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Live Imaging of Xwnt5A-ROR2 Complexes

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...

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Autores principales: Wallkamm, Veronika, Dörlich, Rene, Rahm, Karolin, Klessing, Tina, Nienhaus, Gerd Ulrich, Wedlich, Doris, Gradl, Dietmar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196911/
https://www.ncbi.nlm.nih.gov/pubmed/25313906
http://dx.doi.org/10.1371/journal.pone.0109428
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author Wallkamm, Veronika
Dörlich, Rene
Rahm, Karolin
Klessing, Tina
Nienhaus, Gerd Ulrich
Wedlich, Doris
Gradl, Dietmar
author_facet Wallkamm, Veronika
Dörlich, Rene
Rahm, Karolin
Klessing, Tina
Nienhaus, Gerd Ulrich
Wedlich, Doris
Gradl, Dietmar
author_sort Wallkamm, Veronika
collection PubMed
description 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.
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spelling pubmed-41969112014-10-16 Live Imaging of Xwnt5A-ROR2 Complexes Wallkamm, Veronika Dörlich, Rene Rahm, Karolin Klessing, Tina Nienhaus, Gerd Ulrich Wedlich, Doris Gradl, Dietmar PLoS One Research Article 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. Public Library of Science 2014-10-14 /pmc/articles/PMC4196911/ /pubmed/25313906 http://dx.doi.org/10.1371/journal.pone.0109428 Text en © 2014 Wallkamm et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Wallkamm, Veronika
Dörlich, Rene
Rahm, Karolin
Klessing, Tina
Nienhaus, Gerd Ulrich
Wedlich, Doris
Gradl, Dietmar
Live Imaging of Xwnt5A-ROR2 Complexes
title Live Imaging of Xwnt5A-ROR2 Complexes
title_full Live Imaging of Xwnt5A-ROR2 Complexes
title_fullStr Live Imaging of Xwnt5A-ROR2 Complexes
title_full_unstemmed Live Imaging of Xwnt5A-ROR2 Complexes
title_short Live Imaging of Xwnt5A-ROR2 Complexes
title_sort live imaging of xwnt5a-ror2 complexes
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196911/
https://www.ncbi.nlm.nih.gov/pubmed/25313906
http://dx.doi.org/10.1371/journal.pone.0109428
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