Fig. 4.

Calcium transients on dendritic protrusions depend on membrane-associated Wnts. (A) Schematic of the generated mCherry-tagged membrane-tethered NOTUM construct (memNOTUM). mCh, mCherry. (B) Co-transfection of the membrane marker mem-mCherry and WNT7A-GFP shows localisation of WNT7A-GFP to cytoneme tips (yellow circles), whereas co-transfection of memNotum and WNT7A-GFP reduces WNT7A-GFP-positive filopodia. (C) Quantification of mCherry nuclear expression in the receiving cell population identified the ability of memNotum to significantly reduce Wnt-mediated paracrine signalling to a similar level as secreted NOTUM (transfection of full-length untethered human NOTUM). Statistical significance was addressed using one-way ANOVA with Dunnett's multiple comparison test to compare relevant controls within groups and an unpaired, one-tailed Student's t-test to compare specific combinations. *P<0.05; **P<0.01; ***P<0.005. ns, not significant. Experiments were performed in biological triplicate, with three fields per group analysed. (D,E) SH-SY5Y differentiated neurons transfected with WNT7A-GFP and mem-mCherry followed by post-staining with anti-LRP6 show strong colocalisation (D), which is reduced when neurons are transfected with memNotum (E). (F-H) SH-SY5Y neurons transfected with FingR-PSD95 and memNotum and post-stained with anti-WNT7A also show reduced colocalisation of the PSD95/WNT7A proteins compared to the mem-mCherry control (F,G) signal. Quantification is shown in H. signal. Two-way ANOVA with Bonferroni's post-hoc test for multiple comparisons was performed for statistical comparisons between each group. **P<0.01.