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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Phosphatidylinositol (PI) is a constituent of biomembranes and a precursor of all phosphoinositides (PIPs). A prominent characteristic of PI is that its sn-2 position is highly enriched in polyunsaturated fatty acids (PUFAs), such as arachidonic acid or eicosapentaenoic acid. However, the biological significance of PUFA-containing PI remains unknown. We previously identified Caenorhabditis elegans (C. elegans) mboa-7 as an acyltransferase that incorporates PUFAs into the sn-2 position of PI. In this study, we performed an RNAi enhancer screen against PI kinases and phosphatases using mboa-7 mutants that have a reduced PUFA content in PI. Among the genes tested, knockdown of vps-34, a catalytic subunit of class III PI 3-kinase that produces PI 3-phosphate (PI3P) from PI, caused severe growth defects in mboa-7 mutants. In both vps-34 RNAi-treated wild-type worms and mboa-7 mutants, the size of PI3P-positive early endosomes was significantly decreased. We also performed an RNAi enhancer screen against PI3P-related genes and found that, like knockdown of vps-34, knockdown of autophagy-related genes caused severe growth defects in mboa-7 mutants. Finally, we showed that autophagic clearance of protein aggregates is impaired in mboa-7 mutants. Taken together, these results suggest that the PUFA chain in PI has a role in some PI3P signaling.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Polyunsaturated fatty acids (PUFAs) are major components of cellular membrane phospholipids and participate in a variety of biological functions such as the production of lipid mediators and the maintenance of membrane physical properties. In mammals, PUFA depletion causes various abnormalities including sterility, ulceration and dermatitis (Williard et al. 2001; Stoffel et al. 2008; Stroud et al. 2009). PUFAs are also required for the development and function of the brain and central nervous system (Kotani et al. 2003; Dijck-Brouwer et al. 2005; Dyall & Michael-Titus 2008; Maekawa et al. 2009; Schuchardt et al. 2010). A certain level of fatty acid unsaturation is needed for efficient membrane traffic such as vesicle budding and fusion (Chernomordik et al. 1997, 1999; Ben Gedalya et al. 2009).

Phosphatidylinositol (PI) is a component of membrane phospholipids and participates in various physiological processes through distinct phosphorylated derivatives of the inositol head group (PI phosphates, PIPs) (Di Paolo & De Camilli 2006; Sasaki et al. 2009). A prominent characteristic of PI is that its sn-2 position is highly enriched in PUFAs, such as arachidonic acid or eicosapentaenoic acid (EPA) (Patton et al. 1982; Tanaka et al. 2003; Lee et al. 2008). However, the role of PUFAs in PI or PIPs in PIP signaling, if any, remains unknown. Many studies have shown that PUFAs are incorporated into PI by the sequential actions of phospholipase A2 and lysoPI acyltransferase after de novo synthesis of PI (Holub & Kuksis 1971; Baker & Thompson 1973; Luthra & Sheltawy 1976; Yamashita et al. 2003). Although a high level of lysoPI acyltransferase activity was detected in various mammalian tissues by in vitro assays (Baker & Thompson 1973; Inoue et al. 1984; Sanjanwala et al. 1989; Yashiro et al. 1995), the gene responsible for the activity was not identified until recently. In an RNA interference (RNAi)-based genetic screen using Caenorhabditis elegans (C. elegans), we identified mboa-7 as an acyltransferase that selectively incorporates PUFAs such as arachidonic acid or EPA into the sn-2 position of PI (Lee et al. 2008). We also reported that deletion of the mboa-7 gene significantly decreased the content of EPA, the predominant PUFA in C. elegans, in PI. However, the biological significance of PUFA-containing PI remains unknown. Because PI is a precursor of PIPs, we hypothesized that certain PIP signaling pathways would be impaired by the reduced content of PUFAs in PI and PIPs. In this study, we conducted an RNAi-based genetic enhancer screen against PI kinases and phosphatases using mboa-7 mutants that have a reduced PUFA content in PI. We found that knockdown of vps-34, a class III PI 3-kinase responsible for PI 3-phosphate (PI3P) synthesis, caused severe growth defects in mboa-7 mutants and demonstrated that PI3P-related events such as early endosome morphology and autophagy are impaired in the mutants.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Generation of mboa-7 mutants

We generated two deletion alleles, mboa-7 (tm3536) and mboa-7 (tm3645), and obtained two others, mboa-7 (gk399) and mboa-7 (ok1028), from the Caenorhabditis Genetics Center (CGC). The mboa-7 (gk399) and mboa-7 (ok1028) alleles lack exons 1-3 and exons 1-4, respectively (Fig. 1A). The mboa-7 (tm3536) allele lacks exon 8, which contains the conserved histidine residue that is essential for acyltransferase activity (Lee et al. 2008). The deletion in the mboa-7 (tm3645) allele causes a frame shift that yields a new stop codon before the conserved histidine. All four alleles exhibited almost no lysoPI acyltransferase activity with arachidonoyl-CoA as an acyl donor (Fig. 1B), indicating that they were null or strong loss-of-function alleles. Growth of all four of the mboa-7 mutant alleles was similar to that of wild type (Fig. 1C).

image

Figure 1. Generation of mboa-7 mutants. (A) Genomic structure of mboa-7 (F14F3.3). The mboa-7 gene is located on chromosome X. Gray boxes indicate coding exons, and white boxes indicate 5′ and 3′ untranslated sequences. The positions of the ATG initiation codon, stop codon (TAG), the poly(A) tail and the conserved histidine (red) are shown. The extent of the deletion in each allele is indicated by a horizontal line. (B) [14C]Arachidonoyl-CoA:lysoPI acyltransferase activity in the membrane fractions of wild-type animals or mboa-7 mutants. Results are expressed as means ± SEM from three independent experiments. (C) Representative pictures of wild-type and mboa-7 mutant worms. Embryos were placed onto culture plates, incubated at 20 °C, and photographed after 60 h of growth. Scale bars, 200 μm.

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Knockdown of class III PI 3-kinase causes growth defects in mboa-7 mutants

Phosphoinositides are synthesized by PI kinases and phosphatases and play crucial roles in the regulation of a wide variety of cellular processes via specific interactions of PIP-binding proteins (Di Paolo & De Camilli 2006; Sasaki et al. 2009). To identify PIP signaling that is affected by reduced PUFA content in PI, we performed an RNAi screen against PI kinases and phosphatases (Table S1 in Supporting Information). We found that among the genes tested, knockdown of vps-34, a catalytic subunit of class III PI 3-kinase, caused severe growth defects in mboa-7 mutants (Fig. 2A,C). When vps-34 was knocked down in mboa-7 mutants and wild-type animals, the former displayed small, skinny and pale appearance and did not grow to adult size, whereas the latter grew to adult size and laid eggs. In an RNAi-hypersensitive eri-1 background (Kennedy et al. 2004), knockdown of vps-15 (ZK930.1), a regulatory subunit of vps-34, also caused growth defects in mboa-7 mutants (Fig. 2B,C). Class III PI 3-kinase produces PI3P by phosphorylating the D-3 position of the inositol head group of PI (Backer 2008). Thus, reduction in the PI3P level may induce severe growth defect in mboa-7 mutants. Neuronal expression of MBOA-7::GFP was sufficient to rescue the growth defects of mboa-7 mutants (Fig. 3), suggesting that PUFAs in PI have an important role in PI3P signaling in neurons.

image

Figure 2. Knockdown of class III PI 3-kinase causes growth defects in mboa-7 mutants. (A) Representative pictures of wild-type and mboa-7 mutant worms treated with mock, vps-34, age-1 or piki-1 RNAi. Knockdown of vps-34 causes growth defects in mboa-7 (gk399) and mboa-7 (tm3536) alleles, but not in wild-type animals. Knockdown of either age-1 (class I PI 3-kinase) or piki-1 (class II PI 3-kinase) does not cause growth defects in mboa-7 mutants. Scale bars, 200 μm. (B) Representative pictures of eri-1 and eri-1; mboa-7 mutant worms treated with mock or vps-15 RNAi. Knockdown of vps-15 causes growth defects in mboa-7 mutants in the RNAi-hypersensitive eri-1 background. Scale bars, 200 μm. (C) Percentage of animals that exhibit growth defects. At least 50 worms were counted for each experiment. **P < 0.01; ***P < 0.001 (chi-squared test).

