Junctional Tension Research Articles

Cell cycle–dependent active stress drives epithelia remodeling

Epithelia have distinct cellular architectures which are established in development, reestablished after wounding, and maintained during tissue homeostasis despite cell turnover and mechanical perturbations. In turn, cell shape also controls tissue function as a regulator of cell differentiation, proliferation, and motility. Here, we investigate cell shape changes in a model epithelial monolayer. After the onset of confluence, cells continue to proliferate and change shape over time, eventually leading to a final architecture characterized by arrested motion and more regular cell shapes. Such monolayer remodeling is robust, with qualitatively similar evolution in cell shape and dynamics observed across disparate perturbations. Here, we quantify differences in monolayer remodeling guided by the active vertex model to identify underlying order parameters controlling epithelial architecture. When monolayers are formed atop an extracellular matrix with varied stiffness, we find the cell density at which motion arrests varies significantly, but the cell shape remains constant, consistent with the onset of tissue rigidity. In contrast, pharmacological perturbations can significantly alter the cell shape at which tissue dynamics are arrested, consistent with varied amounts of active stress within the tissue. Across all experimental conditions, the final cell shape is well correlated to the cell proliferation rate, and cell cycle inhibition immediately arrests cell motility. Finally, we demonstrate cell cycle variation in junctional tension as a source of active stress within the monolayer. Thus, the architecture and mechanics of epithelial tissue can arise from an interplay between cell mechanics and stresses arising from cell cycle dynamics.

Glycolysis Inhibition Regulates Endothelial Junctions by Perturbing Actin and Focal Adhesions

One of the central functions of the endothelium is to maintain a vascular barrier that prevents fluid leakiness and immune cell influx from the circulating blood into the tissue. The barrier integrity of the endothelium is largely controlled by adherens junctions (AJs) and the key AJ molecule VE-cadherin, which maintains cell cohesion via homotypic trans-interaction with VE-cadherin molecules on neighboring endothelial cells. Tension is required to maintain junction homeostasis, but little is known about the role of endothelial metabolism and bioenergetics in regulating junctional forces. Because glycolysis is the main source of ATP generation in endothelial cells, we examined the bioenergetic control of the mechanics of VE-cadherin junctions, by focusing on the glycolysis regulatory enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Results from traction force imbalance measurements and a VE-cadherin tension sensor revealed that inhibiting PFKFB3 significantly reduced the average junctional tension and the force on VE-cadherin complexes. The decrease in tension was largely due to mechanical changes distal from the cell-cell contacts. Specifically, inhibiting glycolysis perturbs focal adhesions and disrupts actin organization, directly impacting the net force on intercellular contacts. These findings identify a critical role of cellular metabolism for the mechanics and integrity of vascular endothelial junctions, by maintaining global cell mechanics.

Vinculin Controls Endothelial Cell Junction Dynamics During Vascular Lumen Formation

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors, which depend on dynamic cell-cell interactions. Remodeling of endothelial cell-cell junctions promote morphogenetic cellular events while preserving vascular integrity. Here, we have analyzed the dynamics of endothelial cell-cell junctions during lumen formation in angiogenic sprouts. By live-imaging of the formation of intersegmental blood vessels in zebrafish, we demonstrate that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Formation and maintenance of these junctional fingers are positively regulated by blood pressure whereas inhibition of blood flow prevents their formation. Using fluorescent reporters, we show that the tension-sensor Vinculin localizes to junctional fingers. Furthermore, loss of vinculin function, in vinculin a and -b double knockouts, prevents junctional finger formation in angiogenic sprouts, whereas endothelial expression of a vinculin transgene is sufficient to restore junctional fingers. Taken together, our findings suggest a mechanism in which lumen expansion during angiogenesis leads to an increase in junctional tension, which triggers recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells may employ force-dependent junctional remodeling to react to changes in external forces to protect cell-cell contacts and to maintain vascular integrity during sprouting angiogenesis.

Tracking collective cell motion by topological data analysis.

