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De novo lumen formation necessitates the precise segregation of junctional proteins from apical surfaces, yet the underlying mechanisms remain unclear. Using a zebrafish model, we develop a series of molecular reporters, photo-convertible and optogenetic tools to study the establishment of apical domains. Our study identifies Rasip1 as one of the earliest apical proteins recruited, which suppresses actomyosin contractility at junctional patches by inhibiting NMII, thereby allowing for the sustained outward flow of junctional complexes. Following the establishment of apical compartments, Rasip1 shuttles between junctions and the apical compartments in response to local high tension. Rasip1 confines Cdh5 to junctions by suppressing apical contractility. Conversely, the recruitment of Rasip1 to junctions is regulated by Heg1 and Krit1 to modulate contractility along junctions. Overall, de novo lumen formation and maintenance depend on the precise control of contractility within apical compartments and junctions, orchestrated by the dynamic recruitment of Rasip1., (© 2024. The Author(s).)

Background: Latrophilin-2 (Lphn2), an adhesive GPCR (G protein-coupled receptor), was found to be a specific marker of cardiac progenitors during the differentiation of pluripotent stem cells into cardiomyocytes or during embryonic heart development in our previous studies. Its role in adult heart physiology, however, remains unclear., Methods: The embryonic lethality resulting from Lphn2 deletion necessitates the establishment of cardiomyocyte-specific, tamoxifen-inducible Lphn2 knockout mice, which was achieved by crossing Lphn2  flox/flox mice with mice having MerCreMer (tamoxifen-inducible Cre [Cyclization recombinase] recombinase) under the α-myosin heavy chain promoter., Results: Tamoxifen treatment for several days completely suppressed Lphn2 expression, specifically in the myocardium, and induced the dilated cardiomyopathy (D-CMP) phenotype with serious arrhythmia and sudden death in a short period of time. Transmission electron microscopy showed mitochondrial abnormalities, blurred Z-discs, and dehiscent myofibrils. The D-CMP phenotype, or heart failure, worsened during myocardial infarction. In a mechanistic study of D-CMP, Lphn2 knockout suppressed PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and mitochondrial dysfunction, leading to the accumulation of reactive oxygen species and the global suppression of junctional molecules, such as N-cadherin (adherens junction), DSC-2 (desmocollin-2; desmosome), and connexin-43 (gap junction), leading to the dehiscence of cardiac myofibers and serious arrhythmia. In an experimental therapeutic trial, activators of p38-MAPK (p38 mitogen-activated protein kinases), which is a downstream signaling molecule of Lphn2, remarkably rescued the D-CMP phenotype of Lphn2 knockout in the heart by restoring PGC-1α and mitochondrial function and recovering global junctional proteins., Conclusions: Lphn2 is a critical regulator of heart integrity by controlling mitochondrial functions and cell-to-cell junctions in cardiomyocytes. Its deficiency leads to D-CMP, which can be rescued by activators of the p38-MAPK pathway., Competing Interests: None.

