SNX3 controls Wingless/Wnt secretion through regulating retromer-dependent recycling of Wntless

Drosophila Wingless (Wg) acts as a morphogen during development. Wg secretion is controlled by a seven-pass transmembrane cargo Wntless (Wls). We have recently identified retromer as a key regulator involved in Wls trafficking. As sorting nexin (SNX) molecules are essential components of the retromer complex, we hypothesized that specific SNX(s) is required for retromer-mediated Wnt secretion. Here, we generated Drosophila mutants for all of the eight snx members, and identified Drosophila SNX3 (DSNX3) as an essential molecule required for Wg secretion. We show that Wg secretion and its signaling activity are defective in Dsnx3 mutant clones in wing discs. Wg levels in the culture medium of Dsnx3-depleted S2 cells are also markedly reduced. Importantly, Wls levels are strikingly reduced in Dsnx3 mutant cells, and overexpression of Wls can rescue the Wg secretion defect observed in Dsnx3 mutant cells. Moreover, DSNX3 can interact with the retromer component Vps35, and co-localize with Vps35 in early endosomes. These data indicate that DSNX3 regulates Wg secretion via retromer-dependent Wls recycling. In contrast, we found that Wg secretion is not defective in cells mutant for Drosophila snx1 and snx6, two components of the classical retromer complex. Ectopic expression of DSNX1 or DSNX6 fails to rescue the Wg secretion defect in Dsnx3 mutant wing discs and in Dsnx3 dsRNA-treated S2 cells. These data demonstrate the specificity of the DSNX3-retromer complex in Wls recycling. Together, our findings suggest that DSNX3 acts as a cargo-specific component of retromer, which is required for endocytic recycling of Wls and Wg/Wnt secretion.     

Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.     

A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.     

Protein kinase CK2 is required for Wntless internalization and Wnt secretion

Wnt proteins are lipid modified signaling molecules that have essential functions in development and adult tissue homeostasis. Secretion of Wnt is mediated by the transmembrane protein Wntless, which binds Wnt and transports it from the endoplasmic reticulum to the cell surface for release. To maintain efficient Wnt secretion, Wntless is recycled back to the Golgi and the endoplasmic reticulum through endocytosis and retromer dependent endosome to Golgi transport. We have previously identified protein kinase CK2 (CK2) in a genome-wide screen for regulators of Wnt signaling in Caenorhabditis elegans. Here, we show that CK2 function is required in Wnt producing cells for Wnt secretion. This function is evolutionarily conserved, as inhibition of CK2 activity interferes with Wnt5a secretion from mammalian cells. Mechanistically, we show that inhibition of CK2 function results in enhanced plasma membrane localization of Wls in C. elegans and mammalian cells, consistent with the notion that CK2 is involved in the regulation of Wls internalization.     

The retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-Golgi network

Secreted Wnt proteins play essential roles in many biological processes during development and diseases. However, little is known about the mechanism(s) controlling Wnt secretion. Recent studies have identified Wntless (Wls) and the retromer complex as essential components involved in Wnt signaling. While Wls has been shown to be essential for Wnt secretion, the function(s) of the retromer complex in Wnt signaling is unknown. Here, we have examined a role of Vps35, an essential retromer subunit, in Wnt signaling in Drosophila and mammalian cells. We provide compelling evidence that the retromer complex is required for Wnt secretion. Importantly, Vps35 colocalizes in endosomes and interacts with Wls. Wls becomes unstable in the absence of retromer activity. Our findings link Wls and retromer functions in the same conserved Wnt secretion pathway. We propose that retromer influences Wnt secretion by recycling Wntless from endosomes to the trans-Golgi network (TGN).     

