Tools and methods to analyze autophagy in C. elegans.

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9 December 2014 Methods xxx (2014) xxx–xxx 1

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Tools and methods to analyze autophagy in C. elegans

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Céline Jenzer, Elena Simionato, Renaud Legouis ⇑ Centre de Génétique Moléculaire, CNRS UPR3404, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, 1 Avenue de la Terrasse, 91198 Gif sur Yvette, France

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a r t i c l e

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i n f o

Article history: Received 2 October 2014 Received in revised form 24 November 2014 Accepted 25 November 2014 Available online xxxx

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Keywords: Allophagy Aggrephagy Development Cell death Pathologies Aging

a b s t r a c t For a long time, autophagy has been mainly studied in yeast or mammalian cell lines, and assays for analyzing autophagy in these models have been well described. More recently, the involvement of autophagy in various physiological functions has been investigated in multicellular organisms. Modification of autophagy flux is involved in developmental processes, resistance to stress conditions, aging, cell death and multiple pathologies. So, the use of animal models is essential to understand these processes in the context of different cell types and during the whole life. For ten years, the nematode Caenorhabditis elegans has emerged as a powerful model to analyze autophagy in physiological or pathological contexts. In this article, we present some of the established approaches and the emerging tools available to monitor and manipulate autophagy in C. elegans, and discuss their advantages and limitations. Ó 2014 Published by Elsevier Inc.

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1. Autophagy in Caenorhabditis elegans physiology

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Autophagy is a highly dynamic process, essential to maintain cellular homeostasis in response to the variations of environmental conditions. Autophagy is the main catabolic pathway that allows the degradation of cytoplasmic constituents by the formation of double membrane vesicles, the autophagosomes. Induction of autophagy is done from a cup-shaped membrane structure called phagophore which progressively extends to engulf cytoplasmic components into autophagosome before fusing with lysosome to form autolysosome. Autophagy could also be a selective process which degrades specific cargoes or organelles like mitochondria or peroxysomes (mitophagy, pexophagy). During the last decade, an increasing number of reports revealed that autophagy functions in a large variety of physiological and developmental processes. Identification of the yeast autophagic Atg (autophagy-related) proteins and their mammalian homologs has revealed a globally well conserved machinery impliQ3 cated in autophagosome formation (Table 1). In this regard, animal models are essential to understand the multiple functions of autophagy. Among them, the nematode C. elegans which allows genetic, biochemical and microscopy analyses is a powerful model to identify novel actors and new functions of autophagy during the whole life cycle. In particular, C. elegans was used to study autoph⇑ Corresponding author at: Centre de Génétique Moléculaire, CNRS UPR3404, 1

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Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. E-mail address: [email protected] (R. Legouis).

agy in the context of development, cell survival and cell death, aging, stress response and various pathologies (Fig. 1).

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1.1. Development

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Studies on C. elegans have shown that autophagy displays multiple roles during development, from fertilization and embryogenesis to larval stages (Fig. 1). The earliest developmental process involving autophagy is triggered upon fertilization of the oocyte by the spermatozoon. C. elegans spermatozoa have no flagellum but contain mitochondria as well as nematode-specific membranous organelles, both of which can enter in the ooplasm during fertilization. We and other have shown that the degradation of these paternal organelles is dependant of the formation of autophagosomes few minutes after fertilization [1,2]. The process of the degradation of sperm components by selective autophagy was named allophagy (allogenic organelles autophagy) and is linked to poly-ubiquitination of these organelles [3]. Later on during early embryogenesis, selective autophagy is involved in the removal of protein aggregates. Protein aggregation is a continuous process in living cells, and the selective degradation of ubiquitinated protein aggregates by macroautophagy is called aggrephagy. Recent studies have shown that a variety of maternally inherited germline specific components are selectively removed by autophagy in somatic cells, establishing C. elegans as a multicellular genetic model for understanding the physiological roles of aggrephagy [4]. This selective process, independent of starvation-induced autophagy and strictly regulated during

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http://dx.doi.org/10.1016/j.ymeth.2014.11.019 1046-2023/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: C. Jenzer et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.11.019

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Table 1 Genes used to analyze the autophagic pathway.

*

C. elegans genes

Mammalian homologs

Mutant*/RNAi

References

lgg-1 lgg-2 lgg-3 epg-1 epg-2 epg-3 epg-4 epg-5 epg-6 epg-7 epg-8 epg-9 atg-2 atg-3 atg-4.1 atg4.2 atg-5 atg-7 atg-9 atg-10 atg-18 bec-1 unc-51 rab-7 vps-39 vps-41 sqst-1 sepa-1 pgl-3 let-363

Gabarap Lc3 Atg12 KIAA0652 ? Vpm1 Ei24 mEpg5 Wipi3/4 Fip200 Atg14 Atg101 Atg2 Atg3 Atg4A Atg4B Atg4C Atg4D Atg5 Atg7 Atg9 Atg10 Wipi3/4 Beclin1 Ulk Rab7 Vps39 Vps41 Sqstm1/p62 ? ? Tor

tm348/RNAi tm5755/RNAi RNAi bp414 bp444/RNAi bp405/RNAi bp425/RNAi tm3425/RNAi bp242 tm2508 bp251 bp320 bp576 bp412/RNAi bp410 tm3948 bp546/RNAi bp422/RNAi bp564/RNAi bp588/RNAi gk378/RNAi ok691/RNAi e369/RNAi ok511/RNAi tm2253 ep402 ok2892 bp456 bp439 h98/RNAi

