Biology subjects

Conway, D. E.

Publications and source records attributed to Conway, D. E..

15 recordsLinked to original sources

bioRxiv2026

The Desmoplakin Hinge Domain Confers a Reversible Conformational Change in Response to Acute External Force

Desmosomes provide mechanical integrity to tissues that experience strain, yet how mechanical force is sensed and distributed within the desmosomal plaque remains poorly understood. Here we identify the hinge within the desmoplakin (DP) head domain is required for repositioning the DP tail in response to acute uniaxial mechanical stretch. This repositioning occurs within 30 seconds of applied stretch and reverses with release, indicating a dynamic reversible switch rather than a stable structural transition. We show this hinge-dependent repositioning correlates with the propagation of mechanical force to the desmosomal cadherins, consistent with the hinge's role in buffering tension at the cadherin interface during mechanical loading. Deletion or shortening of the hinge abolishes the stretch-induced response and compromises desmosome mechanical integrity. The repositioning of the DP tail in the calcium-dependent to hyper-adhesive transition was not impacted by alterations to the hinge, demonstrating that DP repositioning due to acute force or adhesive state is mechanistically distinct. Together, these findings define a reversible, hinge-dependent mechanotransduction pathway through which DP senses and relays mechanical force within the desmosome. This mechanism could act as a mechanical buffer by reducing the impulse of forces on the cadherins, and play a central role in mechanical resilience.

cell biology↗

bioRxiv2026

Deep Invaginations of Nuclear Envelope Coordinate Spatial Organization of Chromatin in Epithelium

Cell nuclei are often used to assess cell health, but how their shapes vary in normal tissues and how they respond to mechanical forces is not well understood. Here, we describe deep invaginations of the nuclear envelope (DINEs) as common features of epithelial cell nuclei. After their formation, DINEs exist independently of the cytoskeleton, depend on A-type lamins, and emerge in response to cell crowding, contact inhibition, and tissue maturation. High-resolution imaging shows that, in contrast to the peripheral nuclear lamina, DINEs contain densely packed chromatin with regions of active gene transcription. They also remodel dynamically during confined migration, allowing nuclei to adapt to physical constraints. Mechanistically, DINE formation is linked to suppression of MAPK signaling, while activation of growth-promoting pathways reduces their occurrence. These findings reveal DINEs as intrinsic, mechanosensitive structures that coordinate nuclear shape, chromatin organization, and gene activity, providing new insight into how epithelial cells integrate mechanical and biochemical cues to maintain tissue homeostasis. TeaserDeep nuclear envelope invaginations organize chromatin and gene activity in response to epithelial crowding.

cell biology↗

bioRxiv2025

A novel FRET-force biosensor for nucleoporin gp210 reveals that the nuclear pore complex experiences mechanical tension

The nuclear pore complex (NPC) is a large multi-protein structure that enables movement of macromolecules, such as mRNA and proteins, between the nucleoplasm and cytoplasm. There has been great interest in how the physical state of the NPC can influence nuclear-cytoplasmic transport. The hypothesis that the NPC may be mechanosensitive is supported by prior reports showing that the diameter of the NPC increases with nuclear envelope stretch as well as increased ECM stiffness. We therefore sought to develop a biosensor-based approach to determine if the NPC experiences mechanical tension. Using a previously developed FRET-force biosensor, known as TSmod, we developed a gp210 tension sensor. gp210 is a transmembrane nucleoporin, which may serve to anchor the NPC into the nuclear envelope. Using a CRISPR knock-in strategy, we developed a HeLa cell line which expresses the gp210 tension sensor at endogenous levels. Using this sensor, we observed that the biosensor exhibited FRET changes that were consistent with increased force in response to osmotically induced nuclear swelling. Cell attachment, the nuclear LINC complex, ECM stiffness, chromatin condensation, and actomyosin contractility were all observed to influence gp210 forces. Surprisingly, gp210 forces were increased with chromatin relaxation and myosin light chain kinase inhibition, indicating that NPC forces may be differentially regulated from forces on the LINC complex. Our data support a hypothesis where nuclear strain, rather than cytoskeletal forces, is the predominant source for NPC forces. Our studies provide the first direct measurements of protein-level mechanical forces on the NPC. We anticipate that the gp210 force sensor will be of use for future studies of NPC mechanobiology.

biophysics↗

bioRxiv2025

Epithelial eversion, a collective rearrangement from apical-in to apical-out polarity, is initiated by α6β4 integrins and sustained by increased cell proliferation and anchorage-independence

