Opposite Cell Poles Research Articles

Immunodetection of tubulin and centromeric histone H3 (CENH3) proteins in Glycine species.

The centromeres appear as primary constrictions on monocentric metaphase chromosomes; where sister chromatids are held together and assemble the proteinaceous kitechore complex at which microtubule proteins attach during nuclear divisions for pulling sister chromatids to opposite cell poles. The movement of chromosomes is usually governed by structural proteins that are either species-specific or highly conserved, such as the centromere-specific histone H3 (CENH3) and tubulin proteins, respectively. We aimed to detect these proteins across eight different Glycine species by an immunofluorescence assay using specific antibodies. Furthermore, with the α-tubulin antibody we traced the dynamics of microtubules during the mitotic cell cycle in Glycine max. With two-color immunofluorescence staining, we showed that both proteins interact during nuclear division. Finally, we proved that in different diploid and tetraploid Glycine species CENH3 can be detected in functional centromeres with spatial proximity of microtubule proteins.

To let go or not to let go: how ParA can impact the release of the chromosomal anchoring in Caulobacter crescentus.

Chromosomal maintenance is vital for the survival of bacteria. In Caulobacter crescentus, chromosome replication initiates at ori and segregation is delayed until the nearby centromere-like region parS is replicated. Our understanding of how this sequence of events is regulated remains limited. The segregation of parS has been shown to involve multiple steps including polar release from anchoring protein PopZ, slow movementand fast ParA-dependent movement to theopposite cell pole. In this study, we demonstrate that ParA's competing attractions from PopZ and from DNA are critical for segregation of parS. Interfering with this balance of attractions-by expressing a variant ParA-R195E unable to bind DNA and thus favoring interactions exclusively between ParA-PopZ-results in cell death. Our data revealed that ParA-R195E's sole interactions with PopZ obstruct PopZ's ability to release the polar anchoring of parS, resulting in cells with multiple parS loci fixed at one cell pole. We show that the inability to separate and segregate multiple parS loci from the pole is specifically dependent on the interaction between ParA and PopZ. Collectively, our results reveal that the initial steps in chromosome segregation are highly regulated.

Unorthodox regulation of the MglA Ras-like GTPase controlling polarity in Myxococcus xanthus.

Motile cells have developed a large array of molecular machineries to actively change their direction of movement in response to spatial cues from their environment. In this process, small GTPases act as molecular switches and work in tandem with regulators and sensors of their guanine nucleotide status (GAP, GEF, GDI and effectors) to dynamically polarize the cell and regulate its motility. In this review, we focus on Myxococcus xanthus as a model organism to elucidate the function of an atypical small Ras GTPase system in the control of directed cell motility. M.xanthus cells direct their motility by reversing their direction of movement through a mechanism involving the redirection of the motility apparatus to the opposite cell pole. The reversal frequency of moving M.xanthus cells is controlled by modular and interconnected protein networks linking the chemosensory-like frizzy (Frz) pathway - that transmits environmental signals - to the downstream Ras-like Mgl polarity control system - that comprises the Ras-like MglA GTPase protein and its regulators. Here, we discuss how variations in the GTPase interactome landscape underlie single-cell decisions and consequently, multicellular patterns.

Genus-Specific Interactions of Bacterial Chromosome Segregation Machinery Are Critical for Their Function.

Most bacteria use the ParABS system to segregate their newly replicated chromosomes. The two protein components of this system from various bacterial species share their biochemical properties: ParB is a CTPase that binds specific centromere-like parS sequences to assemble a nucleoprotein complex, while the ParA ATPase forms a dimer that binds DNA non-specifically and interacts with ParB complexes. The ParA-ParB interaction incites the movement of ParB complexes toward the opposite cell poles. However, apart from their function in chromosome segregation, both ParAB may engage in genus-specific interactions with other protein partners. One such example is the polar-growth controlling protein DivIVA in Actinomycetota, which binds ParA in Mycobacteria while interacts with ParB in Corynebacteria. Here, we used heterologous hosts to investigate whether the interactions between DivIVA and ParA or ParB are maintained across phylogenic classes. Specifically, we examined interactions of proteins from four bacterial species, two belonging to the Gram positive Actinomycetota phylum and two belonging to the Gram-negative Pseudomonadota. We show that while the interactions between ParA and ParB are preserved for closely related orthologs, the interactions with polarly localised protein partners are not conferred by orthologous ParABs. Moreover, we demonstrate that heterologous ParA cannot substitute for endogenous ParA, despite their high sequence similarity. Therefore, we conclude that ParA orthologs are fine-tuned to interact with their partners, especially their interactions with polarly localised proteins are adjusted to particular bacterial species demands.