[1,2,5,7,18,19,21,24,29,30,32,34,36,45,52,54,58,68,77,78] [1,5,7,19,32,45,56,68,78] [5,78] [21,30,31,48,51–54] [30,52] [7,30,52,54] [7,21,30,52,54] [21,26,30,52–54] [30,48,53,54] [52,54] [30,31,53–55] [53] [21,30,31,52–54] [5,21,30,31,48,52–54,78] [5,30,52,78,79] [30,48,78,79] [21,30,52,54,78] [1,2,5,7,11,22,28,30,34,37,38,51,52,56,66,68,80] [30,53,54,12,78,79,81] [5,30,52,78] [2,5,21,22,26,28,30,31,38,51–54,12,78,82] [9,11,13–16,20,23,24,27–32,34,37,38,58,12,62,66,72,78,79,82–86] [2,5,18,22–24,28,30,31,36,40,52,54,58,12,77,78,82–85,87] [1,2,7,45,56] [7] [7] [54] [51,52] [51] [13,18,24,45,54,56,57,12,88]

Most used allele is indicated.

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development, is essential for the restriction of germ-cell determinants in the germline precursor cells [5]. Autophagy is also involved in differentiation and organogenesis and mutations in several autophagy genes result in an embryonic lethality [6]. In particular, autophagy appears to play a critical role in the remodeling of several tissues during organogenesis [7], similarly to what have been observed in other organisms [8]. From the first report on autophagy in C. elegans [9], it is known that autophagy is important for several larval developmental stages. Under starvation treatment, the hatched larvae stop their development and remain viable for 1–2 weeks, thank to activation of autophagy. Autophagy also plays a critical role in the differentiation of the dauer larvae, an alternate diapause larval stage which is able to survive for several months in unfavorable environmental conditions [10].

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1.2. Aging

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With an average lifespan of 2–3 weeks, C. elegans is a convenient model to study longevity by using long-lived mutants. Indeed, different mutants lead to a lifespan extension such as dietary restriction (eat-2), germline-less (gpl-1), inhibition of TOR signaling pathway (let-363), reduced activity of the insulin/IGF-1 signaling (daf-2), decreased mitochondrial respiration (clk-1) and reduced translation (rsks-1). Autophagy is essential in the extended lifespan of these mutants. Inactivation of several autophagic genes (lgg-1, atg-18, bec-1, atg-9, atg-7, vps-34) (Table 1) suppresses the long-lived phenotype of dietary restriction mutants [11–13]. Autophagy is also required for the extension of lifespan in daf-2 mutants, TOR inhibition, clk-1 mutants or in deficient calcineurin mutants [9,12,14,15]. Besides, Tavernarakis and colleagues demonstrated that the effect on lifespan extension in animal mutant for cep-1, the worm homolog of p53 (Table 1), is also mediated by autophagy [16]. Modification of cellular lipid content can affect

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lifespan extension in an autophagy dependent manner [17]. In germline-deficient animals, autophagy in concert with lipid homeostasis may modulate the lifespan extension [18]. Lastly, HLH-30, the worm homolog of the transcription factor TFEB, controls the expression of autophagic genes to regulate the lifespan of several longevity models [18,19]. Numerous evidences indicate that in C. elegans, autophagy has a central role in modulating the longevity process but little is known about the molecular mechanisms of this regulation.

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1.3. Cell death

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Cell death is crucial for many aspects of normal animal development and tissue homeostasis, including the removal of damaged or aged cells, tissue sculpting and morphogenesis. Cells may die by a genetically defined programmed cell death (apoptosis) or by necrosis upon a traumatic injury. C. elegans is a well-established model to study the relationships between apoptosis and autophagy. More than 10% of generated cells undergo programmed cell death during embryogenesis and larval development. Cell death also occurs in adult gonads, where a large number of germ cells die by apoptosis. However, interactions between autophagic and apoptotic pathways are complex and not fully understood. BEC-1, the C. elegans ortholog of human Beclin-1 interacts with the antiapoptotic protein CED-9/BCL2 and its inactivation causes an increased number of apoptotic cell corpses in embryos [20]. In addition, several genetic or pharmacological modifications, which cause embryonic lethality, trigger autophagy as part of the cell death response [21,22]. Autophagy is also required for the physiological germ cell death when the apoptotic pathway is impaired [21]. Several studies have demonstrated that autophagy is also required during necrotic cell death and becomes upregulated in the early phase of necrosis [23,24]. Specifically, inactivation of

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Fig. 1. Schematic representation of C. elegans life cycle at 20 °C. The autophagy is essential for various physiological processes during whole life.