Epithelial cells primarily segregate transmembrane proteins to apical or basal surfaces, establishing apical-basal polarity. For 3D tissues, apical proteins face inwards and basal proteins are on the outer surfaces (in contact with ECM). Apical-basal polarity can become inverted such that the apical proteins are on the outer surfaces. Originally this polarity inversion was assumed to always occur due to changes in protein trafficking. However, recent work by our group showed that increased RhoA activation causes epithelial spheroids to invert apical-basal polarity via a collective rearrangement of cells, a process we and others have termed eversion. We hypothesized that specific integrin-ECM interactions could facilitate the collective cellular migration required for eversion. Using multiple integrin knockout lines, we observed that epithelial cells lacking either 6 or {beta}4 integrins do not develop apical-out polarity in response to RhoA activation. Similarly, 6 blocking antibody or culturing spheroids in collagen (lacking laminin) also inhibited the formation apical-out polarity. These data indicate that 6{beta}4-laminin interactions are required for eversion. Next, we examined the role of cell proliferation and anchorage independence in maintaining apical-out polarity of everted spheroids. Inhibition of cell proliferation (with DNA synthesis inhibitor aphidicolin) or inhibition of anchorage-independence (with FAK inhibitor 14) were sufficient to restore apical-in polarity to RhoA treated spheroids, indicating that both proliferation and anchorage-independence maintain spheroids in an apical-out state. We also observed that apical-out spheroids can revert to apical-in polarity through apoptotic cavitation of cells located in the center of the spheroids. Lastly, through RNA sequencing we demonstrate that apical-out spheroids have unique gene expression profiles. This study provides new mechanistic insights into the biochemical and biophysical mechanisms that drive eversion and maintain apical-out polarity. This work supports the concept that changing from apical-in to apical-out polarity be an important marker for phenotypic switch in epithelial cells.

cell biology↗

bioRxiv2025

Nucleus softens during herpesvirus infection

Nuclear mechanics is remodeled not only by extracellular forces but also by internal modifications, such as those induced by viral infections. During herpes simplex virus type 1 infection, the nuclear structures undergo drastic reorganization, but little is known about how nuclear mechanobiology changes as a result. We show that the nucleus softens dramatically during the infection. To understand the phenomenon, we used advanced microscopy and computational modeling. We discovered that the enlarged viral replication compartment had a low biomolecular density, partially explaining the observed nuclear softening. The mobility of the nuclear lamina decreased, which suggests increased rigidity and an inability to induce softening. However, computational modeling supported by experimental data showed that reduced outward forces, such as cytoskeletal pull and intranuclear osmotic pressure acting both on and within the nucleus, can explain the decreased nuclear stiffness. Our findings reveal that during infection, the nucleus is subject to changes in multiple mechanical forces, leading to decreased nuclear stiffness. Author SummaryDNA viruses take over the host cell nucleus, inducing dramatic structural modifications. There is currently very little knowledge of how the progression of viral infection modifies the mechanical properties of the nucleus, which are essential for various cellular processes, including gene expression and cell migration. Here, we show that the nucleus softens when herpesvirus infection progresses. We discovered that the viral replication compartment established in the central parts of the nucleus had a low biomolecular density, which may contribute to the nuclear softening. The shape and motion of the nuclear lamina suggested that it became more rigid, indicating that another mechanism was involved in the decreased elasticity. Our mechanical simulations and experiments showed that a reduction in outward forces, such as actin cytoskeleton pull or osmotic pressure, is the most likely factor in the nuclear softening. Our study provides new insights into the effects of DNA viruses on the mechanics of host cell nuclei, significantly expanding the knowledge of viral infection mechanobiology.

cell biology↗

bioRxiv2024

Desmosomal Cadherin Tension Loss in Pemphigus Vulgaris Mediated by the Inhibition of Active RhoA at Cell-Cell Adhesions

