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Published in final edited form as: Cold Spring Harb Protoc. 2016 Jan 4;2016(1):pdb.prot086496. doi: 10.1101/pdb.prot086496

Detection of Autophagy in Caenorhabditis elegans

Nicholas J Palmisano 1,2, Alicia Meléndez 1,2,*
PMCID: PMC5292878  NIHMSID: NIHMS841850  PMID: 26729905

Abstract

Autophagy is a dynamic and catabolic process that results in the breakdown and recycling of cellular components through the autophagosomal-lysosomal pathway. Many autophagy genes identified in yeast and mammals have orthologs in C. elegans. In recent years, gene inactivation, by RNAi and/or chromosomal mutations, has been useful to probe the functions of autophagy in C. elegans, and a role for autophagy has been shown in multiple processes such as, the adaptation to stress, longevity, cell death, cell growth control, clearance of aggregate prone proteins, degradation of P granules during embryogenesis, and apoptotic cell clearance. Here we discuss some of these roles and describe methods that can be used to study autophagy in C. elegans. Specifically, we summarize how to visualize autophagy in embryos, larva, or adults, how to detect the lipidation of LGG-1 by western blot, and how to inactivate autophagy genes by RNAi.

INTRODUCTION

Autophagy in C. elegans

Autophagy is a lysosomal-mediated pathway resulting in the degradation and recycling of long-lived proteins, protein aggregates, as well as damaged and old organelles (Klionsky, 2004). It is highly conserved and has been shown to be a fundamental catabolic process in eukaryotes that is required for key developmental and pathological events. Autophagy was first described in mammals, through morphological studies of rat liver cells (Deter et al., 1967). However, it was in yeast where many autophagy genes (atg) were discovered, by screening for mutations that decreased the survival of yeast cells under starvation, as well as mutations that disrupted the cytoplasm-to-vacuole targeting (cvt) process (Harding, 1996; Harding et al., 1995; Hutchins and Klionsky, 2001; Klionsky et al., 2003; Thumm et al., 1994; Tsukada and Ohsumi, 1993).

The process of autophagy is composed of several distinct steps: formation of a phagophore (also referred to as an isolation membrane or preautophagosomal structure); elongation and closure of the phagophore to form the double membrane autophagosome; transport and fusion of the autophagosome with a lysosome; and finally, degradation of the autophagosomal contents, and recycling of degraded material (FIG. 1) (Mizushima, 2007; Nakatogawa et al., 2009; Xie and Klionsky, 2007). In addition to fusing with a lysosome, an autophagosome may also fuse with an endosome to form a hybrid organelle called the amphisome (Jing and Tang, 1999; Liou et al., 1997). When an amphisome or autophagosome fuses with a lysosome, it is referred to as an autophagolysosome (or an autolysosome).

Figure 1.

Figure 1

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Autophagy in C. elegans A. The process of autophagy has been delineated by studies in yeast and mammalian cells. We presume that induction of autophagy begins with the activation of UNC-51. B. Autophagosome formation requires the integral protein ATG-9, thought to contribute membrane to the developing autophagosome. C. Nucleation requires the Class III PI3K complex, which recruits downstream autophagy proteins to the isolation membranes (IM) in mammals or pre-autophagosomal structure (PAS) in yeast, through the production of PI3P (light purple). D. Two conjugation complexes (LGG-1 and ATG-12) are required for elongation of the isolation membranes and completion of the developing autophagosome. LGG-1 conjugated to phospatidylethanolamine (PE, red) binds to both the inner and outer membranes of the autophagosome. LGG-1 also has the ability to bind to the autophagic adaptor proteins, such as SQST-1 which bind poly-ubiquitinated aggregates. E. The complete autophagosome eventually fuses with the lysosome leading to the degradation of cargo within the autophagosome.

