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Vol. 17, Issue 7, 3062-3074, July 2006
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Departments of *Molecular Pathology and ||Developmental Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale U596, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7104, Université Louis Pasteur de Strasbourg, Collège de France, 67404 Illkirch, France; Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721; Oncogenesis and Signaling in Hematopoietic Cells, Institut National de la Santé et de la Recherche Médicale U563, Centre de Physiopathologie de Toulouse Purpan, Institut Fédératif de Recherche 30, Hôpital Purpan, 31059 Toulouse, France; and Department of Molecular, Cell, and Development Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064
Submitted December 13, 2005;
Revised April 13, 2006;
Accepted April 18, 2006
Monitoring Editor: Jean Gruenberg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Several lipid-binding protein domains have been described (Simonsen et al., 2001). For example, phosphatidylinositol-3,4,5-triphosphate recruits Akt/protein kinase B through its pleckstrin homology domain at the plasma membrane, leading to phosphorylation of effectors and activation of downstream pathways, whereas phosphatidylinositol 3-monophosphate [PtdIns(3)P] binds to the FYVE domains of early endosomal antigen EEA1 and Hrs, proteins implicated respectively in early endosome fusion and in receptor sorting in multivesicular bodies (MVBs) (Gruenberg and Stenmark, 2004). PtdIns(3)P is found mainly on early endosomes and internal membranes of MVBs, whereas phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] is on the external membrane of MVBs. These PIs regulate endocytosis and thus protein transport and degradation. For example, growth factors receptors are internalized after stimulation and their signals are down-regulated by their degradation in lysosomes (Miaczynska et al., 2004). Receptors are first endocytosed into early endosomes where they are sorted: they are either recycled back to the plasma membrane or internalized into the lumen of maturing MVBs. Lysosomes could be formed either through maturation of late endosome/MVB by Rab conversion as in the case of the maturation from early to late endosomes (Rink et al., 2005), through fusion into a hybrid organelle and fission (Treusch et al., 2004), or by kiss-and-run between late endosomes and existing lysosomes (Bright et al., 2005). Numerous and highly regulated exchanges exist between different compartments. Transported proteins are concentrated on microdomains, and membrane exchange is initiated by budding and fission. In the case of the endosome-to-Golgi retrograde transport, membrane retrieval is regulated by the retromer complex (Griffin et al., 2005; Seaman, 2005). This is controlled at least by the Vps34p, a phosphoinositide-3 kinase, indicating a role for PIs in retrograde transport (Burda et al., 2002).
The phosphorylation status of PIs is regulated by lipid kinases and phosphatases. Mammalian PIKfyve, the phosphatidylinositol-3-phosphate 5-kinase type III (PIP5K3), transforms PtdIns(3)P into PtdIns(3,5)P2 (Shisheva, 2001). These two PIs are substrates of myotubularin phosphatases, which are mutated in several neuromuscular disorders (Laporte et al., 1996; Bolino et al., 2000; Azzedine et al., 2003; Laporte et al., 2003; Senderek et al., 2003). In addition, PIKfyve was recently found mutated in patients with a corneal dystrophy (Li et al., 2005). PIKfyve was shown to be implicated in endomembrane integrity and in the control of fluid phase endocytosis (Ikonomov et al., 2002, 2003a). Overexpression of a kinase-defective PIKfyveK1831E mutant in mammalian cells produced abnormal vesicles, which were described as enlarged MVBs. However, this did not affect epidermal growth factor receptor degradation. In 3T3-L1 adipocytes, PIKfyve enhanced insulin-stimulated GLUT4 vesicle translocation to the plasma membrane (Berwick et al., 2004). The importance of this phosphoinositide pathway in membrane trafficking has also been highlighted in yeast by the study of Fab1p, the orthologue of PIKfyve (Efe et al., 2005). Fab1p is important for vacuole size regulation, protein sorting at MVBs, and vacuole acidification (Gary et al., 1998; Odorizzi et al., 1998). The yeast vacuole, which is analogous to the mammalian lysosome, was enlarged in Fab1p mutants and defects in vacuolar inheritance have also been noted (Efe et al., 2005). In wild-type yeast, PtdIns(3,5)P2 increased upon hyperosmotic shock (Dove et al., 1997), leading to vacuolar fragmentation (Bonangelino et al., 2002), suggesting that Fab1p is also implicated in osmoregulation. Part of the roles of PIKfyve/Fab1p are transduced by PtdIns(3,5)P2 effectors. These include Ent3p, Ent5p, and mVps24 for MVB sorting (Friant et al., 2003; Whitley et al., 2003) and Atg18p for membrane retrograde transport and partitioning of the vacuole (Dove et al., 2004). Atg18p has also been implicated in membrane and protein retrieval from the preautophagosomal structures (Reggiori et al., 2004). Discrepancies still exist between yeasts and mammalian cell systems concerning the roles of PIKfyve/Fab1p on receptor degradation and signal transduction.
