CRYO CORE

低温核心

• 批准号: 7507581
• 负责人: Peijun Zhang
• 金额: $ 50.72万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-27 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7507581
• 关键词: 3-Dimensional; Algorithms; Architecture; Back; Biological; Cell Communication; Cell Nucleus; Cells; Cellular Morphology; Cellular Structures; Complex; Condition; Confocal Microscopy; Crowding; Crystallography; Cytoplasm; Data; Discipline; Dose; Electron Microscope; Electron Microscopy; Electrons; Event; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Freezing; Frozen Sections; Genetic; HIV; HIV Infections; Image; Individual; Label; Life; Locales; Mammalian Cell; Methodology; Methods; Microscope; Microscopic; Molecular; Multiprotein Complexes; Nature; Nuclear Import; Organelles; Pathogenesis; Proteins; Radiation; Resolution; Roentgen Rays; Series; Signal Transduction; Specimen; Staging; Standards of Weights and Measures; Structure; System; Techniques; Technology; Temperature; Thick; Time; Tomogram; Viral; Viral Pathogenesis; Virion; Work; base; cell type; cellular imaging; computerized; cryogenics; data acquisition; density; design; electron tomography; fluorescence imaging; improved; insight; interest; intracellular protein transport; light microscopy; macromolecular assembly; macromolecule; novel; particle; pressure; protein localization location; single molecule; structural biology; time interval; tomography; tool

The emerging discipline of electron tomography provides new and unprecedented opportunities to determine three-dimensional cellular architecture at resolutions of ~50A or better, i.e., potentially high enough to identify individual macromolecules such as proteins in a 3-D volume of a cell289. Electron tomography is especially applicable for the structural analysis of cellular organelles and other macromolecular assemblies that are too heterogeneous to be investigated by NMR or X-ray crystallographic techniques or even electron microscope-based methods that involve averaging of multiple copies of the same object to improve signal-tonoise ratios. Electron tomography bridges the gap between high resolution structure determination of protein complexes by NMR or X-ray crystallographic techniques and single-particle living cell imaging by light microscopy using fluorescent probes. It extends the resolution of cellular imaging by one to two orders of magnitude over what is currently achieved using light microscopy. The fundamental principles underlying electron tomography and the strategy to use back-projection algorithms to extract 3-D information from a series of 2-D images recorded at different orientations were clearly articulated nearly four decades ago290'291. However, this field has seen a significant burst of activity in the last five years, principally propelled by the availability of tools for automated data acquisition using modern computerized microscopes. Efforts at imaging complex assemblies at room temperature as well as cryogenic temperatures have rapidly begun to provide many new insights into the 3-D architecture of cells289'292. During the immediate post-entry stages in HIV pathogenesis, the viral particle or, more precisely, its contents must traverse through the cytoplasm of the host cell prior to nuclear import of the pre-integration complex (PIC). Identification and characterization of these cellular events and viral and host interactions are central to understanding the molecular events that occur during HIV pathogenesis. NMR or X-ray crystallographic methods will provide high resolution structures of the viral and host protein complexes, while confocal microscopy in combination with fluorescent-labeling techniques allows tracking of viral particles and assessing the approximate localization of individual proteins in living cells. However, advancement of electron microscopic techniques, particularly electron tomography, will be essential to obtain higher resolution snapshots of the molecular arrangement of multiprotein complexes in the context of the host cellular structure and to provide the structural information necessary to fill the gap between NMR or X-ray crystallographic structural determination and live cell imaging. We, therefore, expect that our work to develop approaches for 3- D cellular imaging will have a direct impact on cellular and structural studies of HIV infection.

新兴的电子断层摄影学科提供了新的和前所未有的机会， 以~ 50 A或更高的分辨率确定三维细胞结构，即，潜在地足够高 以识别单个大分子，如细胞3D体积中的蛋白质289。电子断层扫描是 特别适用于细胞器和其他大分子组装体的结构分析 它们太不均匀，以至于不能用核磁共振或X射线晶体学技术甚至电子 基于显微镜的方法，包括对同一物体的多个副本进行平均，以改善信号色调 比率。电子断层扫描技术弥补了蛋白质高分辨率结构测定之间的差距 通过NMR或X射线晶体学技术和通过光的单颗粒活细胞成像的复合物 使用荧光探针的显微镜。它将细胞成像的分辨率提高了一到两个数量级， 这是目前使用光学显微镜所能实现的。 电子层析成像的基本原理和反投影技术的应用策略 从不同方向记录的一系列二维图像中提取三维信息的算法清楚地表明， 290 '291.然而，这一领域的活动在过去几年中出现了显著的爆发。 五年，主要是通过使用现代化的自动数据采集工具的可用性推动的。 计算机化显微镜在室温和低温下对复杂组件成像的努力 温度已经迅速地开始提供许多对细胞3D结构的新认识289 ′ 292。 在HIV发病机制的进入后阶段，病毒颗粒，或者更准确地说，其 内容物必须在整合前的核输入之前穿过宿主细胞的细胞质 复合物（PIC）。这些细胞事件以及病毒和宿主相互作用的鉴定和表征是 对理解HIV发病过程中发生的分子事件至关重要。NMR或x射线 晶体学方法将提供病毒和宿主蛋白质复合物的高分辨率结构， 与荧光标记技术相结合的共聚焦显微镜允许跟踪病毒颗粒， 评估活细胞中单个蛋白质的近似定位。然而，电子的进步 显微技术，特别是电子层析成像技术，将是获得更高分辨率的必要条件 在宿主细胞结构背景下多蛋白复合物分子排列的快照 并提供必要的结构信息，以填补NMR或X射线晶体学之间的差距 结构测定和活细胞成像。因此，我们希望我们的工作能够为3- 三维细胞成像将对HIV感染的细胞和结构研究产生直接影响。