SPATIAL AND TEMPORAL ASPECTS OF INSP3 SIGNALING

INSP3 信号的空间和时间方面

• 批准号: 2749919
• 负责人: IAN PARKER
• 金额: $ 19.14万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749919
• 关键词: Xenopus oocyte; biological signal transduction; calcium flux; calcium indicator; chloride channels; confocal scanning microscopy; fluorescent dye /probe; image processing; inositol phosphates; intracellular membranes; membrane permeability; membrane transport proteins; phospholipase C; photolysis; protein structure function; video microscopy; voltage /patch clamp

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.

肌醇1，4，5-三磷酸（InsP3）几乎被所有细胞利用， 控制许多信号通路中的细胞内信使 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。这一途径的中断 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。 InsP3主要通过释放Ca2+发挥作用 离子被隔离在细胞内储存。此外， 用于监测胞质Ca 2+的技术已经揭示了巨大的复杂性 在其释放模式中; Ca2+可能作为局部"喷流"释放， 或者作为重复的圆形和螺旋形波。这些时空 InsP3信号传导的各个方面对于确定 Ca2+信号是否保持定位于亚细胞区域或起作用， 在全球范围内，对于信息的"数字"编码， 重复波，以及信号在内部和之间的传播 耦合细胞作为Ca2+波。 在InsP 3存在下，细胞质充当可兴奋介质， 由多个离散的、自主的释放"量子"的位点组成 以再生的方式响应双重阳性和阴性 Ca2+反馈。因此，Ca2+波类似于化学动作电位， 它们的特性将由三个因素决定： 释放位点的功能特性，Ca2+离子作为 地点内和地点之间的扩散信使， 释放现场的组织。我们的总体目标是研究 这些方面，目的是阐明它们如何有助于 InsP3信号转导诱发的最终Ca2+动态。我们将使用非洲爪蟾卵母细胞 作为一个方便和良好表征的模型细胞系统，利用非- 可代谢的InsP3类似物和笼状InsP3的光解以引起Ca2 + 解放，然后将监测与高空间和时间 分辨率通过视频率共聚焦显微镜。 释放位点的功能研究将包括Ca2+的作用 反馈在唤起分级和再生释放，随机 再生反应的触发，激活的基础过程， 释放失活和释放位点之间的变异性。的 InsP3和Ca2+的迁移率和作用范围将在 完整的细胞，我们将研究内源性和外源性 缓冲剂对Ca2+扩散和随后的剂量依赖性变化， 喷流和波浪的动力学。自发Ca 2+的高分辨率成像 由光释放的InsP3引起的抽吸和"热点"将允许绘制 Ca 2+释放位点的三维分布和形态， 并且它们的存在将与InsP3受体的存在相关， 急诊室结构最后，与理论小组合作，这些 定量数据将用于模拟Ca2+迁移率的影响， 细胞上离散释放位点的空间组织 InsP3介导的Ca2+信号转导的动力学。