Figure 4

Figure 4

Acylation of Wnt3 facilitates its partitioning into more ordered plasma membrane environments. (A) Membrane flotation assay for wt and mutant Wnt constructs. Western blots show soluble and detergent-resistant fractions of plasma membranes derived from zebrafish embryos injected with 1 ng capped sense RNA of wt Wnt3-GFP or Wnt3S212A-GFP. DRMs and soluble fractions are marked by endogenous TfR2 and Caveolin-1 (Cav1), respectively. All Wnt proteins are detected by their GFP tags. Eighty-five percent of wt Wnt3-GFP is detected in the DRM fractions while only sixteen percent of mutant Wnt3S212A-GFP is detected in DRMs with the rest of it shifting to soluble phases. Percentages represent mean ± standard deviation (SD) of three independent experiments. (B) TIRF images of HEK293T cells expressing wt Wnt3-GFP and Wnt3S212A-GFP (C) ITIR-FCS measurements. Signal is recorded from the basolateral membrane of the cells and correlated with different bin size to form varying observation areas. The diffusion time positively correlates with the size of the area and determines the diffusion mode of the molecule as free or hindered. wt Wnt3-GFP undergoes domain-like diffusion (positive y-axis intercept) while Wnt3S212A-GFP tends to diffuse freely after cholesterol extraction. MßCD, methyl-β-cyclodextrin. Transit time and SD are obtained from three independent experiments.