Biology subjects

Villard, C.

Publications and source records attributed to Villard, C..

17 recordsLinked to original sources

bioRxiv2025

Snooping helices: The elastic path finding algorithm of growing hyphae

How living organisms utilize physical mechanisms to sense their environments and make informed decisions is an open question at the interface of biology and physics. In filamentous organisms like fungal hyphae, the decisions are taken by their growing tip cells and later imprinted onto the rest of the multi-cellular filament. Here we report on the growth and pathfinding of hyphae from the opportunistic fungal pathogen Candida albicans, whose ability to cross intestinal epithelial layers is associated to severe systemic infections in humans. It has been sporadically reported that C. albicanss hyphae display helical growth inside or on top of agar gels, helicity turning in the latter case into two-dimensional oscillatory shapes. We provide an extended description of oscillatory C. albicans hyphal growth modalities, revealed under various physical confinements thanks to the use of dedicated microfluidic devices and quantitative time-lapse imaging-based analysis. These include sudden sliding events accompanied by curvature switching of the tip portion, resulting in a final oscillatory morphology of the entire filament, and stable curved tips moving against vertical microfluidic channels walls. These behaviors are unified under the formalism of growing squeezed helices, in which the final hyphal curved shapes result from an elastic energy minimization of a spatially confined helical portion at the tip followed by a continuous solidification front. Ultimately, the combination of our experimental results and theoretical framework provide an insight into the penetration strategy of C. albicans hyphae, which is essential for the virulence of this fungal microorganism. Significance StatementProprioception is the integrated sense of self-movement and body position in complex organisms. Here we describe a novel, mechanical form of proprioception driving directional choice making in tip-growing helical organisms. We show that C. albicans hyphae utilize their built-in helicity as an environment-scanning mechanism to explore their surrounding and find target surfaces for invasion. When confined to surfaces, hyphae continue producing in-plane oscillatory shapes that promote further invasive behavior. C. albicans inherent mechanical instabilities regulate the switching of growth direction and their abrupt directional decisions can be understood as elastic bifurcations of squeezed, confined helices.

biophysics↗

bioRxiv2025

Brain biomechanics governs mitotic fidelity of embryonic neural progenitors

Accurate chromosome segregation is essential to maintain genetic stability and prevent the onset of diseases such as developmental disorders, infertility, and cancer. While many intrinsic factors and processes involved in mitosis have been extensively characterized, less is known about how extrinsic factors and tissue properties contribute to mitotic fidelity. In this study, using both in vivo and ex vivo systems, in combination with pharmacological perturbations and high-resolution microscopy, we investigated mitosis in apical radial glial (aRG) cells--the primary neural progenitors in the developing mammalian brain. We found that the high cell density typical of early neurogenic stages enhances microtubule polymerization rates from the spindle poles, thereby influencing chromosome segregation. Mechanistically, our results indicate that cortical actin and elevated cortical tension function as sensors of biomechanical stress during mitosis. These findings identify biomechanics as a threat to mitotic fidelity in the embryonic brain.

cell biology↗

bioRxiv2025

Introducing furanocoumarin biosynthetic genes in tomato results in coumarins accumulation and impacted growth

Over the past three decades, eeorts to decipher plant metabolism have shed light on key enzymes driving specialized metabolite biosynthesis. Although only few pathways have been completely investigated to date, their characterization paves the way for exploring the potential eeects of specialized metabolites on plant physiology. Among them is the linear furanocoumarin pathway, which was recently completed to produce up to psoralen. In this study, we report the first metabolic engineering of the linear furanocoumarin pathway to enable artificial psoralen production in tomato, through the integration of four genes coding for the enzymes: Umbelliferone Synthase, Demethylsuberosin Synthase, Marmesin Synthase and Psoralen Synthase. Interestingly, coumarins were produced instead of furanocoumarins. Using morphophysiological, metabolomic, and transcriptomic analyses, we suggest how coumarins, particularly scopoletin, can impact growth and aeect plant physiology, even at low concentrations. As coumarins have increasingly attracted interest for agricultural applications due to their minimal environmental impact, this work both expands and challenges their potential by highlighting the physiological costs and benefits they may impose on tomato. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/663522v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1df0bdeorg.highwire.dtl.DTLVardef@843d9borg.highwire.dtl.DTLVardef@1e898b6org.highwire.dtl.DTLVardef@18205eb_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

plant biology↗

bioRxiv2025

Every Piece of the Puzzle Matters: A Novel Zinc-Binding Site in the Luminal Domain of STIM1 Drives Clustering

