PROJECT SUMMARY Airway motile cilia play an important role in mucociliary clearance, the primary innate defense mechanism of the lung, by propelling inhaled pathogens trapped in mucus up and out of the respiratory tract. Impaired ciliary function is commonly found in patients with chronic pulmonary diseases, and genetic mutations that result in aberrant cilia formation and loss of function often lead to the development of genetically heterogenous, multisystem disorders called ciliopathies. The current body of literature suggests that the unique composition and morphology of the ciliary membrane that envelopes the cilium are involved in maintaining cilia function and homeostasis. However, the molecular players and mechanisms underlying the formation and maintenance of the ciliary membrane remain poorly understood. The overarching objective of this proposal is to define the physiological roles of the membrane-binding proteins ciBAR1 and 2 (formerly known as FAM92A and B) in ciliogenesis and airway ciliated cell differentiation by using novel knockout mouse models. Both ciBAR proteins possess a single Bin/Amphiphysin/Rvs (BAR) domain, which allows them to dimerize and form crescent- shaped structures to preferentially sense and sculpt curved membranes. Previous studies have shown that ciBAR1 and 2 bind to the ciliary protein Chibby1 (Cby1), which acts in preciliary vesicle recruitment and basal body docking during early stages of ciliated cell differentiation. ciBAR 1 and 2 colocalize with Cby1 at the ciliary base and play crucial roles in ciliogenesis, but their in vivo functions are currently unknown. The central hypothesis of this proposal is that ciBAR1 and 2 play key roles in ciliogenesis and differentiation of airway ciliated cells via their membrane-binding and shaping activity. To address this hypothesis, I will use novel ciBAR1-/- and ciBAR2-/- knockout mouse models developed by our laboratory and propose the following Specific Aims. In Aim 1, I will investigate if the loss of ciBAR1 or ciBAR2 in mice results in ciliary defects in the trachea. In Aim 2, I will examine the roles of ciBAR proteins in formation and maintenance of ciliary membranes during ciliogenesis using primary cell cultures, biochemical methods, and cell biological approaches. Collectively, my proposed project will shed light on the in vivo functions of ciBAR1 and 2 in ciliogenesis and airway ciliated cell differentiation and advance our existing knowledge of how the ciliary membrane is generated and maintained throughout the process of ciliogenesis.