|
|
|
|
|
|
|
||
Article |
The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans
Correspondence to Michel Labouesse: lmichel{at}igbmc.u-strasbg.fr
 Top Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Polarized intracellular trafficking in epithelia is critical in development, immunity, and physiology to deliver morphogens, defensins, or ion pumps to the appropriate membrane domain. The mechanisms that control apical trafficking remain poorly defined. Using Caenorhabditis elegans, we characterize a novel apical secretion pathway involving multivesicularbodies and the release of exosomes at the apical plasma membrane. By means of two different genetic approaches, we show that the membrane-bound V0 sector of the vacuolar H+-ATPase (V-ATPase) acts in this pathway, independent of its contribution to the V-ATPase proton pump activity. Specifically, we identified mutations in the V0 "a" subunit VHA-5 that affect either the V0-specific function or the V0+V1 function of the V-ATPase. These mutations allowed us to establish that the V0 sector mediates secretion of Hedgehog-related proteins. Our data raise the possibility that the V0 sector mediates exosome and morphogen release in mammals.
S. Liégeois's present address is Institut de Biologie Moléculaire et Cellulaire, F-67000 Strasbourg, France.
Abbreviations used in this paper: DIC, differential interference contrast; mRFP, monomeric red fluorescent protein; MVB, multivesicular body; SEM, scanning electron microscopy; TEM, transmission electron microscopy; V-ATPase, vacuolar H+-ATPase.
 |
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Basolateral sorting signals usually correspond to tyrosine or dileucine residues found in the COOH terminus of proteins. They are recognized by basolateral-specific adaptor complexes (Bonifacino and Lippincott-Schwartz, 2003; Rodriguez-Boulan et al., 2005), such as AP-1B in epithelial cells (Folsch et al., 1999). Before membrane fusion and SNARE action, vesicles are thought to be tethered to the basolateral membrane by the exocyst complex (Whyte and Munro, 2002), which was initially identified in yeast (TerBush and Novick, 1995). In metazoans, the exocyst is required for basolateral delivery of the LDL receptor in MDCK cells (Grindstaff et al., 1998; Yeaman et al., 2001), of E-cadherin in the Drosophila melanogaster notum (Langevin et al., 2005), and for Rhodopsin1 transport in D. melanogaster photoreceptor cells (Beronja et al., 2005). Recent results suggest that AP-1B and the exocyst act primarily in recycling endosomes (Ang et al., 2004; Beronja et al., 2005; Langevin et al., 2005; Lock and Stow, 2005; Satoh et al., 2005), which underlines the central role of this organelle in sorting processes. Indeed, recycling endosomes may be compartmentalized into apical- and basolateral-related domains, or even divided into distinct organelles, suggesting that they could also play a critical role in apical trafficking (Hoekstra et al., 2004; Rodriguez-Boulan et al., 2005).
Aside from this possible role of recycling endosomes, all other aspects of sorting along the basolateral and apical routes seem to differ. Apical signals are more diverse and often correspond to posttranslational adducts, such as lipids or glycans (Schuck and Simons, 2004; Rodriguez-Boulan et al., 2005). For instance, the Hedgehog morphogen is secreted apically upon cholesterol addition, but basolaterally otherwise (Gallet et al., 2003). No specific apical cytosolic complex, akin to AP-1B or the exocyst, has been identified so far. Instead, protein clustering, possibly through lipid rafts, is thought to mediate the sorting and transport of apical cargoes (Schuck and Simons, 2004; Rodriguez-Boulan et al., 2005). In particular, glycosyl phosphatidylinositol–linked proteins appear to form oligomers that are directly targeted to the apical membrane (Paladino et al., 2004, 2006; Hua et al., 2006). Several proteins have been proposed to play an active role in apical protein clustering, raft formation, and/or apical delivery, such as caveolins (Kurzchalia et al., 1992), annexin 13b (Fiedler et al., 1995), and the tetraspan protein VIP17/MAL (Cheong et al., 1999; Puertollano et al., 1999). However, their mechanistic roles have not been fully elucidated, or their implication has been questioned (Manninen et al., 2005). In addition to the limited understanding of apical secretion at the molecular level, it is not clear whether the terminal fusion process involves small vesicles, such as those defined at synapses, or larger organelles, such as secretory lysosomes (Blott and Griffiths, 2002).
Hence, despite the many critical findings originating from tissue culture cells (Rodriguez-Boulan et al., 2005), investigations with other systems and other cargo proteins could help to elucidate the mechanisms involved in apical exocytosis. Caenorhabditis elegans, which has contributed to decipher the mechanisms controlling vesicular trafficking (Nurrish, 2002), provides such an in vivo model. We have chosen to analyze apical secretion of cuticle proteins by the epidermis. The cuticle includes glycosylated collagens, glycosyl phosphatidylinositol–linked cuticlins, and lipid-modified Hedgehog-related peptides (McMahon et al., 2003; Sapio et al., 2005; Zugasti et al., 2005). We previously suggested that the gene che-14 is required for cuticle secretion (Michaux et al., 2000). The CHE-14 protein is the C. elegans orthologue of Drosophila Dispatched, which participates in apical targeting of cholesterol-modified Hedgehog (Burke et al., 1999; Gallet et al., 2003).