The evolutionary conservation of autophagy genes between yeast and C. elegans allowed for the identification of genes that encode core components of the autophagic machinery in C. elegans, on the basis of genomic sequence homology (Table 1) (Meléndez and Levine, 2009; Meléndez et al., 2003). Genetic screens for mutations that disrupt the degradation of P granules, has recently discovered autophagy genes not previously identified in C. elegans on the basis of sequence homology, including: epg-1, the ortholog of yeast ATG13, and epg-8, the ortholog of yeast ATG14 (Table 1) (Tian et al., 2009; Yang and Zhang, 2011). Although, the similarities between S. cerevisiae, mammals, and C. elegans autophagy proteins suggest that the molecular mechanisms of autophagosome formation may be conserved (FIG. 1 and Table 1) (Meléndez and Levine, 2009), genes recently identified in C. elegans that do not have a yeast ortholog may indicate that autophagy involves more complex membrane dynamics in higher eukaryotes. It is important to uncover further details about the roles of autophagy genes in autophagosome formation and maturation in C. elegans, and the role of these genes in different settings where autophagy is required.

Table 1:

C. elegans autophagy genes

C. elegans Atg gene Allele Yeast/Mammalian ortholog Phenotype in C. elegans Reference
let-363R h98 TOR1,2/mTOR Let, LL (Brown et al. 1994; Noda and Ohsumi 1998; Vellai et al. 2003; Jia et al. 2004; Hansen et al. 2007; Hansen et al. 2008)
unc-51R e369 ATG1/Ulk1/2 Unc, AbD, Pg, Egl (Hedgecock et al. 1985; Ogura et al. 1994; Matsuura et al. 1997; Kuroyanagi et al. 1998; Meléndez et al. 2003; Zhang et al. 2009)
epg-1R bp414 ATG13/Atg13 Dv, Pg (Funakoshi et al. 1997; Chan et al. 2009; Tian et al. 2009)
bec-1R ok691 ATG6,VPS30/beclin 1 Let, AbD, St, SL, Pg pQ (Seaman et al. 1997; Kametaka et al. 1998; Kihara et al. 2001; Meléndez et al. 2003; Takacs-Vellai et al. 2005; Jia et al. 2007; Hansen et al. 2008; Zhao et al. 2009; Ruck et al. 2011)
let-512/vps-34R h797 VPS34/Vps34 Let, SL, Pg (Seglen and Gordon 1982; Volinia et al. 1995; Roggo et al. 2002; Zhao et al. 2009; Ruck et al. 2011)
ZK930.1R ok3132 VPS15/p150 ND (Panaretou et al. 1997; Kihara et al. 2001; Kovács et al. 2003)
epg-8R bp251 ATG14/Atg14L, Barkor Dv, Pg (Kihara et al. 2001; Obara et al. 2006; Sun et al. 2008); (Fan et al. 2011; Yang and Zhang 2011)
epg-6 bp242 -/WIPI4 Dv, Pg (Lu et al. 2011)
epg-3R bp405 -/VMP1 Dv, Pg (Tian et al. 2010)
epg-4R bp425 -/EI24,PIG8 Dv, Pg (Tian et al. 2010)
atg-3 bp412 ATG3/Atg3 Pg (Tanida et al. 2002; Zhang et al. 2009)
atg-4.1*R tm3949 ATG4/Atg4 Pg (Kirisako et al. 2000; Tanida et al. 2004; Zhang et al. 2009)
atg-4.2*R tm3948 ATG4/Atg4 ND (Kirisako et al. 2000; Tanida et al. 2004; Zhang et al. 2009)
atg-5 bp545 ATG5/Atg5 ND (Mizushima et al. 1998; Mizushima et al. 2001; Tian et al. 2010)
atg7/M7.5R tm831 ATG7/Atg7 AbD, SL, Pg, pQ (Kim et al. 1999; Tanida et al. 2001; Meléndez et al. 2003)
Igg-1R bp500, tm3489 ATG8/LC3 Let, Dv, AbD, SL, Pg (Kirisako et al. 2000; He et al. 2003; Meléndez et al. 2003); (Zhang et al. 2009; Alberti et al. 2010)
Igg-2R - ATG8/LC3 Let, Dv, AbD, SL, Pg (Kirisako et al. 2000; He et al. 2003; Meléndez et al. 2003; Zhang et al. 2009; Alberti et al. 2010)
atg-10R bp588 ATG10/Atg10 ND (Takahiro Shintani 1999; Mizushima et al. 2002; Meléndez et al. 2003; Tian et al. 2010)
Igg-3R gk1857 ATG12/Atg12 SL, Pg (Mizushima et al. 1998; Meléndez et al. 2003; Hars et al. 2007)
atg-16.1*R - ATG16/Atg16L1 ND (Kuma et al. 2002; Mizushima et al. 2003; Tian et al. 2010)
atg-16.2*R ok3224 ATG16/Atg16L1 ND (Kuma et al. 2002; Mizushima et al. 2003; Tian et al. 2010)
atg-2 bp576 ATG2/Atg2 (Shintani 2001; Wang et al. 2001; Lu et al. 2011)
atg-9R bp564 ATG9/Atg9 (Noda et al. 2000; Yamada et al. 2005; Reggiori 2006)
atg-18R gk378 ATG18/WIPI1/2 Let, AbD, Pg, pQ (Barth 2001; Meléndez et al. 2003; Jia et al. 2007); (Polson et al. 2010; Tian et al. 2010)
epg-2 bp287 -/- Pg (Tian et al. 2010)
epg-5R bp450 -/ KIAA1632 Dv, Pg (Tian et al. 2010)
sepa-1 bp402 -/- Pg (Zhang et al. 2009)
T12G3.1 ok2892 -/p62(SQSTM1) (Tian et al. 2010; Lu et al. 2011)
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Let= Lethal; Unc= uncoordinated; Dv = Decreased viability of L1s during starvation; AbD= Abnormal Dauer; St= Sterile; LL= Long lifespan; SL= Short Lifespan; Pg= P granule accumulation; Egl= Egg laying defective; pQ= polyQ expansion susceptibility; ND= Not determined

