In the past, we investigated the role of the Notch pathway during wing development of Drosophila as a paradigm for its role in organ formation. This process takes place in an epithelial monolayer, the wing imaginal disc. This disc consists of initially undifferentiated precursor cells that become determined in a stepwise fashion. Together with several other groups, we could show that cell communication mediated by the Notch pathway is continuously required in a small stripe of cells at the dorso-ventral (D/V) boundary to orchestrate the sequential induction of genes required for pattern formation and definition of the wing field. Wing development has become one of the processes where the role of the Notch pathway is particular well understood and we are now using our knowledge of this process to investigate its regulation.

The Notch pathway is activated trough ligand induced proteolytic cleavage of Notch. The released intracellular domain (Nintra) translocates in the nucleus and activates the expression of the target genes in collaboration with Suppressor of Hairless (Su(H)). During the release of Nintra, Notch is first cleaved in the extra-cellular region close to the membrane through Kuzbanian (Kuz) in a ligand-dependent manner. The membrane anchored intermediate Notch fragment (NEXT) is immediately further processed by the y-secretase complex producing Nintra. This cleavage is ligand-independent and occurs constitutively on all NEXT-like Notch variants inserted in the plasma membrane.

The current focus of our investigations is the role of the endosomal pathway during Notch signalling. This fundamental pathway has gained attention because of its involvement in the regulation of the activity of the Notch pathway as well as the degradation of the Notch receptor. Endosomal trafficking of Notch is initiated by endocytosis through addition of single ubiquitines to lysines of the intra-cellular domain of Notch. After abscission the early endosomal vesicles undergo homotypic fusion to form the early endosome (EE). Notch is located in the limiting membrane (LM) of the EE with the intracellular domain reaching into the cytoplasm. In order to be degraded, the intra-cellular domain has to be transported into the lumen of the endosome during its maturation into a late endosome (LE). This is achieved by concentrating Notch at certain regions of the LM and subsequent inside budding of this region into the lumen of the endosome. The consequence is the formation of intra-luminal vesicles (ILV) with high concentration of Notch in maturing endosomes. The ILV containing endosomes are also called multi-vesicular bodies (MVB) and their formation is controlled by the activity of four in sequence acting ESCRT protein complexes (ESCRT-0-III) (Williams and Urbe, 2007). The MVBs eventually fuse with the lysosome where the luminal content is degraded.

One of the main projects of the lab is the characterization of the function of the tumour-suppressor-gene lethal (2) giant discs (lgd) during endosomal trafficking of the Notch-receptor. lgd encodes a member of a so far uncharacterised protein family whose hallmarks are four tandem repeats of the so far uncharacterised DM14 and a C2-domain. Lgd has two human orthologs which are involved in several disease, e. g. autism spectrum disorder, cancer and dementia.

Loss of lgd function in Drosophila results in the activation of the Notch pathway in a novel, ligand-independent manner in several tissues. The observed uncontrolled activation of Notch is the cause of the observed over-proliferation of lgd mutant imaginal discs. We could show that Lgd is a regulator of the ESCRT machinery, which is required for fundamental processes, such as cell division, formation of intraluminal vesicles and membrane repair. It is also hijacked by viruses such as HIV and Ebola to exit infected cells. At the moment, we are resolving the atomic structure of Lgd and also its ESCRT interaction partners.

Endocytosis plays also a crucial role during activation of the Notch signalling pathway by its ligands. The endocytosis of the bound ligands exerts a conformation-changing pulling force onto the Notch receptor which is required for intracellular signal transduction to the nucleus. As a second main project, we are investigating the requirements for ubiquitylation of the intracellular domains by the E3 ligases Mindbomb1 and Neuralized to initiate the activating endocytosis of the ligands. To this end, we are also using humanized flies to include the human orthologs of the Notch components in our analysis.

Other projects in the lab investigate

• the role of Notch during formation of the sensory organ precursor cells.
• the role of the endocytosis pathway during epithelial polarity