While searching for che-14 alleles (Michaux et al., 2000), we uncovered several additional mutations inducing che-14–like phenotypes and reasoned that they might identify new components of the apical trafficking pathway. Two such mutations, mc37 and mc38, proved to be small deletions behaving as genetic-null alleles of the gene vha-5 (unpublished data). The gene vha-5 encodes one of the four C. elegans "a" subunits of the V0 sector of the vacuolar H+-ATPase (V-ATPase), and is required for development beyond the L2 larval stage (Oka et al., 2001; Pujol et al., 2001). The V-ATPase is a multisubunit protein complex consisting of two subcomplexes called the V0 and V1 sectors (Fig. 1 A). The cytosolic V1 sector hydrolyses ATP and provides the energy to pump protons through the transmembrane proteolipid pore formed by the V0 sector (Nishi and Forgac, 2002). The V-ATPase is the main proton pump establishing a pH gradient in the secretory and endocytic pathways. It generates a proton-motive force that is essential to load synaptic vesicles with neurotransmitters before secretion (Amara and Kuhar, 1993). The V-ATPase is also found at the apical plasma membrane of polarized cells, where it is essential for osmoregulation in animal excretory systems (Nishi and Forgac, 2002). More recently, biochemical and genetic data suggested that the V0 sector can play a role independently from the V1 sector. In Saccharomyces cerevisiae, vacuoles deficient for the "a" subunit Vph1p do not fuse efficiently (Peters et al., 2001; Bayer et al., 2003). In D. melanogaster, neurons lacking the "a" subunit Vha100 accumulate vesicles in synaptic terminals (Hiesinger et al., 2005). In both cases, the defects were independent of the proton gradient and placed downstream of SNARE function (Peters et al., 2001; Bayer et al., 2003; Hiesinger et al., 2005).
|
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
105-kD band in these extracts, but detected an 135-kD band (Fig. 1 C, lane b). These results are consistent with vha-5(mc38) being a small deletion associated with a frameshift (Fig. 1 B and not depicted) and with the presence of 257 additional residues in the GFP-fusion protein. We conclude that the VHA-5 antiserum is specific and that vha-5(mc38) is a molecular null mutation. In agreement with previously published observations (Oka et al., 2001; Pujol et al., 2001), we found that VHA-5 is expressed in the H-shaped excretory cell corresponding to the C. elegans kidney-like organ (Fig. 1, D and E). It is also expressed in the main epidermal syncytium (Fig. 1, D–F), which had previously been overlooked. The excretory cell extends long processes called excretory canals where osmoregulation takes place (Nelson and Riddle, 1984), whereas the epidermis controls body length and apical cuticle secretion (White, 1988). VHA-5 colocalized apically with the V1 subunit VHA-8 in both tissues (Fig. 1 E; note that VHA-5 is not expressed in the lateral epidermis). VHA-5 was localized at the level of apical membrane stacks by immunogold staining (Fig. 1 G). Consistent with VHA-5 distribution and a role of the V-ATPase in osmoregulation (Nishi and Forgac, 2002), vha-5(mc38) larvae filled with fluid and died at the L1 stage (unpublished data), which corresponds to the phenotype observed after laser ablation of the excretory cell (Nelson and Riddle, 1984). In addition, vha-5(mc38) L1 larvae had a severe malformation of the lateral cuticular specializations known as alae (Fig. 1 D and Fig. 2 A), which are primarily synthesized by the lateral seam cells. Although VHA-5 is not expressed in these cells, the main epidermal syncytium also contributes to their morphogenesis (Sapio et al., 2005). Because VHA-5 is transmembraneous and not cuticular, the simplest interpretation for this phenotype is that vha-5 mutations compromise the secretion of proteins needed for alae formation.
