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The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal-adhesion (bFA) sites to power gliding motility. Using total internal reflection fluorescence and force microscopies, we identify the von Willebrand A domain-containing outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling adhesin of the gliding transducer (Glt) machinery at bFAs. Biochemical and genetic analyses reveal that CglB localizes to the cell surface independently of the Glt apparatus; once there, it is recruited by the OM module of the gliding machinery, a heteroligomeric complex containing the integral OM β barrels GltA, GltB, and GltH, as well as the OM protein GltC and OM lipoprotein GltK. This Glt OM platform mediates the cell-surface accessibility and retention of CglB by the Glt apparatus. Together, these data suggest that the gliding complex promotes regulated surface exposure of CglB at bFAs, thus explaining the manner by which contractile forces exerted by inner-membrane motors are transduced across the cell envelope to the substratum.

Many species, such as fish schools or bird flocks, rely on collective motion to forage, prey, or escape predators. Likewise,Myxococcus xanthusforages and moves collectively to prey and feed on other bacterial species. These activities require two distinct motility machines enabling adventurous (A) and social (S) gliding, however when and 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: (1) foragers and swarms can comprise A- and S-motile cells, with single cells exchanging frequently between these groups; (2) A-motility is critical to ensure the directional movement of both foragers and swarms; (3) the combined action of A- and S-motile cells within swarms leads to increased predation efficiencies. These results challenge the notion that A- and S-motilities are exclusive to foragers and swarms, and show that these machines act synergistically to enhance predation efficiency.

In mammals, insulators contribute to the regulation of loop extrusion to organize chromatin into topologically associating domains. InDrosophilathe role of insulators in 3D genome organization is, however, under current debate. Here, we addressed this question by combining bioinformatics analysis and multiplexed chromatin imaging. We describe a class ofDrosophilainsulators enriched at regions forming preferential chromatin interactions genome-wide. Notably, most of these 3D interactions do not involve TAD borders. Multiplexed imaging shows that these interactions occur infrequently, and only rarely involve multiple genomic regions coalescing together in space in single cells. Finally, we show that non-border preferential 3D interactions enriched in this class of insulators arise before TADs and transcription duringDrosophiladevelopment. Our results are inconsistent with insulators forming stable hubs in single cells, and instead suggest that they fine-tune existing 3D chromatin interactions, providing an additional regulatory layer for transcriptional regulation.

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), for example, enhancers and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. In the present study, we employ Hi-M, a single-cell spatial genomics approach, to detect CRM–promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM–promoter interactions in defining transcriptional states, as well as distinct cell types. Fil: Espínola, Sergio Martín. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Université de Montpellier. Centre de Biologie Structurale; Francia Fil: Götz, Markus. Université de Montpellier. Centre de Biologie Structurale; Francia. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; Francia Fil: Bellec, Maelle. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; Francia. Université de Montpellier. Centre de Biologie Structurale; Francia. Institut National de la Santé et de la Recherche Médicale; Francia Fil: Messina, Olivier. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; Francia. Université de Montpellier. Centre de Biologie Structurale; Francia. Institut National de la Santé et de la Recherche Médicale; Francia Fil: Fiche, Jean Bernard. Université de Montpellier. Centre de Biologie Structurale; Francia. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; Francia Fil: Houbron, Christophe. Université de Montpellier. Centre de Biologie Structurale; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Centre National de la Recherche Scientifique; Francia Fil: Dejean, Matthieu. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; Francia Fil: Reim, Ingolf. Universitat Erlangen Nuremberg; Alemania Fil: Cardozo Gizzi, Andres Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Alberto C. Taquini de Investigaciones en Medicina Traslacional - Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas "Prof. Dr. Alberto C. Taquini". Instituto Alberto C. Taquini de Investigaciones en Medicina Traslacional; Argentina Fil: Lagha, Mounia. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; Francia Fil: Nollmann, Marcelo. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Université de Montpellier. Centre de Biologie Structurale; Francia

Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). We describe the technical details for handling and imaging the samples for sptPALM, with a particular emphasis on the specificity of imaging plant cells, such as their thick cell walls or high degree of autofluorescence. We then provide a practical guide from data collection to image analyses. In particular, we introduce our sptPALM_viewer software and describe how to install and use it for analyzing sptPALM experiments. Finally, we report an R statistical analysis pipeline to analyze and compare sptPALM experiments. Altogether, this protocol should enable plant researchers to perform sptPALM using a benchmarked reproducible protocol. Routinely, the procedure takes 3–4 h of imaging followed by 3–4 d of image processing and data analysis. This protocol describes how to perform single-particle tracking photoactivated localization microscopy (sptPALM) of membrane proteins in living plant tissues. The procedure covers all stages, from sample preparation to data analysis.

Polycomb (Pc) group proteins are transcriptional regulators with key roles in development, cell identity and differentiation. Pc-bound chromatin regions form repressive domains that interact in 3D to assemble repressive nuclear compartments. Here, we used multiplexed chromatin imaging to investigate whether Pc compartments involve the clustering of multiple Pc domains during Drosophila development. Notably, 3D proximity between Pc targets is rare and involves predominantly pairwise interactions. These 3D proximities are particularly enhanced in segments where Pc genes are co-repressed. In addition, segment-specific expression of Hox Pc targets leads to their spatial segregation from Pc repressed genes. Finally, non-Hox Pc targets are proximal in regions where they are co-expressed. These results indicate that long-range Pc interactions are temporally and spatially regulated during differentiation and development but do not involve clustering of multiple distant Pc genes.

Multiplexed sequential and combinatorial imaging enables the simultaneous detection of multiple biological molecules, e.g. proteins, DNA, or RNA, enabling single-cell spatial multi-omics measurements at sub-cellular resolution. Recently, we designed a multiplexed imaging approach (Hi-M) to study the spatial organization of chromatin in single cells. In order to enable Hi-M sequential imaging on custom microscope setups, we developed Qudi-HiM, a modular software package written in Python 3. Qudi-HiM contains modules to automate the robust acquisition of thousands of three-dimensional multicolor microscopy images, the handling of microfluidics devices, and the remote monitoring of ongoing acquisitions and real-time analysis. In addition, Qudi-HiM can be used as a stand-alone tool for other imaging modalities.

The spatial organization of chromatin at the scale of topologically associating domains (TADs) and below displays large cell-to-cell variations. Up until now, how this heterogeneity in chromatin conformation is shaped by chromatin condensation, TAD insulation, and transcription has remained mostly elusive. Here, we used Hi-M, a multiplexed DNA-FISH imaging technique providing developmental timing and transcriptional status, to show that the emergence of TADs at the ensemble level partially segregates the conformational space explored by single nuclei during the early development of Drosophila embryos. Surprisingly, a substantial fraction of nuclei displayed strong insulation even before TADs emerged. Moreover, active transcription within a TAD led to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affected insulation to the neighboring TAD. Overall, our results indicate that multiple parameters contribute to shaping the chromatin architecture of single nuclei at the TAD scale.

Cell motility and predation are important for the dynamics of many multi-cellular ecosystems, such as the gut or the soil. Approaches to image cell dynamics in such complex systems are scant, and high-throughput analysis methods to segment and track single-cell behaviors are currently lacking. Here, we addressed these limitations by implementing a fast fluorescence microscopy technique enabling the high-resolution acquisition of cell movement over large areas and long time periods. Next, we applied deep learning to semantically segment two different bacteria species within complex micro-environments . We implemented a method to build single cell traces by combining the cell masks from different time points to follow the dynamics of single cells with high spatial and temporal resolutions and over long periods of time. We applied and validated these methods by characterizing the dynamics of Escherichia coli predation by Myxococcus xanthus.