STIM1 is pivotal in the tightly regulated mechanism controlling calcium homeostasis in the ER. It is activated by calcium dissociation from its EF-hand domain when ER calcium levels decrease, leading to its interaction with the ORAI channel to initiate calcium influx. Despite advancements in understanding the complex STIM1 machinery, including its domain organization and structural rearrangements upon activation, many aspects of this process remain poorly understood. In this study, we focused on a small conserved region situated upstream to the EF-hand, which has been previously shown to be involved in the modulation of STIM1 by ROS. Our findings reveal that this region binds zinc and plays a pivotal role in STIM1 activation by promoting its clustering, a process essential for the activation of calcium influx. These results revealed the functional importance of this domain and added a crucial piece to the puzzle of how calcium and zinc signaling are interconnected. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/659323v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@16efeborg.highwire.dtl.DTLVardef@1e15e96org.highwire.dtl.DTLVardef@108cad0org.highwire.dtl.DTLVardef@3b58b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Using lineage-specific patterns to understand convergence of enzymatic functions led to the identification of Moraceae-specific P450s involved in furanocoumarin biosynthesis

O_LISpecialized metabolites are molecules involved in plants interaction with their environment. Elucidating their biosynthetic pathways is a challenging but rewarding task, leading to societal applications and ecological insights. Furanocoumarins emerged multiple times in Angiosperms, raising the question of how different enzymes evolved into catalyzing identical reactions. C_LIO_LITo identify enzymes producing lineage-specific metabolites, an evolutionary-based approach was developed and applied to furanocoumarin biosynthesis in Ficus carica (Moraceae). This led to the characterization of CYP71B129-131a, three P450 enzymes whose evolution of the function was investigated using phylogenetics, structural comparisons and site-directed mutagenesis. C_LIO_LICYP71B129 and CYP71B130,131a were found to hydroxylate umbelliferone (coumarin) and xanthotoxin (furanocoumarin), respectively. Results suggest that CYP71Bs xanthotoxin hydroxylase activity results from duplications and functional divergence of umbelliferone hydroxylase genes. Structural comparisons highlighted an amino acid affecting CYP71Bs substrate specificity, which may play a key role in allowing xanthotoxin hydroxylation in several P450 subfamilies. C_LIO_LICYP71B130-131a characterization validates the proposed enzyme-discovery approach, which can be applied to different pathways and help to avoid the classic bottlenecks of specialized metabolism elucidation. The CYP71Bs also exemplify how furanocoumarin-biosynthetic enzymes can stem from coumarin-biosynthetic ones and provides insights into the molecular mechanisms underlying the multiple emergences of xanthotoxin hydroxylation in distant P450 subfamilies. C_LI

plant biology↗

bioRxiv2023

Natural gene variation in Cannabis sativa unveils a key region of cannabinoid synthase enzymes

