Precise and rapid DNA segregation is required for proper inheritance of genetic material. In most bacteria archaea, this process assured by a broadly conserved mitotic-like apparatus in which NTPase (ParA) displaces the partition complex. Competing observations models imply starkly different 3D localization patterns components machinery during segregation. Here we use super-resolution microscopies to localize each component with respect bacterial chromosome. We show that Par proteins locate...

Chromatin insulators are genetic elements implicated in the organization of chromatin and regulation transcription. In Drosophila, different insulator types were characterized by their locus-specific composition proteins co-factors. Insulators mediate specific long-range DNA contacts required for three dimensional interphase nucleus transcription regulation, but mechanisms underlying formation these is currently unknown. Here, we investigate molecular associations between components...

Studies of bacterial communities, biofilms and microbiomes, are multiplying due to their impact on health ecology. Live imaging microbial communities requires new tools for the robust identification cells in dense 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 complex images interacting with little parameter adjustment, independent microscopy settings...

Superresolution light microscopy allows the imaging of labeled supramolecular assemblies at a resolution surpassing classical diffraction limit. A serious limitation superresolution approach is sample heterogeneity and stochastic character labeling procedure. To increase reproducibility results, we apply multivariate statistical analysis methods 3D reconstruction approaches originally developed for cryogenic electron single particles. These allow reference-free nanomolecular structures from...

Membrane partition and remodeling play a key role in numerous cell mechanisms, especially viral replication cycles where viruses subvert the plasma membrane to enter escape from host cell. Specifically assembly release of HIV-1 particles require specific cellular components, which are recruited egress site by protein Gag. We previously demonstrated that alters both partitioning dynamics tetraspanins CD9 CD81, players many infectious processes, forming enriched areas virus buds. In this study...

Many species, such as fish schools or bird flocks, rely on collective motion to forage, prey, escape predators. Likewise, Myxococcus xanthus forages and moves collectively prey feed other bacterial species. These activities require two distinct motility machines enabling adventurous (A) social (S) gliding, however when how these mechanisms are used has remained elusive. Here, we address this long-standing question by applying multiscale semantic cell tracking during predation. We show that:...

Rho is a ring-shaped, ATP-fueled motor essential for remodeling transcriptional complexes and R-loops in bacteria. Despite years of research on this fundamental model helicase, key aspects its mechanism translocation remain largely unknown. Here, we used single-molecule manipulation fluorescence methods to directly monitor the dynamics RNA by Rho. We show that efficiency activation strongly dependent force applied but that, once active, able translocate against large opposing (at least 7 pN)...

Abstract SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed explain mechanism by which interact with DNA. The loading model proposes oligomerize exclusively on SpoIIIE recognition sequence/orienting polar (SRS/KOPS) accomplish...

We demonstrate a fast and direct calibration method for systems using single laser optical tweezers particle position detection. The takes advantage of back-focal-plane interferometry measuring not an absolute but differential position, i.e. the trapped relative to center tweezers. Therefore, step-wise motion yields impulse response particle. Calibration parameters such as detector's spatial temporal spring constant then follow readily from fitting measured response.

An optimized chemistry turns a small green dye into useful label for single-molecule experiments where steric hindrance is an issue. Thanks to reducing and oxidizing system combined oxygen depletion, single Bodipy (boron-dipyrromethene) FL fluorophore emits, on average, 20 times more photons around 510 nm (see picture) its lifetime before photobleaching increased by the same amount, reaching several seconds.

We present a new method for calibrating an optical-tweezer setup that does not depend on input parameters and is less affected by systematic errors like drift of the setup.It based inference approach uses Bayesian probability to infer diffusion coefficient potential felt bead trapped in optical or magnetic trap.It exploits much larger amount information stored recorded trajectory than standard calibration approaches.We demonstrate this outperforms equipartition power-spectrum required (bead...

Abstract Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates actively and specifically organized sub-cellular space by which mechanisms. Here, we address this question focusing on ParAB S DNA segregation system, composed a centromeric-like sequence ( parS ), DNA-binding protein (ParB) motor (ParA). We show that -ParB associate form nanometer-sized, round condensates. ParB molecules...

We present a study of the photophysical properties BODIPY-FL fluorescent dye at single molecule scale using total internal reflexion fluorescence microscopy. characterized saturation intensity and maximum number photons for in usual buffer. then studied influence buffer depletion oxygen addition redox agents on brightness lifetime before photobleaching BODIPY-FL. The great improvement these two factors such medium allows use single-molecule biophysics experiments.

Protein synthesis is a complex multistep process involving many factors that need to interact in coordinated manner properly translate the messenger RNA. As translating ribosomes cannot be synchronized over elongation cycles, single-molecule studies have been introduced bring deeper understanding of prokaryotic translation dynamics. Extending this approach eukaryotic very appealing, but initiation and specific labeling are much more complicated. Here, we use noncanonical based on internal...

Abstract Studies of microbial communities by live imaging require new tools for the robust identification bacterial cells in dense and often inter-species populations, sometimes over very large scales. Here, we developed MiSiC, a general deep-learning-based segmentation method that automatically segments wide range spatially structured with little parameter adjustment, independent modality. Using predator-prey interaction model, demonstrate MiSiC enables analysis interspecies interactions,...

Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates actively and specifically organized sub-cellular space by which mechanisms. Here, we address this question focusing on ParABS DNA segregation system, composed a centromeric-like sequence (parS), DNA-binding protein (ParB) motor (ParA). We show that parS-ParB associate form nanometer-sized, spherical droplets. ParB molecules diffuse...