*

Paralogs in C. elegans

R

RNAi clone available

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The role of autophagy genes in C. elegans development has emerged from studies using chromosomal mutations or RNA interference against autophagy genes. Chromosomal mutations exist for many of the autophagy genes found in C. elegans and many RNAi clones are available (Table 1).

Autophagy in C. elegans Development and Aging

L1 arrest after starvation:

Autophagy plays a role mediating the developmental changes associated with survival during extracellular and/or intracellular stress, such as starvation (Levine and Klionsky, 2004). In the absence of food, L1 larvae undergo a reversible developmental arrest and can survive for 1–2 weeks (Johnson et al., 1984). The insulin/IGF-1 signaling pathway, composed of the insulin-like/IGF-1 receptor daf-2 and the FOXO transcription factor daf-16, is involved in regulating L1 arrest triggered by starvation (Baugh and Sternberg, 2006; Fukuyama et al., 2006; Gems, 1998). Interestingly, reduced levels of autophagy have been shown to greatly decrease the survival of starved L1 larvae, emphasizing the importance of autophagy during early stages of development (Alberti et al., 2010; Kang et al., 2007; Lu et al., 2011; Tian et al., 2009; Tian et al., 2010; Yang and Zhang, 2011).

Dauer development:

During the first larval molt, animals that are exposed to a limited food supply develop into an alternate L3 larval stage termed dauer (Albert et al., 1981). Dauer development is associated with morphological and behavioral changes that allow for survival under harsh conditions and stress (Cassada and Russell, 1975; Golden and Riddle, 1984). The regulation of dauer development has been well characterized and requires the IGF-1/insulin-like, guanylyl cyclase, and TGF-β signaling pathways, as mutations in any of these pathways can result in a dauer constitutive phenotype (Daf-c) or a dauer defective phenotype (Daf-d) (Birnby et al., 2000; da Graca et al., 2004; Estevez et al., 1993; Gottlieb and Ruvkun, 1994; Inoue and Thomas, 2000; Patterson et al., 1997; Ren, 1996; Schackwitz, 1996; Thomas et al., 1993). Dauer development is associated with an increase in autophagy, which appears to be required for the cell remodeling associated with proper dauer formation (Meléndez et al., 2003).

