InteractiveFly: GeneBrief

Vacuolar protein sorting 35: Biological Overview | References

BIOLOGICAL OVERVIEW

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described in this study, Crb was determined to be an interaction partner and cargo of the retromer complex (See Retromer-mediated sorting). Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals. Loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. These data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. A novel function is also shown for retromer in maintaining epithelial cell polarity (Pocha, 2011).

This study aimed to identify factors that interact with the cytoplasmic domain of the type I transmembrane protein Crumbs (Crb) and are involved in its trafficking. A strategy was devised to present the Crb cytoplasmic tail on liposomes, a method uniquely suited to recruit and identify coats, because it mimics the native configuration of a receptor tail at the membrane/cytosol interface (Pocha, 2011).

Proteoliposomes have been used successfully to identify coat complexes and their accessory proteins; however, these studies were restricted to short, chemically synthesized peptides, which severely limited the length of the cytoplasmic tail. To overcome this, this study redesigned the recruitment assay enabling the use of tails expressed and purified from E. coli. A bacterial expression plasmid was designed containing an N-terminal tandem affinity tag followed by a tobacco etch virus (TEV) protease cleavage site and a single cysteine for the chemical coupling to liposomes, to which the cytoplasmic tail of mouse Crb2 (amino acids R1246 to I1282) was fused (Pocha, 2011).

Because the levels of many transmembrane proteins are regulated by sorting decisions in the early (sorting) endosome, phosphatidylinositol 3-phosphate, the predominant inositol phospholipid of early endosomes, was incorporated into proteoliposomes to selectively enrich endosomal trafficking proteins. These proteoliposomes were used for recruiting cytosolic coat components and other interactors from brain extract], followed by protein identification by tandem mass spectrometry (MS/MS). Crb2 was chosen, because it is the predominant Crb gene expressed in the vertebrate brain. Importantly, the tails of all Crb proteins are highly conserved, suggesting that their trafficking mechanisms may also be conserved. Mass spectroscopic analysis confirmed that large amounts of Crb2 (∼600 MS2 spectra) were coupled onto the liposomes. The most abundant protein isolated (as determined by MS2 spectra) with an established role in the recognition and trafficking of transmembrane cargoes was the retromer subunit Vps35. In addition, Vps26B was identified. Western blotting confirmed the presence of Vps35 in our Crb2 recruitment reactions and showed it to be highly enriched relative to two independent controls (Pocha, 2011).

The mammalian retromer is composed of a cargo recognition subcomplex containing Vps35, Vps26, and Vps29 and a membrane interacting subcomplex consisting of SNX1/SNX2 and SNX5/SNX6 heterodimers. Because both Vps35 and Vps26 are crucial for cargo recognition and binding, the recruitment data suggest that Crb2 is a retromer cargo (Pocha, 2011).

To probe the hypothesis that Crb is a retromer cargo, internalization assays were performed by overexpressing Flag-hCrb2 in HeLa cells and analyzing the uptake of anti-Flag antibodies, visualizing compartments through which Crb2 traffics. Previous studies using the classical retromer cargo, the cation-independent mannose-6-phosphate receptor (ciMPR), have shown that retromer subunits and cargo decorate tubules that emanate from endosomes and travel toward the trans-Golgi network (TGN). This study observed colocalization of Crb2 with Vps35 on intracellular vesicles and tubules as well as an overlap with ciMPR- and galactosyltransferase (GalT) label. These data suggest that in HeLa cells, Crb2 travels in retromer-decorated tubules and can traffic via the TGN. However, it should be noted that it does not accumulate there like other retromer cargoes (e.g., ciMPR). Instead, Crb2 appears to undergo rapid transport back to the plasma membrane. RNA interference (RNAi) suppression of Vps35 in HeLa cells displays enhanced localization of Crb2 in lysosomal structures positive for Lamp-I, a phenotype described previously for other retromer cargoes. These data are all in line with Crb being a potential retromer cargo (Pocha, 2011).

To study the functional interaction between Crb and retromer in Drosophila, a previously generated null allele of Vps35, Vps35^MH20 was used. As a result of strong maternal contribution, animals homozygous for this allele reach the third larval instar, allowing analysis of Crb in homozygous mutants. Because retromer is required for the retrieval of receptors from endosomes and thus the prevention of their lysosomal degradation, total Crb levels were analyzed and found to be reduced in Vps35^MH20 heterozygote third-instar larvae compared to stage-matched wild-type (WT) larvae and dramatically reduced in Vps35^MH20 homozygotes. Analysis of the mRNA levels of Crb showed that loss of Vps35 has very little effect on crb transcripts, suggesting that the dramatic reduction in Crb protein that was seem is due to posttranscriptional regulation of Crb by Vps35 (Pocha, 2011).

This led to an investigation of Crb at a cellular level. For this, two different epithelia, wing discs of third-instar larvae and the follicle epithelium, were chosen. Clones of Vps35^MH20 mutant cells in wing disc epithelia, labeled with GFP using the mosaic analysis with a repressible cell marker (MARCM) system, were induced by heat shock-Flp at early larval stages. Crb localizes to the subapical region of wing disc epithelial cells. In agreement with results from western blot analysis, Crb staining is decreased in Vps35^MH20 clones. Quantification of the fluorescence intensity in the clone and in surrounding tissues revealed that there is an ∼50% reduction in Crb signal within Vps35^MH20 clones. The wing discs of Vps35^MH20 homozygous animals are small and show variable morphological defects, presumably as a result of defective Wingless secretion. Analysis of Crb localization (by immunofluorescence) and protein levels (by western blotting) in Vps35^MH20 hetero- and homozygous wing discs corroborated the data that were obtained using Vps35^MH20 clones and larval lysate, respectively (Pocha, 2011).

The stability of the cargo-selective retromer subcomplex is dependent on the presence of all of its components [8 and 16]. To show that the loss of Crb is due to loss of retromer function rather than just the loss of Vps35, the effect was compared of Vps26 and Vps35 knockdown in the posterior compartment of the wing disc using engrailed-Gal4 to drive UAS-Vps26RNAi and UAS-Vps35RNAi. Hedgehog expression, which is unperturbed by loss of retromer, served to label the posterior compartment. Expression of either RNAi construct resulted in a clear reduction of Crb staining in the posterior compartment (∼50% reduction in fluorescence). Expression of engrailed-Gal4 alone had no effect on Crb. From these data, it is concluded that the retromer cargo recognition subcomplex is required for the maintenance of Crb levels (Pocha, 2011).

