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eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled

2020; Elsevier BV; Volume: 295; Issue: 26 Linguagem: Inglês

10.1074/jbc.ra120.012892

1083-351X

Janine Wesslowski, Paweł Kozielewicz, Xianxian Wang, Haijun Cui, Hannes Schihada, D Kranz, Pradhipa Karuna M, Pavel A. Levkin, Julia Christina Gross, Michael Boutros, Gunnar Schulte, Gary Davidson,

RNA Research and Splicing

The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD1–10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity. The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD1–10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity. In metazoans, cell-to-cell communication is predominantly controlled by the activation of specific cell surface receptors localized within the plasma membrane. Receptor-activating ligands are secreted from (paracrine) or presented on the surface (cell contact) of neighboring cells or presented as a humoral ligand with systemic effects (endocrine). Ligand-receptor interaction initiates conformational changes in the receptor to activate diverse intracellular signal transduction events that alter cellular behavior in a context-specific manner. The Wnt signaling system is an evolutionarily conserved, tightly regulated signaling network comprised of multiple transmembrane receptors and secreted ligands that play complex roles in embryonic development and tissue homeostasis (1Niehrs C. The complex world of WNT receptor signalling.Nat. Rev. Mol. Cell Biol. 2012; 13 (23151663): 767-77910.1038/nrm3470Crossref PubMed Scopus (972) Google Scholar, 2Clevers H. Nusse R. Wnt/beta-catenin signaling and disease.Cell. 2012; 149 (22682243): 1192-120510.1016/j.cell.2012.05.012Abstract Full Text Full Text PDF PubMed Scopus (3993) Google Scholar, 3Wiese K.E. Nusse R. van Amerongen R. Wnt signalling: conquering complexity.Development. 2018; 145 (29945986): dev16590210.1242/dev.165902Crossref PubMed Scopus (126) Google Scholar). Ten different G protein–coupled receptors of the class F (FZD1–10) engage with 19 different lipidated Wnt proteins with unknown ligand-receptor selectivity to transduce Wnt signaling (1Niehrs C. The complex world of WNT receptor signalling.Nat. Rev. Mol. Cell Biol. 2012; 13 (23151663): 767-77910.1038/nrm3470Crossref PubMed Scopus (972) Google Scholar, 4Schulte G. Wright S.C. Frizzleds as GPCRs—more conventional than we thought!.Trends Pharmacol. Sci. 2018; 39 (30049420): 828-84210.1016/j.tips.2018.07.001Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 5Kozielewicz P. Turku A. Schulte G. Molecular pharmacology of class F receptor activation.Mol. Pharmacol. 2020; 97 (31591260): 62-7110.1124/mol.119.117986Crossref PubMed Google Scholar, 6Schulte G. International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors.Pharmacol. Rev. 2010; 62 (21079039): 632-66710.1124/pr.110.002931Crossref PubMed Scopus (154) Google Scholar). In addition to FZDs, coreceptors for Wnts are usually associated with specific branches of the pathway, such as low-density-lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) for Wnt/β-catenin signaling (1Niehrs C. The complex world of WNT receptor signalling.Nat. Rev. Mol. Cell Biol. 2012; 13 (23151663): 767-77910.1038/nrm3470Crossref PubMed Scopus (972) Google Scholar). Pathway selectivity of Wnts is dictated by the relative availability of specific receptors, coreceptors, and Wnts, as well as the relative binding affinities of ligand-receptor interactions (7Rulifson E.J. Wu C.H. Nusse R. Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless.Mol. Cell. 2000; 6 (10949033): 117-12610.1016/S1097-2765(05)00018-3Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Various biochemical assays have been used to investigate the binding affinities of specific Wnt-FZD pairs, and Kd values ranging from 5–100 nm have been reported in Drosophila, which have 5 Wnt and 4 FZD genes (7Rulifson E.J. Wu C.H. Nusse R. Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless.Mol. Cell. 2000; 6 (10949033): 117-12610.1016/S1097-2765(05)00018-3Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 8Wu C.-H. Nusse R. Ligand receptor interactions in the Wnt signaling pathway in Drosophila.J. Biol. Chem. 2002; 277 (12205098): 41762-4176910.1074/jbc.M207850200Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Such studies in higher vertebrates have proven more challenging because of the increased ligand-receptor diversity as well as difficulties associated with the biochemistry and lipophilicity of Wnt proteins resulting in low specific activity or a large proportion of nonspecific binding. Nevertheless, binding affinities ranging from 100 nm (Wnt-4/FZD2-CRD) down to 1.5 nm (Wnt-3a/FZD8-CRD) have also been reported (9Dijksterhuis J.P. Baljinnyam B. Stanger K. Sercan H.O. Ji Y. Andres O. Rubin J.S. Hannoush R.N. Schulte G. Systematic mapping of WNT-Frizzled interactions reveals functional selectivity by distinct WNT-Frizzled pairs.J. Biol. Chem. 2015; 290 (25605717): 6789-679810.1074/jbc.M114.612648Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 10Hsieh J.C. Rattner A. Smallwood P.M. Nathans J. Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.Proc. Natl. Acad. Sci. U S A. 1999; 96 (10097073): 3546-355110.1073/pnas.96.7.3546Crossref PubMed Scopus (289) Google Scholar). All of these studies used membrane-tethered Wnts (7Rulifson E.J. Wu C.H. Nusse R. Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless.Mol. Cell. 2000; 6 (10949033): 117-12610.1016/S1097-2765(05)00018-3Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 8Wu C.-H. Nusse R. Ligand receptor interactions in the Wnt signaling pathway in Drosophila.J. Biol. Chem. 2002; 277 (12205098): 41762-4176910.1074/jbc.M207850200Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and/or the soluble, isolated cysteine-rich domain (CRD) of FZDs (8Wu C.-H. Nusse R. Ligand receptor interactions in the Wnt signaling pathway in Drosophila.J. Biol. Chem. 2002; 277 (12205098): 41762-4176910.1074/jbc.M207850200Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 9Dijksterhuis J.P. Baljinnyam B. Stanger K. Sercan H.O. Ji Y. Andres O. Rubin J.S. Hannoush R.N. Schulte G. Systematic mapping of WNT-Frizzled interactions reveals functional selectivity by distinct WNT-Frizzled pairs.J. Biol. Chem. 2015; 290 (25605717): 6789-679810.1074/jbc.M114.612648Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 10Hsieh J.C. Rattner A. Smallwood P.M. Nathans J. Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.Proc. Natl. Acad. Sci. U S A. 1999; 96 (10097073): 3546-355110.1073/pnas.96.7.3546Crossref PubMed Scopus (289) Google Scholar), which fail to fully recapitulate native conditions for Wnt interaction with the full-length FZD on live cells in real time. Fluorescent labeling of proteins is a widely used and valued technique because of the range of biophysical and biochemical detection options it offers (11Rodriguez E.A. Campbell R.E. Lin J.Y. Lin M.Z. Miyawaki A. Palmer A.E. Shu X. Zhang J. Tsien R.Y. The growing and glowing toolbox of fluorescent and photoactive proteins.Trends Biochem. Sci. 2017; 42 (27814948): 111-12910.1016/j.tibs.2016.09.010Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). However, the introduction of a large fluorescent moiety into a protein often compromises the stability and functionality of the target protein. A major limitation for the study of Wnt signaling and receptor binding originates from the lipophilicity of the Wnts themselves, which results in poor solubility and their recalcitrance to epitope tagging. Only a few attempts to generate functional, fluorescently tagged Wnt proteins have met with some degree of success, such as Xenopus Wnt-2b (12Holzer T. Liffers K. Rahm K. Trageser B. Ozbek S. Gradl D. Live imaging of active fluorophore labelled Wnt proteins.FEBS Lett. 2012; 586 (22554900): 1638-164410.1016/j.febslet.2012.04.035Crossref PubMed Scopus (30) Google Scholar), Xenopus Wnt-5a (13Wallkamm V. Dorlich R. Rahm K. Klessing T. Nienhaus G.U. Wedlich D. Gradl D. Live imaging of Xwnt5A-ROR2 complexes.PLoS ONE. 2014; 9 (25313906): e10942810.1371/journal.pone.0109428Crossref PubMed Scopus (13) Google Scholar), zebrafish Wnt-8 (14Rhinn M. Lun K. Luz M. Werner M. Brand M. Positioning of the midbrain-hindbrain boundary organizer through global posteriorization of the neuroectoderm mediated by Wnt8 signaling.Development. 2005; 132 (15703279): 1261-127210.1242/dev.01685Crossref PubMed Scopus (86) Google Scholar), and chick Wnt-1 (15Galli L.M. Santana F. Apollon C. Szabo L.A. Ngo K. Burrus L.W. Direct visualization of the Wntless-induced redistribution of WNT1 in developing chick embryos.Dev. Biol. 2018; 439 (29715461): 53-6410.1016/j.ydbio.2018.04.025Crossref PubMed Scopus (4) Google Scholar), all of which are C-terminally tagged. Farin et al. elegantly demonstrated that successful epitope tagging of a mammalian Wnt is indeed possible after reporting that mice harboring an internal HA tag inserted at residue Q41 of the Wnt-3a gene were viable and could produce a fully functional, tractable Wnt-3a protein (16Farin H.F. Jordens I. Mosa M.H. Basak O. Korving J. Tauriello D.V. de Punder K. Angers S. Peters P.J. Maurice M.M. Clevers H. Visualization of a short-range Wnt gradient in the intestinal stem-cell niche.Nature. 2016; 530 (26863187): 340-34310.1038/nature16937Crossref PubMed Scopus (325) Google Scholar). Wnt-3a with Flag inserted at the same position (Wnt-3a–iFlag) is secreted from cells and binds to FZDs and LRP6 (17). More recently, an N-terminal GFP-tagged version of mouse Wnt-3a (GFP-Wnt-3a) was reported that is secreted from cells and shows partial activity (18Takada R. Mii Y. Krayukhina E. Maruyama Y. Mio K. Sasaki Y. Shinkawa T. Pack C.G. Sako Y. Sato C. Uchiyama S. Takada S. Assembly of protein complexes restricts diffusion of Wnt3a proteins.Commun. Biol. 2018; 1 (30320232): 16510.1038/s42003-018-0172-xCrossref PubMed Scopus (13) Google Scholar). Despite these advancements, studies reporting quantitative analysis of the binding of a full-length, soluble Wnt protein to a full-length FZD protein on cells remain elusive. Here, we describe a novel approach using full-length versions of Wnt-3a and FZDs to study the biology and biophysics of receptor engagement in real time using living cells. A combination of two recent developments enabled this: 1) the availability of a fluorescently tagged Wnt protein that is active, stable, and secreted into the medium of cultured cells, and 2) a highly sensitive proximity-based bioluminescence resonance energy transfer (BRET) assay, termed NanoBRET (19Stoddart L.A. Johnstone E.K.M. Wheal A.J. Goulding J. Robers M.B. Machleidt T. Wood K.V. Hill S.J. Pfleger K.D.G. Application of BRET to monitor ligand binding to GPCRs.Nat. Methods. 