Cannabinoids are well-known specialised metabolites from the plant Cannabis sativa L. (cannabis). They exhibit various therapeutical to intoxicating psychoactive effects and have potential for medicinal applications. Among the enzymes involved in cannabinoid biosynthesis, cannabinoid oxidocyclases such as the tetrahydrocannabinolic acid (THCA) synthase play a key role in determining cannabis chemotype. To improve our understanding of cannabinoid oxidocyclase structure-function relationship, we proposed a new approach to targeted mutagenesis. By reviewing cannabis natural variation, three cannabinoid oxidocyclase mutations (S355N, CONF, G376R) associated to atypical plant chemotypes were selected. In-vitro characterization of THCA synthase mutants demonstrated these mutations significantly impact enzyme activity, correlating with the associated chemotype: S355N nearly inactivated the THCA synthase, CONF impaired CBGA metabolization and altered product specificity, while G376R drastically reduced enzyme activity and altered product specificity. In-silico docking experiments permitted to model the successive steps of THCA synthase substrate metabolization, revealing that the three mutations hamper substrate binding. Collectively, our results demonstrated how plant diversity can be leveraged to guide enzyme targeted mutagenesis, highlighted a key region of cannabinoid oxidocyclases, and permitted the establishment of a new model of the THCA synthase catalytic mechanism. This provides new insights into enzyme function, which can ultimately help developing medicinal cannabis cultivars and cannabinoid biotechnological production.

molecular biology↗

bioRxiv2022

Surface tension drives neuronal sorting in magnetically engineered brain-like tissue

Engineered 3D brain-like models have advanced the understanding of neurological mechanisms and disease, yet their mechanical signature, while fundamental for brain function, remains understudied. The surface tension for instance controls brain development and is a marker of cell-cell interactions. Here, we engineered 3D magnetic brain-like tissue spheroids composed of intermixed primary glial and neuronal cells at different ratios. Remarkably, the two cell types self-assemble into a functional tissue, with the sorting of the neuronal cells towards the periphery of the spheroids, whereas the glial cells constitute the core. The magnetic fingerprint of the spheroids then allows their deformation when placed under a magnetic field gradient, at a force equivalent to a 70 g increased gravity at the spheroid level. The tissue surface tension and elasticity can be directly inferred from the resulting deformation, revealing a transitional dependence on the glia/neuron ratio, with the surface tension of neuronal tissue being much lower. This provides the underlying mechanical explanation for the exclusion of the neurons towards the outer spheroid region, and depicts the glia/neuron organization as a surface tension-driven sophisticated mechanism that should in turn influence brain development and homeostasis.

bioengineering↗

bioRxiv2022

Bending stiffness of Candida albicans hyphae reflects adaptive behavior of the fungal cell wall

The cell wall is a key component of fungi. It constitutes a stiff shell which counteracts internal cell turgor pressure. Its mechanical properties thus contribute to define cell morphology. Measurements of the elastic moduli of the fungal cell wall have been carried out in many species including Candida albicans, a major human opportunistic pathogen. They mainly relied on atomic force microscopy, and mostly considered the yeast form. We developed a parallelized pressure-actuated microfluidic device to measure the bending stiffness of hyphae. We found that the cell wall stiffness lies in the MPa range. We the used three different ways to disrupt cell wall physiology: inhibition of beta-glucan synthesis, a key component of the inner cell wall; application of an hyperosmotic shock triggering a sudden decrease of the hyphal diameter; deletion of two genes encoding GPI-modified cell wall proteins resulting in reduced cell wall thickness. The bending stiffness values were affected to different extents by these environmental stresses or genetic modifications. Overall, our results support the elastic nature of the cell wall and its ability to remodel at the scale of the entire hypha over minutes.

biophysics↗

bioRxiv2021

Quantitative study of alpha-synuclein prion-like spreading in fully oriented reconstructed neural networks reveals non-synaptic dissemination of seeding aggregates