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image

Figure 3. Neuronal expression of MBOA-7 rescues the growth defects of mboa-7 mutants. y-axis indicates the percentage of animals that exhibited growth defects under vps-34 RNAi condition. MBOA-7::GFP was expressed in mboa-7 mutants under the control of tissue-specific promoters: mboa-7 (ubiquitous), unc-119 (neuroblasts and postmitotic neurons), and rgef-1 (postmitotic neurons), dpy-7 (hypodermis), myo-3 (body wall muscle). At least 40 worms were counted. ***P < 0.001 (chi-squared test).

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Decreased size of PI3P-positive early endosomes in mboa-7 mutants

PI3P has been shown to localize mainly in early endosomes, and PI3P-positive early endosomes can be visualized by the PI3P probe 2xFYVE::GFP (Gillooly et al. 2000). To examine whether PI3P-harboring early endosomes are affected in mboa-7 mutants, we used the cdIs85[pcc1::2xFYVE::GFP] strain that expresses 2xFYVE::GFP in coelomocytes (Dang et al. 2004; Luo et al. 2011). Coelomocytes are large scavenger-like cells situated in the pseudocoelomic cavity and suited for the observation of organelles such as endosomes or lysosomes. In wild-type worms subjected to vps-34 RNAi, the size of 2xFYVE::GFP-positive early endosomes was decreased, and 2xFYVE::GFP was diffused in the cytosol of some coelomocytes (Fig. 4A). vps-15 RNAi also caused similar but somewhat less severe defects. These results suggest that reduction in the PI3P level induces smaller PI3P-positive early endosomes and 2xFYVE::GFP dispersion in the cytosol.

image

Figure 4. Loss of MBOA-7 affects early endosomal morphology. (A) Fluorescent images of coelomocytes expressing 2xFYVE::GFP. The adult worms were allowed to lay eggs for 2 h on RNAi plates, and the young (day 1) adult progeny was examined. Mock-treated coelomocytes contain several large (>2 μm) early endosomes labeled with 2xFYVE::GFP. vps-34 RNAi-treated coelomocytes have smaller GFP-positive endosomes or no GFP-positive endosomes with diffuse fluorescence in the cytosol. Scale bars, 5 μm. (B) Number of 2xFYVE::GFP-positive early endosomes per coelomocyte with more than 2 μm in diameter. mboa-7 mutants have fewer 2xFYVE::GFP-positive early endosomes with more than 2 μm in diameter than wild-type animals. At least 30 coelomocytes were measured for each experiment. Results are expressed as means ± SEM from five independent experiments. *P < 0.05 (two-tailed paired t-test). (C) Percentages of coelomocytes with dispersed 2xFYVE::GFP fluorescence. Under vps-34 or vps-15 RNAi conditions, mboa-7 mutants displayed more severely affected coelomocytes than wild-type animals. At least 30 coelomocytes were measured for each group. Results are expressed as means ± SEM from four independent experiments. *P < 0.05 (two-tailed paired t-test).

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Quantification of diameter of the 2xFYVE::GFP-positive vesicles in both wild type and mboa-7 mutants revealed that the number of 2xFYVE::GFP-positive vesicles with more than 2 μm in diameter was significantly lower in mboa-7 mutants than that in wild type (Fig. 4B). Although the number of 2xFYVE::GFP-diffused coelomocytes was not significantly greater in the mboa-7 mutants than in the wild type, RNAi of vps-34 or vps-15 in mboa-7 mutants increased the number of 2xFYVE::GFP-diffused coelomocytes (Fig. 4C). These results suggest that, like vps-34 RNAi worms, the mboa-7 mutants had decreased levels of PI3P.

