Junctional Tension Research Articles

Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions

Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs.

Tight junctions control lumen morphology via hydrostatic pressure and junctional tension.

Formation of fluid-filled lumina by epithelial tissues is essential for organ development. How cells control the hydraulic and cortical forces to control lumen morphology is not well understood. Here, we quantified the mechanical role of tight junctions in lumen formation using MDCK-II cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for the hydraulic inflation of a lumen. However, the depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements of hydrostatic lumen pressure and junctional tension with modeling enabled us to explain lumen morphologies from the pressure-tension force balance. Tight junctions promote lumen inflation by decreasing cortical tension via the inhibition of myosin. In addition, our results suggest that excess apical area contributes to lumen opening. Overall, we provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

Altering Cell Junctional Tension in Spheroids through E-Cadherin Engagement Modulation.

Cadherin-mediated tension at adherens junctions (AJs) is fundamental for cell-cell adhesion and maintaining epithelial integrity. Despite the importance of manipulating AJs to dissect cell-cell interactions, existing three-dimensional (3D) multicellular models have not adequately addressed the precise manipulation of these junctions. To fill this gap, we introduce E-cadherin-modified tension gauge tethers (TGTs) at the junctions within spheroids. The system enables both quantification and modulation of junctional tension with specific DNA triggers. Using rupture-induced fluorescence, we successfully measure mechanical forces in 3D spheroids. Furthermore, mechanically strong TGTs can maintain normal E-cadherin-mediated adhesion. Employing toehold-mediated strand displacement allowed us to disrupt E-cadherin-specific cell-cell adhesion, consequently altering intracellular tension within the spheroids. Our methodology offers a robust and precise way to manipulate cell-cell adhesion and intracellular mechanics in spheroid models.

The Role of ZO-2 in Modulating JAM-A and γ-Actin Junctional Recruitment, Apical Membrane and Tight Junction Tension, and Cell Response to Substrate Stiffness and Topography.

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.

Morphometric and Molecular Assessment of Tor putitora and Neolissochilus hexagonolepis Population in Jia Bhoroli river Ecotone Zone, Assam

Background: Ecotones are areas of overlap between different ecosystems, referred to as ‘junction zones or tension belts’, that harbor a distinct collection of organisms. Tor putitora and Neolissochilus hexagonolepis are two threatened Mahseer species which requires urgent conservation attention. Bhalukpung area in the River Jia Bhoroli is an ideal example of an ecotone, as its ecosystem transforms from resembling a foothill river ecosystem to a river floodplain ecosystem. However, studies on threatened fish populations in ecotone zones, which could be used as conservation sites, are lacking. Therefore, the present study aims to generate firsthand information on the relationship between the populations of these threatened fish and their foothill-floodplain river interface ecotone in the Jia Bhoroli River. Methods: The data on water quality, topography was collected for a period of one year from January to December, 2021 along with fish samples. The mahseer species were identified and catch per unit effort (CPUE) was also calculated. Result: Identification of the collected mahseer fish as T. putitora and N. hexagonolepis were confirmed using both morphometric as well as molecular tools. CPUE was found to be 4.3-12.6 for N. hexagonolepis and 4.1-10.3 for T. putitora. The ecotone zone was found to have a thriving mahseer population and ecotones are believed to play a crucial role as a diverse food source for the fish. Conserving Bhalukpong as a designated ecotone site in the Jia Bhoroli River as a fish sanctuary can help to protect their populations. Further research that encompasses all aspects of ecology is recommended for the future.

Toll-like receptor signalling via IRAK4 affects epithelial integrity and tightness through regulation of junctional tension.

Toll-like receptors (TLRs) in mammalian systems are well known for their role in innate immunity. In addition, TLRs also fulfil crucial functions outside immunity, including the dorsoventral patterning function of the original Toll receptor in Drosophila and neurogenesis in mice. Recent discoveries in flies suggested key roles for TLRs in epithelial cells in patterning of junctional cytoskeletal activity. Here, we address the function of TLRs and the downstream key signal transduction component IRAK4 in human epithelial cells. Using differentiated human Caco-2 cells as a model for the intestinal epithelium, we show that these cells exhibit baseline TLR signalling, as revealed by p-IRAK4, and that blocking IRAK4 function leads to a loss of epithelial tightness involving key changes at tight and adherens junctions, such as a loss of epithelial tension and changes in junctional actomyosin. Changes upon IRAK-4 inhibition are conserved in human bronchial epithelial cells. Knockdown of IRAK4 and certain TLRs phenocopies the inhibitor treatment. These data suggest a model whereby TLR receptors near epithelial junctions might be involved in a continuous sensing of the epithelial state to promote epithelial tightness and integrity.

