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The role of dynein-2 in building a functional cilium.

dynein-2 在构建功能性纤毛中的作用。

基本信息

批准号：
BB/S005390/1
负责人：
Mark Dodding
金额：
$ 106.58万
依托单位：
University of Bristol
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2019
资助国家：
英国
起止时间：
2019 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FS005390%2F1
关键词：
role dynein building functional cilium.

项目摘要

Primary cilia project from the surface of nearly all human cells to serve as signalling platforms. The dynein-2 microtubule motor provides a fundamental link between microtubule motor function, protein trafficking, and cilia function because of its function in driving intraflagellar transport (IFT) within cilia. The Stephens lab was the first to define the subunit composition of the dynein-2 motor in humans. The Roberts lab have made major advances in our understanding of its regulation and are now working to define its structure and mechanisms of function using in vitro reconstitution. A notable point is that, unlike the related (and better understood) dynein-1 motor, dynein-2 is asymmetric with two key proteins, WDR34 and WDR60, associating with the main motor. Despite being part of the same motor complex, our new work shows that these two proteins have distinct functions. Using genome engineering of cells, we have shown that knocking out WDR34 blocks the ability of cells to form cilia. In contrast, cells lacking WDR60 can still form cilia. However, while cells normally form a tight diffusion barrier at the base of the cilia to gate entry and exit of proteins and lipids. This "transition zone" makes the cilia functionally separate from the rest of the cell. WDR60 knockout cells for cilia that have an abnormal structure and no longer have a tight diffusion barrier to physically segregated the cilia from the rest of the cell.Our proposal seeks to provide a complete picture of the molecular interactions of the dynein-2 complex using a combination of molecular cell biology approaches including advanced microscopy and proteomics. Our current BBSRC-funded work has developed proteomics approaches that have identified key interacting proteins that seem to direct the assembly and function of dynein-2. Here we propose to explore the molecular basis for the role of WDR34 and WDR60 in building the cilium, forming and then maintaining the ciliary transition zone. Building on other data, which we show in this proposal, we will also define how and where the dynein-2 motor is assembled from its component parts. We will also develop a new area of our work to study the balance between ciliogenesis and cell cycle. These processes are mutually exclusive because the same key cellular organelle, the centrioles, are required for both. In resting cells, the centrioles build the cilium, in cycling cells, they are used to build the mitotic spindle that segregates chromosomes between the two resulting daughters. Our work has shown that dynein-2 binds to a centriole protein called CEP170 that has been shown to work to control microtubule dynamics during entry to and exit from the cell cycle. This suggests close integration of dynein-2 function with cell cycle control providing an exciting new area of investigation.This is a frontier bioscience project that seeks to understand fundamental processes in cell biology. That said, the formation of cilia, tight control of cilia-based signalling pathways, and the control of entry to and exit from the cell cycle are fundamental to normal health as well as having potential long-term impact on human and animal health. Ciliary signals include those that control early human development as well as others that occur throughout life to control metabolism. Key pharmaceuticals targeting common cancers are also directed against ciliary signalling pathway. A full understanding of the structure and function of cilia is key to a diverse array of fields and has relevance from early human development and throughout life.

初级纤毛从几乎所有人类细胞的表面伸出，作为信号平台。dynein-2微管马达在微管运动功能、蛋白质运输和纤毛功能之间提供了一个基本的联系，因为它在纤毛内驱动鞭毛内运输（IFT）。斯蒂芬斯实验室是第一个确定人类动力蛋白-2马达亚基组成的实验室。罗伯茨实验室在我们对其调控的理解方面取得了重大进展，现在正在努力通过体外重建来确定其结构和功能机制。值得注意的一点是，与相关的（并且更好理解的）动力蛋白-1马达不同，动力蛋白-2与与主马达相关的两个关键蛋白WDR34和WDR60不对称。尽管是同一运动复合体的一部分，但我们的新工作表明，这两种蛋白质具有不同的功能。通过细胞基因组工程，我们发现敲除WDR34阻断了细胞形成纤毛的能力。相反，缺乏WDR60的细胞仍然可以形成纤毛。然而，细胞通常在纤毛基部形成一个紧密的扩散屏障，以限制蛋白质和脂质的进出。这个“过渡区”使纤毛在功能上与细胞的其他部分分离。WDR60敲除纤毛的细胞具有异常结构，并且不再具有将纤毛与细胞其他部分物理分离的紧密扩散屏障。我们的建议旨在结合分子细胞生物学方法，包括高级显微镜和蛋白质组学，提供动力蛋白-2复合物分子相互作用的完整图景。我们目前的工作已经开发出蛋白质组学方法，已经确定了关键的相互作用蛋白，这些蛋白似乎指导了动力蛋白-2的组装和功能。本文旨在探讨WDR34和WDR60在纤毛构建、纤毛过渡区形成和维持过程中作用的分子基础。基于我们在本提案中显示的其他数据，我们还将定义dynein-2电机从其组件组装的方式和位置。我们还将开辟一个新的研究领域，研究纤毛发生与细胞周期之间的平衡。这两个过程是相互排斥的，因为这两个过程都需要相同的关键细胞器——中心粒。在静止细胞中，中心粒构建纤毛；在循环细胞中，中心粒用于构建有丝分裂纺锤体，使两个子代染色体分离。我们的研究表明，dynein-2与一种名为CEP170的中心粒蛋白结合，该中心粒蛋白在进入和退出细胞周期期间控制微管动力学。这表明动力蛋白-2功能与细胞周期控制的紧密结合提供了一个令人兴奋的新研究领域。这是一个前沿生物科学项目，旨在了解细胞生物学的基本过程。也就是说，纤毛的形成、对基于纤毛的信号通路的严格控制以及对细胞周期进入和退出的控制是正常健康的基础，也对人类和动物的健康具有潜在的长期影响。纤毛信号包括控制人类早期发育的纤毛信号，以及在整个生命过程中控制新陈代谢的纤毛信号。针对常见癌症的关键药物也针对纤毛信号通路。对纤毛的结构和功能的充分了解是许多领域的关键，并且与人类早期发育和整个生命相关。