The autophagosomal protein LGG-2 acts synergistically with LGG-1 in ...

Basic Research Paper

Autophagy 6:5, 622-633; July 1, 2010; © 2010 Landes Bioscience

The autophagosomal protein LGG-2 acts synergistically with LGG-1 in dauer formation and longevity in C. elegans Adriana Alberti,† Xavier Michelet,† Abderazak Djeddi† and Renaud Legouis* Centre de Génétique Moléculaire; CNRS FRE3144; Gif-sur-Yvette, France; and Université Paris Sud These authors contributed equally to this work.

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Key words: LC3/Atg8, post-translational modification, dauer, senescence, starvation Abbreviations: GABARAP, γ-aminobutirric-acid-type-A receptor-associated protein; GATE-16, golgi-associated ATPase enhancer of 16 kDa; L1, larval stage 1; LC3, microtubule-associated protein 1 light chain3; PE, phosphatidyl-ethanolamine; SDS, sodium dodecylsulfate; TOR, target of rapamycin

Autophagy has an important function in degrading cytoplasmic components to maintain cellular homeostasis, but is also required during development. The formation of the autophagic vesicles requires the recruitment of the Atg8 ubiquitinlike proteins to the membrane of the nascent autophagosomes. Atg8 is a highly conserved gene which has been duplicated during metazoan evolution. In this report we have investigated, in the nematode C. elegans, the functions and localizations of the two Atg8p homologues LGG-2 and LGG-1. Phylogenetic analyses suggest that LGG-2 is more closely related to the human protein LC3 than LGG-1. LGG-1 but not LGG-2 is able to functionally complement the atg8 mutant yeast. The C-terminal glycine residue of LGG-2 is essential for post-translational modification and localization to the autophagosomes. During C. elegans development the two proteins share a similar expression pattern and localization but LGG-2 is more abundant in the neurons. Using genetic tools to either reduce or increase the autophagic flux we show that both LGG-2 and LGG-1 are addressed to the autophagosomal/lysosomal degradative system. We also demonstrate that the localization of both proteins is modified in several physiological processes when autophagy is induced, namely during diapause “dauer” larval formation, starvation and aging. Finally, we demonstrate that both LGG-2 and LGG-1 act synergistically and are involved in dauer formation and longevity of the worm.

Introduction Autophagy, which usually refers to macroautophagy, is the major ubiquitous catabolic process which allows the bulk degradation of cytoplasmic constituents, generally by nonselective sequestration.1,2 It is essential for survival, differentiation, development and homeostasis. Degradation of long-lived proteins and organelles by autophagy involves the formation of double membrane vesicles, containing a portion of the cytoplasm, the autophagosomes, which finally fuse with the lysosomes.3,4 Genetic studies in mice, flies and worms have shown that mutations in several autophagy genes result in embryonic lethality.5 In particular, autophagy appears to play a critical role in tissue remodeling during development.6 In C. elegans, autophagy is essential for lifespan extension, alternate diapause larval stage development and during starvation.7,8 Autophagy is also involved in cell size,9 cell survival,10-12 and in the clearance of polyglutamine and β-amyloid aggregates in neural cells.13-15 Interestingly, recent reports in C. elegans demonstrated the role of autophagy as a selective pathway

for the degradation of GABA receptors at the neuromuscular junction16 and for removing maternally inherited protein aggregates in the embryo.17,18 Investigations on the yeast autophagic proteins (Atg)19,20 and their mammalian homologues revealed a global conservation of the underlying machinery but with some divergence of the pathway in higher eukaryotes (reviewed in refs. 3, 5, 6 and 21–23). More than 20 Atg proteins are involved in this process forming several complexes whose functions have been extensively studied. Among them, two ubiquitin-like proteins Atg8 and Atg12 are associated with the membrane of the autophagosomes.24-26 Atg8 is present on autophagosome membranes as a phosphatidylethanolamine (PE) conjugated form.26 Atg8 is highly conserved but mammals present four homologues: microtubule-associated protein 1 light chain 3 (LC3); Golgi-associated ATPase enhancer of 16 kDa (GATE-16); γ-aminobutirric-acid-type-A (GABA A) receptor-associated protein (GABARAP) and Atg8L.27-29 In a similar manner to Atg8, these proteins can undergo post-translational modifications carried out by a ubiquitinylation-like

*Correspondence to: Renaud Legouis; Email: [email protected] Submitted: 09/16/09; Revised: 05/03/10; Accepted: 05/05/10 Previously published online: www.landesbioscience.com/journals/autophagy/article/12252 622