Tension Remodeling Regulates Topological Transitions in Epithelial Tissues.

Cell neighbor exchanges play a critical role in regulating tissue fluidity during epithelial morphogenesis and repair. In vivo, these neighbor exchanges are often hindered by the formation of transiently stable fourfold vertices, which can develop into complex multicellular rosettes where five or more cell junctions meet. Despite their importance, the mechanical origins of multicellular rosettes have remained elusive, and current cellular models lack the ability to explain their formation and maintenance. Here we present a dynamic vertex model of epithelial tissues with strain-dependent tension remodeling and mechanical memory dissipation. We show that an increase in cell junction tension upon contraction and reduction in tension upon extension can stabilize higher-order vertices, temporarily stalling cell rearrangements. On the other hand, inducing mechanical memory dissipation via relaxation of junction strain and stress promotes the resolution of higher-order vertices, facilitating cell neighbor exchanges. We demonstrate that by tuning the rates of tension remodeling and mechanical memory dissipation, we can control topological transitions and tissue material properties, recapitulating complex cellular topologies seen in developing organisms.

Dynamics and functions of E-cadherin complexes in epithelial cell and tissue morphogenesis.

Cell-cell adhesion is at the center of structure and dynamics of epithelial tissue. E-cadherin-catenin complexes mediate Ca2+-dependent trans-homodimerization and constitute the kernel of adherens junctions. Beyond the basic function of cell-cell adhesion, recent progress sheds light the dynamics and interwind interactions of individual E-cadherin-catenin complex with E-cadherin superclusters, contractile actomyosin and mechanics of the cortex and adhesion. The nanoscale architecture of E-cadherin complexes together with cis-interactions and interactions with cortical actomyosin adjust to junctional tension and mechano-transduction by reinforcement or weakening of specific features of the interactions. Although post-translational modifications such as phosphorylation and glycosylation have been implicated, their role for specific aspects of in E-cadherin function has remained unclear. Here, we provide an overview of the E-cadherin complex in epithelial cell and tissue morphogenesis focusing on nanoscale architectures by super-resolution approaches and post-translational modifications from recent, in particular in vivo, studies. Furthermore, we review the computational modelling in E-cadherin complexes and highlight how computational modelling has contributed to a deeper understanding of the E-cadherin complexes.

Cell cycle dynamics control fluidity of the developing mouse neuroepithelium

As developing tissues grow in size and undergo morphogenetic changes, their material properties may be altered. Such changes result from tension dynamics at cell contacts or cellular jamming. Yet, in many cases, the cellular mechanisms controlling the physical state of growing tissues are unclear. We found that at early developmental stages, the epithelium in the developing mouse spinal cord maintains both high junctional tension and high fluidity. This is achieved via a mechanism in which interkinetic nuclear movements generate cell area dynamics that drive extensive cell rearrangements. Over time, the cell proliferation rate declines, effectively solidifying the tissue. Thus, unlike well-studied jamming transitions, the solidification uncovered here resembles a glass transition that depends on the dynamical stresses generated by proliferation and differentiation. Our finding that the fluidity of developing epithelia is linked to interkinetic nuclear movements and the dynamics of growth is likely to be relevant to multiple developing tissues.

Mechanobiological Adaptation to Hyperosmolarity Enhances Barrier Function in Human Vascular Microphysiological System.

In infectious disease such as sepsis and COVID-19, blood vessel leakage treatment is critical to prevent fatal progression into multi-organ failure and ultimately death, but the existing effective therapeutic modalities that improve vascular barrier function are limited. Here, this study reports that osmolarity modulation can significantly improve vascular barrier function, even in an inflammatory condition. 3D human vascular microphysiological systems and automated permeability quantification processes for high-throughput analysis of vascular barrier function are utilized. Vascular barrier function is enhanced by >7-folds with 24-48h hyperosmotic exposure (time window of emergency care; >500mOsm L-1 ) but is disrupted after hypo-osmotic exposure (<200mOsm L-1 ). By integrating genetic and protein level analysis, it is shown that hyperosmolarity upregulates vascular endothelial-cadherin, cortical F-actin, and cell-cell junction tension, indicating that hyperosmotic adaptation mechanically stabilizes the vascular barrier. Importantly, improved vascular barrier function following hyperosmotic exposure is maintained even after chronic exposure to proinflammatory cytokines and iso-osmotic recovery via Yes-associated protein signaling pathways. This study suggests that osmolarity modulation may be a unique therapeutic strategy to proactively prevent infectious disease progression into severe stages via vascular barrier function protection.

