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

在动物发育过程中，协同细胞分化形成组织需要在质膜(PM)和底层细胞骨架中建立独特的结构域，这两个结构域共同组成细胞皮质。我的研究重点是这些结构域是如何在不同的细胞谱系中建立起来的。在这个过程中的一个关键因素是Moesin(MoE)，它可逆地将PM与皮质肌动蛋白细胞骨架联系在一起。分子内相互作用在“活性”和“非活性”状态之间循环。MOE被认为在与PM结合时是“活性的”，这一过程依赖于磷酸化和与磷脂酰肌醇4，5-二磷酸(PIP2)的结合。然而，在某些情况下，在没有磷酸化或PIP2结合的情况下，MOE可以是活性的。我们的假设是，MOE与PM相关蛋白的受控相互作用以及棕榈酰化等翻译后修饰也有助于推动MOE进入“活跃”状态。我们的目标是发现这些事件如何影响MOE向细胞皮质的募集，这是在动物发育过程中调节特定组织类型分化所需的细胞黏附和增殖所必需的。一个重要的功能考虑是质膜上的MOE活性与它在细胞连接和分裂沟中的其他作用的区别。因此，我们将比较神经干细胞(NSCs)，其中仅PM组织需要MOE和上皮细胞，其中PM和细胞连接都需要MOE的作用。目标1将确定棕榈酰化如何调节MoE的活性。我们在MOE中确定了两个潜在的棕榈酰化位点。我们发现，突变这些站点会阻止MOE招募到首相我们将在MOE中预测的棕榈酸化位点引入靶向突变，以确定它们如何影响PM在发育中的脑和翼盘的神经干细胞和上皮细胞中的招募。目标2将描述我们确定的几种与MOE相互作用的PM相关蛋白。我们确认这些基因是影响翼盘上皮细胞MoE依赖黏附的基因的正向遗传筛选的一部分，以及神经干细胞中MoE结合蛋白的质谱学筛选的一部分。本科生HQP将确定一名或多名优先候选人的特征，以确定他们如何影响PM的MOE招募以及NSCs、上皮细胞和培养细胞中的细胞连接。他们还将研究这如何影响MOE介导的细胞增殖和分化调节。他们将使用特定标记物和间接免疫荧光检测来比较定向体细胞镶嵌分析对黏附和增殖的影响。这将得到免疫共沉淀的补充，直接探测在PM形成的MoE复合体中这些辅助因子的包含动力学我的计划将继续推进我们对细胞皮质中由MoE等蛋白质组成的亚域的理解，以及这如何协调果蝇的细胞增殖和分化。

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.