Video 7.

The tracheal system of Drosophila embryo develops abnormally upon excessive myosin II phosphorylation by engineered Rok kinase. Time-lapse imaging of the development of the tracheal system with dorso-lateral views of sqh_Sqh::GFP embryos from germband retraction stage until late stages of embryogenesis. Expression of all three active N-Rok variants (N-Rok::vhhGFP4^ZH-86Fb, N-Rok::vhhGFP4^Vi and N-Rok::dGBP1^ZH-86Fb) resulted in the formation of large Sqh::GFP foci and clustering of groups of tracheal cells, leading to a dramatically disorganized tracheal architecture. Variants of the synthetic kinase were expressed in the breathless domain (visualized by co-expression of mCherry-nls). Top panel: control (left): sqh_Sqh::GFP btlGal4 UAS_mCherry-nls/+. Synthetic Rok (middle): sqh_Sqh::GFP btlGal4 UAS_mCherry-nls/+; UAS_N-Rok::vhhGFP4^ZH-86Fb/+. Synthetic Rok with the destabilized nanobody (right): sqh_Sqh::GFP btlGal4 UAS_mCherry-nls/+; UAS_N-Rok::dGBP1^ZH-86Fb/+. Bottom panel: inactive synthetic Rok (left): sqh_Sqh::GFP btlGal4 UAS_mCherry-nls/+; UAS_N-RokDead::vhhGFP4^ZH-86Fb/+. Synthetic N-Rok::vhhGFP4^Vi (left): sqh_Sqh::GFP btlGal4 UAS_mCherry-nls/UAS_N-Rok::vhhGFP4^Vi. Imaging at 10 min intervals, 40× objective, Leica SP5.

Engineered kinases as a tool for phosphorylation of selected targets in vivo

Katarzyna Lepeta, Chantal Roubinet, Milena Bauer, M. Alessandra Vigano, Gustavo Aguilar, Oguz Kanca, Amanda Ochoa-Espinosa, Dimitri Bieli, Clemens Cabernard, Emmanuel Caussinus, and Markus Affolter

DOI: 10.1083/jcb.202106179