Molecular characterization of defects of the nexin-dynein regulatory complex causing Primary Ciliary Dyskinesia (PCD)

原发性睫状体运动障碍(PCD)是一种遗传性异质性疾病，其特征是慢性呼吸道疾病、左右身体不对称的随机性以及由于运动纤毛/鞭毛功能缺陷而导致的男性不育。目前PCD的诊断主要依靠显示呼吸运动纤毛的超微结构缺陷。轴丝成分如外动力蛋白臂、Nexin-dynein调节复合体(N-DRC)或放射状辐条成分的错误定位或缺失会导致纤毛静止或纤毛跳动模式异常，从而干扰粘液纤毛清除。有效的纤毛跳动对于正确清除呼吸道的粘液纤毛是必不可少的，并且需要调节所有涉及的纤毛成分。N-DRC与放射状辐条和中央对一起，负责调节纤毛运动和调节动力蛋白臂。N-DRC对滑移力的抑制将滑移力转化为轴丝的弯曲。我们的团队已经描述了三个编码N-DRC(CCDC164)或与N-DRC相关的基因(CCDC39和CCDC40)。CCDC39和CCDC40功能缺失突变导致轴丝组织紊乱、内动力蛋白臂(IDA)和N-DRC缺陷。有趣的是，CCDC164的功能丧失会导致N-DRC的缺陷，但不影响轴丝组织和IDA。在这项建议中，我们的目标是识别导致人类轴突紊乱和IDA缺陷以及孤立的N-DRC缺陷的其他基因，并通过高速视频显微镜、透射电子显微镜和高分辨率免疫荧光分析来表征这些突变的临床PCD表型和诊断结果。此外，我们计划分析N-DRC和N-DRC相关成分在不同类型的细胞(呼吸道、输卵管、结缔组织纤毛和精子鞭毛)中的组成和分布。由于对N-DRC的组装和对接知之甚少，我们打算在lnks/Ccdc40和Ccdc164突变小鼠中表征N-DRC缺陷。此外，我们将通过使用已知和候选N-DRC和N-DRC相关组件的重组表位标记蛋白进行免疫共沉淀(Co-IP)实验，分析N-DRC组件和负责N-DRC与微管正确对接的组件之间的相互作用。为了补充这一方法，我们将分析N-DRC野生型和N-DRC缺陷细胞裂解物的免疫沉淀物，然后进行质谱分析，以鉴定天然的N-DRC复合体。我们希望更详细地描述N-DRC和N-DRC相关的成分，这应该有助于解释在PCD中观察到的不同表型。综上所述，建议的分析将扩大关于纤毛结构、纤毛跳动调节和PCD的知识，并将为PCD的发病机制提供更多的见解，从而改进对受影响个体的诊断和遗传咨询。

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic airway disease, randomization of left-right (LR) body asymmetry and male infertility due to defects of motile cilia/flagella function. Currently PCD diagnosis mainly depends on demonstration of ultrastructural defects of respiratory motile cilia. Mislocalization or absence of axonemal components like outer dynein arms, nexin-dynein regulatory complex (N-DRC) or radial spoke components result in ciliary immotility or aberrant ciliary beating pattern and thus disturbed mucociliary clearance. Effective ciliary beating is essential for proper mucociliary clearance of the airways and regulation of all involved ciliary components is required. The N-DRC, together with the radial spokes and the central pair, is responsible for modulation of ciliary movement and regulation of dynein arms. Inhibition of the sliding forces by the N-DRC converts the sliding forces into bending of the axoneme. Our group already described three genes that encode components of the N-DRC (CCDC164) or related to the N-DRC (CCDC39 and CCDC40). Loss-of-function mutations in CCDC39 and CCDC40 result in axonemal disorganization, inner dynein arm (IDA) and N-DRC defects. Interestingly, loss-of-function in CCDC164 causes defects of the N-DRC but does not affect axonemal organization and IDA. Within this proposal we aim to identify additional genes responsible for axonemal disorganization and IDA defects as well as isolated N-DRC defects in humans and to characterize the clinical PCD phenotype and diagnostic findings of these mutations by high-speed video microscopy, transmission electron microscopy and high resolution immunofluorescence analyses. Furthermore, we plan to analyze the composition and distribution of N-DRC and N-DRC related components in distinct cell types (respiratory, fallopian tubule and nodal cilia and sperm flagella). Since little is known about the assembly and docking of the N-DRC, we intend to characterize the N-DRC defects in lnks/Ccdc40 and Ccdc164 mutant mice. Additionally, we will analyze the interaction of N-DRC components and components responsible for proper docking of the N-DRC to microtubules by performing co-immunoprecipitation (Co-IP) experiments using recombinant epitope-tagged proteins of known and candidate N-DRC and N-DRC related components. To complement this approach, we will analyze immunoprecipitates of N-DRC wild-type and N-DRC defective cell lysates followed by mass spectrometry analyses to identify native N-DRC complexes. We expect to describe the N-DRC and N-DRC related components in more detail, which should help to explain different phenotypes observed in PCD. Taken together the proposed analyses will expand the knowledge about ciliary structure, ciliary beating regulation and PCD and will give additional insights into the pathogenesis of PCD and thus improving diagnosis and genetic counseling of affected individuals.