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

原发性纤毛运动障碍（PCD）是一种遗传异质性疾病，其特征是慢性气道疾病、随机左右（LR）身体不对称以及由于纤毛/鞭毛运动功能缺陷导致的男性不育。目前PCD的诊断主要依靠呼吸运动纤毛超微结构缺陷的显示。轴突成分如外动力蛋白臂、内链-动力蛋白调节复合体（N-DRC）或径向辐条成分定位错误或缺失导致纤毛不动或纤毛跳动模式异常，从而干扰纤毛黏液清除。有效的纤毛搏动对于气道的纤毛粘液清除和所有相关纤毛成分的调节是必不可少的。N-DRC与径向辐条和中心副一起负责纤毛运动的调节和动力臂的调节。N-DRC对滑动力的抑制将滑动力转化为轴突的弯曲。我们的研究小组已经描述了三个编码N-DRC成分（CCDC164）或与N-DRC相关的基因（CCDC39和CCDC40）。CCDC39和CCDC40的功能缺失突变导致轴突紊乱、内动力臂（IDA）和N-DRC缺陷。有趣的是，CCDC164的功能丧失导致N-DRC缺陷，但不影响轴突组织和IDA。在这项提议中，我们的目标是确定其他基因负责轴突破坏和IDA缺陷以及人类分离的N-DRC缺陷，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表征临床PCD表型和这些突变的诊断结果。此外，我们计划分析N-DRC和N-DRC相关成分在不同细胞类型（呼吸、输卵管、结纤毛和精子鞭毛）中的组成和分布。由于对N-DRC的组装和对接知之甚少，我们打算表征lnks/Ccdc40和Ccdc164突变小鼠的N-DRC缺陷。此外，我们将通过使用已知和候选N-DRC以及N-DRC相关成分的重组表位标记蛋白进行共免疫沉淀（Co-IP）实验，分析N-DRC成分和负责N-DRC与微管适当对接的成分之间的相互作用。为了补充这种方法，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，然后进行质谱分析以鉴定天然N-DRC复合物。我们希望更详细地描述N-DRC和N-DRC相关成分，这将有助于解释在PCD中观察到的不同表型。综上所述，这些分析将扩大对纤毛结构、纤毛跳动调节和PCD的认识，并将进一步深入了解PCD的发病机制，从而提高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.