2015; 12 (26030448): 661-66310.1038/nmeth.3398Crossref PubMed Scopus (177) Google Scholar), which relies on resonance energy transfer from a cell surface-localized Nanoluciferase (Nluc) to a nearby enhanced GFP (eGFP). Compared with the standard luciferase from Renilla reniformis, Nluc from the deep-sea shrimp Oplophorus gracilirostris is smaller and brighter and has a spectrum reduced by ∼20 nm, all of which lends itself well to the precise study of ligand-receptor interactions in the complex milieu of living cells (19Stoddart L.A. Johnstone E.K.M. Wheal A.J. Goulding J. Robers M.B. Machleidt T. Wood K.V. Hill S.J. Pfleger K.D.G. Application of BRET to monitor ligand binding to GPCRs.Nat. Methods. 2015; 12 (26030448): 661-66310.1038/nmeth.3398Crossref PubMed Scopus (177) Google Scholar, 20Hall M.P. Unch J. Binkowski B.F. Valley M.P. Butler B.L. Wood M.G. Otto P. Zimmerman K. Vidugiris G. Machleidt T. Robers M.B. Benink H.A. Eggers C.T. Slater M.R. Meisenheimer P.L. et al.Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.ACS Chem. Biol. 2012; 7 (22894855): 1848-185710.1021/cb3002478Crossref PubMed Scopus (899) Google Scholar). Our analysis of Wnt-3a binding to full-length FZD4 indicates that a high-affinity Wnt-FZD pair presents with a low-nanomolar equilibrium dissociation constant in living cells. Furthermore, we identify Afamin-dependent differences in the association of Wnt-3a with FZD4. Encouraged by recent reports of successful tagging of mouse Wnt-3a (16Farin H.F. Jordens I. Mosa M.H. Basak O. Korving J. Tauriello D.V. de Punder K. Angers S. Peters P.J. Maurice M.M. Clevers H. Visualization of a short-range Wnt gradient in the intestinal stem-cell niche.Nature. 2016; 530 (26863187): 340-34310.1038/nature16937Crossref PubMed Scopus (325) Google Scholar, 17Gerlach J.P. Jordens I. Tauriello D.V.F. van 'T Land-Kuper I. Bugter J.M. Noordstra I. van der Kooij J. Low T.Y. Pimentel-Muinos F.X. Xanthakis D. Fenderico N. Rabouille C. Heck A.J.R. Egan D.A. Maurice M.M. TMEM59 potentiates Wnt signaling by promoting signalosome formation.Proc Natl Acad Sci U S A. 2018; 115: E3996-E400510.1073/pnas.1721321115Crossref PubMed Scopus (30) Google Scholar, 18Takada R. Mii Y. Krayukhina E. Maruyama Y. Mio K. Sasaki Y. Shinkawa T. Pack C.G. Sako Y. Sato C. Uchiyama S. Takada S. Assembly of protein complexes restricts diffusion of Wnt3a proteins.Commun. Biol. 2018; 1 (30320232): 16510.1038/s42003-018-0172-xCrossref PubMed Scopus (13) Google Scholar), we generated eGFP-Wnt-3a with the aim of utilizing this for ligand-receptor interaction studies. We fused eGFP directly to the N terminus of Wnt-3a, which projects away from the Wnt/FZD-CRD binding region (21Hirai H. Matoba K. Mihara E. Arimori T. Takagi J. Crystal structure of a mammalian Wnt-frizzled complex.Nat. Struct. Mol. Biol. 2019; 26 (31036956): 372-37910.1038/s41594-019-0216-zCrossref PubMed Scopus (60) Google Scholar, 22Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337 (22653731): 59-6410.1126/science.1222879Crossref PubMed Scopus (568) Google Scholar), using a short peptide linker (Fig. 1a and Fig. S1a). Our construct is similar to Flag-GFP-Wnt-3a generated by Takada et al. but lacks the Flag epitope tag (18Takada R. Mii Y. Krayukhina E. Maruyama Y. Mio K. Sasaki Y. Shinkawa T. Pack C.G. Sako Y. Sato C. Uchiyama S. Takada S. Assembly of protein complexes restricts diffusion of Wnt3a proteins.Commun. Biol. 2018; 1 (30320232): 16510.1038/s42003-018-0172-xCrossref PubMed Scopus (13) Google Scholar). We generated a stably expressing L-cell