Chromosomes of a broad range of species, from bacteria to mammals, are structured by large topological domains whose precise functional roles and regulatory mechanisms remain elusive. Here, we combine super-resolution microscopies and chromosome-capture technologies to unravel the higher-order organization of the Bacillus subtilis chromosome and its dynamic rearrangements during the cell cycle. We decipher the fine 3D architecture of the origin domain, revealing folding motifs regulated by condensin-like complexes. This organization, along with global folding throughout the genome, is present before replication, disrupted by active DNA replication, and re-established thereafter. Single-cell analysis revealed a strict correspondence between sub-cellular localization of origin domains and their condensation state. Our results suggest that the precise 3D folding pattern of the origin domain plays a role in the regulation of replication initiation, chromosome organization, and DNA segregation.

Drosophila chromosomes are organized in a series of nanocompartments that correspond to topologically associating domains.

Many canonical processes in molecular biology rely on the dynamic assembly of higher-order nucleoprotein complexes. In bacteria, the assembly mechanism of ParABS, the nucleoprotein super-complex that actively segregates the bacterial chromosome and many plasmids, remains elusive. We combined super-resolution microscopy, quantitative genome-wide surveys, biochemistry, and mathematical modeling to investigate the assembly of ParB at the centromere-like sequences parS. We found that nearly all ParB molecules are actively confined around parS by a network of synergistic protein-protein and protein-DNA interactions. Interrogation of the empirically determined, high-resolution ParB genomic distribution with modeling suggests that instead of binding only to specific sequences and subsequently spreading, ParB binds stochastically around parS over long distances. We propose a new model for the formation of the ParABS partition complex based on nucleation and caging: ParB forms a dynamic lattice with the DNA around parS. This assembly model and approach to characterizing large-scale, dynamic interactions between macromolecules may be generalizable to many unrelated machineries that self-assemble in superstructures.

Summary While the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes, or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning laser microscopy (TPSLM) to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We find that neutrophils form both small, transient or large, persistent swarms via a strikingly coordinated migration pattern. We provide evidence that cooperative action of neutrophils and parasite egress from host cells can trigger swarm formation. Neutrophil swarm formation coincides in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses.

Precise and rapid DNA segregation is required for proper inheritance of genetic material. In most bacteria and archaea, this process is assured by a broadly conserved mitotic-like apparatus in which a NTPase (ParA) displaces the partition complex. Competing observations and models imply starkly different 3D localization patterns of the components of the partition machinery during segregation. Here we use super-resolution microscopies to localize in 3D each component of the segregation apparatus with respect to the bacterial chromosome. We show that Par proteins locate within the nucleoid volume and reveal that proper volumetric localization and segregation of partition complexes requires ATPase and DNA-binding activities of ParA. Finally, we find that the localization patterns of the different components of the partition system highly correlate with dense chromosomal regions. We propose a new mechanism in which the nucleoid provides a scaffold to guide the proper segregation of partition complexes.

Summary Various rod-shaped bacteria mysteriously glide on surfaces in the absence of appendages such as flagella or pili. In the deltaproteobacterium Myxococcus xanthus, a putative gliding motility machinery (Agl–Glt) localizes to so-called Focal Adhesion sites (FA) that form stationary contact points with the underlying surface. We discovered that the Agl–Glt machinery contains an inner-membrane motor complex that moves intracellularly along a right-handed helical path, and when it becomes stationary at FA sites, it powers a left-handed rotation of the cell around its long axis. At FA sites, force transmission requires cyclic interactions between the molecular motor and adhesion proteins of the outer membrane via a periplasmic interaction platform, which presumably involves a contractile activity of motor components and possible interactions with the peptidoglycan. This work provides the first molecular model for bacterial gliding motility.

In prokaryotes, the transfer of DNA between cellular compartments is essential for the segregation and exchange of genetic material. SpoIIIE and FtsK are AAA+ ATPases responsible for intercompartmental chromosome translocation in bacteria. Despite functional and sequence similarities, these motors were proposed to use drastically different mechanisms: SpoIIIE was suggested to be a unidirectional DNA transporter that exports DNA from the compartment in which it assembles, whereas FtsK was shown to establish translocation directionality by interacting with highly skewed chromosomal sequences. Here we use a combination of single-molecule, bioinformatics and in vivo fluorescence methodologies to study the properties of DNA translocation by SpoIIIE in vitro and in vivo. These data allow us to propose a sequence-directed DNA exporter model that reconciles previously proposed models for SpoIIIE and FtsK, constituting a unified model for directional DNA transport by the SpoIIIE/FtsK family of AAA+ ring ATPases.The segregation and exchange of genetic material are central to the processes of cell division and evolution. Although mechanisms of genetic transfer between cellular compartments are diverse, all require the movement of DNA across cellular membranes. A dramatic example of intercellular transmembrane DNA transport is the segregation of chromosomes during sporulation in Bacillus subtilis. During this process, newly replicated chromosomes are rearranged into an elongated structure, or axial filament, in which the origin of replication of each chromosome is tethered to opposite cell poles 1,2 . Next, the division plane is relocated to one pole, creating two asymmetric cellular compartments (the forespore and the mother cell) and trapping the origin-proximal 30% of one chromosome within the forespore 3,4 . The septally

26At the kilo-to mega-base pair scales, eukaryotic genomes are partitioned into 27 self-interacting modules or topologically associated domains (TADs) that associate to 28 form nuclear compartments. Here, we combined high-content super-resolution 29 microscopies with state-of-the-art DNA labeling methods to reveal the variability in 30 the multiscale organization of the Drosophila genome. We found that association 31 frequencies within TADs and between TAD borders are below ~10%, independently 32 of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, 33 we were able to visualize nanometer-sized epigenetic domains at the single-cell 34 level. In addition, absolute contact frequencies within and between TADs were to a 35 large extent defined by genomic distance, higher-order chromosome architecture, 36 and epigenetic identity. We propose that TADs and compartments are organized by 37 multiple, small frequency, yet specific interactions that are regulated by epigenetics 38 and transcriptional state. 39 40 41 42 43The multi-scale organization of eukaryotic genomes defines and regulates 44 cellular identity and tissue-specific functions [1][2][3] . At the kilo-megabase scales, 45 genomes are partitioned into self-interacting modules or topologically associated 46 domains (TADs) 4-6 . TAD formation seems to require specific looping interactions 47 between TAD borders 7,8 , while the association of TADs can lead to the formation of 48 active/repressed compartments 9 . These structural levels were often seen as highly 49 stable over time, however, recent single-cell Hi-C studies have reported different 50 degrees of heterogeneity 10,11 . Other studies have reported that genomes also display 51 stochasticity in their association with the nuclear lamina 12 , in the formation of 52 chromosome territory neighborhoods 13 , and in gene kissing 14 . However, access to 53 single-cell absolute probability contact measurements between loci and efficient 54 detection of low-frequency, long-range interactions are essential to quantify the 55 stochastic behaviour of chromatin at different scales. 56Here, we combined high-content super-resolution microscopy with state-of-57 the-art DNA labeling methods to reveal the variability in the multiscale organization of 58 chromosomes in different cell-types and developmental stages in Drosophila. 59 Remarkably, we found that stochasticity is present at all levels of chromosome 60 architecture, but is locally modulated by sequence and epigenetic state. Contacts 61 between consecutive TAD borders were infrequent, independently of TAD size, 62 epigenetic state, or cell type. Moreover, long-range contact probabilities between 63 non-consecutive borders, the overall folding of chromosomes, and the clustering of 64 epigenetic domains into active/repressed compartments displayed different degrees 65 of stochasticity that globally depended on cell-type. Overall, our results show that 66 contacts between and within TADs are rare, but can be epigenetical...