Developmental Cell, Volume 28 Supplemental Information The C. elegans LC3 Acts Downstream of GABARAP to Degrade Autophagosomes by Interacting with the HOPS Subunit VPS39 Marion Manil-Ségalen, Christophe Lefebvre, Céline Jenzer, Michael Trichet, Claire Boulogne, Béatrice Satiat-Jeunemaitre, and Renaud Legouis Inventory of Supplementary Materials Figure S1, Characterization of lgg-1 and lgg-2 null mutants, related to Figure 1 Figure S2, LGG-1 and LGG-2 colocalize at the allophagic cluster in 1-cell embryo, related to Figure 2 Figure S3, Analyses of LGG-1 autophagosomes in lgg-2(tm5755) embryo, related to Figure 3 Figure S4, Interactions between lysosomes and the allophagic cluster in control and lgg2(tm5755) embryos, related to Figure 5 Figure S5, Characterization of VPS-39 interaction with LGG-2 and phenotypic analysis, related to Figure 6 Movie 1: Movies from the time series shown in Figure 3I, J. Ppie-1::GFP::LGG-1 allophagic cluster in control (left) and in lgg-2(RNAi) (right) embryo. Movie 2: Movies from the time series shown in Figure 4H, I. GFP::mCherry::LGG-1 in control (left) and in lgg-2(tm5755) mutant (right) embryo. Supplemental Experimental Procedures Strains and transgenes construction Immunofluorescence and dye staining experiments Electron microscopy Yeast Two Hybrid Movies Manil-Ségalen et al. Manil-Ségalen Supplemental Information Legends of supplementary Figures Figure S1: Characterization of lgg-1 and lgg-2 null mutants, related to Figure 1 A) Amino-acid sequence alignment of the entire sequences of LGG-1 and LGG-2 with their homologs. The structural motifs (-helix and -sheets) are indicated with sinusoids and arrows between the two alignments. The conserved residues are shaded in dark blue. The conserved characteristic residues are indicated by a vertical arrowhead (a glycine in position 18 and a basic residue in 40 for GABARAP and an hydrophobic residue in position 20 and a basic residue in 42 for LC3). The conserved C-terminal glycine is boxed in red. B-G) Confocal images of LGG-1 and LGG-2 in lgg-1(tm3489maternal) (B-D) and lgg2(tm5755) (E-G) 1-, 2- and 4-cell stage embryos (control on Fig. 2 and quantification on Fig. 3). Bar is 10µm. H) Quantification of the fertility and the percentage of lethality in the progeny of lgg2(tm5755), lgg-1(tm3489)/+ and double lgg-1(tm3489)/+;lgg-2(tm5755) animals. Only the F1 lgg-1(tm3489) homozygous animals develop normally but are sterile. The double mutants lgg1(tm3489);lgg-2(tm5755), which represent 25% of the progeny, are lethal at the embryonic and first larval stage. lgg-1(tm3489maternal) indicates animals mutant for lgg-1 maternal contribution only. I-L) Nomarski pictures of wild-type embryo (I) and L1 larva (K), and lgg-1(tm3489);lgg2(tm5755) at the corresponding stages of development (J, L). The lgg-1(tm3489);lgg-2(tm5755) animals are arrested during their development between the second half of the embryogenesis and the early L1 stage. The stronger phenotype observed (J) shows organogenesis defects in the epidermis (arrowheads) and the intestine (arrows). Hatched larvae (L) present a strong vacuolisation of the intestine (arrows). Bars are 10 µm (I, J) or 25 µm (K, L). Figure S2: LGG-1 and LGG-2 colocalize at the allophagic cluster in 1-cell embryo, related to Figure 2 A) Schematic representation of the main events occurring after fertilization in the 1-cell stage embryo. B-E) Confocal images of 1-cell stage embryos at the corresponding steps and quantification (F) of the colocalization between LGG-1 and LGG-2. At all stages, the allophagic cluster is positive for both LGG-1 (red) and LGG-2 (green). Bar is 10µm. -1- Manil-Ségalen et al. Figure S3: Analyses of LGG-1 autophagosomes in lgg-2(tm5755) embryo, related to Figure 3 A-C) Confocal images of LGG-1 (A) and GFP::LGG-1 (B, C) in lgg-2(tm5755) 200-cell (A), 500-cell and comma (C) stages. LGG-1 puncta are bigger and brighter compared to control embryos presented in Figure 1L and 2E. Fluorescent image (D) and electron microscopy (E) of an ultrathin section of a 12-cell lgg2(tm5755) embryo expressing Ppie-1::GFP::LGG-1 (green). Fluorescent dots observed in D are indicated by green circles in E and labelled from “g” to “j”. F) Higher magnification of the boxed region in E. The localization of GFP::LGG-1 positive autophagosomes is indicated by black arrows and shown with higher magnification (G-J). Scale bars: 10µm (A, C), 5µm (D, E), 1 µm (F), 200nm (G-J) Figure S4: Interactions between lysosomes and the allophagic cluster in control and lgg2(tm5755) embryos, related to Figure 5 1) Colocalization between GFP::LGG-1 and Lysotracker A-H) Deconvoluted epifluorescence pictures of GFP::LGG-1 (green) and Lysotracker (red) in control (A-D) and lgg-2(tm5755) embryos (E-H). Colocalization between the lysosomes and the allophagic cluster is observed since the 2-cell stage in control embryos (arrows). This colocalization is delayed in lgg-2(tm5755) embryos. Insets are 2 fold magnifications of the allophagic cluster and splitted color pannels. See Fig. 5T for quantifications. 2) Colocalization between GFP::LGG-1 and NUC-1::mCherry Splitted images corresponding to Figure 5. A-H) Deconvoluted epifluorescence pictures of GFP::LGG-1 (green) and NUC-1::mCherry (red) in control (A-D) and lgg-2(tm5755) embryos (E-H). Lysosomes are detected in the allophagic cluster in control (arrows) but not in lgg2(tm5755) embryos. 3) Localization of the lysosomes around the nuclei of early embryos A-F) Deconvoluted epifluorescence images of NUC-1::mCherry (A-C) and Lysotracker (D-F) in 1-, 2- and 4-cell stage control embryos. White arrowheads indicate the enrichment of lysosomes around the nuclei. Bar is 10µm. -2- Manil-Ségalen et al. Figure S5: Characterization of VPS-39 interaction with LGG-2 and phenotypic analysis, related to Figure 6 A) Pairwise two hybrid test validates LGG-2/VPS-39 direct interaction. Interaction pairs were tested in duplicate with independent clones (1 and 2). The diploid yeast cells expressing both bait and prey constructs were spotted on several selective media at several dilutions (10-1, 10-2, 10-3and 10-4). LGG-2 is very weakly autoactivator without 3-AT. LGG-2/VPS-39 interaction resists up to 100mM 3-AT. B-D) Confocal images of LGG-1 (red) and LGG-2 (green) in vps-39(tm2253) embryos at the 1-, 2- and 4-cell stages. Pictures shown are maximum projections corresponding to the whole embryo (1-cell) or 3-5µm (2-cell and 4-cell). White arrows indicate LGG-1 and LGG-2 accumulation around the nuclei. E) Maximum projection of a 4-cell vps-39(tm2253) embryo showing an accumulation of the paternal mitochondria (red) around the nuclei (blue). F-M) Confocal images of LGG-1 (red) and LGG-2 (green) in wild type (F) lgg-2(tm5755) (G), vps39(ok2442) (H), vps-39(tm2253) (I), vps-41(ep402) (J), rab-7(RNAi) (K), rab-7(RNAi);vps39(tm2253) (L) and lgg-2(tm5755);rab-7(RNAi) (M) oocytes. Oocytes are numbered from -1 to -3 according to their relative position to the spermatheca (-1 is the closest to the spermatheca). Allophagic cluster is never observed in these conditions. The LGG-2 signal in the periphery of the oocytes is in somatic cells. Maximum projections of 10µm thick. Bar is 10µm. Movie 