NPHP proteins are binding partners of nucleoporins at the base of the primary cilium

Cilia are microtubule-based organelles that protrude from the surface of eukaryotic cells to generate motility and to sense and respond to environmental cues. In order to carry out these functions, the complement of proteins in the cilium must be specific for the organelle. Regulation of protein entry into primary cilia has been shown to utilize mechanisms and components of nuclear gating, including nucleoporins of the nuclear pore complex (NPC). We show that nucleoporins also localize to the base of motile cilia on the surface of trachea epithelial cells. How nucleoporins are anchored at the cilium base has been unclear as transmembrane nucleoporins, which anchor nucleoporins at the nuclear envelope, have not been found to localize at the cilium. Here we use the directed yeast two-hybrid assay to identify direct interactions between nucleoporins and nephronophthisis proteins (NPHPs) which localize to the cilium base and contribute to cilium assembly and identity. We validate NPHP-nucleoporin interactions in mammalian cells using the knocksideways assay and demonstrate that the interactions occur at the base of the primary cilium using bimolecular fluorescence complementation. We propose that NPHP proteins anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle.

纤毛（Cilia）是基于微管的细胞器，从真核细胞表面伸出，以产生运动并感知、响应环境信号。为行使上述功能，纤毛内的蛋白质组分必须具备该细胞器的特异性。此前研究表明，蛋白质进入初级纤毛（primary cilia）的调控过程依赖核孔门控的机制与组分，其中包括核孔复合物（nuclear pore complex, NPC）的核孔蛋白（nucleoporins）。本研究发现，核孔蛋白同样定位于气管上皮细胞表面的运动纤毛基部。然而，核孔蛋白如何锚定在纤毛基部仍不明确：因为负责在核被膜上锚定核孔蛋白的跨膜核孔蛋白，尚未被发现可定位于纤毛中。本研究通过定向酵母双杂交实验，鉴定出核孔蛋白与肾消耗病蛋白（nephronophthisis proteins, NPHPs）之间的直接相互作用；后者定位于纤毛基部，参与纤毛的组装与身份维持。我们利用反向易位实验（knocksideways assay）在哺乳动物细胞中验证了NPHP与核孔蛋白的相互作用，并通过双分子荧光互补实验（bimolecular fluorescence complementation）证实该相互作用发生在初级纤毛的基部。我们提出，肾消耗病蛋白可将核孔蛋白锚定在初级纤毛基部，从而调控蛋白质进入该细胞器。