|
RNAi against these V1 or V0 subunit genes led to 100% lethality in the progeny of treated animals (Fig. 2 B‘, bottom bars). It is likely that most embryos died because of a defect in yolk endocytosis, which is known to be sensitive to proton pumping (Choi et al., 2003). In agreement, we found that yolk vitellogenin-GFP accumulated in the pseudocoelom of RNAi-treated animals rather than in oocytes and embryos (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200511072/DC1). Despite the strong lethality induced by loss of V-ATPase function, a few L1 larvae hatched and, invariably, died filled with fluid before the L2 larval stage (Fig. 2 B’‘), as observed for vha-5(mc38) larvae. Strikingly, we observed before their death that L1 hatchlings displayed severe alae defects when RNAi targeted the V0 subunits vha-1 or -4, but had wild-type alae after knockdown of the V1 subunits vha-8 or -13 (Fig. 2, B and B’). Consistent with the phenotype of vha-5–null mutants, RNAi against vha-5 also affected alae formation (Fig. 2, B and B‘–C), although lethality was weaker because VHA-5 is not ubiquitously expressed like VHA-4. One trivial explanation for the persistence of normal alae after V1 subunit knockdown could be that RNAi was less efficient than for V0 subunits. It is unlikely, as the lethality rates and the larval osmoregulation defects observed after V1 and V0 subunit knockdown were comparable (Fig. 2, B’ and B‘’), hinting that both RNAi were equally effective. To support this idea, we submitted a vha-8::gfp transgenic strain to vha-8 RNAi and verified that it induced a drastic decrease of VHA-8::GFP fluorescence (Fig. 2 D). We conclude that the V0 sector is required independently from the V1 sector for apical secretion of some cuticle components.
The two functions of V0 are genetically separable
If the V0 sector has two distinct functions, it should be possible to recover vha-5 alleles that affect either its V0-specific secretion function or its V0+V1 proton-pump function. The vha-5 distribution and the aforementioned phenotypes indicate that reducing V0-specific function should affect cuticle secretion, whereas impairing proton pumping should affect the excretory canal responsible for osmoregulation. To identify such mutations, we used a plasmid rescue strategy, whereby we generated mutations by using PCR on a rescuing vha-5::gfp construct, introducing them into vha-5(mc38)/+ animals, and recovering live homozygous vha-5(mc38) animals whenever possible (Fig. 3 A).
We modified charged or large hydrophobic residues, as well as residues previously mutated in the yeast Vph1p (Leng et al., 1996, 1998). We generated 56 mutations (Fig. 3, B and C; and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200511072/DC1); 42 had no obvious phenotype by differential interference contrast (DIC) microscopy (Fig. 3 B, stars), and eight failed to rescue, indicating that those residues are essential for VHA-5 function (Fig. 3 B, white boxes). More interestingly, six substitutions rescued the vha-5(mc38)–induced lethality and affected the cuticle, the excretory canal, or both.
|
|
|
|
|
V0 mediates secretion of hedgehog-like peptides
An important expectation of the cuticle defects described so far is that we should be able to identify cuticular proteins whose secretion depends on VHA-5 activity. Cuticle proteins include collagens and Hedgehog-related peptides (McMahon et al., 2003; Zugasti et al., 2005). We found that the collagen DPY-7 was efficiently secreted in vha-5(mc38)–null animals, in animals carrying cuticle mutations, as well as in che-14(mc35) mutants (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200511072/DC1). We turned our attention onto Hedgehog-related peptides, which appeared as good candidates for three reasons. First, vha-5 alae defects partially resemble those observed in che-14 mutants. Second, CHE-14 is homologous to Dispatched, which is a protein required for Hedgehog release (Burke et al., 1999; Michaux et al., 2000). Third, despite the absence of a Hedgehog homologue in C. elegans, its genome contains several Hedgehog-related peptides required to generate a normal cuticle, although their precise roles remain unknown (Aspock et al., 1999; Zugasti et al., 2005).
We tagged with GFP the secreted domain of the Hedgehog-related peptides WRT-2 and -8 (Fig. 8 A and Fig. S3 E), which are expressed in the epidermis (Aspock et al., 1999). We found that animals bearing cuticle mutations, but not canal mutations, accumulated VHA-5::RFP and WRT-2::GFP or -8::GFP in discrete entities in their epidermis (Fig. 8, B and C; and Fig. S3, D and F). These entities most likely correspond to the dense and hybrid MVBs (Fig. 6, C and D) because VHA-5::RFP also colocalized (Fig. 8 C) with the MVB marker VPS-27::GFP (Roudier et al., 2005). Moreover, both VHA-5 antiserum and a GFP antiserum targeting WRT-2::GFP decorated the MVBs of cuticle mutants (Fig. 8 D and Fig. S5 B, available at http://www.jcb.org/cgi/content/full/jcb.200511072/DC1). Last, in wild-type nontransgenic animals, in addition to membrane stacks (Fig. 1 G), VHA-5 was found at the MVB-limiting membrane, in intralumenal vesicles, and in the cuticle (Fig. 8 E and Fig. S5 A), suggesting that it could act at different steps in the secretion of vesicle (see Discussion). Importantly, the VHA-5 protein with the substitutions L786S (Fig. 8 B) or E830Q (not depicted) could reach the plasma membrane in heterozygous vha-5(mc38)/+ animals, which strongly suggests that their intracellular retention in homozygous mc38 animals is caused by the loss of a trafficking function rather than by misfolding. Consistently, the WRT-2/8 proteins were not retained intracellularly either in heterozygous vha-5(mc38)/+ animals, despite the presence of the L786S (Fig. 8 B), or in E830Q transgenes (not depicted). These results indicate that the V0 sector plays a key role in a specific apical secretion pathway that is taken on by Hedgehog-related proteins, but not by collagens.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
|
What could explain a common requirement for the V0 sector during the C. elegans apical exocytosis, yeast vacuole fusion, and D. melanogaster synaptic transmission? The prevailing view is that a SNAREpin complex initiates membrane fusion once a vesicle has been docked to a proper membrane (Chernomordik and Kozlov, 2003; Jahn et al., 2003). Although the V0 sector is thought to act downstream of SNAREs in yeast and D. melanogaster (Peters et al., 2001; Bayer et al., 2003; Hiesinger et al., 2005), we cannot exclude that it also acts in parallel to SNAREs, at least in C. elegans, to dock MVBs. Another possibility is that V0 transcomplexes initiate the formation of a protein pore, as initially suggested in yeast (Peters et al., 2001). On the other hand, expansion of the fusion pore is considered as the limiting step in membrane fusion, and might require additional catalysts in vivo (Chernomordik and Kozlov, 2003). Such a role could be fulfilled by the V0 sector, either to overcome constraints caused by the big size and/or the specific lipoproteic content of C. elegans epidermal MVBs and yeast vacuoles, or to allow rapid synaptic transmission in D. melanogaster neurons (Hiesinger et al., 2005).
Irrespective of the precise role of the V0 sector in membrane fusion, our findings bear potentially important implications. First, morphogens such as Wingless and Hedgehog in D. melanogaster, or Sonic-Hedgehog at the mouse node, might be secreted through a similar pathway because their secretion involves particles possibly related to exosomes (Greco et al., 2001; Gallet et al., 2003; Panakova et al., 2005; Tanaka et al., 2005). A major objective will be to determine whether CHE-14 and Dispatched act in the aforementioned secretory pathway, and, if so, at which step. Second, several other cell types, such as antigen-presenting cells, reticulocytes, and some epithelial cells, can release exosomes (de Gassart et al., 2004), which might thus also require the V0 sector for their secretion. In particular, the V0 sector might be directly associated with the transmission of some infectious diseases because viruses, such as HIV and the prion protein, can be disseminated through MVBs and the exosome-releasing machinery (de Gassart et al., 2004; Fevrier et al., 2005). Likewise, the aforementioned secretory pathway could be involved in innate immunity because expression of the Hedgehog-related peptide GRD-3 is induced in C. elegans upon bacterial infection (Couillault et al., 2004). Third, our findings raise the issue of the origin of the MVBs. Interestingly, the apical recycling endosomes have been recognized to play an important role in biosynthetic secretory pathways (Hoekstra et al., 2004). Future studies should reveal whether the secretory MVBs that we described could originate from this compartment.
In conclusion, our work shows that trafficking to the apical membrane of at least some lipid-modified proteins involves specific protein complexes (the V-ATPase V0 sector), much as trafficking to the basolateral membrane, and predicts a key role for MVBs in apical exocytosis.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Plasmids
Cloning of the vha-5 coding sequence with a 2.8-kb promoter upstream of the GFP coding sequence in the pPD95.75 vector (Fire kit) generated a rescuing vha-5::gfp construct. A vha-5::mrfp construct was obtained by replacing the GFP with the monomeric red fluorescent protein (mRFP) coding sequence in the vha-5::gfp construct. A vha-8::yfp construct was obtained by cloning the vha-8 coding sequence and a 3-kb promoter upstream of the YFP coding sequence in the pPD136.64 vector (Fire kit). To generate wrt-2::gfp and wrt-8::gfp constructs, we cloned wrt-2 and -8 genomic DNA with their 5' and 3' regulatory sequences into pBSKII-derived plasmids. The GFP coding sequence was inserted in a nonconserved region of the predicted secreted peptide (Fig. 8 A and Fig. S3 D).
Site-directed mutagenesis of VHA-5::GFP
The vha-5::gfp construct was mutated using the QuikChange Site-Directed Mutagenesis kit (Stratagene). Each desired mutation, and the entire vha-5 coding sequence of most important plasmids, was verified. Mutant plasmids were microinjected in heterozygous vha-5(mc38)/unc-5(e53) at 3 ng/µl, along with the marker pRF4 [rol-6(su1006)] at 100 ng/µl, wrt-2::gfp or wrt-8::gfp constructs at 30 ng/µl (when relevant), and pBSKII plasmid at up to 200 ng/µl. Absence of unc-5 animals in the progeny was used as a criterion for rescue. mRFP versions for the mutations W190A, R191A, W327A, L786S, E830Q, and V844F were obtained from GFP derivatives without PCR amplification and resulted in similar phenotypes. At least two independent extrachromosomal lines were initially examined for each mutation. More detailed analysis was performed on a representative line.
