Bacterial scaffold directs pole-specific centromere segregation

Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.The bacterial cytoplasm is a complex mixture of dynamic macromolecules densely packed into a tiny compartment. Recent studies have revealed unexpected levels of organization of bacterial cytoplasmic components, including hundreds of proteins, specific lipids, mRNA molecules, and even the nucleoid itself (1). One strategy used by bacteria to generate subcellular organization of specific macromolecular complexes is active segregation by ParA-mediated molecular partitioning machines. ParA-based partitioning systems are found throughout bacteria and have been shown to spatially organize diverse macromolecular complexes to facilitate their equal distribution to progeny during cell division (2). An important question is how directionality is provided to ParA partitioning machines.One family of highly conserved ParA-based partitioning systems segregates plasmid or chromosomal centromeres to daughter cells during cell division. ParA-mediated DNA partitioning systems (Par systems) are composed of three core components: a centromeric DNA sequence parS, a site-specific DNA binding protein ParB that binds to the centromere parS sequence, and the ATPase ParA. Structural studies demonstrate that the activity of ParA is regulated by a molecular switch in which ATP-bound ParA forms dimers that bind tightly to DNA, and ParB stimulates ATP hydrolysis and release of ADP-bound ParA as monomers (3). During centromere partitioning in vivo, ATP-bound ParA assembles into a multimeric nucleoid-bound structure (4). At the centromere, ParB binds to the parS locus and nearby DNA to create a compact nucleoprotein complex (5). This ParB/parS complex binds to ParA subunits within the ParA/nucleoid structure, stimulating ATP hydrolysis and release of ParA-ADP (6–8). The multivalent ParB/parS complex has thus been proposed to bind to and shorten the ParA superstructure on the nucleoid, moving along a receding track via a dynamic disassembly mechanism (6, 8–10). The result of this process is the movement of the chromosomal centromere (parS) relative to the nucleoid bulk, and therefore to the cell itself.Whereas the fundamental operating principles of ParA-mediated movement seem conserved, how these machines target transfer to specific subcellular destinations is unknown. Many chromosomal Par systems maintain a single origin-proximal ParB/parS complex at the old cell pole and, after replication, move one newly replicated parS locus to the opposite pole (9, 11, 12). Polar protein complexes that interact with chromosome segregation factors have been identified in various bacteria, but the mechanistic consequences of these interactions have not been established (13–15). In Caulobacter, two distinct polar protein factors affect ParA-mediated centromere segregation: the new pole-specific protein TipN (16, 17) and the polar organizing protein PopZ (18, 19). TipN is a large, membrane-anchored, coiled-coil rich protein that localizes to the new pole throughout the cell cycle and, in addition to roles in localization of flagellar synthesis (16, 17), affects processive parS segregation via an unknown mechanism (6, 20).In contrast, PopZ is a small, acidic protein that forms a polymeric network at the cell pole (18, 19). In the prereplicative cell, PopZ localizes exclusively at the old cell pole, where it anchors the ParB-bound parS locus via direct interactions with ParB (18, 19). During chromosome replication initiation, PopZ releases ParB from the old pole and adopts a bipolar PopZ distribution that seems to capture ParB/parS complexes during the segregation process (18, 19). Whereas cells lacking tipN are only mildly elongated, popZ deletion causes severe filamentation (16–19), suggesting that PopZ plays a more important role in the regulation of segregation. However, the molecular mechanism by which PopZ affects segregation has remained elusive.Here we demonstrate that the multifunctional PopZ complex plays a crucial role in pole-directed movement of ParA-mediated chromosome segregation by interacting directly with ParA. We show that PopZ, but not TipN, is required for robust polar recruitment of ParA and demonstrate that a polar PopZ scaffold recruits and concentrates free ParA released during segregation. Recruitment of ParA within the PopZ matrix sequesters free ParA and locally regenerates ParA DNA binding activity. Active ParA complexes are released for recycling into nucleoid-bound structures near the cell pole, which we propose drives centromere segregation toward pole-localized PopZ. Thus, PopZ orchestrates a positive feedback mechanism that forces ParA-mediated centromere transfer to the cell pole. The polar PopZ scaffold complex creates a unique 3D microenvironment at the pole that spatially separates distinct centromere tethering and ParA-modulation activities, enabling coupling between chromosome segregation with the initiation of cell division.