Biology subjects

Arroyo, M.

Publications and source records attributed to Arroyo, M..

18 recordsLinked to original sources

bioRxiv2026

Constitutive ATR Signaling Enforces Domain-Wide Origin Synchrony

Eukaryotic DNA replication relies on organized spatial-temporal domains, yet how neighboring origins are coordinated at the single-molecule level remains unresolved. Combining single-molecule DNA combing with super-resolution microscopy, we demonstrate that during unperturbed early S-phase, adjacent replication origins across diverse vertebrate species fire synchronously as a coordinated unit. This domain-wide temporal harmony is continuously enforced by a baseline, constitutive ATR CHK1 signaling axis that restrains local CDK2 and CDC7 kinase activities. Strikingly, replication stress dismantles this coordination, shifting origin activation toward an asynchronous, replicon-autonomous mode. Mechanistically, stress-induced phosphorylation of the cohesin subunit SMC1A promotes an SMC1A ATR complex that sequesters ATR at stalled replication forks. This localized sequestration creates a regional ATR loss-of-function phenotype, relieving CDK2/CDC7 suppression and driving premature, uncoordinated origin firing. Super-resolution imaging shows that ATR sequestration structurally reorganizes replication foci, converting coordinated clustered foci into fragmented, lower-density clusters. Together, our findings uncover a baseline role for constitutive ATR signaling in enforcing domain synchrony and reveal a molecular switch that dynamically decouples origin activation during genomic stress to safeguard chromosome duplication.

molecular biology↗

bioRxiv2026

Heterochromatin organization and liquid-liquid phase separation: it is not about if but about when

Heterochromatin is a membraneless compartment within the cell nucleus. In recent years, a controversy arose on whether heterochromatin organization is driven by liquid-liquid phase separation or not. While many heterochromatin proteins were shown to undergo liquid-liquid phase separation in vitro, other studies reported that this does not happen in cells. Here, we tested the ability of heterochromatin proteins to generate heterochromatin barrier compartments in cells. We found that, while several proteins (H1.0, H1.4, HP1alpha, HP1beta, Mbd1, Mbd2 and MeCP2) form barrier compartments in mouse and/or human cells this differs between cell types. In addition, not all compartments in the same cell form barriers. We established and experimentally validated a model that predicted the ability to form barrier compartments is dependent on the protein accumulation in heterochromatin followed by the competition between compartments for the nucleoplasm pool of the protein and resulted in larger size for the barrier compartments. These findings resolve the existing controversy and rationalize how in cells heterochromatin compartments form and compete to establish dynamic barriers to the entry and exit of its components. HighlightsHeterochromatin barrier formation differs between proteins, cell lines and heterochromatin compartments within the cell. Barrier formation depends on heterochromatin anchors, including ligands and other scaffolds. Barrier compartments are defined by their larger size and higher protein enrichment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/729812v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a63021org.highwire.dtl.DTLVardef@a20362org.highwire.dtl.DTLVardef@8c2390org.highwire.dtl.DTLVardef@72dde3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Dynamic anatomy of the replisome under stress

When exposed to replication stress, the factors involved in DNA replication display differing responses that in many cases remain unexplained. In this study, we generated a series of genomically engineered embryonic stem cell lines with fluorescently tagged replication factors to track their spatiotemporal dynamics in live-cell microscopy during and after treatment with the replication inhibitors aphidicolin and hydroxyurea. All 3 replicative polymerases were found to accumulate upon stress, with the primase subunits of polymerase alpha retaining catalytic activity. This accumulation was dependent on new origin firing and accompanied by 9-1-1 clamp loading in the vicinity of stalled forks. Pulse-stress-pulse experiments and spatial correlation analyses of replicons indicated that, after stress is removed, replication preferentially restarts at new sites surrounding previously stalled forks. These findings shed light on the replisomes immediate reaction to stalled replication forks and provide comprehensive kinetic maps of the replisome under stress. Highlights- Comprehensible atlas of the replisome under stress - DNA polymerases accumulate during stress, with clamp switch near stalled forks - In the absence of DNA synthesis, new adjacent origins fire with active priming - During stress recovery, replication restart predominantly at new fired origins