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system.30 Soon after translation, the carboxi termini of the proforms of LC-3, GATE-16 and GABARAP are cleaved by the protease Atg4B31 and the newly exposed glycine residue is then PE-conjugated by the sequential action of the E1-like enzyme Atg7 and the E2-like enzyme Atg3. The PE-conjugated forms are named LC3-II, GATE-16-II and GABARAP-II and are bound to the autophagosomal membrane.26-28 LC3, which is thought to be the orthologue of Atg8, is well established as a marker of autophagosomes during starvation-induced autophagy.27 In contrast, even if the forms II of GATE-16 and GABARAP have been found to localize on LC3-II positive autophagosomes, their role during autophagy is still less understood.26-28 The duplication of the atg8 homologues in metazoans could reflect an evolution in the initial function of the protein. Three main categories of changes can occur in duplicated genes, with either a restriction in the expression patterns or the specialization of the functions or the acquisition of new functions unrelated to autophagy. We have investigated the situation in the nematode C. elegans whose genome contains two Atg8 homologous genes named LGG-1 and LGG-2. Initial studies have demonstrated that during dauer larva formation, starvation and senescence, three processes in which autophagy is induced, GFP::LGG-1 changes its localization from diffuse in the cytoplasm to a punctate pattern.7,8,32 For this reason, LGG-1 is thought to be the orthologue of ScAtg8p and is now widely used as autophagic marker in C. elegans. However, LGG-2 localization has not been studied so far. In this report we have characterized the function and localization of LGG-2 and compared it with LGG-1. Our data show that LGG-2 localizes to autophagosomes and displays an expression pattern overlapping with LGG-1. The expression pattern of LGG-2 is modified in autophagy-induced conditions, namely dauer formation, starvation and aging. Moreover, LGG-2 is involved in dauer induction and longevity of the worm and appears to have a synergistic effect with LGG-1 during these processes. Results C. elegans genome contains two genes homologous to atg8. Homology searches with the sequence of the yeast Atg8 protein revealed that the C. elegans genome contains two homologues encoded by C32D5.9 and ZK593.6 (53% and 33% identity, respectively). These two genes encode respectively a 123 amino acid protein named LGG-1 and a 130 amino acid protein, LGG2. RT-PCR experiments confirmed the cDNA sequences reported in wormbase (http://www.wormbase.org/). Orthologues of both genes are also present in other nematode species including C. briggsae, C. remanei, C. brenneri, C. japonica, Oscheius trichinella spiralis, Brugia malayi and Meloidogyne (data not shown). We constructed a phylogenetic tree of the LGG-1 and LGG-2 proteins together with Atg8p and its three human homologues by neighbor joining (Fig. 1A). This analysis revealed that LGG-1 is closer to the yeast protein and to human GABARAP (53% and 83% identity respectively) whereas LGG-2 is closely related to HsLC3A (59% identity), which has been shown to be an autophagosomal marker in mammals.27 To determine if LGG-1 and LGG-2 are the functional homologues of yeast Atg8p, we

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tested their ability to complement the loss of viability of ATG8disrupted yeast (∆atg8) under nitrogen starvation. We found that ∆atg8 yeast transformed with C. elegans lgg-1(+) showed an increase in survival compared to ∆atg8 (Fig. 1B). In contrast, lgg2(+) was not able to enhance survival of ∆atg8 yeast. To check that LGG-2 was correctly expressed we used an antibody directed against human LC3,27 and performed a western blot on total protein extracts from the ∆atg8 mutant transformed with lgg-1 or lgg-2 (Fig. 1C). We detected a single band at the expected size (14 kDa) in mutant transformed with lgg-2 but nothing with lgg1. However, we could not exclude that the inability of LGG-2 to complement ∆atg8 mutant is due to its overexpression or to an absence of lipidation of LGG-2 in yeast. Together these data suggest that LGG-1 and not LGG-2 is the worm orthologue of Atg8p and raises the possibility that the two proteins may have different functions in C. elegans. LGG-2 expression pattern is similar to LGG-1 during C. elegans development. To understand whether LGG-1 and LGG-2 have different or redundant roles, we first analyzed if the genes were expressed in the same tissues. Numerous studies demonstrated that GFP::LGG-1 localization in larvae and adults changes from diffuse to punctate when autophagy is induced7,8,12,32,33 but its expression in the embryo has not been well characterized. In contrast, there is no data on the localization and function of LGG-2. Therefore we generated GFP::LGG-2 transgenic lines expressing an in-frame translational N-terminal GFP fusion construct (Fig. 2) and compared the expression patterns with GFP::LGG-1. Expression of GFP::LGG-2 and GFP::LGG-1 is first detected early during embryogenesis (before the 50 cell stage) with both a punctate and a diffuse cytoplasmic staining (Fig. 2A and B). At the beginning of organogenesis the number of puncta diminishes and the signal becomes mainly restricted to the pharynx, intestine and hypodermis (Fig. 2C and D). After hatching and during larval development GFP::LGG-1 expression has been reported as a diffuse signal predominantly in the lateral hypodermal seam cells and in the pharynx (Fig. 2F). Similarly, the expression of GFP::LGG-2 is high in the seam cells and pharynx and also in the nervous system, especially in some head and tail neurons and the neuron cell bodies in the ventral nerve cord (Fig. 2E and G). Its expression is mainly diffuse in the cytoplasm but a few dots are often detected in neuron cell bodies and seam cells (Fig. 2E). In adults, the tissue distribution of GFP::LGG-2 and GFP::LGG-1 remains diffuse but becomes more ubiquitous in particular the signal is detected in the whole hypodermis, the muscles, the vulva and the spermatheca (Fig. 2H–J and data not shown). Together these data show that lgg-1 and lgg-2 present widely overlapping expression patterns and suggests the presence of numerous autophagosomal structures during embryogenesis. LGG-1 and LGG-2 localize to the autophagosomal/lysosomal degradative pathway. Biochemical analyses on both Atg8p and mammalian LC3 have demonstrated that their localization to the autophagosome is dependent on the reversible conjugation of phosphatidylethanolamine (PE) to a glycine residue after cleavage of their C-terminus by the protease Atg4p.26,28 Sequence alignment of the C-terminal regions of the Atg8p homologues in yeast, human and nematode revealed that the glycine residue

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Figure 1. lgg-1 and lgg-2 are two C. elegans homologues of ScAtg8. (A) Phylogenetic tree realized by neighbor joining using PAUP 4.0 of Saccharomyces cerevisiae (Sc) Atg8 protein, the human (Hs) homologues GABARAP, GATE-16 and MAP1LC-3 and the C. elegans (Ce) homologues LGG-1 and LGG-2. Numbers indicate the percentage of bootstrap support (star is 100%). Of note, LGG-1 is closely related to ScAtg8p and HsGABARAP whereas LGG-2 is more closely related to HsMAP1LC-3. (B) Complementation test of the loss of viability phenotype of the ∆atg8 mutant in nitrogen starvation medium. Wild-type curve has >100% survival because some cells are in the process of dividing when transferred to starvation medium. ∆atg8 transformed with a plasmid overexpressing an Atg8 protein fused to GFP is used as positive control. Note that the rescue is partial, reflecting a possible effect of the GFP tag. Overexpression of lgg-1 in ∆atg8 cells partially alleviates the phenotype whereas ∆atg8 mutant transformed with lgg-2 are less viable than ∆atg8 alone. The graphs are the mean of 2 experiments and bars represent standard error. Statistical analyses using t test is significant for p < 0.05. (C) Western blot of protein extracts from ∆atg8 mutant alone and transformed with lgg-1 or lgg-2 incubated with the human LC3 antibody or the yeast Atp2 antibody as control of loading. Only in ∆atg8 mutants expressing Celgg-2, a protein is detected at about 14 kDa. (D) Aminoacid sequence alignment of the C-terminal segments of LGG-1 and LGG-2 and their homologues. The conserved residues are shaded in black and the arrow indicates the cleavage site in Atg8p and MAPLC3.