The trans-neuronal spread of protein aggregates in a prion-like manner underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinsons disease. Despite being studied actively, the mechanisms of alpha-synuclein (aSyn) aggregates propagation remain poorly understood. This hinders the development of therapeutic approaches aiming at preventing the spatial progression of intracellular inclusions in neural networks. To assess the role of synaptic structures and neuron characteristics in the transfer efficiency of aggregates with seeding propensity, we developed a novel microfluidic culture system which allows for the first time to reconstruct in vitro fully oriented and synaptically connected neural networks. This is achieved by filtering axonal growth with unidirectional "axon valves" microchannels. We exposed the presynaptic compartment of reconstructed networks to well characterized human aSyn aggregates differing in size: Fibrils and Oligomers. Both aggregates were transferred to postsynaptic neurons through active axonal transport, albeit with poor efficiency. By manipulating network maturity, we compared the transfer rate of aggregates in networks with distinct levels of synaptic connectivity. Surprisingly, we found that transfer efficiency was lower in mature networks with higher synaptic connectivity. We then investigated the seeding efficiency of endogenous aSyn in the postsynaptic population. We found that exposure to Fibrils, and not Oligomers, resulted in low efficiency trans-neuronal seeding which was restricted to postsynaptic axons. Finally, we assessed the impact of neuron characteristics and aSyn expression on the propagation of aSyn aggregates. By reconstructing chimeric networks, we found that neuron characteristics, such as the brain region from which they originate or aSyn expression levels, did not significantly impact aggregates transfer, and observed no trans-neuronal seeding where the presynaptic population did not express aSyn. Overall, we demonstrate that this novel platform uniquely allows the quantitative interrogation of original aspects of the trans-neuronal propagation of seeding pathogenic entities.

neuroscience↗

bioRxiv2021

RNA chaperones Hfq and ProQ play a key role in the virulence of the plant pathogenic bacterium Dickeya dadantii

Dickeya dadantii is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the transcription of its genes encoding the main virulence functions, little information is available on the post-transcriptional regulation of these functions. We investigated the involvement of the RNA chaperones Hfq and ProQ in the production of the main D. dadantii virulence functions. Phenotypic assays on the hfq and proQ mutants showed that inactivation of hfq resulted in a growth defect, a modified capacity for biofilm formation and strongly reduced motility, and in the production of degradative extracellular enzymes (proteases, cellulase and pectate lyases). Accordingly, the hfq mutant failed to cause soft rot on chicory leaves. The proQ mutant had reduced resistance to osmotic stress, reduced extracellular pectate lyase activity compared to the wild-type strain, and reduced virulence on chicory leaves. Most of the phenotypes of the hfq and proQ mutants were related to the low amounts of mRNA of the corresponding virulence factors. Complementation of the double mutant hfq-proQ by each individual protein and cross-complementation of each chaperone suggested that they might exert their effects via partially overlapping but different sets of targets. Overall, it clearly appeared that the two Hfq and ProQ RNA chaperones are important regulators of pathogenicity in D. dadantii. This underscores that virulence genes are regulated post transcriptionally by non-coding RNAs.

microbiology↗

bioRxiv2020

Neuron-gated silicon nanowire field effect transistors to follow single spike propagation within neuronal network

Silicon nanowire field effect transistors SiNW-FETs provide a local probe for sensing neuronal activity at the subcellular scale, thanks to their nanometer size and ultrahigh sensitivity. The combination with micro-patterning or microfluidic techniques to build model neurons networks above SiNW arrays could allow monitoring spike propagation and tailor specific stimulations, being useful to investigate network communications at multiple scales, such as plasticity or computing processes. This versatile device could be useful in many research areas, including diagnosis, prosthesis, and health security. Using top-down silicon nanowires-based array, we show here the ability to record electrical signals from matured neurons with top-down silicon nanowires, such as local field potential and unitary spike within ex-vivo preparations and hippocampal neurons grown on chip respectively. Furthermore, we demonstrate the ability to guide neurites above the sensors array during 3 weeks of cultures and follow propagation of spikes along cells. Silicon nanowire field effect transistors are obtained by top-down approach with CMOS compatible technology, showing the possibility to implement them at manufacturing level. These results confirm further the potentiality of the approach to follow spike propagation over large distances and at precise location along neuronal cells, by providing a multiscale addressing at the nano and mesoscales.

bioengineering↗

bioRxiv2020

Parallelized manipulation of adherent living cells by magnetic nanoparticles-mediated forces

