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

影响 Moesin 靶向质膜特定区域的机制

• 批准号: RGPIN-2021-02873
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760758
• 关键词: Mechanisms; affecting; targeting; Moesin; specific

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.

在动物发育过程中，细胞协同分化形成组织需要在质膜（PM）和下面的细胞骨架中建立独特的结构域，它们共同构成细胞皮层。我的研究项目主要集中在这些领域是如何建立在不同的细胞谱系。在这个过程中的一个关键因素是Moesin（莫伊），它可逆地将PM连接到皮质肌动蛋白细胞骨架。莫伊中的分子内相互作用使其在“活性”和“非活性”状态之间循环。当与PM结合时，认为莫伊具有“活性”，这是一个依赖于磷酸化和与磷脂酰肌醇4，5-二磷酸（PIP 2）结合的过程。然而，在某些情况下，莫伊可以在不存在磷酸化或PIP 2结合的情况下具有活性。我们的假设是，调节莫伊与PM相关蛋白的相互作用和翻译后修饰，如棕榈酰化也有助于驱动莫伊进入“活跃”状态。我们的目标是发现这些事件如何影响莫伊招募到细胞皮质，这是介导细胞粘附和增殖所需的分化的特定组织类型在动物发育过程中。一个重要的功能考虑是PM处的莫伊活性与其在细胞连接和卵裂沟处的其他作用的差异。因此，我们将比较神经干细胞（NSC），其中莫伊是PM组织所必需的，而上皮细胞，其中PM和细胞连接作用都需要莫伊。目的1将确定棕榈酰化如何调节莫伊活性。我们在莫伊中鉴定了两个潜在的棕榈酰化位点。我们发现，这些网站的突变块莫伊招聘的PM。我们将在莫伊中预测的棕榈酰化位点引入靶向突变，以确定它们如何分别影响发育中的大脑和翼盘的神经干细胞和上皮细胞中的PM募集。目的2将描述我们鉴定的几种与莫伊相互作用的PM相关蛋白。我们确定这些作为一个正向遗传筛选的一部分，影响翼盘上皮细胞中的Moe依赖性粘附的基因和神经干细胞中的MoE结合蛋白的质谱筛选。本科生HQP将分别表征一个或多个优先的候选人，以确定他们如何影响莫伊招聘到PM和神经干细胞，上皮细胞和培养细胞的细胞连接。他们还将研究这如何影响Moe介导的细胞增殖和分化调节。他们将使用特异性标记和间接免疫荧光检测来比较靶向体细胞嵌合体分析对粘附和增殖的影响。这将通过免疫共沉淀来补充，直接探测PM处莫伊复合物形成中包含这些辅因子的动力学。我的项目将继续推进我们对莫伊等蛋白质在细胞皮层中的亚结构域的组织的理解，以及这种组织如何协调果蝇的细胞增殖和分化。