The Role of Cell-Cell Interactions During Drosophila Development

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Normal development requires the commitment of individual cells to the appropriate cell fate and their subsequent differentiation. The long-term goal of our research program is to understand how individual cell fates are acquired during development. Towards this goal, we have been studying the role of the Notch signaling pathway, which functions to determine the fate of most cell types in all complex animals. Specifically, we have focused on understanding the function of one core member of this pathway, neuralized (neur), which encodes a protein with a C-terminal RING domain and two Neur homology repeats (NHR1 & NHR2). We initially showed that Neur is required for a subset of Notch signaling events during development. We also showed that both NHR and RING finger domains are absolutely required for Neur function in vivo. Specifically, we showed that the RING finger domain in Neur confers E3-ubiquitin ligase activity while the NHR1 domain is required for its interaction with the Notch ligands, Delta & Serrate. We also found that the NHR2 domain mediates formation of intra- and inter-molecular dimers, which regulate Neur activity and hence, Notch activation. Finally, through in vitro binding assays, we identified an interaction between Neur and phosphoinositides (PIPs), membrane lipids that mediate membrane trafficking and signaling. Specifically, we found that the PIP-binding motif in Neur is required for Delta internalization. Altogether, our studies have elucidated the molecular function of Neur and its specific role within the Notch signaling pathway. However, despite intensive analysis, we still know very little as to how Neur itself is regulated. Recently, we have found that Neur is phosphorylated at several key residues flanking the PIP-binding and NHR domains. We also found that Neur can associate with other NHR domain containing proteins suggesting that Neur function may be regulated by protein phosphorylation and oligomerization. Interestingly, we have also uncovered a novel role for Neur in epithelial cell polarity and germ line stem cell maintenance. Importantly, neither Notch nor its ligands Delta and Serrate, are required for these processes, suggesting a novel, Notch-independent function for Neur during development. To uncover the specific roles of Neur in these two developmental processes, we used a combination of biochemical and genetic approaches. To date, we have shown that Neur can bind to another NHR domain containing protein, called CG3894. We also found that loss of function mutations in CG3894 give rise to similar defects in cell polarity as those observed in neur mutants. This raises the exciting possibility that Neur and CG3894 act together to regulate epithelial cell polarity. Finally, we have begun to uncover the potential role of Neur in the male germ line. Specifically, we found that Neur is a direct target of the JAK/STAT pathway, which has previously been shown to play a key role in the maintenance of germ line stem cells. We also found that Neur can bind and ubiquitinate STAT92E, providing additional evidence that the function of Neur in the germ line is mediated by JAK/STAT signaling. In this proposal, we will: 1) Determine how phosphorylation affects Neur function; 2) Determine the role of Neur in epithelial polarity; and, 3) Determine the role of Neur in germline stem cells. Altogether, these experiments will uncover the mechanisms that regulate Neur function in Notch signaling and also reveal novel functions for Neur in cell polarity and germ line stem cell maintenance, two processes that play critical roles in the development of multicellular organisms.