Elementary Events of Intracellular Calcium Signaling

描述(申请人提供)：钙离子从胞外液和内质网(ER)库进入胞浆，几乎被所有类型的细胞用作信号机制，调节各种功能，如电兴奋、分泌、增殖和细胞死亡。改进的光学技术现在可以可视化钙信号事件的层次结构，范围从单通道钙渗透通道的开口(基本事件)、集群通道的协调开口(基本事件)和传播钙波。通过单个和集群通道产生的局部游离[Ca~(2+)]升高具有自主的信号功能，它们的活动可能通过Ca~(2+)扩散和Ca~(2+)诱导的Ca~(2+)释放进一步协调，以传播全局细胞Ca~(2+)波：基础和基本事件从而形成复杂的时空Ca~(2+)信号的分层构件，从而允许分级和选择性地调节细胞功能。因此，阐明它们的产生、相互作用和功能后果对于理解普遍存在的钙信使途径的生理功能及其在疾病中的参与至关重要。我们的总体目标是阐明细胞如何产生钙信号的层次结构，如何利用这些信号对效应器反应进行特定和局部的调节，以及信号通路的中断如何参与疾病的发病机制。通过利用先进的生物光子学工具，包括共焦显微镜、多焦显微镜和全内反射显微镜，结合笼子第二信使和神经递质的光解，我们的目标是：(I)开发改进的光学技术，以便成像完整细胞的质膜和内质网中单个通道的钙通量，(Ii)利用数百个单通道的同时成像来探索它们在门控方面的差异，并研究钙微域对自身和通道间的调制。(Iii)阐明单个IP3R在释放部位的活动是如何被协调以产生基本的钙离子喷发，(Iv)研究IPs/钙信使通路在神经元信号转导中的时空模式层次，以及在帕金森病的发病机制中的作用。钙在人体几乎所有的细胞中都起着生死存亡的作用，调节的过程多种多样，包括心跳和脑细胞之间的突触传递，并与包括阿尔茨海默氏症和帕金森氏症在内的许多疾病有关。我们的目标是阐明钙信号在从单个分子到整个细胞水平上产生的分层机制，以及更好地理解它们的正常功能和钙信号中断如何导致疾病。

DESCRIPTION (provided by applicant): The entry of Ca2+ ions into the cytosol from the extracellular fluid and from endoplasmic reticulum (ER) stores is used as a signaling mechanism by virtually all cell types to regulate functions as diverse as electrical excitability, secretion, proliferation and cell death. Improved optical technology now enables visualization of a hierarchy of Ca2+ signaling events, ranging from openings of single-channel Ca2+-permeable channels ('fundamental' events), concerted openings of clustered channels ('elementary' events) and propagating Ca2+ waves. The localized free [Ca2+] elevations arising through individual and clustered channels serve autonomous signaling functions, and their activity may further be coordinated through Ca2+ diffusion and Ca2+-induced Ca2+ release to propagate global cellular Ca2+ waves: Fundamental and elementary events thus form hierarchical building blocks underlying the complex spatiotemporal Ca2+ signals hat permit graded and selective regulation of cell functions. Elucidation of their generation, interaction and functional consequences is, therefore, pivotal to understand the physiological functioning of the ubiquitous Ca2+ messenger pathway and its involvement in disease. Our overall goals are to elucidate how cells generate the hierarchy of Ca2+ signals, how these are utilized for specific and localized regulation of effector responses, and how disruptions in the signaling pathway may be involved in disease pathogenesis. By utilizing advanced biophotonic tools - including confocal, multi-focal and total internal reflection microscopy, in conjunction with photolysis of caged second messengers and neurotransmitters we aim to: (i) Develop improved optical techniques so as to image Ca2+ flux through individual channels in the plasma membrane and ER of intact cells, (ii) Utilize simultaneous imaging of hundreds of single-channels to explore differences in their gating, and study self- and inter-channel modulation by Ca2+ microdomains. (iii) Elucidate how the activity of individual IP3R at a release site is orchestrated to generate elementary Ca2+ puffs, (iv) Investigate the hierarchy of spatio-temporal patterning of the IPs/Ca2+ messenger pathway in neuronal signaling, and in the pathogenesis of Parkinson's disease. Calcium serves a 'life or death' function in virtually all cells of the body, regulating processes as diverse as the heartbeat and synaptic transmission between brain cells and is implicated in numerous diseases including Alzheimer's and Parkinson's. Our goal is to elucidate the hierarchical mechanisms by which Ca2+ signals are generated at levels from single molecules to the whole cell, with the dual aims of better understanding their normal functioning and how disruptions in Ca2+ signaling, may lead to disease.