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© 2009 John Wiley & Sons A/S doi: 10.1111/j.1600-0854.2009.00871.x
Review
Secretion of Hedgehog-Related Peptides and WNT During Caenorhabditis elegans Development Irina Kolotuev†, Ahmet Apaydin†, Michel Labouesse*
Hedgehog-Related, Patched and Dispatched Homologs in C. elegans
IGBMC, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP.10142, 67400 Illkirch, France *Corresponding author: Michel Labouesse,
[email protected] †These authors contributed equally to this work.
The Hedgehog (Hh) cell–cell signaling pathway is highly conserved between Drosophila and vertebrates where it influences different developmental processes such as the establishment of embryonic and body polarity, cell proliferation and organ patterning (3). In C. elegans this pathway has undergone considerable evolutionary divergence because many core components of the canonical Hh pathway, including Hh and Smoothened, are absent from the C. elegans genome, whereas those still present are involved in completely different processes (4). Nevertheless, the C. elegans genome contains several proteins with sequence similarity to the C-terminus of Hh and many Patched homologs. Analysis of their function has contributed and should keep contributing to our understanding of the canonical Hh pathway.
There is growing awareness that endocytic trafficking plays a critical role in cell–cell communication during animal development. We are beginning to understand how endocytosis can initiate, modulate or terminate signaling. In contrast, our knowledge of the mechanisms involved in secreting signaling peptides remains more limited, particularly when it comes to secretion at the apical surface in epithelial cells. In this study, we review the mechanisms that control secretion in Caenorhabditis elegans, focusing on the role of Patched family members and the V0 complex of the vacuolar-adenosine triphosphatase (V-ATPase) in secreting Hedgehog-related peptides and of MIG-14/Wls and the retromer complex in secreting EGL-20/WNT. Key words: Caenorhabditis elegans, cuticle, Dispatched, epithelial, exosome, Hedgehog, polarized secretion, retromer, V-ATPase, WNT Received 18 November 2008, revised and accepted for publication 11 December 2008 published online 6 February 2009
Secreted proteins of the Hedgehog (Hh) and Wingless (WNT) families regulate many developmental processes (1–3). They can diffuse in the extracellular space and act over long distances to induce concentration-dependent responses in surrounding cells. Genetic and cellular analysis in invertebrate model systems has identified several proteins dedicated to the release of Wingless and Hedgehog morphogens. While we are still far from understanding how these specific proteins act, it is quite likely that they are required to handle the posttranslational modifications imparted on Hh and WNT. Indeed, prior to secretion, WNT and Hh ligands undergo specific lipid modifications, which should cause these proteins to associate with internal and external membranes and should thus potentially hinder their secretion and spreading. In this study, we review how Caenorhabditis elegans has contributed to the understanding of Hh and WNT secretion.
Hh and its vertebrate homologs are unusual proteins that undergo two lipid modifications (2). Their C-terminal domain (or Hint/Hog domain) is a protease that mediates autoproteolytic cleavage of the precursor protein to generate an N-terminal product (the Hedge domain) that is the active signaling entity (Figure 1A). Coincident with autocatalytic cleavage, the new C-terminus is modified by cholesterol addition (2). Its N-terminus is further modified downstream of the signal peptide sequence by palmitoylation, which is catalyzed by a membrane-bound O-acyltransferase called Skinny Hedgehog/Sightless/Raspberry/Central Missing (2). Both lipid modifications are required for normal long-distance Hh signaling activity (2). Interestingly, cholesterol-modified Hh proteins form multimers at the apical membrane (5), which is reminiscent of glycosyl-phosphatidylinositol-linked protein behavior in madine darby canine kidney (MDCK) cells (Figure 4). Secretion of lipid-modified Hh, but not of an artificially generated cholesterol-free form, requires the 12-pass transmembrane protein Dispatched (Disp) (Figure 1B) (5,6). In Drosophila, long-range Hh spreading also requires its association with lipophorin lipoproteins and with the Dlp heparan sulfate proteoglycan (2,7). In receiving cells, Hh binds to the 12-pass transmembrane domain protein Patched (Ptc), which in the absence of Hh represses the activity of the 7-pass transmembrane protein Smoothened (Smo) (Figure 1C) (2,3). The signaling cascade downstream of derepressed Smo includes the complex Cos2, Fused and Su(Fu) and ultimately the transcription factor Ci (Figure 1D,E) (2,3). How Disp acts to promote
