The role of junction related proteins during Drosophila development

在发育过程中，细胞增殖和粘附的基本特性必须协调调节。在这一点上，协调机制仍然不清楚。为了解决这个问题，我们将重点放在果蝇Moesin （Moe）蛋白上，它在细胞中有许多作用，包括增殖调节、肌动蛋白细胞骨架和细胞间的粘附。Moe仅在定位于质膜（PM）时才有活性。目前关于活跃的Moe如何被招募到PM的机制模型有些争议。一些人认为Moe与其他PM驻留蛋白的相互作用受到调控。或者，Moe直接结合PM内的磷酸肌苷。Moe还稳定PM中介导细胞间相互作用的粘附连接（AJ）蛋白复合物。我目前的目标是发现在存在和不存在Moe介导的AJs调节时，活跃的Moe是如何被招募到特定的PM结构域的。***在未来五年内，我们的重点将是确定调节Moe与PM相互作用的因素。我们将比较神经元祖细胞（npc）和翼盘上皮细胞（Moe同时具有PM和AJ特异性活性），前者是PM组织所需的Moe。我推测，通过翻译后脂质侧链的添加以及与细胞类型特异性PM蛋白的相互作用，可以促进Moe向PM的招募。***目的1：我们将确定Moe棕榈酰化在调节Moe活性中的作用。我们之前在Moe中发现了两个棕榈酰化位点。我的硕士学员已经表明，当这些位点发生突变时，Moe不会定位到PM。我们将通过研究转基因果蝇和培养的果蝇细胞来确定Moe棕榈酰化的作用。我们将在标记Moe的预测棕榈酰化位点引入靶向突变，以确定它们分别在NPCs和发育中的脑和翅盘上皮细胞募集到PM中的作用。***目的2：我们将确定在PM中招募或保留它的Moe相互作用蛋白。我们之前在翼盘上皮细胞中进行了前向遗传筛选，在NPCS中进行了质谱筛选，以确定可能与Moe相互作用和调节的基因。本科生实习生将在果蝇组织和培养细胞中表征这38个候选基因，以确定它们如何影响Moe向PM和AJs的募集。我们还将研究它们在moe介导的上皮细胞和npc增殖调节中的作用。我们将通过结合间接免疫荧光和共聚焦显微镜的定向体细胞镶嵌分析来测试候选基因在npc中与翅片相比的功能丧失效应。这将通过直接探测Moe复合体形成的发育动力学的生化实验加以补充。***我的项目将推进我们对果蝇上皮和神经元组织发育过程中Moe如何协调细胞分裂和粘附的理解。*****

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.*****