XB-ART-55393Commun Biol January 1, 2018; 1 165.
Assembly of protein complexes restricts diffusion of Wnt3a proteins.
Members of the Wnt protein family play roles in many aspects of embryogenesis and homeostasis. Despite their biological significance, characteristics of Wnt proteins still remain unclear, mainly due to their insolubility after the removal of serum. Here we examine Wnt proteins in serum-containing media by using analytical ultracentrifugation with a fluorescence detection system. This analysis reveals that Wnt3a assembles into high-molecular-weight complexes that become dissociable by interaction with the extracellular domain of the Frizzled8 receptor or secreted Wnt-binding protein sFRP2. Cross-linking and single-particle analyses of Wnt3a fractionated by gel filtration chromatography show the homo-trimer to be the smallest form of the assembled Wnt3a complexes. Fluorescence correlation spectroscopy and immunohistochemistry reveal that the assembly of Wnt3a complexes restricted their diffusion and signaling range in Xenopus laevis embryos. Thus, we propose that the Wnt diffusion range can be controlled by a balance between the assembly of Wnt complexes and their dissociation.
PubMed ID: 30320232
PMC ID: PMC6179999
Article link: Commun Biol
Species referenced: Xenopus laevis
Genes referenced: btg2 fzd8 gbx2.2 otx2 sfrp2 wnt3a
Article Images: [+] show captions
|Fig. 2. Analytical ultracentrifugation analyses of GFP-tagged Wnt proteins with Fzd8 CRD or sFRP2. a Schematic representation of the analysis. b, c AUC-FDS analysis of conditioned medium of L cells secreting GFP-WntD (n = 7; (b)) and GFP-Wnt3a (n = 8; (c)) co-cultured with HEK293 cells secreting the CRD domain of mouse Fzd8 (pink line) or with control HEK293 cells (black line). For comparison with (e) below, an enlargement of (c) is also shown as (d). e AUC-FDS analysis of conditioned medium of L cells secreting GFP-Wnt3a carrying the C77A mutation cultured with HEK293 cells secreting the CRD domain of mouse Fzd8 (pink line; n = 3) or control HEK293 (gray line; n = 3). A peak with molecular mass corresponding to the Wnt/Fzd8-CRD complex was detected, but its area was smaller comapred with the case of wild-type Wnt3a. f AUC-FDS analysis of conditioned medium of L cells secreting GFP-Wnt3a cultured with HEK293 cells secreting mouse sFRP2 (pink line; n = 8) or control HEK293 cells (black line; n = 8). HMW indicates high-molecular-weight complex. Peaks marked with asterisks in c–f appeared to be derived from fluorescent molecules bound to serum albumin, such as bilirubin|
|Fig. 3. Characterization of Wnt3a fractionated by gel filtration. a Elution profile of gel filtration column chromatography. Affinity-purified FLAG-Wnt3a proteins, prepared from serum-free medium conditioned with FLAG-Wnt3a-secreting L cells, were subjected to gel filtration column chromatography (Superdex200 PC3.2/30, GE Healthcare). b Analysis of fractionated proteins by western blotting with anti-FLAG antibody. Wnt3a proteins are recovered in widely spread fractions, including a fraction corresponded to the void volume (fraction #4). c Analysis of fractionated proteins by silver staining. Protein whose size corresponded to FLAG-Wnt3a is the major component in fractions from fraction #2 to #16. d Analysis of fractionated proteins by western blotting using Blue Native PAGE (Invitrogen), which can separate high-molecular-weight proteins while maintaining their native conformation. This analysis shows that the size of Wnt-3a protein complex is mostly distributed between ~150 kDa and 1000 kDa. Of note, a discrete band corresponding to 150 kDa is detected in fraction 14. An arrow indicates a discrete band whose molecular weight corresponded to the size of a Wnt trimer. e Wnt signaling activity assessed by application of each fraction to mouse L cells. Since the β-catenin protein level is quite low in the absence of Wnt signaling in L cells, we directly monitored Wnt signaling activity by measuring the β-catenin protein level in these L cells. All of the fractions in which FLAG-Wnt3a was detectable increased the β-catenin level, indicating that Wnt3a protein still possessed its signaling activity. f–h Cross-linking analysis with proteins in unfractionated pool (f), in void volume (g), and in fraction 14 (h). Full blot images of b–h are shown in Supplementary Fig. 9|
|Fig. 4. Electron microscopic images of fractionated Wnt3a. a–c EM images of unfractionated sample (a), sample eluted in void volume (b), and sample recovered in fraction 14 (c). The scale bar in a–c represents 500 Å. d–f Image of Wnt3a reconstructed by single-particle analysis. Top view (d), oblique view (e), and side view (f) are shown. The volume enclosed by the isosurface is 100% (cyan) and 150% (dark blue) of the volume estimated from the molecular weight of the Wnt3a trimer|
|Fig. 5. Fluorescence Cross-Correlation Spectroscopy (FCCS) analysis of Wnt3a in the extracellular milieu in Xenopus embryos. a FCCS analyses of the extracellular milieu within almost one-cell diameter from Wnt3a-expressing cells were carried out in Xenopus embryo at the mid-gastrula stage. Through a number of trials of FCCS and FCS (Fig. 6) analyses, we found that the behavior of Wnt proteins was quite different between the inside and outside of cells. Thus, we could ensure the extracellular measurements by examining the dynamics of Wnt proteins. b Molecular interaction between mCherry-Wnt3a and GFP-Wnt3a, secreted GFP or GFP-WntD is evaluated with RCA (relative cross-correlation amplitude), which corresponds to the ratio of interacting molecules to all molecules. c Effects of overexpression of Fzd8-CRD or sFRP2 on molecular interaction between mCherry-Wnt3a and GFP-Wnt3a. Statistical significance (p) was calculated by use of Tukey’s multiple comparisons of means. NS means not significant|
|Fig. 7. Model: Heterogeneity of Wnt complex formation and diffusion range. Wnt trimer is the smallest unit of the HMW complexes. Both the trimer and trimer-assembled larger complexes appear to exist in the extracellular milieu, although it is uncertain as to when the timing of the trimer formation, as well as that of the assembly to the larger HMW complexes, occurs during the process of Wnt secretion. The large HMW complex is less mobile, probably interacting with the plasma membrane, resulting in restriction of the Wnt diffusion range. Some Wnt molecules in the HMW form can be dissociated by local interaction with Frizzled receptor (Fzd), resulting in short-range signal (local action). In contrast, the larger HMW complexes, probably as well as the trimer itself, can also be dissociated by interaction with soluble Wnt-binding protein, such as sFRP2. By this dissociation, Wnt becomes more mobile; and its diffusion range is thus expanded (diffusible action)|
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