Physiological acclimation of plants to an everchanging environment is governed by complex combinatorial signaling networks that perceive and transduce various abiotic and biotic stimuli. Reactive oxygen species (ROS) serve as one of the second messengers in plant responses to hyperosmotic stress. The molecular bases of ROS production and the primary cellular processes that they target were investigated in the Arabidopsis (Arabidopsis thaliana) root. Combined pharmacological and genetic approaches showed that the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) pathway and an additional pathway involving apoplastic ascorbate and iron can account for ROS production upon hyperosmotic stimulation. The two pathways determine synergistically the rate of membrane internalization, within minutes after activation. Live superresolution microscopy revealed at single-molecule scale how ROS control specific diffusion and nano-organization of membrane cargo proteins. In particular, ROS generated by RBOHs initiated clustering of the PLASMA MEMBRANE INTRINSIC PROTEIN2;1 aquaporin and its removal from the plasma membrane. This process is contributed to by clathrin-mediated endocytosis, with a positive role of RBOH-dependent ROS, specifically under hyperosmotic stress.

During development, naïve cells gradually acquire distinct cell fates, through sophisticated mechanisms of precise spatio-temporal gene regulation. Acquisition of cell fate is thought to rely on the specific interaction of remotecis-regulatory modules (e.g. enhancers, silencers) (CRM) and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly in single cells within multicellular developing organisms. Here we employ Hi-M, a single-cell spatial genomics approach, to systematically detect CRM-promoter looping interactions within topological associating domains (TADs) duringDrosophiladevelopment. By comparingcis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses revealed the existence of local interactions between multiple remote CRMs to form hubs. We found that loops and CRM hubs are established early during development, prior to the emergence of TADs. Moreover, CRM hubs are formed via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach offers a new perspective on the role of CRM-promoter interactions in defining transcriptional activation and repression states, as well as distinct cell types.

International audience; 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.

International audience; In the course of their growth and development, plants have to constantly perceive and react to their environment. This is achieved in cells by the coordination of complex combinatorial signaling networks. However, how signal integration and specificity are achieved in this context is unknown. With a focus on the hyperosmotic stimulus, we use live super-resolution light imaging methods to demonstrate that a Rho GTPase, Rho-of-Plant 6 (ROP6), forms stimuli-dependent nanodomains within the plasma membrane (PM). These nanodomains are necessary and sufficient to transduce production of reactive oxygen species (ROS) that act as secondary messengers and trigger several plant adaptive responses to osmotic constraints. Furthermore, osmotic signal triggers interaction between ROP6 and two NADPH oxidases that subsequently generate ROS. ROP6 nanoclustering is also needed for cell surface auxin signaling, but short-time auxin treatment does not induce ROS accumulation. We show that auxin-induced ROP6 nanodomains, unlike osmotically driven ROP6 clusters, do not recruit the NADPH oxidase, RBOHD. Together, our results suggest that Rho GTPase nano-partitioning at the PM ensures signal specificity downstream of independent stimuli.

Super-resolution imaging shows that G1/S transcription factors in budding yeast are spatially organized in small clusters that increase in number, not molecular content, as the transcription factor copy number increases with cell growth in G1. This spatial and temporal organization of the G1/S regulon might help coordinate the Start transition., In budding yeast, the transcription factors SBF and MBF activate a large program of gene expression in late G1 phase that underlies commitment to cell division, termed Start. SBF/MBF are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the questions of how SBF/MBF are dynamically distributed across the G1/S regulon and how this impacts the Start transition. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits in fixed cells revealed each transcription factor was organized into discrete clusters containing approximately eight copies regardless of cell size and that the total number of clusters increased as cells grew through G1 phase. Stochastic modeling using reasonable biophysical parameters recapitulated growth-dependent SBF/MBF clustering and predicted TF dynamics that were confirmed in live cell PALM experiments. This spatio-temporal organization of SBF/MBF may help coordinate activation of G1/S regulon and the Start transition.

The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal adhesion (bFA) sites to power gliding motility. Using TIRF and force microscopy, we herein identify the integrin αI-domain-like outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling protein of the gliding motility complex. Similar to most known OM lipoproteins, CglB is anchored on the periplasmic side of the OM and thus a mechanism must exist to secrete it to the cell surface in order for it to interact with the underlying substratum. We reveal this process to be mediated by a predicted OM β-barrel structure of the gliding complex. This OM platform was found to regulate the conformational activation and secretion of CglB across the OM. These data suggest that the gliding complex promotes surface exposure of CglB at bFAs, thus explaining the manner by which forces exerted by inner-membrane motors are transduced across the cell envelope to the substratum; they also uncover a novel protein secretion mechanism, highlighting the ubiquitous connection between secretion and bacterial motility.

