Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments

Abstract

While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity.

Keywords: Wnt/β-catenin pathway; canonical Wnt; lipid raft; membrane trafficking; plasma membrane lipid.

Figure 1

Membrane order preference of the canonical Wnt ligand and its receptors. (A) Western blot of detergent extracts of the plasma membrane for Fz8 (detected by its Flag tag), Lrp6, Lypd6 (detected by its GFP tag), Wnt3 (detected by its GFP tag), Wnt3a [from conditioned media (CM)], recombinant Wnt3a (Wnt3a rec), Wnt8a, and the controls caveolin1 (Cav1 with known detergent‐resistant fraction preference) and transferrin‐receptor (TfR2 with known detergent soluble fraction preference) with DRM and soluble positions marked. (B) A schematic of SPIM‐FCS measurements. A focal volume is formed with a single plane illumination. Signal is recorded from the apical membrane of the cells. Later, the signal is correlated with different bin sizes to form varying sizes of observation areas. With larger area, the diffusion time is higher while with a smaller area, the diffusion time is lower. The extent of this change depends on the diffusion mode of the molecule, whether it is free or hindered. Dependence of diffusion time to observation area is shown for different diffusion modes; domain, free, and meshwork diffusion. (C) Wnt3 undergoes domain‐like diffusion unlike DiI‐C₁₈ (used as a control for free diffusion). Statistical significance between the two treatments is determined by Kolmogorov–Smirnov (KS) test statistics. Error bars represent the standard error of the mean at each binned area (number of data points is 36 for 1 × 1 binning, 25 for 2 × 2 binning, and 16 for 3 × 3 binning).

Figure 2

Plasma membrane order is altered upon canonical Wnt stimulation. (A) Representative generalized polarization (GP) maps in the plasma membrane of HEK293T cells treated with either control‐ (top) or Wnt3a‐conditioned media (CM) (bottom) using the polarity sensitive dye Di‐4‐AN(F)EPPTEA and confocal microscopy in combination with spectral detection (large GP values indicate high membrane ordering; red in the color scale). (B) Average and standard deviation (error bars) of GP values determined from 10 images (each image contained multiple cells) for both Wnt3a CM and Wnt3a rec. (C) Average and standard deviation (error bars, three independent experiments) of fluorescence emission spectra of SL2 in HEK293T cells treated with either control‐conditioned (black) or Wnt3a‐conditioned (red) media (for panel C, P value was determined using the intensity at λ = 475 nm). Statistical significance was determined using unpaired t‐test. ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 3

Ordered membrane environments are essential for Wnt3a‐mediated β‐catenin signaling. (A) Average and standard deviation (error bars, three independent experiments) values of pBAR luciferase reporter activity monitoring Wnt/β‐catenin signaling activity (normalized to renilla luciferase activity) in HEK293T cells treated with COase, myriocin, or oseltamivir and induced with the control‐ or Wnt3a‐conditioned media. (B, C) GFP or mCherry whole‐mount in situ hybridization showing downregulation of signaling in the two transgenic zebrafish embryos TOPdGFP (B) and 7xTcf:mCherry (C) after treatment with COase, myriocin, and oseltamivir. Arrows highlight the regions where reduction in Wnt/β‐catenin signaling activity is observed. All expression domains detected in the control embryos are downregulated after drug treatment. Note that the drugs appear to influence these domains differently. amd, anterior‐most domain; mhb, midbrain–hindbrain boundary; otv, otic vesicle; llp, lateral line primordium; pmes, posterior mesoderm. (D) Morphological phenotypes at 24 hpf of embryos treated with cholesterol oxidase (3 U·mL⁻¹, 23/24 embryos), myriocin (187.5 μm, 19/21 embryos), or oseltamivir (150 μm, 19/19 embryos) for 19 h. Arrows and the dashed line show the reduction of size in trunk and tail. (E) FCS diffusion law of Wnt3‐EGFP in live transgenic embryos treated with COase, myriocin, or oseltamivir. COase completely diminishes the domain diffusion while myriocin or oseltamivir significantly reduces it. Error bars represent the standard error of the mean at each binned area (number of data points is 36 for 1 × 1 binning, 25 for 2 × 2 binning, and 16 for 3 × 3 binning). Statistical significance is evaluated by unpaired t‐test for luciferase assay (panel A) and by a Kolmogorov‐Smirnov (KS) test, for SPIM‐FCS diffusion data (panel E). ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 4

Wnt/β‐catenin signaling in Niemann–Pick Type C (NPC) cells. Cholesterol distribution in (A) CHO‐wt cells and (B) CHO‐NPC (−/−) cells. Filipin (green) labels cholesterol, lysotracker (magenta) labels lysosomes. Scale bars are 20 μm. (C) Wnt/β‐catenin signaling is reduced in CHO NPC (−/−) cells compared to CHO‐wt cells. (D) cdx4 and axin2 expression levels determined by qRT‐PCR in CHO‐NPC (−/−) cells shown relative to those in CHO‐wt cells. (E) β‐catenin localization in nuclear (marked by Histon H3) and cytoplasmic (marked by β‐tubulin) portions in CHO‐wt and CHO‐NPC (−/−) cells and (F) quantitation of this localization. Nuclear β‐catenin fraction in CHO‐NPC (−/−) cells is reduced (0.3) as compared to CHO‐wt cells (0.5). (G) Wnt/β‐catenin signaling activity in CHO‐NPC (−/−) cells fed with cholesterol, DOPC, sphingomyelin, and GM1. DOPC does not alter the signaling capacity of cells while cholesterol, sphingomyelin, GM1 and a mixture of these three increase the signaling. Statistical significance was evaluated by unpaired t‐test. Error bars are standard deviations of three independent experiments. **P < 0.01, *P < 0.05.