VHA-5 antiserum, Western blots, and immunofluorescence
VHA-5 polyclonal antibodies were raised in rabbits injected with a purified GST fusion protein containing VHA-5 residues I29–M302, which was obtained by cloning a fragment amplified from the cDNA yk458f4 (a gift from Y. Kohara, National Institute of Genetics, Mishima, Japan) into the vector pGEX-2T. Total worm extracts were solubilized in 8 M urea/2% SDS by sonication, before 8% acrylamide gel electrophoresis and Western blotting. VHA-5 antiserum was used at 1:2,000, the actin monoclonal antibody (act-2D7; Institut de Génétique et de Biologie Moléculaire et Cellulaire collection) at 1:4,000; primary antibodies were revealed with a SuperSignal kit (Pierce Chemical Co.). Immunofluorescence was performed using the VHA-5 antiserum at 1:1,000 dilution and the DPY-7 monoclonal antibody (gift from I. Johnstone, Wellcome Centre for Molecular Parasitology, Glasgow, Scotland) at a 1:50 dilution.
DIC and confocal microscopy
Animals were mounted on 4% agarose pads in M9, anaesthetized with 0.2% tricaine/0.02% tetramisole in M9. For DIC imaging, we used a microscope (Axioplan; Carl Zeiss MicroImaging, Inc.) coupled to a camera (CoolSNAP; Roper Scientific) under a 100x objective (PlanApo; Leica). For Fig. 4 A, we took at least 40 pictures of adult worms per strain and used ImageJ (National Institutes of Health) to measure the distance between the rectum and the grinder. Confocal images were captured on a confocal microscope (SP2-AOBS; Leica), scanning every 122 nm for XZ sections through a 100x objective with a 2.15x zoom (Fig. 8, B and C; and Fig. S3 D) or a 4x zoom (Fig. 4 E and Fig. 5). Images were processed with the Tcstk software (McMahon et al., 2001) and edited using Photoshop 7.0 (Adobe). Microscopes were in an air-conditioned room (20–21°C).
TEM and SEM
L4 larvae were transferred onto fresh plates for 24 ± 2 h at 20°C before fixation. For TEM, but not for SEM, animals were sectioned and fixed for at least 24 h in 2.5% glutaraldehyde/2% paraformaldehyde/0.1 M sodium cacodylate, pH 7.2, at 4°C, and then postfixed for 5 h with 2% osmium tetroxide in the same buffer at 4°C, dehydrated in graded alcohol/water mixes, and embedded in Epon. Ultrathin 70-nm sections were contrasted with uranyl acetate and lead citrate. Sections were observed with a microscope (CM12; Philips) operating at 60 kV. Quantification of the excretory canal section area was obtained using the Metamorph software after scanning images were captured at a 17,000x magnification. Quantification of MVBs was performed on 3,600x-magnified images. Quantification of the mean area occupied by organelles (Fig. 6 D) was obtained using ImageJ and dividing the total surface of each organelle subtype by the cytoplasmic surface of the hyp7 epidermis section. At least four animals per mutant strain were examined, and more than nine pictures per animal from different ultrathin sections were analyzed. For SEM, animals were postfixed for 1 h with 2% osmium tetroxide at 4°C, dehydrated, and critical point dried in hexamethyldisilazane. Fixed animals were mounted on stubs, coated with palladium, and observed through a microscope (XL20; Philips). At least 20 animals per strain were analyzed.
High pressure freezing and immunogold labeling
Adult worms were frozen with a high pressure freezing apparatus (EMPACT-2; Leica) in 20% BSA/M9 medium. Cryosubstitution was conducted as in Muller-Reichert et al. (2003). Ultrathin sections were collected on formvar-carbon–coated copper grids and processed for immunogold labeling. Blocking was performed in PBS/glycine 150 mM, and then in PBS/1% BSA/0.1% Cold Water Fish Skin Gelatin (CWFSG; Aurion) for 30 min. Rabbit anti–VHA-5 at 1:1,000 and rabbit anti-GFP at 1:500 (ab6556; AbCam) were incubated for 1 h in PBS/0.1% CWFSG. 10 nm protein A–coupled gold beads (1:50; University Medical Center, Utrecht, Netherlands) were incubated for 1 h in PBS/0.1% CWFSG. Postfixation was achieved in 2.5% glutaraldehyde, contrasted by uranyl acetate/lead citrate. Images were acquired at 60 kV on a microscope (Morgagni; FEI) with a charge-coupled device camera (Megaview III; Soft Imaging System).
