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

 描述(申请人提供)：在呼吸道上皮中发现大量的多纤毛细胞。每个多纤毛细胞上数以百计的活动纤毛有助于信号传递和粘液纤毛清除。这些细胞的分化或纤毛形成的缺陷会导致先天性疾病，如原发性睫状肌运动障碍(PCD)，以及慢性呼吸系统疾病，如囊性纤维化(CF)、哮喘和慢性阻塞性肺疾病。尽管有相当一部分人受到这些慢性肺部疾病以及PCD、CF和类似先天性疾病的破坏性影响，但人们对决定多纤毛细胞分化的分子机制知之甚少。2007年，纤毛发生对纤毛中心体蛋白164(CEP164)的绝对需求被发现。从那时起，随着CEP164被发现是远端附件的一部分，人们对此产生了极大的兴趣。远端附件是一种从中心粒/基底体的远端延伸出来的结构，对于囊泡的募集和基底体的对接至关重要。虽然先前的工作已经在揭示CEP164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啡研究方面取得了进展，但还没有研究在哺乳动物的生理环境中检验其在呼吸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CEP164在正常和病变的呼吸道上皮多纤毛细胞分化过程中的功能。为了实现这一目标，该提案采用了一种新的多纤毛细胞特异性CEP164基因敲除小鼠模型和小鼠气管上皮细胞(MTECs)的原代培养系统。通过利用这两种工具，CEP164、其相互作用的伙伴和远端附件的作用将通过遗传手段、生化分析和直接可视化(扫描电子显微镜、透射电子显微镜、共聚焦和超分辨率显微镜)来阐明。我期望这些方法学将为运动纤毛发生和多纤毛细胞发育过程提供新的见解。我还预计，通过在多纤毛细胞中敲除CEP164，将获得一个强大的PCD小鼠模型。这项工作将确定调控纤毛发生的新分子网络，并为患有功能障碍的运动性纤毛疾病的患者确定潜在的治疗目标。

 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.