Regulation and functional characterization of ciliary calcium signaling

项目摘要 第二信使Ca 2+如何调节像受精这样多种多样的信号传导过程， 增殖、发育、学习和记忆、收缩和分泌？区室化钙 信号传导是一种基本的信号传导原理，通过该原理，Ca 2+离子在细胞中局部地发挥其刺激功能， 精确控制的空间和时间方式。细胞内局部Ca 2+浓度的变化是 通过Ca 2+进入质膜以产生“信号热点”或通过释放 Ca 2+来自细胞内储存，如内质网（ER）、线粒体或内体。变化 Ca 2+浓度由Ca 2+结合蛋白“感知”，其将信息传递到信号输出中。 初级纤毛是一种微管细胞器，从顶端质膜延伸出来， 就像天线一样初级纤毛富含钙渗透离子通道的特定子集，称为 多囊蛋白（PC 1和PC 2）。虽然Ca 2+信号转导领域在理解细胞内Ca 2+信号转导方面取得了开创性的进展， 在细胞器如ER和内体中的区室化Ca 2+信号传导的分子原理，我们是 仍然缺乏对初级纤毛作为Ca 2+信号细胞器的功能理解。突变 多囊蛋白离子通道导致多种人类疾病，从先天性心脏病和 多器官（肝、肾和胰腺）囊肿形成的偏侧性缺陷。Ca 2+可能起着 在所有这些器官中，初级纤毛内的关键第二信使，但纤毛的功能后果 钙信号仍然是个谜，睫状体离子通道的机制也是如此。 监管.本项目的中心目标是了解纤毛离子通道PC 1和PC 2如何调节 睫状体Ca 2+水平，并确定区室化睫状体Ca 2+信号传导的细胞生物学功能。 有三个具体目标。第一个目的是确定PC 1/PC 2通道活动如何影响睫状体Ca 2 + 浓度.第二个目的是检验睫状体钙浓度的变化调节 过渡区的蛋白质的渗透性，初级纤毛基部的扩散屏障。第三 目的是确定PC 1的N-末端，一个3000个氨基酸长的片段，被多个细胞修饰， 粘附结构域，调节PC 1/PC 2离子通道活性。申请人的初步意见包括 记录PC 1/PC 2通道活性和动态调节纤毛Ca 2+的新方法 浓度.该项目的完成将是理解细胞生物学功能的关键的第一步 纤毛钙信号传导。我们的长期目标是了解睫状体Ca 2+动力学失调是如何导致 人类纤毛病

PROJECT SUMMARY How can the second messenger Ca2+ regulate a plethora of signaling processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion? Compartmentalized Calcium signaling is a fundamental signaling principle by which Ca2+ ions exert their stimulatory function locally in a precisely controlled spatial and temporal manner. Changes in local Ca2+ concentration within the cell are regulated through Ca2+ entry across the plasma membrane to generate “signaling hot spots” or by releasing Ca2+ from intracellular stores such as endoplasmic reticulum (ER), mitochondria or endosomes. Changes in Ca2+ concentration are “sensed” by Ca2+ binding proteins which relay the information into a signaling output. The primary cilium is a microtubule based organelle extending from the apical plasma membrane and shaped like an antenna. Primary cilia are enriched in a specific subset of calcium permeating ion channels called polycystins (PC1 and PC2). While the Ca2+ signaling field has made seminal progress in understanding the molecular principles of compartmentalized Ca2+ signaling in organelles such as ER and endosomes, we are still lacking a functional understanding of the primary cilium as a Ca2+ signaling organelle. Mutations in polycystin ion channels result in a variety of human diseases, ranging from congenital heart disease and laterality defects to cyst formation in multiple organs (liver, kidney and pancreas). Ca2+ is likely to function as a critical second messenger within primary cilia in all of these organs, but the functional consequences of ciliary calcium signaling remain mysterious and so do the mechanisms through which ciliary ion channels are regulated. The central goal of this project is to understand how the cilia ion channels PC1 and PC2 regulate ciliary Ca2+ levels and to determine the cell biological function of compartmentalized ciliary Ca2+ signaling. There are three specific aims. The first aim is to determine how PC1/PC2 channel activity affects ciliary Ca2+ concentration. The second aim tests the hypothesis that changes in ciliary calcium concentration regulate the permeability for proteins of the transition zone, a diffusion barrier at the base of the primary cilium. The third aim determines how the N-terminus of PC1, a 3000 amino acid long fragment decorated with multiple cell adhesion domains, regulates PC1/PC2 ion channel activity. The applicants' preliminary observations include novel unpublished methods to r‐ecord PC1/PC2 channel activity and to dynamically regulate ciliary Ca2+ concentration. Completion of this project will be a critical first step in understanding the cell biological function ciliary calcium signaling. Our long term goal is to understand how dysregulation of ciliary Ca2+ dynamics cause human ciliopathies.