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. ? ?
| Ptacin, Jerod L; Shapiro, Lucy (2013) Chromosome architecture is a key element of bacterial cellular organization. Cell Microbiol 15:45-52 |
| Umbarger, Mark A; Toro, Esteban; Wright, Matthew A et al. (2011) The three-dimensional architecture of a bacterial genome and its alteration by genetic perturbation. Mol Cell 44:252-64 |
| Christen, Beat; Abeliuk, Eduardo; Collier, John M et al. (2011) The essential genome of a bacterium. Mol Syst Biol 7:528 |
| Ptacin, Jerod L; Lee, Steven F; Garner, Ethan C et al. (2010) A spindle-like apparatus guides bacterial chromosome segregation. Nat Cell Biol 12:791-8 |
| Bowman, Grant R; Comolli, Luis R; Gaietta, Guido M et al. (2010) Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function. Mol Microbiol 76:173-89 |
| Iniesta, Antonio A; Hillson, Nathan J; Shapiro, Lucy (2010) Polar remodeling and histidine kinase activation, which is essential for Caulobacter cell cycle progression, are dependent on DNA replication initiation. J Bacteriol 192:3893-902 |
| Toro, Esteban; Shapiro, Lucy (2010) Bacterial chromosome organization and segregation. Cold Spring Harb Perspect Biol 2:a000349 |
| Iniesta, Antonio A; Hillson, Nathan J; Shapiro, Lucy (2010) Cell pole-specific activation of a critical bacterial cell cycle kinase. Proc Natl Acad Sci U S A 107:7012-7 |
| Christen, Beat; Fero, Michael J; Hillson, Nathan J et al. (2010) High-throughput identification of protein localization dependency networks. Proc Natl Acad Sci U S A 107:4681-6 |
| Ptacin, Jerod L; Shapiro, Lucy (2010) Initiating bacterial mitosis: understanding the mechanism of ParA-mediated chromosome segregation. Cell Cycle 9:4033-4 |
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