A dicentric bacterial chromosome requires XerC/D site-specific recombinases for resolution

Unlike eukaryotes and archaea, which have multiple replication origins on their chromosomes, bacterial chromosomes usually contain a single replication origin.1 Here, we discovered a dicentric bacterial chromosome with two replication origins, which has resulted from the fusion of the circular and linear chromosomes in Agrobacterium tumefaciens. The fused chromosome is well tolerated, stably maintained, and retains similar subcellular organization and genome-wide DNA interactions found for the bipartite chromosomes. Strikingly, the two replication origins and their partitioning systems are both functional and necessary for cell survival. Finally, we discovered that the site-specific recombinases XerC and XerD2 are essential in cells harboring the fused chromosome but not in cells with bipartite chromosomes. Analysis of actively dividing cells suggests a model in which XerC/D are required to recombine the sister fusion chromosomes when the two centromeres on the same chromosome are segregated to opposite cell poles. Thus, faithful segregation of dicentric chromosomes in bacteria can occur because of site-specific recombination between the sister chromatids during chromosome partitioning. Our study provides a natural comparative platform to examine a bacterial chromosome with multiple origins and a possible explanation for the fundamental difference in bacterial genome architecture relative to eukaryotes and archaea.1.

Migration of Polyphosphate Granules in Agrobacterium tumefaciens

Agrobacterium tumefaciens has two polyphosphate (polyP) kinases, one of which (PPK1_AT) is responsible for the formation of polyP granules, while the other (PPK2_AT) is used for replenishing the NTP pools by using polyP as a phosphate donor to phosphorylate nucleoside diphosphates. Fusions of eYFP with PPK2_AT or of the polyP granule-associated phosin PptA from Ralstonia eutropha always co-localized with polyP granules in A. tumefaciens and allowed the tracking of polyP granules in time-lapse microscopy experiments without the necessity to label the cells with the toxic dye DAPI. Fusions of PPK1_AT with mCherry formed fluorescent signals often attached to, but not completely co-localizing with, polyP granules in wild-type cells. Time-lapse microscopy revealed that polyP granules in about one-third of a cell population migrated from the old pole to the new cell pole shortly before or during cell division. Many cells de novo formed a second (nonmigrating) polyP granule at the opposite cell pole before cell division was completed, resulting in two daughter cells each having a polyP granule at the old pole after septum formation. Migration of polyP granules was disordered in mitomycin C-treated or in PopZ-depleted cells, suggesting that polyP granules can associate with DNA or with other molecules that are segregated during the cell cycle.

Recombination and biased segregation of mitochondrial genomes during crossing and meiosis of different Schizosaccharomyces pombe strains.

During meiosis, tethering of parental mitochondria to opposite cell poles inhibits the mixing of mitochondria with different genomes and ensures uniparental inheritance in thestandard laboratory strain of fission yeast. We here investigate mitochondrial inheritance in crosses between natural isolates using tetrad dissection and next-generation sequencing. We find that colonies grown from single spores can sometimes carry a mix of mitochondrial genotypes, that mitochondrial genomes can recombine during meiosis, that in some cases tetrads do not follow the 2:2 segregation pattern, and that certain crosses may feature a weak bias towards one of the parents. Together, these findings paint a more nuanced picture of mitochondrial inheritance in the wild.

Physical Modeling of a Sliding Clamp Mechanism for the Spreading of ParB at Short Genomic Distance from Bacterial Centromere Sites.

SummaryBacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation are cytosine triphosphate (CTP)-dependent molecular switches. CTP-binding converts ParB dimers to DNA clamps, allowing unidimensional diffusion along the DNA. This sliding property has been proposed to explain the ParB spreading over large distances from parS centromere sites where ParB is specifically loaded. We modeled such a “clamping and sliding” mechanism as a typical reaction-diffusion system, compared it to the F plasmid ParB DNA binding pattern, and found that it can account neither for the long range of ParB binding to DNA nor for the rapid assembly kinetics observed in vivo after parS duplication. Also, it predicts a strong effect on the F plasmid ParB binding pattern from the presence of a roadblock that is not observed in ChIP-sequencing (ChIP-seq). We conclude that although “clamping and sliding” can occur at short distances from parS, another mechanism must apply for ParB recruitment at larger genomic distances.

The effect of hyperhomocysteinemia on the patterns of electron microscopic changes in the liver of adult rats

One of the important tasks of modern science is to find biochemical markers that would be able to reflect the risks of development and the nature of the course of various diseases, as well as to predict their possible consequences. In recent years, a significant number of compounds that can affect the biochemical profile of the organism have been identified. Homocysteine – a product of methionine metabolism, belongs to one of these markers, and the effects of its influece on the structure and function of various organs are being actively studied by modern researchers. The aim of the study is to find the patterns of electron microscopic changes in the liver structure of adult rats with hyperhomocysteinemia. The experimental study was performed on 22 white nonlinear mature male rats, which were divided into a control group and an experimental group. A model of persistent hyperhomocysteinemia was created by administering to rats of experimental group thiolactone homocysteine at a dose of 200 mg/kg body weight intragastrically for 60 days. The study of ultrastructural changes in the liver of rats was performed using an electron microscope PEM-125K. In adult rats with experimental hyperhomocysteinemia at the ultrastructural level, dystrophic and destructive changes in hepatocytes, endotheliocytes in the walls of sinusoids and Kupffer cells were found. These changes were more pronounced than in young rats with experimental hyperhomocysteinemia. Revealed structural changes in decompensation (depletion) of mitochondria – fewer number of cristae and enlightened matrix. In contrast to young rats, adult rats with hyperhomocysteinemia in the perisinusoidal spaces showed elongated Ito cells, a significant proportion of the cytoplasm is occupied by the Golgi complex and granular endoplasmic reticulum tanks, indicating protein synthesis for export. In Ito cells, the content of fat droplets, which are located on opposite poles of cells, is reduced. This morphological picture manifests the transformation of Ito cells into fibroblasts.

Bacterial Division: Journey to the Center of the Cell.

Most bacteria divide by corralling the tubulin-like FtsZ protein to mid-cell, where it assembles into a ring of treadmilling membrane-tethered oligomers. A study in this issue reveals new details about how FtsZ finds its way to the ring.

Chromosome Dynamics in Bacteria: Triggering Replication at the Opposite Location and Segregation in the Opposite Direction.

Maintaining the integrity of the genome is essential to cell survival. In the bacterium Caulobacter crescentus, the single circular chromosome exhibits a specific orientation in the cell, with the replication origin (ori) residing at the pole of the cell bearing a stalk. Upon initiation of replication, the duplicated centromere-like region parS and ori move rapidly to the opposite pole where parS is captured by a microdomain hosting a unique set of proteins that contribute to the identity of progeny cells. Many questions remain as to how this organization is maintained. In this study, we constructed strains of Caulobacter in which ori and the parS centromere can be induced to move to the opposite cell pole in the absence of chromosome replication, allowing us to ask whether once these chromosomal foci were positioned at the wrong pole, replication initiation and chromosome segregation can proceed in the opposite orientation. Our data reveal that DnaA can initiate replication and ParA can orchestrate segregation from either cell pole. The cell reconstructs the organization of its ParA gradient in the opposite orientation to segregate one replicated centromere from the new pole toward the stalked pole (i.e., opposite direction), while displaying no detectable viability defects. Thus, the unique polar microdomains exhibit remarkable flexibility in serving as a platform for directional chromosome segregation along the long axis of the cell.IMPORTANCE Bacteria can accomplish surprising levels of organization in the absence of membrane organelles by constructing subcellular asymmetric protein gradients. These gradients are composed of regulators that can either trigger or inhibit cell cycle events from distinct cell poles. In Caulobacter crescentus, the onset of chromosome replication and segregation from the stalked pole are regulated by asymmetric protein gradients. We show that the activators of chromosome replication and segregation are not restricted to the stalked pole and that their organization and directionality can be flipped in orientation. Our results also indicate that the subcellular location of key chromosomal loci play important roles in the establishment of the asymmetric organization of cell cycle regulators.

