Elucidating the roles of ubiquitylation and ligand-receptor affinity in regulating the membrane distribution and activity of the Notch ligands Dll1 and Dll4

The Notch pathway is the canonical signaling pathway between neighboring cells in metazoans that impacts a plethora of developmental and pathological processes. This pathway is transduced by the interaction between Notch receptors on one cell, with Notch ligands on a neighboring cell. It has been shown that the activation of the Notch receptors by the Notch ligands involves endocytosis of the Notch ligand. This endocytosis is regulated by the ubiquitylation of the intracellular tail of the Notch ligand by the E3 ubiquitin ligase Mind Bomb 1 (Mib1). However, it was recently shown that a synthetic Notch signaling system where the extracellular domains of the receptors and the ligands were replaced by synthetic interaction domains, does not require Mib1 or ubiquitylation. This suggests that a complex interplay between ubiquitylation and receptor-ligand affinity controls Notch activation. Here, we aim to elucidate how different factors such as receptor–ligand affinity, ubiquitylation and endocytosis rates, and cleavage efficiency, govern Notch activation dynamics. In particular, we would like to examine whether higher receptor-ligand affinity can compensate for ubiquitylation. We use a combination of live cell imaging, molecular perturbations, and computational analysis to track the dynamics of the Notch activation sequence. Such dynamic analysis will allow obtaining a quantitative molecular picture of the Notch activation sequence.