Biology subjects

Chalut, K. J.

Publications and source records attributed to Chalut, K. J..

11 recordsLinked to original sources

bioRxiv2026

Basement membrane mechanics drives patterned response to developmental signalling

Precise spatiotemporal patterning of cell fate decisions during development, such as those accompanying gastrulation, has traditionally been attributed to morphogen gradients. Emerging evidence also points to a crucial role for mechanics in regulating cell fate decisions. Here, we uncover that basement membrane mechanics in human pluripotent stem cells (hPSCs) regulates the spatiotemporal response to gastrulation-inducing BMP4. Reducing mechanosensation via soft substrates or chemical inhibitors abolishes stereotypical spatial patterning of the BMP4 response in hPSC colonies. This loss arises from disrupted epithelial polarity and increased permeability, which enhance BMP receptor accessibility. Strikingly, apical exposure to soluble laminin similarly disrupts polarity and suppresses hPSC mechanosensing, phenocopying the soft substrates effect and suggesting feedback between polarity signaling and mechanosensing. Finally, softening the basement membrane in mouse embryos triggers ectopic and premature mesoderm differentiation, disrupting gastrulation patterning. Together, this study describes a mechanochemical feedback mechanism that establishes basement membrane mechanics as a key regulator of developmental patterning.

cell biology↗

bioRxiv2025

Intrinsic mechanotransduction during apical constriction licenses lineage competence in pluripotent stem cells

To acquire the capacity for multi-lineage differentiation, pluripotent stem cells must undergo a transition from naive pluripotency to lineage competency. This transition requires epithelialization and changes in nuclear architecture. We sought to determine whether the cell and tissue mechanics intrinsic to epithelialization drive pluripotency progression and subsequent lineage commitment to neuroectodermal fate. We demonstrate that naive mouse embryonic stem cells (mESCs) undergoing early differentiation in vitro recapitulate features of epithelialization in the peri-implantation epiblast, specifically apical constriction. We further demonstrate that cell contractility during apical constriction induces a distinct nuclear mechanoresponse, notably enrichment of emerin at the outer nuclear membrane, nuclear envelope localization of SUN2, and the global loss of H3K9me3 heterochromatin which is compensated by H3K27me3. Importantly, these nuclear phenotypes and subsequent neuroectodermal lineage priming require myosin II-mediated contractility, an intact LINC complex, and emerin. We demonstrate that LINC-dependent mechanotransduction through emerin regulates H3K27me3 occupancy on the key early neuroectodermal transcription factor gene, Sox1, implicating a mechanical switch in chromatin mediation of neuroectodermal lineage competence. These results indicate that epithelialization-induced nuclear mechanotransduction poises a critical lineage gene for subsequent expression. HighlightsO_LIAdherent naive mESCs in vitro recapitulate in utero apical constriction morphogenesis. C_LIO_LIContractility, the LINC complex and emerin are required for neuroectodermal lineage competence. C_LIO_LIContractility and the LINC complex are required for widespread loss of H3K9me3 and gain of H3K27me3 on Sox1 promoter. C_LI

cell biology↗

bioRxiv2025

Mechanical signalling through collagen I regulates cholangiocyte specification and tubulogenesis during liver development

Cholangiocyte dysfunction accounts for a third of liver transplantations, access to which is limited by a shortage of healthy donor organs. A promising alternative is the therapeutic use of human induced pluripotent stem cell (hiPSC)-derived cholangiocytes. However, the use of hiPSCs is impeded by a lack of knowledge regarding intrahepatic cholangiocyte development, limiting the generation of fully functional cells. In this study, we generate hiPSC-derived tubular cholangiocytes using an approach based in synthetic hydrogels. These hydrogels exert control over stiffness and extracellular matrix (ECM) composition and stability, allowing us to address a critical gap in understanding cholangiocyte development. Our findings reveal that stable collagen I functionalisation, particularly on a soft substrate, enhances cholangiocyte differentiation, largely irrespective of substrate stiffness. Furthermore, high collagen I stability on a soft substrate suppresses hepatic identity whilst promoting biliary identity and duct morphogenesis. Our findings highlight the importance of collagen I mechanical signalling in regulating hepatoblast fate determination. Overall, we propose a mechanism by which the ECM modulates cholangiocyte and bile duct development and present a scalable platform for future clinical applications in the understanding and treatment of cholangiopathies.

