan increased in the absence of EPG-6. Synchronised animals were grown to L4 larval stage and transferred either to NGM E. coli OP50 plates supplemented with FUDR and counted every other day (A), or they were counted and transferred each day to fresh NGM/E. coli OP50 plates without fluorodeoxyuridine (FUDR) (D). The lifespans of wild-type, atg-18(gk378), epg-6(bp242), and atg-18(gk378);epg-6(bp242) worms were measured. Survival plot and statistical analysis of one representative set out of three independent experiments is shown (B,C,E,F). Statistical analysis was performed using basic survival analysis of the “Online Application for the Survival Analysis of Lifespan Assays Performed in Aging” software. For more information see Data S1.
Figure 9
Figure 9
Model for the role of ATG-18 and EPG-6 in autophagy and longevity in C. elegans. Both ATG-18 and EPG-6 are considered to function in autophagy as PI3P effectors on the nascent autophagosome, equivalent to the role of their homologues in mammals [7]. In this context, EPG-6 is considered to function downstream of ATG-18 during autophagosome formation [7,10]. A novel, autophagy-independent role of EPG-6 is postulated in lifespan control, which is likely connected to germline signaling.

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