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Figure 1: Secretion of Hh and Hh-related proteins. Summary of the Hh pathway in Drosophila. In secreting cells, Hh is posttranslationally modified by autocleavage, cholesterol addition at its C-terminus and palmitoylation at its N-terminus (A). This form is secreted with the help of Disp (B). In receiving cells, Hh binds to the Ptc receptor, which releases Smo repression (red cross) (C) to trigger dissociation between the activator form of Gli from the Cos-2/Fu/Su(fu) complex (D) and activate gene expression (E). The Caenorhabditis elegans genome lacks several Hh signaling pathway components but has many Hh-related (61 divided into four groups: Warthog, Quahog, Groundhog and Ground-like) and Ptc-like (3 close Ptc homologs and 24 Ptc-related or ptr) genes (F). Most of them are expressed in epithelial cells, particularly in the epidermis (F). The main C. elegans epidermal cell is the syncytial hyp7 cell, which covers almost the entire body and secretes a protective extracellular matrix (ECM) cuticle layer. Seam epidermal cells (they are surrounded by hyp7) also contribute to another cuticle component, the alae. CHE-14, one of the two C. elegans Disp homologs, is localized at the apical membrane of the epidermis (G). The localization of Ptc-related members is not known (they might be apical, question mark). Secretion of the Hh-related proteins WRT-2 and WRT-8 involves shedding of exosomes from MVBs. Fusion of MVBs with the apical membrane of the hyp7 cell requires the transmembrane V0 complex of the V-ATPase (H). Mutations affecting VHA-5, the largest subunit of the V0 complex, cause severe secretion problems and accumulation of warthog proteins within MVBs.
Hh secretion and how Ptc represses Smo are still unclear (2,3). For both proteins, the presence of a sterol-sensing domain (SSD), named after a motif originally identified in proteins involved in sterol metabolism (8), and their weak homology to bacterial proton-driven pumps of the resistance-nodulation-cell division (RND) family (9), may provide some clues (2). The C. elegans genome harbors 61 sequences predicted to encode secreted proteins that share similarity with the C-terminal autoprocessing Hint/Hog domain of Hh (4,10). Their N-terminus contains a signal peptide sequence and a domain with a characteristic pattern of Cys residues that define four families named Warthog (WRT), Groundhog (GRD), Groundhog like (GRL) and Quahog (QUA), which are collectively referred to as Hedgehog-related proteins
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(Hh-related; Figure 1F) (4,10). Genes comprising a secreted N-terminus and a Hog domain are also found in other nematodes, lophotrochozoa and even protists, indicating that this gene family must have arisen very early in evolution (11,12). The evolutionary distant nematode Trichinella spiralis actually contains a bona fide Hh homolog and a quahog-like gene, even though its genome sequence (>80% complete) lacks Smo, Fu and Cos2 homologs, like C. elegans (11,12). This very striking observation, together with the presence of Hh-related genes in protists, suggests that the ancestral Hh and Hh-related genes probably fulfilled other functions than patterning. Most analyzed Hh-related genes in C. elegans are expressed in the epidermis, which secretes the lipid- and collagen-rich cuticle, in the kidney-like excretory cell as
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SSD-containing genes is a conserved feature of other sequenced nematode genomes (16). Mutations in these genes or RNA interference against most of them (19 of the 27 ptc-/ptr- genes, 1 Disp) induce cuticle patterning and/or molting defects similar to those observed when Hh-related genes are defective (16). Furthermore, at least 10 of these genes are expressed in the epidermis (16). The high similarity in the expression of Hh-related and Ptc-related genes and their related mutant phenotypes suggests that some of them could act in a common process. The function of three Ptc/Disp homologs have been characterized in further detail and are discussed below as they help define the roles of Ptc/Disp proteins.
Figure 2: Example of trafficking defects induced by a partially defective V0 complex. Transmission electron micrographs of the epidermis in a wild type (A) and vha-5 partial mutant (B). Arrows, dark MVBs (they are larger and more numerous in the vha-5 mutant); stars, alae cuticular ridge (they are very abnormal in the vha-5 mutant).
well as in support cells, which create an opening through the cuticle allowing sensory neurons to be exposed to the environment (Figure 1F) (10,13–15). At least two Hhrelated proteins, QUA-1 and WRT-5, are secreted and either incorporated into the cuticle (QUA-1) or released in the luminal space (WRT-5) (13,15). Consistent with a possible role of Hh-related proteins in cuticle formation, RNA interference analysis against some Hh-related genes causes cuticular abnormalities and molting defects (13,15,16). Thus, the ancestral role of Hh-related proteins might have been to contribute to the exoskeleton or the luminal extracellular matrix. Although very little is known about the biogenesis of Hhrelated proteins, the limited information available suggests that they might be modified like Hh (4). Indeed, the C. elegans genome contains two Skinny Hedgehog acyltransferase homologs, one of which is essential for viability (genes hhat-1 and hhat-2, www.wormbase.org), implying that Hh-related proteins could be palmitoylated. Furthermore, at least WRT-1 undergoes autoprocessing in vitro (17), although it is not known whether it becomes attached to a sterol. In addition to the scores of Hh-related genes, C. elegans encodes 2 Disp homologous sequences, 3 close Ptc homologous sequences (ptc- genes) and 24 Ptc-related sequences (ptr- genes) (4,16,18), which are characterized by the presence of a SSD. The presence of multiple
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ptc-1, the first characterized C. elegans Ptc homologue, promotes the formation of a membrane extension between germline nuclei in the germline syncytium in a process akin to cytokinesis (18). The enrichment of PTC-1 protein at the germline plasma membrane is consistent with a role of PTC-1 in trafficking. PTC-1 might promote membrane growth or stabilize the growing membrane furrow during germline cytokinesis. More recently, PTR-2 was also found to be required for somatic cytokinesis (16). The specific trafficking process controlled by PTC-1 and PTR-2 remains to be defined. che-14 encodes one of the two C. elegans Disp homologs (19). Green fluorescent protein reporter studies suggest that the CHE-14 protein is localized at the apical surface of the epidermis, the kidney-like excretory cell and support cells associated with sensory organs (Figure 1G) (19). Mutant che-14 animals show abnormal accumulation of amorphous material beneath the apical membrane surface of the epidermis, and their cuticle is thinner than that in wild-type animals (19). In addition, sensory support cells accumulate numerous large vesicles and fail to provide an opening to sensory neurons (the gene was actually named because the latter phenotype causes a chemosensory behavioral defect) (19,20). These defects strongly suggest an involvement of che-14 in polarized apical secretion in epidermal cells and support cells (19). In che-14 mutants, at least one Hh-related protein accumulates in support cells (A. Benedetto and M. Labouesse, unpublished data), consistent with the notion that CHE-14 helps secrete specific Hh-related proteins. daf-6, which encodes a PTR protein, has a function related to that of che-14. Similar to che-14 mutants, daf-6 mutants have obstructed sensory organs, which affects their response to the environment, in particular to the dauerinducing pheromone (they are dauer formation defective) (20,21). DAF-6 expression is restricted to the apical/luminal surface of tube-forming organs (21). Interestingly, combining mutations in che-14 and daf-6 prevents growth of the excretory canal, a very long collecting tubule extending from the kidney-like excretory cell that runs along the body (21). This phenotype has been interpreted as failure to form the lumen. As DAF-6 is more related to Ptc than to Disp, and since it was proposed that Patched endocytoses
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Hh (22), Perens and Shaham suggested that DAF-6 could act by inhibiting endocytosis (21). In this scenario, lumen formation and expansion of the luminal membrane would require the seemingly opposite activities of CHE-14 to promote exocytosis and DAF-6 to limit endocytosis (21). A role for DAF-6 in endocytosis is plausible because RNA interference (RNAi)-induced inhibition of ptr-4, ptr-18 or ptr-23 compromises endocytosis of the yolk protein YP170 in the germline (16). However, reduction of yolk endocytosis could also result from reduced secretion of the yolk receptor RME-2 at the germline plasma membrane (23). Furthermore, direct proof for an endocytic role of DAF-6 needs to be established. At this point, it seems equally possible that DAF-6 acts redundantly with CHE-14 to promote exocytosis in the excretory cell because many Ptc-related genes appear to act redundantly in cuticle patterning and molting (16). In conclusion, genetic and cellular data in C. elegans strongly support the notion that Ptc, Ptc-related and Disp homologs control trafficking. Expression and phenotypic analysis of Ptcrelated and Hh-related genes indicates that many probably act in the same process. At least CHE-14, and by extension some PTRs, probably control the secretion of Hh-related proteins. What remains to be established is at which step of the biosynthetic secretory pathway they would act. An alternative scenario would be that Ptc-related proteins control the endocytosis of specific factors that in turn help secrete Hh-related peptides. For instance, the absence of LRP-1/megalin, which in vertebrates can endocytose cholesterol as well as Shh, produces a molting phenotype similar to that observed after knockdown of some Ptc-related genes (24). A third possibility raised by the weak similarity between Ptc proteins and bacterial RND pumps would be that Ptc-related proteins promote the efflux of specific lipids necessary for membrane growth or cuticle formation.