By modifying and calibrating an active vertex model to experiments, we have simulated numerically a confluent cellular monolayer spreading on an empty space and the collision of two monolayers of different cells in an antagonistic migration assay. Cells are subject to inertial forces and to active forces that try to align their velocities with those of neighboring ones. In agreement with experiments in the literature, the spreading test exhibits formation of fingers in the moving interfaces, there appear swirls in the velocity field, and the polar order parameter and the correlation and swirl lengths increase with time. Numerical simulations show that cells inside the tissue have smaller area than those at the interface, which has been observed in recent experiments. In the antagonistic migration assay, a population of fluidlike Ras cells invades a population of wild type solidlike cells having shape parameters above and below the geometric critical value, respectively. Cell mixing or segregation depends on the junction tensions between different cells. We reproduce the experimentally observed antagonistic migration assays by assuming that a fraction of cells favor mixing, the others segregation, and that these cells are randomly distributed in space. To characterize and compare the structure of interfaces between cell types or of interfaces of spreading cellular monolayers in an automatic manner, we apply topological data analysis to experimental data and to results of our numerical simulations. We use time series of data generated by numerical simulations to automatically group, track and classify the advancing interfaces of cellular aggregates by means of bottleneck or Wasserstein distances of persistent homologies. These techniques of topological data analysis are scalable and could be used in studies involving large amounts of data. Besides applications to wound healing and metastatic cancer, these studies are relevant for tissue engineering, biological effects of materials, tissue and organ regeneration.

Effect of printing temperature on microstructure, thermal behavior and tensile properties of 3D printed nylon using fused deposition modeling

AbstractThe present investigation aims at the thermal conditions for the printability of nylon using fused deposition modeling (FDM). Dog‐bone like specimens are manufactured under two printing temperatures to measure the tensile performance of 3D printed nylon with respect to the feedstock material properties. Both Scanning Electron Microscopy (SEM) and X‐ray micro‐tomography analysis are conducted to shed more light on the microstructural arrangement of nylon filaments. Finite element computation based on microstructural implementation is considered to study the main deformation mechanisms associated with the nylon filament arrangement and the process‐induced porosity. The results show a narrow temperature range for printability of nylon, and a significant influence of the printing temperature on the thermal cycling, porosity content and mechanical performance. With the support of both numerical and experimental results, complex deformation mechanisms are revealed involving shearing related to the filament sequencing, compression at the junction points and tension within the raster and the frame. All these mechanisms are associated with the particular and regular arrangement of nylon filaments.

In primary airway epithelial cells, the unjamming transition is distinct from the epithelial-to-mesenchymal transition

The epithelial-to-mesenchymal transition (EMT) and the unjamming transition (UJT) each comprises a gateway to cellular migration, plasticity and remodeling, but the extent to which these core programs are distinct, overlapping, or identical has remained undefined. Here, we triggered partial EMT (pEMT) or UJT in differentiated primary human bronchial epithelial cells. After triggering UJT, cell-cell junctions, apico-basal polarity, and barrier function remain intact, cells elongate and align into cooperative migratory packs, and mesenchymal markers of EMT remain unapparent. After triggering pEMT these and other metrics of UJT versus pEMT diverge. A computational model attributes effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, those tissues that are subject to development, injury, or disease become endowed with rich mechanisms for cellular migration, plasticity, self-repair, and regeneration.

DE-cadherin and Myosin II balance regulates furrow length for onset of polygon shape in syncytial Drosophila embryos.

