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bioRxiv2019

The BLIMP1 - EZH2 nexus in a non-Hodgkin lymphoma

Waldenstroms macroglobulinemia (WM) is a non-Hodgkin lymphoma, resulting in antibody-secreting lymphoplasmacytic cells in the bone marrow and pathologies resulting from high levels of monoclonal immunoglobulin M (IgM) in the blood. Despite the key role for BLIMP1 in plasma cell maturation and antibody secretion, its potential role in WM cell biology has not yet been explored. Here we provide evidence of a crucial role for BLIMP1 in the survival of WM cells and further demonstrate that BLIMP1 is necessary for the expression of the histone methyltransferase EZH2 in both WM and multiple myeloma. The effect of BLIMP1 on EZH2 levels is post translational, at least partially through the regulation of proteasomal targeting of EZH2. Chromatin immunoprecipitation analysis and transcriptome profiling suggest that the two factors co-operate in regulating genes involved in cancer cell immune evasion. Co-cultures of natural killer cells and WM cells further reveal that both factors participate directly in immune evasion, promoting escape from natural killer cell mediated cytotoxicity. Together, the interplay of BLIMP1 and EZH2 plays a vital role in promoting the survival of WM cells.

cancer biology↗

bioRxiv2021

A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.

plant biology↗

bioRxiv2022

The PNt-Methodology: a top-down network modelling approach to estimate dose-and time-dependent cell responses to complex multifactorial environments

BackgroundNetwork models are convenient to represent in a mechanistic way the complexity of cell biological activity. Dynamic simulations of such networks might require approximations of equation parameters through reverse engineering, numerous and costly experimental research, and/or have limited capacity to explore cell responses to chronic, dose-dependent stimulus exposure. Here we present a mechanistic methodology allowing the simulation of interrelated cell responses of multicellular systems to multifactorial stimuli with dose-and time dependent network links. Methodsa mathematical framework to approach systems biology research questions is presented, the Parallel Networks (PN)-Methodology. It consists of a novel concept, where multicellular systems are described as many relatively small feed-forward networks acting in parallel. Each parallel network is calculated through a specifically designed ordinary differential equation (ODE). Through a unique approach to feed the ODE with interrelated parameters, a system of decoupled, analytically resolvable ODE was obtained. ResultsApplied to intervertebral disc multicellular systems, virtual environments of multifactorial stimuli and multiple cell responses simulating daily moving activities, and to microgravity could successfully be created. ConclusionsThe PN-Methodology stands for a one-of-a-kind mathematical methodology to approximate dynamics of complex multicellular systems over long periods of time at low computational costs.

systems biology↗

bioRxiv2023

Inverted transflection spectroscopy of live cells using metallic grating on elevated nanopillars

Water absorption of mid-infrared (MIR) radiation severely limits the options for vibrational spectroscopy of the analytes - including live biological cells - that must be probed in aqueous environments. While internal reflection elements, such as attenuated total reflection prisms and metasurfaces, partially overcome this limitation, such devices have their own limitations: high cost, incompatibility with standard cell culture workflows, limited spectral range, and small penetration depth into the analyte. In this work, we introduce an alternative live cell biosensing platform based on metallic nanogratings fabricated atop elevated dielectric pillars. For the MIR wavelengths that are significantly longer than the grating period, reflection-based spectroscopy enables broadband sensing of the analytes inside the trenches separating the dielectric pillars. Because the depth of the analyte twice-traversed by the MIR light excludes the highly absorbing thick water layer above the grating, we refer to the technique as Inverted Transflection Spectroscopy (ITS). We demonstrate the analytic power of ITS by measuring protein concentrations in solution. The ability of ITS to interrogate live cells that naturally wrap themselves around the grating is also exploited to characterize their adhesion kinetics.