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autophagy genes bec-1, unc-51 and lgg-1 (Table 1) partially suppresses necrotic neuronal cell death with toxic ion channel variants [24]. During necrotic cell death, autophagy synergizes with the lysosomal pathway to facilitate necrosis [23]. Furthermore, autophagy is not only involved in cell killing but also in the so-called LC3-associated phagocytosis (LAP). The microtubule-associated protein light-chain 3 (LC3) is an ubiquitin-like protein which plays a key role during the autophagy process (see also Section 2.1). In the LAP process, LC3 is not recruited to a double membrane autophagosome but directly to a single membrane phagosome. This process occurs during the phagocytosis of pathogens, apoptotic and living cells (entosis) [25–27]. LAP is a distinct process from autophagy, but it relies on some members of the classical autophagy pathway. Specifically, Beclin-1, ATG5, ATG7 but not ULK1 are involved in the phagocytosis of dying cells by macrophages [25,27]. In C. elegans adult germline, BEC-1, UNC-51 and ATG-18 are also involved in the clearance of apoptotic cells [27,28]. During larval development, LGG-1, EPG-5, ATG-18 but not UNC-51 (Table 1) are recruited to promote the degradation of apoptotic Q neuroblast [29]. In addition, autophagy genes have also been found to control cell degradation during embryogenesis [30,31]. Mutations in twenty genes acting in various steps of the autophagy pathway result in an increased number of cell corpses and in a delay of their removal during the embryonic development [30,31].

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1.4. Stress and pathologies

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Autophagy mediates the resistance to different kinds of stress such as oxidative stress, anoxia, starvation and pathogens. Inhibition of autophagy genes in C. elegans leads to a reduced survival under certain stress conditions [29,32–35]. During hypoxic injury, inactivation of bec-1, lgg-1, lgg-2 and unc-51 (Table 1) by doublestranded RNA interference (RNAi) or by inhibition of BEC-1 using a phospatidylinositol-3-kinase inhibitor, increases the hypoxiainduced death, arguing for a pro-survival role of autophagy under stress conditions [32]. The autophagy pathway is involved in promoting the degradation of intracellular pathogens and the nematode is a useful system to study host-pathogen interactions in the context of the entire organism. Its rapid life cycle and the fact that C. elegans is bacterivore facilitate the analysis of the host defense response upon an infection. It has been shown that specific pathogens such as Salmonella typhimurium and Staphyloccocus aureus are able to infect and kill the worms [33,34,36]. S. typhimurium is a pathogen that causes a foodborne illness in humans and infects both mammalian intestinal cells and macrophages. Specifically, inactivation by RNAi of autophagy genes (bec-1, atg-7 and lgg-1) decreases the survival rates in S. typhimurium infected animals and abrogates the resistance conferred by the insulin-like signaling pathway [33]. The intestine is the primary site of infection and tissue specific inactivation of bec-1 in intestine, but not in other tissues, reduces the host survival [34]. Recently, autophagy has also been implicated in several pathological conditions. In post-mitotic neurons, autophagy is an essential quality-control system to remove abnormal proteins or aggregates and insures their proper function and survival. Dysregulation of this process causes various neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Amyotrophic Lateral Sclerosis. Abnormal accumulation of autophagosomes is a key feature in several neurodegenerative diseases. C. elegans has been used to study neurodegeneration and to analyze effects of neurotoxic proteins. In a C. elegans model for Alzheimer’s disease, the accumulation of b-amyloid peptide causes an increase of autophagosomes due to the inhibition of autophagosome maturation and/or lysosome function [37]. Furthermore, genetic inactivation of autophagy genes (bec-1, atg-7 and atg-18) (Table 1) increases the accumulation of polyglutamine aggregates inducing muscle toxicity and neurotoxicity [38]. It has also been reported that the autophagosome accumulation observed in neurodegenerative diseases may be due to the disruption of their cellular transport system [39]. Interestingly, in C. elegans, selective autophagy is involved in the degradation of GABA receptors at the neuromuscular junctions [40]. A number of individual reports have shown that autophagy is involved in other cell processes. UNC-51 and BEC-1 function in cell size control and interact genetically with insulin/IGF and transforming growth factor-b (TGF-b) pathways [41]. Autophagy activity also modulates miRNA mediated gene silencing by selectively removing a core component of the miRNA-induced silencing complex [42]. In addition, the autophagy process is activated during the processing of DNA damages through the canonical DNA repair pathways [43].

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2. Visualizing autophagic structures in vivo

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C. elegans is a very convenient system to visualize autophagic structures in vivo. Its small size, 1 mm at adulthood, and transparency combined with the facility to generate transgenic animals explain why in vivo approaches have been widely used. Specific

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fluorescent gene reporters allowed the analysis of both autophagosomal structures, regulatory components, adaptors and cargoes. 2.1. LGG-1 and LGG-2, the C. elegans Atg8/LC3 homologs The ubiquitin-like protein Atg8/LC3 is the only known protein which remains associated to the autophagosome from the early step of biogenesis to the degradation. So, it is the most used marker to monitor autophagy in vivo, from yeast to mammalian cells [44]. While at least 6 homologs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GAB ARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. The first GFP::LGG-1 reporter was generated by Melendez

and colleagues [9] and used to visualize autophagosomes (Fig. 2A). It became rapidly the favorite tool to analyze autophagy in worms (Table 2) [29]. GFP::LGG-2 strain was generated later on, and has been less characterized compared to LGG-1 (Fig. 2B) [45]. Immuno-electron microscopy in embryo revealed that both GFP::LGG-1 and GFP::LGG-2 are bona fide markers of phagophores and autophagosomes (Fig. 3A–D) [7]. GFP::LGG-1 and GFP::LGG-2 present a rather similar, mainly ubiquitous pattern (Fig. 2A and B) with an enrichment of LGG-2 in the nervous system. However, several data demonstrated functional specificities for each gene. First, LGG-1 but not LGG-2 is able to partially complement the yeast atg8 mutant during nitrogen starvation [45]. Second, a genetic analysis of the double mutant revealed a synergistic effect

