A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy

Abstract

Autophagy is important for many physiological processes; and disordered autophagy can contribute to the pathogenesis of a broad range of systemic disorders. C. elegans is a useful model organism for studying the genetics of autophagy, however, current methods for studying autophagy are labor-intensive and not readily amenable to high-throughput procedures. Here we describe a fluorescent reporter, GFP::LGG-1::mKate2, which is useful for monitoring autophagic flux in live animals. In the intestine, the fusion protein is processed by endogenous ATG-4 to generate GFP::LGG-1 and mKate2 proteins. We provide data indicating that the GFP:mKate ratio is a suitable readout for measuring cellular autophagic flux. Using this reporter, we measured autophagic flux in L1 larvae to day 7 adult animals. We show that basal autophagic flux is relatively low during larval development but increases markedly in reproductive adults before decreasing with age. Furthermore, we show that wild-type, eat-2, and daf-2 mutant animals have distinct autophagic flux profiles through post-embryonic development. Finally, we demonstrate the utility of this reporter by performing a high-content small molecule screen to identify compounds that alter autophagic flux in C. elegans.

Keywords: Biomarker; LC3; LGG-1; high-content screening; probe; small molecule.

Figure 1.

AFR is correctly processed and lipidated by the intestinal cells. (A) A schematic showing how the autophagic flux reporter (AFR) is processed. The reporter is expressed as a fusion protein, GFP::LGG-1::mKate2, which is cleaved by ATG-4.1/-4.2 proteases and lipidated by ATG-3 and the conjugation complex. GFP::LGG-1-PE is incorporated into autophagosomal membranes which fuse with lysosomes in an EPG-5-dependent manner. Cleaved mKate2 remains in the cytosol and acts as the internal control. The GFP:mKate2 ratio is a measure of LGG-1 turnover (autophagic flux). (B-C) Western blot analysis of lysates from animals expressing GFP (lane 1), mKate2 (lane 2), GFP::LGG-1::mKate2 (lanes 3), and GFP::LGG-1::mKate2 in the atg-4.1;atg-4.2 double mutant background (lane 4) probed with either anti-GFP (left) or anti-tRFP (right) antibodies. Arrowheads point to the full-length GFP::LGG-1::mKate2 protein band. (*) non-specific band, (#) cleavage product that is only present in the atg-4.1;atg-4.2 double mutants. To avoid signal saturation, a smaller volume of the lysate from animals expressing GFP were loaded (B-C, lane 1). (D) Western blot analysis of lysates of non-transgenic parental worms (lane 1), and transgenic wild-type (lane 2), atg-3(bp412) mutants (lane 3), and epg-5(tm3425) mutants (lane 4) probed with an anti-LGG-1 antibody. Black arrow and arrowhead point to transgenic GFP::LGG-1 protein bands while red arrow and arrowhead points to endogenous LGG-1 protein bands.

Figure 2.

GFP::LGG-1 puncta number is decreased in atg-3(bp412) mutants but increased in size and number in epg-5(tm3425) mutants. Confocal images of the posterior intestine of (A) wild-type, (B) atg-3(bp412) mutants and (C) epg-5(tm3425) mutants expressing the AFR. Insets (lower panels) show higher magnification, single z-slice images of the boxed regions from (a-c). Arrows point to normal GFP::LGG-1 puncta and arrowheads point to large GFP::LGG-1 puncta that only accumulate in epg-5(tm3425) mutants. (D) Quantification of GFP::LGG-1 puncta number per intestinal cell. Error bars denote mean ± SEM. Animals were imaged as day 1 adults. Scale bar in (A) is 50 µm. Scale bar in inset is 10 µm.

Figure 3.

The AFR is sensitive to nutrient deprivation. Starvation induces, a time-dependent decrease in GFP:mKate2 ratio of (A) wild-type animals but not (C) atg-3(bp412) mutants. Each point represents an average of three experiments (~75 animals). Error bars denote mean ± SEM. Western blots of lysates from (B) wild-type or (D) atg-3(bp412) mutants. Each lane contains lysates from exactly 50 day 1 adult animals. Upper panels were probed with an anti-GFP antibody while the lower panels were probed with an anti-tRFP antibody.

Figure 4.

GFP:mKate2 ratio is modulated in autophagy mutants. GFP:mKate2 ratios (A) and representative well images (B) of animals expressing the wild-type AFR or AFR(G116A) mutant reporter. Fed animals are shown in blue. Animals starved for 6 hours are shown in red. GFP:mKate2 ratios of the AFR in (C) various autophagy mutants, (D) let-363(ok3018) mutant and rict-1(ft7) mutant backgrounds. All data are from day 1 adults and were normalized to the fed wild-typed control. All animals carried the AFR transgene. Data are an average of three independent experiments except for lgg-3 and atg-4.2 mutants which are an average of two independent experiments. For let-363(ok3018) which are homozygous lethal at the L3 stage, data were normalized to the L3 stage wild-type control. Error bars denote mean ± SEM.

Figure 5.

Autophagy is differentially regulated during post-embryonic development and aging. Autophagic flux profiles of (A) wild-type and atg-3(bp412) mutants measured from L1 larvae to D7 adults. Autophagic flux profiles of (B) epg-5(tm3425) and (C) atg-9(bp564) mutants. Autophagic flux profiles of (D) daf-2(e1370) mutants and controls upshifted to 25°C for 24 hours. Autophagic flux profiles of (E) eat-2(ad465) mutants. Data were normalized to wild-type L4 stage animals for comparison. Data are an average of three independent experiments. All animals carried the AFR transgene. Error bars denote mean ± SEM.

Figure 6.

A high-content small molecule screen identifies autophagy modulating compounds. (A) GFP:mKate2 ratio of 154 compounds from the Autophagy compounds library (Selleckchem). Each compound was used at a final concentration of 25 µM. There were approximately 30 L4 stage animals per well. Treatment time was 24 hours. Ratios were normalized to the DMSO control. Dots above the blue dotted line represents compounds that increased the GFP:mKate2 ratio above 1.5. Dots below the red dotted line represents compounds that decreased the GFP:mKate2 ratio below 0.5. Representative well images are shown in (B). A complete list of compound names, descriptions and corresponding GFP:mKate2 ratios can be found in Table S2. Eight-point dose response curves of wild-type;AFR animals treated with (C) DMSO, (D) NVP-BGT226, (E) BAY 11-7082, and (F) SRT1720. Eight-point dose response curves of atg-3(bp412);AFR animals treated with (G) DMSO, (H) NVP-BGT226, (I) BAY 11-7082, (J) DMSO, and (K) SRT1720. The SRT1720 drug dose-response experiments on atg-3 mutant animals (J-K) were performed on different dates than the other experiments. As such, we included the DMSO control performed on the same dates for comparison. Graphs are representative of three independent experiments. Error bars denote mean ± SEM. All animals carried the AFR transgene.