Binding of autoantibodies to keratinocyte surface antigens, primarily desmoglein 3 (Dsg3) of the desmosomal complex, leads to the dissociation of cell-cell adhesion in the blistering disorder pemphigus vulgaris (PV). After the initial disassembly of desmosomes, cell-cell adhesions actively remodel in association with the cytoskeleton and focal adhesions. Growing evidence highlights the role of adhesion mechanics and mechanotransduction at cell-cell adhesions in this remodeling process, as their active participation may direct autoimmune pathogenicity. However, a large part of the biophysical transformations after antibody binding remains underexplored. Specifically, it is unclear how tension in desmosomes and cell-cell adhesions changes in response to antibodies, and how the altered tensional states translate to cellular responses. Here, we showed a tension loss at Dsg3 using fluorescence resonance energy transfer (FRET)-based tension sensors, a tension loss at the entire cell-cell adhesion, and a potentially compensatory increase in junctional traction force at cell-extracellular matrix adhesions after PV antibody binding. Further, our data indicate that this tension loss is mediated by the inhibition of RhoA at cell-cell contacts, and the extent of RhoA inhibition may be crucial in determining the severity of pathogenicity among different PV antibodies. More importantly, this tension loss can be partially restored by altering actomyosin based cell contractility. Collectively, these findings provide previously unattainable details in our understanding of the mechanisms that govern cell-cell interactions under physiological and autoimmune conditions, which may open the window to entirely new therapeutics aimed at restoring physiological balance to tension dynamics that regulates the maintenance of cell-cell adhesion.

biophysics↗

bioRxiv2023

Coupling During Collective Cell Migration is Controlled by a Vinculin Mechanochemical Switch

Collective cell migration (CCM) plays important roles in development, physiological, and pathological processes. A key feature of CCM is the dynamic mechanical coupling between cells, which enables both long-range coordination and local rearrangements. This coupling requires the ability of cell adhesions to adapt to forces. Recent efforts have identified key proteins and implicated cellular-scale mechanical properties, but how key proteins give rise to these larger-scale mechanical processes is unclear. Using force-sensitive biosensors, cell migration assays, and molecular clutch models, we sought a molecular understanding of adhesion strengthening that could bridge this gap. We found that the mechanical linker protein vinculin bears substantial loads at AJs, FAs, and in the cytoplasm during epithelial sheet migration, and we identified a switch-like residue on vinculin that regulates its conformation and loading at the AJs during CCM. In vinculin KO-rescue, this switch jointly controlled the speed and coupling length-scale of CCM, which suggested changes in adhesion-based friction. To test this, we developed molecularly detailed friction clutch models of the FA and AJ. They show that open, loaded vinculin increases friction in adhesive structures, with larger affects observed in AJs. Thus, this work elucidates how load-bearing linker proteins can be regulated to alter mechanical properties of cells and enable rapid tuning of mechanical coupling in CCM.

cell biology↗

bioRxiv2022

Loss of mechanical homeostasis drives epithelial acinar eversion

Epithelial cells lining a gland and cells grown in a soft extracellular matrix polarize with apical proteins exposed to the lumen and basal proteins in contact with the extracellular matrix. Alterations to polarity, including an apical-out polarity, occur in human cancers. While some aberrant polarity states may result from altered protein trafficking, recent observations of an extraordinary tissue-level inside-out unfolding suggest an alternate pathway for altered polarity. Because mechanical alterations are common in human cancer including an upregulation of RhoA mediated actomyosin tension in acinar epithelia, we explored whether perturbing mechanical homeostasis could cause apical-out eversion. Acinar eversion was robustly induced by direct activation of RhoA in normal and tumor epithelial acini, or indirect activation of RhoA through blockage of {beta}1 integrins, disruption of the LINC complex, oncogenic Ras activation, or Rac1 inhibition. Furthermore, laser ablation of a portion of the untreated acinus was sufficient to induce eversion. Analyses of acini revealed high curvature and low phosphorylated myosin in the apical cell surfaces relative to the basal surfaces. A vertex-based mathematical model which balances tension at cell-cell interfaces revealed a five-fold greater basal cell surface tension relative to the apical cell surface tension. The model suggests that the difference in surface energy between the apical and basal surfaces is the driving force for acinar eversion. Our findings raise the possibility that a loss of mechanical homeostasis may cause apical-out polarity states in human cancers.

biophysics↗

bioRxiv2022

Nuclear lamina strain states revealed by intermolecular force biosensor

Nuclear lamins have been considered to be an important structural element of the nucleus. The nuclear lamina is thought both to shield DNA from excessive mechanical forces and to transmit mechanical forces onto the DNA. However, to date there is not yet a technical approach to directly measure mechanical forces on nuclear lamins at the protein level. To overcome this limitation, we developed a nanobody-based intermolecular tension FRET biosensor capable of measuring the mechanical strain of lamin filaments. Using this sensor, we were able to show that the nuclear lamina is subjected to significant force. These forces are dependent on nuclear volume, actomyosin contractility, functional LINC complex, chromatin condensation state, cell cycle, and EMT. Interestingly, large forces were also present on nucleoplasmic lamins, indicating that these lamins may also have an important mechanical role in the nucleus. Overall, we demonstrate that nanobody-based approach allows construction of novel force biosensors for mechanobiology studies.