To further analyze the relation between Crb and retromer, the follicular epithelium, which surrounds the germline cysts of the Drosophila ovary, was examined. Previous work has identified key roles for Crb in polarization of the follicular epithelium. Crb localizes to the entire apical membrane of the follicle epithelial cells, with very little detectable in the cytoplasm. Vps35^MH20 clones show strong reduction in Crb staining and protein loss from the apical membrane. Interestingly, although Crb staining at the apical membrane is strongly reduced, it is not detected at increased levels within the cytoplasm, suggesting that Crb is not merely mislocalized but reduced at the protein level, as shown in larvae. The cytoplasmic domain of Crb organizes an apical, membrane-associated protein complex by recruiting the scaffolding proteins Stardust (Sdt), DPATJ, and DLin-7. Therefore, the apical localization of Sdt in the follicular epithelium was assessed, and at was found to be heavily reduced in Vps35^MH20 clones. Probing whole larval lysates from third-instar WT and Vps35^MH20 hetero- and homozygotes for Sdt confirmed that at the protein level, like Crb, Sdt shows a dose dependence on Vps35. Thus, retromer function in maintaining Crb levels and function is conserved between wing and follicle epithelia (Pocha, 2011).

Interestingly, in some Vps35^MH20 clones, the strict monolayer structure of the epithelium is disrupted and the tissue appears multilayered, an indication of polarity defects and characteristic of loss of Crb at early stages of follicle development, whereas loss at later stages results only in the mislocalization of other polarity proteins, without affecting tissue integrity. Multilayering was observed in 19% of Vps35^MH20 clones in follicles between stages 7 and 10 and did not appear to be dependent on clone size or position. Given that various links between Crb and Notch have been reported, tests were performed to see whether the multilayering phenotype observed in the follicle epithelium upon loss of Vps35^MH20 could be the result of defective Notch signaling. The expression of Notch and Hindsight, a transcription factor downstream of Notch signaling that represses proliferation in the follicle epithelium, were examined in Vps35^MH20 mutant clones. Both showed wild-type expression, suggesting that Notch signaling is not affected by loss of retromer, similar to previous findings in the wing disc (Pocha, 2011).

To test whether the loss of Crb in retromer mutants is due to missorting of Crb to the lysosome, follicles harboring Vps35^MH20 clones were incubated in leupeptin, a potent inhibitor of lysosomal proteases. After a 3 hr incubation, a dramatic accumulation of Crb was observed in punctae within the cytoplasm of Vps35^MH20 cells, a phenomenon that was not seen in WT tissue or in follicles containing Vps35^MH20 clones that were incubated in control medium lacking leupeptin. Additionally, colocalization of these intracellular Crb punctae with LysoTracker was observed. Together with the reduction of Crb protein levels and constant crb mRNA levels in Vps35^MH20 larvae and tissue, these data strongly suggest that retromer ablation leads to lysosomal degradation of Crb, as observed for other retromer cargoes (Pocha, 2011).

To test whether retromer functions after endocytosis of Crb, internalization of Crb from the plasma membrane was blocked by expression of a dominant-negative construct of shibire (dynamin) or by incubating follicles in dynasore, a dynamin inhibitor. In Vps35^MH20 clones, this resulted in the accumulation of Crb at the plasma membrane, confirming that retromer is indeed transporting Crb after internalization from the plasma membrane (Pocha, 2011).

Crb is required, together with atypical protein kinase C (aPKC), to restrict Bazooka/Par3 to the zonula adherens, an adhesion belt at the apex of epithelial cells, in the follicle epithelium, and in photoreceptor cells, thus excluding it from the apical membrane and specifying the border between apical and lateral domains. In previous studies, it was shown that the localization of aPKC and Par6 was dependent on Crb. To test whether loss of retromer phenocopies the loss of Crb, aPKC and Par6 localization were examined in follicles containing Vps35^MH20 clones. Indeed, the level of both proteins is reduced at the apical surface in Vps35^MH20 clones. Interestingly, unlike Crb and the Crb complex member Sdt, Par6 and aPKC protein levels are not reduced in Vps35^MH20 mutant larvae. Therefore, it is likely that the loss of Par6 and aPKC from the apical membrane of Vps35^MH20 clones in the follicle epithelium is due to loss of cell polarity in the absence of Crb rather than loss of the proteins themselves (Pocha, 2011).

To test this, Crb was overexpressed in Vps35^MH20 clones. Because overexpression of Crb causes defects in epithelial cell polarity, Crb overexpression was induced using GABFc204 Gal4, a follicle epithelium-specific driver that starts expression late in follicle development (stage 8). Thereby, it was possible to rescue the apical localization of Par6 and Sdt. This rescue did not appear to be dependent on clone size or location. From these data, it is concluded that the loss of polarity observed in retromer mutant clones is the direct result of loss of Crb (Pocha, 2011).

The identification of Crb as a retromer cargo confirms the hypothesis that one crucial step in the regulation of Crb occurs at the early (sorting) endosome and, importantly, fills a gap in the current understanding of Crb trafficking. Previous reports showed that transport of Crb to the plasma membrane is reliant on Rab11, the exocyst and Cdc42 in Drosophila embryonic epithelia. Internalization of Crb from the plasma membrane into endosomes is mediated by the syntaxin Avalanche and Rab5. This study has shown that retromer is responsible for sorting Crb away from the degradative pathway and into a recycling one, thus allowing a high level of control over the amount of cellular Crb, previously shown to be vital for maintaining epithelial polarity and integrity, as demonstrated by numerous loss- and gain-of-function studies. Interestingly, retromer was previously shown to play a role in the apical delivery of the polymeric immunoglobulin receptor (pIgR) in Madin-Darby canine kidney cells. However, as for Crb, it remains unclear whether this transport occurs via the TGN, via recycling endosomes, or through alternative pathways. The exact trafficking itinerary of Crb following recycling by retromer remains unclear and may depend upon the purpose of Crb recycling (Pocha, 2011).

Which function of Crb is the prime target of retromer-driven retrieval? Is this a Crb level-sensing mechanism, in which retromer regulates the amount of protein at the plasma membrane, which is crucial for cell homeostasis? To date, all known functions of Crb require an intact Crb complex. By controlling the recycling of Crb and thereby its level at the plasma membrane, retromer could define the amount of Crb available for complex formation. Alternatively, it is tempting to speculate that Crb, much like Wntless (Wls), acts as a transport receptor and that apical delivery of its (yet to be identified) ligand or many ligands is the main purpose of its recycling to the TGN. These are fascinating hypotheses that will be the focus of future research (Pocha, 2011).

Retromer regulates apical-basal polarity through recycling Crumbs

Epithelial cells are characterized by an 'apical–basal' polarization. The transmembrane protein Crumbs (Crb) is an essential apical determinant which confers apical membrane identity. Previous studies indicated that Crb did not constantly reside on the apical membrane, but was actively recycled. However, the cellular mechanism(s) underlying this process was unclear. This study shows that in Drosophila, retromer, which was a retrograde complex recycling certain transmembrane proteins from endosomes to trans-Golgi network (TGN), regulates Crb in epithelial cells. In the absence of retromer, Crb was mis-targeted into lysosomes and degraded, causing a disruption of the apical–basal polarity. It was further shown that Crb co-localizes and interacts with retromer, suggesting that retromer regulated the retrograde recycling of Crb. These data uncover the role of retromer in regulating apical–basal polarity in epithelial cells and identify retromer as a novel regulator of Crb recycling (Zhou, 2011).