is conserved in both LGG-1 and LGG-2 (Fig. 1D), and corresponds to the last residue of LGG-2. This raises the possibility that LGG-2 does not require exactly the same post-translational modifications. So, we asked if such a PE-conjugation is conserved in LGG-2 and involved in its localization. Western blot analysis of total protein extracts from GFP::LGG-2 and GFP::LGG-1 animals revealed in both samples a majority GFP positive band with apparent molecular weight of 45 kDa corresponding to the expected size of the fusion proteins (Fig. 3A). In addition for both GFP::LGG-1 and GFP::LGG-2, GFP degradation products were detected around 28 kDa which presumably correspond to the stable part of the GFP present in the lysosome.34 This suggests that the GFP::LGG-1 and GFP::LGG-2 fusion proteins can be

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correctly addressed to the autophagosomal/lysosomal degradative system. During starvation, Kang and Avery have detected a minority band for GFP::LGG-1 which has been hypothesized to be a PE modified form.32 In our experimental conditions this band was very weak (Fig. 3A and reviewed in ref. 35) and sometimes not detectable. By contrast, GFP::LGG-2 showed two additional minority bands which could correspond to posttranslational modifications, with respectively higher and lower molecular weight (Fig. 3A). The majority and the two minority bands were respectively named GFP::LGG-2, GFP::LGG-2 high and GFP::LGG-2 low. Previous studies of LC3 have shown that the mutation of the last glycine residue (G120A) abolishes its PE conjugation and localization to the autophagosomes.28,36 To test

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Figure 2. LGG-2 expression pattern is similar to LGG-1 throughout C. elegans development. Projections of confocal images of GFP::LGG-2 (A, C, E and G–J) and GFP::LGG-1 (B, D and F) at different developmental stages. In early embryos, the fusion proteins localize in both a dotted and a diffuse pattern (A and B). At the beginning of organogenesis the number of punctate areas diminishes (C and D). (A’–D’) are the Nomarski pictures corresponding respectively to (A–D). During larval development GFP::LGG-2 and GFP::LGG-1 expression appears intense in the lateral hypodermal seam cells (arrowheads in E and F) and the pharynx. GFP::LGG-2 is also detected in neurons of the head and the tail (G) and in the ventral nerve cord (G, white arrows). GFP::LGG-2 and GFP::LGG-1 localization is mainly diffuse in the cytoplasm but for GFP::LGG-2 one or two dots are often detected in neuron cell bodies and seam cells (E and G). GFP::LGG-1 is also detected to a less extent in the distal tip cell of the gonad (F, white arrows), the intestinal lumen, and some rectal cells. In adults, the tissue distribution of GFP::LGG-2 and GFP::LGG-1 becomes more ubiquitous with an expression in the hypodermis, the muscles (H), the vulva (I) and the spermatheca (J). Scale bars are 10 µm. (K) Western blot of protein extracts from mixed stage GFP::LGG-2 and GFP::LGG-1 transgenic worms incubated with anti-LC3 or anti-GFP antibodies. In GFP::LGG-2 animals, a fusion protein (2 bands) is detected at about 45 kDa with both anti-GFP and anti LC-3 antibodies. In GFP:: LGG-1 animals, a band at about 45 kDa corresponding to the fusion protein is detected with anti-GFP and possibly with anti-GABARAP antibodies, but the signal is very weak and the background noise high.

if the carboxi-terminal glycine of LGG-2 is essential, we generated a GFP::LGG-2(G130A) fusion protein and obtained transgenic strains. GFP::LGG-2(G130A) was detected as a diffuse cytosolic signal (Fig. 3C) and western blot analysis revealed that the two minority bands and the GFP cleaved products almost completely disappeared. These data demonstrate that the glycine 130 residue of LGG-2 is essential for its localization to vesicular structures and the formation of GFP::LGG-2 high. However, we could not definitively conclude that GFP::LGG-2 high corresponds to the PE conjugated form of GFP::LGG-2, because the very weak lower band GFP::LGG-2 low is also affected in


GFP::LGG-2(G130A). We also generated a LGG-2::GFP fusion protein and demonstrated that the C-terminus of LGG-2 could be efficiently cleaved (Fig. S1). In a second step we used an RNAi approach to modify the autophagic process and monitored the modifications of GFP::LGG-1 and GFP::LGG-2 (Fig. 4). CeTOR(RNAi) was used to stimulate the autophagic process by increasing the autophagic flux.21 In addition, we used rab-7(RNAi) to impair the clearance of autophagosomes by reducing their fusion with lysosomes.37 Analysis of GFP::LGG-1 and GFP::LGG-2 by confocal microscopy revealed a strong modification of the localization. In

