REGULATION OF NOTCH SIGNALING BY THE ENDO-LYSOSOMAL SYSTEM IN DROSOPHILA: THE ROLE OF ESCRT-0 AND V-ATPASE
Creators: name: TOGNON, EMILIANA
Publisher: name: Universit� degli Studi di Milano
Year: 2016

description: ABSTRACT
During my Ph.D., I have been involved in two different projects:
1. The study of the role of ESCRT-0 in Notch signaling and tumor suppression. 
2. The study of the regulation of V-ATPase in a subset of Notch-dependent 
developmental processes.
1. Abstract Project 1
Sorting and degradation of ubiquitylated cargoes depends on the endosomal sorting required for transport (ESCRT) machinery. The ESCRT machinery is composed of four multi-subunit ESCRT complexes (ESCRT-0, -I, -II, -III), which act in a sequential fashion to deliver endocytic cargoes into the internal luminal vesicles (ILVs) of the multivescicular endosome (MVE) for subsequent degradation. ESCRTs sort a number of transmembrane proteins including Notch and the JAK/STAT signaling receptor Domeless. In Drosophila epithelial tissue, mutation in ESCRT ?I, -II, -III components results in misregulation of several signaling pathways, loss of epithelial polarity and unrestrained proliferation, suggesting that ESCRT genes act as tumor suppressors. Unexpectedly, Drosophila Hrs, one of the two components of the ESCRT-0 complex that acts upstream of the other ESCRT complexes have been found to be dispensable for tumor suppression. Thus, when I started my Ph.D. it was unclear whether ESCRT-0 had a tumor suppressive function. In my first project, I have found that mutation of Stam, a second ESCRT-0 component or of both Hrs and Stam result in accumulation of ubiquitinated proteins and of the signaling receptors Notch and Domeless. Nevertheless, mutant tissue displays normal tissue architecture, proliferation and Notch signaling activation. Overall, our in vivo data indicate that the ESCRT-0 complex does not play a crucial role in tumor suppression.
2 Abstract Project 2
In mammals, the Transcription Factor EB (TFEB) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether and how these two processes are interconnected. In Drosophila, the Microphthalmia-associated Transcription Factor (Mitf) is the unique homolog of the TFEB family. In my second project I have found that Mitf acts similar to its mammalian counterparts as transcription factor shuttling from lysosomes to the nucleus to regulate V-ATPase expression and lysosomal biogenesis. Interestingly, I found that V-ATPase subunits display diverse expression patterns in the wing imaginal disc, suggesting complex regulation of 
V-ATPase during development. Remarkably, I could show that Mitf cooperates to regulate expression of a key component of the V-ATPase during differentiation of proneural clusters (PNCs), a process that specifies cells with neuronal identity. In addition, I have observed that the PNCs possess a distinctive endo-lysosomal compartment and Notch localization. Finally, I have determined that modulation of V-ATPase and Mitf in the disc alters endo-lysosomal function and PNC development. Overall my in vivo analysis indicates that lysosomal-associated functions regulated by V-ATPase/Mitf axis might play a role in tissue patterning during Drosophila development. 

 In addition to the work described above, I co-wrote a chapter on immunohistochemical tools and techniques to visualize Notch in Drosophila, in Methods in Molecular Biology Vol. 1187 (Tognon &amp; Vaccari, 2014)