line, which secretes biochemically stable eGFP-mWnt-3a into the medium similarly to WT Wnt-3a (Fig. 1b, lower panels, and Fig. S1b). When the conditioned medium (CM) is tested for functional activity using T-cell factor (TCF) reporter assays, eGFP-Wnt-3a behaves like WT Wnt-3a when using NCI-H1703 cells (Fig. 1b, upper graph). H1703 cells were chosen because they display strong Wnt/β-catenin signaling activity (23Winn R.A. Bremnes R.M. Bemis L. Franklin W.A. Miller Y.E. Cool C. Heasley L.E. Gamma-catenin expression is reduced or absent in a subset of human lung cancers and re-expression inhibits transformed cell growth.Oncogene. 2002; 21 (12386812): 7497-750610.1038/sj.onc.1205963Crossref PubMed Scopus (97) Google Scholar) and distinct cell surface expression of overexpressed Wnt receptors. Using HEK293 cells, however, eGFP-Wnt-3a shows only ≈20% activity of WT Wnt-3a (Fig. 1b, upper right graph). Transfection of LRP6 increased the basal Wnt activity of HEK293 cells and, under these conditions, the activity of eGFP-Wnt-3a relative to that of Wnt-3a increased from ≈20% to ≈60% (Fig. S1d). We next tested the ability of eGFP-Wnt-3a CM to induce LRP6 phosphorylation, which occurs in response to acute pathway stimulation (24Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation.Nature. 2005; 438 (16341017): 873-87710.1038/nature04185Crossref PubMed Scopus (653) Google Scholar, 25Davidson G. Wu W. Shen J. Bilic J. Fenger U. Stannek P. Glinka A. Niehrs C. Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction.Nature. 2005; 438 (16341016): 867-87210.1038/nature04170Crossref PubMed Scopus (465) Google Scholar). Similar to Wnt-3a CM, the addition of eGFP-Wnt-3a CM to either HEK293 cells (Fig. 1c) or NCI-H1703 cells (Fig. S1, d–e) promotes phosphorylation of endogenous LRP6, which is accompanied by the characteristic upshift of the mature, cell surface LRP6 protein band (Fig. 1c and Fig. S1d). Further, we validated the activity of eGFP-Wnt-3a in vivo in Xenopus embryos, which provides a physiologically relevant model system for assessing Wnt activity (26Hoppler S. Studying Wnt signaling in Xenopus.Methods Mol. Biol. 2008; 469 (19109718): 319-33210.1007/978-1-60327-469-2_21Crossref PubMed Scopus (8) Google Scholar). Duplication of the primary embryonic axis is well known to be robustly induced by Wnts (27McMahon A.P. Moon R.T. Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads to duplication of the embryonic axis.Cell. 1989; 58 (2673541): 1075-108410.1016/0092-8674(89)90506-0Abstract Full Text PDF PubMed Scopus (392) Google Scholar), and mouse eGFP-Wnt-3a displays activities similar to those of untagged Wnt-3a in the axis duplication assay (Fig. 1d). Wnt-3a/eGFP-Wnt-3a CM was also prepared using transiently transfected HEK293F suspension cells in serum-free medium (Fig. S1e). The serum component Afamin binds to Wnt-3a and aids its secretion and/or release from cells into serum-free medium (28Mihara E. Hirai H. Yamamoto H. Tamura-Kawakami K. Matano M. Kikuchi A. Sato T. Takagi J. Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/alpha-albumin.Elife. 