ELEMENTARY EVENTS OF INTRACELLULAR CALCIUM SIGNALING

几乎所有类型的细胞都利用细胞内储存的钙释放到胞浆中作为一种信号机制来调节各种功能，如分泌、收缩、增殖和细胞死亡。改进的成像技术显示，通过涉及三磷酸肌醇和兰诺定受体/通道的途径释放钙是不连续的，是“基本的”钙释放事件。这些短暂的、局部性的、亚细胞内游离的[Ca~(2+)]升高出现在内质网内形成离散功能释放部位的通道簇上。单个位点可以产生涉及单个或多个通道的自主事件，它们的活动可能通过钙扩散和钙诱导的钙释放来协调，从而传播全球细胞钙波。因此，基本事件形成了复杂的时空钙信号的基本构建块，允许分级和选择性地调节细胞功能。因此，了解它们的产生、相互作用和功能后果是理解普遍存在的钙信使途径的生理和病理功能的关键。我们的具体目标是确定：(I)基本钙事件发生的机制，(H)允许钙波启动和传播的事件之间的协调，以及(Iii)局部钙微域特定激活效应系统的原理。我们将使用非洲爪哇的卵母细胞和心肌细胞作为模型系统，分别研究三磷酸肌醇和兰尼定受体的信号转导。此外，使用培养的细胞系将允许研究由其他受体亚型产生的事件。我们的实验方法包括高分辨率(低于0.5um和1ms)的单光子和双光子共聚焦显微镜，以成像由光释放三磷酸肌醇和钙离子引起的完整细胞中的亚细胞钙事件。这些光学技术提供了一个独特的机会来可视化活细胞中的单通道活动，我们的总体目标是阐明单个钙释放通道如何对细胞钙反应做出贡献。

The liberation of calcium from in intracellular stores into the cytosol is used as a signaling mechanism by virtually all cell types to regulate functions as diverse as secretion, contraction, proliferation and cell death. Improved imaging technology has revealed that calcium liberation through pathways involving both inositol trisphosphate and ryanodine receptor/channels occurs discontinuously, as 'elementary' calcium release events. These transient, localized, subcellular free [Ca2+] elevations arise at clusters of channels that form discrete functional release sites within the endoplasmic reticulum. Individual sites can generate autonomous events involving single or multiple channels, and their activity may be coordinated by calcium diffusion and calcium-induced calcium release to propagate global cellular calcium waves. Elementary events thus form the basic building blocks underlying the complex spatiotemporal calcium signals that permit graded and selective regulation of cell functions. An understanding of their generation, interaction and functional consequences is, therefore, pivotal to understand the physiological and pathological functioning of the ubiquitous calcium messenger pathway. Our specific aims are to determine, (i) the mechanisms underlying the generation of elementary calcium events, (h) the coordination between events allowing the initiation and propagation of calcium waves, and (iii) the principles underlying specific activation of effector systems by local calcium microdomains. We will use Xenopus oocytes and cardiac myocytes as model systems to study signaling by inositol trisphosphate- and ryanodine-receptors, respectively. Furthermore, use of cultured cell lines will allow investigation of events generated by other receptor isoforms. Our experimental methodology involves 1- and 2-photon confocal microscopy with high (less than 0.5 mum and 1 ms) resolution to image subcellular calcium events in intact cells evoked by photorelease of inositol trisphosphate and Ca2+. These optical techniques provide a unique opportunity to visualize single channel activity in living cells, and our overall goal is to elucidate how individual calcium release channels contribute to cellular calcium responses.