Fig. 2. Fluorescent images of C. elegans embryos showing the localization of LGG-1 and LGG-2. Confocal pictures of GFP::LGG-1 (A), GFP::LGG-2 (B), GFP::LGG-1(G116A) (C) and GFP::LGG-2(G130A) (D) in 500-cell stages embryos. (A and B) GFP::LGG-1 and GFP::LGG-2 puncta correspond to autophagosomes. (C and D) The non conjugated form of LGG-1 and LGG-2 presents a diffuse localization pattern. (E) Deconvoluated epifluorescence images of GFP::LGG-1 (green) and lysotracker (red) in a 20-cell stage embryo. White arrow indicates a colocalization between autophagosomes and lysosomes. (F) Epifluorescence picture of GFP::mCherry::LGG-1 in a 2-cell stage embryo. The white arrow shows a yellow puncta corresponding to autophagosome whereas the dotted arrow indicates an autolysosome in red. Confocal images of LGG-1 (G) and LGG-2 (H) in a 200-cell stage embryo. The scale bar represents 10 lm.


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C. Jenzer et al. / Methods xxx (2014) xxx–xxx Table 2 Antibodies and transgenic constructs for visualizing autophagy. Genes

Reporters/antibodies

Localization

Stage

References

lgg-1

GFP, DsRed, GFP::Cherry, Cherry, mRFP/antibody

P

[7,9,23,40,49,52]

lgg-1(g116a) lgg-2

GFP GFP Antibody

D P

lgg-2(g130a) bec-1

GFP GFP, mRFP

D P/Pa

atg-4.1 atg-9 atg-18

GFP GFP GFP

D D P

epg-1

GFP

D

epg-2 epg-3 epg-4

Antibody GFP GFP

P D D

epg-5

GFP

D

epg-6 epg-7

GFP GFP

D P

epg-8 lmp-1 sepa-1

GFP GFP GFP, RFP/antibody

D P P/Pa

sqst-1

GFP

P/Pa

pgl-1 pgl-3

GFP/antibody Antibody

P/Pa P/Pa

Adults (intestine, gonad, seam cells, muscles, neurons) L3–L4 (seam cells, intestine, muscles) L1 (Q cell corpses) early and late embryos Late embryos Adults L3 (seam cells) Early and late embryos Late embryos Adults (intestine, neurons, muscles) L3 (seam cells) Early and late embryos Embryos Late embryos Adults (neurons et intestine) Larvae Late embryos Adults (neurons, muscles, sheath cells) Late embryos Late embryos Late embryos Larvae Late embryos Adults (sheath cells) Larvae Embryos Late embryos Larvae (muscles, neurons) Embryos Late embryos Adults (intestine) L1 Late embryos Adults (intestine) Late embryos Embryos L1 Late embryos

[7] [1,45] [45] [20,40]

[48] [53] [22]

[51] [52] [52] [52] [52]

[53] [54] [55] [28] [5,51] [52] [5] [5]

P: puncta; D: diffuse; Pa: patch.

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of lgg-1 and lgg-2 depletion during embryonic and larval development, but also during starvation and aging. Finally, a detailed analysis of allophagy revealed sequential roles for LGG-1 and LGG-2 during the biogenesis and the maturation of autophagosomes [7,46]. In vivo quantification of autophagosomes is mostly achieved by counting GFP::LGG-1 positive dots and can be performed at any stage of development and in all tissues. However, a particular attention should be given to this approach because a high cytosolic diffuse staining has been observed in some cells, and in particular conditions, GFP::LGG-1 and GFP::LGG-2 could form aggregates independently of autophagosome formation [47]. Mutation of the conserved C-terminal glycine in GFP::LGG-1(G116A) or GFP:: LGG-2(G130A) results in a diffuse cytosolic signal demonstrating that lipidation is essential for localization to the autophagosome membrane (see also Section 3.2). In addition, GFP::LGG-1(G116A) or GFP::LGG-2(G130A) provide good controls for in vivo analysis (Fig. 2C and D). It is not known whether LGG-1 and LGG-2 are conjugated by the same proteins because some genes of the conjugating machinery have been duplicated (e.g. atg-4, atg-16) [48]. Furthermore, several groups generated mCherry and RFP reporters for both LGG-1 and LGG-2 allowing for two colors time-lapse analysis. Because these fluorescent reporters have been less characterized, the possibility of aggregation should be carefully considered. Among the various strains generated, the use of tissue- or stage-specific promoters is interesting to dissect specific autophagic processes. Such approaches have been successfully used to analyze autophagosomes in early embryo, in intestinal cells or in various neuronal populations [2,7,23,39,49,50]. For

instance, the generation of a tandem GFP::mCherry::LGG-1 allowed us to monitor the autophagic flux in the early embryo during allophagy (Fig. 2F) (see also Section 3.3).