The remote actuation of cellular processes such as migration or neuronal outgrowth is a challenge for future therapeutic applications in regenerative medicine. Among the different methods that have been proposed, the use of magnetic nanoparticles appears to be promising since magnetic fields can act at a distance without interactions with the surrounding biological system. To control biological processes at a subcellular spatial resolution, magnetic nanoparticles can be used either to induce biochemical reactions locally or to apply forces on different elements of the cell. Here, we show that cell migration and neurite outgrowth can be directed by the forces produced by a switchable parallelized array of micro-magnetic pillars, following passive uptake of nanoparticles. Using live cell imaging, we first demonstrate that adherent cell migration can be biased toward magnetic pillars and that cells can be reversibly trapped onto these pillars. Second, using differentiated neuronal cells we were able to induce events of neurite outgrowth in the direction of the pillars without impending cell viability. Our results show that the range of forces applied needs to be adapted precisely to the cellular process under consideration. We propose that cellular actuation is the result of the force on the plasma membrane caused by magnetically filled endo-compartments, which exert a pulling force on the cell periphery.

bioengineering↗

bioRxiv2020

A Sequence determinant in 3'UTR of mRNAs for Nuclear Retention by Paraspeckles

Paraspeckles are nuclear membraneless structures composed of a long non-coding RNA, Nuclear-Enriched-Abundant-Transcript-1 and RNA binding proteins, which associate numerous mRNAs. It is therefore believed that their cellular function is to sequester in the nucleus their associated proteins and/or target mRNAs. However, little is known about the molecular determinant in mRNA targets that allow their association to paraspeckles except that inverted repeats of Alu sequences (IRAlu) present in 3UTR of mRNAs may allow this association. While in a previous study we established the list of paraspeckle target RNAs in a rat pituitary cell line, we didnt find however, inverted repeated SINEs, the rat equivalent of primate IRAlus in 3UTR of these RNAs. By developing a candidate gene strategy, we selected a paraspeckle target gene, namely calreticulin mRNA, and we searched for other potential RNA recruitment elements in its 3UTR, since 3UTRs usually contain the sequence recognition for nuclear localization. We found a 15-nucleotide sequence, present as a tandem repeat in 3UTR of this mRNA, which is involved in the nuclear retention by paraspeckles. While being not overrepresented in the 3UTR of the paraspeckle target RNAs, this recruitment element was present in near 30% of all 3UTR mRNAs. In addition, since an oligonucleotide containing this sequence binds the paraspeckle protein component HNRNPK, it is proposed that HNRNPK may constitute a bridging protein between paraspeckles and target mRNAs.

molecular biology↗

bioRxiv2020

Compromised nuclear envelope integrity drives tumor cell invasion

While mutations leading to a fragile envelope of the cell nucleus are well known to cause diseases such as muscular dystrophies or accelerated aging, the pathophysiological consequences of the recently discovered mechanically induced nuclear envelope ruptures in cells harboring no mutation are less known. Here we show that repeated loss of nuclear envelope integrity in nuclei experiencing mechanical constraints promotes senescence in nontransformed cells, and induces an invasive phenotype including increased collagen degradation in human breast cancer cells, both in vitro and in a mouse xenograft model of breast cancer progression. We show that these phenotypic changes are due to the presence of chronic DNA damage and activation of the ATM kinase. In addition, we found that depletion of the cytoplasmic exonuclease TREX1 is sufficient to abolish the DNA damage in mechanically challenged nuclei and to suppress the phenotypes associated with the loss of nuclear envelope integrity. Our results also show that TREX1-dependent DNA damage induced by physical confinement of tumor cells inside the mammary duct drives the progression of in situ breast carcinoma to the invasive stage. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

