The Secreted Signaling Protein Wnt3 Resides in Plasma Membrane Lipid Domains in Vivo: A SPIM-FCS Study

Abstract

Wnt3 is a signaling protein involved in development and disease (oncogenesis and tetraamelia syndrome). Recently, we have shown that Wnt3 regulates cerebellum development using zebrafish Wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP (Teh et al., Development 2015). We identified several fractions of Wnt3: i) an intracellular and ii) a secreted fraction, which comprises of monomeric Wnt3 as well as its complexes, and, iii) a membrane-bound fraction. The membrane-bound O-acyltransferase Porcupine is required for Wnt palmitoylation, secretion and biological activity. It was shown in vitro that for membrane localization in Wnt3-expressing cells Wnt3 undergoes two lipid modifications by palmitoylation at C77 and S209. Such posttranslational modifications have been linked with protein association with cholesterol-dependent lipid domains. This raises the question whether Wnt3 is targeted to these domains in live zebrafish. We therefore used SPIM-FCS and the FCS diffusion laws, which characterize the dependence of the diffusion coefficient on the observed area to deduce deviations from free diffusion, to characterize Wnt3 distribution on plasma membranes in zebrafish embryos.First, we demonstrate that the diffusion laws can be applied in a 3D environment of SPIM-FCS by characterizing lipid distributions in giant unilamellar vesicles (GUVs) of various compositions. Second, we applied this methodology to analyze Wnt3 distribution on plasma membranes in live Wnt3 transgenic embryos. This demonstrated that in vivo the Wnt3 membrane-bound fraction also shows consistent raft partitioning. Third, upon reduction of Wnt3 secretion by a specific inhibitor of Porcupine, Wnt3 partitioning into lipid domains is also reduced. Taken together this demonstrates that in developing zebrafish embryos the plasma membrane fraction of Wnt3 to be secreted needs to be compartmentalized into specialized lipid domains.