Biology subjects

Riviere, C.

Publications and source records attributed to Riviere, C..

12 recordsLinked to original sources

bioRxiv2026

Dynamic membrane organization coordinates protein-O-mannosylation and proteostasis in mycobacteria.

Integral membrane enzymes operate within molecular environments, yet how these are organized remains poorly understood. Using M. smegmatis as a mycobacterial model, we define the membrane environment of the protein O-mannosyltransferase MtPMT through stratified interactomics combining affinity purification-mass spectrometry under four stringencies with a persistence score. This approach resolves a persistent membrane-associated core of 108 proteins from more transient layers, revealing a hierarchically organized landscape enriched in Sec protein translocation, protein biogenesis and envelope proteostasis machineries. Reciprocal affinity purification and in vivo FRET identify YajC and FtsH as neighbors of MtPMT, while structural modelling supports integration within a Sec-associated assembly. Genetic disruption of factors reduces O-mannosylation activity, demonstrating dependence on platform integrity. The MtPMT-associated network is remodelled during growth, indicating an adaptive system. Loss of POM compromises envelope permeability and stress resilience, linking O-mannosylation to fitness. Together, our results establish protein O-mannosylation as an integrated component of a dynamic membrane platform coordinating secretion, membrane protein biogenesis and proteostasis. Our findings support a model in which mycobacterial membranes contain specialized domains dynamically modulated by physiological state.

cell biology↗

bioRxiv2026

CurvoChip: a programmable dynamic curvature-on-chip platform for epithelial mechanobiology

Epithelial tissues continuously remodel their curvature during morphogenesis, homeostasis, regeneration, and disease, yet experimental access to time-varying curvature remains limited. Here, we introduce CurvoChip, a pneumatically actuated microsystem that reversibly deforms confluent epithelial monolayers cultured on a 20-m elastic membrane into concave or convex geometries. The device operates either in a standard incubator or on a microscope stage and provides programmable control over pressure amplitude, direction, and cycling. Analytical scaling, finite-element simulations, and confocal profilometry establish predictable membrane deformation across the operating range, whereas cycling between -400 and +400 mbar for 120 cycles produces stable deflection without detectable drift or residual deformation. We further implement a three-dimensional surface-reconstruction and segmentation workflow to quantify cell and nuclear morphology on curved monolayers. Acute curvature induction produces a marked polarity-dependent response: convex deformation causes greater cell spreading and epithelial thinning than concave deformation, while nuclear projected area, thickness, and volume change in a direction- and position-dependent manner. These results show that epithelial architecture is sensitive not only to curvature magnitude but also to its orientation relative to the apico-basal axis. CurvoChip therefore provides an accessible platform for dissecting how epithelial tissues integrate dynamic geometric cues.

biophysics↗

bioRxiv2026

Integrating 3D Tumor Models and Microfluidics for Precise Metabolic Control

Understanding how metabolic deprivation shapes tumor behavior requires in vitro systems that faithfully reproduce millimeter-scale biochemical gradients. Here, we introduce MilliFlow3D, an open hydrogel-based microfluidic platform that generates controlled metabolic gradients and supports in situ spheroid formation, real-time imaging, and intact retrieval for spatial analyses. Gradients are generated by passive diffusion across a structured agarose microwell array positioned between two perfusion channels. Using a fluorescent tracer and numerical simulations, we show that MilliFlow3D establishes stable linear gradients with local metabolite levels matching those reported in avascular tumor regions. Using HCT116 colorectal cancer spheroids, we demonstrate that a L-glutamine gradient imposes graded effects on growth and proliferation: spheroid expansion decreases from high- to low-glutamine regions, and Ki-67-positive cells progressively shift toward the periphery under glutamine depletion. Thanks to the platforms spatial accessibility, these phenotypic responses could in the future be coupled to molecular readouts, enabling spatial mapping of metabolic pathway activity along the gradient. Altogether, MilliFlow3D provides a robust and versatile platform to investigate how heterogeneous metabolic landscapes sculpt tumor behavior and to identify context-dependent metabolic vulnerabilities with high analytical precision.

bioengineering↗

bioRxiv2025

Transient volume stabilization reveals the key role a PM permeability in pyroptotic cell swelling.