cell biology↗

bioRxiv2019

Glial cell mechanosensitivity is reversed by adhesion cues

Brain tissues demonstrate heterogeneous mechanical properties, which evolve with aging and pathologies. The observation in these tissues of smooth to sharp rigidity gradients raises the question of brain cells responses to both different values of rigidity and their spatial variations. Here, we use recent techniques of hydrogel photopolymerization to achieve stiffness structuration down to micrometer resolution. We investigate primary neuron adhesion and orientation as well as glial cell adhesive and proliferative properties on multi-rigidity polyacrylamide hydrogels presenting a uniform density of adhesive molecules. We first observed that neurons grow following rigidity gradients. Then, our main observation is that glial cell adhesion and proliferation can be enhanced on stiff or on soft regions depending on the adhesive coating of the hydrogel, i. e. fibronectin or poly-L-lysine/laminin. This behavior was unchanged in the presence or not of neuronal cells. In addition, and contrarily to other cell types, glial cells were not confined by sharp, micron-scaled gradients of rigidity. Our observations suggest that their mechanosensitivity could involve adheison-related mechanosensitive pathways that are specific to brain tissues. SIGNIFICANCEBy growing primary brain cells on 2D multi-rigidity polyacrylamide hydrogels, we show that favorable culture conditions for glial cells switch from stiff to soft substrates when changing the adhesive ligands from fibronectin to poly-L-lysine/laminin. Together with neurons, glial cells thus provide a unique example where soft is preferred to stiff, but unlike neurons, this preference can be reversed by changing the nature of the coating. We additionally show that contrarily to other cell types, glial cells are deformed by subcellular gradients of rigidity but cannot be confined by these rigidity gradients. These observations point that glial cell use a very specific, integrin-related machinery for rigidity sensing.

biophysics↗

bioRxiv2019

Blood Flow Dynamics Has a Major Influence on the State of Circulating Tumor Cells

Cancer mortality mainly arises from metastases, due to cells that escape from a primary tumor, circulate in the blood as circulating tumor cells (CTCs), permeate across blood vessels and nest in distant organs. It is still unclear how CTCs overcome the harsh conditions of fluid shear stress and mechanical constraints within the microcirculation. Here, a model of the blood microcirculation was established through the fabrication of microfluidic channels comprising constrictions. Metastatic breast cancer cells of epithelial-like and mesenchymal-like phenotypes were flowed into the microfluidic device. These cells were visualized during circulation, analyzed for their dynamical behavior and retrieved post-circulation. {gamma}-H2AX staining showed significant increase of DNA damage response in epithelial-like SK-BR-3 cells, while gene expression analysis of key regulators of epithelial-to-mesenchymal transition revealed significant increase of Twist2 relative expression in mesenchymal-like MDA-MB-231 cells post-circulation. This work documents first results of the changes at the cellular, subcellular and molecular scales induced by the two main mechanical stimuli arising from circulatory conditions.

cancer biology↗

bioRxiv2018

Topographical cues control the morphology and dynamics of migrating cortical interneurons

In mammalian embryos, cortical interneurons travel long distances among complex three-dimensional tissues before integrating into cortical circuits. Several molecular guiding cues involved in this migration process have been identified, but the influence of physical parameters remains poorly understood. In the present study, we have investigated in vitro the influence of the topography of the microenvironment on the migration of primary cortical interneurons released from mouse embryonic explants.\n\nWe found that arrays of 10 m-sized PDMS micro-pillars, either round or square, influenced both the morphology and the migratory behavior of interneurons. Strikingly, most interneurons exhibited a single and long leading process oriented along the diagonals of the square pillared array, whereas leading processes of interneurons migrating in-between round pillars were shorter, often branched and oriented in all available directions. Accordingly, dynamic studies revealed that growth cone divisions were twice more frequent in round than in square pillars. Both soma and leading process tips presented forward directed movements within square pillars, contrasting with the erratic trajectories and more dynamic movements observed among round pillars. In support of these observations, long interneurons migrating in square pillars displayed tight bundles of stable microtubules aligned in the direction of migration.\n\nOverall, our results show that micron-sized topography provides global spatial constraints promoting the establishment of two different morphological and migratory states. Remarkably, both states belong to the natural range of migratory behaviors of cortical interneurons, highlighting the potential importance of topographical cues in the guidance of these embryonic neurons, and more generally in brain development.

cell biology↗