Abstract
Wnt signalling has important roles during development and in many diseases. As morphogens, hydrophobic Wnt proteins exert their function over a distance to induce patterning and cell differentiation decisions. Recent studies have identified several factors that are required for the secretion of Wnt proteins; however, how Wnts travel in the extracellular space remains a largely unresolved question. Here we show that Wnts are secreted on exosomes both during Drosophila development and in human cells. We demonstrate that exosomes carry Wnts on their surface to induce Wnt signalling activity in target cells. Together with the cargo receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE Ykt6. Our study demonstrates an evolutionarily conserved functional role of extracellular vesicular transport of Wnt proteins.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Neumann, C. J. & Cohen, S. M. Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124, 871–880 (1997).
Zecca, M., Basler, K. & Struhl, G. Direct and long-range action of a wingless morphogen gradient. Cell 87, 833–844 (1996).
Takada, R. et al. Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Dev. Cell 11, 791–801 (2006).
Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452 (2003).
Bartscherer, K. & Boutros, M. Regulation of Wnt protein secretion and its role in gradient formation. EMBO Rep. 9, 977–982 (2008).
Coudreuse, D. Y., Roel, G., Betist, M. C., Destree, O. & Korswagen, H. C. Wnt gradient formation requires retromer function in Wnt-producing cells. Science 312, 921–924 (2006).
Port, F. & Basler, K. Wnt trafficking: new insights into Wnt maturation, secretion and spreading. Traffic 11, 1265–1271 (2010).
Doubravska, L. et al. Fatty acid modification of Wnt1 and Wnt3a at serine is prerequisite for lipidation at cysteine and is essential for Wnt signalling. Cell Signal. 23, 837–848 (2011).
Buechling, T., Chaudhary, V., Spirohn, K., Weiss, M. & Boutros, M. p24 proteins are required for secretion of Wnt ligands. EMBO Rep. 12, 1265–1272 (2011).
Port, F., Hausmann, G. & Basler, K. A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion. EMBO Rep. 12, 1144–1152 (2011).
Bänziger, C. et al. Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125, 509–522 (2006).
Bartscherer, K., Pelte, N., Ingelfinger, D. & Boutros, M. Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125, 523–533 (2006).
Goodman, R. M. et al. Sprinter: a novel transmembrane protein required for Wg secretion and signaling. Development 133, 4901–4911 (2006).
Franch-Marro, X., Wendler, F., Griffith, J., Maurice, M. M. & Vincent, J-P. In vivo role of lipid adducts on Wingless. J. Cell Sci. 121, 1587–1592 (2008).
Herr, P. & Basler, K. Porcupine-mediated lipidation is required for Wnt recognition by Wls. Dev. Biol. 361, 392–402 (2012).
Belenkaya, T. Y. et al. The retromer complex influences Wnt secretion byrecycling wntless from endosomes to the trans-Golgi network. Dev. Cell 14, 120–131 (2008).
Franch-Marro, X. et al. Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex. Nat. Cell Biol. 10, 170–177 (2008).
Port, F. et al. Wingless secretion promotes and requires retromer-dependent cycling of Wntless. Nat. Cell Biol. 10, 178–185 (2008).
Pan, C. L. et al. C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless. Dev. Cell 14, 132–139 (2008).
Yang, P. T. et al. Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells. Dev. Cell 14, 140–147 (2008).
Harterink, M. et al. A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nat. Cell Biol. 13, 1–12 (2011).
Zhang, P., Wu, Y., Belenkaya, T. Y. & Lin, X. SNX3 controls Wingless/Wnt secretion through regulating retromer-dependent recycling of Wntless. Cell Res. 21, 1677–1690 (2011).
Coombs, G. S. et al. WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification. J. Cell Sci. 123, 3357–3367 (2010).
Greco, V., Hannus, M. & Eaton, S. Argosomes: a potential vehicle for the spread of morphogens through epithelia. Cell 106, 633–645 (2001).
Neumann, S. et al. Mammalian Wnt3a is released on lipoprotein particles. Traffic 10, 334–343 (2009).
Panáková, D., Sprong, H., Marois, E., Thiele, C. & Eaton, S. Lipoproteinparticles are required for Hedgehog and Wingless signalling. Nature 435, 58–65 (2005).