The roles of PIKfyve have never been investigated in vivo in higher eukaryotes. We describe here our studies on phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p in Caenorhabditis elegans, a multicellular organism with a complex endocytic machinery. ppk-3 mutants have developmental defects and display a striking enlargement of lysosomes (a "swiss-cheese" like phenotype). These enlarged vacuoles are nonacidic, and protein degradation is not affected. Functional and genetic analyses showed that the defect lies in the maturation of formed lysosomes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The following mutations and rearrangements were used: ppk-3(mc46), vac-2(n2668), vac-2(n2835) (this work), cup-5(ar465) (Fares and Greenwald, 2001), and szT1 (provided by the Caenorhabditis Genetics Center, University of Minnesota, St. Paul, MN), which balances the right half of LG X and the left half of LGI. Transgenic strains used for this study are cdIs36[punc-122::C31E10.7::GFP] for endoplasmic reticulum, cdEx88[pcc1::gyMANS] for Golgi, pwIs50[lmp-1::GFP] for lysosomes, cdEx49[punc-122::GFP::rab-5] and pwIs72[pvha-6::GFP::rab-5] (provided by B. Grant, Rutgers University, Piscataway, NJ; Chen et al., 2006) for early endosomes, bIs33[rme-8::GFP] for late endosomes (provided by B. Grant; Zhang et al., 2001), cdIs67[punc-122::GFP::rab-7] and pwIs429[pvha-6::RFP::rab-7] (provided by B. Grant; Chen et al., 2006) for late endosomes and lysosomes, and Ex[lgg-1::GFP] for autophagosomes [provided by B. Levine (Columbia University College of Physicians and Surgeons, New York); Melendez et al., 2003]. cdEx46[punc- 122::2XFYVE::GFP] expresses a probe which specifically binds PtdIns(3)P. bIs1[vit-2::GFP] (provided by B. Grant), expressing a fusion between the yolk protein YP170 and green fluorescent protein (GFP), was used to monitor yolk degradation in embryos. Coelomocyte endocytosis was assayed using arIs37[pmyo-3::ssGFP] and cdIs5[pmyo-3::ssDsRed2]. ppk-3 genomic DNA (8 kb), including 1.5 kb of the upstream intergenic region and the whole ppk-3 gene, were PCR-amplified using the primers F1 (5'-CATGGATCCTACTGAAATTTGCCATATGGGG-3') and R1 (5'-ATTGGATCCTTATAAGACCTAATTGATCCGAG-3') containing BamHI restriction sites and cloned in the C. elegans pmyo3::YFP vector pPD136-64 [provided by A. Fire (Stanford University School of Medicine, Stanford, CA)]. From this construct, we PCR amplified a fragment containing 1.5 kb of the intergenic region, the ppk-3 genomic DNA fused with yellow fluorescent protein (YFP) in its C terminal part and the 3'-untranslated region of unc-54 using the primers F1 and R2 (5'-AAGGGCCCGTACGGCCGACTAGTAGG-3'). The resulting PCR-amplified ppk-3::YFP fusion (40 ng/µl) was coinjected with pRF4 (100 ng/µl) as a transformation marker into N2 and ppk-3(n2668) young animals to determine ppk-3 expression and to test the rescue of the phenotype.
Lethality assays were done by allowing adult hermaphrodites to lay eggs for 4 h and scoring the number of eggs. For embryonic lethality, larvae were counted 15 h later, and postembryonic lethality was assayed 48 h after egg laying. Lethality percentages were calculated by dividing the number of deduced dead animals (for embryonic lethality, number of eggs laid – number of larvae at 15 h; for postembryonic lethality, number of larvae at 15 h – number of worms alive at 48 h) by the number of eggs laid for each indicated phenotype. The growth delay was assayed by determining the stages of live animals present on the Petri plates 3 and 5 d after egg laying.
