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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 in E. coli cells, imaged by cryo-ET(a) A mutant of ParM that hydrolyses ATP more slowly (D170A) was over-expressed in E. coli cells. Tomographic slices show large bundles of ParM blocking cell division. This experiment was performed two times. (b) The ParMRC operon driven from high-copy number plasmid pDD19. Tomographic slice showing an example of observed doublets. (c) Tomographic slice for a medium-copy number plasmid (pKG321). (d) Tomographic slice for a low-copy number plasmid, emulating the native low-copy number R1 plasmids (pKG491, ‘mini-R1’ replicon) in E. coli (see Videos 5-6 to view entire tomograms). Each experiment with different copy number plasmids was performed once. (e) Schematic depicting proposed asynchronous plasmid DNA segregation. Bipolar ParM spindles are seeded when replication has produced two parC centromeric regions, still in close proximity. Each seeds one unipolar ParM filament that then come together in an antiparallel fashion to form the segregating bipolar spindle. Non-productive unipolar filaments or spindles that lack plasmid attachment will be destroyed through ParM’s dynamic instability. This is in contrast to earlier ideas in which all sister plasmids would be segregated through one bundle of filaments, containing double the number of unipolar filaments as the copy number of the plasmid in the cell 19.
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Figure 4: ParM doublets in E. coli cells, imaged by cryo-ET(a) A mutant of ParM that hydrolyses ATP more slowly (D170A) was over-expressed in E. coli cells. Tomographic slices show large bundles of ParM blocking cell division. This experiment was performed two times. (b) The ParMRC operon driven from high-copy number plasmid pDD19. Tomographic slice showing an example of observed doublets. (c) Tomographic slice for a medium-copy number plasmid (pKG321). (d) Tomographic slice for a low-copy number plasmid, emulating the native low-copy number R1 plasmids (pKG491, ‘mini-R1’ replicon) in E. coli (see Videos 5-6 to view entire tomograms). Each experiment with different copy number plasmids was performed once. (e) Schematic depicting proposed asynchronous plasmid DNA segregation. Bipolar ParM spindles are seeded when replication has produced two parC centromeric regions, still in close proximity. Each seeds one unipolar ParM filament that then come together in an antiparallel fashion to form the segregating bipolar spindle. Non-productive unipolar filaments or spindles that lack plasmid attachment will be destroyed through ParM’s dynamic instability. This is in contrast to earlier ideas in which all sister plasmids would be segregated through one bundle of filaments, containing double the number of unipolar filaments as the copy number of the plasmid in the cell 19.

Mentions: As a first test, we over-expressed a mutant of ParM (D170A) that hydrolysed ATP much more slowly in thin E. coli cells. As observed previously in vitreous sections 19, cryo-ET of these cells (Fig. 4a) allowed unambiguous identification of the over-expressed ParM mutant protein through its tendency to form extremely large bundles.


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

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

ParM doublets in E. coli cells, imaged by cryo-ET(a) A mutant of ParM that hydrolyses ATP more slowly (D170A) was over-expressed in E. coli cells. Tomographic slices show large bundles of ParM blocking cell division. This experiment was performed two times. (b) The ParMRC operon driven from high-copy number plasmid pDD19. Tomographic slice showing an example of observed doublets. (c) Tomographic slice for a medium-copy number plasmid (pKG321). (d) Tomographic slice for a low-copy number plasmid, emulating the native low-copy number R1 plasmids (pKG491, ‘mini-R1’ replicon) in E. coli (see Videos 5-6 to view entire tomograms). Each experiment with different copy number plasmids was performed once. (e) Schematic depicting proposed asynchronous plasmid DNA segregation. Bipolar ParM spindles are seeded when replication has produced two parC centromeric regions, still in close proximity. Each seeds one unipolar ParM filament that then come together in an antiparallel fashion to form the segregating bipolar spindle. Non-productive unipolar filaments or spindles that lack plasmid attachment will be destroyed through ParM’s dynamic instability. This is in contrast to earlier ideas in which all sister plasmids would be segregated through one bundle of filaments, containing double the number of unipolar filaments as the copy number of the plasmid in the cell 19.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493928&req=5

Figure 4: ParM doublets in E. coli cells, imaged by cryo-ET(a) A mutant of ParM that hydrolyses ATP more slowly (D170A) was over-expressed in E. coli cells. Tomographic slices show large bundles of ParM blocking cell division. This experiment was performed two times. (b) The ParMRC operon driven from high-copy number plasmid pDD19. Tomographic slice showing an example of observed doublets. (c) Tomographic slice for a medium-copy number plasmid (pKG321). (d) Tomographic slice for a low-copy number plasmid, emulating the native low-copy number R1 plasmids (pKG491, ‘mini-R1’ replicon) in E. coli (see Videos 5-6 to view entire tomograms). Each experiment with different copy number plasmids was performed once. (e) Schematic depicting proposed asynchronous plasmid DNA segregation. Bipolar ParM spindles are seeded when replication has produced two parC centromeric regions, still in close proximity. Each seeds one unipolar ParM filament that then come together in an antiparallel fashion to form the segregating bipolar spindle. Non-productive unipolar filaments or spindles that lack plasmid attachment will be destroyed through ParM’s dynamic instability. This is in contrast to earlier ideas in which all sister plasmids would be segregated through one bundle of filaments, containing double the number of unipolar filaments as the copy number of the plasmid in the cell 19.
Mentions: As a first test, we over-expressed a mutant of ParM (D170A) that hydrolysed ATP much more slowly in thin E. coli cells. As observed previously in vitreous sections 19, cryo-ET of these cells (Fig. 4a) allowed unambiguous identification of the over-expressed ParM mutant protein through its tendency to form extremely large bundles.

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