Jakub Harnoš, Maria Consuelo Alonso Cañizal, Miroslav Jurásek, Jitender Kumar, Cornelia Holler, Alexandra Schambony, Kateřina Hanáková, Ondřej Bernatík, Zbynêk Zdráhal, Kristína Gömöryová, Tomáš Gybeľ, Tomasz Witold Radaszkiewicz, Marek Kravec, Lukáš Trantírek, Jan Ryneš, Zankruti Dave, Ana Iris Fernández-Llamazares, Robert Vácha, Konstantinos Tripsianes, Carsten Hoffmann, Vítězslav Bryja

• Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by FrizzledDishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.…