Structural Basis of WLS/Evi-Mediated Wnt Transport and Secretion.

Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.

The role of Evi/Wntless in exporting Wnt proteins.

Intercellular communication by Wnt proteins governs many essential processes during development, tissue homeostasis and disease in all metazoans. Many context-dependent effects are initiated in the Wnt-producing cells and depend on the export of lipidated Wnt proteins. Although much focus has been on understanding intracellular Wnt signal transduction, the cellular machinery responsible for Wnt secretion became better understood only recently. After lipid modification by the acyl-transferase Porcupine, Wnt proteins bind their dedicated cargo protein Evi/Wntless for transport and secretion. Evi/Wntless and Porcupine are conserved transmembrane proteins, and their 3D structures were recently determined. In this Review, we summarise studies and structural data highlighting how Wnts are transported from the ER to the plasma membrane, and the role of SNX3-retromer during the recycling of its cargo receptor Evi/Wntless. We also describe the regulation of Wnt export through a post-translational mechanism and review the importance of Wnt secretion for organ development and cancer, and as a future biomarker.

The kinesin motor Klp98A mediates apical to basal Wg transport.

Development and tissue homeostasis rely on the tight regulation of morphogen secretion. In the Drosophila wing imaginal disc epithelium, Wg secretion for long-range signal transduction occurs after apical Wg entry into the endosomal system, followed by secretory endosomal transport. Although Wg release appears to occur from the apical and basal cell sides, its exact post-endocytic fate and the functional relevance of polarized endosomal Wg trafficking are poorly understood. Here, we identify the kinesin-3 family member Klp98A as the master regulator of intracellular Wg transport after apical endocytosis. In the absence of Klp98A, functional mature endosomes accumulate in the apical cytosol, and endosome transport to the basal cytosol is perturbed. Despite the resulting Wg mislocalization, Wg signal transduction occurs normally. We conclude that transcytosis-independent routes for Wg trafficking exist and demonstrate that Wg can be recycled apically via Rab4-recycling endosomes in the absence of Klp98A.

Ykt6-dependent endosomal recycling is required for Wnt secretion in the Drosophila wing epithelium.

Morphogens are important signalling molecules for tissue development and their secretion requires tight regulation. In the wing imaginal disc of flies, the morphogen Wnt/Wingless is apically presented by the secreting cell and re-internalized before final long-range secretion. Why Wnt molecules undergo these trafficking steps and the nature of the regulatory control within the endosomal compartment remain unclear. Here, we have investigated how Wnts are sorted at the level of endosomes by the versatile v-SNARE Ykt6. Using in vivo genetics, proximity-dependent proteomics and in vitro biochemical analyses, we show that most Ykt6 is present in the cytosol, but can be recruited to de-acidified compartments and recycle Wnts to the plasma membrane via Rab4-positive recycling endosomes. Thus, we propose a molecular mechanism by which producing cells integrate and leverage endocytosis and recycling via Ykt6 to coordinate extracellular Wnt levels.

Functional regulation of Wnt protein through post-translational modifications.

Wnts are lipid-modified signaling glycoproteins present in all metazoans that play key roles in development and homeostasis. Post-translational modifications of Wnts regulate their function. Wnts have a unique post-translational modification, O-linked palmitoleation, that is absolutely required for their function. This Wnt-specific modification occurs during Wnt biosynthesis in the endoplasmic reticulum (ER), catalyzed by the O-acyltransferase Porcupine (PORCN). Palmitoleation is required for Wnt to bind to its transporter Wntless (WLS/Evi) as well as to its receptor Frizzled (FZD). Recent structural studies have illustrated how PORCN recognizes its substrates, and how drugs inhibit this. The abundance of WLS is tightly regulated by intracellular recycling and ubiquitylation-mediated degradation in the ER. The function of Wnt glycosylation is less well understood, and the sites and types of glycosylation are not largely conserved among different Wnts. In polarized tissues, the type of glycans can determine whether the route of trafficking is apical or basolateral. In addition, pairing of the 24 highly conserved cysteines in Wnts to form disulfide bonds is critical in maintaining proper structure and activities. Extracellularly, the amino terminus of a subset of Wnts can be cleaved by a dedicated glycosylphosphatidylinositol (GPI)-anchored metalloprotease TIKI, resulting in the inactivation of these Wnt proteins. Additionally, NOTUM is a secreted extracellular carboxylesterase that removes the palmitoleate moiety from Wnt, antagonizing its activity. In summary, Wnt signaling activity is controlled at multiple layers by post-translational modifications.

