Functional architecture of IP3-evoked local Ca2+signals

描述(由申请人提供)：由肌醇三磷酸受体激活引发的细胞质钙离子增加调节着大量的生理事件。相同的IP3刺激的钙释放途径的功能障碍是许多疾病发病的基础。因此，了解IP3受体如何产生与生理相关的钙信号，以及病理事件如何失调IP3受体的行为是很重要的。 我们对IP3诱导的完整细胞内钙信号功能组织的理解已经通过高分辨率成像方法得到了转变。共聚焦成像显示，IP3诱导的钙信号是通过逐渐募集微观钙信号而产生的，被称为“钙泡”，这些信号被解析为高亲和力钙离子指示剂的荧光的瞬时变化。这些功能信号被解释为反映了分布在内质网中的IP3受体小簇的活性，但IP3受体的潜在分布和结构结构尚不清楚。由于全细胞钙信号的空间构型取决于钙释放通道的功能和细胞分布，因此了解控制IP3受体“功能结构”的机制是非常必要的。本研究的目的是确定IP3受体的分布如何影响细胞内钙信号的模式，以验证IP3受体结构受生理和病理信号调控这一中心假设。 为了实现这一目标，我们优化了基于荧光蛋白的工具，这些工具可以解析活细胞中IP3受体的分布。我们将使用这些新的工具以及生化和分子方法来(1)解析1P3诱导的钙信号的不同时空模式下的IP3受体结构；(2)确定在早期发育过程中lP3受体信号功能结构的生理变化机制；以及(3)描述丙型肝炎病毒蛋白NS5A引起的lP3受体信号功能结构的病理变化机制。这些结果将有助于我们理解普遍存在的IP3信号通路在健康和疾病中的作用。

DESCRIPTION (provided by applicant): Cytoplasmic Ca2+ increases triggered by activation of inositol trisphosphate receptors regulate a huge variety of physiological events. Dysfunction of the same IP3-stimulated Ca2+ release pathway underlies the pathogenesis of many disorders. Therefore, it is important to understand how IP3 receptors generate physiologically relevant Ca2+ signals, and how pathological events dysregulate IP3 receptor behavior. Our understanding of the functional organization of IP3-evoked Ca2+ signals in intact cells has been transformed by high resolution imaging methods. Confocal imaging has shown that IP3-evoked Ca2+ signals are generated by progressive recruitment of microscopic Ca2+ signals, known as 'Ca2+ puffs', which are resolved as transient changes in fluorescence of high affinity Ca2+ indicator dyes. These functional signals are interpreted as reflecting the activity of small clusters of IP3 receptors spaced throughout the endoplasmic reticulum, but the underlying distribution, and structural architecture of IP3 receptors is unresolved. As spatial patterning of whole cell Ca2+ signals depends on both the function and the cellular distribution of Ca2+ release channels, it is imperative to understand the mechanisms that control the 'functional architecture' of IP3 receptors. The aim of this proposal is to define how IP3 receptor distribution impacts the patterning of cellular Ca2+ signals to test the central hypothesis that IP3 receptor architecture is modulated by physiological and pathological cues. To achieve this goal, we have optimized fluorescent protein-based tools that resolve IP3 receptor distribution in live cells. We will use these novel tools, as well as biochemical and molecular approaches to (1) resolve IP3 receptor architecture underlying different spatiotemporal patterns of 1P3-evoked Ca2+ signaling; (2) define the mechanisms of physiological change in the functional architecture of lP3 receptor signaling during early development and (3) delineate the mechanisms of pathological change in the functional architecture of lP3 receptor signaling evoked by the hepatitis C viral protein NS5A. Results will aid our understanding of the role of the ubiquitous IP3 signaling pathway in both health and disease.