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Science. Author manuscript; available in PMC Jun 27, 2013.
Published in final edited form as:
PMCID: PMC3694215
EMSID: EMS51090

A Bipolar Spindle Of Antiparallel ParM Filaments Drives Bacterial Plasmid Segregation

Abstract

To ensure their stable inheritance by daughter cells during cell division, bacterial low copy-number plasmids make simple DNA segregating machines that use an elongating protein filament between sister plasmids. In the ParMRC system of Escherichia coli R1 plasmid, ParM, an actin-like protein, forms the spindle between ParRC complexes on sister plasmids. Using a combination of structural work and total internal reflection fluorescence microscopy, we show that ParRC bound and could accelerate growth at only one end of polar ParM filaments, mechanistically resembling eukaryotic formins. The architecture of ParM filaments enabled two ParRC-bound filaments to associate in an antiparallel orientation, forming a bipolar spindle. The spindle elongated as a bundle of at least two antiparallel filaments, thereby pushing two plasmid clusters towards the poles.

During bacterial cell division, equal distribution of replicated plasmids to daughter cells ensures their stable inheritance. Low copy-number plasmids encode the simplest known DNA segregation machines to perform this task. They comprise a nucleotide-driven (cytomotive) protein filament and a centromere-like DNA region, linked by an adaptor protein. The ParMRC segregation system of E. coli R1 plasmid consists of ParM, an actin-like cytomotive protein (1) that forms polar, left-handed double-helical filaments (2), ParR, an adaptor protein, and parC, a centromeric region (3, 4). Dynamic instability of ParM filaments enables plasmid segregation by a ‘search and capture’ mechanism (5, 6), with ParRC (7, 8) stabilizing the filaments. It has been reported that ParRC binds to both ends of a single ParM filament (9, 10). This leads to a conundrum - how does ParRC bind to two different ends of a polar ParM filament?

Here, we provide a comprehensive description of ParM in the monomeric and filament states. An electron cryomicroscopy (cryoEM) reconstruction (11) (Fig. 1A) provided a subnanometer-resolution map of the polar filament of ParM (resolution of 8.5 Å at FSC 0.5; Fig. S1A-D), polymerized in the presence of adenylyl-imidodiphosphate (AMPPNP; a non-hydrolyzable ATP analog). We determined the crystal structures of a non-polymerizing mutant, ParM(L163R) bound to AMPPNP, and ParM(L163A) bound to the C-terminal 17-residue-peptide corresponding to the ParM-interacting region of ParR (8, 10) (ParRpept) and AMPPNP (11). Comparison of the crystal structures, including the previously reported apo- and ADP-bound forms (1), revealed no large differences between the ATP and ADP states of ParM monomers (Figs. 1B-C). In contrast, domains IB and IIB showed a rotation of 9.1° and 10.7° towards the nucleotide-binding pocket (Fig. 1D) in the ParM:ParRpept structure, compared to the free monomer. The domain rotations were reminiscent of the transition from G-actin to F-actin (12, 13). Fitting the monomeric structures of ParM into the cryoEM reconstruction showed that the ParM:ParRpept structure fits best (Figs. 1E-F, S1E; Movie S1). Thus, the filament conformation of ParM is very similar to ParM:ParRpept. The best-fit provided a quasi-atomic model of the ParM filament (Fig. 1G), and implies that binding of ParR or ParRC locks ParM monomers in the filament-like conformation.

Fig. 1
Conformational cycle of ParM

ParRpept was bound in a hydrophobic pocket between domains IA and IIA of ParM (Figs. 2A, S2). The ParM-ParRpept interaction resembled proteins that bind at the barbed-end of actin (Figs. 2A-B). Many actin-binding proteins, including formins (14) and WH2 domain-containing proteins such as Spire (15), insert a helix between the corresponding subdomains 1 and 3 of actin (16). The ParM:ParRpept structure modeled onto the ParM filament highlighted a significant clash between ParRpept and residues 37 to 46 of the next ParM monomer in the protofilament (Fig. 2C). Thus, the ParR-binding site lies within the polymerization interface of ParM, and bound ParR requires to be displaced during elongation. Overlap in the binding site of ParRpept and the polymerization interface occludes all the ParRC-binding sites on the ParM filament except those at the barbed-end, implying that ParRC binds exclusively to this end.