Knockdown of autophagy-related genes causes growth defects in mboa-7 mutants

Class III PI 3-kinase has been shown to regulate a variety of vesicular trafficking pathways including endocytosis, endosome-to-Golgi retrograde transport, autophagy and the target of rapamycin (TOR) signaling pathway (Backer 2008). To identify a causative pathway for the growth defects observed in mboa-7 mutants, we performed an RNAi screen in mboa-7 mutants against PI3P-related genes (Table S2 in Supporting Information). We found that knockdown of autophagy-related genes caused severe growth defects in mboa-7 mutants in an eri-1 mutant background (Fig. 5 and Table S3 in Supporting Information).

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Figure 5. Knockdown of autophagy-related genes causes growth defects in mboa-7 mutants. Percentage of animals that exhibit growth defects. eri-1 and eri-1; mboa-7 mutant worms were treated with RNAi against autophagy-related genes. At least 50 worms were counted for each experiment. **P < 0.01; ***P < 0.001 (chi-squared test) as compared to eri-1 mutants. For more details, see Table S3 in Supporting Information.

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To examine whether autophagy is impaired in mboa-7 mutants, we crossed the adIs2122[GFP::lgg-1] strain, which expresses GFP::LGG-1, with mboa-7 mutants. LGG-1 is a C. elegans ortholog of Atg8/MAP-LC3, and GFP-tagged LGG-1 is often used as a reporter to monitor autophagy (Meléndez et al. 2003). LGG-1 is highly expressed in the seam cells, a type of lateral epidermal cells in C. elegans. Knockdown of autophagy-related genes results in the accumulation of large aggregates of GFP::LGG-1 in seam cells, which is presumably due to impaired clearance of this protein via autophagy (Meléndez et al. 2003; Meléndez & Neufeld 2008; Sigmond et al. 2008). The number of GFP::LGG-1 aggregates was significantly larger in the seam cells of mboa-7 mutants than those of wild type (Fig. 6). When vps-34 was knocked down, GFP::LGG-1 aggregates were also increased in wild-type worms. This observation is consistent with the increased LC3 aggregates in Vps34−/− MEF cells (Jaber et al. 2012). vps-34 knockdown in mboa-7 mutants caused a further increase in the number of GFP::LGG-1 aggregates. These results suggest that autophagic clearance of protein aggregates is impaired in both mboa-7 mutants and vps-34-knocked down wild-type worms.

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Figure 6. Accumulation of GFP::LGG-1 aggregates in seam cells. (A) Confocal fluorescent images of seam cells expressing GFP::LGG-1. Adult worms were allowed to lay eggs for 2 h on RNAi plates, and the young (day 1) adult progeny was examined. Scale bars, 10 μm. (B) Number of GFP::LGG-1 aggregates within 100 μm of the seam cells. vps-34 RNAi caused accumulation of GFP::LGG-1 aggregates in wild-type animals. Further accumulation was observed in mboa-7 mutants. At least 30 animals were measured for each experiment. Results are expressed as means ± SEM from four independent experiments. *P < 0.05 (one-way anova followed by Newman–Keuls test).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, by utilizing an RNAi-based genetic enhancer screen, we found that PI3P-related events such as early endosome morphology and autophagic clearance of aggregated proteins are impaired in mboa-7 mutants, which have a reduced PUFA content in PI.

Vps34, a class III PI 3-kinase that produces PI3P from PI, is responsible for many of the PI 3-kinase-dependent steps in the endocytic system (Backer 2008). Especially, EEA1 is recruited to early endosomes by binding its FYVE domain to PI3P and plays a role in tethering Rab5-positive early endosomes. Suppression of Vps34 activity inhibits EEA1 recruitment to early endosomes (Siddhanta et al. 1998) and inhibits homotypic early endosome fusion (Christoforidis et al. 1999). Smaller PI3P-positive early endosomes observed in coelomocytes of vps-34 RNAi-treated worms may be a result of impaired homotypic early endosomes fusion. The fact that mboa-7 mutants also have small early endosomes suggests that they have decreased PI3P levels.

