Biology subjects

Guerin, C.

Publications and source records attributed to Guerin, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Stress limits the beneficial effects of glutamine in male ob/ob mice

IntroductionObesity is a major health issue associated with metabolic and psychological comorbidities, as well as an increased prevalence of disorders of gut-brain interaction (DGBI). Obesity and DGBI share common mechanisms such as inflammation, gut barrier dysfunction, and alterations of gut microbiota, which are all known to be regulated by stress. Glutamine (Gln), which is essential to maintain intestinal integrity and immune response, may counteract these alterations. This study aimed to evaluate the effects of oral Gln supplementation on stress-induced response in obese mice. MethodsSeven-week-old male leptin-deficient ob/ob mice were assigned to four groups: control, chronic restraint stress (CRS), Gln-supplemented, or both CRS and Gln-supplemented. Gln was administered in drinking water for two weeks, and CRS was performed during the final 4 days. Metabolic parameters, intestinal permeability, inflammatory markers, gene and protein expression, and gut microbiota composition were assessed. ResultsStress increased plasma corticosterone levels but had a limited effect on metabolic parameters. In obese mice without stress, Gln supplementation reduced body weight gain, improved body composition and reduced inflammation in the visceral adipose tissue. These effects were lost under stress conditions, with an increase in fasting glycaemia. Stress reduced occludin protein levels, while Gln exerted context-dependent effects, decreasing gene expression of Tjp3, Cldn15 and Ccl2 in unstressed mice but increasing gene expression of multiple tight junction (Tjp2, Tjp3, Cldn12, Cgn, F11r, Marveld2) and inflammatory markers (Tlr2, Myd88, Irf3) under stress. Interestingly, in unstressed obese mice, Gln altered the composition of the gut microbiota, with changes in key bacterial taxa (Thermodesulfobacteriota and Clostridiaceae). This was associated with decreased levels of cecal short-chain fatty acids and increased levels of branched-chain fatty acids. ConclusionIn conclusion, Gln improves metabolic and adipose inflammatory parameters in genetically obese mice. However, these benefits are no longer observed when mice are under stress conditions. Since, Gln has been found to increase fasting glycaemia and colonic inflammation, in association with alterations of gut microbiota.

pathology↗

bioRxiv2026

Routine FFPE sections support clinically compatible single-nucleus transcriptomics across six human cancer types

Tumor cellular composition--including malignant cell states, immune populations, and stromal populations--is increasingly recognized as a determinant of therapeutic response and resistance to anti-cancer agents, yet comprehensive cellular profiling remains largely confined to research settings. Here, we present a clinically compatible sample-to-report workflow for tumor composition profiling from routine formalin-fixed paraffin-embedded (FFPE) clinical specimens. By combining low-input single-nucleus RNA sequencing with foundation model- based automated cell annotation, this workflow enables prospective sample-by-sample analysis without dedicated research material or cohort-based processing. Across 116 clinical specimens representing six cancer types, we generated reproducible measurements of cellular composition and cell-type-specific gene expression, demonstrated high technical reproducibility, and showed concordance with pathological assessment of immune infiltration. The workflow was similarly applicable to archival FFPE material and ultra-low-input biopsy specimens. Together, these findings establish a practical framework for routine single-cell profiling from standard pathology specimens and open the perspective of prospective evaluation of cellular composition as a clinical biomarker in precision oncology.

cancer biology↗

bioRxiv2026

Chronic Activity-Based Anorexia triggers a glial response in the hippocampus independent of intestinal epithelial Toll-Like Receptor 4

Anorexia nervosa is characterized by maladaptive eating behavior and cognitive dysfunction, which could be explained by a neuroinflammation. A gut dysbiosis could link gastrointestinal alterations to central dysfunctions, particularly via the toll-like receptor 4 (TLR4), which has been shown to play a key role in the activity-based anorexia (ABA) model. We aimed to evaluate the neuroinflammation and its behavioral consequences in the ABA model, and to decipher the role of the microbiota-gut-brain axis, and more specifically of TLR4, in these alterations of the central nervous system. We show that chronic restriction is more strongly associated with gut inflammation, cecal microbiota alteration and neuroinflammatory processes in the hippocampus than acute restriction. The hippocampal glial response is characterized by a loss of astrocyte density, and an increased number of deramified microglia. We further demonstrate that these alterations are independent of TLR4 expressed by intestinal epithelial cells. In conclusion, our results highlight that the chronicity of ABA-associated undernutrition alters the response of glial cells in the hippocampus that is linked with changes in microbiota composition, highlighting the importance of faster diagnosis and treatment of AN.

neuroscience↗

bioRxiv2025

Glutamine alters the response to stress in mice with diet-induced obesity in a sex-dependent manner.

