title: Live imaging of Xwnt5A-ROR2 complexes.
abstract: Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner.
authors.affiliation: Zoological Institute, Department of Cell and Developmental Biology, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Veronika
authors.initials: V
authors.lastname: Wallkamm
authors.affiliation: Institute of Applied Physics and Insitute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Rene
authors.initials: R
authors.lastname: Dörlich
authors.affiliation: Zoological Institute, Department of Cell and Developmental Biology, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Karolin
authors.initials: K
authors.lastname: Rahm
authors.affiliation: Zoological Institute, Department of Cell and Developmental Biology, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Tina
authors.initials: T
authors.lastname: Klessing
authors.affiliation: Institute of Applied Physics and Insitute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: Gerd Ulrich
authors.initials: GU
authors.lastname: Nienhaus
authors.affiliation: Zoological Institute, Department of Cell and Developmental Biology, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Doris
authors.initials: D
authors.lastname: Wedlich
authors.affiliation: Zoological Institute, Department of Cell and Developmental Biology, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Gradl
mesh_terms: D051698:Activating Transcription Factor 2 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D060172:Morpholinos / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D057050:Receptor Tyrosine Kinase-like Orphan Receptors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D051153:Wnt Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071818:Wnt-5a Protein; D014981:Xenopus / Q000254:growth & development; D029867:Xenopus Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D051698:Activating Transcription Factor 2; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D060172:Morpholinos; D012333:RNA, Messenger; D057050:Receptor Tyrosine Kinase-like Orphan Receptors; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D029867:Xenopus Proteins; D000097573:Red Fluorescent Protein; C085096:Wnt5a protein, Xenopus; C485184:enhanced green fluorescent protein; C000598273:Ror2 protein, Xenopus