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: 10490913
• 负责人: Christopher Luis de Graffenried
• 金额: $ 49.41万
• 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: 2021-09-17 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10490913
• 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; 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. 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.

项目总结 锥虫在全世界范围内引起一系列严重的人类疾病。这个 这些寄生虫的成功在很大程度上源于它们调整细胞形态以适应 它们的哺乳动物和昆虫宿主内的环境。广泛观察到的细胞形态 依赖于细胞表面下面的一组微管，称为膜下阵列。这些微管 都是高度交联和非常稳定的，但人们对阵列如何保持其 或者它在细胞分裂过程中如何复制。在最近的一次邻近生物素化筛查中 布鲁氏锥虫，我们鉴定了两种蛋白质，它们是形成阵列并确保它是 在细胞分裂过程中被正确复制。第一个，名为kinesin的孤儿kinesin定位于入口处 沟槽(KLIF)是在细胞分裂结束时将阵列分离成两个不同单元所必需的。KLIF 在体外是一种非常有效的微管捆绑器，这表明其主要功能是组织微管 在阵列内通过将微管正端聚集成极在后面形成新的细胞。另一个，叫做 后腹缘蛋白1(PAVE1)是存在于后部的微管交联物的一种成分 是寄生虫阵列的一部分，对于使阵列变细以产生寄生虫独特的形状是必不可少的。这 Proposal将使用这些蛋白质来了解膜下阵列是如何组装并保持其形状的。 在目标1中，KLIF沿着沟槽进入时的精确轨迹将使用超级 分辨率和活细胞显微镜。我们将使用EM和活细胞成像来研究KLIF RNAi表型 确定微管组织缺陷的具体情况。全长KLIF将被表达以测试其 齐聚状态和功能。在目标2中，PAVE1的微管结合特性及其相互作用 将使用生物物理方法对合作伙伴进行研究。PAVE1对微管+的偏好在细胞结束 将使用脉冲追逐策略结合改变微管的治疗来探测后部 动力学。在目标3中，将使用免疫沉淀和邻近依赖的生物素化来绘制 KLIF和PAVE1的相互作用伙伴，从而参与膜下阵列生物发生的途径可以 被建立起来。这项工作将进一步加深对锥虫如何建立和 传播它们复杂的细胞形态，这是它们生物学的重要组成部分。涉及的途径 这些独特而必要的过程可能成为进一步药物设计的潜在靶点。