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

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Drs. Wilson和奥利弗是细胞生物学家，在通过肥大细胞的高亲和力IgE受体Fc[IgE]RI分析信号转导方面以及在研究受体和信号蛋白的膜形貌的创新电子显微镜方面拥有30多年的经验。我们的工作是第一个描述的变化，在细胞形状和受体拓扑结构诱导的Fc[R]RI交联，描述IP 3受体的快速集群后，细胞内钙离子的升高，最近，建立Fc[R]RI信号发生在多个不同的膜微区。 我们以前曾与生物药理学家和数学建模方面的Ca 2+动员肥大细胞和模型的IgE受体再分布在肥大细胞信号传导。 我们现在有两个不同的建模项目，涉及应用数学和计算专家从UNM部。以及桑迪亚国家实验室的计算科学家。 我们建议使用NCMIR的层析成像资源：1）确定RBL-2 H3细胞中内质网的三维体积。我们先前表明，2型IP 3受体在由受体活化或钙离子载体诱导的钙持续升高的几分钟内在内质网内形成大簇（Wilson等，1998）。 对于我们目前的建模项目，试图预测IP 3受体聚集对ER钙储存的填充状态的影响，我们需要精确测量内质网的体积，形状和分布。 我们对浦肯野细胞中ER的成功重建感到鼓舞（Martone等，1993）。 由于这两种细胞类型是如此不同，我们的建模项目将需要基于RBL细胞中的实际TEM测量。 我们将整合ER体积数据与IP 3集群的数量和分布数据获得的共聚焦显微镜和超低温免疫金标记。

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.