Functional architecture of IP3-evoked local Ca2+ signals

描述(申请人提供)：由肌醇(1，4，5)-三磷酸受体(IP3Rs)引起的胞浆钙离子增加调节许多生理事件。IP3刺激的钙释放途径功能障碍涉及神经退行性疾病和神经系统疾病，以及外分泌、自身免疫和肾脏疾病、心脏异常和癌症。要了解细胞功能是如何由钙信号控制的，以及病理异常如何颠覆IP3R信号通路，我们必须了解IP3R的分布和特性(功能结构)如何控制细胞钙信号的时空组织。在这里，我们将在三个不同的组织水平上解析IP3R的特性，从体外单分子(目标1)到局部、亚细胞(目标2)和活胚胎中的全局组织水平IP3R架构(目标3)。我们将解决：(1)对于单个IP3R，单个IP3分子如何与IP3R结合？(2)IP3R功能是如何在它们所在的内质网结构的动态背景下调节的？(3)在发育中的脊椎动物胚胎中，在区域水平上不断变化的整体钙通道和泵如何影响钙信号的模式和细胞功能。为了应对这些挑战，我们优化了：(I)一种新颖的单分子成像方法，能够解析单个IP3R的特性(目标1)；(Ii)一种双共聚焦显微镜，可以同时解析内质网结构和IP3Rs的功能(目标2)；以及(Iii)设计工具和试剂，用于探索脊椎动物胚胎发育过程中关键的钙通道和泵家族的分布和作用(目标3)。这项工作的广泛意义在于理解控制离子通道动力学的原理，从而理解控制单一、细胞和系统水平反应的钙信号的空间动力学。这些数据将有助于我们理解无处不在的钙信号通路在健康和疾病中的作用。与公共健康相关：这个项目的目标是通过首先了解蛋白质在体外单一水平上如何工作，然后了解这些蛋白质在细胞内和体内发育中的胚胎中如何组织，来了解特定的细胞信号事件在细胞内是如何组织的。其目标是更深入地了解细胞内钙通道的行为以及病理事件如何颠覆它们的功能。

DESCRIPTION (provided by applicant): Increases in cytoplasmic Ca2+ evoked by inositol(1,4,5)-trisphosphate receptors (IP3Rs) regulate many physiological events. Dysfunction of the IP3-stimulated Ca2+ release pathway is involved in neurodegenerative and neurological disease, as well as exocrine, autoimmune and kidney disorders, cardiac abnormalities and cancer. To appreciate how cellular functions are controlled by Ca2+ signals, and how pathological aberrations subvert the IP3R signaling pathway, we must understand how the distribution and properties (the `functional architecture') of IP3Rs control the spatiotemporal organization of cellular Ca2+ signals. Here, we will resolve the properties of IP3Rs at three distinct levels of organization from single molecules in vitro (Aim 1) to local, subcellular (Aim 2) and global, tissue-level IP3R architecture in live embryos (Aim 3). We will address: (1) How, for individual IP3R, does a single molecule of IP3 bind to the IP3R? (2) How is IP3R function modulated within the dynamic context of endoplasmic reticulum structures where they reside? (3) How does a changing global complement of Ca2+ channels and pumps at a regional level in a developing vertebrate embryo impacts the patterning of Ca2+ signals and cell function. To address these challenges, we have optimized: (i) a novel single molecule imaging approach competent to resolve the properties of individual IP3Rs (Aim 1), (ii) a dual confocal microscope to simultaneously resolve ER architecture and the functionality of IP3Rs (Aim 2) and (iii) designed tools and reagents to probe the distribution and role of key families of Ca2+ channels and pumps during vertebrate embryogenesis (Aim 3). The broad significance of this work is in understanding principles controlling ion channel dynamics and thereby the spatial kinetics of Ca2+ signals that control unitary, cellular and systems-level responses. Such data will aid our understanding of the role of ubiquitous Ca2+ signaling pathways in health and disease. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand how particular cell signaling events are organized within cells by understanding first how proteins work at the unitary level in vitro and then appreciating how these proteins work when organized within cells and within developing embryos in vivo. The goal is to provide deeper understand of how intracellular Ca2+ channels behave and how pathological events subvert their function.