1: Movies from the time series shown in Figure 3I,J. Ppie-1::GFP::LGG-1 allophagic cluster in control (left) and in lgg-2(RNAi) (right) embryo. Movie2: Movies from the time series shown in Figure 4H,I. GFP::mCherry::LGG-1 in control (left) and in lgg-2(tm5755) mutant (right) embryo. -3- Manil-Ségalen et al. Supplementary Experimental Procedures Strains and transgenes construction. Mutant alleles were provided by Shohei Mitani at the Japanese National Bioresource Project for Nematode (lgg-1(tm3489), lgg-2(tm5755), vps-39(tm2253)) and the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) (vps-39(ok2442), vps-41(ep402)). The strain DA2123 was a gift from Chahee Kang (University of Texas, Dallas, TX), the Pced-1::nuc-1::mMcherry was kindly provided by Xiaochen Wang (Bejing, China), and VIG-9 was a gift from Vincent Galy (Paris France). The lgg-1(tm3489maternal) was constructed to study the depletion of lgg-1 in the early embryonic stages and to get rid of lethality effect. Briefly, we use the transgenic Plgg-1::gfp::lgg-1, which is expressed in somatic tissue but not in the germ-line to rescue the lgg-1(tm3489) phenotype. Zygotic expression of this construct starts when the embryos reach the 20-cell stage. Homozygous lgg-1(tm3489); adIs2122[Plgg-1::gfp::lgg-1;rol-6(df)] are viable and fertile and immunofluorescence confirms the complete absence of LGG-1 protein during the first cell divisions (Fig. S1). Most of integrated transgenic lines used in this study were generated by microparticle bombardment of unc-119(ed3). To generate the Ppie-1::gfp::lgg-1, Ppie-1::mcherry::lgg-1 and Ppie-1::gfp::mcherry::lgg-1 fusion constructs, the genomic sequence of lgg-1 from pPD117.01 was used (gift from A. Melendez). For the Ppie-1::gfp::mcherry::lgg-1 construct, the mcherry sequence has been added in frame of the lgg-1 genomic sequence. Ppie-1::gfp::lgg-2 has been constructed from the lgg-2 cDNA previously produced (Alberti et al., 2010). The Plgg1::gfp::lgg-1(G116A) was obtained by gtgacgaaagtgtgtatgccggagaggtcgaaaagaa-3’ site and directed mutagenesis using 5’- 5’-ttcttttcgacctctccggcatacacactttcgtcac-3’ forward and reverse primers based on Stratagene strategy (Quick Change Site-Directed Mutagenesis Kit Stratagene, 200518). For every construct, attB1 and attB2 adapters were added by PCR to the boundaries of the entire cloned region. The PCR products were integrated into pDONR221 vector (Invitrogen, 12536017) by BP recombination and then integrated by LR recombination into the destination vector containing unc-119(+) selection marker and attR sites for Gateway cloning. Destination vector used in this study are pID3-01B, containing pie-1 promoter fragment and the gfp coding sequence (Gift from G. Seydoux, used for Ppie1::gfp::lgg-1, Ppie-1::gfp::lgg-2 and Ppie-1::gfp::mcherry::lgg-1 constructs), pID2-01B, containing pie-1 promoter fragment (Gift from G. Seydoux, used for Ppie-1::mcherry::lgg-1 construct) and pSBGW::TAGmut (a gift from M. Mirande, used for the Plgg-1::gfp::lgg-4- Manil-Ségalen et al. 1(G116A) construct). Several independent integrated and non integrated lines were obtained for each construct and presented similar expression patterns. All the strains used in this study are listed below. DA2123 VIG9 RD204 RD216 RD217 RD192 RD197 RD220 RD202 RD155 RD221 RD258 RD227 SY1245 VC2542 CE1463 RD234 RD248 1::gfp::lgg-1] RD271 NL2099 adIs2122[Plgg-1::gfp::lgg-1; rol-6(df)]II unc119(ed3)III; Is[unc-119(+); Plgg-2::gfp::lgg-2] unc119(ed3)III; Is[unc-119(+); Ppie-1::gfp::lgg-1] unc119(ed3)III; Ex[unc-119(+); Ppie-1::gfp::cDNA:lgg-2] unc119(ed3)III; Ex[unc-119(+); Ppie-1::gfp::mcherry::lgg-1] lgg-1(tm3489)II; adIs2122[Plgg-1::gfp::lgg-1; rol-6(df)]II