SPATIAL AND TEMPORAL ASPECTS OF INSP3 SIGNALING

肌醇1，4，5-三磷酸(InsP3)被几乎所有的细胞用作 信号通路中的细胞内信使，控制着许多 多种功能，包括神经递质、激素和生长因子 反应、分泌和肌肉收缩。这条通路的中断 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 随着我们更好地了解它，临床研究肯定会增加。 众所周知，InsP3的功能主要是通过释放钙离子来实现的 离子被隔离在细胞内的存储空间中。此外，在这方面的进展 监测细胞内钙离子的技术揭示了巨大的复杂性 在其释放的模式中，Ca~(2+)可能以局部‘喷’的形式释放， 或者是重复的圆形波和螺旋波。这些空间和时间 InsP3信号的各个方面对于确定 钙信号是否仍局限于亚细胞区域或起作用 在全球范围内，对于作为频率的信息的数字编码 重复波，以及信号在内部和之间的传播 以钙波的形式耦合细胞。 在InsP3存在的情况下，细胞质充当可兴奋的介质， 由多个独立且自治的站点组成，这些站点释放“量子” 以一种再生的方式对双重阳性和阴性作出反应 钙离子的反馈作用。因此，Ca~(2+)波类似于化学作用势， 它们的特点将由三个因素决定； 释放部位的功能特性，即钙离子作为 站点内部和站点之间的可扩散信使，以及空间 发布地点的组织。我们的总体目标是研究每一个 这些方面，目的是阐明它们如何有助于 InsP3信号最终引起的钙动态变化。我们将使用非洲爪哇的卵母细胞 作为一种方便且特性良好的模型细胞系统，利用非 可代谢的InsP3类似物及笼状InsP3的光解激发钙离子 然后将对解放进行高度的空间和时间监控 视频率共聚焦显微镜的分辨率。 释放部位的功能研究将包括钙离子的作用 反馈在唤起分级和再生释放中的随机 触发再生反应，处理潜在的激活和 释放的失活，以及释放地点之间的可变性。这个 InsP3和钙离子的迁移率和作用范围将在#年确定。 完整的细胞，我们将研究内源性和外源性的影响 对钙离子扩散的缓冲以及由此引起的剂量依赖性和 海浪和海浪的动态。自发性钙离子的高分辨率成像 照片发布的InsP3引发的泡芙和热点将允许绘制地图 钙离子释放部位的三维分布和形态， 它们的存在将与InsP3受体和 急诊室。结构。最后，在与理论小组的合作下，这些 定量数据将被用来模拟钙离子移动性和 细胞上离散释药部位的空间组织 InsP3介导的钙信号的动态变化。

Inositol 1,4,5-trisphosphate (InsP3) is utilized by virtually all cells as an intracellular messenger within a signaling pathway controlling many diverse functions including neurotransmitter, hormone and growth factor responses, secretion and muscle contraction. Disruptions of this pathway 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 better. It is well established that InsP3 functions principally by liberating Ca2+ ions sequestered within intracellular stores. Furthermore, advances in techniques for monitoring cytosolic Ca2+ have revealed great complexities in the patterns of its liberation; Ca2+ may be released as local 'puffs', or as repetitive circular and spiral waves. These spatial and temporal aspects of InsP3 signaling are undoubtedly important for determining whether Ca2+ signals remain localized to sub-cellular regions or act globally, for the 'digital' encoding of information as frequency of repetitive waves, and for propagation of signals within and between coupled cells as Ca2+ waves. In the presence of InsP3 the cell cytoplasm acts as an excitable medium, formed from multiple discrete and autonomous sites that release 'quanta' of Ca2 in a regenerative manner in response to dual positive and negative feedback by Ca2+. Ca2+ waves are thus akin to a chemical action potential, and their characteristics will be determined by three factors; the functional properties of release sites, the ability of Ca2+ ions to act as diffusible messengers within and between sites, and the spatial organization of release sites. Our overall goals are to study each of these aspects, with the aim of elucidating how they contribute to the final Ca2+ dynamics evoked by InsP3 signaling. We will use Xenopus oocytes as a convenient and well characterized model cell system, utilizing non- metabolizable InsP3 analogues and photolysis of caged InsP3 to evoke Ca2+ liberation that will then be monitored with high spatial and temporal resolution by video-rate confocal microscopy. Functional studies of release sites will include the roles of Ca2+ feedback in evoking graded and regenerative release, the stochastic triggering of regenerative responses, processes underlying activation and inactivation of release, and variability between release sites. The mobilities and range of action of InsP3 and Ca2+ will be determined in intact cells, and we will study the effects of endogenous and exogenous buffers on Ca2+ diffusion and consequent changes in dose-dependence and dynamics of puffs and waves. High resolution imaging of spontaneous Ca2+ puffs and 'hot spots' evoked by photoreleased InsP3 will allow mapping of the three-dimensional distribution and morphology of Ca2+ release sites, and their presence will be correlated with that of InsP3 receptors and e.r. structure. Finally, in collaboration with theoretical groups, these quantitative data will be used to model the effects of Ca2+ mobility and the spatial organization of discrete release sites on the cellular dynamics of InsP3-mediated Ca2+ signaling.