3D Chromosome/Replisome Positioning in Bacterial Cells

描述(申请人提供)：这是一个由斯坦福大学的工程师和物理学家与高级显微镜专家合作研究的项目，目的是随着细胞周期的进展来确定细菌细胞中染色体和复制体的三维(3D)构型。具体目标是：1.精确定义非复制型细菌细胞(Caulbacter swarmer细胞)内以及在复制染色体的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置。我们还将在活细胞中进行延时荧光显微镜跟踪相同的基因座。2.通过(A)确定已知参与染色体组织的蛋白质中的突变的影响，(B)对错位的离散染色体位点的突变体进行自动高通量筛选，(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模拟和分析染色体运动，以及(E)利用染色体免疫沉淀分析，确定类似肌动蛋白的MreB蛋白是否在细胞周期的不同时间直接或间接与DNA结合，从而确定介导复制和非复制染色体的空间部署的蛋白质。3.确定复制体(复制工厂)在细胞极点组装过程中的空间分布和DNA复制过程中的运动。为此，我们将(A)使用高分辨率(20-100 nm)的EM断层扫描和软X射线断层扫描，(B)使用全内反射(TIR)显微镜来确定移动的复制体在其到达细胞分裂平面的过程中是沿着轴向路径还是螺旋路径，以及(C)使用断层成像来确定复制体是否与沿细胞长轴部署的MreB螺旋共同定位并遵循其路径。

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.