Mulligan, K. A. et al. Secreted wingless-interacting molecule (Swim) promotes long-range signaling by maintaining Wingless solubility. Proc. Natl Acad. Sci. USA 109, 370–377 (2012).
Taelman, V. F. et al. Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes. Cell 143, 1136–1148 (2010).
Korkut, C. et al. Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless. Cell 139, 393–404 (2009).
Koles, K. et al. Mechanism of Evi-exosome release at synaptic boutons. J. Biol. Chem. 20, 16820–16834 (2012).
Théry, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Chapter 3, Unit 3.22. (2006).
Escola, J. M. et al. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J. Biol. Chem. 273, 20121–20127 (1998).
Laulagnier, K. et al. Characterization of exosome subpopulations from RBL-2H3 cells using fluorescent lipids. Blood Cells Mol. Dis. 35, 116–121 (2005).
Stoeck, A. et al. A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44. Biochem. J. 393, 609–618 (2006).
Foreman, J. R. et al. Fractionation of human serum lipoproteins by single-spin gradient ultracentrifugation: quantification of apolipoproteins B and A-1 and lipid components. J. Lipid Res. 18, 759–767 (1977).
Gutwein, P. et al. Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells. Clin. Cancer Res. 11, 2492–2501 (2005).
Koppen, T. et al. Proteomics analyses of Microvesicles released by Drosophila Kc167 and S2 cells. Proteomics 22, 4397–4410 (2011).
Rana, S. & Zoller, M. Exosome target cell selection and the importance of exosomal tetraspanins: a hypothesis. Biochem. Soc. Trans. 39, 559–562 (2011).
Huotari, J. & Helenius, A. Endosome maturation. EMBO J. 30, 3481–3500 (2011).
Tamai, K. et al. Exosome secretion of dendritic cells is regulated by Hrs, an ESCRT-0 protein. Biochem. Biophys. Res. Commun. 399, 384–390 (2010).
Trajkovic, K. et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 319, 1244–1247 (2008).
Sobota, J. A., Back, N., Eipper, B. A. & Mains, R. E. Inhibitors of the V0 subunit of the vacuolar H+-ATPase prevent segregation of lysosomal- and secretory-pathway proteins. J. Cell Sci. 122, 3542–3553 (2009).
Strigini, M. & Cohen, S. M. Wingless gradient formation in the Drosophila wing. Curr. Biol. 10, 293–300 (2000).
Carayon, K. et al. Proteolipidic composition of exosomes changes during reticulocyte maturation. J. Biol. Chem. 286, 34426–34439 (2011).
Simons, M. & Raposo, G. Exosomes–vesicular carriers for intercellular communication. Curr. Opin. Cell Biol. 21, 575–581 (2009).
Vidal, M. J. & Stahl, P. D. The small GTP-binding proteins Rab4 and ARF are associated with released exosomes during reticulocyte maturation. Eur. J. Cell Biol. 60, 261–267 (1993).
Hsu, C. et al. Regulation of exosome secretion by Rab35 and its GTPase-activating proteins TBC1D10A-C. J. Cell Biol. 189, 223–232 (2010).
Ostrowski, M. et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat. Cell Biol. 12, 11–13 (2010).
McNew, J. A. et al. Ykt6p, a prenylated SNARE essential for endoplasmic reticulum-Golgi transport. J. Biol. Chem. 272, 17776–17783 (1997).
Nair, U. et al. SNARE proteins are required for macroautophagy. Cell 146, 290–302 (2011).
Higginbotham, J. N. et al. Amphiregulin exosomes increase cancer cell invasion. Curr. Biol. 21, 779–786 (2011).
Meckes, D. G. Jr et al. Human tumor virus utilizes exosomes for intercellular communication. Proc. Natl Acad. Sci. USA 107, 20370–20375 (2010).
Mittelbrunn, M. et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat. Commun. 2, 282 (2010).
Havel, R. J., Eder, H. A. & Bragdon, J. H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34, 1345–1353 (1955).
Meiringer, C. T., Auffarth, K., Hou, H. & Ungermann, C. Depalmitoylation of Ykt6 prevents its entry into the multivesicular body pathway. Traffic 9, 1510–1521 (2008).
Tai, G. et al. Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the trans-Golgi network. Mol. Biol. Cell 15, 4011–4022 (2004).
Pons, V. et al. Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies. PLoS Biol. 6, e214 (2008).
Nothwehr, S.F., Ha, S.A. & Bruinsma, P. Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p. J. Cell Biol. 151, 297–310 (2000).