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2.2. Other markers and dyes

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The identification of C. elegans ATG ortholog genes facilitated the study of the autophagy process. Indeed, several autophagy fluorescent protein reporters such as GFP::BEC-1, GFP::ATG-4.1, GFP::ATG-9 and GFP::ATG-18 have been generated but mostly used to describe their specific expression patterns (Table 2). Nonetheless, they might represent potential complementary tools to further analyze the autophagy process. A detailed analysis of aggrephagy conducted by Zhang and colleagues led to the development of numerous reporters. Specifically during embryogenesis, the degradation of maternally derived germ P granules can be detected by using several GFP tagged EPG proteins (see also Section 4.1) (Table 2) [51–55]. In addition, these reporters allow the visualization of adaptor proteins or cargoes. For example, the SEPA family and SQST-1 are involved in the specific clearance of P granules or protein aggregates, respectively. Even if these adaptor proteins have been described during aggrephagy, they could be involved in other autophagic processes. Autophagic reporters have been also used in combination with markers for endo-lysosome pathway to detect possible interactions between the two pathways. In particular, we observed the fusion between endosomes and autophagosomes, to generate amphisomes, by using VPS-27::GFP fusion protein which specifically labels the endosomal vesicles [56]. Other markers are

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Fig. 3. Analysis of autophagic structures by electron microscopy experiment. Electron micrographs of GFP::LGG-1 (A) or GFP::LGG-2 (C) embryos incubated with anti-LGG-1 and anti-GFP antibodies, respectively, revealing autophagosomal structures. (B) and (D) are higher magnifications of (A) and (C), respectively. Transmission electron microscopy of hypodermal seam cells in L4 larvae in wild-type (E) and ESCRT-II mutant (F). An accumulation of autophagic structures is observed in ESCRT-II mutant (white arrows). Correlative light and electron microscopy of late embryo expressing GFP::LGG-1 (G-I). (G) represents a merge between bright field and fluorescence images of an ultrathin section of an embryo. (H) and (I) are electron micrographs showing higher magnifications of the boxed region in (G). White arrows point to GFP::LGG-1 autophagosomes. Scale bar: 2 lm (A and C) and 1 lm (E, F, H).

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associated with endosomes at different maturation stages (GFP::RAB-7) or with the lysosomal membrane (LMP-1::GFP or LAAT-1::mCherry) [28,30,57]. Of note, the expression of fluorescent tagged human proteins (DFCP1 or GABARAP) makes of C. elegans an interesting heterologous model to analyze human protein functions [46,52,53]. Thanks to the transparent body of C. elegans, different cellular compartments can also be visualized by using fluorescent dyes. For example, mitochondria can be visualized by using the vital dye MitoTracker [1,2] and lysosomes with Lysotracker, which stains acidic compartment (Fig. 2E) [1,7]. Furthermore, Oil-Red-O, a fixative-based dye that stains lipids, is used to detect defects in lipid storage in autophagy mutants [58]. One major disadvantage in using dyes is the lack of permeability of the eggshell and the cuticle that surrounded the worms which prevents their diffusion in the internal tissues. Interestingly, the analysis of lipid storage has been recently achieved using a dye-free CARS microscopy method [58].

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3. Tools to monitor autophagic flux

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Autophagy is a very dynamic process and is classically analyzed by counting the number of autophagosomes. Nonetheless, autophagosome accumulation may represent either elevated

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autophagy process or blockage of an autophagic step downstream of autophagosome biogenesis.

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3.1. Electron microscopy

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Since the first discovery of autophagy, electron microscopy approaches have always been prominent and essential. The use of electron microscopy for analyzing autophagy in C. elegans is not as developed as in other models. Among several reasons, can be mentioned the fact that the worm is a rather recent model to study autophagy, and the need for strong and time-consuming technical expertise for analyzing autophagic subcellular compartments by transmission electron microscopy (TEM). The main contributions in using TEM for autophagy in C. elegans came from A. Kovacs and collaborators [59–61]. TEM is a very reliable approach to analyze and quantify autophagic compartments. Indeed, TEM allows the visualization of every steps of the autophagic pathway, which are usually classified in three main structures, the phagophore, the autophagosome and the autolysosome. So, TEM is a powerful method to understand at which stages of the flux, the autophagic genes are involved. Observation of several autophagic mutants have revealed the accumulation of different types of autophagic structures, such as myelinated-like membrane whorls indicating defective phagophore formation in unc-51 and bec-1 mutant

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[12], or isolation membranes in epg-3, epg-4, epg-6, atg-2 and atg18 mutants supporting a blockage in autophagosome formation [52,53,12]. TEM microscopy also allows the identification of cargoes and organelles present in selective autophagy process. For instance, a TEM study has revealed the presence of autophagosomes engulfing paternal mitochondria just after the fertilization [1]. Other studies used TEM to analyze the interactions between autophagosomes and endosomes, in mutants defective for endosomal maturation (Fig. 3E and F) [62,63]. Moreover, the analysis of the modifications in both the numbers and the aspect of autophagic structures is a powerful tool to conclude on an increase, a decrease or a blockage of the autophagic flux. For instance, an increase of autophagy has been demonstrated in gpl-1 mutant [18] or upon starvation [64]. Furthermore, in pathological models for muscular and neuronal degeneration, an accumulation of autophagic structures was observed using TEM [38,39,65,66]. Another interesting input of TEM, when combined with immuno-labeling, is to assess the localization of several autophagic proteins to the different stages of autophagic flux. It has been successfully done for the Atg8/LC3 proteins, LGG-1 and LGG-2 (Fig. 3A–D) [7] or the specific autophagic substrate PGL-1 [5]. The development of correlative light and electron microscopy (CLEM) is also a very promising approach to analyze specific populations of autophagosomes (Fig. 3G–I) [7].