biophysics↗

bioRxiv2022

Depletion of Sun1/2 Induces Heterochromatin Accrual in Mesenchymal Stem Cells during Adipogenesis

Critical to the mechano-regulation of mesenchymal stem cells (MSC), Linker of the Nucleoskeleton and Cytoskeleton (LINC) complex transduces cytoskeletal forces to the nuclei. The LINC complex contains outer nuclear membrane Nesprin proteins that associate with the cytoskeleton and their inner nuclear membrane couplers, Sun proteins. In addition to coupling Nesprin-associated cytoskeletal elements to inner nuclear membrane, Sun proteins also function in regulating nuclear mechanics and chromatin tethering to inner nuclear membrane. This suggests that release of LINC-mediated cytoskeletal connections from cell nuclei may have different effects on chromatin organization and MSC differentiation than those due to ablation of intra-nuclear Sun proteins. To test this hypothesis we compared Sun1/2 depletion with expression of the dominant-negative KASH domain (dnKASH) that inhibits Nesprin-Sun association. In cells cultured under adipogenic conditions, disconnecting LINC from the cytoskeleton through dnKASH expression, increased adipogenic gene expression and fat droplet formation; heterochromatin H3K27 and H3K9 methylation was unaltered. In contrast, Sun1/2 depletion inhibited adipogenic gene expression and fat droplet formation; as well the anti-adipogenic effect of Sun1/2 depletion was accompanied by increased H3K9me3, which was enriched on Adipoq, silencing this fat locus. We conclude that releasing the nucleus from cytoskeletal constraints via dnKASH accelerates adipogenesis while depletion of Sun1/2 increases heterochromatin accrual on adipogenic genes in a fashion independent of LINC complex function. Therefore, while these two approaches both disable LINC functions, their divergent effects on the epigenetic landscape indicate they cannot be used interchangeably to study mechanical regulation of cell differentiation.

bioengineering↗

bioRxiv2021

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. While biochemical mechanisms that regulate activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC-45A were decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

cell biology↗

bioRxiv2021

Progerin-Expressing Endothelial Cells are Unable to Adapt to Shear Stress

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare premature aging disease caused by a single-point mutation in the lamin A gene, resulting in a truncated and farnesylated form of lamin A. This mutant lamin A protein, known as progerin, accumulates at the periphery of the nuclear lamina, resulting in both an abnormal nuclear morphology and nuclear stiffening. HGPS patients experience rapid onset of atherosclerosis, with death from heart attack or stroke as teenagers. Progerin expression has been shown to cause dysfunction in both vascular smooth muscle cells and endothelial cells (ECs). In this study we examined how progerin-expressing ECs adapt to fluid shear stress, the principal mechanical force from blood flow. We compared the response to shear stress for progerin-expressing, wild-type lamin A overexpressing, and control ECs to physiological levels of fluid shear stress. Additionally, we also knocked down ZMPSTE24 in ECs, which results in increased farnesylation of lamin A and similar phenotypes to HGPS. Our results showed that ECs either expressing progerin or with ZMPSTE24 knockdown were unable to adapt to shear stress, experiencing significant cell loss at a longer duration of exposure to shear stress (3 days). ECs overexpressing wild-type lamin A also exhibited similar impairments in adaptation to shear stress, including similar levels of cell loss. Quantification of nuclear morphology showed that progerin-expressing ECs had similar nuclear abnormalities in both static and shear conditions. Treatment of progerin-expressing cells and ZMPSTE24 KD cells with lonafarnib and methystat, drugs previously shown to improve HGPS nuclear morphology, resulted in improvements in adaptation to shear stress. Additionally, pre-alignment of cells to shear stress prior to progerin-expression prevented cell loss. Our results demonstrate that changes in nuclear lamins can affect the ability of EC to properly adapt to shear stress.

biophysics↗

bioRxiv2020

Maraviroc inhibits SARS-CoV-2 multiplication and s-protein mediated cell fusion in cell culture

