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

描述（由申请人提供）：布鲁氏锥虫是人类和牛的非洲锥虫病的病原体。这种寄生虫具有高度极化的细胞形状，最适合于逃避哺乳动物宿主的免疫反应，并迁移到其昆虫载体的唾液腺。这种极性的一个显著特征是锥虫的单鞭毛，它在细胞体的后部成核，然后沿着细胞表面向前呈螺旋状延伸。鞭毛的位置是必不可少的适当运动，必须在细胞分裂过程中遗传。有人提出，两个细胞骨架结构，一个在细胞体内部，一个在新鞭毛的尖端，负责确保新鞭毛的正确定位。鞭毛附着区连接（FAZJ）和鞭毛接头（FC）接触到一个预先存在的复制结构副本，并将其作为模板或向导。这个过程被称为结构遗传，是将位置信息从母细胞传递到子细胞的重要过程

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.