PhD Thesis Defense

The ability to understand and predict signaling between different cell types is a major challenge in biology. The Notch pathway enables direct signaling through membrane-bound ligands and receptors and is used in diverse contexts. While its canonical molecular signaling mechanism is well characterized, its many-to-many interacting pathway components, the complexity of their expression patterns, and the presence of same-cell (cis) as well as inter-cellular (trans) receptor-ligand interactions, have made it difficult to predict how a given cell will signal to others. Here, we use a cell-based approach to systematically characterize trans-activation, cis-inhibition, and cis-activation efficiencies across a wide range of ligand expression levels in Chinese hamster ovary (CHO-K1) cells and C2C12 mouse myoblasts. This analysis defines a hierarchy of relative trans-activation and cis-activation strengths for the essential receptors (Notch1 and Notch2) in all pairwise combinations of activating ligands (Dll1, Dll4, Jag1, and Jag2), in the presence of Lunatic Fringe (Lfng) or the enzymatically dead Lfng D289E mutant. All ligands trans-activate Notch1 and Notch2, except for Jag1, which competitively inhibits Notch1 signaling, and whose Notch1 binding strength is potentiated by Lfng. For Notch1, cis-activation is generally weaker than trans-activation, but for Notch2, cis-activation by Delta ligands is much stronger than trans-activation. Cis-inhibition is associated with weak cis-activation, as Dll1 and Dll4 do not cis-inhibit Notch2. Lfng expression potentiates trans-activation of both Notch1 and Notch2 by the Delta ligands and weakens trans-activation of both receptors by the Jagged ligands. The map of receptor-ligand-Fringe interaction outcomes revealed here should help guide rational perturbation and control of the Notch pathway.