developmental biology↗

bioRxiv2024

Hyaluronic Acid and Emergent Tissue Mechanics Orchestrate Digit Tip Regeneration

Tissue regeneration is a dynamic process requiring coordinated cell fate decisions to restore structure and function. For instance, the tips of human and rodent digits fully regrow after amputation, although more proximal injuries fail to regenerate. While biochemical cues have been widely studied in wound healing, the role of the physical microenvironment remains less understood. Here, we discovered that tissue mechanics and extracellular matrix (ECM) composition differ markedly between non-regenerating and regenerating wounds. These differences are due to specific fibroblast sub-types which predominate in the non-regenerating wound and form fibrotic collagen networks. Conversely, bone-forming progenitors in regenerating digits synthesize abundant hyaluronic acid, which mediates collagen fibrillogenesis. By modeling the tissue mechanics of non-regenerative and regenerative wounds using hydrogels, we showed that substrate stiffness regulates the synthesis of fibrotic and regenerative ECM. We further revealed that mimicking the regenerative wound mechanics upregulated bone morphogenic protein signaling and osteogenic differentiation. We ultimately demonstrated that the link protein HAPLN1 promotes hyaluronic acid deposition in vivo, reduces scar formation, and induces bone repair after non-regenerative amputations. These findings emphasize the interplay among the ECM, tissue mechanics, and cell behavior in digit regeneration and point towards ECM modulation as a strategy to improve wound healing. HighlightsO_LINon-regenerating and regenerating digit tips diverge in their cellular and ECM composition C_LIO_LIRegeneration requires hyaluronic acid matrix, which mediates collagen assembly and tissue mechanics C_LIO_LISoft substrates enhance BMP signaling and propagate the synthesis of regenerative ECM C_LIO_LIHAPLN1 overexpression initiates rescue of non-regenerative digit tip amputations C_LI

developmental biology↗

bioRxiv2023

ERK signalling orchestrates metachronous transition from naive to formative pluripotency

Naive epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, precipitating breakdown of the naive state gene regulatory network. Cell variability in pERK dynamics results in metachronous down-regulation of Nanog and naive state exit. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This failure is attributable to loss of expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naive state and preserves pluripotency. These results illustrate that a single signalling pathway can both drive exit from a developmental state and safeguard progression to the successor state.

developmental biology↗

bioRxiv2021

A biomechanical switch regulates the transition towards homeostasis in esophageal epithelium

Epithelial cells are highly dynamic and can rapidly adapt their behavior in response to tissue perturbations and increasing tissue demands. However, the processes that finely control these responses and, particularly, the mechanisms that ensure the correct switch to and from normal tissue homeostasis are largely unknown. Here we explore changes in cell behavior happening at the interface between postnatal development and homeostasis in the epithelium of the mouse esophagus, as a physiological model exemplifying a rapid but controlled tissue growth transition. Single cell RNA sequencing and histological analysis of the mouse esophagus reveal significant mechanical changes in the epithelium upon tissue maturation. Organ stretching experiments further indicate that tissue strain caused by the differential growth of the mouse esophagus relative to the entire body promotes the emergence of a defined committed population in the progenitor compartment as homeostasis is established. Our results point to a simple mechanism whereby the mechanical changes experienced at the whole tissue level are integrated with those "sensed" at the cellular level to control epithelial cell behavior and tissue maintenance.

cell biology↗

bioRxiv2020

Mouse embryonic stem cells switch migratory behaviour during early differentiation

