The role of structural inheritance in the positioning of the T. brucei flagellum

描述(申请人提供)：布氏锥虫是非洲锥虫病的人类病原体和牛的纳加纳病原体。这种寄生虫具有高度极化的细胞形状，这是为了逃避哺乳动物宿主的宿主免疫反应和迁移到昆虫媒介的唾液腺而优化的。这种极性的一个显著特征是锥虫体的单个鞭毛，它在细胞体的后部成核，然后沿着细胞表面向前方螺旋状延伸。鞭毛的位置对于正常的运动是必不可少的，并且必须在细胞分裂过程中遗传。有人提出，两个细胞骨架结构，一个在细胞体内，一个在新鞭毛的顶端，负责确保新鞭毛正确定位。鞭毛附着区连接(FAZJ)和鞭毛连接器(FC)接触复制结构的预先存在的副本，并将其用作模板或引导。这一过程被称为结构遗传，是将位置信息从母细胞传递给子细胞的重要过程 在各种生物体中。虽然已经对Fc和FAZJ进行了形态上的研究，但这两种结构的成分都没有被鉴定出来，这使得确定它们的功能和如何执行它们变得困难。我们最近发现，锥体中的Polo-Kinase同源物(TbPLK)在分裂过程中从细胞的后部转移到细胞的前部时，定位于这两个结构。抑制该激酶会导致鞭毛定位的严重缺陷，包括分离的鞭毛。在进行体内生物素化筛选(BioID)和磷酸化蛋白质组学鉴定TbPLK底物和结合伙伴的过程中，我们发现了Fc和FAZJ的蛋白质。在拟议的工作中，我们将使用荧光和电子显微镜来确定这两种蛋白质在其两个生命周期阶段的定位，以全面描述它们在细胞周期中的位置。我们还将建立RNAi和基因敲除策略耗尽蛋白质的后果，以确定FAZJ和Fc的功能是否与理论一致。我们将使用FAZJ和Fc蛋白进行额外的BioID筛选，以生成这两种结构的蛋白质组。这些蛋白质组将更全面地描述这些结构的组成部分，这将有助于我们解释它们是如何发挥作用的，以及它们是否与其他生物体中的细胞器具有保守的元件。干扰这些结构的组装是一种可行的治疗途径，因为它应该抑制新鞭毛的正确定位。一旦这种位置信息丢失，很可能无法重新建立，这将严重阻碍子细胞的能动性和分裂能力。考虑到治疗锥虫病的新疗法的缺乏，寻找新的药物靶向途径是至关重要的。

DESCRIPTION (provided by applicant): Trypanosoma brucei is the causative agent of African trypanosomiasis in humans and nagana in cattle. The parasite has a highly polarized cell shape that is optimized for evading the host immune response in its mammalian hosts and for migration to the salivary glands of its insect vector. One prominent feature of this polarity is th trypanosome's single flagellum, which nucleates in the posterior of the cell body, then extends adhered along the cell surface towards the anterior in a helical pattern. The positioning of the flagellum is essential for proper motility and must be inherited during cell division. It has been proposed that two cytoskeletal structures, one inside the cell body and one at the tip of the new flagellum, are responsible for ensuring that the new flagellum is positioned correctly. The flagellar attachment zone junction (FAZJ) and flagella connector (FC) contact a preexisting copy of the replicating structure and use it as a template or guide. This process, known as structural inheritance, is an important for conveying positional information from mother cell to daughter cell in a variety of organisms. While the FC and FAZJ have been studied morphologically, no components of either structure have been identified, which has made it difficult to confirm their functions and how they perform them. We have recently shown that the polo-kinase homolog in trypanosomes (TbPLK) localizes to both of these structures as it transits from the posterior to the anterior of the cell during division. Inhibition of the kinase causes severe defects in flagellr positioning, including detached flagella. During the course of performing in vivo biotinylation screens (BioID) and phosphoproteomics to identify TbPLK substrates and binding partners, we discovered proteins that are components of the FC and FAZJ. In the proposed work, we will determine the localization of both proteins in its two life cycle stages using fluorescence and electron microscopy to fully describe their position throughout the cell cycle. We will also establish the consequence of depleting the proteins by RNAi and gene knockout strategies to determine if the functions of the FAZJ and FC are consistent with theory. We will conduct additional BioID screens using the FAZJ and FC proteins to generate proteomes of both structures. These proteomes will more fully describe the components of these structures, which will help us explain how they function and if they have conserved elements with organelles in other organisms. Perturbing the assembly of these structures is a viable therapeutic avenue because it should inhibit the correct positioning of the new flagellum. Once this positional information is lost it is likely that it cannot be reestablished, which will severely hamper the daughter cell's motility and its ability to divide. Considering the lack of novel therapeutics for treating trypanosomiasis, finding novel pathways to exploit for drug targeting is essential.