The role of nephronophthisis proteins in multiciliated cells

NPH蛋白（NPHPs）的突变导致肾单位营养不良，这是一种常染色体隐性遗传的囊性肾病，伴有终末期肾病和多种肾外表现。虽然一些观察表明参与细胞周期和DNA损伤控制，NPHPs主要被视为纤毛过渡区（TZ）内的结构元件，控制货物进入纤毛轴丝。使用多纤毛细胞的非洲爪蟾表皮作为模型系统，我们的初步数据显示，NPHPs形成高度动态的蛋白质复合物已经在基体成熟。我们发现，适配器和肌动蛋白加帽蛋白CD 2AP与MKS 5/NPHP 8相互作用，并招募Pkd 2阳性囊泡的基体之前，他们对接到质膜。爪蟾nphp 8或pkd 2的耗尽减少纤毛发生，并导致TZ的超微结构异常。我们还发现，NPHP 1与SPIRE 1相互作用，这种肌动蛋白成核剂是在爪蟾表皮的多纤毛细胞中形成正常的亚顶端肌动蛋白层所必需的。奇怪的是，这种相互作用是由NPHP 4和NEK 8/NPHP 9控制的，揭示了NPHP 1 -4-8模块，肌动蛋白修饰蛋白，基体对接和完整TZ形成之间的复杂关系。因此，我们建议研究NPHPs在纤毛发生早期阶段的作用。具体来说，我们将1）定义在基体成熟过程中肌动蛋白聚合的动力学，2）分析在纤毛发生过程中钙瞬变的作用，3）定义NPHP蛋白，顶端肌动蛋白细胞骨架和TZ发育之间的联系。为了证实这些发现在哺乳动物细胞中的相关性，我们将4）选择性地消除小鼠运动纤毛中的Pkd 1和Pkd 2，使用floxed等位基因组合Foxj 1CRE小鼠系。总的来说，我们的研究结果将为NPHP和PKD在早期纤毛发生和TZ成熟过程中的功能提供新的分子见解，并有助于理解为什么NPHP/PKD突变患者经常出现呼吸系统症状。

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.