biophysics↗

bioRxiv2020

Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies. Author summaryHuman hematopoietic stem cells and their derivatives of the myeloid and lymphoid lineages are important targets for gene correction or modifications using the CRISPR-Cas9 technology. Among others, this approach can support site-specific insertion of chimeric antigen receptors (CARs) or T cell receptors (TCRs) into primary T cells. Their subsequent adoptive transfer to patient donors is a promising immunotherapeutic concept that may control chronic infection or certain types of cancer. Human B cells have a similar potential but, in contrast to T cells, they are very sensitive, difficult to handle, and short-lived ex vivo precluding their genetic modification. Here, we provide means to manipulate primary human B cells genetically using in vitro assembled Cas9 ribonucleoprotein complexes and electroporation for their delivery. Our study demonstrates near-to-complete loss of a model target gene and provides examples to evaluate a cellular gene with a critical role during infection with Epstein-Barr virus (EBV).

immunology↗

bioRxiv2021

Protein kinase signalling at the Leishmania kinetochore captured by XL-BioID

Elucidating protein kinase signaling pathways is an important but challenging problem in cell biology. Phosphoproteomics has been used to identify many phosphorylation sites, however the spatial context of these sites within the cell is mostly unknown, making it difficult to reconstruct signalling pathways. To address this problem an in vivo proximity capturing workflow was developed, consisting of proximity biotinylation followed by protein cross-linking (XL-BioID). This was applied to protein kinases of the Leishmania kinetochore, leading to the discovery of a novel essential kinetochore protein, KKT26. XL-BioID enabled the quantification of proximal phosphosites at the kinetochore through the cell cycle, allowing the phosphorylation state of the kinetochore to be followed during assembly. A specific inhibitor of kinetochore protein kinases KKT10/KKT19 was used to show that XL-BioID provides a spatially focussed view of protein kinase inhibition, identifying 16 inhibitor-responsive proximal phosphosites, including 3 on KKT2, demonstrating the potential of this approach for discovery of in vivo kinase signalling pathways.

biochemistry↗

bioRxiv2021

DenVar: Density-based Variation analysis of multiplex imaging data

Multiplex immunohistochemistry (mIHC) and multiplexed ion beam imaging (MIBI) platforms have become increasingly popular for studying complex single-cell biology in the tumor microenvironment (TME) of cancer subjects. Studying the intensity of the proteins that regulate important cell-functions, often known as functional markers, in the TME becomes extremely crucial for subject-specific assessment of risks, such as risk of recurrence and risk of death. The conventional approach requires selection of two thresholds, one to define the cells of the TME as positive or negative for a particular functional marker, and the other to classify the subjects based on the proportion of the positive cells. The selection of the thresholds has a large impact on the results and an arbitrary selection can lead to an incomprehensible conclusion. In light of this problem, we present a threshold-free distance between the subjects based on the probability densities of the functional markers. The distance can be used to classify the subjects into meaningful groups or can be used in a linear mixed model setup for testing association with clinical outcomes. The method gets rid of the subjectivity bias of the thresholding-based approach, enabling an easier but interpretable analysis of these types of data. With the proposed method, we analyze a lung cancer dataset from an mIHC platform, finding the difference in the density of functional marker HLA-DR to be significantly associated with the overall survival. The approach is also applied on an MIBI triple-negative breast cancer dataset to analyze effects of multiple functional markers. Finally, we demonstrate the reliability of our method through extensive simulation studies.

bioinformatics↗

bioRxiv2023

3D Nuclei Segmentation by Combining GAN Based Image Synthesis and Existing 3D Manual Annotations

Nuclei segmentation is an important task in cell biology analysis that requires accurate and reliable methods, especially within complex low signal to noise ratio images with crowded cells populations. In this context, deep learning-based methods such as Stardist have emerged as the best performing solutions for segmenting nucleus. Unfortunately, the performances of such methods rely on the availability of vast libraries of ground truth hand-annotated data-sets, which become especially tedious to create for 3D cell cultures in which nuclei tend to overlap. In this work, we present a workflow to segment nuclei in 3D in such conditions when no specific ground truth exists. It combines the use of a robust 2D segmentation method, Stardist 2D, which have been trained on thousands of already available ground truth datasets, with the generation of pair of 3D masks and synthetic fluorescence volumes through a conditional GAN. It allows to train a Stardist 3D model with 3D ground truth masks and synthetic volumes that mimic our fluorescence ones. This strategy allows to segment 3D data that have no available ground truth, alleviating the need to perform manual annotations, and improving the results obtained by training Stardist with the original ground truth data.

bioinformatics↗

bioRxiv2024

Systematic deconstruction of myeloid cell signaling in tuberculosis granulomas reveals IFN-γ, TGF-β, and time are associated with conserved myeloid diversity

Myeloid cells are key constituents of tuberculosis (TB) granulomas. They are the major target of pathogen infection and play central roles in pathogen control, antigen presentation, adaptive immune cell recruitment, and tissue homeostasis. However, the role of myeloid cells in TB has been studied largely through ex vivo experimental approaches that do not capture the dynamic phenotypic and functional states of these cells in the disease environment. To address this gap, we used a combination of bulk and single-cell RNA sequencing (scRNA-seq), computational modeling, and imaging to define the molecular diversity of myeloid cells in granulomas from Mycobacterium tuberculosis-infected nonhuman primates. We observed an increase in myeloid cell diversity in granulomas compared to non-granulomatous lung tissue. This increased transcriptional diversity is defined by a continuum of macrophage differentiation-, metabolism-, and cytokine-regulated transcriptional programs. In vitro experimental modeling of monocyte-to-macrophage differentiation in defined cytokine environments implicates differentiation time, IFN-{gamma}, and TGF-{beta} signaling as candidate drivers of macrophage diversity. We next examined the conservation of these populations across additional experimental models of Mtb infection and found myeloid cell subsets enriched across the TB disease spectrum. To further contextualize these responses, we constructed an atlas of myeloid cells across diverse human lung pathologies, finding myeloid cell subpopulations that were similar between TB and other lung pathologies as well as subpopulations that distinguish between diseases. Collectively, this study identifies points of integration between myeloid cell biology in TB granulomas and other lung diseases that can be used for defining the signals that instruct myeloid cell behavior in TB and other diseases, as well as advance myeloid cell-targeted therapies.

immunology↗

bioRxiv2024

Direct observation of fluorescent proteins in gels: a rapid, cost-efficient and quantitative alternative to immunoblotting

The discovery of Green Fluorescent Protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation. In this study, we report that various green, orange and red FPs can be maintained in a native, fluorescent state during the entire process of protein sample extraction, incubation with sample buffer, loading and migration on SDS-PAGE with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent in immunoblotting such as transfer onto a membrane and antibody incubations. Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable or better to antibody-based detection, a better quantification and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.

biochemistry↗

bioRxiv2025

Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Aggregates of -synuclein (S) are hallmarks of synucleinopathies, including Parkinsons Disease (PD) and Multiple System Atrophy (MSA). We have recently shown that S lysine acetylation in the soluble monomer pool varies between healthy controls, PD, and MSA patients. We used non-canonical amino acid (ncAA) mutagenesis to express and purify all 12 currently known disease-relevant variants of S and studied their binding to membranes as well as their aggregation propensities. We found that acetylation of lysine 12, 43, and 80 had particularly strong effects. To understand the implications for acetylation of monomeric S found in healthy cells, we performed NMR experiments to study protein conformation and fluorescence correlation spectroscopy experiments to quantify lipid binding. We also investigated the effects of acetylation at lysine 12, 43, and 80 on fibril seeding in neurons. Collectively, our biochemical and cell biological investigations indicated that acetylation of K80 could inhibit aggregation without conferring negative effects on monomer function in healthy cells. Therefore, we studied the structures of fibrils with K80 acetylation through cryo-electron microscopy to uncover the structural basis for these effects. Finally, we identified inhibition of HDAC8 as a way of potentially increasing acetylation at K80 and other inhibitory sites for therapeutic benefit.

