3D Chromosome/Replisome Positioning in Bacterial Cells

细菌细胞中的 3D 染色体/复制体定位

• 批准号: 6858425
• 负责人: LUCILLE SHAPIRO
• 金额: $ 38.41万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6858425
• 关键词: DNA binding protein; DNA replication; bacterial DNA; bacterial genetics; bioimaging /biomedical imaging; cell cycle; chromosome movement; cytoplasm; fluorescence microscopy; immunoprecipitation; three dimensional imaging /topography

DESCRIPTION (provided by applicant): This is a program of collaborative research between engineers and physicists at Stanford University together with specialists in advanced microscopy to determine the three dimensional (3D) configuration of the chromosome and the replisome in the bacterial cell as the cell cycle progresses. Specific aims are: 1. To precisely define the dynamic 3D organization of the chromosome within the non-replicating bacterial cell (the Caulobacter swarmer cell) and during the cell cycle while the chromosome is being replicated. To do this, we will perform high-resolution 3D imaging by EM tomography and soft X-ray tomography to locate and map chromosomal loci in the cell. We will also perform time-lapse fluorescent microscopy tracking of the same loci in living cells. 2. To identify the proteins that mediate the spatial deployment of both replicating and non-replicating chromosomes by (a) determining the effect of mutations in proteins known to be involved in chromosome organization, (b) carrying out an automated high throughput screen for mutants that mislocalize discrete chromosomal loci, (c) determining the effect of cell structure by examining the chromosome organization in long filamentous cells, (d) modeling and analyzing the chromosome movement taking account of the hydrodynamic properties of the cytoplasm, and (e) determining if the actin-like MreB protein directly or indirectly binds to DNA at different times in the cell cycle using chromosome immunoprecipitation assays. 3. To define the spatial deployment of the replisome (replication factory) in the Caulobacter celt during its assembly at the cell pole and its movement during DNA replication. To do this, we will (a) use high resolution (20-100 nm) imaging by EM tomography and soft X-ray tomography, (b) use total internal reflection (TIR) microscopy to determine if the moving replisome follows an axial or a spiral path on its way to the cell division plane, and (c) use tomographic imaging to determine if the replisome co-positions with and follows the path of the MreB spiral that is deployed along the long axis of the cell.

描述（由申请人提供）：这是斯坦福大学工程师和物理学家与高级显微镜专家之间的合作研究项目，旨在确定作为细胞周期的细菌细胞中染色体和复制体的三维（3D）结构取得进展。具体目标是： 1. 精确定义非复制细菌细胞（柄杆菌群细胞）内以及染色体复制期间细胞周期中染色体的动态 3D 组织。为此，我们将通过 EM 断层扫描和软 X 射线断层扫描进行高分辨率 3D 成像，以定位和绘制细胞中的染色体位点。我们还将对活细胞中的相同位点进行延时荧光显微镜追踪。 2. 通过以下方式鉴定介导复制和非复制染色体空间部署的蛋白质：(a) 确定已知参与染色体组织的蛋白质突变的影响，(b) 对突变体进行自动高通量筛选错定位离散染色体位点，(c) 通过检查长丝状细胞中的染色体组织来确定细胞结构的影响，(d) 考虑细胞质的流体动力学特性来建模和分析染色体运动，以及 (e) 确定如果使用染色体免疫沉淀分析，肌动蛋白样 MreB 蛋白在细胞周期的不同时间直接或间接与 DNA 结合。 3. 定义柄杆菌细胞中复制体（复制工厂）在细胞极组装过程中的空间部署以及 DNA 复制过程中的运动。为此，我们将 (a) 通过 EM 断层扫描和软 X 射线断层扫描使用高分辨率（20-100 nm）成像，(b) 使用全内反射 (TIR) 显微镜来确定移动复制体是否遵循轴向或(c) 使用断层扫描成像来确定复制体是否与沿细胞长轴部署的 MreB 螺旋共置并遵循其路径。