Biogenesis of the Trypanosoma brucei subpellicular microtubule array

布氏锥虫表膜下微管阵列的生物发生

• 批准号: 10387168
• 负责人: Christopher Luis de Graffenried
• 金额: $ 49.52万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-23 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387168
• 关键词: Africa South of the Sahara; African Trypanosomiasis; Anterior; Binding; Binding Proteins; Biogenesis; Biology; Biotinylation; Blood; Cell division; Cell surface; Cells; Cellular Morphology; Complex; Crowding; Cytokinesis; Defect; Disease; Drug Design; Drug Targeting; Economic Burden; Environment; Eukaryota; Excision; Goals; Human; Immunoprecipitation; In Vitro; Insecta; Kinesin; Leishmania; Length; Livestock; Maintenance; Mammals; Maps; Mediating; Microtubule Bundle; Microtubule-Associated Proteins; Microtubules; Molecular; Morphology; Motor; Names; Organism; Orphan; Parasites; Pathway interactions; Phenotype; Physiologic pulse; Play; Plus End of the Microtubule; Post-Translational Protein Processing; Process; Production; Property; Proteins; RNA Interference; Research; Resolution; Role; Shapes; Site; Structure; System; Testing; Therapeutic; Tissues; Trypanosoma brucei brucei; Trypanosoma cruzi; Viscosity; Work; Yeasts; biophysical techniques; crosslink; daughter cell; health economics; human pathogen; live cell imaging; live cell microscopy; nagana; neglected tropical diseases; preference; protein complex; recruit; scaffold; segregation; stem; success

PROJECT SUMMARY The trypanosomatids cause a broad range of severe human illnesses across the entire world. The success of these parasites stems in large part from their ability to adapt their cellular morphology to suit the environments within their mammalian and insect hosts. The extensive range of observed cellular morphologies rely on a set of microtubules that underlie the cell surface, known as the subpellicular array. These microtubules are heavily crosslinked and remarkably stable, but very little is known about how the array maintains its organization or how it duplicates during cell division. During a recent proximity-dependent biotinylation screen in Trypanosoma brucei, we identified two proteins that are essential for shaping the array and assuring that it is duplicated correctly during cell division. The first, an orphan kinesin named Kinesin Localized to the Ingressing Furrow (KLIF), is essential for the segregation of the array into two distinct units at the end of cell division. KLIF is a very effective microtubule bundler in vitro, which suggests that its primary function is to organize microtubules within the array to form a new cell posterior by gathering microtubule plus-ends into a pole. The other, called Posterior And Ventral Edge Protein 1 (PAVE1) is a component of microtubule crosslinks present at the posterior portion of the array and is essential for tapering the array to produce the parasite’s distinctive shape. This proposal will use these proteins to understand how the subpellicular array is assembled and maintains its shape. In Aim 1, the precise track KLIF takes as it ingresses along the furrow will be established using super- resolution and live-cell microscopy. We will study the KLIF RNAi phenotype using EM and live-cell imaging to determine the specifics of the microtubule organizing defect. Full-length KLIF will be expressed to test its oligomerization state and function. In Aim 2, the microtubule-binding properties of PAVE1 and its interacting partners will be studied using biophysical approaches. PAVE1 preference for microtubule plus ends at the cell posterior will be probed using a pulse-chase strategy in conjunction with treatments that alter microtubule dynamics and posttranslational modifications. In Aim 3, immunoprecipitation and proximity-dependent biotinylation will be employed to map the interacting partners of both KLIF and PAVE1 so that the pathways involved in subpellicular array biogenesis can be established. This work will further the fundamental understanding of how trypanosomatids establish and transmit their complex cellular morphologies, which are essential parts of their biology. Pathways involved in these processes that are unique and essential may be potential targets for further drug design.

项目摘要 锥形剂在全世界引起广泛的严重人类疾病。这 这些寄生虫的成功在很大程度上是从它们适应其细胞形态以适合其的能力 其哺乳动物和昆虫宿主中的环境。大量观察到的细胞形态 依靠一组构成细胞表面的微管，称为亚细胞阵列。这些微管 交联的交联且非常稳定，但对阵列如何保持其保持 组织或在细胞分裂过程中重复的方式。在最近的接近性依赖性生物素化筛网中 布鲁氏锥虫，我们确定了两种蛋白质，这些蛋白质对于塑造阵列并确保它是必不可少的 在细胞分裂期间正确复制。首先，一个名为驱动蛋白的孤儿驱动器定位于入学 Furrow（KLIF）对于在细胞分裂末端将阵列隔离为两个不同的单元至关重要。克里夫 是一个非常有效的微管束体体外，这表明其主要功能是组织微管 在阵列中，通过将微管加末端收集到杆上，形成一个新的单元。另一个叫 后和腹侧边缘蛋白1（PAVE1）是在后部存在的微管交联的组成部分 阵列的一部分，对于缩小阵列以产生寄生虫的独特形状至关重要。这 建议将使用这些蛋白质来了解如何组装细胞阵列并保持其形状。 在AIM 1中，将使用超级 - 分辨率和活细胞显微镜。我们将使用EM和活细胞成像研究KLIF RNAi表型 确定微管组织缺陷的细节。全长KLIF将被表达为测试 寡聚状态和功能。在AIM 2中，Pave1的微管结合特性及其相互作用 合作伙伴将使用生物物理方法进行研究。 Pave1偏爱微管加上细胞处的末端 将使用脉冲练习策略与改变微管的处理结合使用脉冲策略进行探测 动力和翻译后修饰。在AIM 3中，免疫沉淀和接近依赖性 将雇用生物素化来绘制KLIF和PAVE1的相互作用伙伴，以便途径 可以建立参与亚细胞阵列生物发生。这项工作将进一步发展 了解锥虫如何建立和传输其复杂的细胞形态，这是 其生物学的重要部分。这些独特且必不可少的过程中涉及的途径可能是 进一步药物设计的潜在目标。