Porcupine-mediated lipidation is required for Wnt recognition by Wls

Wnt proteins are members of a conserved family of secreted signaling ligands and play crucial roles during development and in tissue homeostasis. There is increasing evidence that aberrant Wnt production is an underlying cause of dysregulated Wnt signaling, however little is known about this process. One protein known to play a role in secretion is the transmembrane protein Wntless (Wls). However, the mechanism by which Wls promotes Wnt secretion is a riddle. It is not known which Wnt family members require Wls and what the structural requirements are that make some of them reliant on Wls for secretion. Here we present a systematic analysis of all known Drosophila Wnt family members with respect to their dependence on Wls function for secretion. We first show that the glycosylation status of Wg at conserved sites does not determine its dependence on Wls. Moreover, in apparent contrast to murine wls, Drosophila wls is not a target gene of canonical Wnt signaling. We then show that all Wnts, with the exception of WntD, require Wls for secretion. All Wnts, with the exception of WntD, also contain a conserved Serine residue (in Wg S239), which we show to be essential for their functional and physical interaction with Wls. Finally, all Wnts, with the exception of WntD, require the acyltransferase Porcupine for activity and for functionally interacting with Wls. Together, these findings indicate that Por-mediated lipidation of the S239-equivalent residue is essential for the interaction with, and secretion by, Wls.     

Inhibition of late endosomal maturation restores Wnt secretion in Caenorhabditis elegans vps-29 retromer mutants

Secretion of Wnt proteins is mediated by the Wnt sorting receptor Wls, which transports Wnt from the Golgi to the cell surface for release. To maintain efficient Wnt secretion, Wls is recycled back to the trans-Golgi network (TGN) through a retromer dependent endosome to TGN retrieval pathway. It has recently been shown that this is mediated by an alternative retromer pathway in which the sorting nexin SNX3 interacts with the cargo-selective subcomplex of the retromer to sort Wls into a retrieval pathway that is morphologically distinct from the classical SNX-BAR dependent retromer pathway. Here, we investigated how sorting of Wls between the two different retromer pathways is specified. We found that when the function of the cargo-selective subcomplex of the retromer is partially disrupted, Wnt secretion can be restored by interfering with the maturation of late endosomes to lysosomes. This leads to an accumulation of Wls in late endosomes and facilitates the retrieval of Wls through a SNX-BAR dependent retromer pathway. Our results are consistent with a model in which spatial separation of the SNX3 and SNX-BAR retromer complexes along the endosomal maturation pathway as well as cargo-specific mechanisms contribute to the selective retrieval of Wls through the SNX3 retromer pathway.     

Wnt signalling requires MTM-6 and MTM-9 myotubularin lipid-phosphatase function in Wnt-producing cells

Wnt proteins are lipid-modified glycoproteins that have important roles in development, adult tissue homeostasis and disease. Secretion of Wnt proteins from producing cells is mediated by the Wnt-binding protein MIG-14/Wls, which binds Wnt in the Golgi network and transports it to the cell surface for release. It has recently been shown that recycling of MIG-14/Wls from the plasma membrane to the trans-Golgi network is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is still poorly understood. In this study, we report the identification of MTM-6 and MTM-9 as novel regulators of MIG-14/Wls trafficking in Caenorhabditis elegans. MTM-6 and MTM-9 are myotubularin lipid phosphatases that function as a complex to dephosphorylate phosphatidylinositol-3-phosphate, a central regulator of endosomal trafficking. We show that mutation of mtm-6 or mtm-9 leads to defects in several Wnt-dependent processes and demonstrate that MTM-6 is required in Wnt-producing cells as part of the MIG-14/Wls-recycling pathway. This function is evolutionarily conserved, as the MTM-6 orthologue DMtm6 is required for Wls stability and Wg secretion in Drosophila. We conclude that regulation of endosomal trafficking by the MTM-6/MTM-9 myotubularin complex is required for the retromer-dependent recycling of MIG-14/Wls and Wnt secretion.     

Secretion of Wnt ligands requires Evi, a conserved transmembrane protein

Wnt signaling pathways are important for multiple biological processes during development and disease. Wnt proteins are secreted factors that activate target-gene expression in both a short- and long-range manner. Currently, little is known about how Wnts are released from cells and which factors facilitate their secretion. Here, we identify a conserved multipass transmembrane protein, Evenness interrupted (Evi/Wls), through an RNAi survey for transmembrane proteins involved in Drosophila Wingless (Wg) signaling. During development, evi mutants have patterning defects that phenocopy wg loss-of-function alleles and fail to express Wg target genes. evi's function is evolutionarily conserved as depletion of its human homolog disrupts Wnt signaling in human cells. Epistasis experiments and clonal analysis place evi in the Wg-producing cell. Our results show that Wg is retained by evi mutant cells and suggest that evi is the founding member of a gene family specifically required for Wg/Wnt secretion.     