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3.2. Immunostaining and Western blotting

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Even though in vivo approaches are favored by C. elegans transparency, they present some limitations. In particular, transgenesis can lead to an overexpression of the fusion protein and make multicolor analyses tricky. In this regards, the use of immunofluorescence experiments, allowing the visualization of endogenous proteins, is a complementary approach that can overcome these problems. Immunostaining with LGG-1 antibodies (Table 2) allows to monitor its localization and to count the number of autophagosomes in worms [52,53]. The antibody against LGG-2 (Table 2) is less used, compared to LGG-1, but it represents a valid alternative marker to study autophagy activity [1,7]. A dotted expression corresponding to the localization on autophagosomes is observed for both LGG-1 and LGG-2 (Fig. 2G and H). The number and the localization of these punctate structures change during C. elegans development. Colocalization experiments demonstrated that LGG-1 positive, LGG-2 positive and double positive populations coexist in the cell [7]. To quantify the autophagic activity, the number of puncta has been often counted, either in hypodermal seam cells at larval stages or in whole embryos. The number of puncta

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can be modified when the autophagic flux is genetically or pharmacologically affected. For example, an increase in the number of LGG-1 or LGG-2 positive dots may result from an elevated autophagy activity or a blockage of autophagy flux due to defects in the fusion between autophagosomes and lysosomes. Moreover, colocalization experiments using antibodies against specific cellular compartments, such as the lysosome, can be performed to study the mechanism of autophagy and to determine at which specific step the proteins of interest are involved. Finally, antibodies against PGL-1 [5], PGL-3 [5], SEPA-1 [5] or SQST-1 [52] allow the observation of the degradation of specific substrates or adaptor proteins in various conditions in which the flux is modified. Western blotting using antibodies against LGG-1 is frequently used [52,53,55,67]. Like Atg8 protein in yeast, LGG-1 is first synthesized as a precursor. Then, it is cleaved by the ATG-4 protease exposing a glycine in C-terminal by which LGG-1 is conjugated to phosphatidyl ethanolamine (PE) at the membrane of autophagosomes. The cytosolic and the lipidated forms of LGG-1 can be separated by Western blotting analyses. The amount of PE-conjugated form usually well correlates with the number of autophagosomes. Measuring the ratio of LGG-1 conjugated to autophagosome allows a quantification of the autophagic activity in different contexts. An alternative approach consists in analyzing GFP::LGG-1 and GFP::LGG-2 on Western blot (Fig. 4) [29,45,56,63,66,68]. A supplemental band corresponding to the cleaved GFP is observed. Indeed, during the fusion between the autophagosome and the lysosome, LGG-1 is degraded but the GFP resists longer at the acidic pH. A lower level of the GFP cleaved band in the lysosome and an accumulation of the lipidated and non lipidated forms have been shown when the formation of autolysosome is altered (Fig. 4A) [7]. Otherwise, PGL-3, a well-characterized specific substrate of autophagy is used as another indicator of autophagic activity [5,52,53]. In addition, Western blotting assays with SQST-1 antibody allow the quantification of degradation of autophagic adaptor proteins when the flux is modulated [53,54,67]. In C. elegans, Western blotting assays permit a global analysis of the autophagic flux but could be less sensitive and informative when analyzing tissue- or stage-specific variations.

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3.3. Others approaches

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Various methods, either not yet frequently used in C. elegans or not specific of autophagy, have been successfully used as complementary approaches to further analyze this process. They are briefly presented here. First, RTqPCR experiment allows analyzing the modification of the autophagic level by quantifying the transcription level of

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Fig. 4. Analysis of the autophagic flux in C. elegans by Western blotting. Western blot analyses of GFP::LGG-1 (A) or GFP::LGG-2 (B) using GFP antibodies. The cleaved GFP corresponds to a product of degradation in autolysosome. Quantification of the cytosolic GFP::LGG-1, the PE conjugated form of GFP::LGG-1 and the cleaved GFP allows the measure of the autophagic flux. Normalization is performed with tubulin.


