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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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Related in: MedlinePlus

ParM bundles and doublets observed in vivo(a-k) E. coli B/R266 cells were transformed with a high-copy (pDD19), or medium-copy (pKG321) plasmid containing the ParMRC locus. Transformed cells were grown to log phase and then prepared for cryo-EM. This figure shows a gallery of ParM bundles (blue arrows) and doublets (yellow arrows) observed in these cells. Panels a, c, e and h show cells transformed with the high-copy number plasmid while panels b, d, f, g, i, j and k show cells transformed with the medium copy number plasmid. Each experiment with different copy number plasmids was performed only once due to low-throughput nature of cryo-ET.
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Figure 11: ParM bundles and doublets observed in vivo(a-k) E. coli B/R266 cells were transformed with a high-copy (pDD19), or medium-copy (pKG321) plasmid containing the ParMRC locus. Transformed cells were grown to log phase and then prepared for cryo-EM. This figure shows a gallery of ParM bundles (blue arrows) and doublets (yellow arrows) observed in these cells. Panels a, c, e and h show cells transformed with the high-copy number plasmid while panels b, d, f, g, i, j and k show cells transformed with the medium copy number plasmid. Each experiment with different copy number plasmids was performed only once due to low-throughput nature of cryo-ET.

Mentions: We then used plasmids with different copy numbers 21, all of which contained the entire ParMRC locus and transformed them in turn into E. coli cells. Cryo-ET of these cells revealed the presence of doublets in all cases (Fig. 4b-d, Videos 5-6, ED Fig. 7, ED Table 3). All doublets were roughly aligned with the long cell axis, and were never observed perpendicular to the cell axis. Although bundles were observed in the high and medium copy number plasmid cases, they were not observed in the low-copy number (mini-R1) case, where partitioning via ParMRC is required for plasmid stability 22. These cryo-ET data are in line with previous immuno-light microscopy data, where single pole-to-pole filaments were only observed in 40 % of cells 1,10 and the other cells showed several localised clusters or more complex patterns.


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

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

ParM bundles and doublets observed in vivo(a-k) E. coli B/R266 cells were transformed with a high-copy (pDD19), or medium-copy (pKG321) plasmid containing the ParMRC locus. Transformed cells were grown to log phase and then prepared for cryo-EM. This figure shows a gallery of ParM bundles (blue arrows) and doublets (yellow arrows) observed in these cells. Panels a, c, e and h show cells transformed with the high-copy number plasmid while panels b, d, f, g, i, j and k show cells transformed with the medium copy number plasmid. Each experiment with different copy number plasmids was performed only once due to low-throughput nature of cryo-ET.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4493928&req=5

Figure 11: ParM bundles and doublets observed in vivo(a-k) E. coli B/R266 cells were transformed with a high-copy (pDD19), or medium-copy (pKG321) plasmid containing the ParMRC locus. Transformed cells were grown to log phase and then prepared for cryo-EM. This figure shows a gallery of ParM bundles (blue arrows) and doublets (yellow arrows) observed in these cells. Panels a, c, e and h show cells transformed with the high-copy number plasmid while panels b, d, f, g, i, j and k show cells transformed with the medium copy number plasmid. Each experiment with different copy number plasmids was performed only once due to low-throughput nature of cryo-ET.
Mentions: We then used plasmids with different copy numbers 21, all of which contained the entire ParMRC locus and transformed them in turn into E. coli cells. Cryo-ET of these cells revealed the presence of doublets in all cases (Fig. 4b-d, Videos 5-6, ED Fig. 7, ED Table 3). All doublets were roughly aligned with the long cell axis, and were never observed perpendicular to the cell axis. Although bundles were observed in the high and medium copy number plasmid cases, they were not observed in the low-copy number (mini-R1) case, where partitioning via ParMRC is required for plasmid stability 22. These cryo-ET data are in line with previous immuno-light microscopy data, where single pole-to-pole filaments were only observed in 40 % of cells 1,10 and the other cells showed several localised clusters or more complex patterns.

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