Retromer Dependent Recycling of the Wnt Secretion Factor Wls Is Dispensable for Stem Cell Maintenance in the Mammalian Intestinal Epithelium

In C. elegans and Drosophila, retromer mediated retrograde transport of Wntless (Wls) from endosomes to the trans-Golgi network (TGN) is required for Wnt secretion. When this retrograde transport pathway is blocked, Wls is missorted to lysosomes and degraded, resulting in reduced Wnt secretion and various Wnt related phenotypes. In the mammalian intestine, Wnt signaling is essential to maintain stem cells. This prompted us to ask if retromer mediated Wls recycling is also important for Wnt signaling and stem cell maintenance in this system. To answer this question, we generated a conditional Vps35 ^fl allele. As Vps35 is an essential subunit of the retromer complex, this genetic tool allowed us to inducibly interfere with retromer function in the intestinal epithelium. Using a pan-intestinal epithelial Cre line (Villin-CreERT2), we did not observe defects in crypt or villus morphology after deletion of Vps35 from the intestinal epithelium. Wnt secreted from the mesenchyme of the intestine may compensate for a reduction in epithelial Wnt secretion. To exclude the effect of the mesenchyme, we generated intestinal organoid cultures. Loss of Vps35 in intestinal organoids did not affect the overall morphology of the organoids. We were able to culture Vps35 ^∆/∆ organoids for many passages without Wnt supplementation in the growth medium. However, Wls protein levels were reduced and we observed a subtle growth defect in the Vps35 ^∆/∆ organoids. These results confirm the role of retromer in the retrograde trafficking of Wls in the intestine, but show that retromer mediated Wls recycling is not essential to maintain Wnt signaling or stem cell proliferation in the intestinal epithelium.     

Roles of N-glycosylation and lipidation in Wg secretion and signaling

Wnt members act as morphogens essential for embryonic patterning and adult homeostasis. Currently, it is still unclear how Wnt secretion and its gradient formation are regulated. In this study, we examined the roles of N-glycosylation and lipidation/acylation in regulating the activities of Wingless (Wg), the main Drosophila Wnt member. We show that Wg mutant devoid of all the N-glycosylations exhibits no major defects in either secretion or signaling, indicating that N-glycosylation is dispensable for Wg activities. We demonstrate that lipid modification at Serine 239 (S239) rather than that at Cysteine 93 (C93) plays a more important role in regulating Wg signaling in multiple developmental contexts. Wg S239 mutant exhibits a reduced ability to bind its receptor, Drosophila Frizzled 2 (dFz2), suggesting that S239 is involved in the formation of a Wg/receptor complex. Importantly, while single Wg C93 or Wg S239 mutants can be secreted, removal of both acyl groups at C93 and S239 renders Wg incapable of reaching the plasma membrane for secretion. These data argue that lipid modifications at C93 and S239 play major roles in Wg secretion. Further experiments demonstrate that two acyl attachment sites in the Wg protein are required for the interaction of Wg with Wntless (Wls, also known as Evi or Srt), the key cargo receptor involved in Wg secretion. Together, our data demonstrate the in vivo roles of N-glycosylation and lipid modification in Wg secretion and signaling.     

Retromer retrieves wntless

Wntless is a sorting receptor required for Wnt secretion. Wntless is retrieved from endosomes to the Golgi by retromer, permitting Wntless reutilization in Wnt transport. In the absence of retromer, Wntless is degraded in lysosomes and Wnt secretion is impaired.     

Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling

The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXφ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls^AEGL was rescued by the insertion of a classical YXXφ motif in the cytoplasmic tail. Significantly, a Drosophila Wls^AEGL mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXφ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins.     

Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex

The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Our analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. We propose that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment.     

Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells

Cell-cell communication via Wnt signals represents a fundamental means by which animal development and homeostasis are controlled. The identification of components of the Wnt pathway is reaching saturation for the transduction process in receiving cells but is incomplete concerning the events occurring in Wnt-secreting cells. Here, we describe the discovery of a novel Wnt pathway component, Wntless (Wls/Evi), and show that it is required for Wingless-dependent patterning processes in Drosophila, for MOM-2-governed polarization of blastomeres in C. elegans, and for Wnt3a-mediated communication between cultured human cells. In each of these cases, Wls is acting in the Wnt-sending cells to promote the secretion of Wnt proteins. Since loss of Wls function has no effect on other signaling pathways yet appears to impede all the Wnt signals we analyzed, we propose that Wls represents an ancient partner for Wnts dedicated to promoting their secretion into the extracellular milieu.     

Wntless/GPR177在小鼠原肠发生和心脏发育中必不可少

小鼠早期胚胎发育包含原肠运动和器官发生等重要发育过程,这些过程受多种信号通路调控,其中有Wnt、BMP、Nodal、FGF等信号通路,它们之间进行精细严密的协调,保证胚胎发育的正确进行。β-联蛋白作为Wnt配体的共同下游信号分子,在小鼠原肠运动和器官发生中发挥至关重要的作用。Wntless/GPR177在以前的研究中已被报道参与调节Wnt配体的成熟、分选和分泌等,小鼠全身剔除Wntless（Wls）将严重影响胚胎体轴形成。在该研究中,Wls被特异性地在上胚层、心血管中胚层和心肌祖细胞中剔除,以探索Wls如何参与到小鼠原肠运动和心血管发育中。我们发现,在上胚层剔除Wls后,明显阻断了上皮-间充质转化过程,这是中胚层迁移中的关键步骤。在Wls条件性剔除的上胚层中,β-联蛋白表达模式发生变化,表达水平明显下降;E-钙黏着蛋白和N 钙黏着蛋白明显上升。此外,被剔除Wls的上胚层中,细胞凋亡明显增加。不论是在心脏中胚层还是在心脏前体细胞中,剔除Wls都导致严重的心血管发育缺陷和胚胎死亡,证明Wls对心脏发育同样十分重要。这些研究结果证明,Wntless在小鼠原肠运动和心脏发育中均发挥十分重要的作用。     

Sailing with the Wnt: Charting the Wnt processing and secretion route

Wnt proteins are members of a highly conserved family of signalling molecules that play a central role in development and disease. During the past years, the different signalling pathways that are triggered by Wnt proteins have been studied in detail, but it is still largely unknown how a functional Wnt protein is produced and secreted. The recent finding that Wnt proteins are post-translationally modified and the discovery of the Wnt binding protein Wntless and its trafficking by the retromer complex show that Wnt secretion is a complex and highly regulated process. In this review, we will give an overview of the Wnt maturation and secretion pathway and discuss how this process may influence the spreading and signalling activity of Wnt.     