Strochlic, T. I., Setty, T. G., Sitaram, A. & Burd, C. G. Grd19/Snx3p functions as a cargo-specific adapter for retromer-dependent endocytic recycling. J. Cell Biol. 177, 115–125 (2007).
Voos, W. & Stevens, T.H. Retrieval of resident late-Golgi membrane proteins from the prevacuolar compartment of Saccharomyces cerevisiae is dependent on the function of Grd19p. J. Cell Biol. 140, 577–590 (1998).
Augustin, I. et al. The Wnt secretion protein Evi/Gpr177 promotes glioma tumourigenesis. EMBO Mol. Med. 4, 38–51 (2012).
Nolo, R., Abbott, L. A. & Bellen, H. J. Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila. Cell 102, 349–362 (2000).
Vaccari, T., Duchi, S., Cortese, K., Tacchetti, C. & Bilder, D. The vacuolar ATPase is required for physiological as well as pathological activation of the Notch receptor. Development 137, 1825–1832 (2010).
Montagne, J. et al. Drosophila S6 kinase: a regulator of cell size. Science 285, 2126–2129 (1999).
Thompson, B. J. & Cohen, S. M. The Hippo pathway regulates the bantam microRNA to control cell proliferation and apoptosis in Drosophila. Cell 126, 767–774 (2006).
Lee, H. H., Elia, N., Ghirlando, R., Lippincott-Schwartz, J. & Hurley, J. H. Midbody targeting of the ESCRT machinery by a noncanonical coiled coil in CEP55. Science 322, 576–580 (2008).
Jullien, J. & Gurdon, J. Morphogen gradient interpretation by a regulated trafficking step during ligand-receptor transduction. Gen. Dev. 19, 2682–2694 (2005).
Horn, T. & Boutros, M. E-RNAi: a web application for the multi-species design of RNAi reagents—2010 update. Nucleic Acids Res. 38, W332-339 (2010).
Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Gen. Biol. 7, R100 (2006).
Rogers, S. L., Rogers, G. C., Sharp, D. J. & Vale, R. D. Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J. Cell Biol. 158, 873–884 (2002).
Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101–1108 (2008).
Slot, J. W. & Geuze, H. J. Cryosectioning and immunolabeling. Nat. Protoc. 2, 2480–2491 (2007).
Acknowledgements
We thank T. Soellner, T. Buechling and D. Kranz for helpful comments on the manuscript. We would like to thank T. Vaccari, S. Eaton, S. Cohen, C. Niehrs, H. Bellen and the Bloomington stock centre for fly strains and reagents. We thank VDRC for the RNAi lines. We are grateful for help from the CellNetworks Electron Microscopy Core Facility (EMCF), the Light Microscopy Core Facility at the DKFZ and the Mass Spectrometry Core Facility at the DKFZ. J.C.G was supported by the CellNetworks postdoctoral fellowship programme. The DFG Wnt Research Group FOR1036 supported research in the laboratory of M.B.
Author information
Authors and Affiliations
Contributions
J.C.G. designed experiments, carried out the biochemical and cell-biological experiments and wrote the manuscript. V.C. designed experiments, carried out the in vivo experiments and wrote the manuscript. K.B. provided essential advice, generated in vivo reagents and contributed to writing the manuscript. M.B. designed experiments and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
Supplementary Information (PDF 1531 kb)
Supplementary Table 1
Supplementary Information (XLS 55 kb)
Supplementary Table 2
Supplementary Information (XLS 38 kb)
Rights and permissions
About this article
Cite this article
Gross, J., Chaudhary, V., Bartscherer, K. et al. Active Wnt proteins are secreted on exosomes. Nat Cell Biol 14, 1036–1045 (2012). https://doi.org/10.1038/ncb2574
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ncb2574
This article is cited by
-
The Ykt6–Snap29–Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles
Scientific Reports (2024)
-
Cytoneme-mediated transport of active Wnt5b–Ror2 complexes in zebrafish
Nature (2024)
-
New insights in ubiquitin-dependent Wnt receptor regulation in tumorigenesis
In Vitro Cellular & Developmental Biology - Animal (2024)
-
A review of the regulatory mechanisms of extracellular vesicles-mediated intercellular communication
Cell Communication and Signaling (2023)
-
Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression
Cell Communication and Signaling (2023)