It is important to understand how apical–basal polarity is regulated during development. This study has uncovered a role of retromer in regulating apical–basal polarity in epithelial cells. The analysis showed that this regulation was achieved through stabilizing the apical determinant Crb, which acted as a novel transmembrane cargo of retromer. It is proposed that retromer regulates apical–basal polarity by mediating the retrograde transportation of Crb from endosomes to TGN. This study analysed the regulation of Crb in Drosophila embryos. Taken together with the studies of Pocha (2011), it is proposed that the recycling of Crb by retromer is a general regulation mechanism in all major epithelial cell types in Drosophila (Zhou, 2011).

A previous study suggested a role of recycling endosomes in regulating Crb recycling (Roeth, 2009). A question of interest lies in the relationship between recycling endosomes- and retromer-mediated Crb recycling. Retromer mediates the transportation from early/late endosomes to TGN. It is as yet unknown whether recycling endosomes- and retromer-mediated recycling are two routes in parallel or whether recycling endosomes serve as a stop during retromer-mediated early/late endosomes to TGN transportation. One intriguing observation is that in Rab11 defective embryos (expressing dominant negative protein), the Crb defect mainly occurred in the ventral ectoderm but much less in the dorsal ectoderm (Roeth, 2009). However, at the same stage, this study found that the retromer mutant embryos had a wider range of Crb defect, which occurred not only in the ventral ectoderm but also largely in the dorsal ectoderm and head epithelium. The weaker Crb defect in Rab11 defective embryos might result from the usage of dominant negative instead of null mutants. However, another interesting possibility is that the retromer-mediated recycling is a general mechanism of Crb recycling in all epithelial cells and the recycling endosome-mediated recycling contributes additionally to Crb recycling in the ventral ectoderm, where a high rate of Crb recycling may occur. In other words, in the ventral ectoderm, the recycling endosome route and the retromer route might work in parallel to recycle Crb. Further work is needed to explore the relationship of recycling endosome- and retromer-mediated recycling (Zhou, 2011).

The retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration

Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, a portion of activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. This study shows that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. It is proposed that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity (Wang, 2014).

Rhodopsins are G protein-coupled receptors that function as light sensors in photoreceptors (PRs), and defective trafficking of rhodopsins often leads to PR degeneration in humans and flies. Because vision is not required for animal survival, previous studies in Drosophila mostly focused on viable mutations that specifically impair PR function. However, it is likely that numerous additional players encoded by essential genes have remained unidentified. This study performed an eye-specific mosaic genetic screen and found that loss of subunits of the retromer causes light-induced PR degeneration (Wang, 2014).

The retromer, a hetero-multimeric protein complex, retrieves specific proteins from endosomes, thereby preventing the degradation of these proteins in lysosomes (Seaman, 2012; Cullen, 2012; Bonifacino, 2008). The retromer is composed of Vps26, Vps29, Vps35, and certain sorting nexins (Snx). Most subunits are evolutionarily conserved. Mutations in some subunits (Vps35 or Snx3) of the retromer have been shown to decrease the abundance of Wntless (Wls) and impair the secretion of Wingless (Wg), a ligand of the Wnt signaling pathway. Wls is a transmembrane protein that binds to Wg and is required for Wg secretion. Impaired retromer function leads to excessive degradation of Wls in lysosomes, severely reducing Wg secretion and signaling. The retromer has also been shown to maintain the levels of Crumbs, a transmembrane protein required for maintaining the apicobasal polarity in some tissues (Zhou, 2011; Pocha, 2011). Mutations in human VPS35 have been shown to cause a dominant inherited form of Parkinson's disease (PD). However, the retromer has not been implicated in neurons of the visual system in flies or vertebrates (Wang, 2014).

The Drosophila compound eye comprises ~800 hexagonal units named ommatidia. Each ommatidium contains eight PRs (R1-R8) that express rhodopsin proteins. Rhodopsin 1 (Rh1) is the major rhodopsin that is primarily expressed in R1-R6. It is synthesized and folded in the endoplasmic reticulum (ER) and transported to rhabdomeres, the stacked membranous structures in PRs, via the secretory pathway. The proper transport of Rh1 from ER to rhabdomeres requires molecular chaperones and Rab GTPases. Binding of opsins to chromophores as well as protein glycosylation and deglycosylation are essential for Rh1 folding and maturation. Mutations in genes involved in Rh1 synthesis, folding, or transport can result in defective PR development or PR degeneration (Wang, 2014).

Phototransduction in the PRs relies on the activation of Rh1 by photons. Active Rh1 (metarhodopsin, M*) activates phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG). DAG or its metabolites can activate Transient Receptor Potential (TRP) and TRP-like cation channels that lead to depolarization of the PRs. Similar to fly PRs, the vertebrate intrinsically photosensitive retinal ganglion cells (ipRGCs) use melanopsin (a homolog of fly Rh1) as the light sensor and requires PLC and TRPC channels for activation. ipRGCs project their axons to specific brain areas to control circadian rhythms or pupillary light reflex (Wang, 2014).

Tight regulation of Rh1 activity upon light exposure is required to maintain the integrity of PR cells. M* can be converted into its inactive form upon exposure to orange light. In addition, a significant portion of active Rh1 can be endocytosed and degraded in lysosomes. Mutations that abolish Rh1 deactivation or impair the endolysosomal system can cause PR degeneration due to Rh1 accumulation. However, it is unknown whether Rh1 can be retrieved from the endolysosomal compartments and whether impaired Rh1 recycling leads to PR degeneration (Wang, 2014).

This study shows that loss of the fly Vps26 or Vps35 causes early-onset PR degeneration. Retromer subunits are expressed in PRs in flies and melanopsin-expressing ipRGCs in the mouse retina. In fly mutant PRs, the numbers of late endosomes and lysosomes are significantly elevated. The PR degenerative phenotypes are dependent on exposure to light and the presence of Rh1. The data indicate that the fly retromer recycles Rh1, preventing Rh1 retention in the PR cell bodies and shunting Rh1 from being degraded in lysosomes, thereby promoting Rh1 redelivery to rhabdomeres. In summary, the retromer recycles Rh1, prevents an overload of the endolysosomal pathway, and salvages a substantial fraction of Rh1 from degradation in flies. It may also play a similar role in ipRGCs in the retina of vertebrates (Wang, 2014).

Although the loss of the retromer does not obviously affect eye development, mutations in Vps26 or Vps35 genes lead to strong light-dependent PR degeneration. The demise of Vps²⁶¹ and Vps35^MH20 PRs is associated with a significant increase in the number of late endosomes and lysosomes upon light exposure, showing that the endolysosomal pathway is strongly affected when Rh1 recycling by the retromer is impaired. Indeed, Rh1 accumulates in late endosomes or lysosomes in the mutant PRs. Although Rh1 can be degraded and the function of Vps²⁶¹ mutant PRs is not abolished upon short light exposure, chronic exposure to light is detrimental to Vps²⁶¹ mutants because persistent Rh1 accumulation in the endolysosomal pathway is toxic to PR cells. Hence, reducing Rh1 endocytosis or Rh1 levels in the rhabdomeres suppresses PR degeneration upon prolonged light exposure. Interestingly, increasing Vps35 or Vps26 in mutants that show PR degeneration due to Rh1 accumulation in the endolysosomal compartments suppresses the degenerative phenotypes. In summary, the retromer is required to retrieve Rh1 from endosomes to maintain PR function and integrity (Wang, 2014).

