SPATIAL AND TEMPORAL ASPECTS OF INSP3 SIGNALLING

肌醇1，4，5-三磷酸（InsP 3）被用作细胞内信使 在一个信号通路中， 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 与包括躁狂抑郁症在内的疾病有关， 肿瘤发生和致畸作用，以及该系统与 临床研究肯定会随着我们对它的了解而增加。 它 现在已经确定InsP 3的功能主要是通过引起 释放在细胞内储存的Ca 2+离子。 然而，在这方面， 最近用于监测细胞内Ca 2+的技术的改进， 揭示了InsP 3介导的Ca ~（2+）的模式非常复杂 解放。 Ca 2+以重复尖峰或振荡的形式释放， Ca 2+释放的波通过细胞传播。 此外，个人 细胞似乎含有许多功能独立的Ca 2+储存， 每一个都以“定量”的、全部或没有的方式释放它们的内容。 InsP 3信号传导的空间和时间方面无疑是 重要的是通过InsP 3介导的信息编码 转导途径 为了研究它们，我们将使用非洲爪蟾卵母细胞作为 模型系统，因为这些细胞具有良好表征的InsP 3途径，并且 他们的大尺寸，以获得良好的空间和时间控制， 细胞内InsP 3，并解决由此产生的升高， 细胞内游离Ca 2 InsP 3将在细胞中通过光分解 “笼状”前体和Ca 2+将通过共聚焦视频显微镜成像 使用长波长指示染料。 “量子”亚细胞钙释放 单位，以确定它们的属性如何导致生成 重复的Ca 2+尖峰和传播的Ca 2+波，并看看如何 Ca 2+释放的空间和时间模式中的信息是 编码为Ca 2+依赖性膜电流的变化。 高分辨率 由光释放的InsP 3诱发的Ca 2+成像将允许绘制 亚细胞InsP 3敏感性Ca ~（2+）释放的分布和形态 位点，并且它们的存在将与InsP 3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 还将研究两者之间Ca 2+信号的极化 卵母细胞的半球。 InsP 3诱发钙释放的功能研究 在各个地点将阐明积极反馈的性质， 引起再生Ca 2+释放，以及正和负作用。 负反馈的钙离子，然后将研究在产生 重复的Ca 2+尖峰和传播的Ca 2+波。 最后，复杂 Ca ~（2+）激活的细胞膜C_1电导对细胞内Ca ~（2+）的依赖性 将通过同步钙离子成像和电压钳测定 录制.

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.