Biochemical Methods to Analyze Wnt Protein Secretion

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.

Neutral sphingomyelinases control extracellular vesicles budding from the plasma membrane

“…MVs and Exos were isolated from fresh cell culture supernatant by differential centrifugation [39] (Figure 1(a)). In brief, after two steps at 750 and 1500 g to pellet cells and debris, the supernatant (SN) was centrifuged at 14,000 g for 35 min and the pellet (P14) washed in PBS.…”

ERAD‐dependent control of the Wnt secretory factor Evi

“…Wnt reporter activity assay was performed as described previously (Glaeser et al , 2016) with some modifications. Wild‐type, Porcn KO1.2 , and Porcn KO1.4 HEK293T cells were transfected in white, flat‐bottom 384‐well plates with 20 ng TCF4‐Firefly Luciferase, 10 ng Actin‐Renilla Luciferase and 20 ng of Wnt3A, Dvl3, or pcDNA3.1 plasmids.…”

“…The original Blue Sepharose protocol (Ross et al , 2014) was modified according to Glaeser et al , 2016. Conditioned medium was recovered ~72 h after cell seeding and ~48 h after plasmid transfection to analyze the secretion of Wnt3A‐KDEL or 48 h after reverse transfection and 24 h after plasmid transfection to analyze the effect of ERAD components.…”

Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

“…The relative amount of Wnts secreted into cell culture supernatant was analyzed using Blue Sepharose precipitation as described (Willert et al, 2003;Glaeser et al, 2016). Shortly, HEK293T cells were transiently transfected in 6-well plates with 1 µg of Wnt3A plasmids.…”