cell biology↗

bioRxiv2025

A keratin bundling transition uncages the nucleus in stretchedepithelia

There is broad consensus that intermediate filaments play a key role in protecting cells and tissues from large deformations. However, little is known about how they fulfil this function. Here we demonstrate that epithelial cells undergo a slow adaptation to stretch that couples a star- bundling transition of keratin filaments and the escape of the nucleus from its keratin cage. The bundling transition begins with a depletion of keratin filaments at tri-cellular junctions followed by a progressive accumulation in thick bundles that bisect cell-cell junctions. Bundling is a cooperative process that initiates in a few scattered cells and propagates to their neighbours through desmosomes, leading to the growth of multicellular clusters that contain a percolated network of thick keratin bundles. Bundling dynamics are slow and strongly influenced by the interaction between actin and keratin. Informed by a computational model, we provide evidence that keratin bundling generates a compressive stress on the nucleus, which is relaxed by nuclear escape from the keratin cage. The topological transitions identified here provide epithelia with a multiscale mechanism to adapt to sustained stretch.

biophysics↗

bioRxiv2025

Multiscale wrinkling dynamics in epithelial shells

Thin shells buckle and wrinkle when compressed. While this behavior is generally detrimental in engineering, it has been widely implicated in epithelial morphogenesis and patterning during development. Yet the rules governing buckling of active viscoelastic shells like epithelia remain unclear. Here we delineate those rules by combining an experimental system that allows us to sculpt epithelial shells and subject them to controlled deflation with a 3D computational model linking cytoskeletal dynamics to tissue mechanics. Experiments and simulations across several orders of magnitude in time and space reveal that buckling emerges for fast deflation relative to the cortexs relaxation time, and is suppressed by high contractility. We show, further, that the tissue develops wrinkle patterns with different degrees of symmetry breaking that depend on its size and viscous confinement. Strikingly, we find that epithelial buckling is a multiscale phenomenon involving long-lived supracellular folds but also short-lived subcellular wrinkles in the actin cortex. Finally, by forming epithelial shells with anisotropic curvature we rationally direct buckling into predictable wrinkle patterns. Our study shows that epithelial tissues can be understood as hierarchical materials with mechanical instabilities that can be harnessed to engineer epithelial morphogenesis.

biophysics↗

bioRxiv2025

Guidance of cellular nematics into shape-programmable living surfaces

Engineering living materials capable of autonomously morphing into predetermined shapes holds great potential for applications ranging from synthetic tissue morphogenesis to soft robotics. In this regard, harnessing the inherent ability of cellular tissues to self-organize and generate robust force fields offers a promising strategy for creating self-shaping living surfaces. However, achieving precise control over tissue mechanics to direct specific morphogenetic outcomes remains a challenge. Here, we introduce a strategy to program tissue shape transformations through the nematic organization of cellular forces. By precisely controlling nematic order and topological defects, we generate millimeter-scale tissues programmed with specific multicellular tension fields. Using a theoretical framework that integrates contractile nematics with thin sheet mechanics, we explore the role of nematically guided tensions in shape morphogenesis. Experimentally, upon tissue detachment, nematically guided tension fields drive out-of-plane deformations, generating reproducible 3D shapes. By integrating tissue contractility and nematic patterning, our approach offers a robust framework for the rational design of shape-programmable living surfaces.

biophysics↗

bioRxiv2025

Theory of multiscale epithelial mechanics under stretch: from active gels to vertex models

Epithelial monolayers perform a variety of mechanical functions, which include maintaining a cohesive barrier or developing 3D shapes, while undergoing stretches over a wide range of magnitudes and loading rates. To perform these functions, they rely on a hierarchical organization, which spans molecules, cytoskeletal networks, adhesion complexes and junctional networks up to the tissue scale. While the molecular understanding and ability to manipulate cytoskeletal components within cells is rapidly increasing, how these components integrate to control tissue mechanics is far less understood, partly due to the disconnect between theoretical models of sub-cellular dynamics and those at a tissue scale. To fill this gap, here we propose a formalism bridging active-gel models of the actomyosin cortex and 3D vertex-like models at a tissue scale. We show that this unified framework recapitulates a number of seemingly disconnected epithelial time-dependent phenomenologies, including stress relaxation following stretch/unstretch maneuvers, active flattening after buckling, or nonreciprocal and non-affine pulsatile contractions. We further analyze tissue dynamics probed by a novel experimental setup operating in a pressure-controlled ensemble. Overall, the proposed framework systematically connects sub-cellular cortical dynamics and tissue mechanics, and ties a variety of epithelial phenomenologies to a common sub-cellular origin.

