Biology subjects

Asnacios, A.

Publications and source records attributed to Asnacios, A..

13 recordsLinked to original sources

bioRxiv2025

Single Root hair growth under constant force: insights into wall mechanics

Tip growth is a tightly regulated process that enables root hairs to explore their surroundings, enhancing plant development, particularly by improving nutrient uptake. While Lockharts viscoplastic framework is widely used to describe this process, it has received limited experimental validation. By integrating optical microscopy with a custom microplate-based rheometer, we created a novel protocol to simultaneously measure, for individual growing root hairs, both the reduction in growth rate and the instantaneous compression in response to a step in applied axial force. The observed growth rate reduction aligns remarkably with a 1D Lockhart viscoplastic model, experimentally validating this framework in tip-growing cells. Additionally, the instantaneous compression upon force application provided an in situ estimate of turgor pressure. Together, these measurements allowed us to determine, for the first time in Arabidopsis root hairs, two critical parameters: the yield turgor pressure and cell wall viscosity. Our approach--including the technique, protocol, and analytical framework--can be readily adapted to other tip-growing species and diverse experimental conditions (e.g., varying nutrient availability or osmotic stress). This opens new opportunities to explore cell wall mechanosensitivity and its role in adapting tip growth to environmental signals. Significance StatementPlant growth relies on their ability to anchor roots in soil and maximize nutrient uptake. This process partly depends on root hairs. These long tubular extensions develop from the root surface, exhibiting a highly directional growth process --tip growth. Understanding how root hair growth adapts to soil mechanics is crucial, especially with climate change and soil hardening. We present a novel, non-invasive technique to probe the mechanics of root hair walls--key to their growth. By applying a feedback-controlled force, we can investigate the effect of mechanical resistance on root hair growth while preventing buckling, thus accessing elusive cell wall features. This method holds broader significance, as tip-driven growth is also used by fungi and yeasts to colonize their environments.

biophysics↗

bioRxiv2025

Synthetic pectin-cellulose nanofiber capsule provides minimal model capturing mechanics of a regenerating plant cell wall

Plant primary cell walls are dynamic supramolecular assemblies composed of layered cellulose, hemicellulose and pectin, progressively built through synthesis and secretion. However, the specific architectural features and structural components sufficient to endow the mechanical properties of the wall remain unclear. Here, we construct a minimal synthetic spherical shell and compare its structural and mechanical properties to those of a plant single-cell system. To eliminate complexities from intercellular connectivity and developmental history, we exploit the ability of plant protoplasts to regenerate cell walls de novo. Compression tests of regenerating protoplasts between parallel plates reveal that wall stiffness increases with wall thickening over time. Despite differences in assembly pathways, architecture, and composition, the synthetic shell exhibits a similar thickness-dependent modulus and similar material stiffness. The synthetic shell, composed of pectin and cellulose layers, mirrors the mechanical behavior of regenerating primary cell walls, suggesting that these components are sufficient to confer key viscoelastic properties in the limit of small deformations. Given the complexity of natural plant cell walls, the synthetic analogue offers a controllable platform to dissect the mechanical contributions of individual wall components.

plant biology↗

bioRxiv2025

Micro-mechanical approaches to characterize tip growth: Insights into Root Hair Elasto-Viscoplastic Properties.

Root hairs are outgrowths of the epidermal cells of plant roots. They increase the roots exchange surface with the soil and provide it with good anchorage in the soil. Root hairs are an emblematic model of apical growth, a process also used by yeasts and hyphae to invade their environment. From a mechanical perspective, the root hair is considered as an elastic cylinder under pressure, closed by a dome that behaves like a yield fluid. We introduce here two innovative mechanical setups and protocols to characterize the mechanical properties of single growing root hairs in Arabidopsis thaliana. In the first setup, root hairs grow against an elastic obstacle until buckling. By measuring the critical buckling force, we determine the surface modulus and estimate the Youngs modulus of the cell wall, which aligns with previous measurements. Using a 1D elasto-viscoplastic model of root hair growth, we assess the excess pressure beyond the yield threshold (the driver of tip growth) and estimate the axial stiffness of the root hair, reflecting its elastic resistance to compression. For the second protocol, we designed a setup where a single root hair grows against a cantilever with variable stiffness, a technique adapted from our earlier work on rigidity sensing by animal cells. This method provides an independent estimate of the root hairs axial stiffness, confirming our initial findings and suggesting that this stiffness primarily involves tip compression and depends mainly on turgor pressure, at least within the low deformation regime explored.

biophysics↗

bioRxiv2025

Intratumoral Heterogeneity of Vimentin Modulates Nuclear Mechanotransduction, DNA Damage Response and Cancer Cell Survival