RationalePatients with class III obesity often suffer from irritable bowel syndrome (IBS) while obesity and IBS share common pathophysiological mechanisms such as altered intestinal barrier function and gut microbiota dysbiosis. Oral glutamine (Gln) supplementation previously showed beneficial effects on gut barrier function in a sex-dependent manner and reduced abdominal pain in IBS patients. Thus, we assessed the sex-dependent response to an oral Gln supplementation in mice with diet-induced obesity and subjected to a chronic restraint stress to mimic IBS. MethodsMale (M) and female (F) C57BL/6 mice received a high fat diet (HFD; 60% kcal from fat) during 14 weeks (W14) and were subjected or not to a restraint stress (S) for the 4 last days. From W12, mice received or not Gln in drinking water (2g/kg/day; n=12/group). Plasma corticosterone, body composition, glucose tolerance (OGTT), intestinal permeability, inflammatory markers in colonic and white adipose tissues, cecal microbiota and short-chain fatty acid (SCFA) composition have been assessed. Within each sex, groups were compared by Kruskal-Wallis test or a 1-way ANOVA test. ResultsIn M-HFD mice, chronic restraint stress was associated with a better glucose tolerance (-15,46% AUC) and a reduced fasting glycemia that was not observed in F-HFD mice. Gln partially prevented body weight loss, reduced plasma resistin, plasma corticosterone and colonic permeability in female stressed obese mice. In male stressed obese mice, Gln limited lean mass loss, reduced colonic permeability and Ccl2 mRNA level in the subcutaneous adipose tissue. Chronic restraint stress and Gln modified cecal microbiota in both sexes but cecal SCFA composition only in male mice. In particular, stress induced an increased abundance of Pseudomonadota in male mice that was partially restored after Gln supplementation. In addition, cecal total SCFA were reduced in Gln-supplemented stressed HFD male mice compared to unstressed HFD mice. In female HFD mice, stress associated to Gln supplementation increased the abundance of Thermodesulfobacteriota and reduced colonic expression of Cxcr3 mRNA. ConclusionsChronic restraint stress has beneficial effects on glycemia control in male HFD mice without additive effects of Gln supplementation. By contrast, Gln reduces stress-induced corticosterone level and body weight loss only in females. These data, as well as the differential impact of Gln on intestinal permeability and gut microbiota according to the sex, deserves further investigations to decipher the underlying mechanisms.

pathology↗

bioRxiv2025

Ovarian development is driven by early spatiotemporal priming of the coelomic epithelium

Ovarian organogenesis relies on the coordinated specification of supporting and steroidogenic lineages from multipotent coelomic epithelium (CE) progenitors. However, it remains unclear how ovarian cellular diversity arises and whether CE progenitors are fate-biased before or after ingression into the gonad. We show that CE cells in fetal mouse and human ovaries are transcriptionally heterogeneous and spatially organized into subdomains already primed toward supporting or steroidogenic identities. CE priming is dynamic, influenced by proximity to the mesonephros, with a transient coexistence of both progenitor types before resolution toward a predominantly supporting-biased CE. In mice, delamination of primed CE cells seeds intragonadal niches that generate pre-granulosa and steroidogenic progenitors. We further demonstrate that fetal steroidogenic progenitors give rise to adult stromal and theca cells, and that granulosa cells have a dual origin from CE-derived and supporting-like cells. Together, these findings reveal a conserved, spatially encoded program of ovarian lineage specification.

developmental biology↗

bioRxiv2025

ModuloStat: An Internet of Things' Path to Continuous Cultures in Mini-Bioreactors

