The role of nephronophthisis proteins in multiciliated cells

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Mutations of NPH proteins (NPHPs) cause nephronophthisis, an autosomal recessive cystic kidney disease accompanied by end-stage renal disease and multiple extrarenal manifestations. Although several observations suggest an involvement in cell cycle and DNA damage control, NPHPs are primarily viewed as structural elements within the transition zone (TZ) of cilia that control access of cargo to the ciliary axoneme. Using multiciliated cells of the Xenopus epidermis as a model system, our preliminary data now reveal that NPHPs form highly dynamic protein complexes already during basal body maturation. We found that the adaptor and actin-capping protein CD2AP interacts with MKS5/NPHP8, and recruits Pkd2-positive vesicles to basal bodies before they dock to the plasma membrane. Depletion of either Xenopus nphp8 or pkd2 reduces ciliogenesis, and results in ultra-structural abnormalities of the TZ. We also discovered that NPHP1 interacts with SPIRE1, and that this actin nucleator is required for the formation of a normal subapical actin layer in multiciliated cells of the Xenopus epidermis. Curiously, this interaction is controlled by NPHP4 and NEK8/NPHP9, revealing a complex relationship between the NPHP1-4-8 module, actin-modifying proteins, basal body docking, and the formation of an intact TZ. We therefore propose to study the role of NPHPs during early steps of ciliogenesis. Specifically, we will 1) define the dynamics of actin polymerization during basal body maturation, 2) analyze the role of calcium transients during ciliogenesis, and 3) define the link between NPHP proteins, apical actin cytoskeleton, and TZ development. To confirm the relevance of these findings in mammalian cells, we will 4) selectively eliminate Pkd1 and Pkd2 in mouse motile cilia, using floxed alleles in combination the Foxj1CRE mouse line. Collectively, our results will provide new molecular insight into the function of NPHPs and PKDs during early ciliogenesis and TZ maturation, and help to understand why patients with NPHP/PKD mutations often develop respiratory manifestations.