Longevity pathways:

In C. elegans, aging is controlled by multiple longevity pathways, such as insulin-like growth factor signaling, TOR signaling, dietary restriction, mitochondrial activity, and germline signaling (Antebi, 2007). Recent genetic studies suggest that autophagy interacts with many of these longevity signals to regulate C. elegans aging (Hansen et al., 2008; Lapierre et al., 2011; Meléndez et al., 2003; Toth et al., 2008). Insulin/IGF-1R/daf-2 mutants display an increase in autophagy, as detected by an increase in the number of punctate structures labeled by the autophagy marker, GFP::LGG-1, in hypodermal seam cells, a cell type commonly used to visualize autophagy in C. elegans (FIG. 2) (Hansen et al., 2008; Meléndez et al., 2003). A reduction in autophagy during development, or only during adulthood, shortens the long lifespan of daf-2 mutants (Hansen et al., 2008; Hars et al., 2007; Meléndez et al., 2003). Reduced food intake without malnutrition, otherwise referred to as dietary restriction, occurs in eat-2 mutants (Avery, 1993). These animals lack a nicotinic acetylcholine receptor specific to the pharynx, thereby exhibiting reduced pharyngeal pumping, and have an extended lifespan phenotype (Lakowski and Hekimi, 1998; Raizen et al., 1995). Consistent with a role for TOR in dietary restriction, eat-2 mutants have reduced TOR signaling, display an increase in autophagy, and require autophagy for their long-lived phenotype (Hansen et al., 2008; Jia and Levine, 2007; Toth et al., 2008). The reduction in mitochondrial respiration in isp-1 mutants extends lifespan (Dillin et al., 2002; Lee et al., 2003), and this phenotype is also dependent on autophagy (Toth et al., 2008). Finally, glp-1/Notch germline-less mutants induce autophagy, and require autophagy for lifespan extension (Lapierre et al., 2011). Interestingly, HLH-30, the ortholog of the mammalian TFEB transcription factor, is required for the lifespan extension associated with the longevity pathways described above, and also regulates autophagy (Lapierre et al., 2013). In conclusion, autophagy is required as part of most longevity pathways in C. elegans, the only exception thus far being the longevity associated with a reduction in protein translation (Hansen et al., 2008; Pan et al., 2007).

Figure 2.

Figure 2

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GFP::LGG-1 expression in hypodermal seam cells of daf-2(e1370) mutants A. daf-2(e1370) mutants grown on OP50 E. coli, at 15°C, display a diffuse localization of GFP::LGG-1. B. daf-2(e1370) mutants grown on OP50 E. coli, at 25°C, display an increase in GFP::LGG-1 positive puncta (up to 12 GFP::LGG-1 positive puncta/seam cell) that represent early autophagic structures or autophagosomes. C. daf-2(e1370) mutants grown on control RNAi E. coli (transformed with empty vector, L4440), at 25°C, display the characteristic GFP::LGG-1 positive punctate structures. D. daf-2(e1370) mutants fed bec-1 RNAi, and raised at 25°C, display an increase in GFP::LGG-1 expression and large GFP::LGG-1 positive aggregates

Degradation of paternal mitochondria:

Directly after fertilization, autophagy is induced resulting in the elimination of spermatozoon specific organelles, including paternal mitochondria (Al Rawi et al., 2011; Sato and Sato, 2011). Whether autophagy also acts in higher eukaryotes to degrade paternal mitochondria is not known, however, an increase in ubiquitination and the localization of LC3 near the sperm mid-piece at the point of entry, may suggest that this is the case in fertilized mouse zygotes (Al Rawi et al., 2011).