Congenital scoliosis (CS) is a type of vertebral malformation for which the etiology remains elusive. The notochord is pivotal for vertebrae development, but its role in CS is still understudied. Here, we generated a zebrafish knockout of ptk7a, a planar cell polarity (PCP) gene that is essential for convergence and extension (C&E) of the notochord, and detected congenital scoliosis-like vertebral malformations (CVMs). Maternal zygotic ptk7a mutants displayed severe C&E defects of the notochord. Excessive apoptosis occurred in the malformed notochord, causing a significantly reduced number of vacuolated cells, and compromising the mechanical properties of the notochord. The latter manifested as a less-stiff extracellular matrix along with a significant reduction in the number of the caveolae and severely loosened intercellular junctions in the vacuolated region. These defects led to focal kinks, abnormal mineralization, and CVMs exclusively at the anterior spine. Loss of function of another PCP gene, vangl2, also revealed excessive apoptosis in the notochord associated with CVMs. This study suggests a new model for CS pathogenesis that is associated with defects in notochord C&E and highlights an essential role of PCP signaling in vertebrae development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Transporting epithelial cells in the gut and kidney rely on protocadherin-based apical adhesion complexes to organize microvilli that extend into luminal space. In these systems, CDHR2 and CDHR5 localize to the distal ends of microvilli, where they form an intermicrovillar adhesion complex (IMAC) that links the tips of these structures, promotes the formation of a well-ordered array of protrusions, and thus maximizes apical membrane surface area. Recently, we discovered that IMACs can also form between microvilli that extend from neighboring cells, across cell-cell junctions. As an additional point of physical contact between cells, transjunctional IMACs are well positioned to impact the integrity of canonical tight and adherens junctions that form more basolaterally. To begin to test this idea, we examined cell culture and mouse models that lacked CDHR2 expression and were unable to form IMACs. CDHR2 knockout perturbed cell and junction morphology, reduced key components from tight and adherens junctions, impaired barrier function, and increased the motility of single cells within established monolayers. These results support the hypothesis that, in addition to organizing apical microvilli, IMACs provide a layer of cell-cell contact that functions in parallel with canonical tight and adherens junctions to promote epithelial functions., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

The formation and organization of complex blood vessel networks rely on various biophysical forces, yet the mechanisms governing endothelial cell-cell interactions under different mechanical inputs are not well understood. Using the dorsal longitudinal anastomotic vessel (DLAV) in zebrafish as a model, we studied the roles of multiple biophysical inputs and cerebral cavernous malformation (CCM)-related genes in angiogenesis. Our research identifies heg1 and krit1 (ccm1) as crucial for the formation of endothelial cell-cell interfaces during anastomosis. In mutants of these genes, cell-cell interfaces are entangled with fragmented apical domains. A Heg1 live reporter demonstrated that Heg1 is dynamically involved in the oscillatory constrictions along cell-cell junctions, whilst a Myosin live reporter indicated that heg1 and krit1 mutants lack actomyosin contractility along these junctions. In wild-type embryos, the oscillatory contractile forces at junctions refine endothelial cell-cell interactions by straightening junctions and eliminating excessive cell-cell interfaces. Conversely, in the absence of junctional contractility, the cell-cell interfaces become entangled and prone to collapse in both mutants, preventing the formation of a continuous luminal space. By restoring junctional contractility via optogenetic activation of RhoA, contorted junctions are straightened and disentangled. Additionally, haemodynamic forces complement actomyosin contractile forces in resolving entangled cell-cell interfaces in both wild-type and mutant embryos. Overall, our study reveals that oscillatory contractile forces governed by Heg1 and Krit1 are essential for maintaining proper endothelial cell-cell interfaces and thus for the formation of a continuous luminal space, which is essential to generate a functional vasculature., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

