Molecular mechanisms underlying the establishment of cell polarity.

项目总结(家长资助)：R35GM145340 真核生物生命的一个基本特征是细胞的建立和维持 两极。分子马达通过运输mRNA，蛋白质，囊泡， 和细胞器连接到细胞内的特定位置。来自单细胞生物的各种生物 酵母到人类使用mRNA定位和翻译调控作为一种方式 不对称地对蛋白质进行分类。这种现象的盛行最好的例证是 发育中的胚胎、神经元和上皮细胞，其中数以千计的mRNA在空间上 本地化。当这个过程受到损害时，可能会导致发育和神经方面的问题。 精神错乱。尽管这一主题很重要，而且这一现象很普遍，但我们缺乏 对信使核糖核酸定位的机械论理解。我们在理解上的一个关键差距是 去往本地化的mRNAs是如何被细胞识别并与非本地化的 本地化的mRNA。除了存在于定位mRNAs中的序列外， 结合这些mRNAs是决定它们细胞命运的关键。这种蛋白质复合体负责 将定位的mRNAs与分子马达连接，并调节它们的翻译。然而， 事实证明，识别这些关键蛋白质是非常具有挑战性的。就其本质而言， 信使核糖核酸的定位是一个高度动态的过程。因此，蛋白质-蛋白质和蛋白质- 组装定位信使核糖核蛋白所需的RNA相互作用 粒子很弱，而且是瞬变的。这使得它们可以用经典的生化方法进行鉴定。 接近几乎不可能；在提纯步骤中，复合体解体。在这 为了解决这一关键的知识鸿沟，我们提出了新的战略。我们的模式是 建议用于这些研究的是具有良好特性的果蝇黑腹果蝇卵 密室。在本申请的目标1中，我们建议使用邻近生物素结扎来定义 传输粒子的核心成分，并考察这些因素的守恒性 在苍蝇和哺乳动物之间。在目标2中，我们建议检验颗粒状的假设 被称为P小体的结构协调了mRNAs的定位及其翻译 监管。

Project Summary (parent grant): R35GM145340 A fundamental feature of eukaryotic life is the establishment and maintenance of cellular polarity. Molecular motors help to establish polarity by transporting mRNAs, proteins, vesicles, and organelles to specific sites within the cell. A variety of organisms from the single-celled yeast to humans use mRNA localization coupled with translational regulation as a way to asymmetrically sort proteins. The prevalence of this phenomenon is best illustrated in developing embryos, neurons and epithelial, in which thousands of mRNAs are spatially localized. When this process is compromised, it can result in developmental and neurological disorders. Despite the importance of this topic and the prevalence of this phenomenon, we lack a mechanistic understanding of mRNA localization. A critical gap in our understanding pertains to how mRNAs destined for localization are recognized by the cell and distinguished from non- localizing mRNAs. In addition to sequences present within localizing mRNAs, the proteins that bind these mRNAs are key to their cellular fate. This complex of proteins is responsible for linking localizing mRNAs with molecular motors and for regulating their translation. However, identifying these critical proteins has proven to be extremely challenging. By its very nature, the process of mRNA localization is highly dynamic. Consequently, the protein-protein and protein- RNA interactions required for assembling localizing messenger ribonucleoprotein (mRNP) particles are weak and transient. This has made their identification using classical biochemical approaches almost impossible; the complex falls apart during the purification step. In this application, we propose novel strategies to address this critical knowledge gap. The model we propose to use for these studies is the well characterized Drosophila melanogaster egg chamber. In Objective 1 of this application, we propose to use proximity biotin ligation to define the core components of transport particles and to examine the conservation of these factors between flies and mammals. In Objective 2, we propose to test the hypothesis that granular structures referred to as P bodies coordinate the localization of mRNAs with their translational regulation.