Wesslowski J et al. (2020), eGFP-tagged Wnt-3a enables functional analysis ...

XB-ART-56968

J Biol Chem 2020 Jun 26;29526:8759-8774. doi: 10.1074/jbc.RA120.012892.

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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.

Wesslowski J , Kozielewicz P , Wang X , Cui H , Schihada H , Kranz D , Karuna M P , Levkin P , Gross JC , Boutros M , Schulte G , Davidson G .

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)/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.

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Species referenced: Xenopus
Genes referenced: ctnnb1 fzd2 fzd4 fzd8 lrp6 pdcd6ip sdcbp wls wnt3a
GO keywords: frizzled binding [+]

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	Figure 1. Generation of functionally active eGFP-Wnt-3a. a) schematic overview of the eGFP-Wnt- 3a fusion protein construct. For more details refer to Fig. S1a. The protein complex model depicts human Wnt-8 (violet) and the mouse FZD8-CRD (bronze) based on the crystal structure (PDB 6AHY). The N terminus, where linker and eGFP are fused, is highlighted in red and the C-terminal region is also indicated. The palmitoleoyl chain (PAM) is shown in yellow. The model was created in PyMOL (The PyMOL Molecular Graphics System, Version 2.0; Schrödinger, LLC). b) Western blot (lower panels, anti-Wnt-3a antibody) and TOPFLASH TCF reporter assays (upper graphs) showing presence of soluble Wnt-3a and eGFP-Wnt-3a in conditioned medium (CM) from stable L-cell lines and activity testing of the indicated CM in either NCI-H1703 or HEK293 cells, respectively. Note that CM is prepared using appropriate medium for the cell lines used (see Experimental procedures for details). Error bars represent ± SD from mean of 4 independent biological samples, represented as solid dots. Experiments were performed 3 times with similar results. c) Western blots showing Wnt-dependent phosphorylation and associated up-shift of endogenous LRP6 protein bands from HEK293 cells after the addition of either Wnt-3a or eGFP-Wnt-3a L-cell CM as indicated. The CM samples shown in (b) were used. Experiments were repeated at least three times with similar results. d) Axis duplication assay in Xenopus laevis embryos. 2.5 ng of either wild-type Wnt-3a or eGFP-Wnt-3a mRNA were injected equatorially in both ventral blastomeres of 4-cell stage embryos. Embryos (n defines total number of embryos evaluated) were scored for presence of a secondary axis the next day (stage 28) and error bars represent ± SD from mean between three independent batches of injected embryos. Arrows indicate the two primary body axes.
	Figure 2. EVI-dependent eGFP-Wnt-3a secretion and association of eGFP-Wnt-3a with exosomes a) Western blots of indicated proteins from medium (secreted) and corresponding cell lysates from wild- type HEK293T cells or EVI knockout HEK293T cells (HEK293T EVI-KO2.9), transfected as indicated. Asterisks indicate non-specific bands. b) Western blot analysis of proteins from purified exosomes derived from HEK293 cells transfected as indicated with Wnt-3a, eGFP-Wnt-3a or a 1:1 combination of both. Exosomes were purified by ultracentrifugation (see Experimental procedures for details). The exosome marker proteins Alix and Syntenin are indicated. c) TCF reporter assay using either wild-type HEK293T cells or ∆FZD1,2,4,5,7,8 HEK293T cells transfected with FZD2, FLUC, RLUC in combination with control (pcDNA), Wnt-3a or eGFP-Wnt-3a. Error bars represent mean ± SD from 5 independent biological samples, represented as solid dots. The experiment was performed 2 times with similar results.
