Functional architecture of IP3-evoked local Ca2+ signals

描述（由申请人提供）：肌醇(1,4,5)-三磷酸受体（IP3Rs）引起的细胞质Ca2+增加调节许多生理事件。ip3刺激的Ca2+释放通路的功能障碍涉及神经退行性和神经系统疾病，以及外分泌、自身免疫和肾脏疾病、心脏异常和癌症。为了了解细胞功能是如何由Ca2+信号控制的，以及病理畸变是如何破坏IP3R信号通路的，我们必须了解IP3R的分布和性质（“功能结构”）是如何控制细胞Ca2+信号的时空组织的。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）和活胚胎中全局、组织水平的IP3R结构（目标3）。我们将讨论：(1)对于单个IP3R，单个IP3分子如何与IP3R结合？(2) IP3R功能是如何在内质网结构的动态背景下被调节的？(3)在发育中的脊椎动物胚胎中，区域水平上Ca2+通道和泵的全球补体变化如何影响Ca2+信号的模式和细胞功能。为了解决这些挑战，我们优化了：(i)一种新的单分子成像方法，能够解决单个IP3Rs的特性（Aim 1）， （ii）双共聚焦显微镜，同时解决内质网结构和IP3Rs的功能（Aim 2）， （iii）设计的工具和试剂，以探测脊椎动物胚胎发生过程中Ca2+通道和泵的关键家族的分布和作用（Aim 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，从而了解Ca2+信号的空间动力学，这些信号控制着单位、细胞和系统水平的反应。这些数据将有助于我们理解无处不在的Ca2+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，首先了解蛋白质在体外的单一水平上是如何工作的，然后了解这些蛋白质在细胞内和体内发育中的胚胎中是如何组织的。目的是提供更深入的了解细胞内Ca2+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.