Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates.

During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and beta-catenin-independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl's role in Wnt signaling, we genome engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for superresolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin-dependent and beta-catenin-independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supramolecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large "puncta," supramolecular condensate formation at physiological protein levels is only observed in a subset of cells with approximately one per cell. We show that, in these condensates, Dvl2 colocalizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt dependent. Single-molecule localization microscopy using photoactivated localization microscopy (PALM) of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle-dependent manner. Our results indicate that the localization of Dvl2 in supramolecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.

HCMV UL8 interaction with ß-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells.

IMPORTANCE: CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/ß-catenin pathway as an important component of viral reactivation. We further define that pUL8 and ß-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with ß-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.

Wnt Signalosome Assembly by DEP Domain Swapping of Dishevelled.

Extracellular signals are often transduced by dynamic signaling complexes ("signalosomes") assembled by oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which is assembled by Dishevelled via reversible head-to-tail polymerization by its DIX domain. Its activity causes stabilization of ß-catenin, a Wnt effector with pivotal roles in animal development and cancer. How Wnt triggers signalosome assembly is unknown. Here, we use structural analysis, as well as biophysical and cell-based assays, to show that the DEP domain of Dishevelled undergoes a conformational switch, from monomeric to swapped dimer, to trigger DIX-dependent polymerization and signaling to ß-catenin. This occurs in two steps: binding of monomeric DEP to Frizzled followed by DEP domain swapping triggered by its high local concentration upon Wnt-induced recruitment into clathrin-coated pits. DEP domain swapping confers directional bias on signaling, and the dimerization provides cross-linking between Dishevelled polymers, illustrating a key principle underlying signalosome formation.

Regulation of Dishevelled DEP domain swapping by conserved phosphorylation sites.

Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signaling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled. During canonical (ß-catenin-dependent) signaling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its Dishevelled and Axin (DIX) domain, which are cross-linked by its Dishevelled, Egl-10, and Pleckstrin (DEP) domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signaling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signaling. Here, we use NMR spectroscopy and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved phosphorylation site upstream of the DEP domain that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. Mutations of this phosphorylation site and its cognate surface on the reciprocal DEP core attenuate DEP-dependent dimerization of Dishevelled and its canonical signaling activity in cells without impeding its binding to Frizzled. We propose that phosphorylation of this crucial residue could be employed to switch off canonical Wnt signaling.

Single-molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation.

Dvl (Dishevelled) is one of several essential nonenzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of ß-catenin degradation. Although this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus of dishevelled2 gene using CRISPR/Cas9-induced homologous recombination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is predominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.12/µm2 to 0.54/µm2 Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The oligomerized Dvl2 complexes have increased dwell time, 2 ∼ 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.

Characterization of the aberrant splicing of DVL2 induced by cancer-associated SF3B1 mutation.

SF3B1, an essential component of the U2 snRNP, is frequently mutated in cancers. Cancer-associated SF3B1 mutation causes aberrant RNA splicing, mostly at 3' splice sites (3'ss). RNA splicing of DVL2, a regulator of Notch signaling, is affected by SF3B1 mutation. Here, we report that the mutated SF3B1 use an alternative branchpoint sequence (BPS) for the aberrant splicing of DVL2, which has a higher affinity to U2 snRNA than the BPS for the canonical splicing of DVL2. Swapping the position of the alternative BPS with the position of the canonical BPS decreased the aberrant splicing of DVL2, suggesting that the mutated SF3B1 prefers to use BPS with high affinity to U2 snRNA for splicing. Additionally, swapping the positions of two BPSs associated with the canonical splicing of DVL2 demonstrated that both the affinity to the U2 snRNA and the distance to the 3'ss are important to the selection of BPS. Importantly, the aberrant splicing of DVL2 does not require the canonical 3'ss and the canonical polypyrimidine tract, which reveals a novel type of aberrant splicing induced by SF3B1 mutation. These findings provide a more comprehensive understanding of the mechanisms underlying aberrant splicing induced by SF3B1 mutation in cancer.