How does Rh1 internalization affect the endolysosomal pathway in retromer mutants? One possibility is that lysosomes in the mutants are unable to cope with the increased levels of internalized Rh1 over time as Rh1 is one of the most abundant proteins in PRs. This in turn triggers an increase in the number of lysosomes, the accumulation of aberrant lysosomes, and the accumulation of endolysosomal intermediates, including late endosomes. Alternatively, loss of the retromer may increase the flux in the endolysosomal pathway, which overpowers the rate of endolysosomal maturation and leads to an adaptive response that eventually leads to the expansion of these compartments. Both pathways can lead to an apparent accumulation of Rh1 in the cell body when stained and analyzed by fluorescence microscopy (Wang, 2014).

Defective regulation of Rh1 can lead to the demise of PRs via apoptosis. Does apoptosis play a critical role in the PR degeneration in retromer mutants? It is argued that this is not the case based on the following observations. First, the retromer mutants exhibit progressive PR degeneration over a 3-wk period, whereas apoptosis typically occurs within hours. Second, mutants that lead to PR loss via apoptosis lose most PRs within ommatidia by the engulfment of surrounding glial cells. However, the degenerating Vps26 and Vps35 mutant PRs are not removed, although their morphology is very severely disrupted. Indeed, they can still be identified in 3-wk-old flies, indicating a lack of engulfment by surrounding cells. Third, overexpressing p35, a pan-caspase inhibitor of apoptosis, fails to suppress the PR degeneration in Vps26 mutants upon light exposure. Indeed, Rh1 accumulation in different subcellular compartments triggers different cellular responses, which leads to PR degeneration of varying severity. Accumulation of Rh1 in the endolysosomal pathway seems particularly toxic to PRs but often does not cause the removal of PRs for many weeks (Wang, 2014).

Loss of Wg affects eye development, whereas loss of Crumbs leads to short and/or fused rhabdomeres. Although the retromer can recycle Wls or Crumbs in some fly tissues, Vps²⁶¹ or Vps35MH20 mutants do not exhibit obvious eye developmental defects. It is therefore very likely that the composition of the eye retromer is different from the wing retromer. Indeed, loss of Snx3 does not cause obvious degenerative phenotypes when compared to loss of Vps26 or Vps35, yet these proteins are all essential for the recycling of Wls and wing development (Wang, 2014).

Many players required for phototransduction in Drosophila are conserved in a phototransduction cascade in vertebrate ipRGCs. These include melanopsin, PLC, and the TRP channels. This phototransduction pathway plays a role in photoentrainment of circadian rhythms and the control of the pupillary light reflex. vps35 is expressed in 92% of the melanopsin-expressing RGCs in the mouse retina. This may be an underestimate due to technical difficulties with β-GAL immunostaining in mouse tissues. In addition, the human Vps26 proteins are able to substitute for the function of the fly homolog in PRs. Because vertebrate melanopsin has very similar photochemical properties to fly rhodopsin, the retromer may play a conserved role in vertebrate ipRGCs. As deletion of vps35 leads to early embryonic lethality, a vps35 conditional KO in ipRGCs will need to be established to address its role in ipRGCs (Wang, 2014).

The retromer has been implicated in human neurodegenerative disease, including Alzheimer's disease (AD) and PD. In AD, a retromer deficiency has been proposed to affect subcellular distribution of β-secretase, which leads to increased amyloid-beta (Aβ) deposits and defective neuronal function. In PD, a missense mutation in ^VPS35 (D620N) has been shown to cause an autosomal dominant late onset form of the disease. The Vps35 D620N mutant protein appears to function as a dominant negative, and Vps35 and LRRK2 (Leucine-Rich Repeat Kinase 2) have been shown to interact. It will therefore be interesting to assess if these mutants affect ipRGCs as PD patients often have sleep issues (Wang, 2014).

Retromer promotes immune quiescence by suppressing Spatzle-Toll pathway in Drosophila

The Toll and Toll-like receptor signaling pathways are evolutionarily conserved pathways that regulate innate immunity in insects and mammals. While efforts have been made to clarify the signal transduction events that occur during infection, much less is known about the components that maintain immune quiescence. This study shows that retromer, an intracellular protein complex known for regulating vesicle trafficking, functions in modulating the Toll pathway in Drosophila melanogaster. In mutant animals lacking retromer function, the Toll pathway but not JAK-STAT or IMD pathway is activated, triggering both cellular and humoral responses. Genetic epistasis and clonal analysis suggest that retromer regulates a component that acts upstream of Toll. The data further show that in the mutant the Toll ligand Spatzle has a processing pattern similar to that of after infection. Together, the results suggest a novel function of retromer in regulating Toll pathway and innate immunity at a step that modulates ligand processing or activity (Zhou, 2014).

Based on previous knowledge that retromer regulates trafficking of transmembrane proteins, one can envisage that retromer normally may regulate the Toll pathway in one of the four following ways: (1) in Toll-responsive cells to transport the Toll-Spz complex for destruction; (2) in Spz secreting cells to suppress the release of active Spz; (3) in certain cells to assist the clearance of active Spz in the hemolymph; or (4) in certain cells to repress Spz through an indirect effect of other yet to be indentified components. In the Vps35 mutant clonal cells in fat bodies no increased Dorsal nuclear localization was observed, indicating that retromer is not simply regulating the Toll pathway cell-autonomously. Epistasis analysis suggests that retromer acts between Toll and SPE. Even though Spz is the only known component in between, there can be many other proteins that regulate the processing, maturation, trafficking or degradation of Spz in normal flies in order to restrict the activity of the Toll signaling pathway prior to infections. The full mechanism of Spz maturation is not yet unveiled and the retromer function in this process requires further investigation. Although the possibility cannot be excluded that retromer has an indirect effect on Spz, a function of retromer in anti-release and/or clearance of active Spz is favored. Retromer has been shown to target transmembrane proteins. It will be intriguing to identify the transmembrane target of retromer in the context of regulating Spz and explore the mechanism of how this transmembrane target suppresses the release and/or assists the clearance of active Spz. Equally important is to examine whether modulating retromer-dependent Spz maturation is part of the activation mechanism of the Toll pathway during infections (Zhou, 2014).

This study has identified a role of retromer in negatively regulating the Toll pathway to maintain immune quiescence. In the absence of infection, the loss of retromer activity alone is capable of activating the Toll pathway and launching both the cellular and humoral immune responses. Furthermore, genetic epistasis and mosaic analysis suggest that retromer acts upstream of Toll and downstream of Spatzle-Processing Enzyme (SPE), and a retromer function was uncovered in restricting the processing/maturation of Spz. In summary, retromer plays a critical role in suppressing the auto-activation of the innate immune system through Spz in the Toll pathway in Drosophila (Zhou, 2014).

