Endocytic regulation of Notch signaling in Drosophila melanogaster neural progenitor cells

Notch signaling is a ubiquitously used signaling pathway that is highly conserved and used throughout metazoan development. Understanding the regulation of Notch signaling is becoming increasingly important in determining the mechanism and treatment for the myriad of human Notch-related diseases. In Drosophila. melanogaster, the development of external sensory organs provides a context in which Notch can be manipulated and phenotypes can be easily interpreted. Here, we expand upon the growing field of Notch regulation through endocytic trafficking by examining the role of Numb and Sara endosomes. Numb is a potent Notch inhibitor whose function is conserved in higher organisms, but whose mechanism of action has remained elusive. In this study, we dispel a previous hypothesis that Numb promotes Notch internalization and instead demonstrate that Numb is a suppressor of Notch endocytic recycling. In support of this, we show that Numb is necessary and sufficient for Notch trafficking to late endosomes/lysosomes to promote degradation. We do this by employing a novel technique that is able to distinguish recycled Notch from other populations within the cell. In addition, we show that the cell fate determinant Lethal (2) Giant Larvae, can also suppress Notch recycling, but at a step upstream of Numb. Results from this study help to answer a long-standing questions in the field of Notch signaling, by demonstrating the role of Numb in Drosophila. We also extended our investigation of endocytic Notch regulation by determining the role of a sub-population of early endosomes positive for Sara. We show that these Sara endosomes are trafficked preferentially to Notch activated cells, but do not contain appreciable levels of Notch. While we conclude that the Sara endosomes do not seem relevant to Notch signaling, we show that the mechanism of Sara endosome trafficking is likely tied to global anterior-posterior cues and not related to cell fate determinants. Results from our studies have important implications in the designing of treatments for Notch related dysfunctions that depend on an exquisite understanding of Notch regulation