Inhibition of microRNA suppression of Dishevelled results in Wnt pathway-associated developmental defects in sea urchin.

MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that regulate gene expressions by binding to the 3' untranslated region of target mRNAs thereby silencing translation. Some miRNAs are key regulators of the Wnt signaling pathways, which impact developmental processes. This study investigates miRNA regulation of different isoforms of Dishevelled (Dvl/Dsh), which encode a key component in the Wnt signaling pathway. The sea urchin Dvl mRNA isoforms have similar spatial distribution in early development, but one isoform is distinctively expressed in the larval ciliary band. We demonstrated that Dvl isoforms are directly suppressed by miRNAs. By blocking miRNA suppression of Dvl isoforms, we observed dose-dependent defects in spicule length, patterning of the primary mesenchyme cells, gut morphology, and cilia. These defects likely result from increased Dvl protein levels, leading to perturbation of Wnt-dependent signaling pathways and additional Dvl-mediated processes. We further demonstrated that overexpression of Dvl isoforms recapitulated some of the Dvl miRNATP-induced phenotypes. Overall, our results indicate that miRNA suppression of Dvl isoforms plays an important role in ensuring proper development and function of primary mesenchyme cells and cilia.

The Daple-CK1ε complex regulates Dvl2 phosphorylation and canonical Wnt signaling.

The canonical Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis and cancer progression. The binding of Wnt ligands to their cognate receptors, the Frizzled (Fzd) family of proteins, recruits Dishevelled segment polarity protein (Dvl) to the plasma membrane and induces its phosphorylation via casein kinase 1 (CK1), which leads to the activation of ß-catenin. Previous studies showed that Dishevelled-associating protein with a high frequency of leucine residues (Daple) is an important component of the Wnt signaling pathway and essential for Dvl phosphorylation. However, the mechanism by which Daple promotes CK1-mediated phosphorylation of Dvl is not fully understood. In this study, we found that Daple overexpression induced CK1ε-mediated Dvl2 phosphorylation at threonine 224 (Thr224). A Daple mutant (Daple ΔGCV) that lacks a carboxyl-terminal motif to associate with Dvl, retained the ability to interact with CK1ε, but did not induce Dvl phosphorylation, suggesting the importance of the Daple/Dvl/CK1ε trimeric protein complex. We further found that Thr224 phosphorylation of Dvl was required for full activation of ß-catenin transcriptional activity. Consistent with this, wild-type Daple promoted ß-catenin transcriptional activity, following dissociation of ß-catenin and axin. Finally, Wnt3a stimulation increased the membrane localization of Daple and its association with Dvl, and Daple knockdown attenuated Wnt3a-mediated ß-catenin transcriptional activity. Collectively, these data suggested a essential role of spatial Daple localization in CK1ε-mediated activation of Dvl in the canonical Wnt signaling pathway.

Dishevelled enables casein kinase 1-mediated phosphorylation of Frizzled 6 required for cell membrane localization.

Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein-coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD6 Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD6 C terminus, is efficiently phosphorylated by casein kinase 1 ϵ (CK1ϵ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1ϵ-mediated FZD6 phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1ϵ-targeted phosphomotif reduced FZD6 surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD6 Phospho-Ser-648 FZD6 immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD6 protein expression, suggesting that FZD6 phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD6 in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD6 rather than asymmetric protein distribution accounts for polarized receptor signaling.

Imaging and Inhibiting: A Dual Function Molecular Flare for Cancer Cells.

The Wnt pathway is dysregulated and activated in many human malignancies. More than 90% of colon cancers have variations in the Wnt pathway. Sulindac, a drug that targets protein Dvl of the Wnt/Dvl/ß-catenin pathway, which regulates cancer gene expression, has been reported to significantly reduce the incidence and the risk of death from colorectal cancer and other types of cancer. Herein, a dual functional compound (SLN) containing Sulindac and a linked fluorophore is first reported, combining the functions of lighting up colon cancer cells as a flare and inhibiting colon tumors as a drug. SLN can not only mark the Dvl protein in the Wnt pathway to recognize tumors layer by layer but also achieve effective inhibition of colon cancer, providing a promising reagent for chemotherapy and a fluorescent indicator for surgery during the removal the colon tumors in situ.

Comparative phosphorylation map of Dishevelled 3 links phospho-signatures to biological outputs.

BACKGROUND: Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported (CK1ε, NEK2, PLK1, CK2α, RIPK4, PKCδ) and newly identified (TTBK2, Aurora A) DVL kinases. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy. RESULTS: We used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced ß-catenin activation. S630-643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization. CONCLUSIONS: We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome. Video Abtract.

Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay.

Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces Wnt signals to cytoplasmic effectors including ß-catenin, to control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies of Dishevelled relied on its Wnt-independent signalling activity resulting from overexpression, which is sufficient to trigger polymerisation, bypassing the requirement for Wnt signals. Here, we generate a human cell line devoid of endogenous Dishevelled (DVL1- DVL3), which lacks Wnt signal transduction to ß-catenin. However, Wnt responses can be restored by DVL2 stably re-expressed at near-endogenous levels. Using this assay to test mutant DVL2, we show that its DEP domain is essential, whereas its PDZ domain is dispensable, for signalling to ß-catenin. Our results imply two mutually exclusive functions of the DEP domain in Wnt signal transduction - binding to Frizzled to recruit Dishevelled to the receptor complex, and dimerising to cross-link DIX domain polymers for signalosome assembly. Our assay avoids the caveats associated with overexpressing Dishevelled, and provides a powerful tool for rigorous functional tests of this pivotal human signalling protein.

Discovery of a small-molecule inhibitor of Dvl-CXXC5 interaction by computational approaches.

The Wnt/ß-catenin signaling pathway plays a significant role in the control of osteoblastogenesis and bone formation. CXXC finger protein 5 (CXXC5) has been recently identified as a negative feedback regulator of osteoblast differentiation through a specific interaction with Dishevelled (Dvl) protein. It was reported that targeting the Dvl-CXXC5 interaction could be a novel anabolic therapeutic target for osteoporosis. In this study, complex structure of Dvl PDZ domain and CXXC5 peptide was simulated with molecular dynamics (MD). Based on the structural analysis of binding modes of MD-simulated Dvl PDZ domain with CXXC5 peptide and crystal Dvl PDZ domain with synthetic peptide-ligands, we generated two different pharmacophore models and applied pharmacophore-based virtual screening to discover potent inhibitors of the Dvl-CXXC5 interaction for the anabolic therapy of osteoporosis. Analysis of 16 compounds selected by means of a virtual screening protocol yielded four compounds that effectively disrupted the Dvl-CXXC5 interaction in the fluorescence polarization assay. Potential compounds were validated by fluorescence spectroscopy and nuclear magnetic resonance. We successfully identified a highly potent inhibitor, BMD4722, which directly binds to the Dvl PDZ domain and disrupts the Dvl-CXXC5 interaction. Overall, CXXC5-Dvl PDZ domain complex based pharmacophore combined with various traditional and simple computational methods is a promising approach for the development of modulators targeting the Dvl-CXXC5 interaction, and the potent inhibitor BMD4722 could serve as a starting point to discover or design more potent and specific the Dvl-CXXC5 interaction disruptors.

Crystal structure of the PDZ domain of mouse Dishevelled 1 and its interaction with CXXC5.

