Biology subjects

Delarue, M.

Publications and source records attributed to Delarue, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Directed evolution of a thermostable family-A DNA polymerase into a reverse transcriptase for 2'-modified RNA

RNA-based therapeutics increasingly rely on nuclease-resistant xeno-nucleic acids (XNAs), but engineering polymerases capable of reading and amplifying these non-canonical substrates remains a major challenge. Natural DNA polymerases cannot process C2' sugar modifications, and the complete structural determinants governing this limitation remain poorly understood, limiting rational design strategies. In this study, we apply directed evolution methods to Thermus thermophilus DNA polymerase using a microfluidic implementation of the Compartmentalized Self-Replication (CSR). We identify an engineered variant, termed E5, capable of reverse transcribing C2'-modified RNA, a previously inaccessible activity for this enzyme. Next-generation sequencing allow us to track evolutionary trajectories across nine rounds of evolution. Through an integrated analysis combining statistical methods, protein language models, and crystallographic data, we reveal unknown epistatic networks controlling polymerase function. This large-scale experimental dataset (>196,000 thermostable enzyme variants) provides a robust basis for predicting sequence-to-function relationships, accelerating development of enzymes for aptamer discovery and synthetic biology applications.

synthetic biology↗

bioRxiv2026

Microfluidic Devices for Imaging and Biochemistry Analysis of Microbes Under Mechanical Pressure

Growth-induced pressure arises when proliferating cell populations are confined within rigid microenvironments and is increasingly recognized as a determinant of microbial physiology in soils, biofilms, and host tissues. We present two complementary microfluidic devices that confine microbes within polydimethylsiloxane (PDMS) chambers and apply defined, optically read-out growth-induced pressure of up to 1.5 MPa. The first, the self-closing (SC) chip, confines cells in multiple small chambers accommodating hundreds of cells; it provides excellent nutrient supply and rapid medium exchange, supports single-cell imaging, and allows multiplexing of cellular or chemical conditions. The second, the pressure-recovery (PR) chip, confines cells in a single 5 cm-long chamber accommodating hundreds of thousands of cells; a scalpel-cut step recovers live cells from the channel within minutes for bulk biochemical assays. The two devices share a single two-layer soft-lithography fabrication process and a common brightfield wall-displacement pressure readout. Together, they enable single-cell imaging and bulk biochemical analysis under matched, defined pressure conditions. We illustrate the protocol with two representative validations: rapid {beta}-estradiol-induced transcription in the SC chip, in which nascent transcription foci appear within 5 min independently of the applied pressure, and the PR chip, which produces uniform growth-induced pressure along the entire 5 cm channel and recovers between 0.1x10^6 and 1x10^6 cells per device in a pressure-tunable manner.

biophysics↗

bioRxiv2026

Cellular integration of surface and nutrient signals during the early stage of filamentous growth in Saccharomyces cerevisiae

Budding yeast cells can transition from vegetative to filamentous growth under specific environmental stimuli. Saccharomyces cerevisiae has served as a model to understand this process, which is linked to the virulence of many fungal pathogens. One manifestation of filamentation in S. cerevisiae is the formation of pseudohyphal cells, which are elongated and divide in a polarized manner. Here, we established a live-cell imaging assay to monitor this morphogenetic transition and developed a set of fluorescent expression reporters to probe the activity of various signaling pathways during the first 10 hours of this transition. This approach allowed us to identify the stimulus that activates the filamentous growth Mitogen-Activated Protein Kinase (fgMAPK) pathway, which has long evaded identification. Our results indicate that the fgMAPK cascade senses mechanical cues and becomes activated when cells grow on a solid surface. In addition, we have observed that the stimulation of the transcription factor Gln3 regulated by the TOR pathway in low ammonium condition correlates with the elongation of the cells.

cell biology↗

bioRxiv2026

Structural basis for continuous DNA-end protection during ligation of double-strand breaks in yeast Non-Homologous End-Joining