biochemistry↗

bioRxiv2025

Stiffening cells with light

Fluorescence microscopy is widely used to observe structures and dynamic processes in living cells and organisms and is often used as if it were purely innocuous to the cells or structures of interest. However, it can lead to phototoxicity, which can affect the cellular behavior and lead to erroneous interpretations of the observations. The major cause of cell damage through phototoxicity is the production of reactive oxygen species (ROS), which can form crosslinks between intracellular molecules, including proteins and nucleic acids. By using profile microindentation and atomic force microscopy, we demonstrate that the excitation of various fluorescent probes leads to a large increase in the stiffness of several cell types within seconds of illumination. The stiffening exhibits a dose-dependent response, where longer exposure times to exciting light are correlated with larger stiffening. This photostiffening effect explains why T cells loaded with the Fluo-4 Calcium probe stop emitting a protrusion within seconds after the excitation light is turned on. We observed photostiffening in different cell types and fluorophores. We showed that repeated cell indentation alone led to cell stiffening as well as excitation with blue or UV light in the absence of a fluoroph ore. However, in the latter case, the stiffening was much smaller than that when the fluorophore was excited. We used both sharp and blunt indenters to show that stiffening occurred not only at the cell cortex level but also deeper in the cell interior and that photostiffening is independent of actin cytoskeleton organization. We correlated the increase in cell stiffness with the production of intracellular reactive oxygen species and reproduced cell stiffening by incubating cells with a ROS inducer, H2O2. The excitation of the photosensitizer Pheophorbide a, which induces a specific type of reactive oxygen species, namely singlet oxygen, also led to cell stiffening. This study reminds the experimentalists that it is crucial to perform controls when using fluorescence. It further allows us to propose exploiting photostiffening as a new method for rapidly quantifying phototoxicity. SignificanceThis study reveals a direct relationship between fluorescence excitation and cell mechanical properties. The generality of th is phenomenon across diverse fluorophores and cell types highlights the importance of controlling phototoxicity in fluorescence experiments, in particular in complex, quantitative cell biology and biophysical experiments, but also reveals an unexpected rol e of intracellular reactive oxygen species production and identifies cell stiffness as a proxy for assessing the efficacy of photodynamic therapy.

biophysics↗

bioRxiv2025

Selection of nanobodies against liponanoparticle-embedded membrane proteins by yeast surface display

Single-domain antibodies, known as nanobodies (Nbs), are widely used in structural biology, therapeutics, and as molecular probes in biology and biotechnology. Nbs towards soluble proteins are routinely developed via alpaca immunization or directed evolution in yeast cell-surface display. However, for membrane proteins, the targets are generally detergent-solubilized, and there remains a need for Nb development methods against membrane proteins in a native-like membrane environment. To address this need, we present a protocol for Nb selection via extraction of membrane proteins into amphiphilic polymers such as styrene-maleic acid to produce purified membrane proteins in stable liponanoparticles. Proof of generality is demonstrated by applying the pipeline to four membrane-resident enzymes of differing fold, oligomerization state, and membrane topology (reentrant membrane helix, transmembrane, membrane-associated). Following screening for optimal stabilization into liponanoparticles, Nbs were selected against four target proteins from glycoconjugate biosynthesis pathways. The selected Nbs showed high affinity and selectivity towards their target proteins with KD apparent values ranging from 15 nM to 200 nM, depending on the Nb-protein conjugate. In accordance with their tight binding, various Nb-protein complexes were found to be stable to size-exclusion chromatography purification. The Nbs were also amenable to sortase-mediated ligation, enabling their conversion into molecular probes for the target membrane protein. The ability to select for such high-affinity Nb against membrane proteins in SMALP will facilitate their widespread application in cell biology and biomedical applications.

biochemistry↗

bioRxiv2025

HIF isoforms contribute distinctly during human NK cell activation

Hypoxia-inducible factors (HIFs) are critical transcriptional regulators that enable cellular adaptation to low oxygen environments. Natural killer (NK) cells are key effectors of innate immunity which frequently operate in hypoxic tissues during viral infection and tumor surveillance. In addition, HIF also play a role in NK cells beyond hypoxic adaptation. However, the distinct and overlapping roles of HIF-1 and HIF-2 in NK cell biology remain incompletely understood. In this study, we investigated the contribution of HIF-1 and HIF-2 to NK cell effector functioning in normoxia using pharmacological inhibition. When HIF-1 was inhibited, we observed a pronounced decline in cytotoxicity as well as IFN{gamma} and TNF production, but not granzyme B, accompanied by elevated mitochondrial reactive oxygen species. In contrast, HIF-2 did not alter these functions, nor did pan-HIF stabilization. These findings indicate a differential role of HIF-1 and HIF-2 in shaping NK cell responses in normoxia, at least during the acute activation phase as investigated here. Still, more mechanistic and contextual insights into the role of HIF isoforms in NK cells is warranted for future therapeutic applications.

immunology↗

bioRxiv2026

On-lamella super-resolution cryo-CLEM for cryo-ET enabled by vacuum-free ultra-stable cryogenic fluorescence microscopy

Cryogenic correlative light and electron microscopy (cryo-CLEM) combines specific fluorescence labelling of proteins inside cells with structural information at the angstrom-level. The introduction of super-resolution fluorescence methods in the field of cryogenic fluorescence microscopy is a necessary step to bridge the large resolution gap between the different imaging modalities. However, there are many challenges hindering the full potential of cryogenic super-resolution correlative light and electron microscopy and seamless integration with structural cell biology. One of the main limiting factors is a lack of dedicated cryogenic fluorescence microscopy systems with sufficient mechanical stability to enable the collection of high-quality super-resolution data and full compatibility with vitrified specimens for cryo-electron tomography. Here, we address this by developing a vacuum-free ultra-stable cryogenic optical microscope (VULCROM). VULCROM is a dedicated super-resolution cryo-CLEM (cryo-SR-CLEM) setup that combines the stability of a vacuum-insulated cryostat with the flexibility and modularity of an open microscopy system. We demonstrate that VULCROM enables detailed investigations of single-molecule cryo-photo-physics across timescales spanning milliseconds to hours. We furthermore demonstrate its suitability for routine cryo-SR-CLEM with a resolution in the 10 nm range in distinct vitrified biological specimen types. We resolve the nanoscale architecture of YFP-labelled PML bodies within the nucleus of mammalian cells and the distribution of ATG9-eGFP in its cellular structural context in a cryo-lift-out lamella of N. benthamiana plant tissue. Owing to its vacuum-free design, VULCROM can be readily adapted for diverse correlative workflows and other cryo-light microscopy applications.

biophysics↗

bioRxiv2026

Autocatalytic growth offset by constant degradation explains mass accumulation during B cell activation

How cells regulate growth is a fundamental question in cell biology. Here we use quantitative phase imaging (QPI) to measure the dry mass of individual naive primary mouse B cells during ex vivo stimulation. B cells undergo an approximately 4-fold increase in mass prior to first division, far exceeding the doubling in size typical of immortalized cell lines and enabling discrimination of growth models that are otherwise indistinguishable. We find that a pure exponential model of mass accumulation exhibits systematic error that is resolved by an offset exponential model, indicating that only an active fraction of cell mass participates in biosynthesis. Monte Carlo simulations establish when these two models can be discriminated as a function of final to initial mass ratio, the magnitude of the degradative pressure, and measurement precision. Analysis of growth rate versus mass independently confirms the offset exponential model and implies that it can be interpreted as indicating a constant degradative pressure that opposes exponential biosynthesis. This model is structurally equivalent to super-exponential growth models derived from autocatalytic ribosome dynamics in bacteria, with the exponential rate reflecting autocatalytic accumulation of biosynthetic machinery and the active mass fraction serving as a proxy for initial ribosomal capacity. Here, the low active fraction in quiescent B cells ([~]20%) is consistent with a limited ribosomal content of resting lymphocytes. Comparison of follicular and marginal zone B cells stimulated with CpG reveals a conserved degradative pressure across subtypes and stimulation conditions. These results demonstrate that deviations from pure exponential growth, previously identified in bacteria, extend to mammalian cells, suggesting that autocatalytic ribosome accumulation offset by constitutive protein turnover is a conserved feature of single-cell growth dynamics across domains of life.

biophysics↗

bioRxiv2018

LION/web: a web-based ontology enrichment tool for lipidomic data analysis

A major challenge for lipidomic analyses is the handling of the large amounts of data and the translation of results to interpret the involvement of lipids in biological systems. We built a new lipid ontology (LION) that associates over 50,000 lipid species to biophysical, chemical and cell biological features. By making use of enrichment algorithms, we used LION to develop a web-based interface (LION/web, www.lipidontology.com) that allows identification of lipid-associated terms in lipidomes. LION/web was validated by analyzing a lipidomic dataset derived from well-characterized sub-cellular fractions of RAW 264.7 macrophages. Comparison of isolated plasma membranes with the microsomal fraction showed a significant enrichment of relevant LION-terms including plasma membrane, headgroup with negative charge, glycerophosphoserines, above average bilayer thickness, and below average lateral diffusion. A second validation was performed by analyzing the membrane fluidity of CHO cells incubated with arachidonic acid. An increase in membrane fluidity was observed both experimentally by using pyrene decanoic acid and by using LION/web, showing significant enrichment of terms associated with high membrane fluidity ('above average, 'very high and 'high lateral diffusion, and 'below average transition temperature). The results demonstrate the functionality of LION/web, which is freely accessible in a platform-independent way.

biochemistry↗

bioRxiv2025

Effects of central dogma processes on the compaction and segregation of bacterial nucleoids

The bacterial cytoplasm is characterized by a distinctive membrane-less organelle, the nucleoid, which harbors chromosomal DNA. We investigate the effects of dynamic processes associated with transcription and translation on the structure of this organelle, using coarsegrained molecular dynamics (MD) simulations implemented with out-of-equilibrium reactions. Our model captures the scale of the entire cell and incorporates a reaction-diffusion system for ribosomes and polyribosomes, combining their out-of-equilibrium dynamics with excluded volume interactions with DNA. Our findings demonstrate that out-of-equilibrium reactions increase the size of the nucleoid. In addition, we show that the nucleoid size increase is proportional to transcriptional activity. Our model reproduces the time-dependent change in nucleoid size observed in rifampicin treatment experiments, where the pool of polyribosomes is depleted. Furthermore, we find these active processes are essential for complete sister chromosome separation and correct nucleoid positioning within the cell. Overall, our study reveals the effects of the central dogma processes on the internal organization and localization of bacterial nucleoids. SignificanceUnderstanding how bacteria organize their chromosomes is fundamental to cell biology. Through our coarse-grained molecular dynamics simulations incorporating out-of-equilibrium processes of transcription and translation, we are able to capture the effects of these central dogma processes on DNA organization and demonstrate that these active biological processes expand the nucleoid and facilitate the separation of daughter chromosomes. Our simulations are compared to experimental measurements and highlight the impact of the out-of-equilibrium conditions of the living cell. These findings point out the crucial interplay between physical forces and biological activity in cellular organization, suggesting that cellular structure depends on non-equilibrium processes.

biophysics↗