Wnts need a p(assport)24 to leave the ER

Wnts are secreted through a dedicated exocytic pathway, which has been only partly characterized. Here, Palmer and colleagues comment on two recent reports by the groups of K. Basler and M. Boutros, respectively, in which they show that p24 proteins take part in this exocytic route and are required for Wnt exit from the ER to the Golgi.EMBO Rep (2011) advance online publication. doi:10.1038/embor.2011.212Wnt signalling proteins regulate diverse cellular processes during development and homeostasis. Since misregulation of Wnt signalling is associated with cancer, most research has been aimed at characterizing the signal transduction pathway. Recently, attention has focused on Wnt production due to the identification of factors required specifically for Wnt secretion. For instance, the specific requirement of Evi, also known as Wntless or Sprinter, suggested that Wnts might follow a specialized secretory route (Banziger et al, 2006; Bartscherer et al, 2006). Two recent papers published in EMBO reports by the groups of Konrad Basler in last month''s issue, and Michael Boutros in this issue, show that Wnt secretion requires the activity of p24 family members (Buechling et al, 2011; Port et al, 2011). Therefore, the specialized route might begin at endoplasmic reticulum (ER) exit sites.Wnts are secreted glycoproteins that can act many cell diameters from their source of production. Most, but not all Wnt proteins, are acylated and thus associate tightly with cellular membranes. Despite this association, acylated Wnts can be released from secreting cells and spread in the extracellular space (Bartscherer & Boutros, 2008; Port & Basler, 2010). Acylation of Wnts, which occurs in the ER, is thought to be mediated by the N-acetyl transferase encoded by porcupine (porc; van den Heuvel et al, 1993). After acylation, Wnt proteins associate with Evi, a multipass transmembrane protein found mostly at the Golgi and the plasma membrane (Fig 1A). This association is essential for the secretion of the Wnts that are acylated since, in the absence of Evi, they accumulate on internal membranes (Banziger et al, 2006; Bartscherer et al, 2006). It is therefore thought that acylated Wnts require Evi to exit the Golgi and progress to the cell surface (Port et al, 2008). This does not seem to be a requirement for non-lipidated Wnts, such as Drosophila WntD, which are secreted in the absence of Evi (Ching et al, 2008). Similarly, secretion of other signalling proteins proceeds normally without Evi. After reaching the cell surface, Evi is likely to have a choice between various routes. One such route, which involves the retromer complex, takes it back to the Golgi where it can participate in another round of Wnt secretion (Fig 1A). Alternatively, Evi can be targeted to lysosomes (Bartscherer & Boutros, 2008; Port & Basler, 2010). The factors that determine Evi transport remain poorly understood. Nevertheless, these studies highlight the essential and specific role of Evi for the secretion of lipidated Wnts.Open in a separate windowFigure 1Model summarizing the suggested roles of p24 proteins in endoplasmic reticulum to Golgi transport of Wg. (A) Schematic of a cell showing the current model of the Wg secretory pathway. Wg is produced in the ER where it is lipid-modified by Porc, and moved to the Golgi with the assistance of p24 proteins. In the Golgi, Wg joins Evi, which facilitates Wg transport to the cell surface. Evi is then recycled back to the Golgi in a path mediated by the retromer complex. (B)(i) In the absence of p24 proteins, there is no recruitment of Wg to COPII-coated vesicles and therefore a block of secretion in the ER. (ii) Port et al (2011) propose a similar model in which CHOp24/Emp24 and Éclair are involved in Wg recruitment, although only CHOp24/Emp24 binds to Wg. (iii) According to Buechling et al (2011), Opm recruits Wg to COPII-coated vesicles for movement to the Golgi. CHOp24 and p24-1 are also required for this process. ER, endoplasmic reticulum.Two groups have now reported the use of cell-based RNA interference (RNAi) screens to identify further proteins required for the secretion of Wingless (Wg), the main Drosophila Wnt (Buechling et al, 2011; Port et al, 2011). They found that p24 family members, a group of proteins previously implicated in both retrograde and anterograde transport between the ER and Golgi (Strating & Martens, 2009), are required for Wg secretion by S2 cells. Similarly, knockdown by transgenic RNAi shows that p24 proteins are required for normal levels of Wg secretion in Drosophila wing imaginal discs (Buechling et al, 2011; Port et al, 2011). As with Evi, this requirement seems to be relatively specific, since general secretion and the secretion of other signalling proteins, including the lipid-modified morphogen Hedgehog, are unaffected by p24 knockdown. Buechling et al also assessed the role of p24 proteins in WntD secretion. They found that RNAi against opossum (opm), one of the p24 members, prevents WntD secretion in cultured cells. They also