Autophagy in apoptosis, necrosis and cell clearance:

Although autophagy has a role in homeostasis as an important pro-survival mechanism in response to stress, an excess in autophagy may result in cell death (Kang et al., 2007). Autophagy is also required for necrotic cell death, a type of cell death characterized by the loss of plasma membrane integrity (Samara et al., 2008; Toth et al., 2007). Additionally, similar to mammals, BEC-1, a component of the class III Phosphatidylinositol 3-Kinase (PI3K) complex (FIG. 1), interacts with the anti-apoptotic ortholog of Bcl-2, CED-9, suggesting cross-talk between autophagy and apoptosis (Erdelyi et al., 2011; Takacs-Vellai et al., 2005). Autophagy proteins have been shown to play a role in the proper degradation of apoptotic cell corpses in C. elegans, since in autophagy deficient animals, apoptotic cells are internalized, but not properly degraded (Li et al., 2012; Ruck et al., 2011). Interestingly, rescue experiments indicate that autophagy genes are required within the engulfing cell to promote apoptotic cell degradation (Li et al., 2012).

Detecting autophagy in C. elegans:

Autophagy can be monitored by transmission electron microscopy (TEM), fluorescent image analysis of the GFP::LGG-1 reporter or other autophagy reporters (Table 2), and by western blot, evaluating LGG-1 lipidation. It should be noted that an increase in the number of autophagosomes does not necessarily reflect an induction of autophagy (Klionsky, 2012), and is therefore important to distinguish between induction of autophagy, an increase in autophagic flux, and the accumulation of autophagosomes due to inefficient or blocked autophagy (Klionsky, 2012). Usually, it is useful to infer the turnover of autophagosomes in the presence and absence of lysosomal degradation. In C. elegans, this may be achieved by RNAi knockdown of genes with lysosomal function, such as cup-5 (Fares and Greenwald, 2001; Kostich et al., 2000; Sun et al., 2011), or the addition of inhibitors such as bafilomycin A1, or chloroquine, routinely used in mammalian cells, which have also been successful in C. elegans (Ji et al., 2006; Oka and Futai, 2000; Pivtoraiko et al., 2010). Clearly, the use of multiple assays to verify an increase in functional autophagy is recommended. A comprehensive list of guidelines was recently reported (Klionsky et al., 2012). Here we describe four protocols for the basic study of autophagy in C. elegans: detection of autophagy using GFP::LGG-1, autophagy in embryos, western blotting to evaluate lipidation of LGG-1, and RNAi as a method to target the knockdown of autophagy genes.

Table 2.

Fluorescent reporters for monitoring Autophagy in C. elegans

Protein Function Tissue Expression¥ TransgenesΦ References
LGG-1 Microtubule – associated protein-1/Ubiquitin-like protein Intestine, Hypodermis, Muscle, Pharynx, Neurons, Vulva, Somatic Gonad, Germline adIs2122[Plgg-1::GFP::LGG-1; rol-6(su1006)] izEx1[Plgg-1::GFP::LGG-1; rol-6(su1006)] izEx5[Plgg-1::GFP::LGG-1; Podr-1::RFP] vkEx1093[Pnhx-2::mCherry::LGG-1] dkIs399[Ppie-1::GFP::lgg-1, unc-119 (+)] Is(Ppie-1::GFP::mCherry::LGG-1; unc-119(+)) Ex[Plgg-1::DsRED::LGG-1; Pmyo-2::GFP] (Melendez et al. 2003; Kang et al. 2007; Samara et al. 2008; Gosai et al. 2010; Manil-Segalen et al. 2014)
LGG-2 Ubiquitin-like protein Hypodermis, Intestine, Vulva, Pharynx, Neurons, Muscle RD108 Ex[Plgg-2::GFP::LGG-2; rol-6(su1006)] RD217 unc119(ed3)III; Ex[unc-119(+); Ppie-1::gfp::mcherry::lgg-1] VIG9 unc119(ed3)III; Is[unc-119(+); Plgg-2::gfp::lgg-2] (Alberti et al. 2010; Manil-Segalen et al. 2014)
DFCP1 Double FYVE-Containing Protein Head, Tail, Vulva, Neurons bpIs168[Pnfya-1::DFCP1::GFP; unc-76(+)] (Derubeis et al. 2000; Cheung et al. 2001; Axe et al. 2008; Tian et al. 2010)
ATG-16.1 WD repeat-containing protein Intestine, Head, Pharynx, Muscle, Neurons [Patg-16.1::ATG-16.1::GFP; rol-6(su10006)] (Zhang et al. 2013)
ATG-16.2 WD repeat-containing protein Intestine, Head, Pharynx, Muscle, Neurons [Patg-16.2::ATG-16.2::GFP; rol-6(su10006)] (Zhang et al. 2013)
ATG-9 Integral Membrane Protein Head, Tail, Vulva, Neurons bpIs211[Pnfya-1::ATG-9::GFP; unc-76(+)] (Noda et al. 2000; Lu et al. 2011; Liang et al. 2012; Lin et al. 2013)
EPG-1 Atg13 homolog Neurons, Pharynx, Muscle bpIs175[Pepg-1::EPG-1::GFP; rol-6(su1006)] (Tian et al. 2009)
EPG-9 Atg101 homolog Intestine, Pharynx, Neurons bpIs214[Pepg-9::EPG-9::GFP; unc-76(+)] (Liang et al. 2012)
BEC-1 Coiled-Coil protein Intestine, Hypodermis, Vulva, Neurons, Somatic Gonad swEx520 [Pbec-1::BEC-1::GFP; rol-6(su1006)] grEx129[Pbec-1::BEC-1::mRFP; lin-15(+)] Ex[Pced-1::mCherry::BEC-1; rol-6(su1006)] Ex[Pegl1-::mCherry::BEC-1; rol-6(su1006)] (Takacs-Vellai et al. 2005; Rowland et al. 2006; Ruck et al. 2011; Huang et al. 2012)
SQST-1 p62/Autophagy adaptor protein Hypodermis, Neurons, Intestine, Vulva, Muscle bpIs151[Psqst-1::SQST-1::GFP; unc-76(+)] (Hunt-Newbury et al. 2007; Pankiv et al. 2007; Tian et al. 2010)
SEPA-1 Autophagy adaptor protein Intestine, Head, Tail bpIs131[Psepa-1::SEPA-1::GFP; unc-76(+)] (Zhang et al. 2009; Tian et al. 2010)
PGL-1 RNA-binding protein/P-granule component P-granules, Intestine bnIs1[Ppie-1::GFP::PGL-1; unc-119(+)] bnIs26[Pelt-2::PGL-1::GFP; Pmyo-2::mCherry] (Cheeks et al. 2004; Zhang et al. 2009; Updike et al. 2011)
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¥

Tissue expression may vary depending on the specific promoter used

Φ

Transgenes shown are those found in autophagy studies or those which may be beneficial in autophagy studies; Additional transgenes may be available for each gene

Alberti A, Michelet X, Djeddi A, Legouis R. 2010. The autophagosomal protein LGG-2 acts synergistically with LGG-1 in dauer formation and longevity in C. elegans. Autophagy 6: 622–633.

Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, Griffiths G, Ktistakis NT. 2008. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. The Journal of cell biology 182: 685–701.

Cheeks RJ, Canman JC, Gabriel WN, Meyer N, Strome S, Goldstein B. 2004. C. elegans PAR proteins function by mobilizing and stabilizing asymmetrically localized protein complexes. Current biology : CB 14: 851–862.

Cheung PC, Trinkle-Mulcahy L, Cohen P, Lucocq JM. 2001. Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi. The Biochemical journal 355: 113–121.

Derubeis AR, Young MF, Jia L, Robey PG, Fisher LW. 2000. Double FYVE-containing protein 1 (DFCP1): isolation, cloning and characterization of a novel FYVE finger protein from a human bone marrow cDNA library. Gene 255: 195–203.

Gosai SJ, Kwak JH, Luke CJ, Long OS, King DE, Kovatch KJ, Johnston PA, Shun TY, Lazo JS, Perlmutter DH et al. 2010. Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin alpha1-antitrypsin Z. PloS one 5: e15460.

Huang S, Jia K, Wang Y, Zhou Z, Levine B. 2012. Autophagy genes function in apoptotic cell corpse clearance during C. elegans embryonic development. Autophagy 9.