Fig. 2
ParRC binds at the barbed-end of ParM filaments

Our ParM-ParRpept structure supports a formin-like mechanism for the processive movement of ParRC (Fig. S3). Ten ParR dimers bind parC repeats (7, 8), forming a scaffold that allows a formin-like stair-stepping mechanism (Fig. S3B, C) (17). The C-terminal helices from the twenty ParRs are localized in a confined area. This probably facilitates ParM filament nucleation by ParRC (18). It also ensures ParRC-bound ParM monomer recruitment upon ParR displacement from the filament (Fig. S3D), explaining end-tracking of ParM filaments by ParRC through insertional polymerization (6, 19). The ParRC cap locks the terminal monomers in the filament-conformation, thus protecting the filament from dynamic instability.

To confirm the proposed single-end binding of ParRC, we examined the effect of ParRC on ParM filament elongation using TIRF microscopy (11). The experiments were performed with non-hydrolyzable AMPPNP to prevent dynamic instability. ParM-AMPPNP elongated symmetrically at both ends from an initial seed of the ParM filament (Fig. 3A; Movie S2) (5). In the presence of unlabeled ParRC, one end of the filaments grew faster, resulting in asymmetric growth (Figs. 3B, S4A; Movie S3). The rates of growth were 9.4 ± 4.1 and 2.5 ± 1.9 monomers/s, for fast and slow growing ends (n = 32; Table S3). Experiments with labeled ParRC showed that ParRC accelerated growth and recruited ParM monomers at the ParRC-bound filament end only (Figs. 3C, S4B; Movie S4), reconfirming insertional polymerization (6). The unidirectional elongation by ParRC leads us to the key question – what enables bipolar plasmid segregation?

Fig. 3
ParRC accelerates growth at one end of ParM filaments

Frequent condensation events were observed between ParM filaments, both with ATP and AMPPNP (Fig. 4A, Movies S5S8). Furthermore, continuous motion due to interfilament sliding occurred within filament bundles (Figs. 4B, S4C; Movies S9S11). A molecular model for the filament sliding and condensation was generated based on two monomers in the crystal packing of ParM:ParRpept (Fig. S5A). ParM filament subunits, when superposed sequentially onto the ParM monomers, produced sliding and an antiparallel packing of filaments (Figs. 4C, S5A). Mutation of residues within loop 18-21 (S19R, G21R) at the proposed antiparallel filament interface (Figs. 4C-D, S5B) prevented interfilament sliding (Movie S12; Figs. 4E, S4D), due to stronger interactions between filaments caused by alternating charges.

Fig. 4
A bipolar spindle comprises at least two antiparallel ParM filaments

The helical geometry of ParM filament, with 12 subunits per turn, is compatible with a hexagonal or square packing of ParM filaments in a bundle (Fig. S5C) (4), in contrast to non-bundling actin filaments with a 13-monomer repeat. Bundles of ParM filaments have been observed in E. coli cells expressing ParM at wild-type levels (20) and during plasmid partitioning in vivo (21). Also, bundles of antiparallel ParM filaments have been described in vitro (22).

Antiparallel pairing explains observations of ParRC at both ends of ParM filaments (9, 10). Of the 826 filaments we counted, 104, 540 and 182 filaments were observed with ParRC at 0, 1 and 2 ends, respectively, consistent with single-end binding and pairing. Bipolar spindles of ParM were observed by TIRF microscopy using ATP. Stable filaments were seen only as elongating spindles with ParRC at both ends, pushing plasmids apart at a rate of 22.6 ± 4.8 monomers/s (n = 40; Movies S13-S14; Table S3). Upon loss of ParRC at limiting concentrations of ParM (350 nM – 500 nM), dynamic instability caused spindle disassembly at a rate of 100.3 ± 18.7 monomers/s (n = 61; Figs. 4F, S4E; Movies S15-S16; Table S3).

