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

ABSTRACT: The differential activation of Wnt pathways (canonical: Wnt/β-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca²⁺) 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.

SUBMITTER: Neuhaus J

PROVIDER: S-EPMC8584856 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters measured in this report.

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

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