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targeted autophagy genes. For example, mRNA level of the autophagy genes unc-51, bec-1, atg-7, lgg-1 and atg-18 (Table 1) is increased in daf-2 long-lived mutant [69]. The increase of the autophagy process is suggested to cause an expansion of the lifespan. Others studies demonstrated that the transcription factors HLH-30/TFEB and NHR-62 up-regulate the expression of autophagic genes in different conditions [36,57,70]. Classical approaches to identify protein–protein interactions (yeast two hybrid, in vitro pull down assay or co-immunoprecipitation) have been used to better understand the exact mechanisms of regulation of the autophagic pathway or the degradation of specific adaptors and substrates. These methods allowed the dissection of an interaction of EPG-7 with ATG-9, LGG-1 and ATG-18 proteins [54], EPG-6 with ATG-2 [53] and EPG-1 with UNC-51 [51]. Interactions between proteins of interest and specific autophagy substrates, as the PGL-3 protein [5] or with adaptor protein like SEPA-1 [5] or SQST-1 [54] have been similarly demonstrated. Furthermore, the use of the protein–protein interaction assays may permit the discovery of new actors in the autophagic process. For instance, an interaction between LGG-2 and a specific subunit of the HOPS complex, VPS-39, was recently reported [7]. Finally, a GFP::mCherry::LGG-1 fusion protein, similar to the classical Tandem, widely used in other models, has been recently developed in C. elegans to analyze the autophagic flux (Fig. 2F) [7]. This transgenic line allows the discrimination between autophagosomes and autolysosomes based on the distinct chemical properties of GFP and mCherry fluorophores. As a matter of fact, at cytoplasmic pH, both GFP and mCherry fluoresce and autophagosomes appear in yellow, whereas in acidic pH, only the mCherry fluoresces and acidic autophagosomes are red. This construct was expressed specifically in early embryo, allowing to monitor the autophagic flux by quantifying the number of autophagosomes/autolysosomes when the autophagic pathway is modified.

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4. Methods to modify autophagic flux

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4.1. Genetic approaches

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Almost two decades ago, ATG genes have been identified through genetic screens in the yeast Saccaromyces cerevisiae. Most of the ATG genes have a single ortholog in C. elegans with almost no duplications, and play key roles during the autophagy process. For example, bec-1 is one of the first characterized autophagy genes in C. elegans. Depletion of bec-1 by RNAi causes defects in dauer formation and life-span extension [9]. Then, the identification of new C. elegans autophagy genes has been performed by Zhang and colleagues who carried out non-lethal genetic screens to isolate mutants defective in the degradation of autophagy aggregates during embryogenesis [52]. These screens allowed the identification of numerous new alleles of well-known autophagic genes but interestingly, they also uncovered previously uncharacterized genes named epg (ectopic PGL granules) (Table 1). The epg genes are either distantly related to ATG genes or exist only in higher eukaryotes [51,52]. These screens made a great contribution to study autophagy in C. elegans by generating new tools, such as mutants or fluorescent reporters. In addition, a collection of knocked-out alleles of autophagy genes is also available thanks to public consortia, the C. elegans Gene Knockout Consortium in the U.S. and Canada (ok alleles), and the National BioResource Project in Japan (tm alleles). In 2014, a genome wide RNAi screen was designed to identify signaling pathways that modulate the autophagy process in C. elegans [71]. The results of these screenings confirmed C. elegans as a powerful model to genetically dissect the autophagy pathway. The combination between the well described

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autophagy process together with the availability of mutants and genetic tools allows to investigate many aspects of this process in vivo. Worm carrying mutations in genes involved in distinct steps throughout the autophagy process, from the early phase of autophagy induction to lysosome degradation, are widely used (Table 1). For example, epg-6 mutant animals are defective in an early step of the aggrephagy process, specifically during the progression from phagophores to autophagosomes [53]. Both mutants in the classical autophagy pathway and mutants involved in the regulation of autophagy such as HLH-30, TOR or adaptor proteins (SEPA, SQST-1) have been described so far (Table 1) [30,51,52,57]. Mutations in some autophagy genes, such as bec-1 or lgg-1, can lead to lethality or sterility, which can preclude the analysis of their functions at later stages of development. To circumvent this problem, RNAi has been efficiently used to observe the effects of gene knocked down in larvae or adult (Table 1). A genetic approach has been used to study the depletion of lgg-1 in the early embryonic stages overstepping its lethality effect. lgg-1(tm3489maternal) strain was generated by using the transgene Plgg-1::gfp::lgg-1, which is expressed in somatic tissue but not in the germ-line to rescue the lethality of lgg-1(tm3489) [7]. The expression of this construct only starts when the embryos reach the 20-cell stage. Thus, in lgg-1(tm3489maternal), LGG-1 is depleted in oocyte and embryo until 20-cell stage allowing the analysis of its inactivation in early embryo. One of the possible limitation of classical mutants is the analysis of tissue-specific requirement of autophagy genes. Recently, a tissue specific RNAi of bec-1 was used to show that intestinal autophagy activity is essential during S. typhimurium infection [34]. In this study, a C. elegans strain where RNAi is limited to the intestine shows an important decrease of lifespan in bec-1 RNAi treated worms infected with salmonella.

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In mammalian cells, drugs are commonly used to quantify the autophagic flux. Moreover, given the central role of autophagy in pathological processes such as neurodegenerative diseases and cancer, it is important to identify drugs that modulate the autophagic pathway. These molecules can be divided in two main categories based on their mechanisms of action, the autophagy activators or inhibitors. Among the autophagy activators, starvation and ER stressing drugs stimulate the induction step. Other activators are targeting the autophagosome maturation in a mTOR dependent or independent manner. Several inhibitors are blocking the induction step (class III PI3K inhibitor and protein synthesis inhibitor) whereas others prevent the autophagosome degradation (vacuolar-type H(+)-ATPase inhibitors, lysosomal lumen alkalizers and acid protease inhibitors). Some of these compounds have been used only recently to modulate autophagy in C. elegans. Inactivation of autophagy by using the inhibitors of type III PI3K, 3-methyladenine and Wortmannin, causes an increased hypoxic lethality in adult worms [32]. On the other hand, resveratrol and spermidine, two potent inducers of autophagy prolong the lifespan in an autophagy dependent fashion [72,73]. Autophagy can be also induced in animals treated with the cell death inducers 5-fluorouracil and methotrexate [22]. Pharmacological approaches have not been much developed in C. elegans due to several limitations. In particular, C. elegans is surrounded by a protective cuticle which compromised the penetration and diffusion of drugs. In addition, it has been suggested that the amount of an active drug is lowered down by the metabolism of the worms. So, higher concentrations of active compounds have been used to treat worms compared to cell cultures [74]. Despite these limitations, drug screens and pharmacological studies on autophagic processes have been recently validated in

