Electron Tomography Of Cellular Structures

细胞结构的电子断层扫描

• 批准号: 7319073
• 负责人: Richard D Leapman
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7319073
• 关键词: Electron; Tomography; Cellular; Structures

We have implemented the technique of automated electron tomography in a 300 kV field-emission TEM equipped with a post-column energy-filter to determine the three-dimensional arrangement of supramolecular assemblies and subcellular cellular organelles. Series of unfiltered or energy-filtered digital images each containing 2048 x 2048 pixels were acquired using a cooled CCD camera over a specimen tilt range from -70 degrees to +70 degrees and for dual tilt axes rotated by 90 degrees. These image series were analyzed using the IMOD program (University of Colorado) to obtain three-dimensional reconstructions. We have applied electron tomography to determine the three-dimensional arrangement of proteins in post-synaptic densities (PSDs) of cultured neurons from rat brain prepared by high-pressure freezing, freeze-substitution and heavy metal staining. The structures of interest associated with the PSDs include CAM kinase II, scaffolding proteins as well as cytoskeletal fibers. To complement studies being conducted by NIDDK investigators to understand defects in the endocytic trafficking pathway that occur in Niemann-Pick Type C Disease, we are determining the cellular ultrastructure of endosomal vesicles and endosomal tubules in cultured fibroblasts and CHO cellls containing wild-type or mutant NPC protein. The reconstructions show multi-lamellar tubules that appear to correlate with late endosomal tubules previously observed in fluorescent light microscopy of labeled cells.

我们已经在配备了柱后能量过滤器的300 kV场发射TEM中实施了自动电子断层扫描技术，以确定超分子组件和亚细胞细胞器细胞器的三维布置。一系列未经过滤或能量过滤的数字图像每个包含2048 x 2048像素的像素均使用冷却的CCD摄像机在样品倾斜范围内从-70度到+70度，对于双倾斜轴，旋转90度。使用IMOD计划（科罗拉多大学）分析这些图像系列，以获得三维重建。 我们已经应用电子断层扫描来确定通过高压冻结，冷冻终止和重金属染色制备的大鼠大脑中培养神经元的蛋白质的三维布置。与PSD相关的感兴趣结构包括CAM激酶II，脚手架蛋白以及细胞骨架纤维。 NIDDK研究人员正在进行补体研究，以了解Niemann-Pick型C疾病中发生的内吞运输途径中的缺陷，我们确定了内体囊泡和内体肾小管的细胞超微结构，内体肾小管中培养的成纤维细胞和含有野生野生野生型或突变的蜂窝状的细胞超微结构NPC蛋白。重建显示了多层小管，似乎与标记细胞的荧光光学显微镜中观察到的晚期内体小管相关。