C. elegans srGAP is an α-catenin M domain-binding protein that strengthens cadherin-dependent adhesion during morphogenesis.

The cadherin-catenin complex (CCC) is central to embryonic development and tissue repair, yet how CCC binding partners function alongside core CCC components remains poorly understood. Here, we establish a previously unappreciated role for an evolutionarily conserved protein, the slit-robo GTPase-activating protein SRGP-1/srGAP, in cadherin-dependent morphogenetic processes in the Caenorhabditis elegans embryo. SRGP-1 binds to the M domain of the core CCC component, HMP-1/α-catenin, via its C terminus. The SRGP-1 C terminus is sufficient to target it to adherens junctions, but only during later embryonic morphogenesis, when junctional tension is known to increase. Surprisingly, mutations that disrupt stabilizing salt bridges in the M domain block this recruitment. Loss of SRGP-1 leads to an increase in mobility and decrease of junctional HMP-1. In sensitized genetic backgrounds with weakened adherens junctions, loss of SRGP-1 leads to late embryonic failure. Rescue of these phenotypes requires the C terminus of SRGP-1 but also other domains of the protein. Taken together, these data establish a role for an srGAP in stabilizing and organizing the CCC during epithelial morphogenesis by binding to a partially closed conformation of α-catenin at junctions.

Extracellular matrix regulates force transduction at VE-cadherin junctions.

Increased tension on VE-cadherin (VE-cad) complexes activates adaptive cell stiffening and local cytoskeletal reinforcement--two key signatures of intercellular mechanotransduction. Here we demonstrate that tugging on VE-cad receptors initiates a cascade that results in downstream integrin activation. The formation of new integrin adhesions potentiates vinculin and actin recruitment to mechanically reinforce stressed cadherin adhesions. This cascade differs from documented antagonistic effects of integrins on intercellular junctions. We identify focal adhesion kinase, Abl kinase, and RhoA GTPase as key components of the positive feedback loop. Results further show that a consequence of integrin involvement is the sensitization of intercellular force transduction to the extracellular matrix (ECM) not by regulating junctional tension but by altering signal cascades that reinforce cell-cell adhesions. On type 1 collagen or fibronectin substrates, integrin subtypes α2β1 and α5β1, respectively, differentially control actin remodeling at VE-cad adhesions. Specifically, ECM-dependent differences in VE-cad force transduction mirror differences in the rigidity sensing mechanisms of α2β1 and α5β1 integrins. The findings verify the role of integrins in VE-cad force transduction and uncover a previously unappreciated mechanism by which the ECM impacts the mechanical reinforcement of interendothelial junctions.

Analysis of the Drosophila Ajuba LIM protein defines functions for distinct LIM domains.

The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.

Image-based parameter inference for epithelial mechanics.

Measuring mechanical parameters in tissues, such as the elastic modulus of cell-cell junctions, is essential to decipher the mechanical control of morphogenesis. However, their in vivo measurement is technically challenging. Here, we formulated an image-based statistical approach to estimate the mechanical parameters of epithelial cells. Candidate mechanical models are constructed based on force-cell shape correlations obtained from image data. Substitution of the model functions into force-balance equations at the cell vertex leads to an equation with respect to the parameters of the model, by which one can estimate the parameter values using a least-squares method. A test using synthetic data confirmed the accuracy of parameter estimation and model selection. By applying this method to Drosophila epithelial tissues, we found that the magnitude and orientation of feedback between the junction tension and shrinkage, which are determined by the spring constant of the junction, were correlated with the elevation of tension and myosin-II on shrinking junctions during cell rearrangement. Further, this method clarified how alterations in tissue polarity and stretching affect the anisotropy in tension parameters. Thus, our method provides a novel approach to uncovering the mechanisms governing epithelial morphogenesis.

The Lateral Epidermis Actively Counteracts Pulling by the Amnioserosa During Dorsal Closure.

