Development of fast Notch ligand activation system for time resolved dissection of Notch activation

The Notch pathway is a highly conserved signaling pathway between neighboring cells across metazoans that plays an important role during development, homeostasis, and cancer progression. Notch signaling is activated by the interaction between Notch ligands on the membrane of the sender cells and Notch receptors on the membrane of the receiver cells. Despite intensive research, we still lack detailed understanding of the dynamics of the Notch activation process. Here, we describe our progress towards the development of a fast Notch triggerable system that allows initiation of Notch activation within short time scales (seconds). The system is based on the fact that Notch ligand activity depends on the ubiquitylation of the intracellular domain of the Notch ligands (the ligand tail). We designed a system where the Notch ligand tail is expressed separately from the rest of the ligand and binds to it upon addition of a dimerizing inducer. Dimerization is achieved by fusing FKBP and FRB domains to the two parts of the ligand. Addition of rapamycin quickly dimerizes the two parts, leading to a fully functional ligand. We provide data that shows that the inducible ligand tail system is functional and characterize its properties. The inducible Notch ligand tail system will be used for time resolved dissection of single events of Notch activation.