Cell shape morphogenesis, from spherical to polygonal, occurs in epithelial cell formation in metazoan embryogenesis. In syncytial Drosophila embryos, the plasma membrane incompletely surrounds each nucleus and is organized as a polygonal epithelial-like array. Each cortical syncytial division cycle shows a circular to polygonal plasma membrane transition along with furrow extension between adjacent nuclei from interphase to metaphase. In this study, we assess the relative contribution of DE-cadherin (also known as Shotgun) and Myosin II (comprising Zipper and Spaghetti squash in flies) at the furrow to polygonal shape transition. We show that polygonality initiates during each cortical syncytial division cycle when the furrow extends from 4.75 to 5.75 μm. Polygon plasma membrane organization correlates with increased junctional tension, increased DE-cadherin and decreased Myosin II mobility. DE-cadherin regulates furrow length and polygonality. Decreased Myosin II activity allows for polygonality to occur at a lower length than controls. Increased Myosin II activity leads to loss of lateral furrow formation and complete disruption of the polygonal shape transition. Our studies show that DE-cadherin-Myosin II balance regulates an optimal lateral membrane length during each syncytial cycle for polygonal shape transition.This article has an associated First Person interview with the first author of the paper.

Measurement of junctional tension in epithelial cells at the onset of primitive streak formation in the chick embryo via non-destructive optical manipulation.

ABSTRACTDirectional cell intercalations of epithelial cells during gastrulation has, in several organisms, been shown to be associated with a planar cell polarity in the organisation of the actin-myosin cytoskeleton and is postulated to reflect directional tension that drives oriented cell intercalations. We have characterised and applied a recently introduced non-destructive optical manipulation technique to measure the tension in individual epithelial cell junctions of cells in various locations and orientations in the epiblast of chick embryos in the early stages of primitive streak formation. Junctional tension of mesendoderm precursors in the epiblast is higher in junctions oriented in the direction of intercalation than in junctions oriented perpendicular to the direction of intercalation and higher than in junctions of other cells in the epiblast. The kinetic data fit best with a simple viscoelastic Maxwell model, and we find that junctional tension, and to a lesser extent viscoelastic relaxation time, are dependent on myosin activity.

Glycolysis Inhibition Alters Junction Mechanics by Perturbing Actin and Focal Adhesions in Endothelial Cells

In Acute Respiratory Distress Syndrome (ARDS), fluid leakage into alveoli results in hypoxia, a reduction in glycolysis, the disruption of inter endothelial junctions, and increased vascular leakage. This study investigated the impact of glycolysis inhibition on the mechanics and integrity of inter endothelial junctions. Traction force imbalance measurements revealed that inhibiting glycolysis reduces tension at inter endothelial junctions. Inhibitor treatment also altered both the amplitudes and orientational coherence of integrin-mediated traction forces. Both changes contribute to the reduced junctional tension. Immunofluorescence imaging of actin and paxillin further revealed that inhibiting glycolysis perturbed actin organization as well as the focal adhesion size. Prior studies correlated the loss of junction tension or actin disruption with the destabilization of cadherin adhesions. FRAP measurements and immunofluorescence images of VE-cadherin revealed increased VE-cadherin internalization which could also alter junction tension. Using a conformation sensitive antibody against alpha catenin, which is a force transducer in cadherin complexes, we observed less alpha catenin activation at junctions between inhibitor treated cells. Therefore, tension is also reduced at the level of cadherin complexes retained at the junctions. These findings suggest possible mechanisms by which inhibition of glycolysis may contribute to the disruption of endothelial junction integrity.

Desmosomal Junctions Govern Tissue Integrity and Actomyosin Contractility in Apoptotic Cell Extrusion.

During apoptosis, or programmed cell death, a dead cell could be expelled from the tissue by coordinated processes between the dying cell and its neighbors. Apoptotic cell extrusion is driven by actomyosin cable formation and its contraction and lamellipodial crawling of the neighboring cells [1-4]. Throughout cell extrusion, the mechanical coupling of epithelia needs to be maintained in order to preserve tissue homeostasis [1]. Although much is known about the regulation of adherens junctions (AJs) in apoptotic cell extrusion [4-7], the role and dynamics of desmosomal junctions (DJs) during this process remain poorly understood. Here, we show that DJs stay intact throughout and are crucial for cell extrusion. Pre-existing DJs between the apoptotic cell and neighboring cells remain intact, even during the formation of de novo DJs between non-dying cells, suggesting the neighboring cells possess two DJs in the middle of apoptotic cell extrusion. We further found that an actomyosin cable formed in the vicinity of DJs upon apoptosis and subsequently deviated from DJs during its constriction. Interestingly, the departure of the actomyosin cable from DJs coincided with the timing when DJs lost their straightness, suggesting a release of junctional tension at DJs and a mechanical coupling between DJs and actomyosin contractility. The depletion of desmoplakin resulted in defective contractility and an inability to form de novo DJs, leading to a failure of apoptotic cell extrusion. Our study provides a framework to explainhowdesmosomes play pivotal roles in maintaining epithelial sheet integrity during apoptotic cell extrusion.