biophysics↗

bioRxiv2025

Chromatin organization controls nuclear stiffness

Cellular differentiation is driven by epigenetic modifiers and readers, including the methyl CpG binding protein 2 (MeCP2), whose level and mutations cause the neurological disorder Rett syndrome. During differentiation, most of the genome gets densely packed into heterochromatin, whose function has been simplistically viewed as gene silencing. However, gene expression changes reported in mutations leading to Rett syndrome have failed to be a predictor of disease severity. Here, we show that MeCP2 increases nuclear stiffness in a concentration dependent manner and dependent on its ability to cluster heterochromatin during differentiation. MeCP2-dependent stiffness increase could not be explained by changes in the expression of mechanobiology-related genes, but we found it is disrupted by Rett syndrome mutations and correlated with disease severity. Our results highlight the impact of chromatin organization in the mechanical properties of the cell as an alternative or complementary mechanism to changes in cytoskeleton components. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/643219v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@cb077dorg.highwire.dtl.DTLVardef@158c0c6org.highwire.dtl.DTLVardef@1d830e3org.highwire.dtl.DTLVardef@719456_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

TET dioxygenases localize at splicing speckles and promote RNA splicing

The dynamic regulation of RNA metabolism plays a crucial part in cellular function, with emerging evidence suggesting an important role for RNA modifications in this process. This study explores the relationship between RNA splicing and the TET dioxygenase activity, shedding light on the role of hm5C (RNA 5-hydroxymethylcytosine), and TET proteins, in RNA metabolism. Integrating data from mass spectrometry, AlphaFold structural modeling, microscopic analysis, and different functional assays including in vitro splicing, TET proteins were found to regulate splicing. We show that TET1, TET2, and TET3 interact with the splicing factors U2AF1 and U2AF2. Interestingly, TET dioxygenases localize in splicing speckles in mammalian and Drosophila cells. TET speckles association is RNA dependent, as it is TET interaction with splicing factors. Furthermore, in vitro splicing assays revealed that all three TET proteins promote splicing efficiency, and the oxidation of m5C to hm5C can restore splicing efficiency in vitro. The latter highlights the regulatory role of cytosine modifications in RNA metabolism. These findings provide insights into the complex interplay between RNA modifications and splicing, suggesting a multifaceted role for TET proteins in RNA metabolism beyond its canonical DNA demethylation function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/641893v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1a304bborg.highwire.dtl.DTLVardef@18b91d3org.highwire.dtl.DTLVardef@12efeforg.highwire.dtl.DTLVardef@1464355_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- TET1 localizes in splicing speckles in an RNA-dependent manner - TET proteins, especially TET1, interact with the splicing factors U2AF1 and U2AF2 - TET proteins increase splicing efficiency, independent of their catalytic activity - RNA 5-methylcytosine (m5C) oxidation to 5-hydroxymethylcytosine (hm5C) restores splicing efficiently in vitro

cell biology↗

bioRxiv2025

Continuum theory for the mechanics of curved epithelial shells by coarse-graining an ensemble of active gel cellular surfaces

Epithelial tissues undergo complex morphogenetic transformations driven by cellular and cytoskeletal dynamics. To understand the emergent tissue mechanics resulting from sub-cellular mechanisms, we formulate a fully nonlinear continuum theory for epithelial shells that coarse-grains an underlying 3D vertex model, whose surfaces are in turn patches of active viscoelastic gel undergoing turnover. Our theory relies on two ingredients. First, we relate the deformation of apical, basal and lateral surfaces of cells to the continuum deformation of the tissue mid-surface and a thickness director field. We explore two variants of the theory, a Cosserat theory accommodating through-thickness tilt of cells, and a Kirchhoff theory assuming that lateral cell surfaces remain perpendicular to the mid-surface. Second, by adopting a variational formalism of irreversible thermodynamics, we construct an effective Rayleighian functional of the tissue constrained by the cellular-continuum kinematic relations, which therefore depends on continuum fields only. This functional allows us to obtain the governing equations of the continuum theory and is the basis for efficient finite element simulations. Verification against explicit 3D cellular model simulations demonstrates the accuracy of the proposed theory in capturing epithelial buckling dynamics. Furthermore, we show that the Cosserat theory is required to model tissues exhibiting apicobasal asymmetry of active tension. Our work provides a general frame-work for further studies integrating refined subcellular models into continuum descriptions of epithelial mechanobiology.

biophysics↗

bioRxiv2024

Leukemia escapes immunity by imposing a Type-1 regulatory program on neoantigen-specific CD4+ T cells.

