The role of junction related proteins during Drosophila development

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During development, fundamental cellular properties of proliferation and adhesion must be coordinately regulated. At this point, the coordinating mechanisms remain unclear. To address this we focus on the Drosophila Moesin (Moe) protein, which has many roles in the cell including regulation of proliferation, the actin cytoskeleton, and adhesion between cells. Moe is only active when localized to the plasma membrane (PM). The current mechanistic models of how active Moe is recruited to the PM are somewhat controversial. Some suggest regulated interaction of Moe with other PM resident proteins. Alternatively, Moe binds phosphoinositides within the PM directly. Moe also stabilizes adherens junctions (AJ) protein complexes in the PM that mediate cell-cell interactions. My current goal is to discover how active Moe is recruited to specific PM domains in the presence and absence Moe-mediated regulation of AJs.***Over the next five years, our focus will be identification of factors regulating Moe interaction with the PM. We will compare neuronal progenitor cells (NPCs), where Moe is known to be required for PM organization, to wing disc epithelial cells, where Moe has both PM and AJ specific activities. I hypothesize that Moe recruitment to the PM is promoted by post-translational addition of lipid side chains and by interacting with cell type-specific PM proteins.***Objective 1: We will identify what role Moe palmitoylation plays in regulating Moe activity. We previously identified two palmitoylation sites within Moe. My MSc trainee has shown that when these sites are mutated Moe does not localize to the PM. We will determine the role of Moe palmitoylation by studying transgenic Drosophila and cultured Drosophila cells. We will introduce targeted mutations in predicted palmitoylation sites in tagged Moe to determine their role in recruitment to the PM in the NPCs and epithelial cells of the developing brain and wing disc, respectively.***Objective 2: We will identify the Moe interacting proteins that recruit or retain it at the PM. We previously conducted a forward-genetic screen in wing disc epithelial cells and a mass spectrometry screen in NPCS to identify genes potentially interacting with and regulating Moe. Undergraduate trainees will characterize these 38 candidate genes in Drosophila tissue and cultured cells to determine how they affect Moe recruitment to the PM and AJs. We will also examine their role in Moe-mediated regulation of proliferation of epithelial cells and NPCs. We will test the loss of function effects of candidate genes in NPCs as compared to wing discs by targeted somatic mosaic analysis coupled with indirect immunofluorescence and confocal microscopy. This will be complemented by biochemical experiments directly probing developmental dynamics of Moe complex formation.***My program will advance our understanding of how Moe coordinates cell division and adhesion during the development of Drosophila epithelial and neuronal tissues.*****