Extracellular WNTs: Trafficking, Exosomes, and Ligand-Receptor Interaction.

WNT signaling is a key developmental pathway in tissue organization. A recent focus of research is the secretion of WNT proteins from source cells. Research over the past decade on how WNTs are produced and released into the extracellular space has unravelled very specific control mechanisms in the early secretory pathway, specialized trafficking routes, and redundant forms of packaging for delivery to target cells. In this review I discuss the findings that WNT proteins have been found on extracellular vesicles (EVs) such as exosomes and possible functional implications. There is an ongoing debate in the WNT signaling field whether EV are relevant in vivo and can fulfill specific functions, also fueled by the general preconception of EV secretion as cellular garbage disposal. As part of the EV research community, I want to give an overview of what we know and don't know about WNT secretion on EVs and offer a more unifying model that can explain current discrepancies in observations regarding WNT secretion.

Golgi Phosphoprotein 3 Promotes Wls Recycling and Wnt Secretion in Glioma Progression.

BACKGROUND/AIMS: Golgi phosphoprotein 3 (GOLPH3) plays pro-malignancy roles in several types of cancer. However, the molecular mechanism underlying GOLPH3 promoting tumor progression remains poorly understood. METHODS: The expression of GOLPH3 and Wntless (Wls) in glioma tissues was examined by western blotting and immunohistochemistry. EdU incorporation assay and colony formation assay was used to examine the cell growth ability. The effect of GOLPH3 on Wls recycling, Wnt secretion and ß-catenin activity was detected using western blotting, immunofluorescence, RT-PCR, ELISA or luciferase assay. RESULTS: The protein levels of GOLPH3 and Wls were upregulated and positively correlated with each other in human glioma tissues. The promoting effect of GOLPH3 on glioma cell proliferation was partially mediated by Wls. In addition, GOLPH3 interacted with Wls and GOLPH3 down-regulation drove Wls into lysosome for degradation, inhibiting its recycling to golgi and the plasma membrane. Importantly, GOLPH3 down-regulation inhibited Wnt2b secretion and decreased ß-catenin level and transcription activity. CONCLUSIONS: This study provides a brand new evidence that GOLPH3 promotes glioma cell proliferation by facilitating Wls recycling and Wnt/ß-catenin signaling. Our findings suggest a rationale for targeting the GOLPH3-Wls-Wnt axis as a promising therapeutic approach for glioblastoma.

Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche.

Communication between stem and niche supporting cells maintains the homeostasis of adult tissues. Wnt signaling is a crucial regulator of the stem cell niche, but the mechanism that governs Wnt ligand delivery in this compartment has not been fully investigated. We identified that Wnt secretion is partly dependent on Rab8a-mediated anterograde transport of Gpr177 (wntless), a Wnt-specific transmembrane transporter. Gpr177 binds to Rab8a, depletion of which compromises Gpr177 traffic, thereby weakening the secretion of multiple Wnts. Analyses of generic Wnt/ß-catenin targets in Rab8a knockout mouse intestinal crypts indicate reduced signaling activities; maturation of Paneth cells - a Wnt-dependent cell type - is severely affected. Rab8a knockout crypts show an expansion of Lgr5(+) and Hopx(+) cells in vivo. However, in vitro, the knockout enteroids exhibit significantly weakened growth that can be partly restored by exogenous Wnts or Gsk3ß inhibitors. Immunogold labeling and surface protein isolation identified decreased plasma membrane localization of Gpr177 in Rab8a knockout Paneth cells and fibroblasts. Upon stimulation by exogenous Wnts, Rab8a-deficient cells show ligand-induced Lrp6 phosphorylation and transcriptional reporter activation. Rab8a thus controls Wnt delivery in producing cells and is crucial for Paneth cell maturation. Our data highlight the profound tissue plasticity that occurs in response to stress induced by depletion of a stem cell niche signal.

Control of Wnt5b secretion by Wntless modulates chondrogenic cell proliferation through fine-tuning fgf3 expression.