The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating notch receptor trafficking

The correct establishment and maintenance of unidirectional Notch signaling are critical for the homeostasis of various stem cell lineages. However, the molecular mechanisms that prevent cell-autonomous ectopic Notch signaling activation and deleterious cell fate decisions remain unclear. This study shows that the retromer complex (see Drosophila Vps35) directly and specifically regulates Notch receptor retrograde trafficking in Drosophila neuroblast lineages to ensure the unidirectional Notch signaling from neural progenitors to neuroblasts. Notch polyubiquitination mediated by E3 ubiquitin ligase Itch/Su(dx) is inherently inefficient within neural progenitors, relying on retromer-mediated trafficking to avoid aberrant endosomal accumulation of Notch and cell-autonomous signaling activation. Upon retromer dysfunction, hypo-ubiquitinated Notch accumulates in Rab7(+) enlarged endosomes, where it is ectopically processed and activated in a ligand-dependent manner, causing progenitor-originated tumorigenesis. These results therefore unveil a safeguard mechanism whereby retromer retrieves potentially harmful Notch receptors in a timely manner to prevent aberrant Notch activation-induced neural progenitor dedifferentiation and brain tumor formation (Li, 2018).

Unidirectional Notch signaling is a widely used strategy for initiating and maintaining binary cell fates. However, the molecular mechanisms establishing the unidirectionality of Notch signaling in stem cell lineages remain unclear. This study shows that, while asymmetric partition of Numb leads to a biased internalization of the Notch receptor and hence asymmetric dampening of Notch signaling in neural progenitors, it meanwhile poses a high risk of non-canonical endosomal activation of Notch. The retromer complex was found to be the key protein trafficking machinery that resolves this crisis through a timely retrieval of the Notch receptor from its endosomal activation compartments. Upon retromer dysfunction, neural progenitors dedifferentiate into neural stem cell-like status and result in the formation of transplantable tumors. Therefore, retromer acts as a tumor suppressor in Drosophila larval brains. Importantly, mammalian Vps35 physically interacts with Notch, colocalizes with Notch in neural progenitors, and its neuroblast-lineage-specific expression fully rescues neural progenitor-derived brain tumor phenotype in vps35 mutants. Thus, the brain tumor suppressor function of retromer is likely to be conserved in mammals. Intriguingly, downregulation of the retromer complex components has been reported in various human cancers, including glioblastoma. These studies thus provide a new mechanistic link between the retromer complex and carcinogenesis (Li, 2018).

Why the E3 ubiquitin ligase system promoting Notch receptor polyubiquitination and degradation is inherently inefficient in neuroblast lineages? It is speculated that Notch is probably not the only substrate of Su(dx) and Ndfip in neuroblasts or neural progenitors. Therefore, high levels and/or activity of this E3 ubiquitin ligase system above certain threshold may potentially cause imbalanced homeostasis of its critical substrates and hence perturbed neuroblast lineages. Indeed, co-overexpression of Su(dx) and Ndfip led to drastically reduced number of neuroblast lineages and severe tissue atrophy. In this case, a relatively general yet inefficient ubiquitination-degradation system coupled with a highly efficient and selective cargo retrieving system provides a customized regulation of the Notch receptor, ensuring sufficient dampening of Notch signaling in neural progenitors without devastating side effects (Li, 2018).

Intriguingly, previous studies posited that retromer dysfunction causes increased levels of APP (β-amyloid precursor protein) to reside in the endosomes for longer duration than normal, resulting in accelerated processing of APP into amyloid-β, a neurotoxic fragment implicated AD pathogenesis. Furthermore, retromer maintains the integrity of photoreceptors by avoiding persistent accumulation of rhodopsin in endolysosomal compartments that stresses photoreceptors and causes their degeneration. Taken together with this study, these findings indicate that retromer serves as bomb squad to retrieve and disarm harmful or toxic protein fragments from endosomes in a timely manner and thereby safeguard the integrity and fitness of the neuronal lineages (Li, 2018).

How is the Notch receptor ectopically activated in retromer mutants? The idea is favored that Notch is activated in MVBs in a ligand-dependent, cell-autonomous manner, distinct from the majority of non-canonical Notch activation mechanisms. Most of the endosomal Notch activation events identified before, including ectopic Notch signaling activation in ESCRT mutants, BLOS2 mutants, or Rme8 and Vps26 double knockdown background, as well as Hif-alpha-dependent activation of Notch signaling implicated in crystal cell maintenance and survival, are all ligand-independent. It has been proposed that the proteases within the acidifying environment of MVB lumen are sufficient to remove the extracellular domain of Notch, leading to the S3 cleavage of Notch at the limiting membrane. Strongly supporting this notion, blocking the entry of Notch into the ESCRT pathway but not ligand inactivation potently inhibited ectopic Notch activation induced by ESCRT mutations. In sharp contrast to these previously-revealed mechanisms, attenuating ligand activity but not preventing Notch from entering the ESCRT pathway effectively rescues Notch overactivation phenotype caused by retromer dysfunction. Then how Notch signaling is ectopically activated in a ligand-dependent manner in retromer mutants? It is speculated that, upon retromer dysfunction, both Notch and Delta are entrapped in MVBs, where Notch and Delta are presented by limiting membrane and intravesicular membrane respectively and result in ligand-dependent Notch processing and activation, resembling the scenario presented for ligand-dependent Notch signaling activation in Sara endosome. The detailed regulatory mechanisms underlying Notch overactivation in retromer mutants warrants future investigation (Li, 2018).

The ability of vps35 mutant neoplastic neuroblasts to metastasize upon transplantation is intriguing. Metastasis of brain tumor cells derived from neuroblast lineages has never been observed in the developing fly larval brains, likely because the limited time window of fly larval development precludes tumor progression and metastasis. Transplantation assay, however, provides the ectopic microenvironment and allows cancer progression in a much longer time scale (months, or even years upon retransplantation). Importantly, mutations that caused metastasis of fly brain tumor cells upon transplantation have also been implicated in various human cancers. Future studies on the transcriptional profiling of the distal metastatic colonies and stepwise characterization of this long-range metastatic process promise to provide fresh mechanistic insights into the enormously complex process of cancer metastasis (Li, 2018).

Rab9 and retromer regulate retrograde trafficking of luminal protein required for epithelial tube length control

Apical extracellular matrix filling the lumen controls the morphology and geometry of epithelial tubes during development, yet the regulation of luminal protein composition and its role in tube morphogenesis are not well understood. This study shows that an endosomal-retrieval machinery consisting of Rab9, retromer and actin nucleator WASH (Wiskott-Aldrich Syndrome Protein and SCAR Homolog) regulates selective recycling of the luminal protein Serpentine in the Drosophila trachea. Secreted Serpentine is endocytosed and sorted into the late endosome. Vps35, WASH and actin filaments differentially localize at the Rab9-enriched subdomains of the endosomal membrane, where Serpentine containing vesicles bud off. In Rab9, Vps35 and WASH mutants, Serpentine was secreted normally into the tracheal lumen, but the luminal quantities were depleted at later stages, resulting in excessively elongated tubes. In contrast, secretion of many luminal proteins was unaffected, suggesting that retrograde trafficking of a specific class of luminal proteins is a pivotal rate-limiting mechanism for continuous tube length regulation (Dong, 2013).

