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.
Key wordsWnt signaling activity Hydrophobic proteins Wnt secretion Blue Sepharose Purified Wnt Wnts on extracellular vesicles
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.
- 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
- 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.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.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