In continuous culture, a population of microorganisms is propagated in a stable environment over many generations. This is particularly relevant for experimental evolution and metabolic studies. However, continuous culture protocols are difficult to implement, so they are not commonly used in microbiology laboratories. Here, we present the ModuloStat, a modular, open-source framework that facilitates continuous culture in mini-bioreactors. The ModuloStat system is grounded on digital fabrication tools easily accessible in FabLabs and programmable electronics. Maintaining a culture is divided into tasks assigned to dedicated printed circuit boards with a microcontroller connected to a Wi-Fi network. According to Internet of Things principles, each board operates a set of sensors and actuators autonomously and can receive and send information. The boards are stacked to implement complex behaviors and can be modified to accommodate new features. A thermoregulated box holds the components and can be placed on a laboratory bench or transported under a sterile hood for inoculation. Sterility is ensured by autoclaving, after assembly, all components that will come into contact with the culture medium. In-situ optical density monitoring combined with modularity and computer control enables many cultivation modes. Additionally, we present the construction of the Bacillus subtilis strain ZB designed for bioreactor culture that exhibits a zero-biofilm phenotype. To demonstrate the systems versatility, we performed several experimental cultures with this model organism, including chemostat, turbidostat, medium swap, and a cascade of bioreactors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/669317v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@16b3bbborg.highwire.dtl.DTLVardef@11ea708org.highwire.dtl.DTLVardef@1681769org.highwire.dtl.DTLVardef@d91946_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Nano-flow cytometry of single extracellular vesicles reveals subpopulation differences across cell types and pharmacological perturbations

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles released by most cell types, which can transfer signals and cargoes between cells. EVs released by a single donor cell source are increasingly recognized as extremely heterogeneous, in terms of size, intracellular origin, and cargo composition. Analyzing large numbers of EVs at the single vesicle level is therefore the only way to truly decipher their heterogeneity. Here, we developed a reliable pipeline of single EV analysis using a nanoparticle-dedicated flow cytometer, which detects particles and measures their size down to 55 nm in diameter, without the need for vesicle pre-immobilization or fluorescent label. We show that titrating each antibody, eliminating unbound antibodies and using EVs devoid of the analyzed markers as negative controls are required to reliably quantify the proportion of EVs bearing none or any combination of two markers, as well as to measure their sizes. We thus observed, depending on the cell source (human cell lines MDA-MB-231, HeLa, A549), variable proportions of EVs bearing none of the CD9, CD81 and CD63 tetraspanins often used to define EVs, and of single- and double-positive EVs for each of these markers. We also observed CD29 (ITGB1) as a protein detected as frequently on EVs as CD9, while other transmembrane proteins (CD44, SSEA-4, CD98), were detected in a small proportion of EVs, and mostly of relatively large size. Finally, we used this pipeline to uncover differential effects of small molecule drugs on subtypes of EVs, and showed that Homosalate increased the proportion of CD9+/CD63+ EVs while two other drugs, Dipivefrin hydrochloride and Metaraminol bitartrate, instead increased the proportion of CD9-/CD63+ EVs. Overall, nano-flow cytometry allows to reliably quantify proportions of EV subpopulations suggested by bulk analyzes of EV markers, at single EV resolution.

cell biology↗

bioRxiv2025

Fabrication of microcompartments with controlled size and shape for encapsulating active matter

In all living systems, the cytoplasm is separated from the external environment by membranes. This confinement imposes spatial constraints on the self-organization of internal components, filaments and organelles. While reconstituted systems are instrumental for understanding fundamental biological principles, traditional experiments often utilize volumes vastly larger than actual cells. In recent studies, water-in-oil droplets or giant unilamellar vesicles have been widely used to impose confinement. However, these compartments present imaging challenges and make precise protein content control difficult. To address these limitations, we have developed versatile microwells that are straightforward to implement, compatible with different types of imaging, suitable for long-term experiments, and capable of generating large amounts of data. These microwells are compatible with several surface treatments and a wide range of experimental techniques (such as micropatterning) making them a powerful tool for answering key questions in cell biology. We present here a detailed protocol of the fabrication of the microwells as well as characterization of the method to ensure quality throughout the manufacturing process. These microwells support various cytoskeleton-based processes including actin polymerization, dynamic steady-state actin networks, and composite actin-microtubule networks. More broadly, they can be used to encapsulate and study over time any kind of active matter.

biochemistry↗

bioRxiv2025

Fructose malabsorption induces dysbiosis and increases anxiety in Human and animal models

Background & AimsExcessive fructose intake is a growing public health concern, yet many individuals have a lower absorption capacity than the average intake, leading to widespread chronic fructose malabsorption. This results in intestinal fructose spillover, disrupting gut microbiota and triggering peripheral inflammation, which, along with neuroinflammation, plays a key role in mood disorders. This study investigates the connection between fructose malabsorption and mood disorders by examining gut microbiota changes in a human cohort and exploring their links with neuroinflammation in a GLUT5-KO mouse model. MethodsIn a human cohort, fructose malabsorption was assessed using a breath hydrogen test, while plasma lipopolysaccharide (LPS) levels and anxiety traits (measured using the State-Trait Anxiety Inventory, STAI) were analyzed. Gut microbiota composition was characterized through 16S rRNA sequencing, and dietary fructose intake was recorded. In the preclinical study, Glut5-KO mice, which lack intestinal fructose transport, were fed a 5% fructose diet for four weeks. Behavioral assays assessed anxiety- and depressive-like behaviors, while gut microbiota composition and microglia-associated gene expression were analyzed. ResultsAmong the recruited healthy volunteers, 60% exhibited fructose malabsorption, along with elevated plasma LPS levels, increased anxiety traits on the STAI, and distinct gut microbiota alterations, partially linked to fructose intake patterns. The average daily fructose intake was 30 g per individual, with significant variability in dietary sources. In the preclinical model, Glut5-KO mice on a 5% fructose diet displayed increased anxiety- and depressive-like behaviors, pronounced gut microbiota shifts, and altered expression of microglia-associated genes. ConclusionsThese findings highlight the complex interplay between dietary fructose, gut microbiota, and neuroinflammation in shaping mental health. Chronic fructose malabsorption may contribute to mood disorders through gut dysbiosis and microglia-dependent neuroinflammation, warranting further investigation into dietary interventions. HIGHLIGHTSO_LIFructose malabsorption is associated with anxiety traits in healthy volunteers. C_LIO_LIFructose malabsorption enhances anxiety-like behaviors in malabsorptive Glut5-KO mice. C_LIO_LIFructose malabsorption is associated with gut microbiota dysbiosis in human and preclinical mouse model of fructose malabsorption in association with fructose intake C_LIO_LIFructose malabsorption increases neuroinflammation and alters microglia functions in malabsorptive Glut5-KO mice. C_LI

microbiology↗

bioRxiv2025

Filament transport supports contractile steady states of actin networks.

In all eukaryotic cells, the actin cytoskeleton is maintained in a dynamic steady-state. Actin filaments are continuously displaced from cell periphery, where they assemble, towards the cells center, where they disassemble. Despite this constant flow and turnover, cellular networks maintain their overall architecture constant. How such a flow of material can support dynamic yet steady cellular architectures remains an open question. To investigate the role of myosin-based forces in contractile steady-states of actin networks, we used a reconstituted in vitro system based on a minimal set of purified proteins, namely actin, myosin and actin regulators. We found that, contrary to previous bulk experiments, when confined in microwells, the actin network could self-organize into ordered arrangements of contractile bundles, flowing continuously without collapsing. This was supported by three-dimensional fluxes of actin filaments, spatially separated yet balancing each other. Unexpectedly, maintaining these fluxes did not depend on filament nucleation or elongation, but solely on filament transport. Ablation of the contractile bundles abolished the flux balance and led to network collapse. These findings demonstrate that the dynamic steady state of actin networks can be sustained by filament displacement and recirculation, independently of filament assembly and disassembly. Significance StatementCellular structures continuously self-renew, with new material constantly being added and removed while maintaining overall structural stability. This is particularly true for the actin cytoskeleton, whose components are continuously assembled, displaced, and reassembled. Understanding this process is fundamental to uncovering how cells regulate their architecture and adapt to stimuli. Here, we reconstitute an in vitro actomyosin network capable of contracting steadily over time without collapsing, relying solely on myosin-based transport. These findings demonstrate that a minimal system consisting of actin and molecular motors can effectively recapitulate the ability of actin networks to self-organize into stable yet dynamic architectures.

