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

运动纤毛中 PKD2/多囊蛋白-2 的模式形成和功能

• 批准号: 10266797
• 负责人: Karl Lechtreck
• 金额: $ 30.2万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-18 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10266797
• 关键词: Adult; Affect; Area; Autosomal Dominant Polycystic Kidney; Binding; Biochemical; Calcium; Calcium Spikes; Cations; Cells; Chlamydomonas; Cilia; Complex; Cues; Cytoskeleton; Data; Development; Disease; Distal; Ensure; Environment; Event; Feedback; Gene Expression; Glycoproteins; Goals; Hair; Hereditary Disease; Image; Integral Membrane Protein; Invertebrates; Kidney; Label; Learning; Left; Length; Ligand Binding; Light; Mammals; Mechanical Stimulation; Mechanics; Membrane; Membrane Proteins; Microtubules; Modeling; Motion; Motor; Mutation; Nature; Nodal; PKD2 protein; Pattern; Pattern Formation; Phase; Polymers; Positioning Attribute; Process; Proteins; Regulation; Role; Sensory Process; Signal Transduction; Speed; Stimulus; Structure; Surface; Swimming; System; TRP channel; Testing; Touch sensation; Viscosity; calcium indicator; cell motility; cilium motility; extracellular; fluid flow; in vivo; insight; kidney epithelial cell; member; mutant; novel; particle; receptor; response; sound; transmission process

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.

项目总结/摘要 常染色体显性多囊肾病（ADPKD）主要由PKD 1和PKD 2基因突变引起。 PKD 2.这两种跨膜蛋白可能形成受体通道复合物。故障 肾上皮细胞初级纤毛膜中的PKD 1/2复合物被广泛认为是导致肾上皮细胞凋亡的原因。 的疾病。然而，PKD 1/2复合物感受到的体内刺激的性质， 信道开放和下游信令事件仍然不确定。PKD 2是一种瞬时受体， TRP通道也存在于许多原生生物的睫状膜中，表明它具有一种 在纤毛中的保守作用。我们建议使用衣藻作为一个简单的系统来分析组装， PKD 2通道在运动纤毛中的功能。我们的初步数据显示衣原体PKD 2靶向 并将鞭毛体、毛发样糖蛋白聚合物锚定到纤毛的细胞外表面。 一种新的pkd 2无效突变体缺失鞭毛体，其游泳速度降低，表明 PKD 2在运动纤毛中的运动相关功能。值得注意的是，PKD 2只锚定在9个双峰中的2个上， 微管（DMT），即，DMT 4和8，其定位PKD 2-鞭毛体复合体的两行 垂直于纤毛跳动的平面。与细胞骨架和细胞外组分的结合 对于许多机械门控通道是典型的。因此，我们的研究结果表明， 运动纤毛中的PKD 2。在目的1中，我们提出鉴定连接PKD 2与PKD 3的接头的组成。 衣原体的纤毛微管。我们将分析分离的PKD 2复合物的组成， 纤毛，使用体内邻近标记鉴定PKD 2附近的蛋白质，并确定细胞内的 PKD 2的一部分，有助于微管锚定。结果将确定PKD 2是如何针对 九条轴丝中的两条我们期待深入了解细胞如何建立和识别差异 在DMT之间，通常可能需要复杂的纤毛搏动模式。在第二章中，我们将分析 使用高速视频的pkd 2突变体中纤毛运动性如何受到影响。细胞骨架和细胞外 锚可用作在变形时打开通道的门控弹簧。突变体的分离 PKD 2系留的缺陷将使我们能够确定PKD 2锚定和纤毛图案化的作用， 能动性在纤毛的机械刺激过程中粘附细胞的钙成像将用于收集直接信息。 PKD 2的通道功能。最后，我们将确定PKD 2的分布如何适应 细胞环境的变化。总的来说，我们将测试PKD 2感知主动弯曲的假设。 运动的纤毛和定期PKD 2阵列需要这个过程。了解PKD 2在运动中的功能 纤毛可以帮助确定初级纤毛中PKD 2功能的机制，因为感觉到流动诱导的PKD 2功能。 PKD 2引起的纤毛被动弯曲被认为是肾脏中的重要反馈机制，当PKD 2引起纤毛被动弯曲时， 结果是ADPKD。