RNA interference (RNAi) against rab-7 was performed by the feeding method (Timmons and Fire, 1998) and the corresponding clone W03C9.3 is part of the RNAi library of J. Ahringer (University of Cambridge, Cambridge, United Kingdom).
Isolation and Identification of ppk-3 Mutants
The allele ppk-3(mc46) was isolated by PCR screening, after 25 mM ethane methyl sulfonate (EMS)-induced mutagenesis on wild-type L4 hermaphrodites (Wei et al., 2002), by using two pairs of nested primers located around the active site of ppk-3: F1 (5'-TCACCACCGACACAATCTACC-3'), R1 (5'-CAGTATCAAAGCATCAATGATTTC-3'), F2 (5'-ACTAATCAGAATCAATG CTCGC-3'), and R2 (5'-TCACAACAAGTGAATTGGTAGAC-3'). The mutations vac-2(n2668) and vac-2(n2835) were isolated in an EMS screen for larval lethal mutants with defects in morphogenesis and tissue integrity (Chisholm, unpublished results). ok1150 was provided by the Caenorhabditis Genetics Center. The mutations affecting ppk-3(mc46), vac-2(n2668), and vac-2(n2835) alleles were identified by sequencing each ppk-3 exon after PCR amplification. Animals segregating mc46 or n2668 or n2835 were outcrossed eight times to wild-type animals, and mc46 was balanced with szT1. Complementation tests between mc46 and n2668 were performed by crossing n2668/0 males with mc46/mc46 hermaphrodites. The mc46 mutation was followed through genetic crosses by PCR using three primers: two around the deletion F (5'-ACAGATGGATGATGGGGAAA-3') and R (5'-CGGAAAGTACGAGTCGATGG-3'), and one hybridizing into the deletion region F(5'-TGGATAAGGTCAGTGTTTGAAC-3'). n2668 is a missense mutation, which destroys a BanII restriction site. We then followed the mutation by cutting with BanII the fragment PCR-amplified using F (5'-ATGTCACGGTTTGAGATTCAG-3') and R (5'-AGGAAGTGGCTGTCATTGC-3').
Immunofluorescence
For anti-GFP immunostaining of embryos, mixed staged wild-type or mutant embryos were mounted on polylysine-coated slides, freeze cracked in liquid nitrogen, and fixed in successive baths in –20°C methanol, –20°C acetone, and graded acetone:phosphate-buffered saline mixes. Embryos were then incubated overnight with a monoclonal anti-GFP antibody (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Euromedex, Souffelweyersheim, France) diluted 1/1000 in phosphate-buffered saline (PBS)/0.5% Tween/0.1% bovine serum albumin (BSA), washed with PBS/0.5% Tween/0.1% BSA, incubated overnight with a Cy3-coupled secondary antibody, washed with PBS/0.5% Tween containing 1 µg/ml final 4,6-diamidino-2-phenylindole (DAPI) to locate all nuclei, and viewed with a confocal microscope.
Anti-GFP immunostaining of adult worms was done according to the protocol of McIntire with minor modifications (McIntire et al., 1992). Briefly, wild-type and mutant young adults were fixed 1 d in 4% paraformaldehyde and then incubated overnight in 5% 
-mercapthethanol, 0.125 M Tris, pH 6.9, and 1% Triton at 37°C. The cuticle was disrupted by treatment with 1000 U/ml collagenase type IV (Sigma-Aldrich, St. Louis, MO) in 100 mM Tris, pH 7.5, 1 mM CaCl2, for 30 min at 37°C, and membranes were impermeabilized by an incubation of 1 h in 0.125 M Tris, pH 6.9, 1% Triton. Worms were then incubated 24 h with a monoclonal anti-GFP antibody (Institut de Génétique et Biologie Moléculaire et Cellulaire, Euromedex) diluted 1/1000, washed, incubated 4 h with a Cy3-coupled secondary antibody, washed, and viewed with a confocal microscope. Antibodies were diluted in PBS/0.5% Triton/0.5% BSA, and washes were done with PBS/0.5% Triton/0.3% BSA.
