Imaging polycystin-mediated Ca transients in C. elegans

描述(申请人提供)：多囊肾病(ADPKD)是由人类PKD1和PKD2基因突变引起的，这两个基因编码多囊蛋白-1和-2。多囊蛋白被认为作用于肾上皮的初级纤毛，在那里它们通过液体流动将纤毛的机械弯曲转化为细胞质钙信号。囊泡的发生被认为是这一信号通路被破坏的结果。然而，多囊蛋白对机械信号反应的分子机制还不是很清楚。在线虫中，多囊蛋白同源基因LOV-1和PKD-2是21个雄性特异性感觉神经元与两性动物接触所必需的。多囊蛋白定位于这些细胞树突状顶端的初生纤毛。与脊椎动物一样，人们认为这些蛋白质将纤毛的变形转化为下游的钙信号。由于其复杂和快速的实验可及性，线虫模型在探索多囊蛋白信号的分子本质方面具有巨大的潜力。我们在这项应用中的目标是开发和实现一个系统，使用遗传编码的钙指示剂Cameleon在体内直接测量多囊蛋白依赖的钙信号。在第一个目标中，我们将实时测量雄性线虫在各种刺激下的钙瞬变，包括与两性人的接触，人工机械刺激和横向流体流动。通过比较野生型动物和那些携带多囊蛋白零突变的动物之间的钙反应，我们将检验这些反应依赖于多囊蛋白功能的假设。在第二个目的中，我们将应用这一实验来检验最近发现的两个在男性感觉神经元中特异表达的线虫基因CWP-4和CWP-5在多囊蛋白介导的钙信号转导中发挥作用的假设。这些研究将建立秀丽线虫作为一种独特的系统，用于体内遗传和分子分析多囊藻毒素响应机械刺激的信号分子机制。

DESCRIPTION (provided by applicant): Polycystic kidney disease (ADPKD) results from mutations in the human PKD1 and PKD2 genes, which encode the proteins polycystin-1 and -2. The polycystins are thought to act in the primary cilia of the renal epithelium, where they transduce mechanical bending of the cilium by fluid flow into cytoplasmic calcium signals. Cystogenesis is hypothesized to result from the disruption of this signaling pathway. However, the molecular mechanisms by which the polycystins respond to mechanical cues are not well understood. In the nematode C. elegans, the polycystin orthologs LOV-1 and PKD-2 are required for the function of twenty-one male-specific sensory neurons that sense contact with hermaphrodites. The polycystins localize to the primary cilia at the dendritic tips of these cells. As in vertebrates, it is thought that these proteins transduce the deformation of cilia into downstream calcium signals. Because of its sophisticated and rapid experimental accessibility, the nematode model has great potential for exploring the molecular nature of polycystin signaling. Our goal in this application is to develop and implement a system to directly measure polycystin-dependent calcium signaling in vivo using the genetically-encoded calcium indicator cameleon. In the first aim, we will measure calcium transients in C. elegans males in real time in response to a variety of stimuli, including contact with hermaphrodites, artificial mechanical stimuli, and transverse fluid flow. By comparing calcium responses between wild-type animals and those carrying null mutations in the polycystins, we will test the hypothesis that these responses depend on polycystin function. In the second aim, we will apply this assay to test the hypothesis that two recently-identified C. elegans genes specifically expressed in male sensory neurons, cwp-4 and cwp-5, have roles in polycystin-mediated Ca2+ signaling. These studies will establish C. elegans as a unique system for the in vivo genetic and molecular analysis of the molecular mechanisms of signaling by the polycystins in response to mechanical stimuli.