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Review
. 2015;11(1):9-27.
doi: 10.1080/15548627.2014.1003478.

Guidelines for Monitoring Autophagy in Caenorhabditis Elegans

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Free PMC article
Review

Guidelines for Monitoring Autophagy in Caenorhabditis Elegans

Hong Zhang et al. Autophagy. .
Free PMC article

Abstract

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.

Keywords: ASEL, ASE left; ASER, ASE right; ATG, autophagy-related; C. elegans; ER, endoplasmic reticulum; GFP, green fluorescent protein; LC3; MO, membranous organelle; PGL, P-granule abnormality; RER, rough endoplasmic reticulum; SQST, SeQueSTosome related protein; SQSTM1; TEM, transmission electron microscopy; autophagy; development; epg, ectopic PGL granules; lgg-1, LC3, GABARAP and GATE-16 family.

Figures

Figure 1.
Figure 1.
The hierarchical order of autophagy genes in the aggrephagy pathway.
Figure 2.
Figure 2.
LGG-1 pattern during embryogenesis. (A) LGG-1 precursor, LGG-1-I (unlipidated form) and LGG-1-II (lipidated form) in wild-type and atg-4.1, epg-4 and atg-3 mutant embryos. (B–E) LGG-1 forms punctate structures at the ∼100-cell stage ((B)and C), but these structures disappear at the comma stage in wild-type embryos ((D)and E). ((B)and D) DAPI images of the embryos shown in ((C)and E), respectively. ((F)and G) LGG-1 puncta are absent in atg-3 embryos. (F) DAPI image of the embryo shown in (G). (H) LGG-1 accumulates into enlarged cluster-like structures in epg-4 embryos. (I–L) GFP::LGG-1 is largely diffuse in the cytoplasm with some punctate structures. ((I)and K) DIC images of the embryos shown in ((J)and L), respectively. (M–P) GFP::LGG-1 forms aggregates in atg-3 mutant embryos, which colocalize with SQST-1 aggregates. Inserts show magnified views. (M) DAPI image of the embryo shown in (N–P). Scale bar: 5 μm (B-P); 2.5 μm (inserts in N–P). C. elegans embryos remain the same size during embryogenesis. Thus, the scale bar is only shown once in each figure.
Figure 3.
Figure 3.
LGG-2 puncta during embryogenesis. ((A)and B) Confocal images of LGG-1 (red) and LGG-2 (green) in wild-type and epg-3 1-cell embryos. (C) Western blot of protein extracts from mixed-stage GFP::LGG-2 and GFP::LGG-2G130A transgenic worms incubated with anti-GFP antibody. (D) Electron micrographs of GFP::LGG-2 embryos incubated with anti-GFP antibodies. The right panel shows a magnified view of the autophagosome. Scale bar: 200 nm. ((E)and F) Confocal images of GFP::LGG-2 and GFP::LGG-2G130A in 500-cell embryos. GFP::LGG-2 localizes in both a punctate and a diffuse pattern while GFP::LGG-2G130A is diffusely localized in the cytosol. This figure was previously published in references 24 and 25 and is reproduced by permission of Elsevier and Landes Bioscience.
Figure 4.
Figure 4.
Various protein aggregates are degraded by autophagy during embryogenesis. ((A)and B) PGL-1 granules are restricted to germ precursor cells Z2 and Z3 in wild-type embryos. ((C)and D) PGL-1 granules accumulate in somatic cells in atg-3 mutant embryos. ((E)and F) SEPA-1 forms spherical aggregates at the ∼100 cell stage in wild-type embryos. ((G)and H) SEPA-1 aggregates disappear in wild-type comma stage embryos. ((I)and J) SEPA-1 aggregates accumulate in atg-3 mutant embryos throughout embryogenesis. ((K)and L) No SQST-1 aggregates are detected in wild-type embryos. ((M)and N) SQST-1 accumulates into numerous aggregates in atg-3 mutant embryos. (A, C, E, G, I, K, and M) DAPI images of the animals shown in (B, D, F, H, J, L, and N), respectively. Scale bar: 5 μm (A-N).
Figure 5.
Figure 5.
