Deciphering polycystin-dependent electric signaling of primary cilia in the renal system

项目摘要/摘要 初生纤毛是一种独特的细胞器，几乎所有脊椎动物细胞的细胞膜上都有纤毛。有缺陷的 原生纤毛由纤毛形成或信号传递所需蛋白质的突变引起，可导致纤毛疾病， 影响多个器官的多效性疾病。尤其是常染色体显性遗传性多囊肾病 (ADPKD)是导致终末期肾病(ESRD)的最常见的单基因疾病 纤毛病。由跨膜蛋白PC-1和PC-2形成的多囊蛋白通道复合体是 初级纤毛大量表达，PC-1和PC-2突变是ADPKD的主要原因。 到目前为止，我们对多囊蛋白复合体是如何指挥电子的功能了解有限。 由于表征通道的技术挑战，初级纤毛通过阳离子通量(K+、Na+和Ca~(2+))进行信号传递 睫状膜的活性。为了更好地理解，我建立了睫状膜片钳记录来测量 内源性多囊藻毒素通道的活性直接来自睫状膜。我在2020年发表了C- PC-1N-末端的凝集素结构域(CTL)在多囊蛋白激活中起着关键作用，突显了 PC-1亚单位不可或缺的参与。在这份提案中，使用我已建立的化验方法，我将确定 PC-1 N端ADPKD突变如何损害多囊蛋白功能更进一步，我会 建立肾脏器官体内初级纤毛的纤毛膜片钳记录，使我能够 首次在功能上将通道活性受损的分子表型与 包囊形成的宏观表型。在指导的K99阶段，我将描述功能 使用纤毛膜片钳记录研究PC-1亚单位内致病变异的影响。要了解 多囊蛋白复合体的内源性调节，我将确定纤毛的特异性和效力- 富含氧甾醇以激活细胞或睫状膜中的多囊蛋白复合体。为了实现这一目标，我将 在戴尔实验室接受纤毛生物学的进一步培训。同时，共同导师Meyeon Park博士将指导我 了解PC-1致病突变的临床意义。 为了扩大多囊蛋白复合体在更多生理条件下的知识，我计划测量电信号 R00阶段利用肾脏器官传递初级纤毛的信号。肾脏有机化合物提供了一种强大的工具 了解发育和疾病，寻找新的治疗方法和再生方法。 表征有机体中的纤毛离子通道将建立一个重要的里程碑，以了解 原生纤毛在不同节段的肾小管和包囊发育。在此期间，候选人将 在生理系完成指导培训，为独立的R00阶段做准备 华盛顿大学本明明·S·弗里德曼博士的帮助。从这项工作中收集到的信息将 有助于扩大我们对初级纤毛在肾脏生理学中的基础作用的认识。

Project Summary/Abstract Primary cilia are unique organelles that protrude from the cell membrane of almost all vertebrate cells. Defective primary cilia, caused by mutations in proteins required for cilia formation or signaling, can result in ciliopathies, pleiotropic diseases affecting multiple organs. In particular, Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most prevalent monogenic disease leading to end-stage renal disease (ESRD), is considered ciliopathy. The polycystin channel complex, formed by the transmembrane proteins PC-1 and PC-2, is abundantly expressed primary cilia, and mutations in PC-1 and PC-2 account for the vast majority of ADPKD. To date, we only have a limited functional understanding of how the polycystin complex commands the electric signaling of primary cilia via cation flux (K+, Na+, and Ca2+) due to technical challenges in characterizing channel activity of ciliary membranes. To better understand, I established ciliary patch-clamp recordings to measure the activity of endogenous polycystin channels directly from the ciliary membrane. I published in 2020 that the C- type lectin domain (CTL) of the PC-1 N-terminus plays a crucial role in polycystin activation, highlighting the indispensable participation of PC-1 subunits. In this proposal, using my established assays, I will determine how ADPKD-causing mutations within the N-terminus of PC-1 impair polycystin function. Further, I will establish ciliary patch-clamp recordings of primary cilia within kidney organoids, allowing me for the first time to functionally connect the molecular phenotype of impaired channel activity with the macroscopic phenotype of cyst formation. During the mentored K99 phase, I will characterize the functional impact of pathogenic variants within the PC-1 subunit using the ciliary patch-clamp recording. To understand the endogenous regulation of the polycystin complex, I will determine the specificity and potency for the cilia- enriched oxysterols to activate the polycystin complex in the cell or ciliary membrane. To complete this aim, I will receive further training in cilia biology in the Delling lab. In parallel, co-mentor Dr. Meyeon Park will guide me to understand the clinical significance of pathogenic mutants in PC-1. To expand knowledge of polycystin complex under more physiological conditions, I plan to measure electric signaling of primary cilia using kidney organoids during the R00 phase. Kidney organoids provide a powerful tool for understanding development and disease and finding new treatments and regenerative approaches. Characterizing ciliary ion channels in organoids will establish an essential milestone to understand the role of primary cilia in different segments of renal tubules and cyst development. During this time, the candidate will complete mentored training in the Department of Physiology in preparation for the independent R00 phase with the help of Dr. Benjamine S. Freedman at the University of Washington. Information gleaned from this work will be helpful to expand our knowledge of the fundamental role of primary cilia in renal physiology.