Investigating the molecular mechanisms of ciliary dynein motor assembly

研究纤毛动力蛋白运动组件的分子机制

• 批准号: MR/X007219/1
• 负责人: Girish Ram Mali
• 金额: $ 194.19万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FX007219%2F1
• 关键词: Investigating; molecular; mechanisms; ciliary; dynein

Biological motion is a key attribute of life. An entire branch of the eukaryotic tree of life relies on slender hair-like moving structures called cilia to orchestrate motion. Motor proteins called dyneins power ciliary motion and drive fundamental processes such as the swimming of sperm or the clearance of mucus out of lungs by moving the tiny cilia lining our respiratory tracts. Dynein motors are comprised of many parts which must be assembled. A failure in assembly results in motors that do not function resulting in stalled cilia. This mis-assembly lies at the heart of a debilitating lung condition called Primary Ciliary Dyskinesia (PCD) that affects new-borns who are unable to clear their lungs due to static cilia.Here, I propose to understand the dynein assembly process. Nineteen assembly factors build dynein motors in cellular compartments called assembly factories in the same way in which car mechanics might assemble motor engines that power cars in a factory. To fully understand the assembly process, I need to know three things.1) How do the assembly factors work together to meld different parts of the dynein motor and make them fit together? I will use protein-protein interaction studies as well as biochemical assays to study how one DNAAF groups with another DNAAF as well as identify other binding partners which might form larger groups to assemble dyneins. 2) What do these groups of assembly factors look like? Directly looking at the 3D structure of the assembly factors is the most straightforward way of understanding how they work. However, because proteins are a billion times smaller than a human being, taking ultra-high-resolution pictures of proteins requires the use of a powerful instrument called a cryo-electron microscope (cryo-EM). I will use a cryo-EM to snap several thousand pictures of DNAAF complexes and combine these in a computer to generate their 3D models. These 3D reconstructions will provide me with a sufficient level of detail to understand how DNAAFs work together to assemble the various parts of the dynein motors and more importantly, how PCD causing mutations prevent groups of DNAAFs from forming.3) How do the assembly factories work inside a cell? In a healthy situation, assembly factories function smoothly but when assembly gets blocked due to PCD mutations in the assembly factors, this leads to a pile-up of mis-assembled motors inside cells which can be harmful. How assembly factories operate under normal conditions is unclear and it is important to understand this first before trying to resolve the pileups in a diseased condition. To gain deeper insights, I will use imaging techniques to directly observe assembly factories in healthy cells and compare these to diseased cells. This work will point to new ways to disentangle the build-up of motors and restore smooth functioning of assembly factories in patient cells. Overall, this study will provide exciting new insights into how the DNAAF proteins work, helping us understand how cells build biological motors to power the essential movement of cilia in our lungs as well as help sperm cells swim. This work could lead to new ways to kick-start the assembly process to make stalled cilia move again to cure PCD patients.

生物运动是生命的一个关键属性。真核生物生命树的一个完整分支依赖于细长的毛发状运动结构——纤毛来协调运动。被称为动力蛋白的运动蛋白为纤毛运动提供动力，并通过移动附着在呼吸道内的微小纤毛来驱动精子游动或清除肺部粘液等基本过程。动力电机由许多必须组装的部件组成。装配失败会导致电机不能正常工作，导致纤毛失速。这种错误的组装是一种使肺部衰弱的疾病的核心，这种疾病被称为原发性纤毛运动障碍（PCD），它会影响由于静态纤毛而无法清理肺部的新生儿。在这里，我建议了解dynein装配过程。19个装配要素在称为装配工厂的细胞隔间中构建动力马达，就像汽车机械师在工厂中组装汽车发动机一样。为了充分了解装配过程，我需要知道三件事。1)装配因素如何共同工作，将动力电机的不同部分融合在一起，并使它们组装在一起？我将使用蛋白质-蛋白质相互作用研究以及生化分析来研究一个DNAAF组如何与另一个DNAAF组以及识别其他可能形成更大组来组装动力蛋白的结合伙伴。2)这些组合因素是什么样子的？直接观察组装因子的3D结构是理解它们如何工作的最直接的方法。然而，由于蛋白质比人体小10亿倍，拍摄超高分辨率的蛋白质照片需要使用一种叫做冷冻电子显微镜（cryo-EM）的强大仪器。我将使用低温电子显微镜拍摄数千张DNAAF复合物的照片，并将这些照片组合在电脑上生成它们的3D模型。这些3D重建将为我提供足够的细节，以了解DNAAFs如何协同工作以组装动力蛋白马达的各个部分，更重要的是，PCD引起的突变如何阻止DNAAFs组的形成。3)细胞内的组装工厂是如何工作的？在健康的情况下，组装工厂运作顺利，但当组装因装配因子中的PCD突变而受阻时，这将导致细胞内错误组装的马达堆积，这可能是有害的。组装工厂如何在正常条件下运作尚不清楚，在试图解决疾病条件下的堆积之前，了解这一点很重要。为了获得更深入的了解，我将使用成像技术直接观察健康细胞中的组装工厂，并将其与患病细胞进行比较。这项工作将指出新的方法来解开马达的堆积，并恢复患者细胞中组装工厂的正常运作。总的来说，这项研究将为DNAAF蛋白如何工作提供令人兴奋的新见解，帮助我们了解细胞如何建立生物马达，为肺部纤毛的基本运动提供动力，并帮助精子细胞游泳。这项工作可能会带来新的方法来启动组装过程，使停滞的纤毛再次移动，从而治愈PCD患者。