biophysics↗

bioRxiv2025

Sex-dependent effects of intestinal epithelial TLR4 deletion induced before activity-based anorexia

RationaleA role for the microbiota-gut-brain axis in the pathophysiology of anorexia nervosa (AN) has emerged in the last decade. An alteration of intestinal Toll-like receptor type 4 (TLR4) has been reported in the activity-based anorexia (ABA) model with an increase in its expression at the cell surface of colonic epithelial cells. In addition, inducible TLR4 invalidation in intestinal epithelial cells (IECs) was associated with behavioral and energy balance changes in ABA mice. The aim of this study was to assess the intestinal response, e.g. inflammation, gut barrier function and gut microbiota composition, to TLR4 invalidation in IEC in ABA mice. MethodsMale and female Villin-CreERT2-TLR4 LoxP C57Bl/6 mice were injected with tamoxifen to induce a specific invalidation of TLR4 in IECs (TLR4IEC-/- mice). Then, wild-type (wt) and TLR4IEC-/- mice were subjected or not to the ABA protocol which combines an access to a running wheel and a progressively limited access to food. After 12 days, colon samples were collected and the expression of 44 mRNAs encoding proteins involved in inflammatory response, gut barrier function and homeostatic regulation was measured by qPCR. Results were compared by a two-way ANOVA (ABA x TLR4IEC-/-). Gut microbiota composition was analysed by 16S rRNA Illumina sequencing. ResultsIn both male and female ABA TLR4IEC-/- mice, the kinetics of body weight loss was slowed down. In addition, male and female ABA TLR4IEC-/- mice showed an increase and a decrease in food intake, respectively. In males, TLR4 invalidation in IEC was associated with a reduction of Tlr2, Ticam1, Myd88, Tnf, I{kappa}B, Irf3, Cxcr3 and Tgf{beta} mRNA expression and fecal calprotectin levels under control conditions but not in response to the ABA model. In females, Myd88, Il6, Cxcl1 and Ccl2 mRNA levels were increased by TLR4IEC invalidation in control mice but not in ABA, except for Ccl2. TLR4 invalidation also affected the expression of genes involved in gut barrier function in control and ABA mice in a sex-dependent manner. Male mice exhibited more marked alterations. For instance, male CT TLR4IEC-/- showed a decrease of numerous targets (Ocln, Marveld2, F11r, Tjp1, Cldn7, Cldn12, Cldn15). ABA TLR4IEC-/- mice did not exhibit this decrease but other changes were observed such as an increase in Cldn3 and Cldn7 mRNA levels. Finally, TLR4IEC invalidation in control mice, but not in ABA, altered the gut microbiota in a sex dependent manner with an increase in the abundance of Parasutterella and Desulfovibrio genera in females and males, respectively. Interestingly, the ABA model per se induced an increase in the abundance of the Lactobacillus genus in both sexes, which was not observed in ABA TLR4IEC-/-. ConclusionsOur study shows for the first time the impact of inducible TLR4 invalidation in IEC on the intestinal response. We highlighted numerous colonic alterations regarding epithelial permeability, mucosal inflammation and gut microbiota composition, in control and ABA conditions: all were partially reversed in ABA TLR4IEC-/-. TLR4 invalidation in IEC also induced changes in energy homeostasis in response to the ABA model both in female and male mice. Further studies are warranted to deeply evaluate the underlying mechanisms.

physiology↗

bioRxiv2024

The Arp2/3 complex maintains genome integrity and survival of epidermal Langerhans cells

Myeloid cells use intracellular actin networks for key cellular processes, including cell migration and chemotaxis, phagocytosis or macropinocytosis, as well as immune synapse formation. However, whether actin networks play any role in the development and/or survival of myeloid cells in tissues remains open. Here, we found that the Arp2/3 complex, which is responsible for the nucleation of branched actin networks, is needed for in vivo maintenance of epidermal Langerhans cells (LCs) throughout life. Mice harboring a genetic deletion of the Arpc4 subunit of the complex in myeloid cells form LC networks at birth, but these cells decline in numbers following a process reminiscent of premature cellular aging. By combining in vivo analyses of LCs with in vitro experiments on bone-marrow-derived dendritic cells, we found that Arpc4-deficient cells manage to progress through the cell cycle but accumulate DNA damage associated with aberrant nuclear shapes, lamina reduction and events of nuclear envelope rupture. These results provide the first evidence for a physiological role of Arp2/3 in maintenance of genome integrity and survival of immune cells in tissues.

cell biology↗

bioRxiv2024

Sex-dependent effects of a glutamine supplementation on metabolic disorders, intestinal barrier function and gut microbiota in mice with diet-induced obesity.