Hedgehog-Related Peptide Secretion by Means of Exosomes A new twist in our understanding of Hh-related protein secretion has come from the finding that Hh-related peptides could be secreted by means of exosomes (25). Exosomes correspond to intraluminal vesicles of multivesicular bodies (MVBs) released upon exocytic fusion of the MVB with the plasma membrane (26). Looking for mutations that phenocopy che-14 defects, Liegeois et al. found that mutants in the gene vha-5 have cuticular defects and accumulate large dense MVBs in the epidermis (25,27) (Figure. 2). Conversely, they observed fusion events between MVBs and the apical plasma membrane in wildtype animals, which prompted them to propose that a subset of MVBs is secretory in the epidermis (25). The gene vha-5 encodes the large transmembrane subunit of the vacuolar-adenosine triphosphatase (V-ATPase), a proton pump consisting of several transmembrane (the V0 complex) and cytoplasmic (the V1 complex) subunits. Genetic analysis showed that vha-5 trafficking defects were not because of inhibition of the V-ATPase proton pump activity
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and reflected lack of the V0 complex (25). Hence, these authors proposed that the V0 complex mediates the fusion of MVBs with the plasma membrane to allow the release of exosomes containing cuticle proteins (Figure 1H) (25). Interestingly, the V0 complex was also shown to promote membrane fusion independent of its proton pump activity in yeast, Drosophila and zebra fish and to mediate insulin secretion in mouse pancreatic islets (28–31). A search for cuticle proteins whose secretion depends on VHA-5 activity revealed that the Hh-related peptides WRT-2 and WRT-8, but not collagen, accumulate in the MVBs of vha5 mutants (25). Hence, Hh-related peptides can be secreted by means of exosomes. Exosome shedding could provide the lipids necessary to make the cuticle. Does this imply that the V0 complex is involved in Hedgehog release in other systems? The answer is perhaps not or not always. Genetic mapping in zebrafish of a mutant causing optic cup morphogenesis defects identified two closely linked mutations: one was a nonsense Ptc allele and the other a missense mutation in a small V0 complex subunit (32). Subsequent rescue experiments and morpholino assays argued that the eye morphogenetic defects are caused by the Ptc mutation rather than by the V-ATPase mutation (32). Whether Shh secretion in the optic cup field is representative of all Shh secretion events remains to be seen. Assuming vertebrate Hh secretion does not require the V0 complex, what might then be the implications of the C. elegans findings? A possibility is that VHA-5 function in Hh-related peptide secretion reveals an ancestral role of Hh-related peptides in innate immunity and of the V0 complex in mediating exosome release in the immune system. Two indirect arguments support this view. First, the expression of some Hh-related peptides is induced by bacterial infection, indicating that they might be involved in the C. elegans innate immune response (33). Second, exosomes are very common in the immune system, particularly in antigen-presenting cells (26). In addition to promoting the fusion of MVBs with the plasma membrane, the V0 complex seems to provide positional information to instruct where secretion should take place. Indeed, VHA-5 distribution appears to coincide with that of circumferentially oriented actin bundles in the epidermis, specifically at the time of molting (27). These epidermal actin bundles underlie circumferential cuticular structures known as annuli. Hence, the actin cytoskeleton might direct where MVBs should deliver their protein and lipid content, which could in turn help pattern the cuticle.