For anti-PtdIns(3,5)P2 immunostaining, mixed staged embryos of wild-type and mutants were mounted and freeze cracked, incubated in –20°C methanol, fixed overnight in 1% paraformaldehyde, and then incubated overnight with a monoclonal anti-PtdIns(3,5)P2 antibody (Echelon Biosciences, Salt Lake City, UT) diluted 1/40 (12.5 µg/ml final) in PBS/0.1% BSA without any detergent. Embryos were then washed, incubated overnight with a fluorescein isothiocyanate-coupled goat anti-mouse secondary antibody (Beckman Coulter France, Roissy CDG cedex, France), washed with PBS/DAPI, and viewed with a confocal microscope. Experiments were done twice.
Confocal Microscopy
Animals were mounted on 2% agarose pads with 0.2% tricaine/0.02% tetramisole in M9. For differential interference contrast, we used a Zeiss Axioplan microscope coupled to a CoolSNAP camera (Photometrics, Tucson, AZ) under a PlanApo 10x objective. For confocal imaging, we used a Leica SP2-AOBS confocal microscope with an argon 488 excitation to visualize the green dyes and the autofluorescence of worm intestine and a HeNe 543 excitation for the red dyes. Pictures of the same experiment were taken at the same exposure. We then processed images with the Tcstk software (Jean-Luc Vonesch, Institut de Génétique et de Biologie Moléculaire et Cellulaire) and edited pictures using the Dvrtk software (Jean-Luc Vonesch, Institut de Génétique et de Biologie Moléculaire et Cellulaire) and Adobe Photoshop 7.0. Fluorescence intensities were quantified in whole embryos using the Leica Confocal software (Leica, Wetzelar, Germany). Dot-like structures of the anti-PtdIns(3,5)P2 staining in embryos were scored using the MetaMorph software (Molecular Devices, Sunnyvale, CA) in squares of 280 µm2. The average value obtained with 25 different embryos per strain was calculated and difference between wild-type and ppk-3(n2668) mutant was tested with a Student’s t test.
Electron Microscopy
Worms were processed for electron microscopy as described previously (Michaux et al., 2000). Briefly, young adults were sectioned and fixed for 24 h in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, at 4°C. Worms were postfixed for 5 h with 2% osmium tetroxide in the same sodium cacodylate buffer at 4°C. Animals were dehydrated in graded alcohol:water mixes and then embedded in Epon. Ultrathin sections of 70 nm were contrasted with uranyl acetate and lead citrate. Sections were observed with a Philips CM12 electron microscope. At least eight animals per strain were examined.
Endocytosis Assays and Time Course
As described previously (Treusch et al., 2004), Texas-Red BSA (TR-BSA; Sigma) was injected at 1 mg/ml in water into the body cavity of wild-type or n2668 young adult hermaphrodites expressing LMP-1::GFP. At defined time points, animals were mounted on slides, put on ice to stop endocytosis, and fluid-phase internalization of the dye into the coelomocytes was viewed with a confocal microscope. For each time point, similar results were obtained with the coelomocytes of at least six different worms. LysoTracker Red DND-99 (Invitrogen, Carlsbad, CA) is a red fluorescent dye that selectively accumulates in cellular compartments with low internal pH. LysoTracker Red DND-99 was injected into the pseudocoelomic space of wild-type and mutant worms expressing LMP-1::GFP at 0.4 mM in M9 buffer, and its localization inside the coelomocytes was viewed 4 h after injection with a confocal microscope at a 514-nm excitation wavelength and a 580- to 660-nm range emission. The dynamic of the abnormal vacuole biogenesis in ppk-3(mc46) and ppk-3(n2668) mutant coelomocytes was followed through 20 min. Focal plane was changed during experiment to ensure proper visualization of the biggest diameter of the fusing vacuole observed, and pictures were taken at key time points.
Lipid Extraction and Analysis
Worms were collected and washed in phosphate-free RPMI-1640 medium at 20°C and labeled with 1 mCi/ml [32P]orthophosphate (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) during 12 h in phosphate-free RPMI-1640 medium at 20°C. 32P-labeled worms were then washed once in phosphate-free RPMI-1640 medium at 20°C, and lipids were immediately extracted following the modified procedure of Bligh and Dyer (1959). Lipids were then separated by TLC (Merck, Nogent-sur-Marne, France) using CHCl3/CH3COCH3/CH3COOH/H2O [80/30/26/24/14 (vol/vol)] as a solvent. The spots corresponding to [32P]PtdInsP2 were visualized by a PhosphorImager 445 SI (GE Healthcare) using appropriate standards, scraped off, deacylated, and analyzed by high-performance liquid chromatography (HPLC) on a Whatman Partisphere 5 SAX column (Whatman International, Maidstone, United Kingdom) as described previously (Payrastre, 2004).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In neurons, our ppk-3::YFP fusion protein was localized as cytoplasmic dots in cell bodies and axons. In the intestine, PPK-3 displayed a diffuse localization with some punctate staining (Supplemental Figure 2).