The significance of endogenous immune surveillance in acute lymphoblastic leukemia (ALL) remains controversial. Using clinical B-ALL samples and a novel mouse model, we show that neoantigen-specific CD4+ T cells are induced to adopt type-1 regulatory (Tr1) function in the leukemia microenvironment. Tr1s then inhibit cytotoxic CD8+ T cells, preventing effective leukemia clearance. Leukemic cells induce Tr1s by phenocopying hematopoietic stem cells, which normally are subject to effective surveillance by this CD4+ subset. This mechanism effectively redirects Tr1 cells from a role in preventing cancer to maladaptively promoting clinical relapse. In mouse models, inhibition of Tr1 expansion with IL10 receptor (IL10R) blockade is insufficient to improve leukemia control. In contrast, combined therapy with a cytotoxic agent and anti-PDL1 blockade eradicated measurable residual disease. This correlates with polarization of the neoantigen-specific CD4+ T-cell population from Tr1 towards Th1 states. Our findings uncover a mechanism that enables leukemic relapse and resolves existing controversies on the role of immune surveillance towards this cancer type. Therapeutic polarization of neoantigen-specific CD4+ T cells towards Th1 states may improve contemporary immune therapies by reshaping the immune microenvironment towards states permissive for cytotoxic attack of residual leukemia. Key PointsO_LIB-ALL induces neoantigen-specific CD4+ T cells to adopt Type-1 regulatory states, which protect leukemic cells from immune pressure. C_LIO_LIRepolarizing neoantigen-specific CD4+ T-cells towards Th1 states eradicates measurable residual disease. C_LI

cancer biology↗

bioRxiv2024

3D micropatterned traction force microscopy: a technique to control three-dimensional cell shape while measuring cell-substrate force transmission.

Cell shape and function are intimately linked, in a way that is mediated by the forces exerted between cells and their environment. The relationship between cell shape and forces has been extensively studied for cells seeded on flat 2-dimensional (2D) substrates, but not for cells in more physiological three-dimensional (3D) settings. Here, we demonstrate a technique called 3D micropatterned traction force microscopy (3D-TFM) to confine cells in three-dimensional wells of defined shape, while simultaneously measuring the forces transmitted between cells and their microenvironment. This technique is based on the 3D micropatterning of polyacrylamide wells and on the calculation of 3D traction force from their deformation. With 3D-TFM, we show that MCF10A breast epithelial cells exert defined, reproducible patterns of forces on their microenvironment, which can be both contractile and extensile. We further show that cells switch from a global contractile to extensile behaviour as their volume is reduced. Our technique enables the quantitative study of cell mechanobiology with full access to 3D cellular forces while having accurate control over cell morphology and the mechanical conditions of the microenvironment.

bioengineering↗

bioRxiv2023

Patterning of membrane adhesion under hydraulic stress

Hydraulic fracturing plays a major role in the formation of biological lumens during embryonic development, when the accumulation of pressurized fluid leads to the formation of microlumens that fracture cell-cell contacts and later evolve to form a single large lumen. However, the physical principles underpinning the formation of a pattern of microlumens from a pristine adhesion and their subsequent coarsening are poorly understood. Here, we use giant unilamellar vesicles adhered to a supported lipid bilayer and subjected to osmotic stress to generate and follow the dynamics of hydraulic fracturing akin to those in cells. Using this simplified system together with theoretical modelling and numerical simulations, we provide a mechanistic understanding of the nucleation of hydraulic cracks, their spatial patterns and their coarsening dynamics. Besides coarsening, we show that microlumens can irreversibly bud out of the membrane, reminiscent of endocytic vesicles in cell-cell adhesion. By establishing the physics of patterning and dynamics of hydraulic cracks, our work unveils the mechanical constraints for the biological regulation of hydraulically-driven adhesion remodeling.