Dorsal closure is a prominent morphogenetic process during Drosophila embryogenesis, which involves two epithelial tissues, that is, the squamous amnioserosa and the columnar lateral epidermis. Non-muscle myosin II-driven constriction in the amnioserosa leads to a decrease in the apical surface area and pulls on the adjacent lateral epidermis, which subsequently moves dorsally. The pull by the amnioserosa becomes obvious in an elongation of the epidermal cells, especially of those in the first row. The contribution of the epidermal cell elongation has remained unclear to dorsal closure. Cell elongation may be a mere passive consequence or an active response to the pulling by the amnioserosa. Here, we found that the lateral epidermis actively responds. We analyzed tensions within tissues and cell junctions by laser ablation before and during dorsal closure, the elliptical and dorsal closure stages, respectively. Furthermore, we genetically and optochemically induced chronic and acute cell contraction, respectively. In this way, we found that tension in the epidermis increased during dorsal closure. A correspondingly increased tension was not observed at individual junctions, however. Junctional tension even decreased during dorsal closure in the epidermis. We strikingly observed a strong increase of the microtubule amount in the epidermis, while non-muscle myosin II increased in both tissues. Our data suggest that the epidermis actively antagonizes the pull from the amnioserosa during dorsal closure and the increased microtubules might help the epidermis bear part of the mechanical force.

Blood vessel occlusion by Cryptococcus neoformans is a mechanism for haemorrhagic dissemination of infection.

Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis.

Vinculin controls endothelial cell junction dynamics during vascular lumen formation

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving vascular integrity. Here, we analyze the dynamics of endothelial adherens junctions during lumen formation in angiogenic sprouts invivo. Live imaging in zebrafish reveals that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Junctional fingers are positively regulated by blood pressure, whereas flow inhibition prevents their formation. Using fluorescent reporters, we show that junctional fingers contain the mechanotransduction protein vinculin. Furthermore, genetic deletion of vinculin prevents finger formation, a junctional defect that could be rescued by transient endothelial expression of vinculin. Our findings suggest a mechanism whereby lumen expansion leads to an increase in junctional tension, triggering recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells employ force-dependent junctional remodeling to counteract external forces in order to maintain vascular integrity during sprouting angiogenesis.

Morphologies of compressed active epithelial monolayers.

Using a three-dimensional active vertex model, we numerically study the shapes of strained unsupported epithelial monolayers subject to active junctional noise due to stochastic binding and unbinding of myosin. We find that while uniaxial, biaxial, and isotropic in-plane compressive strains do lead to the formation of longitudinal, herringbone pattern, and labyrinthine folds, respectively, the villus morphology characteristic of, e.g., the small intestine appears only if junctional tension fluctuations are strong enough to fluidize the tissue. Moreover, the fluidized epithelium features villi even in the absence of compressive strain provided that the apico-basal differential surface tension is large enough. We analyze several details of the different epithelial forms including the role of strain rate and the modulation of tissue thickness across folds. Our results show that even unsupported, non-patterned epithelia can form nontrivial morphologies.

Cellular mitosis predicts vessel stability in a mechanochemical model of sprouting angiogenesis.

Angiogenesis, the formation of new vessels, occurs in both developmental and pathological contexts. Prior research has investigated vessel formation to identify cellular phenotypes and dynamics associated with angiogenic disease. One major family of proteins involved in angiogenesis are the Rho GTPases, which govern function related to cellular elongation, migration, and proliferation. Using a mechanochemical model coupling Rho GTPase activity and cellular and intercellular mechanics, we investigate the role of cellular mitosis on sprouting angiogenesis. Mitosis-GTPase synchronization was not a strong predictor of GTPase and thus vessel signaling instability, whereas the location of mitotic events was predicted to alter GTPase cycling instabilities. Our model predicts that middle stalk cells undergoing mitosis introduce irregular dynamics in GTPase cycling and may provide a source of aberrant angiogenesis. We also find that cellular and junctional tension exhibit spatial heterogeneity through the vessel, and that tension feedback, specifically in stalk cells, tends to increase the maximum forces generated in the vessel.

Inferring cell junction tension and pressure from cell geometry.

Recognizing the crucial role of mechanical regulation and forces in tissue development and homeostasis has stirred a demand for in situ measurement of forces and stresses. Among emerging techniques, the use of cell geometry to infer cell junction tensions, cell pressures and tissue stress has gained popularity owing to the development of computational analyses. This approach is non-destructive and fast, and statistically validated based on comparisons with other techniques. However, its qualitative and quantitative limitations, in theory as well as in practice, should be examined with care. In this Primer, we summarize the underlying principles and assumptions behind stress inference, discuss its validity criteria and provide guidance to help beginners make the appropriate choice of its variants. We extend our discussion from two-dimensional stress inference to three dimensional, using the early mouse embryo as an example, and list a few possible extensions. We hope to make stress inference more accessible to the scientific community and trigger a broader interest in using this technique to study mechanics in development.