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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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Model of the ParM doublet(a) A cryo-EM image of ParM+AMPPNP + 2% PEG 6000. Instances of doublets are marked with yellow arrowheads. This experiment was repeated 15 times. (b) More examples of ParM doublets observed in cryo-EM. (c) Class averages of the doublets. (d) Directionality assignment of the filaments in the doublet. Individual sub-segments and their assigned directionality are indicated by triangles, coloured based on the cross-correlation score in the alignment procedure: red indicates a poor cross correlation score while green indicates a good score. (e) A schematic model of the anti-parallel ParM doublet. Directionality is indicated with a yellow arrow. (f) The thickest parts of ParM filaments of the doublet (as they appear in projection) are marked with black arrowheads.
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Figure 9: Model of the ParM doublet(a) A cryo-EM image of ParM+AMPPNP + 2% PEG 6000. Instances of doublets are marked with yellow arrowheads. This experiment was repeated 15 times. (b) More examples of ParM doublets observed in cryo-EM. (c) Class averages of the doublets. (d) Directionality assignment of the filaments in the doublet. Individual sub-segments and their assigned directionality are indicated by triangles, coloured based on the cross-correlation score in the alignment procedure: red indicates a poor cross correlation score while green indicates a good score. (e) A schematic model of the anti-parallel ParM doublet. Directionality is indicated with a yellow arrow. (f) The thickest parts of ParM filaments of the doublet (as they appear in projection) are marked with black arrowheads.

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)

Model of the ParM doublet(a) A cryo-EM image of ParM+AMPPNP + 2% PEG 6000. Instances of doublets are marked with yellow arrowheads. This experiment was repeated 15 times. (b) More examples of ParM doublets observed in cryo-EM. (c) Class averages of the doublets. (d) Directionality assignment of the filaments in the doublet. Individual sub-segments and their assigned directionality are indicated by triangles, coloured based on the cross-correlation score in the alignment procedure: red indicates a poor cross correlation score while green indicates a good score. (e) A schematic model of the anti-parallel ParM doublet. Directionality is indicated with a yellow arrow. (f) The thickest parts of ParM filaments of the doublet (as they appear in projection) are marked with black arrowheads.
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Related In: Results  -  Collection

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Figure 9: Model of the ParM doublet(a) A cryo-EM image of ParM+AMPPNP + 2% PEG 6000. Instances of doublets are marked with yellow arrowheads. This experiment was repeated 15 times. (b) More examples of ParM doublets observed in cryo-EM. (c) Class averages of the doublets. (d) Directionality assignment of the filaments in the doublet. Individual sub-segments and their assigned directionality are indicated by triangles, coloured based on the cross-correlation score in the alignment procedure: red indicates a poor cross correlation score while green indicates a good score. (e) A schematic model of the anti-parallel ParM doublet. Directionality is indicated with a yellow arrow. (f) The thickest parts of ParM filaments of the doublet (as they appear in projection) are marked with black arrowheads.
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