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CeTOR(RNAi) animals we observed an increase in the number of punctate structures, mainly in the epidermis and the intestine (Fig. 4C and D and data not shown), while in rab-7(RNAi) animals we also observed a strong accumulation of larger vesicular structures positive for GFP::LGG-2 (Fig. 4E) or GFP::LGG-1 (Fig. 4F). Such an enlargement of autophagosomes in lysosomal fusion mutants has already been observed in mammals.34 Images quantification and western blot analyses of GFP::LGG-2 and GFP::LGG-1 in CeTOR(RNAi) and rab-7(RNAi) animals confirmed the accumulation of autophagosomes (Fig. 4G–J). Together these data validate GFP::LGG-2 as a new marker of the autophagosomal pathway in C. elegans. The localization of LGG-2 and LGG-1 is modified when autophagy is induced. In order to check that LGG-1 and LGG-2 are both markers of autophagy in vivo we analyzed their expression and localization pattern in autophagy-inducing conditions. We first analyzed the process of starvation at the first larval stage (L1) (Fig. 5A–C), during which autophagy is involved.32 The progeny of gfp::lgg-1 or gfp::lgg-2 adults animals were cultured in liquid medium without nutrients, which allows the hatching of L1 animals but not their further development, and GFP::LGG-1 or GFP::LGG-2 was then analyzed every 24 hours during four days by confocal microscopy (Fig. 5A and B) and western blotting (Fig. 5C). After 24 hours of starvation we observed an increase in the expression of both LGG-1 and LGG-2 but the patterns were different (Compare Figs. 5A and B with 2). In normally-fed L1 animals, GFP::LGG-1 is mainly expressed in the lateral epidermis but in 24 hours unfed L1, its expression is increased but becomes more ubiquitous. GFP::LGG-2 is also strongly increased after 24 hours of starvation but in a punctate pattern. From 48 hours to 96 hours of starvation, GFP::LGG-1 expression diminishes and becomes very low but on the contrary, GFP::LGG-2 increases forming intense patches in all tissues which look like aggregates. Quantification by western blotting showed that while GFP::LGG-2 accumulates during starvation, GFP::LGG-2 high first increases after 24 hours and then decreases. The transient accumulation of autophagosomes during starvation suggests a difference between the kinetics of autophagosomal formation and their resolution by lysosomes. We then looked at the formation of dauer larvae, during which autophagy is required.7 To induce dauer formation we used either the e1370 thermosensitive constitutive allele of daf-2 or the purified daumone38 (Fig. 5D–I). As previously described,7 we observed that GFP::LGG-1 becomes more punctated in the lateral epidermis of dauer larvae compared to third-stage larvae and that the intensity of the minority band slightly increased (Fig. S2A). We also noticed that the formation of puncta was stronger in daumone-induced animals compared to daf-2(e1370) (Fig. 5E, G and I). We then analyzed localization of GFP::LGG-2 and observed a decrease of the diffuse signal but a strong increase in the number of positive punctate areas after dauer-induction (Fig. 5D, F and H). This change in the subcellular localization pattern indicates that LGG-2 behaves similarly to LGG-1 and could have a role in dauer formation. Because autophagy has also been reported to be involved in longevity in C. elegans,39,40 we analyzed the localization of GFP::LGG-1 and GFP::LGG-2 from the first day to day 12 of

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Figure 3. The Gly130 of LGG-2 is essential for post-translational modification and sub-cellular localization. (A) Western blot of protein extracts from mixed stage GFP::LGG-1, GFP::LGG-2 and GFP::LGG-2(G130A) transgenic worms incubated with anti-GFP antibody. In GFP::LGG-2 animals, the fusion protein is detected as one majority and two minority bands at about 45 kDa, and GFP degradation products are also visible. In GFP::LGG-2(G130A) the GFP::LGG-2 high and GFP::LGG-2 low are almost completely missing. For GFP::LGG-1, the asterisk indicates the putative PE form according to Kang. 32 (B and C) Projections of confocal images of GFP::LGG-2 (B), and GFP::LGG-2(G130A) (C) in early embryos. GFP::LGG-2 localizes in both a dotted and a diffuse pattern while GFP::LGG-2(G130A) presents a cytosolic diffuse pattern.

adulthood (Fig. 5J–O). Surprisingly, the two proteins present a change in their expression pattern but in an opposite manner. In young adults both GFP::LGG-1 and GFP::LGG-2 present a strong diffuse localization (Fig. 5J and N). However the GFP::LGG-1 diffuse expression strongly decreases during aging and becomes almost restricted to the pharynx in 10 day-old adults (Fig. 5O) where very few puncta are detected. In contrast, we observed a very strong accumulation of GFP::LGG-2 dotted areas during aging in several tissues. In particular, in 10 day-old adults a strong increase of patches and puncta was detected in the pharynx, the muscle cells and the lateral hypodermal seam cells (Fig. 5K and M). Western blot analyses confirmed that all forms