Production and Secretion of WNT Ligands in C. elegans The canonical WNT pathway is well conserved from C. elegans to Drosophila and humans, where it acts to pattern the embryo and organs. WNT proteins undergo two critical posttranslational modifications in the endoplasmic reticulum (ER): N-glycosylation and palmitoylation at two cysteine residues (reviewed in 34,35). These modifications
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are thought to facilitate polarized WNT secretion (Figure 4) and its extracellular diffusion by mediating interaction with heparan sufate proteoglycans (HSPGs). In Drosophila, the transmembrane protein O-acyltransferase Porcupine (Porc) catalyzes WNT palmitoylation (36,37) and facilitates WNT N-glycosylation (36,38). The C. elegans Porcupine orthologue, called mom-1, is required in the early embryo for the MOM-2/WNT signaling process between the P2 and EMS blastomeres that polarizes the EMS blastomere and ultimately specifies endoderm formation (39). Although MOM1 palmitoylation activity has not been tested on any of the five WNT homologs, genetic analysis supports the view that MOM-1 plays the same role as that of Porc.
the Golgi complex (43–46,50). In particular, blocking clathrinmediated endocytosis in C. elegans by means of mutations in the AP-2 m2 subunit DPY-23 causes MIG-14/Wls accumulation at the plasma membrane and induces WNT-type defects (45). Similarly, mutations affecting the retromer subunit VPS-35 dramatically lower MIG-14/Wls and cause its accumulation within MVBs and ultimately in lysosomes, where it gets degraded (45,46). Interestingly, overexpression in C. elegans of MIG-14/Wls in EGL-20/WNT-expressing cells partially rescues the neuronal migration defect of vps35 mutants, suggesting that MIG-14/Wls becomes limiting in the absence of the retromer complex (46). The three groups using Drosophila reached similar conclusions (43,44,50).
Recently, convergent work in Drosophila and C. elegans has identified two novel major players in WNT secretion, the multipass transmembrane protein Wntless (Wls; also known as Evi/Sprinter in Drosophila or MIG-14/MOM-3 in C. elegans) and the retromer complex (1,34,35). Mutations in Wls/Evi/Sprinter, which were identified through different screens for genes acting in the Wingless pathway, lead to Wingless retention in producing cells (40–42). Interestingly, Wls disruption appears to specifically affect WNT signaling (40). Wls coimmunoprecipitates with WNT and is found in the Golgi, the plasma membrane and endosomes of WNTproducing cells (43,44). Basler and coworkers also showed that RNAi against the closest C. elegans Wls homolog compromises the P2/EMS signaling process described above (40). They subsequently found that this homolog corresponds to mig-14/mom-3 (40), originally identified in screens producing defects in processes influenced by Wnt signaling. In particular, mig-14/mom-3 mutations affect processes that are polarized along the anteroposterior axis of the animal: the asymmetric division of the ectodermal blast cell known as V5, the direction of neuronal migration (a few neurons migrate anteriorly or posteriorly in larval development) and neuronal polarity (several neurons extend long axons either anteriorly or posteriorly relative to their cell body) (45,46). MIG-14 is expressed in cells producing the WNT homolog EGL-20 in the tail and is found at the plasma membrane and in distinct intracellular punctae (45,46). Whether MIG-14 physically interacts with any of C. elegans WNT homologs is not yet known.
A model for WNT secretion has been proposed to account for the findings described above, which is presented in Figure 3. WNT homologues are palmitoylated by PORC/ MOM-1 in the ER, where they presumably also become glycosylated (Figure 3A). MIG-14/Wls interacts with WNT in the Golgi and then helps its secretion by a mechanism that remains to be established (Figure 3B,C). Subsequently, MIG-14/Wls gets internalized by AP-2- and clathrin-mediated endocytosis (Figure 3D); this step is defective in AP-2 mutants. Finally, the retromer complex recycles MIG-14/Wls from endosomes to the Golgi complex (Figure 3E) or else enters the lysosomal degradation route (Figure 3F); in the absence of the retromer, MIG-14/ WLS is fated for degradation in lysosomes. In this model, MIG-14/Wls promotes WNT secretion, and contrary to what had been initially proposed (48), it does not modulate long-range versus short-range WNT signaling (43–46,50).