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), for example, enhancers and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. In the present study, we employ Hi-M, a single-cell spatial genomics approach, to detect CRM-promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM-promoter interactions in defining transcriptional states, as well as distinct cell types.

In the course of their growth and development plants have to constantly perceive and react to their environment. This is achieved in cells, by the coordination of complex combinatorial signaling networks. However, how signal integration and specificity are achieved in this context is unknown. With a focus on the hyperosmotic stimulus, we use live super-resolution light imaging methods to demonstrate that a Rho GTPase, Rho-of-Plant 6 (ROP6), forms stimuli-dependant nanodomains within the PM. These nanodomains are necessary and sufficient to transduce production of reactive oxygen species (ROS),that act as secondary messengers and trigger several plant adaptive responses to osmotic constraints. Furthermore, ROP6 activation triggers the nanoclustering of two NADPH oxidases that subsequently generates ROS. ROP6 nanoclustering is also needed for cell surface auxin signaling, but short-time auxin treatment does not induce ROS accumulation. We show that auxin-induced ROP6 nanodomains, unlike osmotically-driven ROP6 clusters, do not recruit the NADPH oxidase, RBOHD. Together, our results suggest that Rho GTPase nano-partitioning at the PM ensure signal specificity downstream of independent stimuli.

Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4–5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1–2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues. Here, the authors provide detailed experimental and analytical procedures for Hi-M, a method that enables simultaneous imaging of 3D genome folding and RNA localization in single cells.

International audience; 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.

Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues.

International audience; Rho GTPases are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. Here, we show that the phospholipid phosphatidylserine acts as a 25 developmentally-controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live super-resolution single molecule imaging revealed that RHO-OF-PLANT6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which is required for auxin signaling. Furthermore, we uncovered that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 30 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work reveals that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development. One Sentence Summary: 35 Phosphatidylserine acts as a developmentally-controlled lipid rheostat that regulates auxin sensitivity and plant development.

The spatio-temporal organization of transcription factor (TF)-promoter interactions is critical for the coordination of transcriptional programs. In budding yeast, the main G1/S transcription factors, SBF and MBF, are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the question of how SBF/MBF are dynamically distributed across the G1/S regulon. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits revealed that 85% were organized into discrete clusters containing ∼8 copies regardless of cell size, while the number of clusters increased with growth. Stochastic simulations with a mathematical model based on co-localization of promoters in clusters recapitulated observed cluster behavior. A prediction of the model that SBF/MBF should exhibit both fast and slow dynamics was confirmed in PALM experiments on live cells. This spatio-temporal organization of the TFs that activate the G1/S regulon may help coordinate commitment to division.

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.

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., In most bacteria and archaea, a broadly conserved mitotic-like apparatus assures the inheritance of duplicated genetic material before cell division. Here, the authors use super-resolution microscopies to dissect the activities required for proper DNA segregation through the nucleoid interior.

Rho GTPases are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. Here, we show that the phospholipid phosphatidylserine acts as a developmentally-controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live super-resolution single molecule imaging revealed that RHO-OF-PLANT6 (ROP6) is stabilized by phosphatidylserine into plasma membrane (PM) nanodomains, which is required for auxin signaling. Furthermore, we uncovered that the PM phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work reveals that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.One Sentence SummaryPhosphatidylserine acts as a developmentally-controlled lipid rheostat that regulates cellular auxin sensitivity and plant development.

With single-molecule localization microscopy (SMLM) it is possible to reveal the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. SMLM remains technically challenging, and frequently its implementation requires tailored experimental conditions that depend on the complexity of the subcellular structure of interest. Here, we describe two simple, robust, and high-throughput protocols to study molecular motors and machineries responsible for chromosome transport and organization in bacteria using 2D- and 3D-SMLM.