Online supplemental material
Fig. S1 provides a control for Fig. 2 B, showing that RNAi against vha-5 was efficient. In addition, it presents the yolk endocytosis defects induced by the loss of V-ATPase activity (yolk proteins are produced by the intestine and are essential for embryonic development); it suggests that RNAi treatment against V0 and V1 subunits was equally effective, and contributes to establish that alae differences described in Fig. 2 are meaningful. Fig. S2 summarizes the main phenotypes observed in vha-5(mc38) animals carrying transgenes with the mutations shown in Fig. 3. Fig. S3 presents the excretory canal, cuticle and MVB phenotypes induced by the mutations R191A, W327A and V844A, which are discussed but not illustrated in the main text, and shows that WRT-8::GFP accumulates in mc38; Ex[vha-5(E830Q)::rfp] animals; it should be viewed along with Figs. 4, 6 and 8. Fig. S4 shows that secretion of the collagen DPY-7 is not affected by vha-5 or che-14 mutations. Fig. S5 provides larger pictures and controls for the immunogold experiments shown in Fig. 8. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200511072/DC1.
|
Acknowledgments |
|---|
S. Liégeois and A. Benedetto were supported by fellowships from the Ministère de la Recherche, and from the FRM. This work was supported by funds from the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, and by a grant from the Ministère de la Recherche (programme ACI).
Submitted: 17 November 2005
Accepted: 15 May 2006
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Amara, S.G., and M.J. Kuhar. 1993. Neurotransmitter transporters: recent progress. Annu. Rev. Neurosci. 16:73–93.[Medline]
Ang, A.L., T. Taguchi, S. Francis, H. Folsch, L.J. Murrells, M. Pypaert, G. Warren, and I. Mellman. 2004. Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells. J. Cell Biol. 167:531–543.
Aspock, G., H. Kagoshima, G. Niklaus, and T.R. Burglin. 1999. Caenorhabditis elegans has scores of hedgehog-related genes: sequence and expression analysis. Genome Res. 9:909–923.
Bayer, M.J., C. Reese, S. Buhler, C. Peters, and A. Mayer. 2003. Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel. J. Cell Biol. 162:211–222.
Beronja, S., P. Laprise, O. Papoulas, M. Pellikka, J. Sisson, and U. Tepass. 2005. Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells. J. Cell Biol. 169:635–646.
Blott, E.J., and G.M. Griffiths. 2002. Secretory lysosomes. Nat. Rev. Mol. Cell Biol. 3:122–131.[CrossRef][Medline]
Bonfanti, L., A.A. Mironov Jr., J.A. Martinez-Menarguez, O. Martella, A. Fusella, M. Baldassarre, R. Buccione, H.J. Geuze, A.A. Mironov, and A. Luini. 1998. Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation. Cell. 95:993–1003.[CrossRef][Medline]
Bonifacino, J.S., and J. Lippincott-Schwartz. 2003. Coat proteins: shaping membrane transport. Nat. Rev. Mol. Cell Biol. 4:409–414.[CrossRef][Medline]
Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics. 77:71–94.
Burke, R., D. Nellen, M. Bellotto, E. Hafen, K.A. Senti, B.J. Dickson, and K. Basler. 1999. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell. 99:803–815.[CrossRef][Medline]
Cheong, K.H., D. Zacchetti, E.E. Schneeberger, and K. Simons. 1999. VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells. Proc. Natl. Acad. Sci. USA. 96:6241–6248.
Chernomordik, L.V., and M.M. Kozlov. 2003. Protein-lipid interplay in fusion and fission of biological membranes. Annu. Rev. Biochem. 72:175–207.[CrossRef][Medline]
Choi, K.Y., Y.J. Ji, B.K. Dhakal, J.R. Yu, C. Cho, W.K. Song, and J. Ahnn. 2003. Vacuolar-type H+-ATPase E subunit is required for embryogenesis and yolk transfer in Caenorhabditis elegans. Gene. 311:13–23.[CrossRef][Medline]
Couillault, C., N. Pujol, J. Reboul, L. Sabatier, J.-F. Guichou, Y. Kohara, and J.J. Ewbank. 2004. TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM. Nat. Immunol. 5:488–494.[CrossRef][Medline]
de Gassart, A., C. Geminard, D. Hoekstra, and M. Vidal. 2004. Exosome secretion: the art of reutilizing nonrecycled proteins? Traffic. 5:896–903.[CrossRef][Medline]
Fevrier, B., D. Vilette, H. Laude, and G. Raposo. 2005. Exosomes: a bubble ride for prions? Traffic. 6:10–17.[CrossRef][Medline]
Fiedler, K., F. Lafont, R.G. Parton, and K. Simons. 1995. Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane. J. Cell Biol. 128:1043–1053.
Folsch, H., H. Ohno, J.S. Bonifacino, and I. Mellman. 1999. A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell. 99:189–198.[CrossRef][Medline]
Gallet, A., R. Rodriguez, L. Ruel, and P.P. Therond. 2003. Cholesterol modification of hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to hedgehog. Dev. Cell. 4:191–204.[CrossRef][Medline]
Grant, B., and D. Hirsh. 1999. Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Mol. Biol. Cell. 10:4311–4326.