Solid-fluid transition and cell sorting in epithelia with junctional tension fluctuations.

Tissues transition between solid-like and fluid-like behavior, which has major implications for morphogenesis and disease. These transitions can occur due to changes in the intrinsic shape of constituent cells and cell motility. We consider an alternative mechanism by studying tissues that explore the energy landscape through stochastic dynamics, driven by turnover of junctional molecular motors. To identify the solid-fluid transition, we start with single-component tissues and show that the mean cell-shape index uniquely describes the effective diffusion coefficient of cell movements, which becomes finite at the transition. We generalize our approach to two-component tissues, and explore cell-sorting dynamics both due to differential adhesion and due to differential degree of junctional fluctuations. We recover some known characteristic scaling relations describing the sorting kinetics, and discover some discrepancies from these relations in the case of differential-fluctuations-driven sorting. Finally, we show that differential fluctuations efficiently sort two solid-like tissues with a fluid intercompartmental boundary.

Reciprocal Association between the Apical Junctional Complex and AMPK: A Promising Therapeutic Target for Epithelial/Endothelial Barrier Function?

Epithelial/endothelial cells adhere to each other via cell–cell junctions including tight junctions (TJs) and adherens junctions (AJs). TJs and AJs are spatiotemporally and functionally integrated, and are thus often collectively defined as apical junctional complexes (AJCs), regulating a number of spatiotemporal events including paracellular barrier, selective permeability, apicobasal cell polarity, mechano-sensing, intracellular signaling cascades, and epithelial morphogenesis. Over the past 15 years, it has been acknowledged that adenosine monophosphate (AMP)-activated protein kinase (AMPK), a well-known central regulator of energy metabolism, has a reciprocal association with AJCs. Here, we review the current knowledge of this association and show the following evidences: (1) as an upstream regulator, AJs activate the liver kinase B1 (LKB1)–AMPK axis particularly in response to applied junctional tension, and (2) TJ function and apicobasal cell polarization are downstream targets of AMPK and are promoted by AMPK activation. Although molecular mechanisms underlying these phenomena have not yet been completely elucidated, identifications of novel AMPK effectors in AJCs and AMPK-driven epithelial transcription factors have enhanced our knowledge. More intensive studies along this line would eventually lead to the development of AMPK-based therapies, enabling us to manipulate epithelial/endothelial barrier function.

Arf-GEF localization and function at myosin-rich adherens junctions via coiled-coil heterodimerization with an adaptor protein.

Tissue dynamics require regulated interactions between adherens junctions and cytoskeletal networks. For example, myosin-rich adherens junctions recruit the cytohesin Arf-GEF Steppke, which down-regulates junctional tension and facilitates tissue stretching. We dissected this recruitment mechanism with structure–function and other analyses of Steppke and Stepping stone, an implicated adaptor protein. During Drosophila dorsal closure, Steppke’s coiled-coil domain was necessary and sufficient for junctional recruitment. Purified coiled-coil domains of Steppke and Stepping stone heterodimerized through a hydrophobic surface of the Steppke domain. This mapped surface was required for Steppke’s junctional localization and tissue regulation. Stepping stone colocalized with Steppke at junctions, and was required for junctional Steppke localization and proper tissue stretching. A second conserved region of Stepping stone was necessary and largely sufficient for junctional localization. Remarkably, this region could substitute for the Steppke coiled-coil domain for junction localization and regulation, suggesting the main role of the Steppke coiled-coil domain is linkage to the junctional targeting region of Stepping stone. Thus, coiled-coil heterodimerization with Stepping stone normally recruits Step to junctions. Intriguingly, Stepping stone’s junctional localization also seems partly dependent on Steppke.