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any developmental phenotypes by this RNAi approach, neither in lgg-1(RNAi) nor lgg-2(RNAi), nor by combining both (hereafter referred as lgg-1 + 2(RNAi)). Depleted animals reached adulthood and start to lay eggs like the controls. To check that the depletion of LGG-1 and LGG-2 was effective we performed the same experiments on GFP::LGG-1 and GFP::LGG-2 transgenic animals (Fig. S3). Confocal microscopy and western blot analyses indicate that the expression of GFP::LGG-1 is strongly diminished after lgg-1(RNAi) and lgg-1 + 2(RNAi) (99%) but not lgg-2(RNAi) treatment. However, a very weak expression was still visible in the pharynx. Similarly GFP::LGG-2 is efficiently depleted after lgg-2(RNAi) and lgg-1 + 2(RNAi) (82 and 88%, respectively) but not lgg-1(RNAi) treatment. For GFP::LGG-2 some expression was still detected in the pharynx and the neurons, which are known to be more resistant to RNAi.43 These data show that LGG-1 and LGG-2 depletion is specific and efficient and that under these conditions of RNAi both genes are not essential for C. elegans development and reproduction. During the course of this study the C. elegans knockout service in Japan generated a deletion mutation in lgg-1. The Figure 4. The localization of LGG-2 and LGG-1 is modified in autophagic mutants. (A–F) Confocal allele lgg-1(tm3489) deletes a large part images of epidermal cells in larvae expressing either GFP::LGG-2 (A, C and E) or GFP::LGG-1 (B, D of the lgg-1 coding sequence and is preand F) in control, CeTOR(RNAi) and rab-7(RNAi). In CeTOR(RNAi) the level of both LGG-2 (C) and LGGsumably a null mutant. Our preliminary 1(D) puncta is increased suggesting an accumulation of autophagosomes. In rab-7(RNAi) a strong genetic analysis of fertility and lethalaccumulation of larger autophagosomal structures is detected for GFP::LGG-2 (E) and GFP::LGG-1 (F). Quantifications (shown in G and I) has been performed in the epidermis on areas of 100 µm ity revealed a strong maternal contribulength (n = 8 to 15 on at least 3 animals). Statistical analyses using t test are significant for p < tion for LGG-1 and that homozygous 0.05(*) or p < 0.01 (**). Western blots of total protein extracts from GFP::LGG-2(H) and GFP::LGG-1 lgg-1(tm3489) population is difficult to (J). The ratio of GFP::LGG-2 high or GFP::LGG-1 to tubulin was quantified and normalized with the maintain (Table S1). control. h indicates the minority GFP::LGG-2 high form and * the putative GFP::LGG-1-PE. LGG-1 is required for survival to starvation. Autophagy is involved in starvation of LGG-2 accumulate during aging while the degradation of survival32 but the roles of neither lgg-1 nor lgg-2 have been anaLGG-1 is increased (Fig. S2B). These data suggest that LGG-2 lyzed, so we explored them using RNAi. Starvation of L1 animals and LGG-1 could have different roles during the physiological was carried out from one to 13 days and we counted the number process of aging. Together these experiments indicate that both of animals able to resume development and reach adulthood in LGG-1 and LGG-2 sub-cellular localization is highly modified presence of nutrients (Fig. 6A). We observed a significant reducin physiological autophagic conditions. tion of starvation survival after lgg-1(RNAi) and lgg-1 + 2(RNAi) LGG-2 and LGG-1 have a synergistic effect in dauer forma- but not lgg-2(RNAi) treatment. However, lgg-2(RNAi) adults tion and longevity. Depletion of lgg-2 and lgg-1 by feeding RNAi from starved animals appeared very sick and died within the next does not affect development or fertility. To characterize the roles of 48 hours (data not shown). Our data suggest a more important lgg-2 and lgg-1 throughout development, we used RNAi deple- role for LGG-1 in starvation survival compared to LGG-2. tion.41 dsRNA corresponding to the coding region of lgg-1 and LGG-2 and LGG-1 are both required for dauer formation. lgg-2 was delivered to adult hermaphrodites in order to inacti- Because LGG-2 and LGG-1 expression patterns and localization vate both the maternal and zygotic contributions in the prog- are modified in dauer larvae we analyzed whether they could have eny. As previously reported by Sigmond42 and in large scale complementary roles at this stage. Melendez et al. have shown RNAi screens (http://www.wormbase.org) we did not observe that LGG-1 is required for dauer formation in the constitutive


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Figure 5. LGG-2 accumulates in starved animals, dauer larvae and aging adults. (A and B) Confocal images of GFP::LGG-2 (A) or GFP::LGG-1 (B) first larval stage animals after 24 to 96 hours-culture in starvation. GFP::LGG-2 expression is strong and ubiquitous after 24 hours of starvation (A) and the protein accumulates even after 96 hours of starvation. (B) In contrast, GFP::LGG-1 first increases after 24 hours of starvation then decreases over time. (C) Western blots of protein extracts from GFP::LGG-2 and GFP::LGG-1 first larval stage animals starved for 24 hours to 96 hours, incubated with anti-GFP antibody or anti-tubulin antibody for control of loading. (D–I) Confocal images of lateral hypodermal seam cells in larvae expressing either GFP::LGG-2 (D, F and H) or GFP::LGG-1 (E, G and I). Both markers present diffuse cytoplasmic expression in control daf-2(e1370) third stage larvae (D and E). A strong increase in the number of GFP::LGG-2 positive punctate areas was observed in daf-2(e1370) dauer larvae (arrows in F). A similar change in the subcellular localization pattern of GFP::LGG-1 is also detected (arrow in G) but the intensity of signal is weaker. In dauer larvae induced by the daumone pheromone both markers display a strong punctate localization (H and I). (J–O) Confocal images of 3 day- and 10 day-old adults expressing GFP::LGG-2 (J–M) or GFP::LGG-1 (N and O). GFP::LGG-2 diffuse expression in young adults strongly accumulates in dotted areas in several tissues of 10 day-old adults. In particular a strong increase of puncta was detected in the pharynx (arrow in K) and the lateral hypodermal seam cells (M compared to L). In contrast GFP::LGG-1 which is ubiquitously diffuse in 3 day-old adults strongly decreases in 10 day-old adults and no punctate pattern is detected.

mutant background daf-2 (e1370). We decided to investigate the functions of lgg-2 and lgg-1 in the dauer formation using daumone.38 Third to fourth larval stage animals were grown on control or RNAi producing bacteria for 48 hours and allowed to lay eggs for several hours on daumone-containing plates. The progeny was grown on these plates for four to six days and the formation of dauer larvae was assessed by morphological analyses. We observed that more than 95% of the control progeny formed thin larvae which presented the cuticular ridges alae characteristic of

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dauer stage. In lgg-1(RNAi), lgg-2(RNAi) or lgg-1 + 2(RNAi) conditions the global morphology of larvae appeared similar and the alae were also formed (Fig. S4). This suggests that LGG-2 and LGG-1 are dispensable for the first steps of induction of the dauer stage. Then we assessed if the lgg-1(RNAi), lgg-2(RNAi) or lgg-1 + 2(RNAi) dauers are physiologically equivalent to wild type by treating them with 1% SDS (Fig. 6B). Wild-type dauer larvae present important modifications of their cuticle which confers a strong resistance to detergents. After SDS treatment only 10.2%

