Limits...
Gene location and DNA density determine transcription factor distributions in Escherichia coli.

Kuhlman TE, Cox EC - Mol. Syst. Biol. (2012)

Bottom Line: Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene.We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid.We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA. tkuhlman@illinois.edu

ABSTRACT
The diffusion coefficient of the transcription factor LacI within living Escherichia coli has been measured directly by in vivo tracking to be D = 0.4 μm(2)/s. At this rate, simple models of diffusion lead to the expectation that LacI and other proteins will rapidly homogenize throughout the cell. Here, we test this expectation of spatial homogeneity by single-molecule visualization of LacI molecules non-specifically bound to DNA in fixed cells. Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene. We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid. Finally, we show that LacI inhomogeneity increases the strength with which targets located proximally to the LacI gene are regulated. We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

Show MeSH

Related in: MedlinePlus

Image averaging procedure. (A) A representative image field with lacI-venus integrated near the origin (atpI locus) imaged at × 133 magnification. (B) Individual cells are found using an automated algorithm, and coordinates are established at the cellular centroid to measure cellular dimensions and the location of each pixel. (C) Because of the narrow distribution of cell widths, correctly identified cells are identified according to correspondence with the appropriate width and binned according to the ratio of the length to width. Alternating gray and white bars indicate each bin. (D) Cells within each bin are rescaled to the size of the average cell within that bin and averaged together on a per-pixel basis and over all orientations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3472691&req=5

f1: Image averaging procedure. (A) A representative image field with lacI-venus integrated near the origin (atpI locus) imaged at × 133 magnification. (B) Individual cells are found using an automated algorithm, and coordinates are established at the cellular centroid to measure cellular dimensions and the location of each pixel. (C) Because of the narrow distribution of cell widths, correctly identified cells are identified according to correspondence with the appropriate width and binned according to the ratio of the length to width. Alternating gray and white bars indicate each bin. (D) Cells within each bin are rescaled to the size of the average cell within that bin and averaged together on a per-pixel basis and over all orientations.

Mentions: We took ∼1000 images for each strain and growth condition. We then used an automated cell-finding algorithm to identify individual cells in each image, binned cells by size, and averaged the fluorescent intensity at each pixel to determine the average spatial distribution of each component (Figure 1). Due to hardware limitations, we were unable to dynamically switch between epifluorescent and TIRF illumination, and this limitation precludes the simultaneous high-throughput measurement and direct correlation of gene location and protein distribution in the same cell. Consequently, imaging of the spatial distribution of each component (DNA, gene, mRNA, and protein) was performed in separate measurements. The absolute orientation of each cell is then ambiguous, and we additionally perform an average over all possible orientations, which introduces symmetries into the resulting average distributions.


Gene location and DNA density determine transcription factor distributions in Escherichia coli.

Kuhlman TE, Cox EC - Mol. Syst. Biol. (2012)

Image averaging procedure. (A) A representative image field with lacI-venus integrated near the origin (atpI locus) imaged at × 133 magnification. (B) Individual cells are found using an automated algorithm, and coordinates are established at the cellular centroid to measure cellular dimensions and the location of each pixel. (C) Because of the narrow distribution of cell widths, correctly identified cells are identified according to correspondence with the appropriate width and binned according to the ratio of the length to width. Alternating gray and white bars indicate each bin. (D) Cells within each bin are rescaled to the size of the average cell within that bin and averaged together on a per-pixel basis and over all orientations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472691&req=5

f1: Image averaging procedure. (A) A representative image field with lacI-venus integrated near the origin (atpI locus) imaged at × 133 magnification. (B) Individual cells are found using an automated algorithm, and coordinates are established at the cellular centroid to measure cellular dimensions and the location of each pixel. (C) Because of the narrow distribution of cell widths, correctly identified cells are identified according to correspondence with the appropriate width and binned according to the ratio of the length to width. Alternating gray and white bars indicate each bin. (D) Cells within each bin are rescaled to the size of the average cell within that bin and averaged together on a per-pixel basis and over all orientations.
Mentions: We took ∼1000 images for each strain and growth condition. We then used an automated cell-finding algorithm to identify individual cells in each image, binned cells by size, and averaged the fluorescent intensity at each pixel to determine the average spatial distribution of each component (Figure 1). Due to hardware limitations, we were unable to dynamically switch between epifluorescent and TIRF illumination, and this limitation precludes the simultaneous high-throughput measurement and direct correlation of gene location and protein distribution in the same cell. Consequently, imaging of the spatial distribution of each component (DNA, gene, mRNA, and protein) was performed in separate measurements. The absolute orientation of each cell is then ambiguous, and we additionally perform an average over all possible orientations, which introduces symmetries into the resulting average distributions.

Bottom Line: Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene.We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid.We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA. tkuhlman@illinois.edu

ABSTRACT
The diffusion coefficient of the transcription factor LacI within living Escherichia coli has been measured directly by in vivo tracking to be D = 0.4 μm(2)/s. At this rate, simple models of diffusion lead to the expectation that LacI and other proteins will rapidly homogenize throughout the cell. Here, we test this expectation of spatial homogeneity by single-molecule visualization of LacI molecules non-specifically bound to DNA in fixed cells. Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene. We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid. Finally, we show that LacI inhomogeneity increases the strength with which targets located proximally to the LacI gene are regulated. We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

Show MeSH
Related in: MedlinePlus