	Figure 3. Diffusion of mCherry-Wnt-3a within spheroids. a) TOPFLASH TCF reporter assay showing the comparative activities of mCherry-Wnt-3a and eGFP-Wnt-3a, when applied to HEK293 cells as L cell conditioned medium (CM). Error bars represent ± SD from mean of 4 independent biological samples, represented as solid dots. The experiment was performed 2 times with similar results. Western blot analysis showing eGFP-Wnt-3a and mCherry-Wnt-3a proteins in the CM is shown in lower panel. b) HEK293T cell spheroid fusion assay for visualisation of paracrine Wnt/β-catenin signaling. mCherry-Wnt-3a or control LacZ transfected HEK293 cells were used to form spheroids, which were then combined with spheroids prepared from a stable TOP-GFP HEK293 reporter cell line. 24 or 48 hours later, merged spheroids were imaged using laser scanning confocal microscopy for activation of Wnt/β-catenin signaling in the TOP-GFP reporter cells (green cells). The two lower arrows mark mCherry-Wnt3a producing cells flanking a highly-activated TOP-GFP reporter cell (smaller upper arrow). BF, bright-field image showing fused spheroids with the dashed line demarking the border between the Wnt secreting/control cells and the TOP-GFP reporter cells (TOP). The presented images are representative of findings obtained from 3 independent experiments. c) Bar graph showing the relative fluorescent intensities measured in the TOP‐GFP spheroids shown in Fig. 3b, calculated from at least 9 merged spheroids in each of the assays conditions. Error bars represent mean ± SD from 10 independent fluorescence intensity values, represented as solid dots. The fluorescence intensity of the 24 h control TOP-GFP spheroids is set to 1.
	Figure 4. Association of eGFP-Wnt-3a with different FZDs. a) Laser scanning confocal microscopy images of living NCI-H1703 cells with stable integration of the indicated mouse FZD-mCherry gene, incubated for 3 hours with eGFP-Wnt-3a conditioned medium (CM) derived from L cells. b) Laser scanning confocal microscopy images of living ∆FZD1-10GFP-free HEK293 cells transiently transfected with the indicated mouse FZD-mCherry gene and incubated for 1 hour with eGFP-Wnt-3a CM derived from L cells. A direct comparison of the fluorescent intensities between the two cells lines in (a) and (b) is provided in Fig. S3b. c) TOPFLASH TCF reporter assay responses in ∆FZD1-10 HEK293 cells, showing the relative Wnt/β-catenin signaling activity of the indicated mouse FZDs as well as their mCherry tagged counterparts. Error bars represent ± SD from mean of 4 independent biological samples, represented as solid dots. The experiment was performed 3 times with similar results.
	Figure 5. eGFP-Wnt-3a binding to Nluc-FZD4. a) Schematic illustration of the NanoBRET setup to detect eGFP-Wnt-3a binding to Nluc-FZD-tagged receptors. b) Association kinetics of the HEK293F suspension cell-derived eGFP-Wnt-3a to Nluc-FZD4 were determined by the detection of NanoBRET in transiently-overexpressing living ΔFZD1-10 HEK293 cells over time. The eGFP-Wnt-3a was produced either with (left graph) or without (right graph) cellular co-expression of Afamin. NanoBRET was sampled once per 60 sec for 180 min. Data points are presented as mean ± SD (+Afamin) or mean ± SEM (-Afamin) from n=3 individual experiments, fitting a one-phase association model. The kobs values from the individual eGFP-Wnt-3a association curves were plotted over eGFP-Wnt-3a concentration. c) Saturation curves are presented as sigmoidal curves with logarithmic eGFP-Wnt-3a concentrations (the left panel for eGFP-Wnt-3a + Afamin and the right panel for eGFP-Wnt-3a - Afamin). Graphs present raw NanoBRET values obtained following 2 h ligand exposure to living ΔFZD1-10 HEK293 cells transiently-overexpressing Nluc-FZD4. d) Raw BRET ratio values for eGFP-Wnt-3a binding from c were normalized to 0 and 100 % to emphasize the difference in affinity. Data points are presented as mean ± SEM from n=3 individual experiments.

References [+] :

Arthofer, WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα12/13. 2016, Pubmed