We screened by PCR for ppk-3 mutants generated by EMS mutagenesis and isolated a deletion mutant (mc46) lacking most of the kinase domain (
5213-6077 on the genomic sequence) (Figure 1). This mutant displayed large vacuoles and by comparison with this swiss-cheese phenotype, we identified two other mutants, n2668 and n2835, mapping to the same chromosomal region as the ppk-3 gene, which were obtained by EMS mutagenesis and screened for larval lethal mutants with defects in morphogenesis and tissue integrity. n2668 displayed enlarged vacuoles similar to mc46 as monitored by Nomarski optics (Figure 2B). ok1150 (4027–5640 on the genomic sequence) was created by the C. elegans Gene Knockout Consortium. There was no allelic complementation between mc46 and n2668 (Figure 2A) nor between n2668 and n2835. n2668 causes a serine-to-phenylalanine (S1356F) change within the conserved catalytic loop DLKGS of the kinase domain, close to the aspartate mutated in the S. cerevisiae kinase dead Fab1p mutant (D2134R; Gary et al., 1998). n2835 causes a nonsense mutation (Q1487X), which removes the last 11 amino acids conserved in mammals (Figure 1).
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Abnormal Vacuoles in ppk-3 Mutants Are Enlarged Lysosomes
To characterize these abnormal vacuoles and their origin, we used electron and fluorescent microscopy. The vacuoles were of different sizes and could reach up to 30% of the worm width (Figure 3A). This striking vacuolar phenotype did not prevent 65% of the worms from reaching adulthood in the n2668 mutant. The vacuoles were surrounded by a membrane and were either empty, filled with membrane whirls in huge vacuoles, or electron-dense deposits in smaller vacuoles. We noted numerous pictures of aggregation and fusion between the vacuoles (Figure 3B). Normal multivesicular bodies were visible in the epidermis of the ppk-3 mutant (Figure 3C). Most of the coelomocytes were filled with a unique huge vacuole that leads to an increase of the cell size of approximately two- to fourfold (Figure 3E). These results suggested a defect of membrane trafficking in ppk-3 mutants.
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). We chose the n2668 mutant, which is viable and has a missense alteration in the catalytic loop of the kinase domain, to focus on the impact of PI dysregulation. We crossed n2668 with transgenic worms expressing specific markers of different endocytosis and membrane compartments. There was no difference in Golgi (mannosidase II) and endoplasmic reticulum (C31E10.7 cytochrome b5) patterns between wild-type and mutant (our unpublished data). The endocytic markers, RAB-5 for early endosomes, and RME-8 for late endosomes (Zhang et al., 2001), did not label the enlarged vacuole (Figure 4). In contrast, LMP-1, a lysosomal marker orthologous to human lysosomal-associated membrane protein 1 (Kostich et al., 2000), always stained the membrane of the enlarged vacuoles, strongly suggesting a lysosomal identity. We obtained similar results for LMP-1 in the mc46 mutant. RAB-7 weakly labeled the enlarged vacuoles of the mutant (Supplemental Figure 3A). In C. elegans, RAB-7 labels not only late endosomes but also lysosomes because all the vacuoles accumulating the fluid-phase marker Texas-Red–coupled BSA after 24 h were positive for RAB-7 and LMP-1, whereas they were negative for RME-8 (Supplemental Figure 3B).
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), showed aggregations distinct from the abnormal vacuoles in the intestine of the ppk-3 mutant (Figure 4). Thus, ppk-3 mutants have enlarged lysosomes and a possible accumulation of autophagosomes.
