| Autor: |
Neuhaus, Jochen, Weimann, Annett, Berndt-Paetz, Mandy |
| Jazyk: |
angličtina |
| Rok vydání: |
2021 |
| Předmět: |
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| Druh dokumentu: |
Článek |
| Popis: |
The differential activation of Wnt pathways (canonical: Wnt/-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–coreceptor 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 -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/-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 |
| Databáze: |
Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
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