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: 10096638
• 负责人: Karl Lechtreck
• 金额: $ 29.71万
• 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/10096638
• 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.

项目摘要/摘要 常染色体显性多囊性肾脏疾病（ADPKD）主要是由PKD1突变引起的 PKD2。这两种跨膜蛋白可能形成接收器通道复合物。故障 肾上皮细胞原发性纤毛的膜中的PKD1/2复合物被普遍认为是原因 疾病。但是，PKD1/2复合物感受到体内刺激的性质， 通道打开和下游信号事件仍然不确定。 PKD2是瞬态接收器 许多生物的睫状膜中也存在的电势（TRP）通道，表明它具有 在纤毛中保守的作用。我们建议使用衣原体作为一个简单的系统来分析组装和 PKD2通道在纤毛母亲中的功能。我们的初步数据表明衣原体PKD2目标 并锚定肥大，头发样糖蛋白聚合物到纤毛的细胞外表面。 新型的PKD2空突变体缺少乳腺素，该突变体以降低的速度游泳，表明 PKD2在纤毛中的运动相关功能。值得注意的是，PKD2仅锚定在九个双打中的两个 微管（DMTS），即DMTS 4和8，它位于PKD2-Mastigoneme复合物的两行 垂直于睫状节的平面。与细胞骨架和细胞外成分的关联 对于许多机械封闭的通道来说是典型的。这，我们的发现表明了机理感官的作用 PKD2在Motile Cilia中。在AIM 1中，我们建议确定将PKD2连接到的连接器的组成 衣原体中的睫状微管。我们将分析从中分离出的PKD2复合物的组成 纤毛，使用体内接近标记在PKD2附近鉴定蛋白质，并确定细胞内 PKD2的一部分有助于微管锚定。结果将确定PKD2的目标 九个轴突双峰中的两个。我们希望洞察细胞如何建立和识别差异 在DMT之间，通常对复杂的睫状节模式的可能要求。在AIM 2中，我们将分析 使用高速视频在PKD2突变体中如何影响睫状运动。细胞骨架和细胞外 锚可以作为门控弹簧的作用，在变形时打开通道。突变体的隔离 PKD2绑定中有缺陷将使我们能够确定PKD2锚定和图案的作用 运动。纤毛机械刺激期间，粘附细胞的钙成像将用于收集直接 洞悉PKD2的频道功能。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感应的假设 该过程需要纤毛和常规PKD2阵列。了解母亲的PKD2功能 纤毛可以帮助确定原发性纤毛中PKD2功能的机制，因为流动诱导的敏感性 PKD2对纤毛的被动弯曲被认为是肾脏中的重要反馈机制， ADPKD的不对劲结果。