title: The Secreted Signaling Protein Wnt3 Is Associated with Membrane Domains In Vivo: A SPIM-FCS Study.
abstract: Wnt3 is a morphogen that activates the Wnt signaling pathway and regulates a multitude of biological processes ranging from cell proliferation and cell fate specification to differentiation over embryonic induction to neural patterning. Recent studies have shown that the palmitoylation of Wnt3 by Porcupine, a membrane-bound O-acyltransferase, plays a significant role in the intracellular membrane trafficking of Wnt3 and subsequently, its secretion in live zebrafish embryos, where chemical inhibition of Porcupine reduced the membrane-bound and secreted fractions of Wnt3 and eventually led to defective brain development. However, the membrane distribution of Wnt3 in cells remains not fully understood. Here, we determine the membrane organization of functionally active Wnt3-EGFP in cerebellar cells of live transgenic zebrafish embryos and the role of palmitoylation in its organization using single plane illumination microscopy-fluorescence correlation spectroscopy (SPIM-FCS), a multiplexed modality of FCS, which generates maps of molecular dynamics, concentration, and interaction of biomolecules. The FCS diffusion law was applied to SPIM-FCS data to study the subresolution membrane organization of Wnt3. We find that at the plasma membrane in vivo, Wnt3 is associated with cholesterol-dependent domains. This association reduces with increasing concentrations of Porcupine inhibitor (C59), confirming the importance of palmitoylation of Wnt3 for its association with cholesterol-dependent domains. Reduction of membrane cholesterol also results in a decrease of Wnt3 association with cholesterol-dependent domains in live zebrafish. This demonstrates for the first time, to our knowledge, in live vertebrate embryos that Wnt3 is associated with cholesterol-dependent domains.
authors.affiliation: Department of Chemistry, National University of Singapore, Singapore, Singapore; Center for BioImaging Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Xue Wen
authors.initials: XW
authors.lastname: Ng
authors.affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore.
authors.forename: Cathleen
authors.initials: C
authors.lastname: Teh
authors.affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore; Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Korzh
authors.affiliation: Department of Chemistry, National University of Singapore, Singapore, Singapore; Center for BioImaging Sciences, National University of Singapore, Singapore, Singapore; Department of Biological Sciences, National University of Singapore, Singapore, Singapore. Electronic address: twohland@nus.edu.sg.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wohland
mesh_terms: D000818:Animals; D044482:Benzeneacetamides / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008853:Microscopy; D019308:Palmitic Acid / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D060508:Wnt3 Protein / Q000378:metabolism*; D015027:Zebrafish
chemical_list: C579131:2-(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridin-3-yl)phenyl)acetamide; D044482:Benzeneacetamides; D011725:Pyridines; C555507:WNT3 protein, human; D060508:Wnt3 Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid