Fig. 7: Wnt3a mutant defective in heterodimer formation with sFRP2 prevents re-secretion of Wnt3a on exosomes.

Analysis with AUC-FDS of the CS from co-culture of GFP-Wnt3a (a) or GFP-Wnt3a (C77A) (b) expressing L cells with sFRP2-expressing or control HEK293 cells. In the presence of sFRP2, a shift in the peak position of control GFP-Wnt3a was observed, whereas peak positions of GFP-Wnt3a (C77A) remained largely unchanged. Thus, in contrast to control GFP-Wnt3a, heterodimers of GFP-Wnt3a (C77A) and sFRP2 were not formed. c Western blot for detection of FLAG-Wnt3a in lysate of MDCK cells treated with exosome-depleted CS of FLAG-Wnt3a or FLAG-Wnt3a (C77A) for indicated periods. d–g Distribution of GFP-Wnt3a (C77A) in sFRP2-expressing cells. To examine whether the Wnt3a (C77A) mutant is defective in distribution in the presence of sFRP2, GFP-Wnt3a- (d, e) or GFP-Wnt3a (C77A; f, g) producing L cells were co-cultured with sFRP2-expressing cells (e, g), or control HEK293 cells (d, f) from 2 days before observation. All images were processed by maximum intensity projection. Images of GFP-Wnt3a (d–g), bright field (d’–g’), and their merged images (d”–g”) are shown. Scale bar; 50 μm. Magnified images of areas surrounded by white lines in (e, g), are also shown in (e”’, g”’). h Examination of exosome-mediated secretion of Wnt3a (C77A) following Re-secretion assay #1. Western blot for detection of Wnt3a recovered in S10, S100 and P100 fractions collected from CS of MDCK cells cultured with S100 of non-tagged Wnt3a/L or FLAG-Wnt3a (C77A)/L co-cultured with sFRP2-expressing or control HEK293 cells are shown. i Exosomal Wnt re-secretion model.