Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells

The differential activation of Wnt pathways (canonical: Wnt/β-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca<sup>2+</sup>) depends on the cell-specific availability and regulation of Wnt receptors, called Frizzled (FZD). FZDs selectively recruit co-receptors to activate vario...

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Autores principales:	Jochen Neuhaus, Annett Weimann, Mandy Berndt-Paetz
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	Wnt signaling receptors interaction proximity ligation assay PC-3 cells Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/dd4fda3d52b94cf18250f1608701f034
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spelling	oai:doaj.org-article:dd4fda3d52b94cf18250f1608701f0342021-11-11T17:27:16ZImmunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells10.3390/ijms2221120571422-00671661-6596https://doaj.org/article/dd4fda3d52b94cf18250f1608701f0342021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/12057https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067The differential activation of Wnt pathways (canonical: Wnt/β-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca<sup>2+</sup>) depends on the cell-specific availability and regulation of Wnt receptors, called Frizzled (FZD). FZDs selectively recruit co-receptors to activate various downstream effectors. We established a proximity ligation assay (PLA) for the detection of endogenous FZD–co-receptor interactions and analyzed time-dependent Wnt pathway activation in cultured cells. Prostate cancer cells (PC-3) stimulated by Wnt ligands (Wnt5A, Wnt10B) were analyzed by Cy3-PLA for the co-localization of FZD6 and co-receptors (canonical: LRP6, non-canonical: ROR1) at the single-cell level. Downstream effector activation was assayed by immunocytochemistry. PLA allowed the specific (siRNA-verified) detection of FZD6–LRP6 and FZD6–ROR1 complexes as highly fluorescent spots. Incubation with Wnt10B led to increased FZD6–LRP6 interactions after 2 to 4 min and resulted in nuclear accumulation of β-catenin within 5 min. Wnt5A stimulation resulted in a higher number of FZD6–ROR1 complexes after 2 min. Elevated levels of phosphorylated myosin phosphatase target 1 suggested subsequent Wnt/PCP activation in PC-3. This is the first study demonstrating time-dependent interactions of endogenous Wnt (co-)receptors followed by rapid Wnt/β-catenin and Wnt/PCP activation in PC-3. In conclusion, the PLA could uncover novel signatures of Wnt receptor activation in mammalian cells and may provide new insights into involved signaling routes.Jochen NeuhausAnnett WeimannMandy Berndt-PaetzMDPI AGarticleWnt signalingreceptorsinteractionproximity ligation assayPC-3 cellsBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12057, p 12057 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Wnt signaling receptors interaction proximity ligation assay PC-3 cells Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	Wnt signaling receptors interaction proximity ligation assay PC-3 cells Biology (General) QH301-705.5 Chemistry QD1-999 Jochen Neuhaus Annett Weimann Mandy Berndt-Paetz Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells
description	The differential activation of Wnt pathways (canonical: Wnt/β-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca<sup>2+</sup>) depends on the cell-specific availability and regulation of Wnt receptors, called Frizzled (FZD). FZDs selectively recruit co-receptors to activate various downstream effectors. We established a proximity ligation assay (PLA) for the detection of endogenous FZD–co-receptor interactions and analyzed time-dependent Wnt pathway activation in cultured cells. Prostate cancer cells (PC-3) stimulated by Wnt ligands (Wnt5A, Wnt10B) were analyzed by Cy3-PLA for the co-localization of FZD6 and co-receptors (canonical: LRP6, non-canonical: ROR1) at the single-cell level. Downstream effector activation was assayed by immunocytochemistry. PLA allowed the specific (siRNA-verified) detection of FZD6–LRP6 and FZD6–ROR1 complexes as highly fluorescent spots. Incubation with Wnt10B led to increased FZD6–LRP6 interactions after 2 to 4 min and resulted in nuclear accumulation of β-catenin within 5 min. Wnt5A stimulation resulted in a higher number of FZD6–ROR1 complexes after 2 min. Elevated levels of phosphorylated myosin phosphatase target 1 suggested subsequent Wnt/PCP activation in PC-3. This is the first study demonstrating time-dependent interactions of endogenous Wnt (co-)receptors followed by rapid Wnt/β-catenin and Wnt/PCP activation in PC-3. In conclusion, the PLA could uncover novel signatures of Wnt receptor activation in mammalian cells and may provide new insights into involved signaling routes.
format	article
author	Jochen Neuhaus Annett Weimann Mandy Berndt-Paetz
author_facet	Jochen Neuhaus Annett Weimann Mandy Berndt-Paetz
author_sort	Jochen Neuhaus
title	Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells
title_short	Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells
title_full	Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells
title_fullStr	Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells
title_full_unstemmed	Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells
title_sort	immunocytochemical analysis of endogenous frizzled-(co-)receptor interactions and rapid wnt pathway activation in mammalian cells
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/dd4fda3d52b94cf18250f1608701f034
work_keys_str_mv	AT jochenneuhaus immunocytochemicalanalysisofendogenousfrizzledcoreceptorinteractionsandrapidwntpathwayactivationinmammaliancells AT annettweimann immunocytochemicalanalysisofendogenousfrizzledcoreceptorinteractionsandrapidwntpathwayactivationinmammaliancells AT mandyberndtpaetz immunocytochemicalanalysisofendogenousfrizzledcoreceptorinteractionsandrapidwntpathwayactivationinmammaliancells
_version_	1718432092034957312

Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells

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