title: Polarized translocation of fluorescent proteins in Xenopus ectoderm in response to Wnt signaling.
abstract: Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination is visualized in Xenopus ectodermal cells to study Wnt signal transduction. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.
authors.affiliation: Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine.
authors.forename: Keiji
authors.initials: K
authors.lastname: Itoh
authors.forename: Sergei Y
authors.initials: SY
authors.lastname: Sokol
mesh_terms: D000818:Animals; D016764:Cell Polarity / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000378:metabolism*; D004475:Ectoderm / Q000378:metabolism*; D005260:Female; D051157:Frizzled Receptors / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism*; D008845:Microinjections / Q000379:methods; D012313:RNA / Q000008:administration & dosage / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism*; D015398:Signal Transduction; D051153:Wnt Proteins / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins / Q000032:analysis / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D003598:Cytoskeletal Proteins; D051157:Frizzled Receptors; D008164:Luminescent Proteins; D012313:RNA; D011993:Recombinant Fusion Proteins; D051153:Wnt Proteins; D029867:Xenopus Proteins; D000097573:Red Fluorescent Protein; C464904:ANKRD6 protein, Xenopus