Functional architecture of IP3-evoked local Ca2+ signals

描述（由申请人提供）：（1，4，5）-三磷酸肌醇受体（IP 3R）引起的细胞质Ca 2+增加调节许多生理事件。IP 3刺激的Ca 2+释放途径的功能障碍涉及神经变性和神经系统疾病，以及外分泌、自身免疫和肾脏疾病、心脏异常和癌症。为了理解细胞功能是如何被Ca 2+信号控制的，以及病理性畸变是如何破坏IP 3R信号通路的，我们必须了解IP 3R的分布和性质（“功能结构”）是如何控制细胞Ca 2+信号的时空组织的。在这里，我们将解决IP 3R在三个不同层次的组织从单分子在体外（目标1）的本地，亚细胞（目标2）和全球，组织水平的IP 3R架构在活胚胎（目标3）的属性。我们将解决：（1）如何，对于个别IP 3R，一个单一的IP 3分子结合到IP 3R？(2)IP 3R的功能是如何在其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，钙离子通道和泵的全球补体变化如何影响钙离子信号和细胞功能的模式。为了应对这些挑战，我们优化了：（i）能够解析单个IP 3R的性质的新型单分子成像方法（目标1），（ii）双重共聚焦显微镜，以同时解析ER结构和IP 3R的功能（目的2）和（iii）设计工具和试剂，以探测脊椎动物胚胎发生期间Ca 2+通道和泵的关键家族的分布和作用（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，从而理解控制单元、细胞和系统水平响应的Ca 2+信号的空间动力学。这些数据将有助于我们了解普遍存在的Ca 2+信号通路在健康和疾病中的作用。公共卫生相关性：该项目的目标是了解特定的细胞信号事件是如何在细胞内组织的，首先了解蛋白质如何在体外单位水平上工作，然后了解这些蛋白质在细胞内和体内发育胚胎内组织时如何工作。目的是提供更深入的了解细胞内钙离子通道的行为和病理事件如何破坏其功能。

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.