3D Chromosome/Replisome Positioning in Bacterial Cells

描述（由申请人提供）：这是斯坦福大学的工程师和物理学家以及高级显微镜专家之间的合作研究项目，旨在确定细菌细胞中染色体和复制体在细胞周期过程中的三维（3D）结构。具体目标是：1.；精确定义染色体在非复制细菌细胞（Caulobacter swarm cell）内和在染色体被复制的细胞周期内的动态三维组织。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。我们还将在活细胞中进行延时荧光显微镜跟踪相同的位点。2. 通过(a)确定已知参与染色体组织的蛋白质突变的影响，(b)对离散染色体位点错误定位的突变进行自动化高通量筛选，(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，从而确定介导复制和非复制染色体空间部署的蛋白质。(d)考虑到细胞质的流体动力学特性，对染色体运动进行建模和分析，(e)使用染色体免疫沉淀法确定肌动蛋白样MreB蛋白是否在细胞周期的不同时间直接或间接地与DNA结合。3. 定义茎状杆菌细胞中复制体（复制工厂）在细胞极组装期间的空间部署及其在DNA复制期间的运动。为此，我们将(a)使用EM断层扫描和软x射线断层扫描的高分辨率（20-100 nm）成像，(b)使用全内反射（TIR）显微镜来确定移动的复制体是否沿着细胞分裂平面的轴向路径或螺旋路径，以及(c)使用断层成像来确定复制体是否与沿着细胞长轴部署的MreB螺旋路径共位并遵循路径。

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.