RationaleObesity is often associated with sex-dependent metabolic complications, in which altered intestinal barrier function and gut microbiota contribute. Glutamine (Gln) supplementation previously showed beneficial effects on gut barrier function and glycemic control. We thus aimed to characterize in mice the sex-dependent effects of a Gln supplementation during high fat diet induced obesity. MethodsMale and female C57BL/6 mice received a standard (SD) or high fat diet (HFD; 60% kcal from fat) during 14 weeks (W14). From W12, mice received or not Gln in drinking water (2g/kg/day; n=12/group). Body composition, glucose tolerance, insulin sensitivity, intestinal permeability, colonic expression of 44 genes encoding factors involved in inflammatory response and gut barrier function, cecal microbiota and inflammatory/endocrine adipose response have been assessed. Data were analyzed using t-test or Mann-Whitney test (HFD effect), and two-way ANOVA (HFD x Gln) followed by Bonferroni post-tests. ResultsIn both male and female mice, Gln supplementation failed to improve body weight and body composition. However, Gln reduced glucose intolerance in HFD males (AUC reduced by 14.57%, p<0.05) that was associated to a partial restoration of plasma resistin and insulin and to a trend for a limitation of adipose inflammatory response. In males, Gln did not affect gut microbiota composition and colonic response. To the opposite, in females fed HFD, Gln supplementation led to gut microbiota changes (increase of Bacteroidota and Pseudomonadota phyla; increase of Muribaculaceae and Tannerellaceae families), increased colonic inflammatory markers (TNF, IL-1{beta}, TLR4, Myd88, Irf3) that were associated to increased inflammatory response in subcutaneous adipose tissue and increased HOMA-IR. ConclusionsHigh fat diet mice exhibit sex-dependent response to glutamine supplementation with protective effects in males and harmful effects in females. The role of gut microbiota should be deeply deciphered in further investigations.

pathology↗

bioRxiv2024

Sex-dependent effects of a high-fat diet on the hypothalamic response in mouse

Sex differences in rodent models of diet-induced obesity are still poorly documented, particularly regarding how central mechanisms vary between sexes in response to an obesogenic diet. Here, we wanted to determine whether obesity phenotype and hypothalamic response differed between male and female C57Bl/6J mice when exposed to a high-fat diet (HFD). Mice were exposed either a free 60% HFD or standard diet first for both a long-(14 weeks) and shorter-periods of time (3, 7, 14 and 28 days). Analysis of the expression profile of key neuronal, glial and inflammatory hypothalamic markers was performed using RT-qPCR. In addition, astrocytic and microglial morphology was examined in the arcuate nucleus. Monitoring of body weight and composition revealed that body weight and fat mass gain appeared earlier and was more pronounced in male mice. After 14 weeks of HFD exposure, normalized increase of body weight reached similar levels between male and female mice. Overall, both sexes under HFD displayed a decrease of orexigenic neuropeptides expression and an increase in POMC gene expression was observed only in female mice. In addition, changes in the expression of hypothalamic inflammatory markers were relatively modest. We also reported that the glial cell markers expression and morphology were affected by HFD in a sex- and time dependent manner, suggesting a more pronounced glial cell activation in female mice. Taken together, these data show that male and female mice responded differently to HFD exposure, both on short- and long-term and suggest that a strong inflammatory hypothalamic profile is not systematically present in DIO models. Nevertheless, in addition to these present data, the underlying mechanisms should be deciphered in further investigations.