show that the phenotypes of opm mutants and WntD mutant embryos resemble each other (Buechling et al, 2011). Therefore, while Evi is specifically required for the secretion of acylated Wnts, p24 proteins could contribute to the secretion of all Wnts. This function is likely to be conserved since the mammalian homologue of Opm, TMED5, is required for Wnt1 signalling, at least in a mammalian cell culture assay (Buechling et al, 2011).To gain understanding of the role of p24 proteins in Wnt secretion, both groups analysed the subcellular localization of Wg following p24 knockdown. They found accumulation in the ER and concomitant depletion in the Golgi, as indicated by reduced co-localization with Golgi markers (Fig 1Bi). They also found that p24 knockdown prevents Wg from stabilizing Evi in producing cells, suggesting that the stabilizing influence of Wg requires its exit from the ER (Buechling et al, 2011; Port et al, 2011). These results lead the authors to propose that the loss of p24 prevents the transport of Wg from the ER to the Golgi. Importantly, immunoprecipitation experiments suggest that Wg might interact physically with Opm and Emp24 (also known as CHOp24). This led both sets of authors to postulate a model whereby p24 proteins act as cargo receptors to escort Wnt proteins from the ER to the Golgi, whereupon they can bind to Evi, which will escort them to the plasma membrane. Thus, in this context, p24 proteins seem to have an anterograde function.Although both studies highlight the role of p24 proteins in Wnt secretion, they disagree on the relative importance of the various family members. Among the nine predicted p24 proteins encoded by the Drosophila genome, only Éclair and Emp24/CHOp24 were found to be required for Wg secretion by Port et al (2011; Fig 1Bii). By contrast, Buechling et al found that Opm, Emp24/CHOp24 and p24-1 all play a role in Wg secretion (fig 1Biii). Thus, only Emp24/CHOp24 is found by both groups to be essential for Wg secretion. Although functional redundancy among p24 proteins could explain why the removal of a single p24 protein has a relatively weak phenotype, there is no simple explanation as to why the very similar assays used by the two groups do not lead to identical conclusions. These differences could be worked out by the exchange of reagents and protocols.Regardless of the discrepancies, the two studies provide an important step in our understanding of Wnt secretion by demonstrating that Wnts engage with specialized components of the secretory machinery as early as in the ER. It might be relevant that the anterograde function of p24 proteins is directed at glycophosphatidylinositol (GPI)-anchored proteins, which have been shown to partition in raft-like microdomains (Strating & Martens, 2009). It is conceivable that GPI-anchored proteins, as well as Wnts, gather in a subdomain of the ER where they could both interact with p24 proteins and set off along their specialized secretory pathways. Wnt targeting to specialized membrane domains could in principle be mediated by their lipid moieties (Bartscherer & Boutros, 2008; Port & Basler, 2010). However, the process might turn out to be more complex if it is confirmed that p24 proteins are also required for the secretion of non-acylated Wnts (for example, WntD), as suggested by Buechling et al. In any case, it will be interesting to determine the precise molecular mechanism underlying the functional interaction between Wnts and p24 proteins as it is likely to explain how Wnts are allowed to exit the ER and start their journey out of the cell.     

C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. Here we report that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on our observations, we propose that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion.     

Wnt signaling establishes anteroposterior neuronal polarity and requires retromer in C. elegans

Secreted Wnt proteins influence neural connectivity by regulating axon guidance, dendritic morphogenesis and synapse formation. We report a new role for Wnt and Frizzled proteins in establishing the anteroposterior polarity of the mechanosensory neurons ALM and PLM in C. elegans. Disruption of Wnt signaling leads to a complete inversion of ALM and PLM polarity: the anterior process adopts the length, branching pattern and synaptic properties of the wild-type posterior process, and vice versa. Different but overlapping sets of Wnt proteins regulate neuronal polarity in different body regions. Wnts act directly on PLM via the Frizzled LIN-17. In addition, we show that they are needed for axon branching and anteriorly directed axon growth. We also find that the retromer, a conserved protein complex that mediates transcytosis and endosome-to-Golgi protein trafficking, plays a key role in Wnt signaling. Deletion mutations of retromer subunits cause ALM and PLM polarity, and other Wnt-related defects. We show that retromer protein VPS-35 is required in Wnt-expressing cells and propose that retromer activity is needed to generate a fully active Wnt signal.