Greco, V., M. Hannus, and S. Eaton. 2001. Argosomes: a potential vehicle for the spread of morphogens through epithelia. Cell. 106:633–645.[CrossRef][Medline]
Grindstaff, K.K., C. Yeaman, N. Anandasabapathy, S.C. Hsu, E. Rodriguez-Boulan, R.H. Scheller, and W.J. Nelson. 1998. Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell. 93:731–740.[CrossRef][Medline]
Hiesinger, P.R., A. Fayyazuddin, S.Q. Mehta, T. Rosenmund, K.L. Schulze, R.G. Zhai, P. Verstreken, Y. Cao, Y. Zhou, J. Kunz, and H.J. Bellen. 2005. The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila. Cell. 121:607–620.[CrossRef][Medline]
Hoekstra, D., D. Tyteca, and S.C. van Ijzendoorn. 2004. The subapical compartment: a traffic center in membrane polarity development. J. Cell Sci. 117:2183–2192.
Hua, W., D. Sheff, D. Toomre, and I. Mellman. 2006. Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging. J. Cell Biol. 172:1035–1044.
Jahn, R., T. Lang, and T.C. Sudhof. 2003. Membrane fusion. Cell. 112:519–533.[CrossRef][Medline]
Kamath, R.S., A.G. Fraser, Y. Dong, G. Poulin, R. Durbin, M. Gotta, A. Kanapin, N. Le Bot, S. Moreno, M. Sohrmann, et al. 2003. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 421:231–237.[CrossRef][Medline]
Kawasaki-Nishi, S., T. Nishi, and M. Forgac. 2001. Yeast V-ATPase complexes containing different isoforms of the 100-kDa a-subunit differ in coupling efficiency and in vivo dissociation. J. Biol. Chem. 276:17941–17948.
Kurzchalia, T.V., P. Dupree, R.G. Parton, R. Kellner, H. Virta, M. Lehnert, and K. Simons. 1992. VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles. J. Cell Biol. 118:1003–1014.
Langevin, J., M.J. Morgan, J.B. Sibarita, S. Aresta, M. Murthy, T. Schwarz, J. Camonis, and Y. Bellaiche. 2005. Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-cadherin trafficking from recycling endosomes to the plasma membrane. Dev. Cell. 9:355–376.[Medline]
Leng, X.H., M.F. Manolson, Q. Liu, and M. Forgac. 1996. Site-directed mutagenesis of the 100-kDa subunit (Vph1p) of the yeast vacuolar (H+)-ATPase. J. Biol. Chem. 271:22487–22493.
Leng, X.H., M.F. Manolson, and M. Forgac. 1998. Function of the COOH-terminal domain of Vph1p in activity and assembly of the yeast V-ATPase. J. Biol. Chem. 273:6717–6723.
Lock, J.G., and J.L. Stow. 2005. Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin. Mol. Biol. Cell. 16:1744–1755.
Luzio, J.P., B.M. Mullock, P.R. Pryor, M.R. Lindsay, D.E. James, and R.C. Piper. 2001. Relationship between endosomes and lysosomes. Biochem. Soc. Trans. 29:476–480.[CrossRef][Medline]
Manninen, A., P. Verkade, S. Le Lay, J. Torkko, M. Kasper, M. Füllekrug, and K. Simons. 2005. Caveolin-1 is not essential for biosynthetic apical membrane transport. Mol. Cell. Biol. 25:10087–10096.
McMahon, L., R. Legouis, J.L. Vonesch, and M. Labouesse. 2001. Assembly of C. elegans apical junctions involves positioning and compaction by LET-413 and protein aggregation by the MAGUK protein DLG-1. J. Cell Sci. 114:2265–2277.[Medline]
McMahon, L., J.M. Muriel, B. Roberts, M. Quinn, and I.L. Johnstone. 2003. Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix. Mol. Biol. Cell. 14:1366–1378.
Michaux, G., A. Gansmuller, C. Hindelang, and M. Labouesse. 2000. CHE-14, a protein with a sterol-sensing domain, is required for apical sorting in C. elegans ectodermal epithelial cells. Curr. Biol. 10:1098–1107.[CrossRef][Medline]
Muller-Reichert, T., H. Hohenberg, E.T. O'Toole, and K. McDonald. 2003. Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure. J. Microsc. 212:71–80.[Medline]
Nelson, F.K., and D.L. Riddle. 1984. Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery. J. Exp. Zool. 231:45–56.[CrossRef][Medline]
Nishi, T., and M. Forgac. 2002. The vacuolar (H+)-ATPases–nature's most versatile proton pumps. Nat. Rev. Mol. Cell Biol. 3:94–103.[CrossRef][Medline]
Nurrish, S.J. 2002. An overview of C. elegans trafficking mutants. Traffic. 3:2–10.[CrossRef][Medline]
Oka, T., T. Toyomura, K. Honjo, Y. Wada, and M. Futai. 2001. Four subunit a isoforms of Caenorhabditis elegans vacuolar H+-ATPase. Cell-specific expression during development. J. Biol. Chem. 276:33079–33085.