neuroscience↗

bioRxiv2024

Balancing limited resources in actin networks competition

In cells, multiple actin networks coexist in a dynamic manner. These networks compete for a common pool of actin monomers and actin-binding proteins. Interestingly, this competition does not result in the mere survival of the more consuming networks. Moreover, the co-existence of networks with various strengths is key to cell adaption to external changes. However, a comprehensive view of how these networks coexist in this competitive environment, where resources are limited, is still lacking. To address this question, we used a reconstituted system, in closed microwells, consisting of beads propelled by actin polymerization or micropatterns functionalized with lipids capable of initiating polymerization close to a membrane. This system enabled us to build dynamic actin architectures, competing for a limited pool of proteins, over a period of hours. We demonstrated the importance of protein turnover for the coexistence of actin networks, showing it ensures resource distribution between weak and strong networks. However, when competition becomes too intense, turnover alone is insufficient, leading to a selection process that favors the strongest networks. Consequently, we emphasize the importance of competition strength, which is defined by the turnover rate, the amount of available protein, and the number of competing structures. More generally, this work illustrates how turnover allows biological populations with various competition strengths to coexist despite resource constraints. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/612098v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@370343org.highwire.dtl.DTLVardef@1b75a2forg.highwire.dtl.DTLVardef@81e55dorg.highwire.dtl.DTLVardef@1f97452_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cell biology↗

bioRxiv2023

The mutational landscape of Bacillus subtilis conditional hypermutators suggests how proofreading inherently skews polymerase error rates

Polymerase errors during DNA replication are a major source of point mutations in genomes. The resulting rate of spontaneous mutation also depends on the counteracting activity of DNA repair mechanisms, with mutator phenotypes appearing constantly and allowing for periods of rapid evolution in nature and in the laboratory. Here, we use the Gram-positive model bacterium Bacillus subtilis to disentangle the contributions of DNA polymerase initial nucleotide selectivity, DNA polymerase proofreading, and mismatch repair (MMR) to the mutation rate. To achieve this, we constructed several conditional hypermutators with a proofreading-deficient allele of polC and/or a deficient allele of mutL and performed mutation accumulation experiments. With their wide range of mutation rates and contrasting mutation profiles, these conditional hypermutators enrich the B. subtilis synthetic biology toolbox for directed evolution. Using mathematical models, we investigated how to interpret the apparent probabilities with which errors escape MMR and proofreading, highlighting the difficulties of working with counts that aggregate potentially heterogeneous mutations and with unknowns about the pathways leading to mutations in the wild-type. Aware of these difficulties, the analysis shows that proofreading prevents partial saturation of the MMR in B. subtilis and that an inherent drawback of proofreading is to skew the net polymerase error rates by amplifying intrinsic biases in nucleotide selectivity.

genomics↗

bioRxiv2023

Microtubules under mechanical pressure can breach dense actin networks

The crosstalk between actin network and microtubules is key to the establishment of cell polarity. It ensures that the asymmetry of actin architec ture along cell periphery directs the organization of microtubules in cell interior. In particular, the way the two networks are physically inter-twined regulates the spatial organization and the distribution of forces in the microtubule network. While their biochemical crosstalk is getting uncovered, their mechanical crosstalk is still poorly understood. Here we designed an in vitro reconstitution assay to study the physical interaction between dynamic microtubules with various structures made of actin fil aments. We found that microtubules can align and move by their polymerization force along linear bundles of actin filaments. But they cannot enter dense and branched actin meshworks, such as those found in the lamellipodium along cell periphery. However, when microtubules are immobilized, by their crosslinking with actin structures or others means, the force of polymerization builds up pressure in the microtubules that is sufficient to allow them to breach and penetrate these dense actin meshworks. This mechanism may explain the final progression of microtubules up to cell periphery through the denser parts of the actin network.

biophysics↗

bioRxiv2023

Tunable DNMT1 degradation reveals cooperation of DNMT1 and DNMT3B in regulating DNA methylation dynamics and genome organization

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present untransformed and cancer cell models that allow inducible and reversible global modulation of DNAme through DNMT1 depletion. By dynamically assessing the effects of induced passive demethylation through cell divisions at both the whole genome and locus-specific level, we reveal a cooperative activity between DNMT1 and DNMT3B to maintain and control DNAme. Moreover, we show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin abundance, compartmentalization, and peripheral localization. DNA methylation loss coincided with a gradual reduction of cell fitness due to G1 arrest, but with minor level of mitotic failure. Altogether, this powerful system allows DNMT and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNA methylation dysfunction and human disease.

molecular biology↗