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	Fig. 1. Analytical ultracentrifugation analyses of GFP-tagged Wnt3a proteins secreted from cultured cells. a Schematic representation of the analysis using AUC. b–f AUC-FDS analysis of secreted GFP (n = 3; (b)), GFP-tagged Drosophila WntD (GFP-WntD; n = 7; (c)), and GFP-tagged mouse Wnt3a (GFP-Wnt3a; n = 20; (d, e)), secreted from stably transformed mouse L cells. As a negative control, AUC-FDS analysis of conditioned medium (C.M.) from neomycin-resistant L cells is also indicated (n = 4; (f)); e is an enlarged version of d. HMW indicates high-molecular-weight complex. The closed arrowhead in e indicates the HMW complex with the smallest molecular mass (~9.6 S corresponding to ~200 kDa). g AUC-FDS analysis of GFP-Wnt3a in culture media with mock-treated (black; n = 3) or afamin pre-depleted (orange; n = 3) serum. The relative area size of the Wnt3a/afamin complex peak, which was normalized by the whole area size obtained in each measurement, was significantly decreased by afamin pre-depletion (relative area of ~ 7.0 peaks was decreased from 35.1 ± 6.6% to 14.1 ± 1.5% by the pre-depletion (mean ± s.d., p < 0.05); whereas that of the HMW peaks was increased from 51.1 ± 4.8% to 69.6 ± 1.2% (mean ± s.d., p < 0.05, n = 3, paired t-test). h AUC-FDS analysis of GFP-Wnt3a secreted from HEK293 cells (n = 4). i Wnt signaling activity of GFP-Wnt3a in culture media with mock-treated (black) or afamin pre-depleted (orange) serum. Wnt activity monitored by use of the SuperTOPFlash assay was normalized by the fluorescence intensity of GFP. Peaks marked with asterisks in d–h appeared to be derived from fluorescent molecules bound to serum albumin, such as bilirubin (Supplementary Fig. 3f)
	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. 6. Expansion of Wnt3a distribution range by expression of sFRP2 in Xenopus embryos. a–f Fluorescence Correlation Spectroscopy (FCS) analysis of GFP-Wnt3a. Schematic representation of the FCS analyses is presented (a). GFP-Wnt3a mRNA was injected into a blastomere (b, c), and GFP-Wnt3a and sFRP2 mRNAs were injected into the same blastomere (d) or into different blastomeres (e) in Xenopus eggs at the 4-cell stage, and then the extracellular milieu within almost one-cell diameter from Wnt3a-expressing cells was analyzed by FCS at the mid-gastrula stage. CPM and Dslow measured in each time period (10 seconds) are plotted (b, d, e). Distribution of Dslow values shown in (b) is indicated in increments of 3.5 CPM (c). The pattern of distribution of Dslow was clearly divided at 7.0 CPM. Statistical significance (p) was calculated by using the Wilcoxon rank sum test. Summarized representation of the high and low CPM populations is also shown (f). Multiple comparisons using Bonferroni correction were performed with Fisher’s exact test (two-sided; ***p < 0.001). g, h Distribution of GFP-Wnt3a in the absence (n = 4; (g)) or presence (n = 4; (h)) of sFRP2 expression. GFP-Wnt3a and sFRP2 mRNAs were injected into different blastomeres in Xenopus embryos at the 4-cell stage, and the distribution of GFP-Wnt3a was determined by GFP-Fluorescence at stage 10.5. The distribution range of GFP-Wnt3a was expanded in the presence of sFRP2. The scale bars in (g), (h) represent 100 μm. i Expansion of the signaling range of Wnt3a by sFRP2. Wnt3a was expressed by injecting a DNA expression construct (pCS2 + Wnt3a, 8.25 pg/embryo) to avoid affecting the early Wnt signaling involved in the dorsal determination, together with nβ-gal (pCS2 + nβ-gal, 100 pg/embryo). sFRP2 mRNA (1 pg/embryo) was injected in the blastomere diagonal to the Wnt3a-injected one, as illustrated. Wnt3a source cells were stained in magenta. In situ hybridization of Wnt target genes, gbx2 or otx2, was examined in st. 12 Xenopus embryos. Double-headed arrows indicate expansion of Wnt3a effect on gbx2 activation or otx2 inhibition. Representative images are presented (numbers of embryos with similar staining patterns/total embryos examined are as indicated at the right bottom of panels)
	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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