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）储存库进入细胞质，被用作几乎所有细胞类型的信号传导机制，以调节各种功能，如电兴奋性、分泌、增殖和细胞死亡。改进的光学技术现在可以可视化Ca2+信号事件的层次结构，包括单通道Ca2+可渗透通道的打开（“基本”事件），集群通道的协调打开（“基本”事件）和传播Ca2+波。通过个体和集群通道产生的局部自由[Ca2+]升高服务于自主信号功能，其活性可能进一步通过Ca2+扩散和Ca2+诱导的Ca2+释放来传播全球细胞Ca2+波进行协调：基本和基本事件因此形成了复杂时空Ca2+信号的分层构建块，允许细胞功能的分级和选择性调节。因此，阐明它们的产生、相互作用和功能后果对于理解无处不在的Ca2+信使通路的生理功能及其在疾病中的作用至关重要。我们的总体目标是阐明细胞如何产生Ca2+信号的层次结构，这些信号如何用于特定和局部的效应反应调节，以及信号通路的中断如何参与疾病发病机制。通过利用先进的生物光子工具-包括共聚焦，多焦点和全内反射显微镜，结合笼子第二信使和神经递质的光解，我们的目标是：(i)发展改进的光学技术，以便通过完整细胞的质膜和内质网中的单个通道成像Ca2+通量；（ii）利用数百个单通道的同时成像来探索其门控的差异，并研究Ca2+微域对自身和通道间的调制。（iii）阐明个体IP3R在释放位点的活性如何被精心安排以产生基本的Ca2+泡；（iv）研究神经元信号通路中IPs/Ca2+信使通路的时空模式层次，以及帕金森病的发病机制。钙在人体几乎所有细胞中都起着“生死攸关”的作用，调节着各种各样的过程，如心跳和脑细胞之间的突触传递，并与许多疾病有关，包括阿尔茨海默氏症和帕金森症。我们的目标是阐明Ca2+信号在从单分子到整个细胞水平上产生的分层机制，具有更好地理解其正常功能和Ca2+信号中断如何导致疾病的双重目的。

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.