Experimental validation of force inference in epithelia from cell to tissue scale

Morphogenesis relies on the active generation of forces, and the transmission of these forces to surrounding cells and tissues. Hence measuring forces directly in developing embryos is an essential task to study the mechanics of development. Among the experimental techniques that have emerged to measure forces in epithelial tissues, force inference is particularly appealing. Indeed it only requires a snapshot of the tissue, as it relies on the topology and geometry of cell contacts, assuming that forces are balanced at each vertex. However, establishing force inference as a reliable technique requires thorough validation in multiple conditions. Here we performed systematic comparisons of force inference with laser ablation experiments in four epithelial tissues from two animals, the fruit fly and the quail. We show that force inference accurately predicts single junction tension, tension patterns in stereotyped groups of cells, and tissue-scale stress patterns, in wild type and mutant conditions. We emphasize its ability to capture the distribution of forces at different scales from a single image, which gives it a critical advantage over perturbative techniques such as laser ablation. Overall, our results demonstrate that force inference is a reliable and efficient method to quantify the mechanical state of epithelia during morphogenesis, especially at larger scales when inferred tensions and pressures are binned into a coarse-grained stress tensor.

Tissue Fluidity Promotes Epithelial Wound Healing.

SummaryThe collective behaviour of cells in epithelial tissues is dependent on their mechanical properties. However, the contribution of tissue mechanics to wound healing in vivo remains poorly understood. Here we investigate the relationship between tissue mechanics and wound healing in live Drosophila wing imaginal discs and show that by tuning epithelial cell junctional tension, we can systematically alter the rate of wound healing. Coincident with the contraction of an actomyosin purse string, we observe cells flowing past each other at the wound edge by intercalating, reminiscent of molecules in a fluid, resulting in seamless wound closure. Using a cell-based physical model, we predict that a reduction in junctional tension fluidises the tissue through an increase in intercalation rate and corresponding reduction in bulk viscosity, in the manner of an unjamming transition. The resultant fluidisation of the tissue accelerates wound healing. Accordingly, when we experimentally reduce tissue tension in wing discs, intercalation rate increases and wounds repair in less time.

Cytocortex-dependent dynamics of Drosophila Crumbs controls junctional stability and tension during germ band retraction.

During morphogenesis, epithelia undergo dynamic rearrangements, which requires continuous remodelling of junctions and cell shape, but at the same time mechanisms preserving cell polarity and tissue integrity. Apico-basal polarity is key for the localisation of the machinery that enables cell shape changes. The evolutionarily conserved Drosophila Crumbs protein is critical for maintaining apico-basal polarity and epithelial integrity. How Crumbs is maintained in a dynamically developing embryo remains largely unknown. Here, we applied quantitative fluorescence techniques to show that, during germ band retraction, Crumbs dynamics correlates with the morphogenetic activity of the epithelium. Genetic and pharmacological perturbations revealed that the mobile pool of Crumbs is fine-tuned by the actomyosin cortex in a stage-dependent manner. Stabilisation of Crumbs at the plasma membrane depends on a proper link to the actomyosin cortex via an intact FERM-domain-binding site in its intracellular domain, loss of which leads to increased junctional tension and higher DE-cadherin (also known as Shotgun) turnover, resulting in impaired junctional rearrangements. These data define Crumbs as a mediator between polarity and junctional regulation to orchestrate epithelial remodelling in response to changes in actomyosin activity.This article has an associated First Person interview with the first author of the paper.

