3-D RECONSTRUCTION OF RBL-2H3 ER & 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。 我们使用NCMIR和NBCR的断层摄影资源：(1)确定RBL-2H3细胞内质网的三维体积；(2)重建“典型”静息和激活的RBL-2H3细胞的三维视图，反映表面形貌(和潜在体积)的巨大变化。我们以前已经证明，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。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。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。我们以前使用免疫金标记膜片来绘制受体和相关信号分子在质膜的离散微域中的分布。奥利弗小组之前的工作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粒子成像。

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.