In biology, the transport of ions across lipid membranes is crucial and is generally performed by membrane proteins. Deficiencies in transport are at the origin of various diseases, such as cystic fibrosis. In this context, synthetic anion carriers incorporated within the lipid bilayer could play a remedial role. They extract ions from one side of the membrane, move across, and release the ions on the other side.

Vladimír Šindelář Research Group

Significance

The exchange of chloride and bicarbonate across lipid bilayers is an important biological process. Synthetic molecules can act as mobile carriers for these anions, although most show little selectivity. Here we report on three bambus[6]uril macrocycles functionalized with fluorinated benzyl groups, which are ableto exchange Cl^-and HCO₃^-efficiently. Remarkably, rates for Cl^-/NO₃^- exchangeare two orders of magnitude lower. The higher rates of Cl^-/HCO₃^-transport can be explained by the ability of the bambusurils to complex Cl^- and HCO₃^-simultaneously, facilitating their exchange at the bilayer interface.

Furthermore, the exceptionally high affinity and selectivity of these systemsfor NO₃^-appear to contribute to the poor Cl^-/NO₃^-exchange. This worknot only demonstrates the importance of anion binding characteristics onanion transport but also the potential relevance of bambusurils for aniontransport applications considering the high rate observed for Cl^-/HCO₃^-exchange.

Valkenier, H., Akrawi, O., Jurcek, P., Sleziakova, K., Lizal, T., Bartik, K. and Sindelar, V.: Fluorinated Bambusurils as Highly Effective and Selective Transmembrane Cl^-/HCO₃^-Antiporters, Chem(2019) 5, 429-444. doi:10.1016/j.chempr.2018.11.008 

Summary showing CK1ε role in DVL3 conformational dynamics. A summarizing model which proposes at least three DVL conformations in vivo: (i) a closed (CK1ε present and inactive), (ii) open (CK1ε active), and (iii) non-physiological open, which occurs when CK1ε is absent or the DVL-CK1ε interaction is disrupted. Position of insertion of FlAsH III binding tag is indicated. The CK1-induced phosphorylation events are depicted as P in red circle and the C-terminus of DVL as red thick line. The molecular distance analysed in the FRET FlAsH sensor III is shown as a dashed red line; ECFP, enhanced cyan fluorescent protein

Vítězslav Bryja Research Group

Significance

Dishevelled (DVL) is the key component of the Wnt signalling 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 modelling 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.

Harnoš, J., Cañizal, M.C.A., Jurásek, M., Kumar, J., Holler, C., Schambony, A., Hanáková, K., Bernatík, O., Zdráhal, Z. Gömöryová, K.,Gybel’,T.,  Radaszkiewicz, T.W., Kravec, M., Trantírek, L., Ryneš, J., Dave, Z., Fernández-Llamazares, A.I., Vácha, R., Tripsianes, K., Hoffmann, C., and Bryja, V.: Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1, Nat. Commun. (2019) 10, 1804, 1-18.  doi.org/10.1038/s41467-019-09651-7