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Figure 6. Synergistic effects of lgg-1(RNAi) and lgg-2(RNAi) in dauer formation and life span. (A) Starvation survival curves of first stage larvae in control, lgg-1(RNAi), lgg-2(RNAi), lgg-1(RNAi) + lgg-2(RNAi), at 20°C. Both lgg-1(RNAi) and lgg-1(RNAi) + lgg-2(RNAi) present a decrease in starvation survival (p < 0.05 when applying Log Rank test). NS: nonsignificant. (B) Synergistic effect of lgg-1(RNAi) and lgg-2(RNAi) on daumone-induced dauer formation. lgg-1(RNAi), lgg-2(RNAi) and lgg-1(RNAi) + lgg-2(RNAi) dauer larvae are less resistant to 1% SDS treatment than control. The mean percentages have been calculated from two independent experiments with a number of animals superior to 400. Bars indicate the standard error deviation. **Khi-2 is significant for α = 0.01. (C and D) Synergistic effect of lgg-1(RNAi) and lgg-2(RNAi) on life span. Survival curves of control, lgg-1(RNAi), lgg-2(RNAi), lgg-1(RNAi) + lgg-2(RNAi), have been done at 20°C. The Kaplan-Meier analysis represents the percentage of animals remaining alive according to their age. RNAi was applied either during the larval development and adulthood (C) or during adulthood only (D). Statistical data are presented in Table S2.

of the control dauers presented a lethality while lgg-1(RNAi), lgg-2(RNAi) showed 29.6 and 23.3% of lethality respectively. These data indicate that both LGG-1 and LGG-2 are important to achieve the formation of physiological dauer larvae. We then tested whether the combination of both lgg-1(RNAi) and lgg-2(RNAi) could increase this phenotype, and observed that 43.3% of lgg-1 + 2(RNAi) dauers die after SDS treatment (Fig. 6B). This indicates that LGG-1 and LGG-2 could have partially redundant functions in dauer physiology. Synergistic effect of lgg-2(RNAi) and lgg-1(RNAi) on C. elegans longevity. We then analyzed whether lgg-1(RNAi) and lgg2(RNAi) could affect life span in a synergistic manner (Fig. 6C and D, and Table S2). L3 to L4 wild-type animals were grown on RNAi producing bacteria for 48 hours and allowed to lay eggs on fresh RNAi plates. This progeny which had been fed continuously on RNAi was then assessed for life span. Both lgg-1(RNAi) and lgg-2(RNAi) presented a highly significant life-span shortening compared to the control (mean life span of 13.4, 12.8 and


16.6 days respectively). Moreover, the double inactivation of lgg-1 and lgg-2 resulted in a stronger reduction of longevity (mean life span of 8.6 days). As the inactivation was done during whole life, the effect could be due to both a developmental and aging role of the genes. So, we then assessed for life-span animals which have been fed on RNAi only during adulthood (Fig. 6D). A significant life-span shortening was observed for both lgg-1(RNAi) and lgg-2(RNAi) with stronger effect in the double inactivation (mean life span of 15.7, 15.9 and 11.3 days respectively compared to 19.5 for control). These data indicate that both LGG-1 and LGG-2 are involved in longevity in C. elegans and their depletion appears to have synergistic effects on life-span reduction. Discussion In this report we have analyzed the roles of LGG-2 and LGG-1 the two Atg8 homologues in the nematode C. elegans. We have shown that LGG-1 is a functional homologue of the yeast Atg8

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and that LGG-2 appears to be closer to the mammalian LC3 protein. Our work is the first characterization of LGG-2 and three data indicate that LGG-2 is a “bona fide” autophagosomal marker. (i) The conserved C-terminal glycine is essential for the formation of the LGG-2 modified forms and its localization to vesicles. (ii) The localization pattern of LGG-2 is affected both in autophagic mutants and in physiological conditions of increased autophagy. (iii) The depletion of LGG-2 by RNAi affects the longevity and the formation of dauer and this effect is synergistic with the knockdown of the well-characterized autophagosomal gene lgg-1. One of the conserved characteristics of Atg8/LC3 proteins is their conjugation with a PE by a ubiquitin-like modification machinery, which is essential for their association with the autophagosomal membrane. Our western blot analyses revealed a post-translational modification essential for LGG-2 localization and which could be linked to the lipidation of the C-terminal glycine. Surprisingly, the GFP::LGG-2 high form migrates with a higher apparent molecular weight than GFP::LGG-2, which is different from reports for Atg8, LC-3, GATE-16 and GABARAP.28,36 One can hypothesize that this difference relies either on the intrinsic nature of LGG-2 or in an additional posttranslational modification not present in other Atg8/LC3 homologues. Further biochemical experiments should determine the precise nature of GFP::LGG-2 high and in particular whether it corresponds to the PE conjugated form. Of note, western blotting of the drosophila ATG8 has been unsuccessful in identifying PE modification.34 Until now GFP::LGG-1 was the unique marker used in C. elegans to quantify autophagosomes, mainly by counting the numbers of dots per cell.7 However it could be difficult to analyze puncta when there are large number per cell and in adults where GFP::LGG-1 signal is very strong and diffuse (reviewed in refs. 7 and 8). Another caveat is the formation of punctate structures independently of autophagy due to the overexpression of the fusion protein.44 In this respect, the use of GFP::LGG-2 which can be analyzed both by in vivo observation and by western blotting will be helpful to analyze autophagy in C. elegans. Our analysis of LGG-2 and LGG-1 expression during the whole development revealed widely overlapping patterns. We do not know yet whether LGG-2 and LGG-1 label all autophagosomes or define different populations. In the latter hypothesis, this could indicate either specific autophagosomal functions or different stages in autophagosomal formation in C. elegans. Colocalization experiments will be necessary to answer this question. The main difference between LGG-2 and LGG-1 localization patterns concerns the neuromuscular system where LGG-2 is more strongly expressed and could play a particular role. Interestingly, a role of autophagy in the specific degradation of GABA receptors at the neuromuscular junction has been reported.16 Another striking observation is the presence in the embryo of numerous puncta positive for GFP::LGG-1 or GFP::LGG-2 which suggests a particular role for autophagy during embryogenesis. The massive reduction of these puncta in GFP::LGG2(G130A) animals strongly support that they correspond to autophagosomal structures. Recent reports described the role of

