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

结构遗传在布氏鞭毛定位中的作用

基本信息

批准号：
9094424
负责人：
Christopher Luis de Graffenried
金额：
$ 20.31万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9094424
关键词：
African Trypanosomiasis Anterior Binding Biochemical Biology Biotinylation Cattle Cell Cycle Cell Line Cell Shape Cell Survival Cell division Cell surface Cells Cytokinesis Defect Drosophila polo protein Drug Targeting Electron Microscopy Elements Ensure Eukaryota Familiarity Flagella Fluorescence Microscopy Funding Mechanisms Generations Genetic Health Homologous Gene Human Immune response Immunofluorescence Microscopy Infection Inherited Insect Vectors Length Life Life Cycle Stages Methods Molecular Morphology Mothers Organelles Organism PLK1 gene Parasites Pathogenesis Pathway interactions Pattern Periodicity Phosphotransferases Positioning Attribute Process Proteins Proteome Proteomics RNA Interference Role Salivary Glands Staging Structure Substrate Interaction Techniques Testing Therapeutic Trypanosoma Trypanosoma brucei brucei Trypanosomiasis Validation Work base cell motility cell type daughter cell drug development drug discovery in vivo knockout gene migration nagana new therapeutic target novel novel therapeutics pathogen phosphoproteomics polarized cell prevent research study screening theories

项目摘要

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.

性状（由申请方提供）：布氏锥虫是人类非洲锥虫病和牛长角线虫病的病原体。该寄生虫具有高度极化的细胞形状，其被优化用于逃避其哺乳动物宿主中的宿主免疫应答和用于迁移至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个鞭毛，它在细胞体的后部成核，然后以螺旋状方式沿着细胞表面向前部延伸粘附。鞭毛的位置对于适当的运动性是必不可少的，并且必须在细胞分裂期间遗传。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，并将其用作模板或指导。这一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在各种各样的生物体中。虽然FC和FAZJ已经在形态学上进行了研究，但尚未鉴定出任何结构的组分，这使得难以确认它们的功能以及它们如何执行它们。我们最近发现，polo激酶同系物在锥虫（TbPLK）定位于这两个结构，因为它从后面到前面的细胞分裂过程中的过渡。激酶的抑制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以鉴定TbPLK底物和结合伴侣的过程中，我们发现了FC和FAZJ的组成蛋白。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，以充分描述它们在整个细胞周期中的位置。我们还将建立通过RNAi和基因敲除策略消耗蛋白质的结果，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如何发挥作用，以及它们是否具有其他生物体中细胞器的保守元件。扰乱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，就很可能无法重建，这将严重阻碍子细胞的运动性和分裂能力。考虑到缺乏治疗锥虫病的新疗法，寻找新的途径来利用药物靶向是至关重要的。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Alternate histories of cytokinesis: lessons from the trypanosomatids.

DOI：
10.1091/mbc.e19-12-0696
发表时间：
2020-11-15
期刊：
Molecular biology of the cell
影响因子：
3.3
作者：
Campbell PC;de Graffenried CL
通讯作者：
de Graffenried CL

Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesis.

DOI：
10.1111/mmi.13986
发表时间：
2018-08
期刊：
Molecular microbiology
影响因子：
3.6
作者：
Hilton NA;Sladewski TE;Perry JA;Pataki Z;Sinclair-Davis AN;Muniz RS;Tran HL;Wurster JI;Seo J;de Graffenried CL
通讯作者：
de Graffenried CL

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins.