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Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Bharat TA, Murshudov GN, Sachse C, Löwe J - Nature (2015)

Bottom Line: Growing ParMRC spindles push sister plasmids to the cell poles.The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions.Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

ABSTRACT
Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

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ParM doublets formed in vitro(a) Cryo-EM images of ParM doublets formed in vitro with crowding agent PEG 6000. This experiment was repeated 15 times. (b) Slice through an electron cryotomogram (cryo-ET) showing clear lack of super-helicity in the doublets (see Video 3). (c) A 2D class average of the ParM doublet. The thickest parts of double helical ParM filaments have been indicated with yellow arrowheads (see ED Fig. 5). (d) Model of the doublet, shown in the same orientation as the class average in c) (see Video 4). (e) An orthogonal, magnified view of the doublet cut at the plane shown as a dashed line in d). (f) Atomic model of the doublet. Residues shown in red in one ParM filament interact with residues in orange in the other filament (see ED Table 2). (g) An orthogonal view of the doublet, with the filament axes going into the plane of the paper. One of the residues (S19) that forms the doublet interface has been highlighted (see ED Fig. 6).
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Figure 3: ParM doublets formed in vitro(a) Cryo-EM images of ParM doublets formed in vitro with crowding agent PEG 6000. This experiment was repeated 15 times. (b) Slice through an electron cryotomogram (cryo-ET) showing clear lack of super-helicity in the doublets (see Video 3). (c) A 2D class average of the ParM doublet. The thickest parts of double helical ParM filaments have been indicated with yellow arrowheads (see ED Fig. 5). (d) Model of the doublet, shown in the same orientation as the class average in c) (see Video 4). (e) An orthogonal, magnified view of the doublet cut at the plane shown as a dashed line in d). (f) Atomic model of the doublet. Residues shown in red in one ParM filament interact with residues in orange in the other filament (see ED Table 2). (g) An orthogonal view of the doublet, with the filament axes going into the plane of the paper. One of the residues (S19) that forms the doublet interface has been highlighted (see ED Fig. 6).

Mentions: Having described the structure of the ParM filaments, we then wished to put the structural data in context of the bipolar spindles that segregate plasmid DNA in cells. For bipolar spindles to form, filamentous ParM subunits must engage in another interaction, inter-filament contacts, formed between double-helical filaments. It was known that incubation of ParM filaments with a crowding agent causes them to bundle 19. However, bundles are not amenable to high-resolution cryo-EM analysis because of their heterogeneity 20. To obtain a more defined sample, we titrated ParM+AMPPNP with varying amounts of crowding agent. When 2 % poly ethylene glycol (PEG) 6000 was added to ParM+AMPPNP, we found that ParM filaments dimerised to form ‘doublets’, containing two double-helical filaments (Fig. 3a, ED Fig. 5a-b). In raw cryo-EM images, doublets appeared as two roughly parallel lines, with no evidence of supercoiling or twisting. Electron cryotomography (cryo-ET) of the doublet specimen confirmed that the filaments do not twist around each other (Fig. 3b, Video 3).


Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Bharat TA, Murshudov GN, Sachse C, Löwe J - Nature (2015)

ParM doublets formed in vitro(a) Cryo-EM images of ParM doublets formed in vitro with crowding agent PEG 6000. This experiment was repeated 15 times. (b) Slice through an electron cryotomogram (cryo-ET) showing clear lack of super-helicity in the doublets (see Video 3). (c) A 2D class average of the ParM doublet. The thickest parts of double helical ParM filaments have been indicated with yellow arrowheads (see ED Fig. 5). (d) Model of the doublet, shown in the same orientation as the class average in c) (see Video 4). (e) An orthogonal, magnified view of the doublet cut at the plane shown as a dashed line in d). (f) Atomic model of the doublet. Residues shown in red in one ParM filament interact with residues in orange in the other filament (see ED Table 2). (g) An orthogonal view of the doublet, with the filament axes going into the plane of the paper. One of the residues (S19) that forms the doublet interface has been highlighted (see ED Fig. 6).
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Figure 3: ParM doublets formed in vitro(a) Cryo-EM images of ParM doublets formed in vitro with crowding agent PEG 6000. This experiment was repeated 15 times. (b) Slice through an electron cryotomogram (cryo-ET) showing clear lack of super-helicity in the doublets (see Video 3). (c) A 2D class average of the ParM doublet. The thickest parts of double helical ParM filaments have been indicated with yellow arrowheads (see ED Fig. 5). (d) Model of the doublet, shown in the same orientation as the class average in c) (see Video 4). (e) An orthogonal, magnified view of the doublet cut at the plane shown as a dashed line in d). (f) Atomic model of the doublet. Residues shown in red in one ParM filament interact with residues in orange in the other filament (see ED Table 2). (g) An orthogonal view of the doublet, with the filament axes going into the plane of the paper. One of the residues (S19) that forms the doublet interface has been highlighted (see ED Fig. 6).
Mentions: Having described the structure of the ParM filaments, we then wished to put the structural data in context of the bipolar spindles that segregate plasmid DNA in cells. For bipolar spindles to form, filamentous ParM subunits must engage in another interaction, inter-filament contacts, formed between double-helical filaments. It was known that incubation of ParM filaments with a crowding agent causes them to bundle 19. However, bundles are not amenable to high-resolution cryo-EM analysis because of their heterogeneity 20. To obtain a more defined sample, we titrated ParM+AMPPNP with varying amounts of crowding agent. When 2 % poly ethylene glycol (PEG) 6000 was added to ParM+AMPPNP, we found that ParM filaments dimerised to form ‘doublets’, containing two double-helical filaments (Fig. 3a, ED Fig. 5a-b). In raw cryo-EM images, doublets appeared as two roughly parallel lines, with no evidence of supercoiling or twisting. Electron cryotomography (cryo-ET) of the doublet specimen confirmed that the filaments do not twist around each other (Fig. 3b, Video 3).

Bottom Line: Growing ParMRC spindles push sister plasmids to the cell poles.The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions.Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

ABSTRACT
Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

Show MeSH
Related in: MedlinePlus