Elucidating divergent regulation of mammalian Notch ligands by Mindbomb1 using humanized Drosophila

点击翻译按钮获取中文摘要

The Notch signalling pathway mediates short-range cell communication in many developmental processes and homeostasis in probably all eukaryotes. It is therefore not surprising that the Notch pathway was found to play an important role in the pathogenesis of an increasing number of diseases (e. g. cancer) and in ageing. During signalling, the ligands, belonging to the DSL family, in the signal-sending cells bind and activate Notch receptors on the signal-receiving side. In mammals, there are 5 Notch ligands and 4 Notch receptors that can all interact with each other. While it has been shown that different ligands have different functional roles in vivo, it is unclear how their different activities are encoded molecularly. Notch ligand activity has been shown to depend on endocytosis initiated by the E3 ligases Mindbomb1 (Mib1), which catalyses the ubiquitylation (ubi) of the intracellular domains (ICDs) of the ligands, and creates a pulling force that induces an activating conformational change of the receptor. We hypothesize that differences in ligand activities are encoded by the interaction between MIB1 and the mammalian ligands’ ICDs, as well as by the resulting ubi patterns of the ICDs. We will test this hypothesis by answering the following questions: 1. Do the ICDs of mammalian ligands differ in their ability to activate the Notch pathway? 2. How do the ligands ICDs interact with MIB1? 3. What is the consequence of these interactions for function? 4. Which amino acids in the ICDs of the ligands are ubiquitylated and important for signalling and endocytosis? 5. Can the ligands also act in a ubi-independent manner, as has been found for Drosophila ligand Delta? To answer these questions, we will use ‘humanized’ flies by replacing the Drosophila Delta ligand ICD with the ICDs of all mammalian ligands, and the Drosophila Mib1 with the mammalian MIB1. This will allow exploiting the vast array of techniques available in Drosophila (e.g. genetics, imaging – performed in the TK lab) and test the functional differences in an in vivo settings. In parallel, we will use state of the art in vitro and cell culture assays including FRET and live imaging endocytosis assays to provide a complementary analysis of ligand tail activity in mammalian cells (DS lab). The results will reveal the fundamental rules for the interaction of Mib1 with the DSL ligands and how these interactions promote variable Notch activity in different contexts.