biophysics↗

bioRxiv2022

Mapping mechanical stress in curved epithelia of designed size and shape

The function of organs such as lungs, kidneys and mammary glands relies on the three-dimensional geometry of their epithelium. To adopt shapes such as spheres, tubes and ellipsoids, epithelia generate mechanical stresses that are generally unknown. Here we engineered curved epithelial monolayers of controlled size and shape and mapped their state of stress. We designed pressurized epithelia with circular, rectangular and ellipsoidal footprints. We developed a computational method to map the stress tensor in these epithelia. This method establishes a direct correspondence between epithelial shape and mechanical stress without assumptions of material properties. In epithelia with spherical geometry spanning more than one order of magnitude in radius, we show that stress weakly increases with areal strain in a size-independent manner. In epithelia with rectangular and ellipsoidal cross-section we found pronounced stress anisotropies consistent with the asymmetric distribution of tractions measured at the cell-substrate contact line. In these anisotropic profiles, cell shape tended to align with the direction of maximum principal stress but this alignment was non-universal and depended on epithelial geometry. Besides interrogating the fundamental mechanics of epithelia over a broad range of sizes and shapes, our approach will enable a systematic study of how geometry and stress influence epithelial fate and function in three-dimensions.

biophysics↗

bioRxiv2022

The laminin-keratin link shields the nucleus from mechanical deformation and signalling

The mechanical properties of the extracellular matrix (ECM) dictate tissue behaviour. In epithelial tissues, laminin is both a very abundant ECM component, and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111, unlike fibronectin or collagen I, impairs cell response to substrate rigidity, and YAP nuclear localization. Blocking the laminin-specific integrin {beta}4 increases nuclear YAP ratios in a rigidity dependent manner, without affecting cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that {beta}4 integrins establish a mechanical linkage between the substrate and the keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects nuclear YAP mechanoresponses and chromatin methylation. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.

cell biology↗

bioRxiv2021

A mechanosensing mechanism mediated by IRSp53 controls plasma membrane shape homeostasis at the nanoscale

As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nano-scale topography. Here we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nano-scale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by the I-BAR protein IRSp53, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios. TeaserCell stretch cycles generate PM evaginations of {approx}100 nm which are sensed by IRSp53, triggering a local event of actin polymerization that flattens and recovers PM shape.

biophysics↗

bioRxiv2020

Dynamic Mechanochemical feedback between curvedmembranes and BAR protein self-organization

In many physiological situations, BAR proteins interact with, and reshape, pre-existing curved membranes, contributing to essential cellular processes. However, the non-equilibrium and timedependent process of reshaping, and its dependence on initial membrane shape, remains largely unknown. Here we explain, both experimentally and through modelling, how a BAR protein dynamically interacts with mechanically bent lipid membranes. We capture protein binding to curved membranes, and characterize a variety of dynamical reshaping events depending on membrane shape and protein arrangement. The events can be generally understood by an isotropic-to-nematic phase transition, in which low curvature templates with isotropic protein orientation progress towards highly curved lipid tubes with nematic protein arrangement. Our findings also apply in cells, where mechanical stretch triggers BAR-protein-membrane interactions that enable potential mechanotransduction mechanisms. Our results characterize and broaden the reshaping processes of BAR proteins on mechanically constrained membranes, demonstrating the interplay between membrane mechanical stimuli and BAR protein response.

biophysics↗

bioRxiv2020

Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration

Intestinal organoids capture essential features of the intestinal epithelium such as folding of the crypt, spatial compartmentalization of different cell types, and cellular movements from crypt to villus-like domains. Each of these processes and their coordination in time and space requires patterned physical forces that are currently unknown. Here we map the three-dimensional cell-ECM and cell-cell forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the ECM and folds through apical constriction, whereas the transit amplifying zone pulls the ECM and elongates through basal constriction. Tension measurements establish that the transit amplifying zone isolates mechanically the stem cell compartment and the villus-like domain. A 3D vertex model shows that the shape and force distribution of the crypt can be largely explained by cell surface tensions following the measured apical and basal actomyosin density. Finally, we show that cells are pulled out of the crypt along a gradient of increasing tension, rather than pushed by a compressive stress downstream of mitotic pressure as previously assumed. Our study unveils how patterned forces enable folding and collective migration in the intestinal crypt.

cell biology↗