Dishevelled (Dvl) plays a crucial role in Wnt signaling by interacting with membrane-bound receptors and downstream molecules through its PDZ domain. CXXC5 is one of the key molecules that interacts with Dvl and negatively regulates the Wnt/ß-catenin pathway in osteoblast differentiation. Recently, the Dvl-CXXC5 interaction has been identified as an excellent target for osteoporosis treatment. Therefore, it is desirable to have detailed structural information for the Dvl-CXXC5 interaction. Although solution structures of the Dvl1 PDZ domain have been reported, a high-resolution crystal structure would provide detailed sidechain information that is essential for drug development. Here, we determined the first crystal structure of the Dvl-1 PDZ domain at a resolution of 1.76 Å, and compared it with its previously reported solution structure. The Dvl1 PDZ domain crystal belonged to the space group H32 with unit-cell parameters a = b = 72.837, c = 120.616, α = ß = 90.00, γ = 120.00. The crystal structure of Dvl1 PDZ shared its topology with the previously reported structure determined by nuclear magnetic resonance (NMR); however, the crystal structure was quite different from the solution structure in both the secondary structural region and the ligand-binding pocket. Molecular modeling based on NMR and X-ray crystallographic data yielded detailed information about the Dvl1/CXXC5 interaction, which will be useful for designing inhibitors.

Identification of small-molecule compounds targeting the dishevelled PDZ domain by virtual screening and binding studies.

The Dishevelled (Dvl) protein, which conveys signals from receptors to the downstream effectors, is a critical constituent of the Wnt/ß-catenin signaling pathway. Because the PDZ domain of Dvl protein functions through associations with a wide range of protein partners, Dvl protein involved in the Wnt signaling pathway has been considered to be therapeutic targets in cancers. In this study, we performed structure-based pharmacophore model of the Dvl PDZ domain to discover novel small-molecule binders and identified eight compounds with micromolar affinity. The most potent compound identified, BMD4702, efficiently bound to the Dvl PDZ domain with 11.2µM affinity and had a 0.186µM KD value according to surface plasmon resonance and fluorescence spectroscopy, respectively. Combining both structural-kinetic relationship analyses and docking studies, we fourmulated that the ligand-binding site is composed of three H-bonds and three hydrophobic features. Thus, our approach led to the identification of potent binders of the Dvl PDZ domain and the findings provide novel insights into structure-based approaches to design high-affinity binders for the Dvl PDZ domain.

Dishevelled2 activates WGEF via its interaction with a unique internal peptide motif of the GEF.

The Wnt-planar cell polarity (Wnt-PCP) pathway is crucial in establishing cell polarity during development and tissue homoeostasis. This pathway is found to be dysregulated in many pathological conditions, including cancer and autoimmune disorders. The central event in Wnt-PCP pathway is the activation of Weak-similarity guanine nucleotide exchange factor (WGEF) by the adapter protein Dishevelled (Dvl). The PDZ domain of Dishevelled2 (Dvl2PDZ) binds and activates WGEF by releasing it from its autoinhibitory state. However, the actual Dvl2PDZ binding site of WGEF and the consequent activation mechanism of the GEF have remained elusive. Using biochemical and molecular dynamics studies, we show that a unique "internal-PDZ binding motif" (IPM) of WGEF mediates the WGEF-Dvl2PDZ interaction to activate the GEF. The residues at P2, P0, P-2 and P-3 positions of IPM play an important role in stabilizing the WGEFpep-Dvl2PDZ interaction. Furthermore, MD simulations of modelled Dvl2PDZ-WGEFIPM peptide complexes suggest that WGEF-Dvl2PDZ interaction may differ from the reported Dvl2PDZ-IPM interactions. Additionally, the apo structure of human Dvl2PDZ shows conformational dynamics different from its IPM peptide bound state, suggesting an induced fit mechanism for the Dvl2PDZ-peptide interaction. The current study provides a model for Dvl2 induced activation of WGEF.

Head-to-Tail Complex of Dishevelled and Axin-DIX Domains: Expression, Purification, Crystallographic Studies and Packing Analysis.

