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 ²⁺ ) 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.