Mechanism and Regulation of Axonemal Dynein Arm Assembly in Motile Ciliated Epithelial Cells

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Motile cilia beat rhythmically to propel cell movement or drive extracellular fluid flow. The functional importance of cilia motility in human health is highlighted by primary ciliary dyskinesia (PCD), a genetic disease caused by cilia motility defects. Patients with PCD display left-right asymmetry defect, reduced fertility and progressive lung disease. Currently there is no specific therapy for PCD and management of symptoms has been the main approach. The dynein arms that power cilia motility comprise multiple components that are pre-assembled in the cytosol and many genes associated with PCD encode components of these dynein arms. Intriguingly, however, a separate group includes proteins that reside in the cytosol and appear to be involved in the assembly of dynein arm subunits and they are called dynein arm assembly factors (DNAAFs). Interestingly, multiple DNAAFs are localized in droplet shaped cytosolic foci. However, the precise function of these foci and the precise molecular function of most DNAAFs remain poorly understood. In addition to the assembly of protein components, how mRNAs and the translation machinery are coordinated to supply dynein arm components stoichiometrically is also largely unknown. Based on extensive preliminary and published data, our central hypothesis is that the co- chaperone proteins Pontin and Reptin are core components of a novel membraneless cytosolic assemblage distinct from droplets formed through LLPS; they function to coordinate the translation, folding and assembly of axonemal dynein arm components and prevent aggregate formation. In this project, we will combine zebrafish genetics, mouse genetics and cultured tracheal cells to test our central hypothesis. We propose two specific aims to achieve this goal. In the first aim, we will dissect the molecular and cellular function of Pontin-Reptin foci in discrete steps in dynein arm assembly. In the second aim, we will characterize Pontin-Reptin foci and investigate the mechanism of foci formation. Successful completion of this project will not only provide a molecular framework for dynein arm assembly and the etiology of PCD, but also lay the foundation for future investigation into the regulation, and possible intervention, of dynein arm assembly and cilia motility under diverse physiological and disease conditions.