DNA-Membrane Anchor Facilitates Efficient Chromosome Translocation at a Distance in Bacillus subtilis.

Chromosome segregation in sporulating Bacillus subtilis involves the tethering of sister chromosomes at opposite cell poles. RacA is known to mediate chromosome tethering by interacting with both centromere-like elements in the DNA and with DivIVA, a membrane protein which localizes to the cell poles. RacA has a secondary function in which it assists in nucleoid condensation. Here we demonstrate that, in addition to positioning and condensing the chromosome, RacA contributes to efficient transport of DNA by the chromosome segregation motor SpoIIIE. When RacA is deleted, one-quarter of cells fail to capture DNA in the nascent spore, yet 70% of cells fail to form viable spores without RacA. This discrepancy indicates that RacA possesses a role in sporulation beyond DNA capture and condensation. We observed that the mutant cells had reduced chromosome translocation into the forespore across the entire length of the chromosome, requiring nearly twice as much time to move a given DNA locus. Additionally, functional abolition of the RacA-DivIVA interaction reduced translocation to a similar degree as in a racA deletion strain, demonstrating the importance of the RacA-mediated tether in translocation and chromosome packaging during sporulation. We propose that the DNA-membrane anchor facilitates efficient translocation by SpoIIIE, not through direct protein-protein contacts but by virtue of physical effects on the chromosome that arise from anchoring DNA at a distance.IMPORTANCE To properly segregate their chromosomes, organisms tightly regulate the organization and dynamics of their DNA. Aspects of the process by which DNA is translocated during sporulation are not yet fully understood, such as what factors indirectly influence the activity of the motor protein SpoIIIE. In this work, we have shown that a DNA-membrane tether mediated by RacA contributes to the activity of SpoIIIE. Loss of RacA nearly doubles the time of translocation, despite the physically distinct locations these proteins and their activities occupy within the cell. This is a rare example of an explicit effect that DNA-membrane connections can have on cell physiology and demonstrates that distant changes to the state of the chromosome can influence motor proteins which act upon it.

Characterization of Siglec-8 Expression on Lavage Cells after Segmental Lung Allergen Challenge

Background: Siglec-8 is present at a high level on human blood eosinophils and low level on blood basophils. Engagement of Siglec-8 on blood eosinophils causes its internalization and results in death. Siglec-8 is a potential therapeutic target in eosinophilic asthma. Objectives: The aim of this study was to determine Siglec-8 levels on eosinophils and basophils recruited during lung inflammation. Method: We analyzed surface Siglec-8 by flow cytometry on cells obtained by bronchoalveolar lavage (BAL) 48 h after segmental lung allergen challenge of human subjects with mild allergic asthma and used confocal microscopy to compare Siglec-8 distribution on BAL and blood eosinophils. Results: Like their blood counterparts, BAL eosinophils had high unimodal surface Siglec-8, while BAL basophils had lower but detectable surface Siglec-8. BAL macrophages, monocytes, neutrophils, and plasmacytoid dendritic cells did not express surface Siglec-8. Microscopy of freshly isolated blood eosinophils demonstrated homogeneous Siglec-8 distribution over the cell surface. Upon incubation with IL-5, Siglec-8 on the surface of eosinophils became localized in patches both at the nucleopod tip and at the opposite cell pole. BAL eosinophils also had a patchy Siglec-8 distribution. Conclusions: We conclude that 48 h after segmental allergen challenge, overall levels of Siglec-8 expression on airway eosinophils resemble those on blood eosinophils, but with a patchier distribution, a pattern consistent with activation. Thus, therapeutic targeting of Siglec-8 has the potential to impact blood as well as lung eosinophils, which may be associated with an improved outcome in eosinophilic lung diseases.

New insights into the function of a versatile class of membrane molecular motors from studies of Myxococcus xanthus surface (gliding) motility.