In an effort to identify therapeutic intervention strategies for the treatment of COVID-19, we have investigated a selection of FDA-approved small molecules and biologics that are commonly used to treat other human diseases. A investigation into 18 small molecules and 3 biologics was conducted in cell culture and the impact of treatment on viral titer was quantified by plaque assay. The investigation identified 4 FDA-approved small molecules, Maraviroc, FTY720 (Fingolimod), Atorvastatin and Nitazoxanide that were able to inhibit SARS-CoV-2 infection. Confocal microscopy with over expressed S-protein demonstrated that Maraviroc reduced the extent of S-protein mediated cell fusion as observed by fewer multinucleate cells in the context of drugtreatment. Mathematical modeling of drug-dependent viral multiplication dynamics revealed that prolonged drug treatment will exert an exponential decrease in viral load in a multicellular/tissue environment. Taken together, the data demonstrate that Maraviroc, Fingolimod, Atorvastatin and Nitazoxanide inhibit SARS-CoV-2 in cell culture.

microbiology↗

bioRxiv2019

A Predictive Model of Intercellular Tension and Cell-Matrix Mechanical Interactions in a Multicellular Geometry

Epithelial cells form continuous sheets of cells that exist in tensional homeostasis. Homeostasis is maintained through cell-to-cell adhesions that distribute tension and balance forces between cells and their underlying matrix. Disruption of tensional homeostasis can lead to Epithelial-Mesenchymal Transition (EMT), which is a transdifferentiation process in which epithelial cells adopt a mesenchymal phenotype, where cell-cell adhesion is lost and individual cell migration is acquired. This process is critical during embryogenesis and wound healing, but is also dysregulated in many disease states. To further understand the role of intercellular tension in spatial patterning of epithelial cell monolayers, we developed a multicellular computational model of cell-cell and cell-substrate forces. This work builds on a hybrid Cellular Potts-finite element model to evaluate cell-matrix mechanical feedback of an adherent multicellular cluster. Thermodynamically-constrained cells migrate by generating traction forces on a finite element substrate to minimize the total energy of the system. Junctional forces at cell-cell contacts balance these traction forces, thereby producing a mechanically stable epithelial monolayer. Simulations were compared to in vitro experiments using fluorescence-based junction force sensors in clusters of cells undergoing EMT. Results indicate that the multicellular CPM model can reproduce many aspects of EMT, including epithelial monolayer formation dynamics, changes in cell geometry, and spatial patterning of cell geometry and cell-cell forces in an epithelial colony.\n\nAuthor summaryEpithelial cells line all organs of the human body and act as a protective barrier by forming a continuous sheet. These cells exert force on both their neighboring cells as well as the underlying extracellular matrix, which is a network of proteins that creates the structure of tissues. Here we develop a model that encompasses both cell-cell forces and cell-matrix forces in an epithelial cell sheet. The model accounts for cell migration and proliferation, and regulates how cell-cell adhesions are formed. We demonstrate how the interplay between cell-cell forces and cell-matrix forces can regulate the formation of the epithelial cell sheet, the organization of cells within the sheet, and the pattern of cell geometries and cell forces within the sheet. We compare computational results with experiments in which epithelial cell sheets are disrupted and cell-cell junction forces are measured, and demonstrate that the model captures many aspects of epithelial cell dynamics observed experimentally.

cell biology↗

bioRxiv2019

Mechanochemical coupling and junctional forces during collective cell migration

Cell migration, a fundamental physiological process in which cells sense and move through their surrounding physical environment, plays a critical role in development and tissue formation, as well as pathological processes, such as cancer metastasis and wound healing. During cell migration, dynamics are governed by the bidirectional interplay between cell-generated mechanical forces and the activity of Rho GTPases, a family of small GTP-binding proteins that regulate actin cytoskeleton assembly and cellular contractility. These interactions are inherently more complex during the collective migration of mechanically coupled cells, due to the additional regulation of cell-cell junctional forces. In this study, we present a minimal modeling framework to simulate the interactions between mechanochemical signaling in individual cells and interactions with cell-cell junctional forces during collective cell migration. We find that migration of individual cells depends on the feedback between mechanical tension and Rho GTPase activity in a biphasic manner. During collective cell migration, waves of Rho GTPase activity mediate mechanical contraction/extension and thus synchronization throughout the tissue. Further, cell-cell junctional forces exhibit distinct spatial patterns during collective cell migration, with larger forces near the leading edge. Larger junctional force magnitudes are associated with faster collective cell migration and larger tissue size. Simulations of heterogeneous tissue migration exhibit a complex dependence on the properties of both leading and trailing cells. Computational predictions demonstrate that collective cell migration depends on both the emergent dynamics and interactions between cellular-level Rho GTPase activity and contractility, and multicellular-level junctional forces.

biophysics↗