The endocytic requirements for mechanosensitive Notch activation in vivo

Abstract. Notch is a conserved transmembrane receptor that has important functions in animal development and its dysregulation causes diverse human disorders and cancers. An understanding of the initiation and regulation of Notch signal transduction is therefore important to human health. A key event in Notch activation is the exertion of mechanical force on the receptor ectodomain which triggers a proteolytic cleavage of Notch that ultimately precipitates the intracellular domain, a transcriptional activator, translocating to the nucleus. This activating force is provided by ligand endocytosis into the signal sending cell, but remarkably only a select route of endocytosis involving the endocytic adapter protein Epsin can generate this force. Furthermore, in the Drosophila wing, only a small proportion of the Notch ligand Delta enters the cell via this route. Here we propose to resolve the critical aspects of Epsin-mediated ligand endocytosis that allows the exertion of activating force across a ligand/Notch bridge. We will extend our existing in vivo tools and genetic protocols to study ligand/receptor interactions to incisively control signaling from mutant cells, monitor transendocytosis of the ligand/receptor bridge, and conduct biochemical assays. First, we will confirm that the primary role of Epsin is to link Notch ligands to the Clathrin route of endocytosis, and identify the endocytic route responsible for the internalization of Delta excluded from the Clathrin route. Second, we will probe the force generating capacity of Epsin and non-Epsin routes of ligand-endocytosis. Finally, we will investigate the endocytic behavior of the Drosophila ligand Serrate that is largely endocytosed via the Epsin route and is potentially itself mechanosensitive. If successful, the proposed experiments will identify additional steps in Notch activation, new possibilities that could regulate the sending of signals to Notch, and general principles relevant to multiple juxtacrine signaling molecules subject to Clathrin-mediated endocytosis and proteolysis.