3 D RECONSTRUCTION OF RBL 2H3 ER & PLASMA MEMBRANES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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。我们以前已经证明，在受体激活或钙离子载体引起的钙升高和维持的几分钟内，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。我们对Purkinje细胞内质网的成功重建感到鼓舞。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。2)重建“典型”静息和激活的RBL-2H3细胞的三维图像，反映表面形态(和潜在体积)的戏剧性变化。我们最近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。此前，奥利弗小组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粒子成像。我们的目标是建立一个RBL-2H3大鼠肥大细胞形状的几何模型，并在其上叠加选定的跨膜蛋白的地形分布。该模型将通过交联I型IgE受体FceRI来对比静息和信号启动后细胞上信号分子的分布。该模型将是一个统计重建，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，以及针对FceRI、膜适配器、信号蛋白和内吞机械的免疫金粒子。我们设想，这两个目标的初步数据都可以使用相同的系列厚切片和倾斜系列来实现。已经制备了样品(其中ER使用福布斯等人1977年的亚铁氰化Os方法选择性地染色)，并被嵌入EPON中。2001年10月开始了初步工作，购置了倾斜系列，随后进行了重建。这项工作被证明是有希望的，还需要在切片染色方面进行额外的工作。2001年12月，亚历克斯·史密斯参观了NCMIR，收集了几个倾斜序列，并介绍了重建技术。目前，我们正在将数据转换为适合建模软件的形式。该项目的工作将继续进行，史蒂芬·杰特将于2004年春天访问。他将收集更多数据，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