Maturation of the tracheal tube involves diameter expansion triggered by a cell-intrinsic programme of luminal material secretion and apical membrane growth, and the tube’s longitudinal growth is negatively regulated by the presumed conversion of chitin to chitosan through deacetylation by chitin deacetylase. Each step involves temporally regulated apical secretion, but the regulatory mechanism underlying this selective cargo secretion has not been understood. This study identified Serp as a novel cargo for the Rab9-mediated retrograde recycling pathway. Using time-lapse imaging, it was demonstrated that Serp in the endocytic compartment is sorted out by the budding of actin- and WASH-enriched portions of the LEs. The similarity of the Rab9, Vps35, WASH and Serp tracheal tube-length phenotypes, and the colocalization of those in the Serp-containing endosomal-budding sites in S2 cells, suggest that Serp is one of the major cargoes of the retrograde trafficking mediated by Rab9, Vps35 and WASH. It was also found that association of WASH and actin filaments with retrograde cargo is Rab9 and Vps35 independent. Modular organization of machineries for cargo retrieval (retromer) and membrane remodelling (WASH) may allow flexibility in endosomal sorting systems. This work connects retrograde trafficking to luminal retention of the key rate-limiting protein required to restrict the tracheal tube length to the proper level and has identified a new regulatory process for tracheal tube size (Dong, 2013).

Previous works in mammalian cells revealed that recruitment of the cargo recognition subcomplex of retromer (Vps35, Vps29 and Vps26) to the endosomal membrane requires Rab5 and Rab7. Rab7 has been shown to bind directly to Vps35/29/26 complex, but no evidence is available for association of Rab5 or Rab9 with those components. This analysis of the Drosophila counterparts have shown that conversion of early to LE is accompanied by gradual reduction of relative abundance of GFP-Rab5 to RFP-Rab9 signals. Throughout the process, Vps35-mRFP is present as distinct puncta associated with endosomes. The following order of Rab9-Vps35 assembly is proposed. In early endosome, Rab9 recruits Vps35 to the endosomal membrane through physical binding. This process requires Rab5. After initial recruitment of Vps35, endosomal maturation proceeds with exchange of Rab5 with Rab7, which promotes retromer assembly and cargo concentration, followed by membrane scission induced by WASH and F-actin. Together, these results indicate that Rab5, Rab9 and Rab7 acts on retromer assembly at different stages of endosomal maturation and tubular membrane formation, where Serp and other cargos are retrieved for retrograde pathway (Dong, 2013).

In contrast to the tracheal diameter expansion, which is triggered by a cell autonomous burst of exocytosis, axial elongation is a slow and continuous process spanning stage 14–16. Therefore, any mechanism that restricts elongation is expected to be active throughout the elongation process. Previous studies showed that the two chitin deacetylases Serp and Verm act additively in tube length control and the overexpression of either one causes the same tube length defects, indicating that the amount of luminal chitin deacetylase must be precisely maintained. Furthermore, the expression of Serp mRNA in the trachea declines at stage 16, when luminal Serp protein is still abundant, implying that Serp synthesized in the previous stage must be effectively retained and reused. This study has shown that Serp is endocytosed and associates with GFP-Rab9. The requirement for Rab9 and Vps35 for the luminal retention of Serp suggests that the endocytosed Serp is sorted from LEs to the TGN for secretion into the lumen, and this recycling pathway actively maintains the steady-state level of Serp to optimize the tube elongation process. Moreover, the retrieval transport through the TGN might help promote the modification of the endocytosed Serp, to recover its activity or to bind to an adaptor for its subsequent polarized secretion (Dong, 2013).

In rab9, vps35 and wash mutants, Serp was mislocalized at the apical cortex of tracheal cells. This mislocalization may have been due to the trapping of Serp in endosomes marked with Rab7 or Rab11 which are apically localized in a Rab9-independent manner. Consistent with this idea, a recent report showed that the suppression of Vps35 expression in NLT cells and neurons causes the accumulation of its cargo protein β-secretase (BACE1) in endosomes. The second possibility is that the loss of Rab9 diverts the trafficking route of Serp from the endosomes to the cell surface, as observed in Rab9-deficient HeLa cells. In support of this idea, an enhancement of Serp accumulation at the surface of the epidermis was observed in rab9 mutants. The third possibility is that a defect in endocytosis arrested Serp at the apical membrane, because Vps35 has been shown to be involved in endocytosis in S2 cells. Further analysis will be required to understand the diverse influences of retrograde trafficking on endosomal dynamics (Dong, 2013).

The apical localization of Crb depends on the retromer complex, but it was unaffected in rab9 mutants. One possible explanation for this finding is that the alternative recycling pathway of Crb, which involves Rab11, compensates for the defect in retrograde trafficking in rab9 mutants. Another possibility is that Crb-retromer and Serp-retromer complexes use different trafficking pathways distinguished by sorting nexins, which organize endosomal membranes into distinct morphological and functional regions for transport to diverse destinations. Further study of the different sorting nexins should uncover the role of Rab9 in retromer cargo specificity (Dong, 2013).

The formin Dia and motor protein Myosin V (Didum) are required for the secretion of a number of markers, including 2A12 antigen, Pio and artificial ANF-GFP, into the tracheal lumen. On the other hand, the secretion of Serp and Verm and the cell-surface localization of Crb are normal in Dia mutants, indicating that a Dia-independent secretory pathway regulates these protein localizations in the trachea8. The spectrum of secreted protein localizations affected in the rab9 and vps35 mutants was nearly complementary to that of Dia, suggesting that the Dia-dependent and retrograde trafficking-dependent mechanisms are the two major apical secretory pathways in the trachea. Verm localization was not affected in the rab9 and vps35 mutants, suggesting that this putative chitin deacetylase behaves differently from Serp. The difference in cargo specificity for each pathway allows cells versatility in controlling the localization of different proteins according to distinct schedules, so that the secretory burst that triggers tube diameter expansion and the continuous recycling of chitin deacetylases in axial elongation are controlled separately. The results provide a molecular basis for the roles of distinct trafficking pathways in controlling tubule growth and geometry (Dong, 2013).

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. This study examined a role of Vps35, an essential retromer subunit, in Wnt signaling in Drosophila and mammalian cells. Compelling evidence is provided 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. These findings link Wls and retromer functions in the same conserved Wnt secretion pathway. It is proposed that retromer influences Wnt secretion by recycling Wntless from endosomes to the trans-Golgi network (TGN) (Belenkaya, 2008).

This study provided convincing evidence for mechanistic roles of retromer and Wls in Wnt secretion. The retromer complex is involved in Wnt secretion in both Drosophila and mammalian cells. Wls stability is regulated by Dynamin-mediated endocytosis, and retromer plays an essential role in maintaining Wls protein levels, possibly via the retrieval of Wls from endosomes to the Golgi. Together, these findings have linked retromer and Wls into the same conserved Wnt secretion pathway. It is proposed that retromer influences Wnt secretion by recycling Wntless from endosomes to the TGN (Belenkaya, 2008).

One main finding of this work is the demonstration of a retromer requirement for Wnt secretion. First, it was shown that Wg protein accumulated inside the Wg-producing cells, whereas Wg levels in the receiving cells were reduced in the absence of the retromer activity. Second, secretion of Wg, as well as of Wnt3a and Wnt5a proteins, is inhibited in cultured cells, in which retromer activity is depleted by RNAi. Finally, a role of retromer in controlling the levels of Wls protein, which is essential for Wnt secretion, is demonstrated (Belenkaya, 2008).

Previously, Coudreuse (2006) found that vps-35 mutant in C. elegans did not affect the level of the Wnt, EGL-20, within producing cells when examined using an EGL-20-GFP fusion protein. Coudreuse also failed to detect Wnt3a secretion defects in Vps35-depleted mammalian cells. These differences could be due to variations in experimental procedures. In the current experiment, mosaic clones were used to compare Wg accumulation in Vps35 mutant cells with surrounding wild-type cells, whereas Coudreuse used homozygous Vps35 mutant embryos to compare with wild-type embryos. Furthermore, Coudreuse used a relatively short time period (maximum 5 hr) to collect Wnt3a protein in Vps35-depleted cells, which perhaps was not long enough to detect a decrease in Wnt3a secretion. Finally, EGL-20-GFP and Wnt3a-proteinA chimeras used previously might be less dependent on retromer activity for secretion compared with the native forms of these proteins. In the current experiments, nontagged Wg, Wnt3a, and Wnt5a proteins were used to avoid this potential complication (Belenkaya, 2008).

Consistent with a role of retromer in Wg secretion, reduced Wg short-range and long-range signaling activities were observed in the absence of retromer activity in the wing discs. The data argue strongly that diminished levels of Wg protein contribute to the reduced short- and long-range signaling activities of Wg. In this regard, it is important to mention that the short-range signaling activity of EGL-20 was only mildly affected, while the long-range signaling of EGL-20 was fully impaired in C. elegans (Coudreuse, 2006). The distinct responses of target genes are attributed to differences in their sensitivity to extracellular Wnt protein levels. Indeed, it was noticed that while Sens is strikingly reduced, only relatively weak reduction of Dll expression is observed in the absence of retromer activity (Belenkaya, 2008).

Several models have been proposed for the function of retromer in Wnt-gradient formation. These models include roles of retromer in (1) Wnt maturation by directly interacting with other enzymes such as Porcupine and (2) facilitating the interaction of Wnt protein with lipoprotein particles (Coudreuse, 2006). Although these mechanisms of retromer's role in Wnt activity cannot be ruled out completely, in light of the data, it is argued strongly that the main function of retromer in Wnt signaling is to maintain Wls protein levels. It was shown that Wls is substantially reduced in the absence of retromer activity and it interacts with retromer in cells. In further support of this view, it was shown that overexpression of Wls can restore Wg secretion defects in Dvps35 depleted wing discs. Since Wls is likely to be required for the secretion of all of Wnt members (Banziger, 2006; Hausmann, 2007), it is suggested that retromer may be essential for the secretion of all Wnt proteins (Belenkaya, 2008).

One main issue related to Wls function is its subcellular distribution. While Bartscherer (2006). identified Wls at the plasma membrane of Drosophila and HEK293T cells, Banziger (2006) showed localization of Wls in the Golgi and in vesicles between the Golgi and the surface of mammalian cells. To clarify this issue, Wls distribution was carefully examined in wing discs and in HeLa cells. The results showed that Wls protein localizes on plasma membrane, TGN, and vesicles. Data from the biotinylation experiment, the extracellular staining of Wls, and colocalization with plasma membrane markers provide compelling evidence that some of the Wls protein is present on the plasma membrane. Importantly, the data revealed colocalization of Wls protein with early endosome markers including Rab5, EEA1, and Hrs. Thus, it is likely that Wls is trafficking from the TGN onto cell membrane and is then subsequently endocytosed from the cell surface. A previous study showed that Wls is able to bind Wnt (Banziger, 2006). Therefore, it is likely that Wls acts as a Wnt cargo receptor for the delivery of Wnt ligands from the Golgi onto the cell surface for secretion. Consistent with this view, accumulation of Wg in the Golgi was observed in the absence of Vps35 activity. Perhaps, the absence of retromer activity causes depletion of Wls in the Golgi, thereby resulting in Wg accumulation in the Golgi (Belenkaya, 2008).

These experiments further demonstrate that Wls is actively internalized and degraded through Dynamin-mediated endocytosis. Since Wls is enhanced in endosomes in the absence of Hrs activity, the data also suggest that Wls is likely to undergo the lysosomal degradation. Endocytosis-mediated protein degradation has been shown to be a mechanism for regulation of a number of signaling receptors such as Patched, Thickveins, and EGF receptor. Thus, this work builds on the known principle that lysosomal targeting of receptors regulates signaling in the responding cell, by showing that lysosomal targeting of a putative cargo receptor can also attenuate the production and presentation of ligand in the first place (Belenkaya, 2008).

Extensive colocalization of Wls with Vps35 protein was found in endosomes in HeLa cells, arguing that endosomes are the main sites of retromer activity. Studies in both yeast and mammalian cells suggest an essential role of retromer in retrieving membrane proteins from endosomes back to TGN (Seaman, 2005). In light of the data, it is suggested that the retromer complex is involved in recycling Wls from endosomes to TGN for its further function in Wnt secretion. This is likely mediated by the interaction of Wls with the retromer complex. Consistent with this view, it was found that cell surface Wls can be internalized and returned to the Golgi. In the absence of retromer activity, internalized Wls is likely to be sorted into lysosomes for degradation. In support of this view, it was demonstrated that overexpression of Vps35 can significantly enhance levels of Wls in mammalian cells even in the absence of Wnt ligand (Belenkaya, 2008).

On the basis of these findings, the following model is proposed. At the ER, Wnt protein is dually lipid-modified by the Porc. The modified Wnt exits from the ER and enters the Golgi, where it binds Wls. Wls carries and sends Wnt proteins from TGN onto the cell surface for secretion. Wnt is delivered to the cell surface, the unloaded Wls protein on the plasma membrane will subsequently be internalized through a Dynamin-mediated endocytosis process. In the endosomes, the internalized Wls can have two fates. (1) The retromer complex interacts with Wls and retrieves Wls from endosomes back to TGN, thereby maintaining the normal levels of Wls protein. (2) In the absence of retromer activity, Wls protein is subsequently delivered into lysosomes for degradation. In this model, Wls acts as a Wnt cargo receptor. More experiments will be needed to define the mechanism by which Wls protein transports Wnt from TGN onto the cell surface for secretion. In addition, it also remains to be determined whether Wls is required for Wnt modification or its interaction with other proteins such as lipoprotein particles before its secretion (Belenkaya, 2008).

