Elucidating the Role of the Distal Appendage Protein CEP164 in Multiciliated Cell Differentiation

 描述（由申请方提供）：在呼吸道上皮中发现大量多纤毛细胞。每个多纤毛细胞上的数百个活动纤毛有助于信号传导过程和粘液纤毛清除。这些细胞的分化或纤毛形成的缺陷导致先天性疾病，如原发性纤毛运动障碍（PCD）和慢性呼吸道疾病，如囊性纤维化（CF）、哮喘和慢性阻塞性肺病。尽管受这些慢性肺部疾病以及PCD、CF和类似先天性疾病的破坏性性质影响的人口的显著部分，但对决定多纤毛细胞分化的分子机制知之甚少。2007年，发现了纤毛蛋白质中心体蛋白164（CEP 164）在初级纤毛发生中的绝对需求。从那时起，人们对CEP 164产生了极大的兴趣，因为CEP 164被发现是远端附属物的一部分，这是一种从中心粒/基体的远端部分延伸的结构，对于囊泡募集和基体对接至关重要。虽然先前的工作已经在揭示CEP 164在培养细胞中初级纤毛形成中的作用以及在斑马鱼中的吗啉代研究方面取得了进展，但是还没有研究在哺乳动物的生理环境中检查其在气道多纤毛细胞中的功能 模型系统该提案的总体目标是确定CEP 164在正常和患病气道上皮的多纤毛细胞分化过程中的功能。为了实现这一目标，该提议采用了一种新的多纤毛细胞特异性CEP 164敲除小鼠模型和小鼠气管上皮细胞（MTEC）的原代培养系统。通过利用这两种工具，CEP 164的作用，其相互作用的合作伙伴，和远端附件将通过遗传手段，生化测定和直接可视化（SEM，TEM，共聚焦和超分辨率显微镜）阐明。我希望这些方法将提供新的见解运动纤毛和多纤毛细胞发育的过程。我还预计，通过敲除多纤毛细胞中的CEP 164，将获得一个强大的PCD小鼠模型。这项工作将确定新的分子网络调节纤毛和潜在的治疗目标的患者患有疾病的功能障碍运动纤毛。

 DESCRIPTION (provided by applicant): Multiciliated cells are found in abundance in the respiratory epithelium. The hundreds of motile cilia on each multiciliated cell aids in both signaling processes and mucociliary clearance. Defects in the differentiation of these cells or in cilia formation result in congenital disorders, such as primary ciliary dyskinesia (PCD), and chronic respiratory conditions, such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease. Despite the significant portion of the population affected by these chronic pulmonary conditions as well as the devastating nature of PCD, CF, and similar congenital disorders, little is known about the molecular mechanisms dictating multiciliated cell differentiation. In 2007, an absolute requirement in primary ciliogenesis was discovered for the ciliary protein centrosomal protein 164 (CEP164). Since this time, great interest has followed as CEP164 was revealed to be part of the distal appendage, a structure that extends from the distal portion of the centriole/basal body and is critical for vesicle recruitment and basal body docking. While prior work has made progress in uncovering the role of CEP164 in the formation of primary cilia in cultured cells and morpholino studies in zebrafish, no study has yet to examine its functionality in airway multiciliated cells in the physiological setting of a mammalian model system. The overall goal of this proposal is to define the function of CEP164 during multiciliated cell differentiation in normal and diseased airway epithelium. To accomplish this goal, this proposal employs a novel multiciliated cell-specific CEP164 knockout mouse model and a primary culture system of mouse tracheal epithelial cells (MTECs). By harnessing both of these tools, the roles of CEP164, its interacting partners, and the distal appendage will be elucidated through genetic means, biochemical assays, and direct visualization (SEM, TEM, confocal, and super-resolution microscopy). I expect that these methodologies will provide novel insights into the processes of motile ciliogenesis and multiciliated cell development. I also anticipate that, by knocking out CEP164 in multiciliated cells, a powerful mouse model of PCD will be obtained. This work will identify novel molecular networks regulating ciliogenesis and potential therapeutic targets for patients afflicted with diseases of dysfunctional motile cilia.