Directed surface motility of metazoan cells and protozoan parasites involves substratum engagement by surface-exposed integrin(-like) adhesins, directionally transported by molecular motors via coupling to the internal cytoskeleton. The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal adhesions to power gliding motility. However, the mechanisms of gliding machinery-substratum coupling and force mechanotransduction between innermembrane motors and substratum are unknown. Herein, we use bead force spectroscopy and TIRF microscopy to characterize CglB as the essential substratum-coupling integrin αI-domain-like adhesin. Protease susceptibility reveals that CglB interacts with a globular-protein-accessorized β-barrel OM display platform, which regulates the cell-surface conformational state and accessibility of the adhesin. Surface retention of CglB is further regulated by a cell-surface metalloprotease, a phenomenon also modulated by the OM display platform. These data depict a complex mechanism for bacterial gliding adhesin secretion, cell-surface anchoring, and processing, with conserved themes between prokaryotic and eukaryotic cell motility.

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome organization while maintaining spatiotemporal information. Here, we present a multiplexed, sequential imaging approach (Hi-M) that permits the simultaneous detection of chromosome organization and transcription in single nuclei. We used Hi-M to study chromosome organization during the awakening of the zygotic genome in intact Drosophila embryos. By visualizing chromosome architecture and gene expression simultaneously in single nuclei, we were able to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome un-pairing. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture in time and space will be key to understand the mechanisms and consequences of the 4D organization of the genome.

With single-molecule localization microscopy (SMLM) it is possible to reveal the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. SMLM remains technically challenging, and frequently its implementation requires tailored experimental conditions that depend on the complexity of the subcellular structure of interest. Here, we describe two simple, robust, and high-throughput protocols to study molecular motors and machineries responsible for chromosome transport and organization in bacteria using 2D- and 3D-SMLM.

At the kilo- to mega-base pair scales, eukaryotic genomes are partitioned into self-interacting modules or topologically associated domains (TADs) that associate to form nuclear compartments. Here, we combined high-content super-resolution microscopies with state-of-the-art DNA labeling methods to reveal the variability in the multiscale organization of the Drosophila genome. We found that association frequencies within TADs and between TAD borders are below ~10%, independently of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, we were able to visualize nanometer-sized epigenetic domains at the single-cell level. In addition, absolute contact frequencies within and between TADs were to a large extent defined by genomic distance, higher-order chromosome architecture, and epigenetic identity. We propose that TADs and compartments are organized by multiple, small frequency, yet specific interactions that are regulated by epigenetics and transcriptional state.

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here, we present a multiplexed, sequential imaging approach (Hi-M) that permits the simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome un-pairing during the awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understand the mechanisms and consequences of the 4D organization of the genome.

International audience; We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm.

In order to study the detailed assembly and regulation mechanisms of complex structures and machineries in the cell, simultaneous in situ observation of all the individual interacting components should be achieved. Multi-color Single-Molecule Localization Microscopy (SMLM) is ideally suited for these quantifications. Here, we build on previous developments and thoroughly discuss a protocol for two-color SMLM combining PALM and STORM, including sample preparation details, image acquisition and data postprocessing analysis. We implement and evaluate a recently proposed colocalization analysis method (aCBC) that allows single-molecule colocalization quantification with the potential of revealing fine, nanometer-scaled, structural details of multicomponent complexes. Finally, using a doubly-labeled nuclear factor (Beaf-32) in Drosophila S2 cells we experimentally validate the colocalization quantification algorithm, highlight its advantages and discuss how using high molecular weight fluorescently labeled tags compromises colocalization precision in two-color SMLM experiments.

International audience; Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a " precise color " MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.

International audience; 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.

Bacteria have evolved complex, multi-component cellular machineries to carry out fundamental cellular processes such as cell division/separation, locomotion, protein secretion, DNA transcription/replication, or conjugation/competence. Diffraction of light has so far restricted the use of conventional fluorescence microscopy to reveal the composition, internal architecture and dynamics of these important machineries. This review describes some of the more recent advances on single-molecule super-resolution microscopy methods applied to bacteria and highlights their application to chemotaxis, cell division, DNA segregation, and DNA transcription machineries. Finally, we discuss some of the lessons learned from this approach, and future perspectives.