Polarized translocation of fluorescent proteins in Xenopus ectoderm in response to Wnt signaling

J Vis Exp. 2011 May 26:(51):2700. doi: 10.3791/2700.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Video-Audio Media

MeSH terms

  • Animals
  • Cell Polarity / physiology
  • Cytoskeletal Proteins / analysis
  • Cytoskeletal Proteins / metabolism*
  • Ectoderm / metabolism*
  • Female
  • Frizzled Receptors / metabolism*
  • Humans
  • Luminescent Proteins / analysis
  • Luminescent Proteins / metabolism*
  • Microinjections / methods
  • RNA / administration & dosage
  • RNA / genetics
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / metabolism*
  • Red Fluorescent Protein
  • Signal Transduction
  • Wnt Proteins / metabolism*
  • Xenopus
  • Xenopus Proteins / analysis
  • Xenopus Proteins / metabolism*

Substances

  • ANKRD6 protein, Xenopus
  • Cytoskeletal Proteins
  • Frizzled Receptors
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Wnt Proteins
  • Xenopus Proteins
  • RNA