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C. Jenzer et al. / Methods xxx (2014) xxx–xxx Table 3 Methods for analyzing autophagy in C. elegans. Methods

Applications

Limitations

In vivo imaging

Visualization and quantification of autophagic structures, adaptor proteins and cargoes Analysis of autophagic flux Visualization and quantification of autophagic structures, adaptor proteins and cargoes Co-localization studies Analysis of all cell compartments Analysis of autophagic flux Identification of selective organelles of autophagy Quantification of autophagic structures, adaptor proteins and cargoes Modulation of autophagic flux Modulation of autophagic flux

Overexpression of fusion proteins Risks of aggregates Compromised penetration and diffusion of dyes Fixed tissues

Immunostaining

Electron microscopy

Western blotting Genetic approach Pharmacology

588

C. elegans [49,75,76]. The alpha-1-antitrypsin deficiency is a liver disease associated with hepatic fibrosis and hepatocellular carcinoma caused by an aberrantly accumulation of a secretory protein, alpha-1-antitrypsin Z (ATZ), in the endoplasmic reticulum. Autophagy plays a central role in the removal of the insoluble ATZ from the endoplasmic reticulum. A high-throughput drug screening showed that fluphenazine, a human mood stabilizer and a potent autophagy enhancer, can reduce the proteotoxicity of ATZ accumulation in C. elegans [49]. This result confirms that C. elegans could represent a complementary animal model to cell culture and mice in biomedical studies.

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5. Concluding remarks

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Despite its relatively recent use as a model for autophagy, C. elegans presents numerous advantages to study the roles of autophagy in physiological and pathological conditions. This genetic model, which allows in vivo analysis of the whole animal during its entire life is particularly well adapted to further understand the functions and processes of selective autophagy. Because autophagy is a very dynamic and versatile process, quantitative analyses must be further develop, even if they are not always easy to set up (Table 3). Moreover, because each method presents potential limitations and caveats, it is absolutely essential to combine several approaches before concluding on the autophagic flux. If biochemical and pharmacological approaches have not yet been used extensively in C. elegans, the strong potentials of optic and electron microscopies and the increasing number of autophagic markers, are a promise of success for the worm in the quickly growing field of autophagy.

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Acknowledgements

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The authors would like to thank their colleagues for helpful discussion and particularly Emmanuel Culetto and Marion ManilSégalen for reading this manuscript and Céline Largeau for providing EM pictures. We are also grateful to the C. elegans autophagy community for sharing informations and apologize if some data could not be mentioned in this review due to size limitations. The Legouis’ group is supported by the Agence National de la Recherche (project EAT, ANR-12-BSV2-018) and the Association pour la Recherche contre le Cancer (SFI20111203826). C.J. is a recipient of fellowship from the ‘‘Actions IDEX Paris-Saclay 2012Initiative Doctorale Interdisciplinaire’’.

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Methods to assess subcellular compartments of muscle in C. elegans.

Tissue-specific autophagy responses to aging and stress in C. elegans.

Lightweight object oriented structure analysis: tools for building tools to analyze molecular dynamics simulations.

Thoughtful Methods to Increase Evidence Levels and Analyze Nonparametric Data.

Kinetic studies in isolated organs: tools to design analgesic peptides and to analyze their receptor effects.

Non-microfluidic methods for imaging live C. elegans.

How to analyze mitochondrial morphology in healthy cells and apoptotic cells in Caenorhabditis elegans.

Intestinal Autophagy Improves Healthspan and Longevity in C. elegans during Dietary Restriction.

Correction: Intestinal Autophagy Improves Healthspan and Longevity in C. elegans During Dietary Restriction.

Autophagy activity contributes to programmed cell death in Caenorhabditis elegans.

Effectiveness and Usability of Bioinformatics Tools to Analyze Pathways Associated with miRNA Expression.

A Survey of Computational Tools to Analyze and Interpret Whole Exome Sequencing Data.

Guidelines for monitoring autophagy in Caenorhabditis elegans.

Approaches for Studying Autophagy in Caenorhabditis elegans.

The two C. elegans ATG-16 homologs have partially redundant functions in the basal autophagy pathway.

Methods for skin wounding and assays for wound responses in C. elegans.

Methods to study chaperone-mediated autophagy.

Novel methods to rapidly and sensitively analyze antigenic peptide binding to MHC class II molecules.

New imaging methods and tools to study vascular biology.

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Methods to analyze bone regenerative response to different rhBMP-2 doses in rabbit craniofacial defects.

Biophysical and computational methods to analyze amino acid interaction networks in proteins.

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