Polycystins, cilia, and extracellular vesicles in C. elegans

项目摘要 常染色体显性多囊肾病（ADPKD）是一种常见的、危及生命的疾病，影响 1/400-1/1000. ADPKD是由PKD 1和PKD 2突变引起的，PKD 1和PKD 2编码多囊蛋白-1， 多囊蛋白-2（PC 1和PC 2）。值得注意的是，近30年来，多囊蛋白的功能仍然是个谜。 在克隆后的20年，以及在肾原纤毛上发现后的20年。除纤毛外，PC 1和 PC 2也存在于其他亚细胞位置，包括细胞外囊泡（EV）。尿液EV可以 可用作肾脏疾病包括ADPKD的生物标志物。这些携带多囊蛋白的电动汽车是否是 睫状体起源以及EV在健康和患病肾脏中发挥的作用仍然未知。模型中 有机体C.在线虫和哺乳动物中，多囊蛋白LOV-1/PC 1和PKD-2/PC 2在结构上是 相似，在相同的遗传途径中起作用，在感觉能力中起作用，定位于初级/感觉纤毛， 并且在电动车中脱落，这表明古代的保护。我们将使用现有的C。elegans模型和 荧光标记的睫状EV货物PC 1（LOV-1）和PC 2（PKD-2）以研究睫状EV脱落， 生物活性和靶向活体动物。超分辨率，真实的时间成像显示，LOV-1和 PKD-2共定位于纤毛EV上，纤毛从两个不同的位点-- 睫状体顶端和睫状体基部。睫状体尖端EV脱落由机械刺激和功能触发 动物之间的交流。例如，雄性将携带存款PKD-2的EV沉积到哺乳动物的外阴上。 雌雄同体。我们还使用PKD-2：：GFP来富集和分析C.秀丽隐杆线虫 蛋白质组，更具体地说，多囊蛋白EV信号体。我们假设携带多囊蛋白的 EV充当离散的信号传导单元并且携带用于生物发生、货物分选、信号传导和运输的特定货物。 面向.在目标1中，我们将定义多囊蛋白和多囊蛋白组分之间的关系， 带着电动车在目标2中，我们将确定多囊蛋白相关EV货物的功能，并测试多囊蛋白相关EV货物的功能。 假设多囊蛋白和相关蛋白在纤毛EV中起信号体的作用， 纤毛感觉神经元在目标3中，我们通过研究PKD 1，关注EV在健康和疾病中的功能 和PKD 2致病性突变和变异的不确定的意义，在我们的C。elegans模型这 竞争性的更新应用将揭示纤毛EV生物学的基本原理和纤毛EV的功能。 纤毛和EV上的多囊藻毒素。我们的研究将提供关于正常和病理性的生物学的见解。 需要EV或由EV调节的过程，包括纤毛病。

Project Summary Autosomal dominant polycystic kidney disease (ADPKD) is a common, life threatening disease that affects 1/400-1/1000. ADPKD is caused by mutations in PKD1 and PKD2, which encode polycystin-1 and polycystin-2 (PC1 and PC2). Remarkably, the function of the polycystins remains enigmatic almost 30 years after their cloning and 20 years after their discovery on renal primary cilia. Besides cilia, PC1 and PC2 are also found in other subcellular locations including extracellular vesicles (EVs). Urinary EVs can be used as biomarkers of renal disease including ADPKD. Whether these polycystin-carrying EVs are of ciliary origin and what role EVs play in healthy and diseased kidneys remains unknown. In the model organism C. elegans and mammals, the polycystins LOV-1/PC1 and PKD-2/PC2 are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to primary/sensory cilia, and are shed in EVs, suggesting ancient conservation. We will use our established C. elegans model and fluorescently labeled ciliary EV cargoes PC1 (LOV-1) and PC2 (PKD-2) to study ciliary EV shedding, bioactivity, and targeting in living animals. Super resolution, real time imaging reveals that LOV-1 and PKD-2 co-localize on ciliary EVs and that cilia shed polycystin-carrying EVs from two distinct sites - the ciliary tip and the ciliary base. Ciliary tip EVs shedding is triggered by mechanical stimulation and functions in animal-to-animal communication. For example, males deposit PKD-2-carrying EVs onto the vulva of the hermaphrodite during mating. We also used PKD-2::GFP to enrich and profile the C. elegans EV proteome, and, more specifically, the polycystin EV signalosome. We hypothesize that polycystin-carrying EVs act as discrete signaling units and carry specific cargo for biogenesis, cargo sorting, signaling, and targeting. In Aim 1, we will define the relationships between polycystins and the components of polycystin- carrying EVs. In Aim 2, we will ascertain the function of polycystin-associated EV cargo and test the hypotheses that the polycystins and associated proteins function as a signalosome in ciliary EVs and in ciliated sensory neurons. In Aim 3, we focus on EV function in health and disease by studying PKD1 and PKD2 pathogenic mutations and variants of uncertain significance in our C. elegans model. This competing renewal application will uncover the fundamentals of ciliary EV biology and the functions of the polycystins on cilia and EVs. Our studies will provide insight about the biology of normal and pathological processes that require or are modulated by EVs including ciliopathies.