Cell motility is a central function of living cells, as it empowers colonization of new environmental niches, cooperation, and development of multicellular organisms. This process is achieved by complex yet precise energy-consuming machineries in both eukaryotes and bacteria. Bacteria move on surfaces using extracellular appendages such as flagella and pili but also by a less-understood process called gliding motility. During this process, rod-shaped bacteria move smoothly along their long axis without any visible morphological changes besides occasional bending. For this reason, the molecular mechanism of gliding motility and its origin have long remained a complete mystery. An important breakthrough in the understanding of gliding motility came from single cell and genetic studies in the delta-proteobacterium Myxococcus xanthus. These early studies revealed, for the first time, the existence of bacterial Focal Adhesion complexes (FA). FAs are formed at the bacterial pole and rapidly move towards the opposite cell pole. Their attachment to the underlying surface is linked to cell propulsion, in a process similar to the rearward translocation of actomyosin complexes in Apicomplexans. The protein machinery that forms at FAs was shown to contain up to seventeen proteins predicted to localize in all layers of the bacterial cell envelope, the cytosolic face, the inner membrane (IM), the periplasmic space and the outer membrane (OM). Among these proteins, a proton-gated channel at the inner membrane was identified as the molecular motor. Thus, thrust generation requires the transduction of traction forces generated at the inner membrane through the cell envelope beyond the rigid barrier of the bacterial peptidoglycan.

Chromosome search and rescue

![Figure][1] A time-lapse series shows how two homologous loci (red) pair up for homologous recombination and then resegregate to opposite cell poles. [Badrinarayanan et al.][2] describe how regions of bacterial chromosomes come together to repair DNA breaks and then move back to their

PilB localization correlates with the direction of twitching motility in the cyanobacterium Synechocystis sp. PCC 6803.

Twitching motility depends on the adhesion of type IV pili (T4P) to a substrate, with cell movement driven by extension and retraction of the pili. The mechanism of twitching motility, and the events that lead to a reversal of direction, are best understood in rod-shaped bacteria such as Myxococcus xanthus. In M. xanthus, the direction of movement depends on the unipolar localization of the pilus extension and retraction motors PilB and PilT to opposite cell poles. Reversal of direction results from relocalization of PilB and PilT. Some cyanobacteria utilize twitching motility for phototaxis. Here, we examine twitching motility in the cyanobacterium Synechocystis sp. PCC 6803, which has a spherical cell shape without obvious polarity. We use a motile Synechocystis sp. PCC 6803 strain expressing a functional GFP-tagged PilB1 protein to show that PilB1 tends to localize in 'crescents' adjacent to a specific region of the cytoplasmic membrane. Crescents are more prevalent under the low-light conditions that favour phototactic motility, and the direction of motility strongly correlates with the orientation of the crescent. We conclude that the direction of twitching motility in Synechocystis sp. PCC 6803 is controlled by the localization of the T4P apparatus, as it is in M. xanthus. The PilB1 crescents in the spherical cells of Synechocystis can be regarded as being equivalent to the leading pole in the rod-shaped cells.

Choreography of the Mycobacterium replication machinery during the cell cycle.

ABSTRACTIt has recently been demonstrated that bacterial chromosomes are highly organized, with specific positioning of the replication initiation region. Moreover, the positioning of the replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis, a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of chromosome replication in relation to the cell cycle, as well as cell division and chromosome segregation events, revealed that chromosomal origin-of-replication (oriC) regions segregate soon after the start of replication. Moreover, our data demonstrate that organization of the chromosome by ParB determines the replisome choreography.

A model for spatio-temporal dynamics in a regulatory network for cell polarity

Cell polarity in Myxococcus xanthus is crucial for the directed motility of individual cells. The polarity system is characterised by a dynamic spatio-temporal localisation of the regulatory proteins MglA and MglB at opposite cell poles. In response to signalling by the Frz chemosensory system, MglA and MglB are released from the poles and then rebind at the opposite poles. Thus, over time MglA and MglB oscillate irregularly between the poles in synchrony but out of phase. A minimal macroscopic model of the Mgl/Frz regulatory system based on a reaction–diffusion PDE system is presented. Mathematical analysis of the steady states derives conditions on the reaction terms for formation of dynamic localisation patterns of the regulatory proteins under different biologically-relevant regimes, i.e. with and without Frz signalling. Numerical simulations of the model system produce either a stationary pattern in time (fixed polarity), periodic solutions in time (oscillating polarity), or excitable behaviour (irregular switching of polarity).

