3 D RECONSTRUCTION OF RBL 2H3 ER & PLASMA MEMBRANES

RBL 2H3 ER 的 3D 重建

• 批准号: 7358070
• 负责人: Bridget S Wilson
• 金额: $ 0.41万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7358070
• 关键词: RECONSTRUCTION; RBL; 2H3; ER; PLASMA

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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。Drs。Wilson和Oliver是细胞生物学家，在通过高亲和IgE受体Fc[epsilon]RI分析肥大细胞的信号转导以及创新的电子显微镜研究受体和信号蛋白的膜形貌方面拥有30多年的经验。我们的工作首次描述了Fc[epsilon]RI交联诱导的细胞形状和受体地形的变化，描述了细胞内钙升高后IP3受体的快速聚集，最近，我们确定了Fc[epsilon]RI信号传导发生在多个不同的膜微域中。我们之前曾与生物物理学家和数学建模者合作，研究肥大细胞中Ca2+动员的各个方面，以及肥大细胞信号传导过程中IgE受体再分配的建模。我们现在有两个不同的建模项目，涉及来自新墨西哥大学数学系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。我们建议利用NCMIR的层析成像资源：1)确定RBL-2H3细胞内质网的三维体积。我们之前的研究表明，在受体激活或钙离子载体诱导的钙升高和维持升高的几分钟内，2型IP3受体在内质网内形成大簇。在我们目前的建模项目中，我们试图预测IP3受体聚类对内质网钙储存填充状态的影响，我们需要精确测量内质网的体积、形状和分布。我们对浦肯野细胞成功重建内质网感到鼓舞。由于两种细胞类型如此不同，我们的建模项目将需要基于RBL细胞的实际TEM测量。我们将整合ER体积数据与共聚焦显微镜和超低温免疫金标记获得的IP3簇数和分布数据。2)重建一个“典型”休眠和激活的RBL-2H3细胞的三维视图，反映表面形貌（以及潜在的体积）的巨大变化。我们最近使用膜片的免疫金标记来绘制受体和相关信号分子在质膜离散微域的分布。此前，奥利弗小组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-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。该模型将对比信号分子在静止细胞上的分布，以及通过交联I型IgE受体（FceRI）引发信号后的分布。该模型将是一个统计重建，通过整合扫描和透射电子显微镜图像，从膜的两侧标记细胞，免疫金颗粒特异性FceRI，膜适配器，信号蛋白和内吞机制。我们设想，这两个目标的初步数据可以通过使用同一组连续厚切片和倾斜序列来完成。样品已经制备好（使用Forbes等人，1977年的亚铁氰化锇方法对ER进行选择性染色），并包埋在Epon中。初步工作于2001年10月开始，获得了一个倾斜系列和随后的重建。这项工作被证明是有希望的，需要在切片染色上做额外的工作。2001年12月，Alex Smith访问了NCMIR，收集了几个倾斜序列并运行了重建技术。目前，我们正在将数据转换为适合建模软件的形式。该项目的工作将于2004年春季由Stephen Jett访问后继续进行。他将收集更多的数据，学习断层扫描重建使用