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 锥虫在全世界引起广泛的严重人类疾病。的 这些寄生虫的成功在很大程度上源于它们能够适应其细胞形态，以适应 它们在哺乳动物和昆虫宿主中的环境。观察到的细胞形态的广泛范围 依赖于一组位于细胞表面的微管，称为表膜下阵列。这些微管 是高度交联和非常稳定的，但很少有人知道如何阵列保持其 组织或它在细胞分裂过程中如何复制。在最近的邻近依赖性生物素化筛选中， 通过对布氏锥虫的研究，我们确定了两种蛋白质，这两种蛋白质对于形成阵列并确保其 在细胞分裂过程中正确复制第一个是一个孤儿驱动蛋白，名为Kinesin Localized to the Ingressing。 沟（KLIF）是在细胞分裂结束时将阵列分离成两个不同单位所必需的。KLIF 是一种非常有效的体外微管组装剂，这表明它的主要功能是组织微管 在阵列内通过将微管正末端聚集成极而形成新的细胞后部。另一个，叫 后部和后部边缘蛋白1（PAVE 1）是存在于后部的微管交联的组分。 它是阵列的一部分，并且对于使阵列逐渐变细以产生寄生虫的独特形状至关重要。这 这项提案将利用这些蛋白质来了解薄膜下阵列是如何组装和保持其形状的。 在目标1中，KLIF沿着犁沟进入时的精确轨迹将使用超 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具体情况。将表达全长KLIF以检测其 低聚状态和功能。在目的2中，研究了PAVE 1的微管结合特性及其与微管的相互作用。 将使用生物物理方法对伙伴进行研究。PAVE 1对细胞中微管加末端的偏好 将使用脉冲追踪策略结合改变微管的治疗来探测后壁。 动力学和翻译后修饰。在目标3中，免疫沉淀和邻近依赖性 生物素化将用于绘制KLIF和PAVE 1的相互作用伴侣， 参与膜下阵列生物发生。这项工作将进一步促进基本的 了解锥虫如何建立和传播其复杂的细胞形态， 生物学的重要组成部分。参与这些过程的途径是独特的和必要的， 进一步药物设计的潜在目标。