Wnts and Fgfs regulate various tissues development in vertebrates. However, how regional Wnt or Fgf activities are established and how they interact in any given developmental event is elusive. Here, we investigated the Wnt-mediated craniofacial cartilage development in zebrafish and found that fgf3 expression in the pharyngeal pouches is differentially reduced along the anteroposterior axis in wnt5b mutants and wntless (wls) morphants, but its expression is normal in wnt9a and wnt11 morphants. Introducing fgf3 mRNAs rescued the cartilage defects in Wnt5b- and Wls-deficient larvae. In wls morphants, endogenous Wls expression is not detectable but maternally deposited Wls is present in eggs, which might account for the lack of axis defects in wls morphants. Secretion of endogenous Wnt5b but not Wnt11 was affected in the pharyngeal tissue of Wls morphants, indicating that Wls is not involved in every Wnt secretion event. Furthermore, cell proliferation but not apoptosis in the developing jaw was affected in Wnt5b- and Wls-deficient embryos. Therefore, Wnt5b requires Wls for its secretion and regulates the proliferation of chondrogenic cells through fine-tuning the expression of fgf3 during jaw cartilage development.

Wntless spatially regulates bone development through ß-catenin-dependent and independent mechanisms.

BACKGROUND: Canonical and noncanonical Wnt signaling pathways both play pivotal roles in bone development. Wntless/GPR177 is a chaperone protein that is required for secretion of all Wnt ligands. We previously showed that deletion of Wntless within mature osteoblasts severely impaired postnatal bone homeostasis. RESULTS: In this study, we systemically evaluated how deletion of Wntless in different stages of osteochondral differentiation affected embryonic bone development, by crossing Wntless (Wls)-flox/flox mice with strains expressing cre recombinase behind the following promoters: Osteocalcin, Collagen 2a1, or Dermo1. Ex vivo µCT and whole-mount skeletal staining were performed to examine skeletal mineralization. Histology and immunohistochemistry were used to evaluate cellular differentiation and alterations in Wnt signaling. In this work, we found that Wntless regulated chondrogenesis and osteogenesis through both canonical and noncanonical Wnt signaling. CONCLUSIONS: These findings provide more insight into the requirements of different Wnt-secretion cell types critical for skeletal development.

Enrichment and Detection of Wnt Proteins from Cell Culture Media.

Wnt proteins are secreted, lipid-modified growth factors with a wide range of activities across all metazoan species. Their production, secretion, and signaling range are under tight cellular control such that detection of Wnt proteins in biological samples is often extremely difficult. In this chapter, we describe a protocol to detect secreted Wnt proteins in the culture medium of cell lines that ectopically or endogenously express Wnt genes. This protocol uses an affinity resin, called Blue Sepharose, that binds and thereby enriches Wnt proteins, followed by immunoblotting for the Wnt protein of interest. This method for detecting Wnt proteins will aid in the isolation of biologically active Wnt proteins, provide an assay to study the molecular basis of Wnt secretion, and potentially offer a means to detect trace amounts of Wnt proteins associated with pathological states.

Visualization and Quantitation of Wg trafficking in the Drosophila Wing Imaginal Epithelium.

Secretory Wnt trafficking can be studied in the polarized epithelial monolayer of Drosophila wing imaginal discs (WID). In this tissue, Wg (Drosophila Wnt-I) is presented on the apical surface of its source cells before being internalized into the endosomal pathway. Long-range Wg secretion and spread depend on secondary secretion from endosomal compartments, but the exact post-endocytic fate of Wg is poorly understood. Here, we summarize and present three protocols for the immunofluorescence-based visualization and quantitation of different pools of intracellular and extracellular Wg in WID: (1) steady-state extracellular Wg; (2) dynamic Wg trafficking inside endosomal compartments; and (3) dynamic Wg release to the cell surface. Using a genetic driver system for gene manipulation specifically at the posterior part of the WID (EnGal4) provides a robust internal control that allows for direct comparison of signal intensities of control and manipulated compartments of the same WID. Therefore, it also circumvents the high degree of staining variability usually associated with whole-tissue samples. In combination with the genetic manipulation of Wg pathway components that is easily feasible in Drosophila, these methods provide a tool-set for the dissection of secretory Wg trafficking and can help us to understand how Wnt proteins travel along endosomal compartments for short- and long-range signal secretion. Graphic abstract: Figure 1. Visualization of extracellular and intracellular Wg trafficking in Drosophila wing imaginal discs. While staining of extracellular Wg without permeabilization exclusively visualizes Wg bound to the extracellular surface (left), Wg uptake and endosomal trafficking can be visualized using an antibody uptake assay (middle). Dynamic Wg release can be visualized by performing a non-permeabilizing staining at a permissive temperature that sustains secretory Wg transport (right).

