3 D RECONSTRUCTION OF RBL 2H3 ER & PLASMA MEMBRANES

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。Wilson博士和奥利弗博士是细胞生物学家，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受体在内质网内形成大簇，在几分钟内升高并维持由受体活化或钙离子载体诱导的钙升高。对于我们目前的建模项目，它试图预测的影响IP 3受体聚集的ER钙储存的填充状态，我们需要准确的测量内质网的体积，形状和分布。我们对浦肯野细胞中ER的成功重建感到鼓舞。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。 2)重建一个“典型的”静息和活化的RBL-2 H3细胞的三维视图，反映表面形貌（和潜在的体积）的巨大变化。我们最近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质膜的离散微区。此前，奥利弗团队已经通过扫描电子显微镜绘制了受体分布图，使用背散射电子检测进行金颗粒成像。我们的目标是建立一个RBL-2 H3大鼠肥大细胞形状的几何模型，并在其上显示所选跨膜蛋白的拓扑分布。该模型将对比静止时和通过交联I型IgE受体FceRI的信号起始后细胞上信号分子的分布。该模型将是通过整合从膜两侧标记的细胞的扫描和透射电子显微镜图像而生成的统计重建，所述细胞具有对FceRI、膜衔接子、信号传导蛋白和内吞机制特异性的免疫金颗粒。 我们设想，这两个目标的初步数据可以使用同一套连续厚切片和倾斜系列来完成。已经制备了样品（其中使用福布斯等人的亚铁氰化锇方法选择性地染色ER，1977年），并被嵌入Epon。2001年10月开始了初步工作，获得了一个倾斜系列，随后进行了重建。这项工作被证明是有前途的，需要额外的工作切片染色。2001年12月，Alex Smith访问了NCMIR，收集了几个倾斜系列并通过了重建技术。目前，我们正在将数据转换为适合建模软件的形式。2004年春季，斯蒂芬·杰特将访问该项目，继续进行该项目的工作。他将收集更多的数据，并学习使用

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 more than 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 raising and sustaining elevations in calcium induced by receptor activation or calcium ionophore. For our current modeling project, which 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. 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. 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 have recently used immunogold labeling of membrane sheets to map distributions of receptors and associated signaling molecules in discrete microdomains of the plasma membrane. Previously, the Oliver group has mapped receptor distribution by scanning electron microscopy, using backscattered electron detection for gold particle imaging. We aim to develop a geometric model of the shape of the RBL-2H3 rat mast cell and superimpose upon it the topographical distribution of selected transmembrane proteins. The model will contrast the distribution of signaling molecules on cells at rest and following signal initiation by crosslinking the Type I IgE receptor, FceRI. The model will be a statistical reconstruction generated through the integration of scanning and transmission electron microscopy images of cells labeled from either side of the membrane with immunogold particles specific for FceRI, membrane adaptors, signaling proteins and endocytic machinery. We envision that preliminary data for both of these aims can be accomplished using the same set of serial thick sections and tilt series. Samples have already been prepared (where the ER is stained selectively using the osmium ferrocyanide method of Forbes et al., 1977) and are embedded in Epon. Preliminary work began in October 2001 with the acquisition of a tilt series and subsequent reconstruction. This work proved promising, with additional work needed on section staining. In December 2001, Alex Smith visited the NCMIR, collected several tilt series and ran through the reconstruction techniques. Currently, we are converting the data into a form appropriate for the modeling software. Work on this project will continue with a visit by Stephen Jett in Spring 2004. He will collect more data and learn tomography reconstruction using