Structure and function of the prokaryotic cytoskeleton

原核细胞骨架的结构和功能

• 批准号: 7019874
• 负责人: GRANT J JENSEN
• 金额: $ 34.26万
• 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/7019874
• 关键词: Caulobacter; Escherichia coli; Mycoplasma pneumoniae; actins; bioimaging /biomedical imaging; cell cycle; cell morphology; cryoelectron microscopy; microfilaments; microorganism reproduction; molecular /cellular imaging; plasmids; prokaryote; three dimensional imaging /topography; tomography

DESCRIPTION (provided by applicant): 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 powerful new 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.

描述（由申请人提供）：仅在过去几年中，荧光显微镜已经显示，在细菌中，许多蛋白质在时间和空间上都是动态调节的，某些染色体位点是严格定位的，并且存在许多细胞骨架丝。因此，即使是原核细胞，也不是简单的酶“袋”，而是具有使其生命周期得以实现的实质性内部组织。这种组织的分子基础仍然不清楚，但细菌细胞骨架的两个关键功能已经被假设：细胞分裂期间染色体的分离和细胞形状的确定。为了支持这些假设，在细胞质膜内形成细胞骨架丝的蛋白质中发现了几种改变形状的突变，包括MreB，Mbl，克雷斯和FtsZ。此外，现在很清楚，至少有一些细菌质粒被原核肌动蛋白同源物分离，形成丝状有丝分裂机制，并提出了特定的分子模型。越来越多的证据表明，原核生物的染色体也被蛋白质丝主动分离和定位。在这些发现的同时，电子冷冻断层扫描已经成为一种强大的新工具，可以将完整的小细胞的三维结构可视化到“分子分辨率”（约4-8 nm），处于类似生命的状态。利用最近在加州理工学院安装的一台独一无二的、最先进的电子低温显微镜，我们去年第一次直接在完整的细胞内观察到了细菌的细胞骨架丝。在这里，我们建议扩展这些结果，并测试上述假设，通过确定在几个模式物种的细菌细胞骨架的结构，在其整个生命周期的电子cryotomography。我们相信，最令人兴奋的结果将是直接可视化的有丝分裂机构参与细菌染色体分离。由于这项工作将利用原型新仪器，一个重要组成部分的努力将是技术开发。这将包括优化和完善收集冷冻水合细胞双轴倾斜系列的策略，以及开发软件，以最佳方式将图像合并为三维重建。