Cargando…
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...
Autores principales: | , , , , , , |
---|---|
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 |
_version_ | 1782339545724878848 |
---|---|
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. |
format | Online Article Text |
id | pubmed-4196911 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT wallkammveronika liveimagingofxwnt5aror2complexes AT dorlichrene liveimagingofxwnt5aror2complexes AT rahmkarolin liveimagingofxwnt5aror2complexes AT klessingtina liveimagingofxwnt5aror2complexes AT nienhausgerdulrich liveimagingofxwnt5aror2complexes AT wedlichdoris liveimagingofxwnt5aror2complexes AT gradldietmar liveimagingofxwnt5aror2complexes |