BACKGROUND: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/ß- catenin signalling pathway, but this interaction mechanism is still unknown. OBJECTIVE: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation. METHODS: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them. RESULTS: The crystal of the DAX-DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wntsignalosome model. CONCLUSION: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.

High-resolution structure of a Y27W mutant of the Dishevelled2 DIX domain.

Dishevelled (Dvl) is a positive regulator of the canonical Wnt pathway that downregulates the phosphorylation of ß-catenin and its subsequent degradation. Dvl contains an N-terminal DIX domain, which is involved in its homooligomerization and interactions with regulators of the Wnt pathway. The crystal structure of a Y27W mutant of the Dishevelled2 DIX domain (DIX-Y27W) has been determined at 1.64 Šresolution. DIX-Y27W has a compact ubiquitin-like fold and self-associates with neighbouring molecules through ß-bridges, resulting in a head-to-tail helical molecular arrangement similar to previously reported structures of DIX domains. Glu23 of DIX-Y27W forms a hydrogen bond to the side chain of Trp27, corresponding to the Glu762...Trp766 hydrogen bond of the rat Axin DIX domain, whereas Glu23 in the Y27D mutant of the Dishevelled2 DIX domain forms a salt bridge to Lys68 of the adjacent molecule. The high-resolution DIX-Y27W structure provides details of the head-to-tail interaction, including solvent molecules, and also the plausibly wild-type-like structure of the self-association surface compared with previously published Dvl DIX-domain mutants.

A direct heterotypic interaction between the DIX domains of Dishevelled and Axin mediates signaling to ß-catenin.

The Wnt-ß-catenin signaling pathway regulates embryonic development and tissue homeostasis throughout the animal kingdom. Signaling through this pathway crucially depends on the opposing activities of two cytoplasmic multiprotein complexes: the Axin destruction complex, which destabilizes the downstream effector ß-catenin, and the Dishevelled signalosome, which inactivates the Axin complex and thus enables ß-catenin to accumulate and operate a transcriptional switch in the nucleus. These complexes are assembled by dynamic head-to-tail polymerization of the DIX domains of Axin or Dishevelled, respectively, which increases their avidity for signaling effectors. Axin also binds to Dishevelled through its DIX domain. Here, we report the crystal structure of the heterodimeric complex between the two DIX domains of Axin and Dishevelled. This heterotypic interface resembles the interfaces observed in the individual homopolymers, albeit exhibiting a slight rearrangement of electrostatic interactions and hydrogen bonds, consistent with the heterotypic interaction being favored over the homotypic Axin DIX interaction. Last, cell-based signaling assays showed that heterologous polymerizing domains functionally substituted for the DIX domain of Dishevelled provided that these Dishevelled chimeras retained a DIX head or tail surface capable of binding to Axin. These findings indicate that the interaction between Dishevelled and Axin through their DIX domains is crucial for signaling to ß-catenin.

Developmentally regulated GTP-binding protein 1 modulates ciliogenesis via an interaction with Dishevelled.

Cilia are critical for proper embryonic development and maintaining homeostasis. Although extensively studied, there are still significant gaps regarding the proteins involved in regulating ciliogenesis. Using the Xenopus laevis embryo, we show that Dishevelled (Dvl), a key Wnt signaling scaffold that is critical to proper ciliogenesis, interacts with Drg1 (developmentally regulated GTP-binding protein 1). The loss of Drg1 or disruption of the interaction with Dvl reduces the length and number of cilia and displays defects in basal body migration and docking to the apical surface of multiciliated cells (MCCs). Moreover, Drg1 morphants display abnormal rotational polarity of basal bodies and a decrease in apical actin and RhoA activity that can be attributed to disruption of the protein complex between Dvl and Daam1, as well as between Daam1 and RhoA. These results support the concept that the Drg1-Dvl interaction regulates apical actin polymerization and stability in MCCs. Thus, Drg1 is a newly identified partner of Dvl in regulating ciliogenesis.