To demonstrate that the spindles comprised at least two antiparallel filaments, we introduced negatively charged residues within loop 18-21 (S19E, G21E), (Fig. 4D) to weaken the interfilament interaction through repulsive electrostatic forces. We observed spindles (n = 65) that split into the constituent filaments (Movie S17, Fig. 4G). This confirms that spindles are not formed by a single ParM filament, with ParRC attached at both ends (9, 10) (Fig. 4H). ParM filaments in a spindle were protected from dynamic instability by binding of ParRC at the barbed-end and pairing with another ParRC-bound filament at the pointed-end (Movies S15-S19; Figs. 4F-H, S4E-F). The trigger for disassembly was either the loss of ParRC (Movies S15S16) or exposure of pointed-ends due to unwinding of the antiparallel bundle (Movies S17S19).

These observations, and previously published work (3, 4), provide a comprehensive model of ParMRC-mediated plasmid segregation (Fig. S6): a critical concentration of ATP-bound monomers in the cell nucleates ParM filaments (or nucleation is ParRC-mediated) (18). ParRC–binding rescues the dynamic filaments from disassembly at the barbed-end only. ParRC speeds up the growth at the barbed-end by a formin-like mechanism. The free pointed-end remains prone to disassembly unless it pairs up antiparallel with another ParM filament bound to ParRC, probably shortly after plasmid replication. Thus, a bipolar spindle of two antiparallel filaments is the minimum unit in plasmid segregation. R1 plasmid has a copy number of about six, which is approximately the number of filaments within bundles in plasmid-segregating cells (20). ParM bundles are stronger than single filaments, which is advantageous when pushing plasmids through the cytoplasm. A single bundle will also ensure concerted segregation of all sister plasmids, as observed in vivo (21).

The lateral interaction among dynamic filaments, as in ParMRC, may also facilitate contraction in other cytomotive filament systems such as FtsZ in bacterial cell division (23). Our model of antiparallel actin-like ParM filaments, without the necessity of bundling factors or motor proteins provides an attractive conceptual precursor for actin contractile systems, such as muscle.

One Sentence Summary: A bipolar spindle, formed by antiparallel actin-like filaments, pushes sister plasmids apart during bacterial plasmid segregation.

Supplementary Material

Movie S1

Movie S18

Movie S19

Movie S2

Movie S3

Movie S4

Movie S5

Movie S6

Movie S7

Movie S8

Movie S9

Movie S10

Supplementary Data

Movie S11

Movie S12

Movie S13

Movie S14

Movie S15

Movie S16

Movie S17

Acknowledgments

We acknowledge N. Barry and C. Johnson (MRC-LMB, help with TIRF microscopy and ITC), beamline support at ID29, ID14-2 (ESRF, France) and I02 (Diamond Light Source, UK), L. Amos, S. Bullock, A. Carter and R. Gasper-Schönenbrücher (MRC-LMB, critical reading of the manuscript), K. Gerdes (Newcastle University) and A. Toste Rêgo (MRC-LMB) (provision of reagents). Support by the Medical Research Council (U105184326 to JL), Grants-in-Aid for Scientific Research (21227006 to KN), and FP7-IIF-2008 fellowship (PG) is acknowledged.

Footnotes

Protein Data Bank (PDB) accession codes: ParM-AMPPNP (4A61), ParM-AMPPNP-ParRpept (4A62). The EM reconstruction has been deposited in the EMDB (EMD-1980), fitted coordinates in the PDB (4A6J).

Supplementary Materials: Materials and Methods

Figs. S1 to S6

Tables S1 to S3

Movies S1 to S19

References (24 – 41)

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