Autophagy is a membrane trafficking event initiated by the formation of an autophagosome, which then fuses with a lysosome to become an autolysosome, where the contents are degraded by lysosomal hydrolases (Meléndez & Neufeld 2008; Inoue & Klionsky 2010; Mizushima & Komatsu 2011; Mizushima et al. 2011). It is well known that PI 3-kinase is necessary for autophagy. For example, genetic ablation of yeast, Drosophila and mammalian Vps34, inhibits autophagy at the earliest stages (Kihara et al. 2001; Byfield et al. 2005; Juhász et al. 2008). Wortmannin or 3-methyladenine, both of which inhibit Vps34 activity, have similar effects on inhibiting the induction of autophagy (Blommaart et al. 1997; Petiot et al. 2000). Although the exact function of PI3P in autophagy is unclear, it is possible that PI3P recruits PI3P-binding proteins that are important for autophagosome formation, such as Atg18 (Yoshimori & Noda 2008). Accumulation of GFP::LGG-1 large aggregates, which results from impaired autophagy in C. elegans, in both vps-34 RNAi worms and mboa-7 mutants also supports the idea that the PI3P level is decreased in mboa-7 mutants.

Previous in vitro studies have suggested that alteration of the fatty acyl chains of PI and PIPs affects the substrate recognition of some PI kinases and phosphatases (Shirai et al. 1999; Schmid et al. 2004). Although the substrate preference of VPS-34 for the fatty acid species of PI is unknown, VPS-34 may prefer PUFA-containing PI as a substrate. mboa-7 mutants have low levels of PUFA-containing PI and, therefore, may have low levels of PI3P.

Because of the low content of PI3P in cells, it is very difficult to measure the fatty acid species of PI3P. Previous studies reported that the fatty acid species of PI mono-phosphate (PIP) and PI bis-phosphate (PIP2) are very similar to those of PI (Wenk et al. 2003; Imae et al. 2012). Moreover, acyltransferase activity with arachidonoyl-CoA as an acyl donor toward lysoPIP or lysoPIP2 is negligible compared with that toward lysoPI (Palmer 1986). These results suggest that PUFA-containing PIPs are produced from PUFA-containing PI. Based on this circumstantial evidence, it is evident that the fatty acyl chains of PI3P are changed similarly to those of PI in mboa-7 mutants. As mentioned previously, PI3P exerts its function through PI3P-binding proteins such as EEA1 and Atg18. The reduced content of PUFAs in PI3P might impair the binding of these proteins to PI3P, which leads to defects in early endosomes and autophagy. Alternatively, PUFAs in PI3P may affect the microdomain localization of PI3P. PI3P is enriched in the inner surface of isolation membranes and in the region next to the elongating tips of isolation membranes (Obara et al. 2008). In addition, PI3P-enriched ER subdomains, called omegasomes, are proposed to be potential precursors of autophagosomes (Axe et al. 2008). Decreased PUFA content in PI3P may induce mislocalization of PI3P in endomembranes.

We showed that mboa-7/vps-34 double inhibition led to the growth defects and that neuronal expression of MBOA-7::GFP was sufficient to rescue the defects. Recent studies indicate that suppression of autophagy in neuronal cells causes growth defects in mice (Hara et al. 2006; Komatsu et al. 2006), possibly because autophagy is required for preventing neurodegeneration through clearing out toxic and aggregation-prone proteins. Thus, suppression of autophagy in neurons may cause growth defects in C. elegans. vps-34 knockdown should cause the decrease in PI3P level. As discussed above, mboa-7 depletion may suppress PI3P production from PI and/or induce mislocalization of PI3P in endomembranes. Both vps-34 knockdown and mboa-7 depletion should synergistically impair the PI3P signaling in neural autophagy and cause growth defects.

In conclusion, this study demonstrated that deficiency of lysoPI acyltransferase (which incorporates PUFAs into PI) impairs some PI3P-related signaling such as the signaling involved in early endosome formation and autophagy. The next challenge will be to determine whether the change in PUFA content in PI affects PI3P production, and whether the change in PUFA content in PI3P affects the interaction with PI3P-binding proteins and the localization of PI3P in endomembranes.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Materials

[1-14C]-labeled arachidonoyl-CoA was purchased from Moravec Biochemicals, Inc. (Brea, CA, USA). Arachidonoyl-CoA was obtained from SIGMA. LysoPI from porcine liver was purchased from Serdary Research Laboratories (London, ON, Canada).