Non-homologous end joining (NHEJ) repairs DNA double-strand breaks by synapsing and ligating DNA ends. In vertebrates, DNA-PKcs promotes end alignment and processing, yet several eukaryotes - including budding yeast - perform NHEJ without DNA-PKcs, and the underlying mechanism remains unclear. Here we report cryo-electron microscopy structures of reconstituted Saccharomyces cerevisiae NHEJ synaptic complexes assembled on DNA ends bearing either 4-bp terminal microhomologies or blunt termini. On the microhomology substrate with a single 5-P, we capture a ligation-competent short-range complex in which a single Dnl4 catalytic core is fully ordered and engaged at the 5'-adenylated nick. When two 5-P are present, we resolve two predominant, coexisting DNA-aligned protective states in which both Dnl4 DBD-NTD modules occupy the break region with reciprocal geometries, supporting an alternating engagement model for sequential sealing of the two strands while maintaining continuous end association. In contrast, blunt-ended substrates yield a non-aligned protective configuration in which two Dnl4 catalytic modules constrain the DNA ends [~]30 [A] apart in a ligation-incompatible arrangement, providing a structural explanation for slow blunt-end joining in yeast. Together, these structures define architectural and mechanistic principles of DNA-PKcs-independent NHEJ.

molecular biology↗

bioRxiv2026

Selective Laser Etching technology for reconfigurable microfluidic and electrochemistry-on-chip

Polydimethylsiloxane (PDMS) is widely used in academic microfluidics due to its favorable biocompatible properties and compatibility with soft lithography. Moreover, the recent developments of reconfigurable microfluidics rely on the microfabrication of sliding elements, which are 3D objects insertable inside a microfluidic chip to provide a given function. However, the complexity of microfluidic device geometries or sliding elements remains largely limited by the traditional microfabrication methods such as, among others, on SU-8 photolithography or dry epoxy films. Such methods are suited for simple, planar "2.5D" structures with uniform depths, yet struggle to produce more advanced architectures due to material and alignment constraints. To address these limitations, we propose to investigate advanced laser-based approaches, such as Direct Laser Writing (DLW) and Selective Laser Etching (SLE), which enable the construction of high-resolution, 3D designs. We further explore the use of SLE to fabricate sliding elements that enhance chip functionality, including chamber reconfiguration and biological sample manipulation. As a proof of concept, we show that these elements can be functionalized into pH microsensors, paving the way to reusable and reconfigurable electrochemistry-on-chip.

biophysics↗

bioRxiv2025

Mutants of p53 sustain tumor growth under mechanical compression

ContextSolid tumors are subjected to mechanical stimuli arising from their growth in confined environments. Growth-induced pressure builds up in tumors such as pancreatic cancer and rises alongside the occurrence of genetic alterations during tumorigenesis. This study aims to understand the so far unknown relationship between genetic alterations and cancer cell behavior under compressive stress. ResultsUsing isogenic cell lines with engineered p53 mutations, we showed that the p53 background influences cell response to compression. Tumor growth under compression increased in cells harboring a mutated-truncated p53 form. This mutation blocked caspase 3 cleavage and promoted survival and growth through PI3K-AKT activation and dysregulation of c-FOS and FOSB transcription factors network. Mutated-truncated p53 cells displayed a unique behavior and heightened an activation state under compression. ConclusionMechanical compression and p53 mutations together drive tumor growth. p53 status could be a biomarker for predicting tumor adaptation to mechanical stress and efficiency of therapies targeting mechanosensitive pathways. TeaserMechanical compression and p53 mutations together enhance cancer cell survival and growth, driving solid tumor progression.

cancer biology↗

bioRxiv2025

Proportional modulation of proliferation and motility under 2D compressive stress depends on mesenchymal phenotype

Tumor development is accompanied by strong physico-chemical modifications. Among them, compressive stress can emerge in both the epithelial and stromal compartments. Using a simple two-dimensional compression assay which consisted in placing an agarose weight on top of adherent cells, we studied the impact of compressive stress on cell proliferation and motility in different pancreatic cancer cell lines. We observed a proportional reduction of both proliferation and motility in all tested cell types, with genotypes displaying a more "mesenchymal" phenotype (high velocity-to-proliferation ratio) and others related to a more "epithelial" phenotype (low velocity-to-proliferation ratio). Moreover, "mesenchymal" cells seemed more sensitive to compression, a result that was further suggested by a TGF{beta}1 induction of epithelial-to-mesenchymal transition. Finally, we measured that the change in cell proliferation was associated with a change in intracellular macromolecular crowding, which could modulate a plethora of biochemical reactions. Our results together suggest a mechanism in which all biochemical reactions related to proliferation and motility can be modulated by a change in macromolecular crowding, itself depending on the phenotype, leading to differential sensitivity to pressure.