lgg-1(tm3489)/dpy-10(e128) unc-4(e120)II lgg-2(tm5755)IV unc119(ed3)III; Is[unc-119(+); Plgg-1::gfp::lgg-1(G116A)] unc119(ed3)III; Ex[unc-119(+); Plgg-2::gfp::lgg-2(G130A)] lgg-2(tm5755)IV; lgg-1(tm3489); adIs2122[Plgg-1::gfp::lgg-1; rol-6(df)] lgg-2(tm5755)IV; lgg-1(tm3489)/dpy-10(e128) unc-4(e120)II qxIs257[Pced-1::nuc-1::mcherry]; Is[unc-119(+); Ppie-1::gfp::lgg-1] vps-39(tm2253)V vps-39(ok2442) V/nT1[qIs51](IV;V) vps-41(ep402)unc-6(e78)/dpy-8(e130)unc-6(e78)X lgg-2(tm5755)IV; Ex[unc-119(+); Ppie-1::gfp::mcherry::lgg-1] lgg-2(tm5755)IV; qxIs257[Pced-1::nuc-1::mcherry]; Is[unc-119(+); Ppielgg-2(tm5755)IV; adIs2122[Plgg-1::gfp::lgg-1; rol-6(df)]II rrf-3(pk1426)II Immunofluorescence and dye staining experiments Immunostaining assays were performed on permeabilized and fixed embryos. After a saturation step with 1X PBS containing 5% of milk and 0.5% of Tween20 for 40 min and two washing steps in 1X PBS Tween20 0,5%, samples were incubated in 1X PBS, Tween20 0.5% buffer overnight at 4°C (or 2 hours at room temperature (20°C)) with the following primary antibodies. After two washing steps of 30 min in 1X PBS 0.5% Tween 20, samples were incubated in the same buffer 1h30 at room temperature (20°) with the secondary antibody and the DNA labelling. Final mountings have been made in DABCO (Sigma). Primary antibodies used in this study: mouse monoclonal anti-GFP at 1:250 (Roche, 1814460), rabbit anti-GFP at 1:200 (Invitrogen, A11122), mouse anti-LGG-1 at 1:100 (kindly provided by Zhang H.), rat anti-LGG-1 at 1:500 (gift from Zhang lab, Beijing, China, (Tian et al., 2010)., rabbit anti-LGG2 at 1:250 (Al Rawi et al., 2011), rabbit anti-HGRS-1/VPS-27 at 1:250 (Roudier et al., 2005) and rabbit anti-VPS-32.1 at 1:500 (Michelet et al., 2009). As secondary conjugated antibodies, Alexa Fluor® 488, Alexa Fluor® 568 and Alexa Fluor® 633 (Molecular Probes) were used at a dilution of 1:500. DNA was labeled using either Hoescht at 1/500 or TO-PRO®3-iodide at 10µM (Molecular Probes). -5- Manil-Ségalen et al. For lysosome staining, NGM agar plates were supplemented with LysoTracker Red (Molecular Probes) at 2 μM. L4 stage animals were placed on LysoTracker plates and incubated in the dark at 20°C. For staining paternal mitochondria, males were transferred to 150 μl of M9 buffer containing 2 μM of MT CMXRos (Molecular probes) and incubated for 4 h in the dark, before mating. LysoTracker or Mitotracker was analyzed in embryos either directly or after freezecrack and methanol fixation for 5 min. Electron microscopy For immuno electron microscopy, embryos were transferred to 200-mm deep flat carriers, followed by cryo-immobilization in the EMPACT-2 HPF apparatus (Leica Microsystems) and cryo-substitution. Immunostaining were made in PHEM Buffer (Pipes 30mM, Hepes 12.5mM, EGTA 10mM, MgCl2 1mM, pH6.9). Ultrathin sections on formvar- or carbon-coated copper grids were sequentially treated for 15 minutes with 1% BSA, labelled with the primary antibody for 1 hour and, after washes, labelled with the secondary antibody for 45 minutes. After extensive washes, sections were contrasted with 2% uranyl acetate for 7 minutes and with lead citrate for 2 minutes and observed with a Jeol 1400 TEM at 120 kV. Antibodies used in this study are rabbit anti-GFP (1:3000 dilution, Abcam), Rat anti-LGG-1 (1:10 or 1:50 dilutions), donkey anti rabbit coupled to 10nm colloidal gold particles (1μg/ml dilution, Aurion, Biovalley) and goat anti rat (1μg/ml dilution, Aurion, Biovalley). Yeast Two Hybrid The