General methods and strains

General methods for maintaining C. elegans are described by Brenner (Brenner 1974). The C. elegans wild-type strain was Bristol N2, and E. coli HT115 was used as the sole food source. The following mutations and transgenes were used: mboa-7 (gk399), mboa-7 (ok1028), mboa-7 (tm3536), mboa-7 (tm3645), eri-1 (mg366) (Kennedy et al. 2004), cdIs85[pcc1::2xFYVE::GFP], adIs2122[GFP::lgg-1], xhEx4072[Pmboa-7::mboa-7cDNA::GFP], xhEx4077[Punc-119::mboa-7cDNA::GFP], xhEx4079[Pdpy-7::mboa-7cDNA::GFP], xhEx4084[Pmyo-3::mboa-7cDNA::GFP], and xhEx4085[Prgef-1::mboa-7cDNA::GFP]. Some of the strains used in this work were obtained from Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN). All mutations were backcrossed at least five times before further analysis.

Feeding RNAi

Feeding RNAi was performed as described previously (Kamath et al. 2001). RNAi feeding strains from the Ahringer library (Ashrafi et al. 2003) or from the ORFeome RNAi collection (Rual et al. 2004) were tested on wild type and mboa-7 mutants. For the assessment of growth, adult P0 worms were allowed to lay eggs for 2-3 h on RNAi plates, and after incubation at 20 °C for 72 h, the F1 progeny was transferred to fresh RNAi plates. Then, the F1 adult worms were allowed to lay eggs for 2–3 h on RNAi plates, and their F2 progeny was observed. Worms that displayed small, skinny and pale appearance and did not grow to adult size after 60 h of growth were counted as growth defects. For observations of coelomocytes and seam cells, adult worms were mounted on the RNAi plates and allowed to lay eggs for 2 h on RNAi plates, and the young (day 1) adult progeny was observed.

Preparation of transgenic worms

For each construct, more than two independent transgenic lines were analyzed. DNA injection into the C. elegans germ-line was carried out as described previously (Mello et al. 1991). The array xhEx4072[Pmboa-7::mboa-7cDNA::GFP] contained the plasmid pLH015 (Pmboa-7::mboa-7cDNA::GFP). The array xhEx4077[Punc-119::mboa-7cDNA::GFP] contained the plasmid pLH017 (Punc-119::mboa-7cDNA::GFP). The array xhEx4079[Pdpy-7::mboa-7cDNA::GFP] contained the plasmid pLH019 (Pdpy-7::mboa-7cDNA::GFP). The array xhEx4084[Pmyo-3::mboa-7cDNA::GFP] contained the plasmid pLH020 (Pmyo-3::mboa-7cDNA::GFP). The array xhEx4085[Prgef-1::mboa-7cDNA::GFP] contained the plasmid pLH029 (Prgef-1::mboa-7cDNA::GFP). The transgenes were injected into mboa-7 (tm3536) mutants with a marker Pges-1::DsRedm, which provides red fluorescence in gut. pLH015, pLH017, pLH019, pLH020 and pLH029 were prepared as follows.

pLH015

mboa-7 promoter (4000 base pairs upstream of the initiation codon) was subcloned by polymerase chain reaction (PCR) from pKE1 vector (Lee et al. 2008) and was cloned into the pPD95.67 vector (NLS-; a kind gift from Dr. Andrew Fire, Stanford University School of Medicine) at the PstI and XbaI sites, yielding a vector pLH001. Next, Full-length mboa-7 cDNA was amplified by PCR from a C. elegans cDNA library with primers Ke49, 5′-TCTAGAGGATCCCCGGGATGGAAAATATCCTTGGCTTG-3′ and Ke50, 5′-CCTTTGGCCAATCCCGCGGCCGCTCCGATTTTTGAGCTTTTTTC-3′ and integrated in frame with the green fluorescent protein (GFP) gene into pLH001 vector at the SmaI site using an In-Fusion Dry-Down PCR cloning kit (Clontech, Mountain View, CA).