biophysics↗

bioRxiv2025

Structure and dynamics of a multidomain ligand-gated ion channel revealed under acidic conditions

Pentameric ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution, including key targets of biophysical studies and therapeutic development. However, the intrinsically allosteric, polymodal, modular nature of gating in this protein family presents persistent challenges to biophysical characterization. The bacterial channel DeCLIC constitutes a provocative model system for structure, function, and dynamics in this family, including a modulatory N-terminal domain (NTD). Previous closed structures of DeCLIC support a rationale for its inhibition by calcium via a site of conserved relevance in other family members; however, mechanisms of gating in DeCLIC and properties of its open state have remained unclear. Here we integrated structure-function methods including cryogenic electron microscopy (cryo-EM), molecular dynamics simulations and small-angle neutron scattering under acidic conditions to characterize a previously unreported conformation of DeCLIC with a fully hydrated pore. In contrast to previous structures, the low-pH open conformation captured by cryo-EM was stable and permeable in simulations, and consistent with solution-phase behavior as measured by small-angle scattering. We further captured an alternative closed state of the channel, evidently promoted by depletion of modulatory calcium at low pH, exhibiting dynamic rearrangements in the N-terninal domain. The expanded-pore structure evidently corresponds to a functional open state of DeCLIC, while calcium-site and NTD dynamics drive channel closure, providing a detailed template for biophysical characterization of modulatory mechanisms in ligand-gated ion channels and related systems. Significance StatementThe bacterial protein DeCLIC is a provocative representative of the pentameric ligand-gated ion-channel family. Like its eukaryotic relatives, DeCLIC is sensitive to external calcium, and incorporates a possibly disordered modulatory domain. But as for many ion channels, the details of DeCLIC activation have remained unclear, as no stable open state has been characterized. Combining biophysical methods, including cryogenic electron microscopy, molecular dynamics simulations, and small-angle neutron scattering, allows us to capture, validate, and characterize an evidently open state of DeCLIC, as well as a closed state with a disordered modulatory region. Details of these structures, their function, and their dynamics allow us to propose a mechanism for DeCLIC gating, as well as a generalized scheme for the larger protein family.

biophysics↗

bioRxiv2024

Dynamic cytoplasmic fluidity during morphogenesis in a human fungal pathogen

The molecular crowding of the cytoplasm impacts a range of cellular processes. Using a fluorescent microrheological probe (GEMs), we observed a striking decrease in molecular crowding during the yeast to filamentous growth transition in the human fungal pathogen Candida albicans. This decrease in crowding is due to a decrease in ribosome concentration that results in part from an inhibition of ribosome biogenesis, combined with an increase in cytoplasmic volume; leading to a dilution of the major cytoplasmic crowder. Moreover, our results suggest that inhibition of ribosome biogenesis is a trigger for C. albicans morphogenesis.

cell biology↗

bioRxiv2024

Template-Independent Enzymatic RNA Synthesis

A route to prepare ribonucleoside triphosphates featuring a 3-aminoxy (3-O-NH2) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Pol{theta}) and mimiviral PrimPol, accept these triphosphates as substrates to add single nucleotides to an RNA primer under engineered conditions. Cleaving the O-N bond in the 3-O-NH2 group within the extended primer regenerates the 3-OH group, facilitating subsequent polymerase cycles that add a second, selected, nucleotide. These enzymes and triphosphates together enable template-independent enzymatic RNA synthesis (TIERS) exploiting a cyclic reversible termination framework. The study shows that this process is ready for instrument adaptation by using it to add three ribonucleotides in three cycles using an engineered Pol{theta}. This work creates a new way to synthesize RNA with a de novo defined sequence, without requiring the protecting groups, hazardous solvents, and sensitive reagents that bedevil phosphoramidite-based RNA synthesis.

