| The DSL-Notch signaling pathway is a conserved signaling mechanism for cell-cell communication that dictates a wide range of developmental and physiological processes in organisms ranging from nematodes to humans. Delta/Serrate/Lag2 (DSL) ligands and their receptor, Notch, are single-pass transmembrane proteins. DSL ligands on the surface of signal-sending cells activate Notch on the surface of signal-receiving cells by triggering successive proteolytic cleavages of the receptor. Ligand-induced cleavage leads to the release and translocation of the Notch intracellular domain (NICD), a transcriptional activator, into nucleus, thereby transducing the signal. The mechanism by which DSL ligand binding activates Notch is not known.; Herein, using transgenes that express altered forms of Delta (Dl), I demonstrate that signal-sending cells must endocytose DSL ligands in order to activate Notch in signal-receiving cells. I also show that mono-ubiquitination of DSL ligands is sufficient to target them for endocytosis, and is normally essential for their signaling activity.; To better understand the requirements for mono-ubiquitination and endocytosis in DSL signaling, I have defined the roles of two genes, liquid facets (lqf), and mind bomb (dmib) that encode, respectively, the conserved endocytic protein Epsin and a ubiquitin ligase; both of these genes are selectively required in signal-sending cells for activating Notch in signal-receiving cells. By using mutations and transgenes to manipulate the functions of Epsin, Dmib, and a functionally related ubiquitin ligase, Neuralized (Neur), I demonstrate that under normal conditions, (i) mono-ubiquitination of DSL ligands by either Dmib or Neur is required to target them for endocytosis by Epsin, and (ii) DSL ligands enter the cell by multiple endocytic pathways, but only those that are internalized via the action of Epsin activate Notch. These findings indicate that signaling activity does not depend on endocytosis of DSL ligands, per se, but rather on entry of the ligands into a select, Epsin-mediated endocytic pathway. I propose two general classes of models to explain the essential role of Epsin-mediated endocytosis in DSL-Notch signaling, involving the recruitment of DSL ligands to a special class of endocytic structures, or to a special recycling pathway. |
