A moving ParA gradient on the nucleoid directs subcellular cargo transport via a chemophoresis force

Abstract

DNA segregation is a critical process for all life, and although there is a relatively good understanding of eukaryotic mitosis, the mechanism in bacteria remains unclear. The small size of a bacterial cell and the number of factors involved in its subcellular organization make it difficult to study individual systems under controlled conditions in vivo. We developed a cell-free technique to reconstitute and visualize bacterial ParA-mediated segregation systems. Our studies provide direct evidence for a mode of transport that does not use a classical cytoskeletal filament or motor protein. Instead, we demonstrate that ParA-type DNA segregation systems can establish a propagating ParA ATPase gradient on the nucleoid surface, which generates the force required for the directed movement of spatially confined cargoes, such as plasmids or large organelles, and distributes multiple cargos equidistant to each other inside cells. Here we present the critical principles of our diffusion-ratchet model of ParA-mediated transport and expand on the mathematically derived chemophoresis force using experimentally-determined biochemical and cellular parameters.

Keywords: ATP, adenosine triphosphate; DNA, deoxyribonucleic acid; Par, partition; ParA ATPase; RD, reaction diffusion; Sop, stability of plasmid; bacterial chromosome segregation; intracellular transport; plasmid partition; subcellular organization.

Figure 1.

ParA-type cargo transport on a DNA-carpet. (A) Schematic of the reconstitution setup, where a magnet above the flowcell confined sopC-coated magnetic beads on to the DNA-carpet. Fluorescent labeled components of the system were visualized by TIRFM. (B) A freeze-frame image series of SopA (green) on the DNA-carpet and SopB (red) on a bead traveling from right to left. Figure panels with permission from Vecchiarelli et al.

Figure 2.

Comparison of experimental and simulated SopA-SopB driven motion. (A) Position as a function of time for SopB coated beads moving on a random DNA surface with bound SopA from Vecchiarelli et al. (red lines) and 50 simulated trajectories (gray lines) based on the chemophoresis force (Equation 1) and the reaction diffusion expression (Equation 2) for parameters listed in Table 1 (Simulation 1) for which the average velocity of the simulated traces (0.09 ± 0.01 μm s⁻¹ (SEM)) was the same as the experimental traces (0.1 ± 0.02 μm s⁻¹ (SEM)). The experimental trajectories correspond to the maximum projection of the motion, which was highly directional. The simulated trajectories were oriented so that the average velocity for each trajectory was positive. Note the frequent reversals in the direction of motion of the simulated trajectories. (B) Same as in (A) except that the SopB density was 5-fold less (parameter set 2 in Table 1). The average velocity of the simulated traces was 0.089 ± 0.005 μm s⁻¹ (SEM). (C) The mean square displacements (MSD) of the trajectories in panel (A) plotted as a function of the time interval. (D) The mean square displacements (MSD) of the trajectories in panel (B) plotted as a function of the time interval.

Figure 3.

Simulations resemble experimentally-observed ParA-mediated cargo dynamics. Time-lapse sequence of the simulated 2-dimensional motion of a SopB-coated particle on a SopA-coated surface. Scale bar = 10 μm. Also see Movie 2 and SI Methods for simulation details.