The significance of MIG-14/Wls role in WNT secretion was illuminated by the discovery of a role for the retromer complex in WNT signaling. The retromer complex, which was originally identified in yeast, acts to recycle proteins from endosomes to the Golgi complex (reviewed in 47). Using C. elegans, Korswagen, Clark and their collaborators first showed that proper retromer function is required in WNT-producing cells for WNT secretion and formation of the EGL-20/WNT gradient in the events that determine the direction of neuronal migration and neuronal polarity (48,49). Building on these findings, two C. elegans and three Drosophila papers established that MIG-14/Wls reaches endosomes after clathrin-mediated endocytosis and that the retromer complex retrieves it from early endosomes to
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Figure 3: Hypothetical trafficking route of Caenorhabditis elegans WNTs. See section ‘‘Production and Secretion of WNT Ligands in C. elegans’’ for further details. Cis-G, cis-Golgi; End, endosome; Lys, lysosome; TGN, trans Golgi network.
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which might mean that WNTs are secreted through exosomes or that MIG-14/Wls clears unsecreted WNT from the plasma membrane.
Conclusions
Figure 4: Secretion in polarized epithelia. Most of our knowledge on trafficking along the polarized biosynthetic secretory pathway comes from studies using polarized MDCK cells. Five main trafficking routes have been recognized. Basolateral routes: Basolateral sorting signals often correspond to tyrosine or dileucine motifs. A) Basolateral proteins can first be addressed to endosomes by the AP-1 clathrin adaptor and from there to the basolateral membrane. B) Alternatively, they can be directly sorted from the trans Golgi network (TGN) to the basolateral membrane or C) by a transcytosis mechanism first to the apical membrane and then to the basolateral membrane, presumably after sorting in endosomes. Apical routes: Apical sorting signals often correspond to posttranslational adducts, such as a glycosyl-phosphatidylinositol (GPI) anchor, N- or O-glycosylation. D) GPI-linked proteins often assemble into oligomers, which are thought to form sphingomyelin-rich and cholesterol-rich lipid rafts (51). E) N- or O-glycosylation-modified proteins can be sorted based on interaction with specific proteins such as galectin (52,53); they can be associated with lipid rafts as well. How GPI-linked oligomers or glycosylated proteins become recognized, transported and secreted apically remains unclear, although a few proteins such as the MAL proteolipid have been shown to facilitate apical sorting. For further details, see Mellman and Nelson (54).
It is intriguing that despite the absence of a canonical Hh signaling pathway, C. elegans and other nematodes have several Ptc-related, Disp and Hh-related proteins. It is likely that many of these genes act in related processes, such as cuticle formation and possibly innate immunity. Genetics and cellular analysis in C. elegans has strengthened the notion that CHE-14/Disp promotes secretion, although the subcellular membrane where it acts remains to be defined. It has revealed that Hh-related peptides can be secreted through exosomes. Although during eye morphogenesis Shh does not require the V0 complex for secretion, additional studies are necessary to rule out the involvement of exosomes for Hh/Shh secretion in all Shh-mediated events. C. elegans contributed to our understanding of WNT secretion by revealing the role of the retromer complex and helped clarify that of MIG-14/Wls in parallel to studies in Drosophila. The major challenge ahead both for Hh-related peptide and for WNT secretion is to figure out what is the specific role of the apparently dedicated secretion factors CHE-14/Disp and MIG-14/Wls.
Acknowledgments We apologize to our colleagues whose work could not be cited owing to space limitations. I. K. is supported by the Centre National de la Recherche Scientifique (CNRS) and A. A. by a Ministe`re de la Recherche fellowship. Our work is supported by funds from the CNRS and Institut National de la Sante´ et de la Recherche Me´dicale and by grants from the Institut National du Cancer and the Ligue Nationale contre le Cancer.
References Several aspects of this model still remain unclear. The most critical issue is to determine the precise role of MIG-14/Wls in WNT secretion. For instance, it could be a transporter for WNT proteins, it could interact with the posttranslational adducts to promote WNT oligomerization or it could interact with the secretion machinery (syntaxin, Sec18, etc.) to facilitate WNT exocytosis. In other words, how MIG-14/Wls interacts with WNT and in which subcellular membrane(s) this interaction is functionally important are open questions. The mechanism of WNT exocytosis and its polarity are also unknown, although evidence from fly research point to an apical secretion route (reviewed in 1). The presence of WNT in endosomes and MVBs of WNT-producing cells (reviewed in 1) raises several possibilities: (i) endosomes could act as sorting platforms for the recruitment of MIG-14/Wls and (ii) MIG-14/Wls could be required to reach MVBs,
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