coding sequence of the C. elegans lgg-1 fragment (aa. 1-115, GenBank accession number gi:193204203) was PCR-amplified and cloned in frame with the LexA DNA binding domain (DBD) into the inducible plasmid pB31 (N-LexA-lgg-1-C). pB31 was constructed by inserting LexA into the pFL39 backbone under the control of Met25 promoter. The DBD construct was checked by sequencing the entire insert. Hybrigenics reference for this bait is hgx3100v1_pB31. The coding sequence of the C. elegans lgg-2 fragment (aa. 1-129, GenBank accession number gi:71999766) was PCR-amplified and cloned in frame with the LexA DNA binding domain (DBD) into plasmid pB27 (N-LexA-lgg-2-C), derived from the original pBTM116. The DBD construct was checked by sequencing the entire insert. Hybrigenics reference for this bait is hgx3101v1_pB27. The prey fragment for the C. elegans vps-39 (GenBank accession number gi: 212646543) was extracted from the ULTImate Y2H™ screening of lgg-2 (aa. 1-129) against the C. elegans Mixed Stage cDNA library. The prey fragment was cloned in frame with the Gal4 Activation Domain (AD) into plasmid pP6, -6- Manil-Ségalen et al. derived from the original pGADGH. The AD construct was checked by sequencing. The prey Hybrigenics reference is CEMS_RP1_hgx3101v1_pB27_B-83.The pP7 prey plasmid used in the control assay is derived from the pP6 plasmid. Bait and prey constructs were transformed in the yeast haploid cells, respectively L40 Gal4 (mata) and YHGX13 (Y187 ade2-101::loxP-kanMX-loxP, mat) strains. The diploid yeast cells were obtained using a mating protocol with both yeast strains. These assays are based on the HIS3 reporter gene (growth assay without histidine). For the pB31bait construction, the growth assay was done with selection medium lacking histidine, and methionine. The following interaction pairs were tested: i) Smad3 / Smurf1 (Hybrigenics’ positive control, ii) empty inducible LexA bait vector (pB31)/ empty prey vector, pP7 (negative control), iii) empty inducible LexA bait vector (pB31) / AD-vps-39 (negative control), iv) LexA-lgg-1 / empty prey vector (negative control), v) LexA-lgg-1 / AD-vps-39, vi) Smad3 / Smurf1 (Hybrigenics’ positive control, vii) empty LexA bait vector (pB27) / AD-vps-39 (negative control), viii) LexAlgg-2 / empty prey vector (negative control), ix) LexA-lgg-2 / AD-vps-39. Interaction pairs were tested in duplicate as two independent clones from each diploid were picked for the growth assay. For each interaction, several dilutions (10-1, 10-2, 10-3and 10-4) of the diploid yeast cells (culture normalized at 5Å~104 cells) and expressing both bait and prey constructs were spotted on several selective media. The DO-2 selective medium lacking tryptophan and leucine was used as a growth control and to verify the presence of both the bait and prey plasmids. The different dilutions were also spotted on a selective medium, DO-3-Met (without tryptophan, leucine, methionine and histidine) for testing the LGG-1 interaction; and DO-3 (without tryptophan, leucine, and histidine) for testing the LGG-2 interaction. Four different concentrations of 3-AT, an inhibitor of the HIS3 gene product, were added to the DO3 plates to increase stringency and reduce autoactivation by LGG-2. The following 3-AT concentrations were tested: 1, 5, 10, 50, 100, and 200mM. Movies For each movie, a z-stack acquisition was performed every 1 min on a Zeiss Axio Oberver Z1 microscope equipped with Evolve EMCCD camera. All movies were assembled with NIH ImageJ software using single slice (Movie 1) or 6 slices projection (Movie 2) and saved in mp4 format. -7-