Structure and function of the prokaryotic cytoskeleton by electron cryotomography

电子冷冻断层扫描原核细胞骨架的结构和功能

• 批准号: 7325689
• 负责人: GRANT J JENSEN
• 金额: $ 32.64万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-15 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7325689
• 关键词: Actins; Arts; Bacteria; Bacterial Proteins; California; Caulobacter crescentus; Cell Shape; Cell division; Cell membrane; Cells; Chromosome Segregation; Chromosomes; Collection; Compatible; Computer software; Cytoskeletal Filaments; Cytoskeleton; Data; Development; Electrons; Entomoplasma; Enzymes; Escherichia coli; Filament; Freezing; Genes; Genetic; Grant; Helium; Homologous Gene; Image; Imagery; Institutes; Life; Life Cycle Stages; Liquid substance; Mitotic; Modeling; Molecular; Mutation; Mycoplasma pneumoniae; Plasmids; Positioning Attribute; Prokaryotic Cells; Proteins; Research Personnel; Resolution; Series; Shapes; Structure; Technology; Test Result; Testing; Time; Work; base; computerized data processing; data acquisition; image processing; improved; instrumentation; lens; light microscopy; molecular modeling; prototype; reconstruction; software development; technology development; three dimensional structure; tool

In just the last few years, fluorescent light microscopy has shown that in bacteria, many proteins are dynamically regulated in both time and space, certain chromosomal loci are strictly positioned, and numerous cytoskeletal filaments exist. Thus instead of being simple "bags" of enzymes, even prokaryotic cells have substantial internal organization that enables their life cycle. The molecular basis of this organization is still unclear, but two key functions of"the bacterial cytoskeleton have been hypothesized: determination of cell shape and segregation of chromosomes during cell division. In support of these hypotheses, several shape-altering mutations have been found in proteins which form cytoskeletal filaments just inside the cytoplasmic membrane, including MreB, Mbl, CreS, and FtsZ. In addition, it is now clear that at least some bacterial plasmids are segregated by prokaryotic actin homologs that form filamentous mitotic machineries, and specific molecular models have been proposed. A growing body of evidence suggests that prokaryotic chromosomes are also actively segregated and positioned by protein filaments. Concurrent to these discoveries, electron cryotomography has emerged as a powerfulnew tool to visualize the three- dimensional structure of intact, small cells to "molecular resolution" (~4-8 nm) in a life-like state. Capitalizing on the recent installation of a one-of-a-kind, state-of-the-art electron cryomicroscope at the California Institute of Technology, we have, for the first time just last year, visualized bacterial cytoskeletal filaments directly within intact cells. Here we propose to extend these results and test the hypotheses above by determining the structure of the bacterial cytoskeleton in several model species throughout their life cycles by electron cryotomography. We believe the most exciting result will be direct visualization of mitotic machineries involved in bacterial chromosome segregation. Because this work will exploit prototype new instrumentation, a significant component of the effort will be technology development. This will include optimizing and refining strategies for collecting dual-axis tilt-series of frozen-hydrated cells as well as development of software to optimally merge the images into a three-dimensional reconstruction.

就在过去的几年里，荧光显微镜已经表明，在细菌中，许多蛋白质是 在时间和空间上动态调节，某些染色体位点被严格定位， 存在许多细胞骨架丝。因此，即使是原核生物， 细胞具有使它们的生命周期得以进行的实质性内部组织。这种现象的分子基础 组织仍然不清楚，但细菌细胞骨架的两个关键功能已经被假设： 在细胞分裂过程中确定细胞形状和染色体分离。为支持这些 在形成细胞骨架丝的蛋白质中发现了几种形状改变突变 就在细胞质膜内，包括MreB、Mbl、克雷斯和FtsZ。此外，现在很清楚， 至少一些细菌质粒被原核肌动蛋白同源物分离， 机制和具体的分子模型已经提出。越来越多的证据表明， 原核染色体也被蛋白质丝主动分离和定位。同时 这些发现，电子冷冻断层扫描已经成为一个强大的新工具，以可视化的三个- 将完整的小细胞的三维结构提高到“分子分辨率”（约4-8 nm），呈类生命状态。资本化 最近在加州安装了一台独一无二的最先进的电子低温显微镜 去年，我们第一次看到了细菌的细胞骨架丝 直接在完整的细胞内。在这里，我们建议扩展这些结果，并测试上述假设， 确定细菌细胞骨架的结构在几个模式物种在其整个生命周期 通过电子冷冻断层扫描。我们相信最令人兴奋的结果将是直接显示有丝分裂 参与细菌染色体分离的机器。因为这项工作将利用新的原型 仪器，努力的一个重要组成部分将是技术开发。这将包括 优化和完善收集双轴倾斜系列冷冻水化细胞的策略， 开发软件，以最佳方式将图像合并为三维重建。