SPATIAL AND TEMPORAL ASPECTS OF INSP3 SIGNALLING

肌醇1，4，5-三磷酸(InsP3)被用作细胞内信使 在用于控制许多不同细胞的信号通路中 功能，包括神经递质和激素反应，分泌， 肌肉收缩和光传导。这种信号的紊乱有 与躁郁症等疾病有牵连， 肿瘤发生和畸形发生，以及这一系统与 随着我们对它了解得更多，临床研究肯定会增加。它 现在已经确定InsP3的功能主要是通过引起 钙离子的释放被隔离在细胞内的储存库中。然而， 细胞内钙离子监测技术的最新进展 揭示了InsP3介导的钙离子模式的巨大复杂性 解放。Ca2+以重复的尖峰或振荡的形式释放，并且 钙释放波在细胞内传播。此外，个人 细胞似乎含有许多功能独立的钙离子储存， 每个网站都以“量化”的方式发布内容，要么全有，要么全不发布。 InsP3信号的空间和时间方面无疑是 对InsP3介导的信息编码很重要 转导途径。为了研究它们，我们将使用非洲爪哇的卵母细胞作为 模型系统，因为这些细胞具有很好的特征的InsP3途径，并且 它们的大小可以获得良好的空间和时间控制 细胞内InsP3，以及由此产生的高程在 细胞内的游离钙离子InsP3将通过光解在细胞内形成 笼中前驱体，钙离子将通过共聚焦视频显微镜成像 使用长波指示染料。亚细胞内“量子”钙释放 单位，以确定它们的属性如何导致生成 重复性的钙尖峰和传播的钙波，以及如何 钙释放的空间和时间模式中的信息是 编码为钙依赖的膜电流的变化。高分辨率 光释放的InsP3诱发的钙成像将允许映射 亚细胞InsP3敏感钙释放的分布和形态 它们的存在将与InsP3受体的存在相关 和内质网标记蛋白。在整个细胞水平上，我们 我还将研究两者之间的钙信号极化 卵母细胞的半球。InsP3诱导的钙释放的功能研究 将阐明积极反馈的性质，即 引起再生性钙释放，以及正性和 然后将研究钙离子的负反馈在生成 重复性的钙尖峰和传播的钙波。最后，这个情结 钙激活膜C_1电导与细胞内钙离子的关系 将通过同时进行钙离子成像和电压钳来确定 正在录音。

Inositol 1,4,5-trisphosphate (InsP3) is used as an intracellular messenger within a signalling pathway that serves to control many diverse cellular functions, including neurotransmitter and hormone responses, secretion, muscle contraction and phototransduction. Disorders of this signaling have been implicated in disorders including manic depressive illness, tumorigenesis and teratogenesis, and the relevance of this system to clinical studies will certainly grow as we come to understand it more. It is now well established that InsP3 functions principally by causing the liberation of Ca2+ ions sequestered within intracellular stores. However, recent improvements in techniques for monitoring intracellular Ca2+ have revealed great complexities in the patterns of InsP3-mediated Ca2+ liberation. Ca2+ is released as repetitive spikes or oscillations, and waves of Ca2+ release propagate through the cell. Furthermore, individual cells appear to contain many functionally independent Ca2+ stores, that each release their contents in a 'quantal', all-or-none manner. The spatial and temporal aspects of InsP3 signalling are undoubtedly important for the encoding of information by the InsP3-mediated transduction pathway. To study them we will employ Xenopus oocytes as a model system, as these cells have a well characterized InsP3 pathway, and their large size to obtain good spatial and temporal control of intracellular InsP3, and resolution of the resulting elevations in intracellular free Ca2 InsP3 will be formed in the cell by photolysis of a 'caged' precursor, and Ca2+ will be imaged by confocal video microscopy using long-wavelength indicator dyes. 'quantal' subcellular Ca2+ release units, to determine how their properties lead to the generation of repetitive Ca2+ spikes and propagating Ca2+ waves, and to see how information in the spatial and temporal patterns of Ca2+ liberation is encoded as changes in Ca2+-dependent membrane currents. High resolution imaging of Ca2+ evoked by photoreleased InsP3 will allow mapping of the distribution and morphology of subcellular InsP3-sensitive Ca2+ release sites, and their presence will be correlated with that of InsP3 receptors and endoplasmic reticulum marker proteins. At the whole cell level, we will also study the polarization of Ca2+ signalling between the two hemispheres of the oocyte. Functional studies of InsP3-evoked Ca2+ release at individual sites will elucidate the nature of the positive feedback that gives rise to regenerative Ca2+ release, and the roles of positive and negative feedback by Ca2+ will then be investigated in the generation of repetitive Ca2+ spikes, and propagating Ca2+ waves. Finally, the complex dependence of Ca2+-activated membrane C1- conductance on intracellular Ca2+ will be determined by simultaneous Ca2+ imaging and voltage-clamp recording.