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

Abstract

Active segregation of E. coli low-copy number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments 1-6. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments 3,7-9. Growing ParMRC spindles push sister plasmids to the cell poles 9,10. Here, using modern electron cryomicroscopy methods we have investigated 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 determined structures of ParM filaments in different nucleotide states. The structure of filaments bound to AMPPNP was 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 reconstructed 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.