Structural basis of dynein-driven ciliary motility

研究策略： 摘要： 纤毛和鞭毛是进化上保守的感觉细胞外的类似附属器的细胞器。 环境，驱动单个细胞的运动，或运输液体。睫毛功能缺陷导致 许多疾病被称为纤毛病，这些疾病会导致各种先天性疾病，并引起广泛的 一系列症状。外臂动力蛋白(OAD)是一种关键的马达蛋白，它产生了大多数机械 通过三磷酸腺苷的水解力驱动纤毛跳动。在超过一半的原发肿瘤中发现了OAD突变 睫状肌运动障碍(PCD)患者。这些突变在藻类和纤毛虫中有同源基因，这也导致了 纤毛/鞭毛功能障碍，表明低等物种和人类在 纤毛运动的机制。然而，缺少大多数睫毛成分的原子模型一直是主要的 阻碍了我们对纤毛系统的理解。我们将使用T.thermophila和C.reinhardtii的模型系统 阐明纤毛组装和动力蛋白驱动的纤毛运动在接下来的几年中，重点是 OAD及其监管。我们的目标是通过低温EM/ET的组合来揭示原子细节的机制， 相关光学和电子显微镜(Clem)、生物化学、细胞生物学、单分子生物物理学以及 计算建模等。我们接下来几年的目标是了解OAD阵列是如何在 纤毛，OAD在跳动过程中如何相互协调，OAD活动如何被其他纤毛调节 组件(如中央对)和细胞外信号，并建立轴丝的原子模型。我们会 共同开发低温电磁/ET方法，以解决长期存在的问题。已揭示的机制将提供 为我们未来在哺乳动物系统和人类疾病模型中的突变提供更准确的信息。

Research Strategy: Summary: Cilia and flagella are evolutionarily conserved appendage-like organelles that sense the extracellular environment, drive the movement of individual cells, or transport fluids. Defects of ciliary functions lead to numerous diseases termed the ciliopathies, which result in a variety of congenital disorders and cause a broad spectrum of symptoms. The outer-arm dynein (OAD) is a key motor protein that generates most mechanical forces to power the ciliary beating by ATP hydrolysis. OAD mutations were found in over half of the primary ciliary dyskinesia (PCD) patients. These mutations have orthologs in algae and ciliates, which also lead to cilia/flagellar dysfunctions, suggesting that the lower species and humans have important commonalities on the mechanisms of ciliary motility. However, lacking an atomic model of most ciliary components has been a main barrier to our understanding the cilium system. We will use the model systems T. thermophila and C. reinhardtii to elucidate the cilium assembly and dynein-driven ciliary motility in the following years, with an emphasis on OAD and its regulation. We aim to reveal the mechanisms in atomic details by a combination of cryo-EM/ET, correlative light and electron microscopy (CLEM), biochemistry, cell biology, single-molecule biophysics, and computational modeling etc. Our aims for the following years are to understand how OAD arrays are formed in cilia, how OADs coordinate with each other during beating, how the OAD activity is regulated by other ciliary components (such as central pair) and extracellular signals, and build an atomic model of axoneme. We will co-develop cryo-EM/ET methods to address long-standing problems. The revealed mechanisms will provide more accurate information for our future mutagenesis in mammalian systems and human disease models.