The role of junction related proteins during Drosophila development

连接相关蛋白在果蝇发育过程中的作用

• 批准号: RGPIN-2019-06338
• 负责人: Hughes, Sarah
• 金额: $ 2.62万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=686837
• 关键词: role; junction; related; proteins; during

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.*****

在发育过程中，必须协调调节增殖和粘附的基本细胞特性。在这一点上，协调机制仍不清楚。为了解决这个问题，我们集中在果蝇Moesin（莫伊）蛋白，它在细胞中有许多作用，包括调节增殖，肌动蛋白细胞骨架，细胞间的粘附。莫伊仅在定位于质膜（PM）时才有活性。目前的机制模型，积极莫伊是如何招募到PM有些争议。一些建议调节相互作用的莫伊与其他PM居民蛋白。或者，莫伊直接结合PM内的磷酸肌醇。莫伊还稳定PM中介导细胞-细胞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调节的情况下，活跃的莫伊是如何被招募到特定的PM结构域的。在接下来的五年里，我们的重点将是识别调节莫伊与PM互动的因素。我们将比较神经元祖细胞（NPC），其中莫伊是PM组织所需的，翼盘上皮细胞，其中莫伊具有PM和AJ特异性活动。我推测，莫伊向PM的募集是通过脂质侧链的翻译后添加以及通过与细胞类型特异性PM蛋白的相互作用而促进的。*目的1：我们将确定莫伊棕榈酰化在调节莫伊活性中的作用。我们以前确定了两个棕榈酰化位点内莫伊。我的硕士实习生已经证明，当这些位点发生突变时，莫伊不会定位于PM。我们将通过研究转基因果蝇和培养的果蝇细胞来确定莫伊棕榈酰化的作用。我们将在标记的莫伊中预测的棕榈酰化位点中引入靶向突变，以确定它们分别在发育中的大脑和翼盘的NPC和上皮细胞中招募到PM中的作用。目的2：我们将确定莫伊相互作用的蛋白质，招募或保留在PM。我们以前进行了一个正向遗传筛选翅盘上皮细胞和质谱筛选NPCS，以确定潜在的相互作用和调节莫伊的基因。本科学员将描述果蝇组织和培养细胞中的这38个候选基因，以确定它们如何影响莫伊对PM和AJs的招募。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。我们将测试的候选基因的功能损失的影响，在NPC相比，翅盘有针对性的体细胞嵌合体分析加上间接免疫荧光和共聚焦显微镜。这将通过直接探索莫伊复合物形成的发育动力学的生化实验来补充。*我的计划将推进我们对莫伊如何在果蝇上皮和神经元组织发育过程中协调细胞分裂和粘附的理解。*