Reconstitution of DNA segregation driven by assembly of a prokaryotic actin homolog

Abstract

Multiple unrelated polymer systems have evolved to partition DNA molecules between daughter cells at division. To better understand polymer-driven DNA segregation, we reconstituted the three-component segregation system of the R1 plasmid from purified components. We found that the ParR/parC complex can construct a simple bipolar spindle by binding the ends of ParM filaments, inhibiting dynamic instability, and acting as a ratchet permitting incorporation of new monomers and riding on the elongating filament ends. Under steady-state conditions, the dynamic instability of unattached ParM filaments provides the energy required to drive DNA segregation.

Fig. 1

In vitro reconstitution of the R1 plasmid spindle. (A) Individual ParR/parC-coated beads with radiating fluorescently labeled ParM asters (red, Cy3-labeled DNA; green, Alexa 488–labeled ParM). (B) Left: Time-lapse sequence of an Alexa 488–labeled ParM aster. Right: Maximum intensity projection of the time-lapse sequence, illustrating fall-off in fluorescence at 3 µm. (C) Inhibition of ParM dynamic instability with AMP-PNP produces substantially larger asters. (D) Two-color images and time-lapse sequence of bipolar ParM spindles segregating ParR/parC-coated beads. (E) Individual ParM filaments run from bead to bead, as shown by EM of negatively stained R1 spindles. (F) Time-lapse series of bipolar spindle elongation. (G) Elongation of a multipolar ParM structure.

Fig. 2

ParM filaments are stabilized when bound at each end by the ParR/parC complex. (A) ParM spindles form by “search and capture.” Sequence shows isolated ParM asters forming bipolar spindles that subsequently push the beads apart. Red arrows indicate capture events. (B) ParM filament bundles are stabilized a both ends by interaction with ParR/parC beads. Cutting a ParM spindle by laser irradiation (red box) results in depolymerization of severed ends. (C) The R1 spindle finds the long axis of a channel. Time-lapse sequence shows R1 spindles confined within a microfabricated channel.

Fig. 3

R1 spindles elongate by insertion of ParM monomers at the ParR/parC complex. (A) Photobleaching (indicated by red arrows) of ParM spindles reveals symmetrical, bipolar elongation at the bead surface. (B) ParM spindles elongate at each end at the same rate as free filament ends. The distance between the two photobleached stripes or distance between the stripe and the bead in (A) was measured on a per-frame basis, and the change in distance was plotted against time. (C) By speckle microscopy, all new ParM monomers add to the spindle at the ParR/parC interface. Green, Alexa 488–ParM; red, rhodamine-ParM doped at 1:1000 to produce fluorescent speckles. Left: Time-lapse sequence of an elongating spindle end (80 s per frame). Right: Kymograph of the time-lapse sequence.

Fig. 4

Dynamic instability of free ParM filaments provides the energy for R1 spindle elongation. (A) The ParR/parC complex stabilizes ParM filaments down to the ParM-ATP critical concentration. Spindle assembly reactions were performed at the indicated concentrations of Alexa 488–labeled ParM. (B) By abolishing the difference in critical concentration between free and ParR/parC-bound ParM filaments, AMP-PNP eliminates sustained polymerization on ParR/parC beads. (C) Small amounts of ATP added into AMP-PNP restore sustained polymerization on ParR/parC beads. (D) Hydrolysis-dependent ParM tail elongation occurs specifically at filament ends bound to ParR/parC complexes and not at free filament ends. Assembly reactions as in (C) were initiated with Alexa 488–ParM (green); at indicated times, the reactions were spiked with Cy3-ParM (red). Results were visualized after 50 min. Two examples of each time point are shown. (E) Rates of ParM tail elongation scale with the fraction of hydrolyzable nucleotide. Reactions as in (C) were performed at various ATP/AMP-PNP ratios. The rate of tail elongation was plotted against the percent of ATP within the nucleotide mixture.