As a key planar cell polarity protein, Van Gogh-like 2 (Vangl2) is essential for mammalian spermatogenesis. As a decapod crustacean, Eriocheir sinensis exhibits distinct spermatogenic processes due to its unique seminiferous tubule morphology and hemolymph-testis barrier (HTB). To determine whether Vangl2 performs analogous functions in E. sinensis, we identified the Es-Vangl2. Es-Vangl2 exhibited high expression and wide distribution in the testes, indicating its crucial involvement in spermatogenesis. Following targeted knockdown of Es-Vangl2in vivo, the structure of seminiferous tubules was disrupted, characterized by vacuolization of the germinal zone and obstruction of spermatozoon release. Concurrently, the integrity of the HTB was compromised, accompanied by reduced expression and aberrant localization of junction proteins. More importantly, the regulatory influence of Es-Vangl2 was manifested through modulating the organization of microfilaments, a process mediated by epidermal growth factor receptor pathway substrate 8 (Eps8). Further studies demonstrated that these phenotypes resulting from Es-Vangl2 knockdown were attributed to the inhibition of Rock signaling pathway activity, which was verified by the Es-Rock interference and Y27632 inhibition assays. In summary, the findings highlight the pivotal role of Es-Vangl2 in stabilizing HTB integrity by regulating Eps8-mediated actin remodeling through the Rock signaling pathway in the spermatogenesis of E. sinensis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The T cell receptor (TCR) is thought to be a mechanosensor, meaning that it transmits mechanical force to its antigen and leverages the force to amplify the specificity and magnitude of TCR signalling. Although a variety of molecular probes have been proposed to quantify TCR mechanics, these probes are immobilized on hard substrates, and thus fail to reveal fluid TCR-antigen interactions in the physiological context of cell membranes. Here we developed DNA origami tension sensors (DOTS) which bear force sensors on a DNA origami breadboard and allow mapping of TCR mechanotransduction at dynamic intermembrane junctions. We quantified the mechanical forces at fluid TCR-antigen bonds and observed their dependence on cell state, antigen mobility, antigen potency, antigen height and F-actin activity. The programmability of DOTS allows us to tether these to microparticles to mechanically screen antigens in high throughput using flow cytometry. Additionally, DOTS were anchored onto live B cells, allowing quantification of TCR mechanics at immune cell-cell junctions., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: Declaration of interests A.L.M. serves on Current Biology’s editorial advisory board., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The food-borne pathogen Listeria monocytogenes uses actin-based motility to generate plasma membrane protrusions that mediate the spread of bacteria between host cells. In polarized epithelial cells, efficient protrusion formation by L. monocytogenes requires the secreted bacterial protein InlC, which binds to a carboxyl-terminal Src homology 3 (SH3) domain in the human scaffolding protein Tuba. This interaction antagonizes Tuba, thereby diminishing cortical tension at the apical junctional complex and enhancing L. monocytogenes protrusion formation and spread. Tuba contains five SH3 domains apart from the domain that interacts with InlC. Here, we show that human GTPase Dynamin 2 associates with two SH3 domains in the amino-terminus of Tuba and acts together with this scaffolding protein to control the spread of L. monocytogenes . Genetic or pharmacological inhibition of Dynamin 2 or knockdown of Tuba each restored normal protrusion formation and spread to a bacterial strain deleted for the inlC gene (∆ inlC ). Dynamin 2 localized to apical junctions in uninfected human cells and protrusions in cells infected with L. monocytogenes . Localization of Dynamin 2 to junctions and protrusions depended on Tuba. Knockdown of Dynamin 2 or Tuba diminished junctional linearity, indicating a role for these proteins in controlling cortical tension. Infection with L. monocytogenes induced InlC-dependent displacement of Dynamin 2 from junctions, suggesting a possible mechanism of antagonism of this GTPase. Collectively, our results show that Dynamin 2 cooperates with Tuba to promote intercellular tension that restricts the spread of ∆ inlC Listeria . By expressing InlC, wild-type L. monocytogenes overcomes this restriction., Competing Interests: The authors declare no conflict of interest.

Cells depend on precisely regulating barrier function within the vasculature to maintain physiological stability and facilitate essential substance transport. Endothelial cells achieve this through specialized adherens and tight junction protein complexes, which govern paracellular permeability across vascular beds. Adherens junctions, anchored by vascular endothelial (VE)-cadherin and associated catenins to the actin cytoskeleton, mediate homophilic adhesion crucial for barrier integrity. In contrast, tight junctions composed of occludin, claudin, and junctional adhesion molecule A interact with Zonula Occludens proteins, reinforcing intercellular connections essential for barrier selectivity. Endothelial cell-cell junctions exhibit dynamic conformations during development, maturation, and remodeling, regulated by local biochemical and mechanical cues. These structural adaptations play pivotal roles in disease contexts such as chronic inflammation, where junctional remodeling contributes to increased vascular permeability observed in conditions from cancer to cardiovascular diseases. Conversely, the brain microvasculature's specialized junctional arrangements pose challenges for therapeutic drug delivery due to their unique molecular compositions and tight organization. This commentary explores the molecular mechanisms underlying endothelial cell-cell junction conformations and their implications for vascular permeability. By highlighting recent advances in quantifying junctional changes and understanding mechanotransduction pathways, we elucidate how physical forces from cellular contacts and hemodynamic flow influence junctional dynamics.