synthetic biology↗

bioRxiv2024

Bacterial growth under confinement requires transcriptional adaptation to resist metabolite-induced turgor pressure build-up

Bacterial proliferation often occurs in confined spaces, during biofilm formation, within host cells, or in specific niches during infection, creating mechanical constraints. We investigated how spatial confinement and growth-induced mechanical pressure affect bacterial physiology. Here, we found that, when proliferating in a confining microfluidic-based device with access to nutrients, Escherichia coli cells generate forces in the hundreds of kPa range. This pressure decouples growth and division, producing shorter bacteria with higher protein concentrations. This leads to cytoplasmic crowding, which ultimately arrests division and stalls protein synthesis. In this arrested state, the pressure produced by bacteria keeps increasing. A minimal theoretical model of bacterial growth predicts this novel regime of steady pressure increase in the absence of protein production, that we named overpressurization. In this regime, the Rcs pathway is activated and that abnormal shapes appear in rcs mutant populations only when they reach the overpressurized state. A uropathogenic strain of E. coli displayed the same confined growth phenotypes in vitro and requirement for Rcs in a mice model of urinary tract infection, suggesting that these pressurized regimes are relevant to understand the physiopathology of bacterial infections.

microbiology↗

bioRxiv2024

Intracellular dry mass density increases under growth-induced pressure

Cells that proliferate in confined environments develop mechanical compressive stress, referred to as growth-induced pressure, which inhibits growth and division across various organisms. Recent studies have shown that in these confined spaces, the diffusivity of intracellular nanoparticles decreases. However, the physical mechanisms behind this reduction remain unclear. In this study, we use quantitative phase imaging to measure the refractive index and dry mass density of Saccharomyces cerevisiae cells proliferating under confinement in a microfluidic bioreactor. Our results indicate that the observed decrease in diffusivity can be at least attributed to the intracellular accumulation of macromolecules. Furthermore, the linear scaling between cell content and growth-induced pressure suggests that the concentrations of macromolecules and osmolytes are maintained proportionally under such pressure in S. cerevisiae.

biophysics↗

bioRxiv2024

Mechanisms Ensuring Fidelity of Family X DNA Polymerases in Programmed DNA rearrangements in Paramecium tetraurelia

ABSTRACTRepairing programmed DNA double-strand breaks (DSBs) is crucial in the lifecycle of Paramecium tetraurelia, especially during its sexual reproduction phase when its somatic highly polyploid macronucleus is lost. The formation of a new macronucleus involves Programmed Genome Rearrangements, introducing DNA DSBs at approximately 45,000 loci. P. tetraurelia employs a Non-Homologous End Joining (NHEJ)-related mechanism for the systematic repair of these DSBs. Four genes encoding DNA polymerases of family X are present in the genome, one of which was found recently to colocalize with other proteins of NHEJ. The question arises as to how they make almost no error. Here we show that these enzymes are most similar to metazoan DNA polymerase {lambda} and exhibit high fidelity through two different molecular mechanisms. Using X-ray structure determination of polymerase lambda mutants recapitulating sequence determinants of P. tetraurelia PolXs, we find both a local conformational change that involves exchanging partners in a crucial salt bridge in the active site upon binding of correct dNTPs, and a larger conformational change involving the closure of Loop3. This stabilizes the template DNA in the active site, only in the presence of the correct incoming dNTP. Differences with human pol {lambda} and pol {beta} are discussed. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/605286v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@b44352org.highwire.dtl.DTLVardef@afd472org.highwire.dtl.DTLVardef@1a4a77eorg.highwire.dtl.DTLVardef@f05fc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Vestibular modulation by stimulant derivatives in a pentameric ligand-gated ion channel

