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Biochemical Methods to Analyze Wnt Protein Secretion

  • Kathrin Glaeser
  • Michael Boutros
  • Julia Christina GrossEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1481)

Abstract

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.

Key words

Wnt signaling activity Hydrophobic proteins Wnt secretion Blue Sepharose Purified Wnt Wnts on extracellular vesicles 

Abbreviations

EV

Extracellular vesicles

MV

Microvesicles

MVBs

Multivesicular bodies

Notes

Acknowledgments

We would like to thank Suat Özbek for the mouse S209A Wnt3A overexpression plasmid and the CellNetworks Electron Microscopy Core Facility Heidelberg. This work was supported by the DFG Wnt research group FOR1036.

References

  1. 1.
    Clevers H, Loh KM, Nusse R (2014) Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science 346:1248012CrossRefPubMedGoogle Scholar
  2. 2.
    Tanaka K, Okabayashi K, Asashima M, Perrimon N, Kadowaki T (2000) The evolutionarily conserved porcupine gene family is involved in the processing of the Wnt family. Eur J Biochem 267:4300–4311CrossRefPubMedGoogle Scholar
  3. 3.
    Takada R, Satomi Y, Kurata T, Ueno N, Norioka S, Kondoh H, Takao T, Takada S (2006) Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Dev Cell 11:791–801CrossRefPubMedGoogle Scholar
  4. 4.
    Bänziger C, Soldini D, Schütt C, Zipperlen P, Hausmann G, Basler K (2006) Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125:509–522CrossRefPubMedGoogle Scholar
  5. 5.
    Bartscherer K, Pelte N, Ingelfinger D, Boutros M (2006) Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125:523–533CrossRefPubMedGoogle Scholar
  6. 6.
    Goodman RM, Thombre S, Firtina Z, Gray D, Betts D, Roebuck J, Spana EP, Selva EM (2006) Sprinter: a novel transmembrane protein required for Wg secretion and signaling. Development 133:4901–4911CrossRefPubMedGoogle Scholar
  7. 7.
    Janda CY, Waghray D, Levin AM, Thomas C, Garcia KC (2012) Structural basis of Wnt recognition by Frizzled. Science 337:59–64CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Panakova D, Sprong H, Marois E, Thiele C, Eaton S (2005) Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435:58–65CrossRefPubMedGoogle Scholar
  9. 9.
    Neumann S, Coudreuse DY, van der Westhuyzen DR, Eckhardt ER, Korswagen HC, Schmitz G, Sprong H (2009) Mammalian Wnt3a is released on lipoprotein particles. Traffic 10:334–343CrossRefPubMedGoogle Scholar
  10. 10.
    Hsiung F, Ramirez-Weber FA, Iwaki DD, Kornberg TB (2005) Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic. Nature 437:560–563CrossRefPubMedGoogle Scholar
  11. 11.
    Stanganello E, Hagemann AI, Mattes B, Sinner C, Meyen D, Weber S, Schug A, Raz E, Scholpp S (2015) Filopodia-based Wnt transport during vertebrate tissue patterning. Nat Commun 6:5846CrossRefPubMedGoogle Scholar
  12. 12.
    Gross JC, Chaudhary V, Bartscherer K, Boutros M (2012) Active Wnt proteins are secreted on exosomes. Nat Cell Biol 14:1036–1045CrossRefPubMedGoogle Scholar
  13. 13.
    Menck K, Klemm F, Gross JC, Pukrop T, Wenzel D, Binder C (2013) Induction and transport of Wnt 5a during macrophage-induced malignant invasion is mediated by two types of extracellular vesicles. Oncotarget 4:2057–2066CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Beckett K, Monier S, Palmer L, Alexandre C, Green H, Bonneil E, Raposo G, Thibault P, Le Borgne R, Vincent JP (2013) Drosophila S2 cells secrete wingless on exosome-like vesicles but the wingless gradient forms independently of exosomes. Traffic 14:82–96CrossRefPubMedGoogle Scholar
  15. 15.
    Yanez-Mo M, Siljander PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, Buzas K, Casal E, Cappello F, Carvalho J, Colas E, Cordeiro-da Silva A, Fais S, Falcon-Perez JM, Ghobrial IM et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066CrossRefPubMedGoogle Scholar
  16. 16.
    Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR III, Nusse R (2003) Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423:448–452CrossRefPubMedGoogle Scholar
  17. 17.
    Demir K, Kirsch N, Beretta CA, Erdmann G, Ingelfinger D, Moro E, Argenton F, Carl M, Niehrs C, Boutros M (2013) RAB8B is required for activity and caveolar endocytosis of LRP6. Cell Rep 4:1224–1234CrossRefPubMedGoogle Scholar
  18. 18.
    Ross J, Busch J, Mintz E, Ng D, Stanley A, Brafman D, Sutton VR, Van den Veyver I, Willert K (2014) A rare human syndrome provides genetic evidence that WNT signaling is required for reprogramming of fibroblasts to induced pluripotent stem cells. Cell Rep 9:1770–1780CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Thery C, Amigorena S, Raposo G, Clayton A (2014) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3:Unit 3 22Google Scholar
  20. 20.
    Liu J, Pan S, Hsieh MH, Ng N, Sun F, Wang T, Kasibhatla S, Schuller AG, Li AG, Cheng D, Li J, Tompkins C, Pferdekamper A, Steffy A, Cheng J et al (2013) Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974. Proc Natl Acad Sci U S A 110:20224–20229CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Kathrin Glaeser
    • 1
  • Michael Boutros
    • 1
  • Julia Christina Gross
    • 2
    Email author
  1. 1.Division Signaling and Functional Genomics and Heidelberg University, Department for Cell and Molecular Biology, German Cancer Research Center (DKFZ)Medical Faculty MannheimHeidelbergGermany
  2. 2.Haematology and Oncology and Developmental BiochemistryUniversity Medicine Göttingen, Göttingen Center for Molecular Biosciences (GZMB)GöttingenGermany

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