The adhesion receptor vascular endothelial (VE)-cadherin transduces an array of signals that modulate crucial lymphatic cell behaviors including permeability and cytoskeletal remodeling. Consequently, VE-cadherin must interact with a multitude of intracellular proteins to exert these functions. Yet, the full protein interactome of VE-cadherin in endothelial cells remains a mystery. Here, we use proximity proteomics to illuminate how the VE-cadherin interactome changes during junctional reorganization from dis-continuous to continuous junctions, triggered by the lymphangiogenic factor adrenomedullin. These analyses identified interactors that reveal roles for ADP ribosylation factor 6 (ARF6) and the exocyst complex in VE-cadherin trafficking and recycling. We also identify a requisite role for VE-cadherin in the in vitro and in vivo control of secretion of reelin-a lymphangiocrine glycoprotein with recently appreciated roles in governing heart development and injury repair. This VE-cadherin protein interactome shines light on mechanisms that control adherens junction remodeling and secretion from lymphatic endothelial cells., (© 2024. The Author(s).)

Fluid shear stress (FSS) from blood flow, sensed by the vascular endothelial cells (ECs) that line all blood vessels, regulates vascular development during embryogenesis, controls adult vascular physiology and determines the location of atherosclerotic plaque formation. Although a number of papers have reported a crucial role for cell-cell adhesions or adhesion receptors in these processes, a recent publication has challenged this paradigm, presenting evidence that ECs can very rapidly align in fluid flow as single cells without cell-cell contacts. To address this controversy, four independent laboratories assessed EC alignment in fluid flow across a range of EC cell types. These studies demonstrate a strict requirement for cell-cell contact in shear stress sensing over timescales consistent with previous literature and inconsistent with the newly published data., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Epithelial-mesenchymal transitions (EMTs) are thought to promote metastasis via downregulation of E-cadherin (also known as Cdh1) and upregulation of mesenchymal markers such as N-cadherin (Cdh2) and vimentin (Vim). Contrary to this, E-cadherin is retained in many invasive carcinomas and promotes collective cell invasion. To investigate how E-cadherin regulates metastasis, we examined the highly metastatic, E-cadherin-positive murine 4T1 breast cancer model, together with the less metastatic, 4T1-related cell lines 4T07, 168FARN and 67NR. We found that 4T1 cells display a hybrid epithelial/mesenchymal phenotype with co-expression of epithelial and mesenchymal markers, whereas 4T07, 168FARN, and 67NR cells display progressively more mesenchymal phenotypes in vitro that relate inversely to their metastatic capacity in vivo. Using RNA interference and constitutive expression, we demonstrate that the expression level of E-cadherin does not determine 4T1 or 4T07 cell metastatic capacity in mice. Mechanistically, 4T1 cells possess highly dynamic, unstable cell-cell junctions and can undergo collective invasion without E-cadherin downregulation. However, 4T1 orthotopic tumors in vivo also contain subregions of EMT-like loss of E-cadherin. Thus, 4T1 cells function as a model for carcinomas with a hybrid epithelial/mesenchymal phenotype that promotes invasion and metastasis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Mitral valve (MV) disease including myxomatous degeneration is the most common form of valvular heart disease with an age-dependent frequency. Genetic evidence indicates that mutations of the human transcription factor FOXC1 are associated with MV defects, including MV regurgitation. In this study, we sought to determine whether murine Foxc1 and its closely related factor, Foxc2 , are required in valvular endothelial cells (VECs) for the maintenance of MV leaflets, including VEC junctions and the stratified trilaminar ECM (extracellular matrix)., Methods: Adult mice carrying tamoxifen-inducible, vascular endothelial cell (EC), and lymphatic EC-specific, compound Foxc1;Foxc2 mutations (ie, EC- Foxc -DKO and lymphatic EC- Foxc -DKO mice, respectively) were used to study the function of Foxc1 and Foxc2 in the maintenance of MVs. The EC and lymphatic EC mutations of Foxc1/c2 were induced at 7 to 8 weeks of age by tamoxifen treatment, and abnormalities in the MVs of these mutant mice were assessed via whole-mount immunostaining, immunohistochemistry/RNAscope, Movat pentachrome/Masson Trichrome staining, and Evans blue injection., Results: EC deletions of Foxc1 and Foxc2 in mice resulted in abnormally extended and thicker MVs by causing defects in the regulation of ECM organization with increased proteoglycan and decreased collagen. Notably, reticular adherens junctions were found in VECs of control MV leaflets, and these reticular structures were severely disrupted in EC- Foxc -DKO mice. PROX1 (prospero homeobox protein 1), a key regulator in a subset of VECs on the fibrosa side of MVs, was downregulated in EC- Foxc1/c2 mutant VECs. Furthermore, we determined the precise location of lymphatic vessels in murine MVs, and these lymphatic vessels were aberrantly expanded and dysfunctional in EC- Foxc1/c2 mutant MVs. Lymphatic EC deletion of Foxc1/c2 also resulted in similar structural/ECM abnormalities as seen in EC- Foxc1/c2 mutant MVs., Conclusions: Our results indicate that Foxc1 and Foxc2 are required for maintaining the integrity of the MV, including VEC junctions, ECM organization, and lymphatic vessel formation/function to prevent myxomatous MV degeneration., Competing Interests: None.

