Pattern formation and function of PKD2/polycystin-2 in motile cilia

项目摘要/摘要 常染色体显性遗传性多囊肾病(ADPKD)主要由PKD1和 PKD2。这两种跨膜蛋白可能形成受体-通道复合体。电源发生故障 肾上皮细胞原生纤毛细胞膜上的PKD1/2复合体被广泛认为是原因 这种疾病的危害。然而，PKD1/2复合体感受到的体内刺激的性质，其机制是 通道开放和下行信令事件仍不确定。PKD2是一种短暂性受体 潜在的(Trp)通道，也存在于许多原生动物的睫状膜中，表明它具有 纤毛中的保守角色。我们建议使用衣藻作为一种简单的系统来分析组装和 PKD2通道在运动纤毛中的作用。我们的初步数据显示衣藻PKD2靶标 并将毛发状的糖蛋白聚合物--生殖器锚定在纤毛的细胞外表面。 一种新的PKD2缺失突变体缺失了乳腺体，该突变体游泳速度减慢，表明 PKD2在运动纤毛中的运动相关功能。值得注意的是，PKD2只锚定在九对中的两对上 微管(DMT)，即DMT 4和8，其定位两排PKD2-复合体 垂直于纤毛跳动的平面。与细胞骨架和胞外成分的关系 对于许多机械门控通道来说是典型的。因此，我们的发现表明，机械感觉的作用 运动纤毛中的PKD2。在目标1中，我们建议确定连接PKD2和PKD2的连接子的组成 衣藻中的纤毛微管。我们将分析从中国分离的PKD2复合体的组成 纤毛，利用体内邻近标记鉴定PKD2附近的蛋白质，并确定细胞内 有助于微管锚定的PKD2部分。结果将确定PKD2如何定位于 九对轴突中的两对。我们期待对细胞如何建立和识别差异的深入了解 在DMT之间，一般可能需要复杂的纤毛搏动模式。在目标2中，我们将分析 使用高速视频研究PKD2突变体的纤毛运动如何受到影响。细胞骨架和细胞外 锚可以起到浇注弹簧的作用，在变形时打开通道。突变体的分离 PKD2系链的缺陷将使我们能够确定PKD2在纤毛的锚定和构图中的作用 能动性。机械刺激纤毛时贴壁细胞的钙成像将用于直接采集 洞察PKD2的S通道功能。最后，我们将确定PKD2的分布如何适应 细胞环境的变化。总体而言，我们将测试PKD2感知到的主动弯曲的假设 这一过程需要可移动纤毛和常规的PKD2阵列。了解PKD2在运动中的作用 纤毛可以帮助确定PKD2在初级纤毛中的作用机制，因为感知到了流动诱导 PKD2对纤毛的被动弯曲被认为是肾脏的一种重要反馈机制，当 失误导致ADPKD。

Project Summary/Abstract Autosomal dominant polycystic kidney disease (ADPKD) is predominately caused by mutations in PKD1 and PKD2. These two transmembrane proteins likely form a receptor-channel complex. Malfunction of the PKD1/2 complex in the membrane of primary cilia of kidney epithelial cells is widely thought to be the cause of the disease. However, the nature of the in vivo stimulus sensed by the PKD1/2 complex, the mechanism of channel opening and the down-stream signaling events remain uncertain. PKD2 is a transient receptor potential (TRP) channel that is also present in the ciliary membrane of many protists indicating that it has a conserved role in cilia. We propose to use Chlamydomonas as a simple system to analyze the assembly and function of the PKD2 channel in motile cilia. Our preliminary data show that Chlamydomonas PKD2 targets and anchors mastigonemes, hair-like glycoprotein polymers, to the extracellular surface of cilia. Mastigonemes are missing from a novel pkd2 null mutant, which swims with reduced velocity indicating a motility related function of PKD2 in motile cilia. Remarkably, PKD2 is anchored on just two of the nine doublet microtubules (DMTs), i.e., DMTs 4 and 8, which positions the two rows of PKD2-mastigoneme complexes perpendicular to the plane of the ciliary beating. Association to the cytoskeleton and extracellular components are typical for many mechanically gated channels. Thus, our findings suggest a mechanosensory role of PKD2 in motile cilia. In Aim 1, we propose to identify the composition of the linker that connects PKD2 to the ciliary microtubules in Chlamydomonas. We will analyze the composition of PKD2 complexes isolated from cilia, identify proteins in the vicinity of PKD2 using in vivo proximity labeling, and determine the intracellular parts of PKD2 that contribute to microtubule anchoring. The results will establish how PKD2 is targeted to just two of the nine axonemal doublets. We expect insights into how cells establish and identify differences between the DMTs, a likely requirement for complex ciliary beat patterns in general. In Aim 2, we will analyze how ciliary motility is affected in the pkd2 mutant using high speed video. Cytoskeletal and extracellular anchors can function as gating springs that open channels upon deformation. The isolation of mutants defective in PKD2 tethering will allow us to determine the role of PKD2 anchoring and patterning for ciliary motility. Calcium imaging of adhered cells during mechanical stimulation of cilia will be used to gather direct insights into PKD2’s channel function. Finally, we will determine how the distribution of PKD2 adapts to changes in the cell’s environment. Overall, we will test the hypothesis that PKD2 senses the active bending of motile cilia and that regular PKD2 arrays are required for this process. Understanding PKD2 function in motile cilia could aid in determining the mechanism of PKD2 function in primary cilia since sensing of flow-induced passive bending of cilia by PKD2 is thought to be an important feedback mechanism in kidneys, that when amiss results in ADPKD.