3-D RECONSTRUCTION OF RBL-2H3 ER & 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。 我们使用NCMIR和NBCR的断层成像资源：（1）确定RBL-2 H3细胞中内质网的三维体积，（2）重建“典型”静息和活化RBL-2 H3细胞的三维视图，反映表面形貌（和潜在体积）的显著变化。我们以前表明，2型IP 3受体在内质网内形成大簇内的持续升高的钙受体激活或钙离子载体诱导的分钟。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。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。我们以前使用免疫金标记的膜片映射分布的受体和相关的信号分子在离散的细胞膜的微区。奥利弗小组以前的工作已经通过扫描电子显微镜绘制了受体的分布图，使用背散射电子检测进行金颗粒成像。

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. We use the NCMIR's and NBCR's tomographic resources to: (1) determine the 3-dimensional volume of the endoplasmic reticulum in RBL-2H3 cells and (2) reconstruct a 3-dimensional view of a "typical" resting and activated RBL-2H3 cell, reflecting the dramatic changes in surface topography (and potentially volume). 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. 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. 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. We previously used immunogold labeling of membrane sheets to map distributions of receptors and associated signaling molecules in discrete microdomains of the plasma membrane. Previous work from the Oliver group has mapped distribution of receptors by scanning electron microscopy, using backscattered electron detection for gold particle imaging.