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autophagy in the embryo for the specific degradation of maternally inherited granules in somatic cells. Both LGG-1 and LGG-2 are essential for this process but the double depletion was not documented in these studies.17,18 Our functional analysis of LGG-2 and LGG-1 indicates that both proteins play a role in several autophagic processes. Interestingly, when both genes are depleted we have shown a strong increase in the defects on longevity and dauer formation. These additive effects suggest that LGG-2 and LGG-1 act in parallel, but we cannot exclude a partial redundancy of the proteins or a compensatory mechanism by transcriptional regulation. However we did not observe any obvious upregulation of LGG-1 and LGG-2 protein levels in lgg-2(RNAi) and lgg-1(RNAi) animals, respectively. Our data are the first report of a role of LGG-2 during longevity and dauer formation. Depletion of LGG-1 and LGG-2 by an RNAi approach has already been performed and results in heterogenous results which could be in part explained by technical differences. In their founding paper, Melendez and colleagues reported an embryonic lethality after injection of lgg-1 or lgg-2 RNAi.7 Such an embryonic lethality is also supported by our genetic data on lgg-1(tm3489) mutant. After injection of a two-fold diluted RNAi these authors observed a defect in dauer formation for lgg-1 but not lgg-2. All other published data were based on RNAi feeding and no lethality has been reported.14,17,45-47 lgg-2 has been also reported to be involved in hypoxic injury 46 and granule removal.17 Such differences could be explained by threshold effects linked to the RNAi delivery methods and to possible partial redundancy between LGG-1 and LGG-2. Altogether these data indicate that the autophagic genes lgg-1 and lgg-2 are essential for multiple developmental and physiological processes in C. elegans. C. elegans appears to be a good paradigm to understand the evolution of Atg8 gene functions in eukaryotes. The presence of the two homologues lgg-1 and lgg-2 is an intermediate situation between the unique gene of yeast and the complex situation of mammals with four genes displaying several isoforms. Among them LC3 is the best characterized as an autophagosome marker in mammalian cells and its induction level is tissue dependent.48 In rat tissues, the patterns of expression of LC3, GABARAP and GATE-16 are quite different,29 suggesting tissue-specific autophagic functions for these homologues. However the functional relations between these homologues are not known. The sequence conservation between C. elegans LGG-1 and LGG-2 and their mammalian orthologues suggest that they might have similar activities. Further analyses of the precise functions of both genes in the nematode will be useful to better understand the complexity of their mammalians’ counterparts. Materials and Methods C. elegans strains and genetic methods. Nematode strains were grown on NGM plates seeded with E. coli strain OP50 and handled as described.49 The wild-type parent strain used was the C. elegans Bristol variety strain N2. Mutant strains CB1370 [daf-2(e1370)III] and unc-119(ed3)III were provided by the Caenorhabditis Genetics Center at the University of Minnesota.

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lgg-1(tm3489) was outcrossed four times and maintained as a heterozygous strain of the following genotype: lgg-1(tm3489)/ dpy-10(e128)unc-4(e120). DA2123 strain carrying the integrated array adIs2122[gfp::lgg-1; rol-6(su1006)] was a gift from C. Kang (University of Texas). RD108 and RD155 strains correspond to Ex[gfp::lgg-2; rol-6(su1006)] and Ex[gfp::lgg-2(G130A); rol6(su1006)] respectively. Yeast complementation. The S. cerevisiae strains used in this study were JMG276 (MATa leu2∆0 met15∆0 ura3∆0 his3∆0) and the isogenic mutant strain JMG1028 (∆atg8::kanMX4). Yeast growth media were YPD, SD (1.7 gl-1 of yeast nitrogen base, 2% glucose with ammonium sulphate and appropriate aminoacids supplements for auxotrophic strains) and SD(N) for starvation experiments. lgg-1 and lgg-2 c-DNAs were RT-PCR amplified from total RNA of C. elegans N2 strain and cloned in the plasmid pRS416 to generate pRD110 and pRD111 respectively. These plasmids and pJMG437 which contains a GFP::ATG8 fusion construction under the control of the ATG8 promoter were used to transform JMG1028 strain as described.50 For starvation survival experiments, wild-type and transformed strains were grown to mid-log phase and transferred to SD(-N) medium. Aliquots were removed each day and spread onto YPD plates in triplicate. Colonies were counted after two or three days. Yeast strains and pJMG437 were kindly provided by J.M. Galan. Fluorescence tagged protein constructs and transgenes. To generate an N-terminal GFP::LGG-2 fusion protein, first we cloned a 2.5 kb fragment containing the lgg-2 coding sequence downstream of the GFP coding sequence into the ApaI-EcoRI sites of plasmid pPD117.01 (provided by A. Fire) and a 2.7 kb lgg-2 promoter fragment upstream of the GFP coding sequence into the SalI-KpnI sites of plasmid pPD117.01 to yield the plasmid pRD114. This plasmid was microinjected at ∼30 ng/ml as described51 and [Rol] transgenic strains were obtained. To generate GFP::LGG-2 transgenic lines by biolistic transformation,52 attB1 and attB2 adapters were added by PCR to the boundaries of the entire cloned region in plasmid pRD114. The 6.1 kb generated product was integrated into pDONRTM221 vector (Invitrogen, 12536017) by BP recombination and then integrated by LR recombination into the pSB_GW_TAG_mut destination vector (a gift from M. Mirande) which contains the unc-119(+) gene. gfp::lgg-2(g130a) was obtained by site directed mutagenesis using the Quik Change Site-Directed Mutagenesis Kit (Stratagene, 200518) and transgenic lines were obtained by biolistic transformation. Several independent nonintegrated lines were obtained and presented a similar expression pattern. To limit the variability at least 50 animals were observed for each experiment and those with stronger or weaker signals (less than 10%) were systematically discarded. Analysis of dauer animals. Strains daf-2(e1370)III; adIs2122[gfp::lgg-1; rol-6(df)] and daf-2(e1370)III; Ex[gfp::lgg-2; rol-6(su1006)] were generated using standard genetic procedures. Dauer larvae formation was induced by shifting L1 larvae at 25°C as previously described.7 Dauer induction by daumone38 was performed according to the manufacturer’s instructions (KDR BIOTECH, DA-1-010). For survival of dauer in 1% SDS