Mechanical control of cell biology. Effects of cyclic mechanical stretch on cardiomyocyte cellular organization

ObjectivesThe aim of our study was to elucidate how cyclic mechanical stretch is sensed by cardiomyocytes and in which way it affects cytoskeletal organization. MethodsNeonatal rat cardiomyocytes, cultured on flexible membranes, were subjected to cyclic mechanical stretch (1 Hz, 10% elongation) for 24 h using either round or rectangular loading posts for equibi-axial or uni-axial stretch, respectively, using the FlexCell stretch system. Cells were treated either with vehicle, the focal adhesion kinase (FAK) inhibitor PF-573,228 (200 nM), or the stretch-activated ion channel blocker gadolinium (Gd3+; 100 μM). ResultsCyclic mechanical stretch (36 mm diameter silicone membrane, equibi-axial stretch, 10% elongation, 1 Hz) induced elongation of the cardiomyocytes together with accentuation of Cx43 at the cell poles, and with an orientation of the cell axis between the radial axis and the circumferential axis (mean deviation: 11° from the circumference). Moreover, stretch resulted in ca. 1.4 fold increased Cx43 expression. FAK was found to be phosphorylated at the edges of the cells. In order to find out, how cardiomyocytes might sense stretch, we investigated possible effects of Gd3+and PF-573,228. Gd3+ had no effect on elongation or polarization and did not affect stretch-induced Cx43 expression. Interestingly, the FAK inhibitor completely antagonized the stretch-induced elongation, orientation and Cx43-polarization. However, the stretch-induced Cx43 expression was insensitive to this treatment. In order to clarify our result that the cells in equibi-axial stretch did not exactly organize to the circumference or to the radial axis, we decided to use a uni-axial stretch protocol. In uni-axially stretched cells, we found that the cardiomyocytes also showed elongation, Cx43 polarization, and orientation near to the stretch axis, but not exactly in the stretch axis but ca. 25° oblique to it. Furthermore, we investigated the tubular system, the Golgi apparatus, the SR and the nucleus. After 24 h stretch the microtubules were localized nearly (but not completely) parallel to the stretch axis (i.e. in longitudinal cell axis). Moreover, the localization of nucleus and the Golgi was also changed: while under static conditions, the Golgi was distributed more or less around the nucleus, after stretch the Golgi was accentuated at one site of the nucleus facing a cell pole with the nucleus facing the opposite cell pole. The plus motor protein kinesin accentuated at the cell poles and at the cell periphery, while the minus motor protein dynein was found near to the Golgi apparatus. ConclusionsThe stretch signal sensing is mediated via FAK and leads to intracellular re-organization and orientation. The oblique orientation of the cell with regard to the direction of stretch may define a directed force vector which could allow the cell to orientate.

DNA Segregation and Partitioning in Caulobacter Crescentus: Super-Resolving Protein Colocalization at the Cell Pole

Biological function relies on biochemical reactions that require spatial proximity between biomolecules. We are investigating how DNA segregation and partitioning is driven by the spatial recruitment of proteins to the cell pole in the bacterial model system Caulobacter crescentus. The polar organizing protein PopZ assembles as a polymeric network at one and then the opposite cell pole where it respectively anchors the original and the newly replicated DNA. Previous super-resolution imaging experiments in our lab have determined the cytosolic locations of the partitioning system proteins, ParAB, during directional DNA segregation (Ptacin et al., 2010), but the role of the PopZ network in the biochemical reaction cycle of these proteins has not yet been explored. To determine precise positioning of the partitioning system proteins in relation to the PopZ polymeric network, we utilized quantitative two-color 3D super-resolution microscopy in live Caulobacter cells. We measured the shapes and volumes of the PopZ networks with tens of nanometer resolution and, by counting the localized PopZ proteins within each network, we determined that PopZ localizes with a globally conserved volume density. These results indicate that PopZ may serve as a homogeneous scaffold that spatially compartmentalizes the biochemical interactions of the partitioning system proteins. This model is supported by two-color super-resolution images that clearly reveal where and how the Par system proteins overlap with the PopZ networks. These data thus provide key mechanistic insights into the spatial regulation of ParAB protein activity during DNA segregation and partitioning.