2016; 5: e1162110.7554/eLife.11621Crossref PubMed Scopus (107) Google Scholar), and Afamin also improved eGFP-Wnt-3a secretion (Fig. S1f). Taken together, these results confirm that synthesis, secretion, stability, and signaling activity of eGFP-Wnt-3a are largely preserved upon fusion of the 25-kDa eGFP protein to the N terminus of Wnt-3a. Wnt proteins are acylated in the endoplasmic reticulum by the palmitoyltransferase porcupine (29Gao X. Hannoush R.N. Single-cell imaging of Wnt palmitoylation by the acyltransferase porcupine.Nat. Chem. Biol. 2014; 10 (24292069): 61-6810.1038/nchembio.1392Crossref PubMed Scopus (120) Google Scholar), and the Wnt-specific chaperone EVI/Wntless mediates subsequent transport through the secretory pathway, which is necessary for correct cell surface transportation and release of lipidated Wnts from cells (30Bartscherer K. Boutros M. Regulation of Wnt protein secretion and its role in gradient formation.EMBO Rep. 2008; 9 (18787559): 977-98210.1038/embor.2008.167Crossref PubMed Scopus (80) Google Scholar, 31Herr P. Basler K. Porcupine-mediated lipidation is required for Wnt recognition by Wls.Dev. Biol. 2012; 361 (22108505): 392-40210.1016/j.ydbio.2011.11.003Crossref PubMed Scopus (136) Google Scholar). Since eGFP-Wnt-3a is efficiently secreted from mammalian cells, we investigated whether secretion depends on EVI/Wntless using ΔEVI mutant cells. Expression levels of WT and eGFP-Wnt-3a are similar; however, neither is secreted in ΔEVI mutant HEK293 cells (Fig. 2a). Another characteristic of lipidated Wnt proteins is their trafficking in exosomes, which is one mechanism that accounts for Wnt secretion (32Zhang L. Wrana J.L. The emerging role of exosomes in Wnt secretion and transport.Curr. Opin. Genet. Dev. 2014; 27 (24791688): 14-1910.1016/j.gde.2014.03.006Crossref PubMed Scopus (59) Google Scholar, 33Gross J.C. Chaudhary V. Bartscherer K. Boutros M. Active Wnt proteins are secreted on exosomes.Nat. Cell Biol. 2012; 14 (22983114): 1036-104510.1038/ncb2574Crossref PubMed Scopus (680) Google Scholar). In a further confirmation of the functional similarity between WT and fluorescently tagged Wnt-3a, exosomes purified from conditioned medium associate with both Wnt-3a and eGFP-Wnt-3a (Fig. 2b and Fig. S2). This was the case for exosomes purified using either ultracentrifugation (Fig. 2b and Fig. S2) or magnetic-activated cell sorting antibody-based sorting (Fig. S2). Exosomes were prepared from either HEK293 adherent cells in serum-containing medium (Fig. 2b) or HEK293F suspension cells in serum-free medium (Fig. S2). Wnt-3a acts through FZDs and LRP5/6 to transduce Wnt/β-catenin signaling manifested in the phosphorylation of LRP5/6, phosphorylation of the scaffold protein Disheveled (DVL), stabilization of β-catenin, and transcriptional regulation of TCF/LEF-dependent genes. Although endogenously expressed FZDs in HEK293 cells are sufficient to mediate WNT/β-catenin pathway activation, removal of FZD expression blunts Wnt-induced signaling (34Eubelen M. Bostaille N. Cabochette P. Gauquier A. Tebabi P. Dumitru A.C. Koehler M. Gut P. Alsteens D. Stainier D.Y.R. Garcia-Pino A. Vanhollebeke B. A molecular mechanism for Wnt ligand-specific signaling.Science. 2018; 361 (30026314): eaat117810.1126/science.aat1178Crossref PubMed Scopus (116) Google Scholar, 35Voloshanenko O. Gmach P. Winter J. Kranz D. Boutros M. 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To define the role of FZDs in the eGFP-Wnt-3a-induced WNT/β-catenin response, we compared the transcriptional response induced by untagged Wnt-3a and eGFP-Wnt-3a in WT HEK293 cells or HEK293 cells lacking FZD1,2,4,5,7,8 (35Voloshanenko O. Gmach P. Winter J. Kranz D. Boutros M. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.FASEB J. 2017; 31 (28733458): 4832-484410.1096/fj.201700144RCrossref PubMed Scopus (68) Google Scholar). The complete absence of a Wnt response in FZD mutant HEK293 cells emphasizes that Wnt/β-catenin signaling activated by eGFP-Wnt-3a is transduced in an FZD-dependent manner (Fig. 2c). Transfection of FZD2 in the FZD mutant cells restores the ability of both untagged Wnt-3a and eGFP-Wnt-3a to transduce WNT/β-catenin signaling (Fig. 2c). Extracellular dispersal of Wnt proteins contributes to signaling events (38Pani A.M. Goldstein B. Direct visualization of a native Wnt in vivo reveals that a long-range Wnt gradient forms by extracellular dispersal.Elife. 