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). By 30 min, Texas-Red BSA was mostly in LMP-1–negative vesicles, which are late endosomes as verified with a time course using RME-8::GFP. From 30 min, it started to accumulate in lysosomes labeled with LMP-1::GFP (Figure 6B; our unpublished data). By 1 h and after 24 h of internalization, Texas-Red BSA had accumulated into most of the lysosomes (Figure 6B) and was absent from the RME-8::GFP late endosomes (Supplemental Figure 3B). In ppk-3 mutants, the fluid phase endocytosis followed the same kinetics. Texas-Red BSA first localized to LMP-1–negative endosomes with concentrations in subdomains. By 30 min and 1 h, the Texas-Red BSA accumulated in normal LMP-1–positive compartments and in the LMP-1 large vacuoles. This is consistent with our finding that the large vacuoles are competent to fuse with other compartments. The fluorescent marker localization to the enlarged vacuole increased during the time course, indicating the progressive accumulation of the tracer (Figure 6B). Thus, lack of ppk-3 activity does not prevent the fluid phase endocytosis to lysosomes, suggesting that the biogenesis of lysosomes is normal.
We further tested the maturation status of the enlarged lysosomes. We used worm strains expressing GFP or DsRed2 in the body wall muscle under the myo-3 muscle-specific promoter. These fluorescent proteins are secreted into the body cavity and nonspecifically endocytosed primarily by coelomocytes. It is known that GFP is degraded, or bleached by the low pH, in mature lysosomes, whereas DsRed2 is stable (Fares and Grant, 2002; Patton et al., 2005). The enlarged lysosomes of the ppk-3(n2668) mutant were strongly positive for DsRed2, whereas there was no accumulation of the GFP signal (Figure 7A). As a control, we used coelomocyte uptake defective (cup)-5 mutants, which have a defect in lysosome formation (Treusch et al., 2004). cup-5 encodes a calcium-permeable channel orthologous to h-mucolipin-1, mutated in human mucolipidosis type IV (Bargal et al., 2000). In contrast to ppk-3 mutants, cup-5 mutants accumulated the GFP in aberrant late endosome/lysosome hybrid organelles. To confirm that ppk-3 lies downstream of cup-5, we generated a double mutant cup-5(ar465);ppk-3(n2668). GFP accumulated similarly in this double mutant compared with the single cup-5 mutant (Figure 7A). CUP-5:: GFP accumulated around the enlarged vacuoles of the ppk-3 mutants, and lethality increased in the double cup-5(ar465); ppk-3(n2668) (supplementary Figure 4), suggesting that these two proteins are implicated in the same process, PPK-3 being downstream of CUP-5. In conclusion, enlarged vacuoles of the ppk-3 mutants are maturing lysosomes. It suggests that PPK-3 is not essential for lysosome formation but plays a role in lysosome maturation.
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As a second test for protein degradation, we monitored the presence and level of the yolk protein vitellin-2 (YP170) in embryos (Britton and Murray, 2004). Yolk proteins are synthesized by intestinal cells and transported to the oocytes to be used as amino acid source by the developing eggs. It was previously reported that cathepsin-L1 mutants and worms fed with RNAi against rab-7, two genes implicated in lysosome formation and function, are defective for yolk protein processing and degradation, leading to an accumulation of YP170::GFP in abnormal vacuoles (Grant and Hirsh, 1999; Britton and Murray, 2004), as shown for rab-7 RNAi in Figure 8A. We compared the fluorescence intensity of YP170::GFP in wild-type and ppk-3 mutant transgenic embryos. No detectable difference was noted (p = 0.07) (Figure 8, B and C). To rule out possible bleaching of the GFP fluorescence due to an acidic environment, we performed an anti-GFP immunostaining and obtained similar results (p = 0.29) (Figure 8, B and C). This was sustained by the presence of autofluorescent granules in the intestine of ppk-3 mutants, believed to correspond to lysosomes containing lipofuscin, a degradation product (Clokey and Jacobson, 1986).
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Defect of PtdIns(3,5)P2 Production in ppk-3 Mutants
We monitored the localization of PtdIns(3)P and PtdIns(3,5)P2, substrate and product of the PIKfyve/Fab1p kinase activity, respectively, in the ppk-3(n2668) mutant, which has a S1356F missense in the catalytic loop. PtdIns(3)P was specifically detected with a probe consisting of a tandem repeat of human HRS FYVE domain (Gillooly et al., 2000; Dang et al., 2004) under the control of a coelomocyte promoter. The limiting membrane of the enlarged lysosome was not labeled for the majority of the coelomocytes, although other vesicles were positive for this probe; one-third of the coelomocytes contain an abnormal vacuole faintly positive for the 2XFYVE probe (Figure 9A). PtdIns(3,5)P2 was detected with a specific mouse monoclonal antibody in embryos and occurred as cytoplasmic dots. The number of dot-like structures in ppk-3(n2668) embryos was highly significantly decreased compared with wild-type (p = 2 x10–7) (Figure 9, B and C). The remaining PtdIns(3,5)P2 staining in the mutant might be due either to nonspecific labeling, or to residual production of PtdIns(3,5)P2 by the mutated PPK-3 or synthesis by an uncharacterized pathway.
