Investigating the molecular mechanisms of ciliary dynein motor assembly

生物运动是生命的一个关键属性。真核生物生命树的整个分支依靠细长的毛发状运动结构，称为纤毛来协调运动。被称为动力蛋白的马达蛋白质为纤毛运动提供动力，并通过移动呼吸道内的微小纤毛来驱动基本过程，如精子的游泳或将粘液清除出肺部。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无法工作，从而导致纤毛停滞。这种错误的组装是一种名为原发性纤毛运动障碍(PCD)的衰弱肺部疾病的核心，这种疾病会影响由于纤毛静止而无法清理肺部的新生儿。在这里，我建议了解动力蛋白的组装过程。19个组装因素在称为组装工厂的蜂窝隔间中制造动力引擎，就像汽车修理工可能在工厂组装为汽车提供动力的发动机一样。为了完全理解组装过程，我需要知道三件事1)组装因素如何共同作用，将动力蛋白马达的不同部件融合在一起，并使它们安装在一起？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如何与另一个DNAAF结合，并寻找其他可能形成更大基团的结合伙伴来组装动力蛋白。2)这些组装系数组是什么样子的？直接查看装配要素的3D结构是了解其工作原理的最直接方式。然而，由于蛋白质比人类小10亿倍，拍摄蛋白质的超高分辨率照片需要使用一种名为冷冻电子显微镜(Cryo-EM)的强大仪器。我将使用低温电子显微镜拍摄数千张DNAAF复合体的照片，并将这些照片组合在计算机中生成它们的3D模型。这些3D重建将为我提供足够的详细信息，以了解DNAAFs如何协同工作来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DNAAFs组的形成。3)组装工厂如何在细胞内工作？在健康的情况下，组装厂运作顺利，但当组装因PCD突变而受阻时，这会导致细胞内错误组装的马达堆积在一起，这可能是有害的。组装厂在正常情况下如何运作尚不清楚，在试图解决疾病情况下的连环问题之前，首先了解这一点是很重要的。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，并将这些工厂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，恢复患者细胞中组装工厂的平稳运行。总体而言，这项研究将为DNAAF蛋白的工作方式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。这项工作可能会带来启动组装过程的新方法，使停滞的纤毛再次移动，从而治愈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.