Pyroptosis, an inflammatory form of cell death, is characterized by massive cell swelling and plasma membrane rupture. Although swelling was recently shown to occur in two steps, the molecular and biophysical mechanisms driving this process remained unclear. Using fast quantitative microscopy, we reveal that between the two swelling phases, cell volume transiently stabilizes despite sustained plasma membrane permeability to ions and small molecules. From a biophysical perspective, the existence of such a plateau is puzzling, as ion pumps should not be able to regulate cell volume under these conditions. To address this, we developed a physical model based on an ion pump and leak framework that incorporates the dynamics of non-selective pore formation. Experimentally, we demonstrate that the plateau phase is controlled by the dynamics of the GSDMD pore enlargement, which is modulated by Ninj1 activation, possibly through intracellular calcium. Ninj1-mediated lesions are also required for the second swelling phase. We further show that fully opened GSDMD pores display an effective hydrodynamic radius slightly above 1.9 nm, providing an insitu upper bound for pore size. Together, our findings demonstrate that pyroptotic volume dysregulation emerges from the successive and interdependent actions of GSDMD and Ninj1, each imparting distinct permeability regimes associated with increased water filtration and decreased ion selectivity due to pore opening. These insights bridge molecular and biophysical perspectives on lytic cell death and may inform the broader understanding of membrane rupture in inflammatory and pathological contexts. Significance StatementAmong programmed modes of lytic cell death, pyroptosis mediated by gasdermin D (GSDMD) and ninjurin-1 (Ninj1) involves dramatic changes in cell shape and large volume fluctuations, fundamentally altering the cells physical properties. By combining optogenetics, quantitative microscopy, and modeling, we show that a progressive increase in plasma membrane pore size drives cell swelling and membrane lysis through successive and interdependent actions of GSDMD and Ninj1, each imparting distinct permeability regimes associated with increased water filtration and decreased ion selectivity. A deeper understanding of these dynamic cell modifications will shed light on the molecular and biophysical mechanisms driving different forms of cell death.

biophysics↗

bioRxiv2024

MAJOR METABOLITES FROM HYPERICUM PERFORATUM L., HYPERFORIN AND HYPERICIN, ARE BOTH ACTIVE AGAINST HUMAN CORONAVIRUSES

COVID-19 pandemic has highlighted the need of antiviral molecules against coronaviruses. Plants are an endless source of active compounds. In the current study, we investigated the potential antiviral effects of Hypericum perforatum L.. Its extract contained two major metabolites belonging to distinct chemical classes, hypericin (HC) and hyperforin (HF). First, we demonstrated that HC inhibited HCoV-229E at the entry step by directly targeting the viral particle in a light-dependent manner. While antiviral properties have already been described for HC, the study here showed for the first time that HF has pan-coronavirus antiviral capacity. Indeed, HF was highly active against Alphacoronavirus HCoV-229E (IC50 value of 1.10 {micro}M), and Betacoronaviruses SARS-CoV-2 (IC50 value of of 0.24 to 0.98 {micro}M), SARS-CoV (IC50 value of 1.01 {micro}M) and MERS-CoV (IC50 value of 2.55 {micro}M). Unlike HC, HF was active at a post-entry step, most likely the replication step. Antiviral activity of HF on HCoV-229E and SARS-CoV-2 was confirmed in primary human respiratory epithelial cells. Furthermore, in vitro combination assay of HF with remdesivir showed that their association was additive, which was encouraging for a potential therapeutical association. As HF was active on both Alpha- and Betacoronaviruses, a cellular target was hypothesized. Heme oxygenase 1 (HO-1) pathway, a potential target of HF, has been investigated but the results showed that HF antiviral activity against HCoV-229E was not dependent on HO-1. Collectively, HF is a promising antiviral candidate in view of our results and pharmacokinetics studies already published in animal models or in human.

microbiology↗

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↗

bioRxiv2023

Mitosis down-regulates nuclear volume and resets nuclear envelope folding of cancer cells under prolonged confinement

During their life, mammalian cells are subjected to numerous mechanical constraints, especially in pathological contexts such as cancer. Recent studies have highlighted the central role of the nucleus in sensing mechanical cues, but they only focus on short periods of time, and so far, whether cells can adapt to prolonged confinement remains unknown. Here, we reveal the unsuspected role of mitosis in the long-term adaptation of nuclei to prolonged uniaxial confinement. For the colorectal cancer cell line investigated, following the first confined cell division, a new homeostatic state was reached by nuclei: they were smaller, and had reset the tension of their envelope. This adaptation through mitosis relied both on the nuclear tension sensor cPLA2 and the contractility machinery. We report for the first time a mechano-adaptation during mitosis, a process that could be crucial to adapt to stresses in the tumor microenvironment. We therefore anticipate that our work could provide new insight into cancer cell plasticity and cancer relapse. Significance StatementMost cell types undergo significant deformation throughout their life cycles. Immune cells must deform to navigate through dense matrices, while cancer cells in solid tumors experience squeezing from neighboring cells. The nucleus, central for many cell function, is the stiffest and largest organelle. Understanding its long-term response to spatial constraints is hence crucial yet largely unexplored. In this study, we investigate how a colorectal cancer cell line adapts to prolonged confined environments, with a particular focus on nuclear dynamics under continuous squeezing. Our groundbreaking findings reveal for the first time a mechano-adaptation during mitosis leading to a decrease in nuclear size. This research contributes to the fundamental understanding of cellular mechanosensing, opening new avenues for cancer biology research.

biophysics↗

bioRxiv2021

Clearing spheroids for 3D fluorescent microscopy:combining safe and soft chemicals with deep convolutional neural network

