Investigating the molecular mechanisms of ciliary dynein motor assembly

生物运动是生命的一个重要属性。真核生物生命之树的一个完整的分支依赖于一种叫做纤毛的细长的毛发状运动结构来协调运动。被称为动力蛋白的运动蛋白为纤毛运动提供动力，并通过移动我们呼吸道上的微小纤毛来驱动基本过程，如精子的游动或肺部粘液的清除。动力蛋白发动机由许多必须组装的部件组成。组装失败导致马达不起作用，从而导致纤毛停滞。这种错误的组装是一个衰弱的肺部疾病的核心，称为原发性纤毛运动障碍（PCD），影响新生儿谁是无法清除他们的肺部由于静态纤毛。在这里，我建议了解动力蛋白的组装过程。19个组装因子在称为组装工厂的细胞隔间中构建动力蛋白发动机，就像汽车机械师在工厂中组装为汽车提供动力的发动机一样。为了充分理解组装过程，我需要知道三件事：1）组装因素如何共同作用，以融合动力蛋白马达的不同部分，并使它们配合在一起？我将使用蛋白质-蛋白质相互作用的研究，以及生化分析，研究如何一个DNAAF组与另一个DNAAF，以及确定其他结合伙伴，可能形成更大的群体组装动力蛋白。2)这些组装因子组是什么样的？直接查看装配因子的3D结构是了解它们如何工作的最直接方法。然而，由于蛋白质比人类小十亿倍，因此拍摄蛋白质的超高分辨率照片需要使用称为冷冻电子显微镜（cryo-EM）的强大仪器。我将使用冷冻电镜拍摄数千张DNAAF复合物的照片，并在计算机中将这些联合收割机组合起来，生成它们的3D模型。这些3D重建将为我提供足够的细节水平，以了解DNAAF如何一起工作，以组装动力蛋白马达的各个部分，更重要的是，PCD如何导致突变阻止DNAAF组的形成。在健康的情况下，装配工厂运行顺利，但当装配因装配因子中的PCD突变而受阻时，这会导致细胞内错误装配的电机堆积，这可能是有害的。装配厂在正常情况下如何运作尚不清楚，重要的是要先了解这一点，然后再试图解决在患病条件下的堆积。为了获得更深入的见解，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积聚，并恢复患者细胞中组装工厂的顺利运作。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构建生物马达来为我们肺部纤毛的基本运动提供动力，并帮助精子细胞游泳。这项工作可能会导致新的方法来启动组装过程，使停滞的纤毛再次移动，以治愈PCD患者。

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.