GFP::LGG-1 pattern at post-embryonic stages. (A and B) GFP::LGG-1 is diffusely localized in the hypodermis of wild-type animals. (A) DIC image of the animal shown in (B). (C) GFP::LGG-1 forms numerous small punctate structures in the hypodermis after the animals are starved for 4 h. (D and E) GFP::LGG-1 is diffusely localized in seam cells of wild-type animals. (D) DIC image of the animal shown in (E). (F) GFP::LGG-1 forms many small punctate structures in the seam cells after the animals are starved for 4 h. Inserts show magnified views. (G) Numerous GFP::LGG-1 punctate structures are observed in the intestine of epg-5 mutants. L4 larvae are shown in (A-G). (H and I) A large number of GFP::LGG-1 puncta accumulate in atg-3 mutants at the L1 larval stage (H), but largely disappear at late larval stage (I). Scale bars: 20 μm (A–C, G-I); 10 μm (D–F); 10 μm (inserts in (B and C); 5 μm (inserts in (E and F).
Figure 6.
Figure 6.
Bafilomycin A1 treatment increases GFP::LGG-1 puncta in seam cells of adult animals. (A) A few GFP::LGG-1 puncta form in seam cells in DMSO-treated wild-type day 1 adult animals. (B) The number of GFP::LGG-1 puncta dramatically increases after bafilomycin A1 (Baf A) treatment for 2 h in wild-type day 1 adult animals. Arrows point to puncta. Scale bar: 5 μm.
Figure 7.
Figure 7.
GFP::LGG-1 pattern in L3 larva using different mounting methods. (A) GFP::LGG-1 is largely diffuse in seam cells of L3 larvae mounted with M9 medium on a 2% agarose pad. These animals are not paralyzed. (B) GFP::LGG-1 is largely diffuse in seam cells of fully anesthetized L3 larvae mounted in a final concentration of 0.1 % (w/v; ∼150 mM) NaN3 in M9 medium on a 2% agarose pad with the same concentration of NaN3. (C–D) GFP::LGG-1 forms many different-sized punctate structures (arrows) in the seam cells (and other tissues) of fully anesthetized L3 larvae mounted in a final concentration of 0.2 mM (C), or 2 mM (D) tetramisole in M9 medium on a 2% agarose pad. Punctate structures start appearing 5 min after mounting. (E–F) GFP::LGG-1 forms many different-sized punctate structures (arrows) in the seam cells (and other tissues) of fully anesthetized LC3 larvae mounted in a final concentration of 0.2 mM (C) or 2 mM (D) levamisole in M9 medium on a 2% agarose pad. Punctate structures start appearing 5 min after mounting. Scale bar: 5 μm.
Figure 8.
Figure 8.
Expression of SQST-1 aggregates at larval stages. (A and B) SQST-1::GFP is weakly expressed in wild-type L4 larvae. bpIs151(sqst-1p:: sqst-1::gfp) was used. (C and D) In epg-9 mutants, SQST-1::GFP accumulates into aggregates in multiple tissues. (A and C) DAPI images of the animals shown in (B and D), respectively. Scale bar: 20 μm.
Figure 9.
Figure 9.
Accumulation of SQST-1 aggregates in the intestine in rpl-43 mutants. (A and B) SQST-1::GFP accumulates into a large number of aggregates in the intestine in rpl-43 mutant L4 larvae. (C-F) Accumulation of SQST-1 aggregates in rpl-43 mutant L4 larvae is suppressed by elevated autophagy activity induced by starvation (C and D) or by lin-35 inactivation (E and F). (A, C, and E) DIC images of the animals shown in (B, D, and F), respectively. Scale bar: 20 μm (A–F).
Figure 10.
Figure 10.
HLH-30::GFP translocates to the nucleus upon LET-363/TOR inactivation in adult animals. (A) HLH-30::GFP shows diffuse localization in the cytoplasm in the intestine of day 1 animals. (B) HLH-30::GFP translocates to the nucleus following whole-life let-363/TOR RNAi treatment. Scale bar: 300 μm.
Figure 11.
Figure 11.
Electron microscopy images of autophagic structures in wild-type and autophagy mutants. (A–D) The 3 main autophagic structures (subcompartments): (A) phagophore; (B) autophagosome; (C) light-type, actively digesting autolysosome; (D) autolysosomes with dark undigested content—they may frequently have irregular shapes. (E) Complex myelinated autophagic structure in a hypodermal cell of an unc-51(e361) mutant. (F–H) Three types of secretory vacuoles, which are similar to autolysosomes. Scale bars: 500 nm.

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