In life sciences, there are increasing interest in 3D culture models to better reproduce the 3D environment encountered in-vivo. Imaging of such 3D culture models is instrumental for drug discovery, but face several issues before its use becomes widespread. Extensive microscopic investigation of these 3D cell models faces the challenge of light penetration in depth in opaque biological tissues. To overcome this limit, diverse clearing techniques have emerged over the past decades. However, it is not straightforward to choose the best clearing protocols, and assess quantitatively their clearing efficiency. Focusing on spheroids, we propose a combination of fast and cost-effective clearing procedure for such medium-sized samples. A generic method with local contrast metrics and deep convolutional neural network-based segmentation of nuclei is proposed to quantify the efficiency of clearing. We challenged this method by testing the possibility to transfer segmentation knowledge from a clearing protocol to another. The later results support the pertinence of training deep learning algorithms on cleared samples to further use the segmentation pipeline on non-cleared ones. This second step of the protocol gives access to digital clearing possibilities applicable to live and high-throughput optical imaging.

bioinformatics↗

bioRxiv2021

Quantifying nanotherapeutics penetration using hydrogel based microsystem as a new 3D in-vitro platform

The huge gap between 2D in vitro assays used for drug screening, and the in vivo 3D-physiological environment hampered reliable predictions for the route and accumulation of nanotherapeutics in vivo. For such nanotherapeutics, Multi-Cellular Tumour Spheroids (MCTS) is emerging as a good alternative in vitro model. However, the classical approaches to produce MCTS suffer from low yield, slow process, difficulties in MCTS manipulation and compatibility with high-magnification fluorescent optical microscopy. On the other hand, spheroid-on-chip set-ups developed so far require a microfluidic practical knowledge difficult to transfer to a cell biology laboratory. We present here a simple yet highly flexible 3D-model microsystem consisting of agarose-based microwells. Fully compatible with the multi-well plates format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging including live high-resolution optical microscopy can be performed in-situ, with no manipulation of spheroids. As a proof-of-principle of the relevance of such in vitro platform for nanotherapeutics evaluation, this study investigates the kinetic and localization of nanoparticles within colorectal cancer MCTS cells (HCT-116). The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI (AGuIX(R), currently implicated in clinical trials as effective radiosensitizers for radiotherapy) and confocal microscopy after addition of Cy 5.5. We show that the amount of AGuIX(R) nanoparticles within cells is largely different in 2D and 3D. Using our flexible agarose-based microsystems, we are able to resolve spatially and temporally the penetration and distribution of AGuIX(R) nanoparticles within MCTS. The nanoparticles are first found in both extracellular and intracellular space of MCTS. While the extracellular part is washed away after few days, we evidenced intracellular localisation of AGuIX(R), mainly within lysosomes compartment, but also occasionally within mitochondria. Our agarose-based microsystem appears hence as a promising 3D in vitro user-friendly platform for investigation of nanotherapeutics transport, ahead of in vivo studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/427020v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@bd48adorg.highwire.dtl.DTLVardef@ee7502org.highwire.dtl.DTLVardef@143ee60org.highwire.dtl.DTLVardef@d6f5ca_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

bioengineering↗

bioRxiv2020

Mechanical-control of cell proliferation increases resistance to chemotherapeutic agents

While many cellular mechanisms leading to chemotherapeutic resistance have been identified, there is an increasing realization that tumor-stroma interactions also play an important role. In particular, mechanical alterations are inherent to solid cancer progression and profoundly impact cell physiology. Here, we explore the impact of compressive stress on the efficacy of chemotherapeutics in pancreatic cancer spheroids. We find that increased compressive stress leads to decreased drug efficacy. Theoretical modeling and experiments suggest that mechanical stress leads to decreased cell proliferation which in turn reduces the efficacy of chemotherapeutics that target proliferating cells. Our work highlights a mechanical-form of drug resistance, and suggests new strategies for therapy.

cancer biology↗

bioRxiv2020

Development of a soft cell confiner to decipher the impact of mechanical stimuli on cells

Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all tested cell lines. The soft cell confiner is thus a valuable tool to decipher the effect of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY A unique tool to analyze the role of long-term effect of mechanical confinement in normal and pathological conditions

biophysics↗

bioRxiv2018

Dehydrojuncusol, a Natural Phenanthrene Compound Extracted from Juncus maritimus Is a New Inhibitor of Hepatitis C Virus Replication

Recent emergence of direct acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, search for new inhibitors and with lower cost of production should be pursued. In this context, crude extract of Juncus maritimus Lam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed isolating and identifying the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (EC50 = 1.35 {micro}M). This antiviral activity was confirmed with a HCV subgenomic replicon and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. No in vitro toxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit replication of two frequent daclatasvir resistant mutants (L31M or Y93H in NS5A). Finally, resistant mutants to dehydrojuncusol were obtained and showed that HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted from J. maritimus, inhibits infection of different HCV genotypes by targeting NS5A protein and is active against HCV resistant variants frequently found in patients with treatment failure.\n\nImportanceTens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of the treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to appearance of viral resistance. Here we report on the identification of a new HCV inhibitor, dehydrojuncusol that targets HCV NS5A and is able to inhibit replication of replicons harboring resistance mutations to anti-NS5A \nDAAs used in current therapy. Dehydrojuncusol is a natural compound isolated from Juncus maritimus, a halophilic plant species very common in the coastlines worldwide. This molecule might serve as a lead for the development of new therapy more accessible to hepatitis C patients in the future.

microbiology↗