First person – Anna Bajur

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Anna Bajur is first author on ‘Cytocortex-dependent dynamics of Drosophila Crumbs controls junctional stability and tension during germ band retraction’, published in JCS. Anna conducted the research described in this article while a PhD student in Prof. Elisabeth Knust's lab at Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. She is now a Research associate/Postdoc in the lab of Dr Katelyn Spillane at King's College London, Department of Physics, UK, investigating the interplay between mechanical forces and the plasma membrane, especially in the context of signalling and modulation of cellular behaviour.

Involvement of the Areae Compositae of the Heart in Endemic Pemphigus Foliaceus.

A new variant of endemic pemphigus foliaceus in El Bagre (El Bagre-EPF), Colombia, South America, shares features with Senear-Usher syndrome and occurs in an endemic fashion. Patients affected by El Bagre-EPF have heterogeneous antigenic reactivity not only to the skin but to other organs, including the heart. Here we test for autoantibodies to the areae compositae of the heart (structure consisting of typical desmosomal amalgamated fascia adherens molecules) and evaluate any possible clinical correlation. A case-control study comparing 45 patients and 45 controls from the endemic area, matched by demographics including age, gender, weight, work activities, and comorbidities, was performed. Direct and indirect immunofluorescence, immunohistochemistry, confocal microscopic studies, and echocardiogram studies were completed. The main clinical abnormally seen in the El Bagre-EPF patients was left ventricular hypertrophy in 15/45 patients, compared with no such findings in the control population (P < 0.1). Seventy percent of El Bagre-EPF patients and none of the controls displayed polyclonal autoreactivity using different immunoglobulins and complement to the areae compositae of the heart using different methods and antibodies (P < 0.1). Patients affected by El Bagre-EPF demonstrated autoantibodies to the areae compositae of the heart. This finding was associated with left ventricular hypertrophic cardiomyopathy. The areae compositae may play a role in cell junction tension and the El Bagre-EPF patients' autoantibodies possibly disrupting these junctions and thereby contributing to the left ventricular hypertrophy.

Anillin regulates epithelial cell mechanics by structuring the medial-apical actomyosin network.

Cellular forces sculpt organisms during development, while misregulation of cellular mechanics can promote disease. Here, we investigate how the actomyosin scaffold protein anillin contributes to epithelial mechanics in Xenopus laevis embryos. Increased mechanosensitive recruitment of vinculin to cell-cell junctions when anillin is overexpressed suggested that anillin promotes junctional tension. However, junctional laser ablation unexpectedly showed that junctions recoil faster when anillin is depleted and slower when anillin is overexpressed. Unifying these findings, we demonstrate that anillin regulates medial-apical actomyosin. Medial-apical laser ablation supports the conclusion that that tensile forces are stored across the apical surface of epithelial cells, and anillin promotes the tensile forces stored in this network. Finally, we show that anillin's effects on cellular mechanics impact tissue-wide mechanics. These results reveal anillin as a key regulator of epithelial mechanics and lay the groundwork for future studies on how anillin may contribute to mechanical events in development and disease.

Response of Drosophila epithelial cell and tissue shape to external forces in vivo.

How actomyosin generates forces at epithelial adherens junctions has been extensively studied. However, less is known about how a balance between internal and external forces establishes epithelial cell, tissue and organ shape. We used the Drosophila egg chamber to investigate how contractility at adherens junctions in the follicle epithelium is modulated to accommodate and resist forces arising from the growing germ line. We found that between stages 6 and 9, adherens junction tension in the post-mitotic epithelium decreases, suggesting that the junctional network relaxes to accommodate germline growth. At that time, a prominent medial Myosin II network coupled to corrugating adherens junctions develops. Local enrichment of medial Myosin II in main body follicle cells resists germline-derived forces, thus constraining apical areas and, consequently, cuboidal cell shapes at stage 9. At the tissue and organ level, local reinforcement of medial junction architecture ensures the timely contact of main body cells with the expanding oocyte and imposes circumferential constraints on the germ line guiding egg elongation. Our study provides insight into how adherens junction tension promotes cell and tissue shape transitions while integrating the growth and shape of an internally enclosed structure in vivo.