Paladino, S., D. Sarnataro, R. Pillich, S. Tivodar, L. Nitsch, and C. Zurzolo. 2004. Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins. J. Cell Biol. 167:699–709.
Paladino, S., T. Pocard, M.A. Catino, and C. Zurzolo. 2006. GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells. J. Cell Biol. 172:1023–1034.
Panakova, D., H. Sprong, E. Marois, C. Thiele, and S. Eaton. 2005. Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature. 435:58–65.[CrossRef][Medline]
Peters, C., M.J. Bayer, S. Buhler, J.S. Andersen, M. Mann, and A. Mayer. 2001. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature. 409:581–588.[CrossRef][Medline]
Porter, J.A., S.C. Ekker, W.J. Park, D.P. von Kessler, K.E. Young, C.H. Chen, Y. Ma, A.S. Woods, R.J. Cotter, E.V. Koonin, and P.A. Beachy. 1996. Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell. 86:21–34.[CrossRef][Medline]
Puertollano, R., F. Martin-Belmonte, J. Millan, M.C. de Marco, J.P. Albar, L. Kremer, and M.A. Alonso. 1999. The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells. J. Cell Biol. 145:141–151.
Pujol, N., C. Bonnerot, J.J. Ewbank, Y. Kohara, and D. Thierry-Mieg. 2001. The Caenorhabditis elegans unc-32 gene encodes alternative forms of a vacuolar ATPase a subunit. J. Biol. Chem. 276:11913–11921.
Raiborg, C., T.E. Rusten, and H. Stenmark. 2003. Protein sorting into multivesicular endosomes. Curr. Opin. Cell Biol. 15:446–455.[CrossRef][Medline]
Roberts, B., C. Clucas, and I.L. Johnstone. 2003. Loss of SEC-23 in Caenorhabditis elegans causes defects in oogenesis, morphogenesis, and extracellular matrix secretion. Mol. Biol. Cell. 14:4414–4426.
Rodriguez-Boulan, E., G. Kreitze, and A. Musch. 2005. Organization of vesicular trafficking in epithelia. Nat. Rev. Mol. Cell Biol. 6:233–247.[CrossRef][Medline]
Roudier, N., C. Lefebvre, and R. Legouis. 2005. CeVPS-27 is an endosomal protein required for the molting and the endocytic trafficking of the low-density lipoprotein receptor-related protein 1 in Caenorhabditis elegans. Traffic. 6:695–705.[CrossRef][Medline]
Sapio, M.R., M.A. Hilliard, M. Cermola, R. Favre, and P. Bazzicalupo. 2005. The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans. Dev. Biol. 282:231–245.[CrossRef][Medline]
Satoh, A.K., J.E. O'Tousa, K. Ozaki, and D.F. Ready. 2005. Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors. Development. 132:1487–1497.
Schuck, S., and K. Simons. 2004. Polarized sorting in epithelial cells: raft clustering and the biogenesis of the apical membrane. J. Cell Sci. 117:5955–5964.
Tanaka, Y., Y. Okada, and N. Hirokawa. 2005. FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left-right determination. Nature. 435:172–177.[CrossRef][Medline]
TerBush, D.R., and P. Novick. 1995. Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol. 130:299–312.
Treusch, S., S. Knuth, S.A. Slaugenhaupt, E. Goldin, B.D. Grant, and H. Fares. 2004. Caenorhabditis elegans functional orthologue of human protein h-mucolipin-1 is required for lysosome biogenesis. Proc. Natl. Acad. Sci. USA. 101:4483–4488.
White, J. 1988. The anatomy. In The Nematode Caenorhabditis elegans. Monograph 17. W.B. Wood and the Community of C. elegans Researchers, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 81–122.
Whyte, J.R., and S. Munro. 2002. Vesicle tethering complexes in membrane traffic. J. Cell Sci. 115:2627–2637.
Yeaman, C., K.K. Grindstaff, J.R. Wright, and W.J. Nelson. 2001. Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells. J. Cell Biol. 155:593–604.
Zhang, Y., B. Grant, and D. Hirsh. 2001. RME-8, a conserved J-domain protein, is required for endocytosis in Caenorhabditis elegans. Mol. Biol. Cell. 12:2011–2021.
Zugasti, O., J. Rajan, and P.E. Kuwabara. 2005. The function and expansion of the Patched- and Hedgehog-related homologs in C. elegans. Genome Res. 15:1402–1410.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
