3-D RECONSTRUCTION OF RBL-2H3 ER AND PLASMA MEMBRANES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 威尔逊博士和奥利弗博士都是细胞生物学家，在通过肥大细胞的高亲和力IgE受体Fc[epsilon]RI进行信号转导分析方面拥有30多年的经验，并在创新的电子显微镜下研究受体和信号蛋白的膜拓扑结构。我们的工作首次描述了Fc[epsilon]RI交联引起的细胞形态和受体形态的变化，描述了IP3受体在细胞内钙升高后的快速聚集，最近，我们建立了Fc[epsilon]RI信号发生在多个不同的膜微域中。我们以前曾与生物物理学家和数学模型师合作，研究肥大细胞中钙离子的动员，以及肥大细胞信号传递过程中IgE受体重新分布的建模。我们现在有两个不同的建模项目，涉及来自UNM部门的应用数学和计算专家。以及桑迪亚国家实验室的计算科学家。我们建议使用NCMIR的断层扫描资源：1)确定RBL-2H3细胞内质网的三维体积。我们以前证明，在受体激活或钙离子载体诱导的钙持续升高的几分钟内，2型IP3受体在内质网内形成大的簇(Wilson等人，1998)。对于我们目前的建模项目，试图预测IP3受体聚集对内质网钙库充盈状态的影响，我们需要准确测量内质网的体积、形状和分布。我们对Purkinje细胞内质网的成功重建感到鼓舞(Marone等人，1993)。由于这两种细胞类型非常不同，我们的建模项目将需要基于RBL细胞中的实际透射电子显微镜测量。我们将把内质网体积数据与通过共聚焦显微镜和超低温免疫金标记法获得的IP3簇数和分布数据相结合。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Drs. Wilson and Oliver are cell biologists with over 30 years experience in the analysis of signal transduction through the high affinity IgE receptor, Fc[epsilon]RI, of mast cells and in innovative electron microscopy to study the membrane topography of receptors and signaling proteins. Our work was the first to describe the changes in cell shape and receptor topography induced by Fc[epsilon]RI crosslinking, to describe the rapid clustering of IP3 receptors following elevations in intracellular calcium and, most recently, to establish that Fc[epsilon]RI signaling occurs in multiple distinct membrane microdomains. We have worked previously with biophysicists and mathematical modelers on aspects of Ca2+ mobilization in mast cells and on the modeling of IgE receptor redistribution during mast cell signaling. We now have two distinct modeling projects, involving applied mathematics and computing specialists from the UNM Dept. of Mathematics as well as computational scientists at Sandia National Laboratories. We propose to use the NCMIR's tomographic resources to:1) Determine the 3-dimensional volume of the endoplasmic reticulum in RBL-2H3 cells. We showed previously that Type 2 IP3 receptors form large clusters within the endoplasmic reticulum within minutes of sustained elevations in calcium induced by receptor activation or calcium ionophore (Wilson et al, 1998). For our current modeling project, that attempts to predict the effects of IP3 receptor clustering on the filling state of the ER calcium store, we need accurate measurements of the endoplasmic reticulum volume, shape and distribution. We are encouraged by the successful reconstruction of the ER in Purkinje cells (Martone et al, 1993). Because the two cell types are so different, our modeling project will need to be based upon actual TEM measurements in RBL cells. We will integrate the ER volume data with IP3 cluster number and distribution data obtained by confocal microscopy and ultra-cryo immunogold labeling.