we proceed as follows. The progeny of control or RNAi treated animals were grown on daumone plates for 6 days, then checked for dauer formation. Dauers were recovered from plates by washing and then exposed to a 1% SDS solution for 10 minutes. After 2 washes, dauers were put on standard plates and dead and living animals were counted after 2 to 4 hours of recovery. RNA mediated interference. RNAi by feeding was performed as described.41,53 Briefly, synchronized worms at L4 larval stage were placed onto IPTG-containing NGM plates seeded with bacteria (E. coli HT115[DE3] carrying the empty vector L4440 (pPD129.36) or the bacterial clones from the J. Ahringer library (lgg-1, WBRNAi00011484; lgg-2, WBRNAi00022071; rab-7, WBRNAi00009246). CeTOR clone was a gift from Malene Hansen. Worms were allowed to lay eggs at 20°C, and the progeny was analyzed. Proteins extraction and western blot analysis. Yeast whole cell lysates were prepared as described.54 To prepare total worm extracts, worm pellets were resuspended in PBS triton and glass beads were added before homogenization using Precellys24 instrument (Bertin Technologies, 03119.200.RD000) by two cycles of 60 sec at 6,000 rpm. After centrifugation at 12,000 rpm, supernatants were mixed with Laemli buffer (final concentration 1.3% SDS, 0.08 M Tris pH 6.8, 0.07% glycerol, 0.1% bromophenol blue). Protein extracts were denatured for 5 min at 100°C and separated by a 15% acrylamide/bisacrylamide (37.5/1) gel electrophoresis with 1% SDS. Proteins were transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, 10401191) and probed with anti-human LC3B or anti-human GABARAP (a gift from T. Ueno, Juntendo University, Japan) anti-GFP (Roche, 1814460) at 1:500 or anti-Tubulin (Sigma, T9026) as described.35 Immunoreactive proteins were revealed with the ECL detection system (SuperSignal pico Chemiluminescent Subsrate, Perbio, 34080). Signals were quantified on a Las3000 photoimager (Fuji) using ImageQuant 5.2 software (Molecular Dynamics). Imaging analysis. Routinely, fluorescent expression patterns and phenotypic analyses were carried out on a Zeiss axioskop 2 plus equipped with Nomarski optics. Confocal images were captured on an inverted Leica SP2 confocal microscope. Z projections were obtained with the ImageJ program (U.S. National Institutes of Health). All images were then processed with Adobe Photoshop Software and Adobe Illustrator. Quantification of GFP::LGG-1 and GFP::LGG-2 dots was carried out on confocal images using the plug-in “analyze particle” from Image J with a size of 0.3 to 4 µm2 and a circularity of 0 to 1. Starvation analysis. Starvation survival analyses were performed as described55 with few modifications. L4 animals were grown in RNAi plates until they start laying eggs, and then bleached to obtain synchronized embryos. L1 larvae were incubated in 0.5 mL sterilized M9 buffer supplemented with fungicide and antibiotic cocktail to prevent contaminations. At each time point, an aliquot from each sample tube was placed on a plate seeded with E. coli OP50. The number of worms surviving to adulthood was estimated three days after starvation rupture by applying this formula: (Number of adults at Day N + 3) * 100/ Number of larvae at Day N.

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Life-span analysis. A first generation of N2 animals have been grown on RNAi plates until adulthood then placed on new RNAi plates for laying. Their progeny which has completed its entire development on RNAi was assessed for life span. Five groups of around 30 adult worms were placed on fresh RNAi plates at their first day of adulthood (day 0). For adulthood RNAi treatment, wild-type young adults were directly placed on plates containing 16 µM 2'fluoro-5'deoxyuridine (Sigma, F0503). Animals were transferred to fresh plates every 2 to 4 days and were examined every day for touch-provoked movement, until death. All experiments have been done in duplicate. Kaplan-Meier method and Log-Rank (Mantel-Cox) test were performed using GraphPad Prism 5.0 software (Graphpad Software INC.,).

the Caenorhabditis Genetic Center for providing reagents. We are grateful to C.J. Herbert and E. Culetto for critical reading of the manuscript. This work was supported by the Agence National de la Recherche, the Association pour la Recherche contre le Cancer, and the Fondation pour la Recherche Médicale. A.D. is a recipient of a fellowship from the Ministère de l’Education et de la Recherche Technologique. The Imaging and Cell Biology facility of the IFR87 (FR-W2251) “La plante et son environnement” is supported by the Action de Soutien à la Technologie et la Recherche en Essonne, Conseil de l’Essonne. The authors declare that they have no competing financial interests.

Acknowledgements

Supplementary materials can be found at: www.landesbioscience.com/supplement/AlbertiAUTO6-5-Sup. pdf

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