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. 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. It is proposed 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 (Franch-Marro, 2008).

Wingless secretion promotes and requires retromer-dependent cycling of Wntless

Wnt ligands are lipid-modified, secreted glycoproteins that control multiple steps during embryogenesis and adult-tissue homeostasis. Little is known about the mechanisms underlying Wnt secretion. Recently, Wntless (Wls/Evi/Srt) was identified as a conserved multi-pass transmembrane protein whose function seems to be dedicated to promoting the release of Wnts. This study describes Wls accumulation in the Golgi apparatus of Wnt/Wingless (Wg)-producing cells in Drosophila, and shows that this localization is essential for Wg secretion. Moreover, Wls localization and levels critically depend on retromer, a conserved protein complex that mediates endosome-to-Golgi protein trafficking in yeast. In the absence of the retromer components Dvps35 or Dvps26, but in presence of Wg, Wls is degraded and Wg secretion impaired. Our results indicate that Wg, clathrin-mediated endocytosis and retromer sustain a Wls traffic loop from the Golgi to the plasma membrane and back to the Golgi, thereby enabling Wls to direct Wnt secretion (Port, 2008).

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). Retromer has been identified as a key regulator involved in Wls trafficking. As sorting nexin (SNX) molecules are essential components of the retromer complex, it was hypothesized that specific SNX(s) is required for retromer-mediated Wnt secretion. Drosophila mutants were generated for all of the eight snx members, and identified Drosophila SNX3 (DSNX3) was identified as an essential molecule required for Wg secretion.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, this study 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, these findings suggest that DSNX3 acts as a cargo-specific component of retromer, which is required for endocytic recycling of Wls and Wg/Wnt secretion (Zhang, 2011).

Drosophila Vps35 function is necessary for normal endocytic trafficking and actin cytoskeleton organisation

To identify novel proteins required for receptor-mediated endocytosis, an RNAi-based screening method was developed in Drosophila S2 cells, based on uptake of a scavenger receptor ligand. Some known endocytic proteins are essential for endocytosis in this assay, including clathrin and alpha-adaptin; however, other proteins important for synaptic vesicle endocytosis are not required. A small screen for novel endocytic proteins identified the Drosophila homologue of Vps35, a component of the retromer complex, involved in endosome-to-Golgi trafficking. Loss of Vps35 inhibits scavenger receptor ligand endocytosis, and causes mislocalisation of a number of receptors and endocytic proteins. Vps35 has tumour suppressor properties because its loss leads to overproliferation of blood cells in larvae. Its loss also causes signalling defects at the neuromuscular junction, including upregulation of TGFbeta/BMP signalling and excessive formation of synaptic terminals. Vps35 negatively regulates actin polymerisation, and genetic interactions suggest that some of the endocytic and signalling defects of vps35 mutants are due to this function (Korolchuk, 2007).

Search PubMed for articles about Drosophila Vps35

Banziger, C., Soldini, D., Schutt, C., Zipperlen, P., Hausmann, G. and Basler, K. (2006). Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125: 509-522. PubMed ID: 16678095

Belenkaya, T. Y., et al. (2008). The retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-Golgi network. Dev. Cell 14(1): 120-31. PubMed ID: 18160348

Bonifacino, J. S., Hurley, J. H. (2008) Retromer. Curr Opin Cell Biol 20: 427-436. PubMed ID: 18472259

Coudreuse, D. Y., Roel, G., Betist, M. C., Destree, O. and Korswagen, H. C. (2006). Wnt gradient formation requires retromer function in Wnt-producing cells. Science 312: 921-924. PubMed ID: 16645052

Cullen, P. J., Korswagen, H. C. (2012) Sorting nexins provide diversity for retromer-dependent trafficking events. Nat Cell Biol 14: 29-37. PubMed ID: 22193161

Dong, B., Kakihara, K., Otani, T., Wada, H. and Hayashi, S. (2013). Rab9 and retromer regulate retrograde trafficking of luminal protein required for epithelial tube length control. Nat Commun 4: 1358. PubMed ID: 23322046

Franch-Marro, X., et al. (2008). Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex. Nat. Cell Biol. 10(2): 170-7. PubMed ID: 18193037

Hausmann, G., Banziger, C. and Basler, K. (2007). Helping Wingless take flight: how WNT proteins are secreted. Nat Rev Mol Cell Biol 8: 331-336. PubMed ID: 17342185

Korolchuk, V. I., Schutz, M. M., Gomez-Llorente, C., Rocha, J., Lansu, N. R., Collins, S. M., Wairkar, Y. P., Robinson, I. M. and O'Kane, C. J. (2007). Drosophila Vps35 function is necessary for normal endocytic trafficking and actin cytoskeleton organisation. J Cell Sci 120: 4367-4376. PubMed ID: 18057029

Li, B., Wong, C., Gao, S. M., Zhang, R., Sun, R., Li, Y. and Song, Y. (2018). The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating notch receptor trafficking. Elife 7. PubMed ID: 30176986

Pocha, S. M., Wassmer, T., Niehage, C., Hoflack, B., Knust, E. (2011). Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs. Curr Biol 21: 1111-1117. PubMed ID: 21700461

Port, F., Kuster, M., Herr, P., Furger, E., Banziger, C., Hausmann, G. and Basler, K. (2008). Wingless secretion promotes and requires retromer-dependent cycling of Wntless. Nat Cell Biol 10: 178-185. PubMed ID: 18193032

Roeth, J. F., Sawyer, J. K., Wilner, D. A. and Peifer, M. (2009). Rab11 helps maintain apical crumbs and adherens junctions in the Drosophila embryonic ectoderm. PLoS One 4: e7634. PubMed ID: 19862327

Seaman, M. N. (2012) The retromer complex - endosomal protein recycling and beyond. J Cell Sci 125: 4693-4702. PubMed ID: 23148298

Wang, S., Tan, K. L., Agosto, M. A., Xiong, B., Yamamoto, S., Sandoval, H., Jaiswal, M., Bayat, V., Zhang, K., Charng, W. L., David, G., Duraine, L., Venkatachalam, K., Wensel, T. G. and Bellen, H. J. (2014). The retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration. PLoS Biol 12: e1001847. PubMed ID: 24781186

Zhang, P., Wu, Y., Belenkaya, T. Y. and Lin, X. (2011). SNX3 controls Wingless/Wnt secretion through regulating retromer-dependent recycling of Wntless. Cell Res 21: 1677-1690. PubMed ID: 22041890

Zhou, B., Wu, Y. and Lin, X. (2011). Retromer regulates apical-basal polarity through recycling Crumbs. Dev Biol 360: 87-95. PubMed ID: 21958744

Zhou, B., Yun, E. Y., Ray, L., You, J., Ip, Y. T. and Lin, X. (2014). Retromer promotes immune quiescence by suppressing Spatzle-Toll pathway in Drosophila. J Cell Physiol 229: 512-520. PubMed ID: 24343480

date revised: 22 March 2022

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