Despite recent studies implicating liquid-like biomolecular condensates in diverse cellular processes, many biomolecular condensates exist in a solid-like state, and their function and regulation are less understood. We show that the tumor suppressor Merlin, an upstream regulator of the Hippo pathway, localizes to both cell junctions and medial apical cortex in Drosophila epithelia, with the latter forming solid-like condensates that activate Hippo signaling. Merlin condensation required phosphatidylinositol-4-phosphate (PI4P)-mediated plasma membrane targeting and was antagonistically controlled by Pez and cytoskeletal tension through plasma membrane PI4P regulation. The solid-like material properties of Merlin condensates are essential for physiological function and protect the condensates against external perturbations. Collectively, these findings uncover an essential role for solid-like condensates in normal physiology and reveal regulatory mechanisms for their formation and disassembly.

Migrating cells preferentially breach and integrate epithelial and endothelial monolayers at multicellular vertices. These sites are amenable to forces produced by the migrating cell and subsequent opening of the junctions. However, the cues that guide migrating cells to these entry portals, and eventually drive the transmigration process, are poorly understood. Here, we show that lymphatic endothelium multicellular junctions are the preferred sites of dendritic cell transmigration in both primary cell co-cultures and in mouse dermal explants. Dendritic cell guidance to multicellular junctions was dependent on the dendritic cell receptor CCR7, whose ligand, lymphatic endothelial chemokine CCL21, was exocytosed at multicellular junctions. Characterization of lymphatic endothelial secretory routes indicated Golgi-derived RAB6+ vesicles and RAB3+/27+ dense core secretory granules as intracellular CCL21 storage vesicles. Of these, RAB6+ vesicles trafficked CCL21 to the multicellular junctions, which were enriched with RAB6 docking factor ELKS (ERC1). Importantly, inhibition of RAB6 vesicle exocytosis attenuated dendritic cell transmigration. These data exemplify how spatially-restricted exocytosis of guidance cues helps to determine where dendritic cells transmigrate., (© 2024. The Author(s).)

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function, and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs), and Plexin D1 located at cell-cell junctions mediates many of these events. However, available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial-specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology, and disease., (© 2024. The Author(s).)

