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.

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.

Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. Here, we investigate the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and develop a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. We show that BEAF32 is able to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, we show that CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. We propose a general model for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division.

Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParAB S DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.

Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParAB S DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.

Studies of bacterial communities, biofilms and microbiomes, are multiplying due to their impact on health and ecology. Live imaging of microbial communities requires new tools for the robust identification of bacterial cells in dense and often inter-species populations, sometimes over very large scales. Here, we developed MiSiC, a general deep-learning-based 2D segmentation method that automatically segments single bacteria in complex images of interacting bacterial communities with very little parameter adjustment, independent of the microscopy settings and imaging modality. Using a bacterial predator-prey interaction model, we demonstrate that MiSiC enables the analysis of interspecies interactions, resolving processes at subcellular scales and discriminating between species in millimeter size datasets. The simple implementation of MiSiC and the relatively low need in computing power make its use broadly accessible to fields interested in bacterial interactions and cell biology.

Membrane partition and remodeling play a key role in numerous cell mechanisms, especially in viral replication cycles where viruses subvert the plasma membrane to enter and escape from the host cell.

Superresolution light microscopy allows the imaging of labeled supramolecular assemblies at a resolution surpassing the classical diffraction limit. A serious limitation of the superresolution approach is sample heterogeneity and the stochastic character of the labeling procedure. To increase the reproducibility and the resolution of the superresolution results, we apply multivariate statistical analysis methods and 3D reconstruction approaches originally developed for cryogenic electron microscopy of single particles. These methods allow for the reference-free 3D reconstruction of nanomolecular structures from two-dimensional superresolution projection images. Since these 2D projection images all show the structure in high-resolution directions of the optical microscope, the resulting 3D reconstructions have the best possible isotropic resolution in all directions.superresolution microscopy | 3D reconstruction | DNA origami | multivariate statistical analysis | structural biology W ith recent advances in electron detection and image processing, electron microscopy (EM) now enables the exploration of supramolecular architectures at nanoscale resolutions (1-3). The resolution in conventional fluorescence light microscopies is limited by diffraction to a few hundred nanometers. Recently, several superresolution microscopy methods were developed to surpass the intrinsic resolution limit of conventional fluorescence microscopy (4). Uniquely, superresolution microscopies enable imaging of man-made materials and biological objects in complex environments with chemical specificity and previously unimagined detail (5-7). However, the dissection of structural heterogeneity, dynamics, and low labeling densities, most noticeable at the nanoscale, limit the full potential of current superresolution analysis methods. Recently, single-particle averaging approaches were successfully applied to superresolution optical microscopy images to unravel ultrastructural details of the nuclear pore complex (8), the endosomal sorting complexes required for transport (ESCRT) machinery at HIV assembly sites (6), and the machinery of the centrosome (9). Typically, these methods relied on the use of structural templates to seed the reconstruction process and used cylindrical or spherical symmetries to reveal information on the radial distribution of components within complexes. These approaches are thus not easily transferable to de novo systems and may lead to structures that are biased toward those of templates.Here, we present a model-free method adapted from conventional EM single-particle reconstruction (EM SPR) algorithms to extract 3D isotropic structural information from 2D singlemolecule localization microscopy (SMLM) (10) images of supramolecular structures. We demonstrate the applicability of this method by solving the 3D structures of DNA origami (11, 12) and of simulated large protein complexes. ResultsSuperresolution Imaging of Model DNA Origami. DNA origami were labeled by DNA-PAINT (13, 14). Equally spaced "docking" strands w...