Multiplexed chromatin imaging reveals predominantly pairwise long-range coordination between Drosophila Polycomb genes.

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 use 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 more 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 induce frequent clustering of multiple distant Pc genes.

Hi-M: A Multiplex Oligopaint FISH Method to Capture Chromatin Conformations In Situ and Accompanying Open-Source Acquisition Software.

The simultaneous observation of three-dimensional (3D) chromatin structure and transcription in single cells is critical to understand how DNA is organized inside cells and how this organization influences or is affected by other processes, such as transcription. We have recently introduced an innovative technology known as Hi-M, which enables the sequential tagging, 3D visualization, and precise localization of multiple genomic DNA regions alongside RNA expression within individual cells. In this chapter, we present a comprehensive guide outlining the creation of probes, as well as sample preparation and labeling. Finally, we provide a step-by-step guide to conduct a complete Hi-M acquisition using our open-source software package, Qudi-HiM, which controls the robotic microscope handling the entire acquisition procedure.

pyHiM: a new open-source, multi-platform software package for spatial genomics based on multiplexed DNA-FISH imaging.

Genome-wide ensemble sequencing methods improved our understanding of chromatin organization in eukaryotes but lack the ability to capture single-cell heterogeneity and spatial organization. To overcome these limitations, new imaging-based methods have emerged, giving rise to the field of spatial genomics. Here, we present pyHiM, a user-friendly python toolbox specifically designed for the analysis of multiplexed DNA-FISH data and the reconstruction of chromatin traces in individual cells. pyHiM employs a modular architecture, allowing independent execution of analysis steps and customization according to sample specificity and computing resources. pyHiM aims to facilitate the democratization and standardization of spatial genomics analysis.

3D chromatin interactions involving Drosophila insulators are infrequent but preferential and arise before TADs and transcription.

In mammals, insulators contribute to the regulation of loop extrusion to organize chromatin into topologically associating domains. In Drosophila the 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 of Drosophila insulators 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 are present before TADs and transcription during Drosophila development. 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.

Multi-scale dynamic imaging reveals that cooperative motility behaviors promote efficient predation in bacteria.

Many species, such as fish schools or bird flocks, rely on collective motion to forage, prey, or escape predators. Likewise, Myxococcus xanthus forages 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.

The receptor kinase FERONIA regulates phosphatidylserine localization at the cell surface to modulate ROP signaling.

Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall-sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations.

Unmasking of the von Willebrand A-domain surface adhesin CglB at bacterial focal adhesions mediates myxobacterial gliding motility.

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 ß 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.

Multiple parameters shape the 3D chromatin structure of single nuclei at the doc locus in Drosophila.

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 display strong insulation even before TADs emerge. Moreover, active transcription within a TAD leads to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affects 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.

Qudi-HiM: an open-source acquisition software package for highly multiplexed sequential and combinatorial optical imaging.

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.

Misic, a general deep learning-based method for the high-throughput cell segmentation of complex bacterial communities.

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.

Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development.

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.

Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues.

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.

A Plasma Membrane Nanodomain Ensures Signal Specificity during Osmotic Signaling in Plants.

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.

G1/S transcription factors assemble in increasing numbers of discrete clusters through G1 phase.

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.

Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M.

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.

Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine.

Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane 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 shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.

Osmotic Stress Activates Two Reactive Oxygen Species Pathways with Distinct Effects on Protein Nanodomains and Diffusion.

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.

Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms.

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 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 unpairing during 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 understanding the mechanisms and consequences of the 4D organization of the genome.

DNA Organization and Superesolved Segregation.

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.

Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions.

At the kilo- to megabase pair scales, eukaryotic genomes are partitioned into self-interacting modules or topologically associated domains (TADs) that associate to form nuclear compartments. Here, we combine 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 find 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 are able to visualize nanometer-sized epigenetic domains at the single-cell level. In addition, absolute contact frequencies within and between TADs are 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.