Development relies on a series of precisely orchestrated cell fate changes. While studies of fate transitions often focus on changes in gene regulatory networks, most transitions are also associated with changes in cell shape and cell behaviour. Here, we investigate changes in migratory behaviour in mouse embryonic stem (ES) cells during their first developmental fate transition, exit from ES cell state. We show that naive pluripotent ES cells cannot efficiently migrate on 2-dimensional substrates but are able to migrate in an amoeboid fashion when placed in confinement. Exit from ES cell state, typically characterised by enhanced cell spreading, is associated with decreased migration in confinement and acquisition of mesenchymal-like migration on 2D substrates. Interestingly, confined, amoeboid-like migration of ES cells strongly depends on Myosin IIA, but not Myosin IIB. In contrast mesenchymal-like migration of cells exiting the ES cell state does not depend on Myosin motor activity but relies on the activity of the Arp2/3 complex. Together, our data suggest that during early differentiation, cells undergo a switch in the regulation of the actin cytoskeleton, leading to a transition from amoeboid-to mesenchymal-like migration. Summary statementNaive mouse embryonic stem cells display amoeboid-like migration in confinement, but switch to mesenchymal-like migration as they exit the ES cell state.

cell biology↗

bioRxiv2019

Cortical cell stiffness is independent of substrate mechanics

Cortical stiffness is an important cellular property that changes during migration, adhesion, and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM onto cells results in a significant deformation of the underlying substrate if it is softer than the cells. This soft substrate effect leads to an underestimation of a cells elastic modulus when analyzing data using a standard Hertz model, as confirmed by finite element modelling (FEM) and AFM measurements of calibrated polyacrylamide beads, microglial cells, and fibroblasts. To account for this substrate deformation, we developed the composite cell-substrate model (CoCS model). Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has significant implications for our interpretation of many physiological and pathological processes.

biophysics↗

bioRxiv2019

Membrane tension mediated mechanotransduction drives fate choice in embryonic stem cells

Changes in cell shape and mechanics frequently accompany cell fate transitions. Yet how mechanics affects the regulatory path-ways controlling cell fate is poorly understood. To probe the interplay between shape, mechanics and fate, we used embryonic stem (ES) cells, which spread as they undergo early differentiation. We found that this spreading is regulated by a beta-catenin mediated decrease in plasma membrane tension. Strikingly, preventing the membrane tension decrease obstructs early differentiation of ES cells. We further find that blocking the decrease in membrane tension inhibits endocytosis of FGF signalling components, which direct the exit from the ES cell state. The early differentiation defects we observed can be rescued by increasing Rab5a-facilitated endocytosis. Thus, we show that a mechanically-triggered increase in endocytosis regulates fate transitions. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling, and therefore cell fate.

cell biology↗

bioRxiv2019

Abscission couples cell division to embryonic stem cell fate

Cell fate transitions are key to development and homeostasis. It is thus essential to understand the cellular mechanisms controlling fate transitions. Cell division has been implicated in fate decisions in many stem cells, including neuronal and epithelial progenitors. In other stem cells, such as embryonic stem (ES) cells, the role of division remains unclear. Here we show that exit from naive pluripotency in mouse ES cells generally occurs after a division. We further show that exit timing is strongly correlated between sister cells, which remain connected by cytoplasmic bridges long after division, and that bridge abscission progressively accelerates as cells exit naive pluripotency. Finally, interfering with abscission impairs exit from naive pluripotency. Altogether, our data indicate that a switch in the division machinery leading to faster abscission is crucial for pluripotency exit. Our study identifies abscission as a key step coupling cell division to fate transitions.

cell biology↗

bioRxiv2019

StemBond hydrogels optimise the mechanical microenvironment for embryonic stem cells

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and tethering of extracellular matrix (ECM) to substrates, making ECM tethering a potentially confounding variable in mechanical signalling investigations. Moreover, poor ECM tethering can lead to weak cell attachment. To address this, we developed StemBond hydrogels, a hydrogel formulation in which ECM tethering is stable and can be varied independently of stiffness. We show that soft StemBond hydrogels provide an optimal format for culturing embryonic stem (ES) cells. We find that soft StemBond substrates improve the homogeneity of ES cell populations, boost their self-renewal, and increase the efficiency of cellular reprogramming. Our findings underline how soft microenvironments impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

cell biology↗