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; Sbrissa et al., 2001). Although ppk-3 mutants consistently incorporated more 32P in phospholipids than wild-type worms, we reproducibly observed that the mutants had a much lower level of PtdIns(3,5)P2 (Figure 9D). We normalized the level of PtdIns(3,5)P2 on PtdIns(4,5)P2 and calculated in the representative experiment Figure 9D that PtdIns(3,5)P2 on PtdIns(4,5)P2 ratio was 1/40 when comparing ppk-3 mutant and wild type. Thus, the enlargement of lysosomes seems to be linked to a defect in PtdIns(3,5)P2 production. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We concentrated our studies on the coelomocytes, which are scavenger cells with pronounced endocytosis and degradative functions (Fares and Grant, 2002). In these cells, mutations in ppk-3 do not have a strong impact on initial uptake and on organelle biogenesis. Studies on mammalian cells transfected with a dominant negative mouse PIKfyve construct (K1831E missense) reported enlargement of MVB-like structures based on electron microscopy (Ikonomov et al., 2003a). In contrast, lysosomes seemed nonaffected. In our ppk-3 mutants, we noted a striking enlargement of the lysosomal compartment, which is more in accordance with the work performed on Fab1p in yeast, where the vacuole compartment is analogous to the lysosome of the higher eukaryotes. Fab1p is implicated in vacuole morphology and homeostasis and is necessary for three different cellular functions (Efe et al., 2005): 1) MVB sorting and protein transport and degradation, 2) vacuole acidification, and 3) membrane recycling from the vacuole. In the ppk-3 mutant, the fluid phase endocytosis was normal and there was no defect of protein transport to the lysosome and degradation, as monitored with fluorescent proteins transport and GFP and YP170 yolk proteins degradation. Thus, C. elegans ppk-3 function is not necessary for protein degradation, in coelomocytes. Although the lysosomal enlargement is the most striking, we noted a slight enlargement of RAB-7–positive LMP-1–negative late endosomes in the intestine.
Enlarged lysosomes of the ppk-3 mutants are not acidic, because they are negative for LysoTracker Red DND-99. This is in accordance with the phenotype described in yeast kinase dead and deleted Fab1 mutants (Gary et al., 1998). In yeast, the pH of the vacuole is regulated by the V-ATPase proton pump, and in Fab1 mutants, the transport and assembly of the V-ATPase is not disrupted (Efe et al., 2005). Together, ppk-3 and Fab1p probably regulate directly, or through its PtdIns(3,5)P2 product, the activity of the V-ATPase. In C. elegans coelomocytes, and despite the acidification defect, protein degradation is normal, as shown by GFP uptake and degradation and yolk proteins degradation (Figures 7B and 8). Similarly, Ikonomov et al. (2003a) noted a normal degradation of the epidermal growth factor receptor in cultured cells overexpressing a dominant negative PIKfyve K1831E mutant (Ikonomov et al., 2003a). Together, ppk-3 acts mainly on the very late stages of lysosomal maturation, and we hypothesize that degradation occurs before the terminal phenotype of enlargement of lysosomes.
In our mutant, we can distinguish two compartments: early lysosomes that are probably sites of protein degradation, and late lysosomes that are nonacidic and enlarged. Indeed, we have observed fusion between lysosomes and the enlarged vacuoles, which confirm that dynamic exchanges exist between lysosomes, as previously noted in cultured mammalian cells (Deng and Storrie, 1988; Patterson and Lippincott-Schwartz, 2002). In wild-type, we suggest the existence of early and late lysosomes. Degradation occurs in the early acidic lysosomes, and late lysosomes are sites of membrane and protein retrieval. The latter may be nonacidic, a prerequisite for further fusion with other compartments, sustaining the idea that lysosomes are not a dead-end but part of a membrane trafficking cycle in cells (Hirota et al., 2004).