Vimentin, a major intermediate filament protein, is essential for maintaining cellular integrity and regulating cytoskeletal dynamics. Its upregulation is a hallmark of epithelial-to-mesenchymal transition (EMT), a process that enhances cancer cell migration, invasion, and metastatic potential. However, single-cell transcriptomic analyses of glioblastoma, the most common and aggressive primary brain tumor, reveal that vimentin expression exhibits significant intratumoral heterogeneity, reflecting diverse cellular subpopulations that may contribute to tumor plasticity, therapy resistance, and disease progression. Here, we show that the absence of vimentin alters nuclear mechanotransduction in response to compression, leading to chromatin remodelling and profound changes in gene expression in cancer cells. Remarkably, we demonstrate that external compressive forces, akin to vimentin deficiency, disrupt DNA damage response pathways. This impairment compromises DNA damage sensing and repair, bypassing DNA damage checkpoints and apoptosis. Consequently, vimentin-negative tumor cells exhibit increased survival in response to physical stress and DNA damage, potentially driving radioresistance and further amplifying intratumoral heterogeneity.

cancer biology↗

bioRxiv2025

Allometric spreading and focal adhesion collective organization are coordinated by cell-scale geometrical constraints

Focal adhesions are protein complexes that transmit actin cytoskeleton forces to the extracellular matrix and serve as signaling hubs that regulate cell physiology. While their growth is achieved through a local force-dependent process, the requirement of sustaining stress at the cell scale suggests a global regulation of the collective organization of focal adhesions. To investigate evidence of such large-scale regulation, we compared changes in cell shape and the organization of focal adhesion-like structures during the early spreading of fibroblasts either on a two-dimensional substrate or confined between two parallel plates, and for cells of different volumes. In this way, we reveal that the areal density of focal adhesions is conserved regardless of cell size or third-dimensional confinement, despite different absolute values of the surface covered by adhesion clusters. In particular, the width of the focal adhesions ring, which fills the flat lamella at the cell front, adapts to cell size and third-dimensional confinement and scales with cell-substrate contact radius. We find that this contact radius also adapts in the parallel-plate geometry so that the cumulated area of cellsubstrate contact is conserved at the cell scale. We suggest that this behavior is the result of 3D cell shape changes which govern spreading transitions. Indeed, because of volume conservation constraints, the evolution of cell-body contact angle adjusts according to cell size and confinement, whereas the rate of early spreading at the cell-substrate contact is not affected by thirddimensional geometry. Overall, our data suggest that a coordination between global and local scales mediates the adaptation of cell-substrate contacts and focal adhesions distribution to large scale geometrical constraints, which allows an invariant cell-substrate adhesive energy.

biophysics↗

bioRxiv2024

Disentangling the contributions of stress fibres and the unbundled actin meshwork to the anisotropy of cortical tension in response to cell shape

Many fundamental biological processes, in particular development and morphogenetic movements, involve tissue and cell deformation, as well as the generation of anisotropic mechanical stresses. They are often accompanied by the appearance of oriented contractile actomyosin structures resembling the stress fibres (SF) observed in vitro. Here, we investigate, at the single cell level, how cell shape -- by itself -- could control the structure and tension of the actomyosin cortex. Using a unique combination of 3D micropatterning, single peripheral SF (PSF) tension measurement, laser ablation and image analysis, we show that cell shape anisotropy, e.g. its 2D aspect ratio, is indeed sufficient to induce anisotropy of the cortical structure and tension. In particular, taking into account the experimentally measured anisotropy of the cortical meshwork, we could quantify cortical tension and decouple the contribution originating from bundled actin (oriented cortical stress fibres, CSF) and the contribution of the unbundled actin meshwork (UAM). We show that the increase of cortical tension anisotropy with the cells aspect ratio depends on the CSF alignment and orientation, the contribution of the isotropic mesh being independent of cell shape. Remarkably, while experimental data from single stress fibre measurements and laser ablation were analysed through different theoretical frameworks, namely that of negative pressure in nematics and hole drilling in prestressed materials, we found quantitatively the same composite material behaviour. In sum, we decipher here the very material properties of the actomyosin cortex, and its sensitivity to cell shape which is at the root of many mechanobiological processes, in particular morphogenesis.

biophysics↗

bioRxiv2022

In vivo measurement of the Young's modulus of the cell wall of single root hairs

Root hairs are cells from the root epidermis that grow as long tubular bulges perpendicular to the root. They can grow in a variety of mechanical or chemical environments. Their mechanical properties are mainly due to their stiff cell wall which also constitutes a physical barrier between the cell and its environment. Thus, it is essential to be able to quantify the cell wall mechanical properties and their adaptation to environmental cues. Here, we present a technique we developed to measure the Youngs (elastic) modulus of the root hair cell wall. In essence, using custom-made glass microplates as cantilevers of calibrated stiffness, we are able to measure the force necessary to bend a single living root hair. From these experiments one can determine the stiffness and Youngs modulus of the root hair cell wall.

biophysics↗

bioRxiv2022

Mechanical resistance of the environment affects root hair growth and nucleus dynamics

Root hair (RH) cells are important for the growth and survival of seedlings. They favor plant-microbe interactions, nutrients, and water uptake. RH cells increase drastically the surface of exchange of the root system with the surrounding environment. To be able to invade the soil, RH cells have to penetrate a dense and porous medium exhibiting a variety of physical properties. The soils physical properties, such as mechanical resistance, impact the growth and survival of plants. Consequently, studying the effect of soil resistance on the growth of RH is essential to improve our understanding of plant growth. Here we investigate the effect of the mechanical resistance of the culture medium on RH-physical and phenotypical parameters such as length, time, and speed of growth. We also analyze the impact of the environment on the positioning, and movement of the nucleus inside the growing cells. To do so, Arabidopsis Thaliana seedlings were cultured in a custom-made microfluidic-like system, in solid media with agar concentrations ranging from 0.5% to 1.25%. We show that the time of growth of RH cells is independent of the mechanical resistance of the surrounding environment, while the RH speed decreases when the mechanical resistance increases. As a consequence, the RH cells are shorter in stiffer environments. Moreover, we show that the speed of the nucleus adapts to the mechanical resistance of the environment and follows the same trend as the average speed of the RH tip. Eventually, during RH growth, the nucleus-to-tip distance was found to decrease when the stiffness of the environment was increased, indicating mechanotransduction from the cell surface to the nucleus.

biophysics↗

bioRxiv2022

Shigella ipaA mediates actin bundling through diffusible vinculin oligomers with activation imprint

Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domain that binds to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.

microbiology↗

bioRxiv2021

Phenotyping Polarization Dynamics Of Immune Cells Using A Lipid Droplet - Cell Pairing Microfluidic Platform

The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intra-cellular components, necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied. Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how ligand concentration, surface fluidity and substrate rigidity impact lysosome polarization, we show that its onset and kinetics depend on the local antigen concentration at the synapse and on substrate rigidity. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

biophysics↗

bioRxiv2021

Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behaviour. Although the contribution of ECM compliance to the control of cell migration or division has been extensively studied, little has been reported regarding infectious processes. We have studied how mechanical properties of the ECM impact invasion of cells by the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identified OPTN as a protein regulated by ECM stiffness whose function is required for bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. We showed that OPTN knockdown cells display enhanced Rac1 activation, strong mechanochemical adhesion signalling and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Despite such features, OPTN knockdown cells displayed defective traction force buildup associated with limited cellular invasion by UTI89. Together, our data indicate that OPTN, through a new role in mechanobiology, supports CNF1-producing uropathogenic E. coli invasion and links HACE1-mediated ubiquitylation of Rac1 to ECM mechanical properties and integrin mechanotransduction.

cell biology↗

bioRxiv2020

Intracellular stiffening competes with cortical softening in glioblastoma cells

Cancer cell softening increases with the progression of the disease, suggesting that mechanical phenotyping could be used as a diagnostic or prognostic method. Here we investigate the cell mechanics of gliomas, brain tumors that originate from glial cells or glial progenitors. We use two microrheology techniques, a single cell parallel plates rheometer to probe whole cell mechanics and optical-tweezers to probe intracellular rheology. We show that cell mechanics discriminates human glioma cells of different grades. When probed globally, grade IV glioblastoma cells are softer than grade III astrocytoma cells, while they are surprisingly stiffer at the intracellular level. We explain this difference between global and local intracellular behaviours by changes in the composition and spatial organization of the cytoskeleton and by changes in nuclear mechanics. Our study highlights the need to combine rheology techniques for potential diagnostic or prognostic methods based on cancer cell mechanophenotyping.

biophysics↗

bioRxiv2019

Vinculin targeting by Shigella IpaA promotes stable cell adhesion independent of mechanotransduction

The Shigella effector IpaA co-opts the focal adhesion protein vinculin to promote bacterial invasion. Here, we show that IpaA triggers an unreported mode of vinculin activation through the cooperative binding of its three vinculin-binding sites (VBSs) leading to vinculin oligomerization via its D1 and D2 head subdomains and highly stable adhesions resisting actin relaxing drugs. Using cross-linking mass spectrometry, we found that while IpaA VBSs1-2 bound to D1, IpaA VBS3 interacted with D2, a subdomain masked to other known VBSs. Structural modeling indicated that as opposed to canonical activation linked to interaction with D1, these combined VBSs interactions triggered major allosteric changes leading to D1D2 oligomerization. A cysteine-clamp preventing these changes and D1D2 oligomerization impaired growth of vinculin microclusters and cell adhesion. We propose that D1D2-mediated vinculin oligomerization occurs during the maturation of adhesion structures to enable the scaffolding of high-order vinculin complexes, and is triggered by Shigella IpaA to promote bacterial invasion in the absence of mechanotransduction. SummaryThe Shigella IpaA effector binds to cryptic vinculin sites leading to oligomerization via its head domain. This vinculin oligomerization mode appears required for the maturation and strengthening of cell adhesion but is co-opted by invasive bacteria independent of actomyosin contractility.

microbiology↗