Mapping the driving forces of chromosome structure and segregation in Escherichia coli

Abstract

The mechanism responsible for the accurate partitioning of newly replicated Escherichia coli chromosomes into daughter cells remains a mystery. In this article, we use automated cell cycle imaging to quantitatively analyse the cell cycle dynamics of the origin of replication (oriC) in hundreds of cells. We exploit the natural stochastic fluctuations of the chromosome structure to map both the spatial and temporal dependence of the motional bias segregating the chromosomes. The observed map is most consistent with force generation by an active mechanism, but one that generates much smaller forces than canonical molecular motors, including those driving eukaryotic chromosome segregation.

Figure 1.

(A) Schematic of the bi-directional replicating circular E. coli chromosome and the chromosome conformation in the cell. Before the initiation of segregation, oriC (red focus) is positioned at mid-cell with the left arm of the chromosome on the left side of the cell and the right arm of the chromosome on the right side of the cell. The chromosomal orientation is measured using the position of a second locus (green focus) on the right arm of the chromosome. The relative long-axis position of foci in the cell is measured relative to cell length from mid-cell. (B) Frame mosaic of a typical cell cycle (every 6th frame shown for clarity). An array of phase-contrast/fluorescence composite images shows the red (oriC) and green (fiducial) fluorescent foci and the cell mask as a function of time (min) since cell division, which is shown in the top left corner of each image. (C) Kymograph for a typical cell shows red fluorescence intensity along the long axis of the cell as a function of time since the splitting of the oriC locus. The black points show the fit oriC long-axis position of the focus relative to mid-cell as a function of time (min). The black dashed line shows the cell poles. (D) Locus position occupancy (heat map) and mean trajectory (black points) of oriC as a function of relative cellular position for 406 independent cell cycles synchronized to the oriC split (t = 0). Dotted horizontal lines show the approximate home positions of the oriC locus before and after segregation. Locus dynamics are organized into four time intervals of motion: (i) Pre-Replication; (ii) Cohesion; (iii) Rapid-Translocation; and (iv) Post-Segregation for analysis.

Figure 2.

(A) MSD for oriC prior to and post-loci splitting both show sub-diffusive motion. In the Pre-Replication interval of motion, the MSD scaling parameter α is 0.39. After oriC splits, α is 0.74 during the Rapid-Translocation and Post-Segregation intervals of motion. Even immediately after the initial locus split, oriC dynamics is characterized by a scaling factor considerably smaller than α = 2 which corresponds to processive motion. (B) The step-size distributions (over 1 min) for oriC for the Pre-Replication and Rapid-Translocation intervals of motion. (In order to capture the bias, we consider only the right moving locus after the split. Other intervals are omitted for clarity.) For each distribution, a Gaussian distribution with the same mean (vertical dotted line) and variance is plotted (dotted curve), representing the step-size distribution for a diffusive model. Both intervals of motion have less than 0.5% more large steps than the diffusive step-size distribution. Biased motion during the Rapid-Translocation interval is the result of a distribution-wide shift to rightward steps rather than a small number of large steps forward biased steps. (Shaded regions represent standard error).

Figure 3.

(A) Spatiotemporal dependence of the drift velocity of oriC. During the Pre-Replication and cohesion intervals of locus motion, there is a restoring drift velocity to the equilibrium position of the locus at mid-cell. Immediately after the oriC loci split, the mid-cell position becomes unstable and equilibrium positions appear at the quarter cell positions. This velocity profile remains qualitatively unchanged for the remainder of the cell cycle. (B) Spatiotemporal dependence of locus occupancy. Higher mean velocity is observed in the Rapid-Translocation interval than in the Post-Segregation interval of motion since the peak occupancies (maxima of the occupancy curves) are further from the equilibrium positions (vertical dotted lines). (Shaded regions represent standard error).

Figure 4.

Schematic model of spatiotemporal drift velocity profile for oriC. During both the Pre-Replication and Cohesion intervals, there is a restoring force (purple arrows) to the equilibrium position (dotted line) at mid-cell. This restoring force is represented by a spring in the physical analogue connecting the loci to mid-cell. Immediately upon oriC locus separation, the equilibrium position moves to quarter cell (dotted lines) and remains qualitatively unchanged for the remainder of the cell cycle, despite significant changes to the nucleoid structure. In the physical analogue system, a spring connects each locus to the quarter cell position. The mean locus velocity is initially high (Rapid-Translocation) since the locus begins far from the equilibrium position (dotted line). Once the locus is close to the equilibrium position, the force is low, corresponding to the Post-Segregation interval of motion.