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

阐明远端附属蛋白 CEP164 在多纤毛细胞分化中的作用

• 批准号: 9356318
• 负责人: Saul Sylvan Siller
• 金额: $ 4.9万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2018-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9356318
• 关键词: Address; Affect; Apical; Asthma; Bardet-Biedl Syndrome; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Brain; Categories; Cell Count; Cell Differentiation process; Cell Line; Cell surface; Cells; Centrioles; Chronic; Chronic Obstructive Airway Disease; Chronic Rhinitis; Chronic Sinusitis; Cilia; Congenital Disorders; Cultured Cells; Cystic Fibrosis; Data; Defect; Development; Disease; Disease model; Distal; Docking; Edema; Epithelial Cells; Fiber; Genes; Genetic; Goals; Growth; Hydrocephalus; Imagery; Individual; Infertility; Injectable; Joubert syndrome; Knock-out; Knockout Mice; Laboratories; Location; Lung; Mammalian Oviducts; Mature Centriole; Methodology; Microscopy; Modeling; Molecular; Movement; Mucociliary Clearance; Mucous body substance; Mus; Mutation; Nasal Epithelium; Nature; Nephronophthisis; Outcome; Pathogenesis; Pathologic; Pathology; Patients; Phenotype; Physiological; Population; Primary Ciliary Dyskinesias; Process; Production; Proteins; Publishing; Recruitment Activity; Reporting; Research; Resolution; Role; Signal Transduction; Structure; Structure of respiratory epithelium; Symptoms; Syndrome; System; Techniques; Time; Tissues; Ventricular; Vesicle; Work; Zebrafish; airway epithelium; appendage; cell motility; ciliopathy; cilium biogenesis; cilium motility; falls; human disease; human tissue; in vivo; insight; interest; kinetosome; mouse model; novel; novel therapeutics; public health relevance; respiratory; therapeutic target; tool

 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.

 描述（由申请人提供）：在呼吸道上皮细胞中发现了大量的多纤毛细胞。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膜纤毛清除。这些细胞分化或纤毛形成的缺陷会导致先天性疾病，如原发性纤毛运动障碍 (PCD) 和慢性呼吸系统疾病，如囊性纤维化 (CF)、哮喘和慢性阻塞性肺病。尽管很大一部分人口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，发现纤毛蛋白中心体蛋白 164 (CEP164) 是初级纤毛发生的绝对必要条件。从那时起，人们对 CEP164 产生了极大的兴趣，因为 CEP164 被发现是远端附属物的一部分，远端附属物是一种从中心粒/基体远端部分延伸的结构，对于囊泡募集和基体对接至关重要。虽然之前的工作在揭示 CEP164 在培养细胞初级纤毛形成中的作用以及斑马鱼吗啡啉研究方面取得了进展，但尚未有研究检验其在哺乳动物生理环境中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 CEP164 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，该提案采用了一种新型多纤毛细胞特异性 CEP164 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，CEP164、其相互作用伙伴和远端附属物的作用将通过遗传手段、生化测定和直接可视化（SEM、TEM、共聚焦和超分辨率显微镜）来阐明。我期望这些方法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，通过敲除多纤毛细胞中的 CEP164，将获得强大的 PCD 小鼠模型。这项工作将确定调节纤毛发生的新分子网络以及患有运动性纤毛功能障碍疾病的患者的潜在治疗靶点。