Elementary Events of Intracellular Calcium Signaling

描述（由申请人提供）：Ca2 离子从细胞外液和内质网 (ER) 库进入细胞质，几乎被所有细胞类型用作信号传导机制，以调节电兴奋性、分泌、增殖和细胞死亡等多种功能。改进的光学技术现在可以实现 Ca2 信号事件层次结构的可视化，范围从单通道 Ca2 渗透通道的打开（“基本”事件）、集群通道的协同打开（“基本”事件）和传播 Ca2 波。通过单个和集群通道产生的局部游离 [Ca2+] 升高具有自主信号功能，并且它们的活动可以通过 Ca2+ 扩散和 Ca2+ 诱导的 Ca2+ 释放进一步协调，以传播全局细胞 Ca2+ 波：基本事件因此形成复杂时空 Ca2+ 信号底层的分层构建块，允许对细胞功能进行分级和选择性调节。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 Ca2 信使途径的生理功能及其与疾病的参与至关重要。我们的总体目标是阐明细胞如何产生 Ca2 信号的层次结构，如何利用这些信号对效应器反应进行特异性和局部调节，以及信号通路的破坏如何参与疾病发病机制。通过利用先进的生物光子工具 - 包括共焦、多焦和全内反射显微镜，结合笼中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，以便对通过完整细胞的质膜和内质网中的各个通道的 Ca2 通量进行成像，（ii）利用数百个单通道的同步成像来探索其门控的差异，并研究 Ca2 微域的自通道和通道间调节。 (iii) 阐明如何协调释放位点的单个 IP3R 的活性以产生基本的 Ca2 泡，(iv) 研究神经元信号传导和帕金森病发病机制中 IPs/Ca2 信使途径的时空模式层次结构。钙在身体的几乎所有细胞中都起着“生或死”的作用，调节心跳和脑细胞之间的突触传递等多种过程，并与包括阿尔茨海默氏症和帕金森氏症在内的多种疾病有关。我们的目标是阐明从单分子到整个细胞水平产生 Ca2 信号的分层机制，其双重目的是更好地了解它们的正常功能以及 Ca2 信号传导的破坏如何导致疾病。

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.