Hunt-Newbury R, Viveiros R, Johnsen R, Mah A, Anastas D, Fang L, Halfnight E, Lee D, Lin J, Lorch A et al. 2007. High-throughput in vivo analysis of gene expression in Caenorhabditis elegans. PLoS biology 5: e237.

Kang C, You YJ, Avery L. 2007. Dual roles of autophagy in the survival of Caenorhabditis elegans during starvation. Genes & development 21: 2161–2171.

Liang Q, Yang P, Tian E, Han J, Zhang H. 2012. The C. elegans ATG101 homolog EPG-9 directly interacts with EPG-1/Atg13 and is essential for autophagy. Autophagy 8: 1426–1433.

Lin L, Yang P, Huang X, Zhang H, Lu Q, Zhang H. 2013. The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery. The Journal of cell biology 201: 113–129.

Lu Q, Yang P, Huang X, Hu W, Guo B, Wu F, Lin L, Kovacs AL, Yu L, Zhang H. 2011. The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes. Developmental cell 21: 343–357.

Manil-Segalen M, Lefebvre C, Jenzer C, Trichet M, Boulogne C, Satiat-Jeunemaitre B, Legouis R. 2014. The C. elegans LC3 acts downstream of GABARAP to degrade autophagosomes by interacting with the HOPS subunit VPS39. Developmental cell 28: 43–55.

Melendez A, Talloczy Z, Seaman M, Eskelinen EL, Hall DH, Levine B. 2003. Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301: 1387–1391.

Noda T, Kim J, Huang WP, Baba M, Tokunaga C, Ohsumi Y, Klionsky DJ. 2000. Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. Journal of Cell Biology 148: 465–480.

Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, Overvatn A, Bjorkoy G, Johansen T. 2007. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. The Journal of biological chemistry 282: 24131–24145.

Rowland AM, Richmond JE, Olsen JG, Hall DH, Bamber BA. 2006. Presynaptic terminals independently regulate synaptic clustering and autophagy of GABAA receptors in Caenorhabditis elegans. The Journal of neuroscience : the official journal of the Society for Neuroscience 26: 1711–1720.

Ruck A, Attonito J, Garces KT, Nunez L, Palmisano NJ, Rubel Z, Bai Z, Nguyen KC, Sun L, Grant BD et al. 2011. The Atg6/Vps30/Beclin1 ortholog BEC-1 mediates endocytic retrograde transport in addition to autophagy in C. elegans. Autophagy 7.

Samara C, Syntichaki P, Tavernarakis N. 2008. Autophagy is required for necrotic cell death in Caenorhabditis elegans. Cell death and differentiation 15: 105–112.

Takacs-Vellai K, Vellai T, Puoti A, Passannante M, Wicky C, Streit A, Kovacs AL, Muller F. 2005. Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans. Current biology : CB 15: 1513–1517.

Tian E, Wang F, Han Ja, Zhang H. 2009. epg-1 functions in autophagy-regulated processes and may encode a highly divergent Atg13 homolog in C. elegans. Autophagy 5: 608–615.

Tian Y, Li Z, Hu W, Ren H, Tian E, Zhao Y, Lu Q, Huang X, Yang P, Li X et al. 2010. C. elegans screen identifies autophagy genes specific to multicellular organisms. Cell 141: 1042–1055.

Updike DL, Hachey SJ, Kreher J, Strome S. 2011. P granules extend the nuclear pore complex environment in the C. elegans germ line. The Journal of cell biology 192: 939–948.

Zhang H, Wu F, Wang X, Du H, Wang X, Zhang H. 2013. The two C. elegans ATG-16 homologs have partially redundant functions in the basal autophagy pathway. Autophagy 9: 1965–1974.

Zhang Y, Yan L, Zhou Z, Yang P, Tian E, Zhang K, Zhao Y, Li Z, Song B, Han J et al. 2009. SEPA-1 mediates the specific recognition and degradation of P granule components by autophagy in C. elegans. Cell 136: 308–321.

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