2018; 7: e3832510.7554/eLife.38325Crossref PubMed Scopus (46) Google Scholar), and it is reported that Wnt-3a forms homotrimeric complexes, with dynamic formation of larger structures controlling the range with which Wnt proteins can diffuse through the extracellular space (18Takada R. Mii Y. Krayukhina E. Maruyama Y. Mio K. Sasaki Y. Shinkawa T. Pack C.G. Sako Y. Sato C. Uchiyama S. Takada S. Assembly of protein complexes restricts diffusion of Wnt3a proteins.Commun. Biol. 2018; 1 (30320232): 16510.1038/s42003-018-0172-xCrossref PubMed Scopus (13) Google Scholar). To study the diffusion of our fluorescently tagged Wnt-3a, we developed a controllable, microdroplet-based cell spheroid formation and fusion assay (see Materials and methods for details), which provides a visual readout of Wnt paracrine signaling. For the fluorescently tagged Wnt-3a, spheroids are prepared from HEK293 cells transfected with mCherry-Wnt-3a, which has an activity similar to that of eGFP-Wnt-3a (Fig. 3a). For the Wnt/β-catenin signaling readout, we prepared spheroids of TOP-GFP HEK293 reporter cells, representing a TCF activity-reporting fluorescent reporter assay (see Supporting information for details). Confocal imaging of spheroids was performed 24 h or 48 h after their fusion, and the activation of TCF activity evoked by mCherry-Wnt3a-secreting cells is evident (Fig. 3b). The strongest TOP-GFP reporter activation is detected in cells that are in direct contact with the mCherry-Wnt-3a-expressing cells (see arrows in Fig. 3b, 24 h); however, distinct activation of TOP-GFP is observed in all spheroid cells, including those located several cell diameters away from mCherry-Wnt-3a-expressing cells. Background activation of fluorescence in TOP-GFP spheroids is low at the 24-h time point, and fluorescence levels are also similar after their fusion with control spheroids prepared from LacZ-transfected cells (Fig. 3b, 24 h), which confirms specific and mCherry-Wnt-3a-dependent activation. At the 48-h time point, background TOP-GFP reporter fluorescence increased only marginally in the control spheroid fusions, whereas the fluorescence intensity in TOP-GFP spheroids fused to mCherry-Wnt-3a-producing spheroids increased substantially (Fig. 3, b and c). These results indicate that fluorescently tagged Wnt-3a can readily diffuse between the tightly packed cells in the spheroids to signal at a distance. Wnts bind to FZDs with some degree of promiscuity (9Dijksterhuis J.P. Baljinnyam B. Stanger K. Sercan H.O. Ji Y. Andres O. Rubin J.S. Hannoush R.N. Schulte G. Systematic mapping of WNT-Frizzled interactions reveals functional selectivity by distinct WNT-Frizzled pairs.J. Biol. 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To assess the relative interaction of eGFP-Wnt-3a to FZDs on live cells, we generated stable NCI-H1703 cell lines expressing moderate levels of FZD1–10 C-terminally tagged with mCherry at the plasma membrane (Fig. 4a and Fig. S3b). Cell lines with broadly similar expression levels of FZD1,2,4,6,7,8,9 at the cell surface were selected for imaging (Fig. 4a, top); however, suitable cell lines for FZD3,5,10 could not be obtained. The application of eGFP-Wnt-3a-containing CM resulted in a strong increase