Allosteric modulation of pentameric ligand-gated ion channels (pLGICs) is critical to the action of neurotransmitters and many psychoactive drugs. However, details of their modulatory mechanisms remain unclear, especially beyond the orthosteric neurotransmitter-binding sites. The recently reported prokaryotic channel sTeLIC, a pH-gated homolog of eukaryotic receptors in the pLGIC family, is thought to be modulated by aromatic compounds via a relatively uncharacterized modulatory site in the extracellular vestibule. Here, we show that sTeLIC is sensitive to potentiation by psychostimulant derivatives. By determining new cryo-EM and X-ray structures in closed and open states, and testing the impact of targeted mutations on electrophysiological behavior, we show that several amphiphilic compounds preferentially bind a vestibular pocket in the contracted open-state extracellular domain. This work provides a detailed structure-function mechanism for allosteric potentiation via a noncanonical lig- and site, with potential conservation in eukaryotic pentameric ligand-gated ion channels.

biophysics↗

bioRxiv2024

Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live Escherichia coli cells

The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-2160 to +1800 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary.

biophysics↗

bioRxiv2023

Parallel on-chip micropipettes enabling quantitative multiplexed characterization of vesicle mechanics and cell aggregates rheology

Micropipette aspiration (MPA) is one of the gold standards to quantify biological samples mechanical properties, which are crucial from the cell membrane scale to the multicellular tissue. However, relying on the manipulation of individual home-made glass pipettes, MPA suffers from low throughput and difficult automation. Here, we introduce the sliding insert micropipette aspiration (SIMPA) method, that permits parallelization and automation, thanks to the insertion of tubular pipettes, obtained by photolithography, within microfluidic channels. We show its application both at the lipid bilayer level, by probing vesicles to measure membrane bending and stretching moduli, and at the tissue level by quantifying the viscoelasticity of 3D cell aggregates. This approach opens the way to high-throughput, quantitative mechanical testing of many types of biological samples, from vesicles and individual cells to cell aggregates and explants, under dynamic physico-chemical stimuli.

biophysics↗

bioRxiv2023

Mechanical characterization of regenerating Hydra tissue spheres

Hydra vulgaris, long known for its remarkable regenerative capabilities, is also a longstanding source of inspiration for models of spontaneous patterning. Recently, it became clear that early patterning during Hydra regeneration is an integrated mechano-chemical process where morphogen dynamics is influenced by tissue mechanics. One roadblock to understand Hydra self-organization is our lack of knowledge about the mechanical properties of these organisms. In this paper, we combined microfluidic developments to perform parallelized microaspiration rheological experiments and numerical simulations to characterize these mechanical properties. We found three different behaviors depending on the applied stresses: an elastic response, a visco-elastic one and tissue rupture. Using models of deformable shells, we quantify their Youngs modulus, shear viscosity as well as the critical stresses required to switch between behaviors. Based on these experimental results, we propose a description of the tissue mechanics during normal regeneration. Our results provide a first step towards the development of original mechano-chemical models of patterning grounded in quantitative, experimental data. Statement of significanceHydra vulgaris is a remarkable organism thanks to its regenerative abilities. One can cut this animal into several pieces which will reform a full Hydra in a few days. In this process, the pieces have to define a new organizing axis. Recently, researchers have shown that this axis definition is under mechanical control. One roadblock to understand the relationship between tissue mechanics and Hydra biology is our lack of knowledge about the mechanical state of this organism. Here, we perform a mechanical characterization using a combination of microaspiration setups and numerical simulations. We finally propose a description of what happens at the mechanical level during Hydra regeneration, allowing quantitative approaches questioning the role of mechanical cues in axis definition.

biophysics↗

bioRxiv2023

Molecular basis for proofreading by the unique exonuclease domain of Family-D DNA polymerases

Replicative DNA polymerases duplicate entire genomes at high fidelity. This feature is shared among the three domains of life and is facilitated by their dual polymerase and exonuclease activities. Family D replicative DNA polymerases (PolD), found exclusively in Archaea, contain an unusual RNA polymerase-like catalytic core, and a unique Mre11-like proofreading active site. Here, we present cryo-EM structures of PolD trapped in a proofreading mode, revealing an unanticipated correction mechanism that extends the repertoire of protein domains known to be involved in DNA proofreading. Based on our experimental structures, mutants of PolD were designed and their contribution to mismatch bypass and exonuclease kinetics was determined. This study sheds light on the convergent evolution of structurally distinct families of DNA polymerases, and the domain acquisition and exchange mechanism that occurred during the evolution of the replisome in the three domains of life.

molecular biology↗