We show that protein degradation and membrane retrieval are uncoupled in the ppk-3(n2668) mutant, and we propose that the enlargement of the lysosomes in our ppk-3 mutant is due to defects in membrane retrieval from matured lysosomes. In yeast, membranes from the vacuole are recycled to the trans-Golgi network (Bryant et al., 1998). We have studied coelomocytes, a cell with highly dynamic endocytosis leading to the degradation of protein substrates retrieved from the pseudocoelom. In another cellular context, PPK-3 may play a role on membrane retrieval from other compartments of the endocytic pathway, as in the intestine where there is a slight concomitant enlargement of late endosomes probably due to the fact that there are more membrane retrieval from earlier endosomal compartments to recycle proteins and receptors that should not be degraded. Similarly, PIKfyve could act on membrane retrieval from late endosomes as described in a mammalian cell system (Ikonomov et al., 2002). There, PIKfyve interacts with the Rab9 effector p40 to facilitate membrane trafficking from late endosomes (Ikonomov et al., 2003b). Both regulators of Fab1p, Vac7 and Vac14, were shown to be implicated in membrane retrograde transport (Bonangelino et al., 2002). Atg18, an effector of PtdIns(3,5)P2, is implicated in this process and was also implicated in autophagy (Dove et al., 2004; Reggiori et al., 2004). The accumulation of autophagosomes that we observed in the ppk-3 mutant could reflect a role of PIKfyve in the maturation of autophagosomes and/or in the retrieval of proteins from these compartments. The action of PPK-3 on membrane retrieval is most probably mediated through its product, PtdIns(3,5)P2. This is sustained by the observation that the n2668 hypomorphic ppk-3 mutant, which has a mutation in the conserved catalytic loop of the kinase domain, shows reduced PtdIns(3,5)P2 levels compared with wild-type. The phenotypes of this specific mutant should mainly reflect the consequence of the dysregulation of PPK-3 PIs product, PtdIns(3,5)P2. Under hyperosmotic shock in yeast, PtdIns(3,5)P2 is up-regulated and is necessary for the fragmentation of the vacuoles (Bonangelino et al., 2002; LaGrassa and Ungermann, 2005). These data suggest that PtdIns(3,5)P2 production is required for fission of the lysosome and that its absence in ppk-3 mutants decreases membrane retrieval, leading to enlargement of this compartment. PtdIns(3,5)P2 could have a direct role on fission by modifying the physicochemical properties of membranes, by recruiting protein effectors implicated in fission, and/or by regulating retrograde machineries (Carlton et al., 2004).
This study describes for the first time the role of PPK-3 in a multicellular organism. Apart from lysosome morphology, PPK-3 loss has also an impact on the physiology of the worm: we noted an embryonic and larval lethality, and a growth delay. Thus, ppk-3 has an important role during development and for physiological functions. Interestingly, mutations in human PIKfyve have been recently found in patients with Francois–Neetens Mouchetée fleck corneal dystrophy (OMIM 121850
[OMIM]
), an autosomal dominant disease (Li et al., 2005). The corneal flecks seem to correspond to keratocytes swollen with poorly characterized enlarged vesicles containing complex lipids and glycosaminoglycans (Nicholson et al., 1977). It is not clear whether the human disease is due to a haplo-insufficiency of PIKfyve function or to a dominant negative effect of the truncated mutations found. Our work suggests that this human disease could be due to a defect in membrane retrieval from late vacuolar compartments. The ppk-3 mutant worms could be a nice model to study the physiopathology of this disease. PPK-3 and myotubularin phosphatases regulate the level of PtdIns(3,5)P2, and some myotubularins are mutated in Charcot–Marie–Tooth neuropathies with anomalies of the myelin sheath (Laporte et al., 2003). The relevance of this PI with respect to myelin formation and stability remains to be determined.
In conclusion, we propose that the enzymatic activity of PPK-3 mainly controls membrane retrieval and not protein degradation. ppk-3 mutants would be valuable tools to identify new regulators of membrane trafficking not present in yeast and to study the poorly understood destiny of lysosomes.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Jocelyn Laporte ( mtm{at}igbmc.u-strasbg.fr)
Abbreviations used: CUP, coelomocyte uptake defective; LGG-1, LC3, GABARAP, and GATE-16; MVB, multivesicular body; PIKfyve, phosphatidylinositol kinase Fab1p, YoTb, Vac1p, EEA1, Fab1p: formation of aploid and binucleate cells; LMP, lysosome-associated membrane protein; PPK-3, phosphatidylinositol phosphate kinase 3; PtdIns(3)P, phosphatidylinositol-3-monophosphate; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; RME, receptor-mediated endocytosis.
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