A Protein Disulfide Isomerase Controls Neuronal Migration through Regulation of Wnt Secretion.

Appropriate Wnt morphogen secretion is required to control animal development and homeostasis. Although correct Wnt globular structure is essential for secretion, proteins that directly mediate Wnt folding and maturation remain uncharacterized. Here, we report that protein disulfide isomerase-1 (PDI-1), a protein-folding catalyst and chaperone, controls secretion of the Caenorhabditis elegans Wnt ortholog EGL-20. We find that PDI-1 function is required to correctly form an anteroposterior EGL-20/Wnt gradient during embryonic development. Furthermore, PDI-1 performs this role in EGL-20/Wnt-producing epidermal cells to cell-non-autonomously control EGL-20/Wnt-dependent neuronal migration. Using pharmacological inhibition, we further show that PDI function is required in human cells for Wnt3a secretion, revealing a conserved role for disulfide isomerases. Together, these results demonstrate a critical role for PDIs within Wnt-producing cells to control long-range developmental events that are dependent on Wnt secretion.

The Mingle-Mangle of Wnt Signaling and Extracellular Vesicles: Functional Implications for Heart Research.

Wnt signaling is an important pathway in health and disease and a key regulator of stem cell maintenance, differentiation, and proliferation. During heart development, Wnt signaling controls specification, proliferation and differentiation of cardiovascular cells. In this regard, the role of activated Wnt signaling in cardiogenesis is well defined. However, the knowledge about signaling transmission has been challenged. Recently, the packaging of hydrophobic Wnt proteins on extracellular vesicles (EVs) has emerged as a mechanism to facilitate their extracellular spreading and their functioning as morphogens. EVs spread systemically and therefore can have pleiotropic effects on very different cell types. They are heavily studied in tumor biology where they affect tumor growth and vascularization and can serve as biomarkers in liquid biopsies. In this review we will highlight recent discoveries of factors involved in the release of Wnts on EVs and its potential implications in the communication between physiological and pathological heart cells.

Biochemical Methods to Analyze Wnt Protein Secretion.

Wnt proteins act as potent morphogens in various aspects of embryonic development and adult tissue homeostasis. However, in addition to its physiological importance, aberrant Wnt signaling has been linked to the onset and progression of different types of cancer. On the cellular level, the secretion of Wnt proteins involves trafficking of lipid-modified Wnts from the endoplasmic reticulum (ER) to Golgi and further compartments via the Wnt cargo receptor evenness interrupted. Others and we have recently shown that Wnt proteins are secreted on extracellular vesicles (EVs) such as microvesicles and exosomes. Although more details about specific regulation of Wnt secretion steps are emerging, it remains largely unknown how Wnt proteins are channeled into different release pathways such as lipoprotein particles, EVs and cytonemes. Here, we describe protocols to purify and quantify Wnts from the supernatant of cells by either assessing total Wnt proteins in the supernatant or monitoring Wnt proteins on EVs. Purified Wnts from the supernatant as well as total cellular protein content can be investigated by immunoblotting. Additionally, the relative activity of canonical Wnts in the supernatant can be assessed by a dual-luciferase Wnt reporter assay. Quantifying the amount of secreted Wnt proteins and their activity in the supernatant of cells allows the investigation of intracellular trafficking events that regulate Wnt secretion and the role of extracellular modulators of Wnt spreading.

RAB and RHO GTPases regulate intestinal crypt cell homeostasis and enterocyte function.

Recent human and mouse genetic studies have highlighted important contributions of several small GTPases, in particular Rab8a, (1) Cdc42, (2-4) and Rab11a, (5-8) to the proper morphogenesis and function of the mature intestinal epithelia. Additional insights about the involvement of these factors in maintaining intestinal stem cell homeostasis have also been obtained. (9,10) These studies suggest a conserved vesicular and membrane trafficking program utilized by the gastrointestinal tissue to support the rapid epithelial cell turnover and the highly sophisticated physiology of mature epithelial cells.

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.