pLH017

unc-119 promoter was subcloned from pGL235 vector (a gift from Giovanni M. Lesa, University College London; Lesa et al. 2003) into pLH015 vector at the PstI and SmaI sites.

pLH019

mboa-7 cDNA was excised from pLH015 vector with SmaI and NotI and inserted into pTK001 vector (a derivative of pHM078, a gift from Dr. H. Moribe, Osaka University; Moribe et al. 2004) under a dpy-7 promoter.

pLH020

myo-3 promoter was subcloned from pGL236 vector (a gift from Dr. Giovanni M. Lesa, University College London; Lesa et al. 2003) into pTK001 vector at the HindIII and BamHI sites, yielding a vector pLH016. Next, mboa-7 cDNA was subcloned into pLH016 vector at the SacI and NotI sites.

pLH029

rgef-1 promoter was excised from pMF344.5 vector (a gift from Dr. Masamitsu Fukuyama, University of Tokyo) with PstI and SmaI and inserted into pLH015 vector.

Acyltransferase assay

Synchronized young adult worms were suspended in 50 mm potassium phosphate buffer (pH 7.0) containing 0.15 m KCl, 0.25 m sucrose, 1 mm EDTA, 1 mm dithiothreitol and 5 μg/mL pepstatin, leupeptin and aprotinin (homogenizing buffer), and they were sonicated three times on ice for 30 s. The homogenate was centrifuged at 10 000 g for 30 min at 4 °C, and the resulting supernatant was further centrifuged at 105 000 g for 60 min. The pellet was suspended in homogenizing buffer (without EDTA, dithiothreitol and protease inhibitors) and used for the enzyme assay described later. [14C]Arachidonoyl-CoA (53 mCi/mm) was diluted with the unlabeled arachidonoyl-CoA to 10 mCi/mm before use. Reaction mixtures contained 10 μm of [14C]arachidonoyl-CoA and 40 μm of lysoPI, and 10 μg of microsomal protein in a total volume of 400 μL assay buffer [0.15 m KCl, 0.25 m sucrose, 50 mm potassium phosphate buffer (pH 6.8)]. After incubation at 20 °C for 5 min, reactions were stopped by the addition of 1 mL of methanol. Total lipid was extracted by the method of Bligh & Dyer (1959) and separated by one-dimensional TLC on silica gel 60 plates (Merck, Darmstadt, Germany) in chloroform:methyl acetate:1-propanol:methanol:0.25% KCl (25:25:25:10:9, v/v). The area of silica gel for PI was scraped off the plate, and radioactivity was measured. The protein concentrations of samples were determined by the BCA assay (Pierce, Rockford, IL, USA).

Microscopy

Young adult worms were mounted on a 5% agar pad on a glass slide and immobilized in 0.02 m azide. For observations of coelomocytes, fluorescence images were obtained from the posterior coelomocytes (ccDL and ccDR) by an Axio Imager M1 (Carl Zeiss MicroImaging, Thornwood, NY, USA) microscope equipped with CCD camera. For observations of seam cells, fluorescence images were obtained by a Zeiss LSM510 META confocal microscope system (Carl Zeiss MicroImaging).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr Andrew Fire (Stanford University School of Medicine) for vector pPD95.67 and pPD95.77, Dr. Giovanni M. Lesa (University College London) for vector pGL235 and pGL236, and Dr Masamitsu Fukuyama (University of Tokyo) for vector pMF344.5. We also gratefully acknowledge technical assistance from Hideko Fukuda and Yuko Funakoshi. Some of the strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Center for Research Resources of the National Institutes of Health. This work was supported by the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (CREST, JST) (to H.A.), the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (to H.A.), Grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science, and Technology, and the Japanese Ministry of Health, Labor, and Welfare (to H.A.), and Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (to H.L.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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gtc1624-sup-0001-Table S1-S2-S3.pdfapplication/PDF352KTable S1 Caenorhabditis elegans PI kinases and phosphatases genes Table S2 A list of PI3P-related genes tested in an RNAi screen Table S3 A list of autophagy-related genes in Caenorhabditis elegans

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