Mechanisms affecting targeting of Moesin to specific domains of the plasma membrane

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Cooperative cell differentiation to form tissues during animal development requires establishment of unique domains in the plasma membrane (PM) and underlying cytoskeleton, which together comprise the cell cortex. My research program focuses on how these domains are established in different cell lineages. A key factor in this process is Moesin (Moe), which reversibly links the PM to the cortical actin cytoskeleton. Intramolecular interactions in Moe cycle it between `active' and `inactive' states. Moe is thought to be `active' when bound to the PM, a process dependent on phosphorylation and binding to phosphatidylinositol 4,5-bisphosphate (PIP2). However, in some cases Moe can be active in the absence of phosphorylation or PIP2 binding. Our hypothesis is that regulated interactions of Moe with PM associated proteins and post-translational modifications like palmitoylation also help drive Moe into an `active' state. Our goal is to discover how these events affect Moe recruitment to the cell cortex that is required for mediating cell adhesion and proliferation needed for differentiation of specific tissue types during animal development. An important functional consideration is the differentiation of Moe activity at the PM versus its other roles at the cell junction and cleavage furrow. Thus, we will compare neural stem cells (NSCs), where Moe is required for PM organization only to epithelial cells, where both PM and cell junction roles for Moe are needed. Objective 1 will identify how palmitoylation regulates Moe activity. We identified two potential palmitoylation sites within Moe. We found that mutating these sites blocks Moe recruitment to the PM. We will introduce targeted mutations in the predicted palmitoylation sites in Moe to determine how they affect PM recruitment in NSCs and epithelial cells of the developing brain and wing disc, respectively. Objective 2 will characterize several PM-associated proteins we identified that interact with Moe. We identified these as part of a forward-genetic screen for genes affecting Moe-dependent adhesion in wing disc epithelial cells and a mass spectrometry screen for Moe binding proteins in NSCs. Undergraduate HQP will each characterize one or more prioritized candidates to determine how they affect Moe recruitment to the PM and cell junctions in NSCs, epithelial, and cultured cells. They will also examine how this affects Moe-mediated regulation of cell proliferation and differentiation. They will compare the effect of targeted somatic mosaic analysis on adhesion and proliferation using specific markers and indirect immunofluorescent detection. This will be complemented by co-immunoprecipitation, directly probing dynamics of the inclusion of these cofactors in Moe complex formation at the PM. My program will continue to advance our understanding of the organization of sub-domains in the cell cortex by proteins like Moe and how this coordinates cell proliferation and differentiation in Drosophila.