In the context of diabetes, endothelial cells frequently exhibit compromised intercellular junctions and accelerated cellular senescence simultaneously. The precise mechanisms underlying these issues and the identification of effective treatments remain largely undefined. Our findings reveal that human umbilical vein endothelial cells (HUVECs) can counteract senescence and uphold the integrity of intercellular junctions under mildly to moderately elevated glucose levels (10 mM and 15 mM) via two primary mechanisms: i) The acetylation of NRF2 at lysine residues K56, K68, and K52 prevents its ubiquitination, enhancing the transcription of antioxidant genes GST, SOD1, and GPX1. This activity diminishes cytoplasmic oxidative stress, thereby mitigating endothelial cell senescence. ii) The interaction between the Neh2 domain of NRF2 and the PAS-B domain of HIF-2α within the nucleus curtails the attachment of HIF-2α to the NOX4/p22phox promoter. This action lessens oxidative stress near the cell membrane, maintaining intercellular junctions by safeguarding the disulfide bonds in occludin and E-cadherin from disruption. However, these protective strategies prove insufficient under severe hyperglycemic conditions (25 mM). Further investigation has identified Oltipraz, an activator of NRF2, as also promoting the degradation of HIF-2α. Through its simultaneous modulation of NRF2 and HIF-2α, Oltipraz significantly reduces cellular senescence and prevents the deterioration of intercellular junctions in HUVECs subjected to high glucose concentrations (25 mM). Our research positions Oltipraz as a promising therapeutic candidate for mitigating diabetes-induced vascular endothelial damage, potentially offering benefits against diabetes-related atherosclerosis and valvular calcification., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: Calcium-sensing receptor (CASR) influences the expression pattern of multiple genes in renal tubular epithelial cells. The objective of this inquiry was to explore the molecular mechanisms of CASR in renal tubular epithelial cells and nephrolithiasis., Methods: HK-2 cells were transfected with lentiviruses carrying either CASR (named CASR) or an empty vector negative control (named NC), as well as shRNA intended to target CASR (named shCASR) or its corresponding negative control (named shNC). CCK-8 assay was used to detect the effect of CASR on the proliferation of HK-2 cells. RNA-Sequencing was applied to explore potential pathways regulated by CASR in HK-2 cells., Results: PCR and western blot results showed that CASR expression was significantly increased in CASR cells and was decreased in shCASR cells when compared to their corresponding negative control, respectively. CCK-8 assay revealed that CASR inhibited the proliferation of HK-2 cells. RNA-Sequencing results suggested that the shCASR HK-2 cells exhibited a significant up-regulation of 345 genes and a down-regulation of 366 genes. These differentially expressed genes (DEGs) were related to cell apoptosis and cell development. In CASR HK-2 cells, 1103 DEGs primarily functioned in mitochondrial energy metabolism, and amino acid metabolism. With the Venn diagram, 4 DEGs (Clorf116, ENPP3, IL20RB, and CLDN2) were selected as the hub genes regulated by CASR. Enrichment analysis revealed that these hub genes were involved in cell-cell junction, and epithelial cell development., Conclusions: In summary, our investigation has the potential to offer novel perspectives on CASR regulating cell-cell junction in HK-2 cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Patients with hemorrhagic shock and trauma (HS/T) are vulnerable to the endotheliopathy of trauma (EOT), characterized by vascular barrier dysfunction, inflammation, and coagulopathy. Cellular therapies such as mesenchymal stem cells (MSCs) and MSC extracellular vesicles (EVs) have been proposed as potential therapies targeting the EOT. In this study we investigated the effects of MSCs and MSC EVs on endothelial and epithelial barrier integrity in vitro and in vivo in a mouse model of HS/T. This study addresses the systemic effects of HS/T on multiorgan EOT., Methods: In vitro, pulmonary endothelial cell (PEC) and Caco-2 intestinal epithelial cell monolayers were treated with control media, MSC conditioned media (CM), or MSC EVs in varying doses and subjected to a thrombin or hydrogen peroxide (H 2 O 2 ) challenge, respectively. Monolayer permeability was evaluated with a cell impedance assay, and intercellular junction integrity was evaluated with immunofluorescent staining. In vivo, a mouse model of HS/T was used to evaluate the effects of lactated Ringer's (LR), MSCs, and MSC EVs on endothelial and epithelial intercellular junctions in the lung and small intestine as well as on plasma inflammatory biomarkers., Results: MSC EVs and MSC CM attenuated permeability and preserved intercellular junctions of the PEC monolayer in vitro, whereas only MSC CM was protective of the Caco-2 epithelial monolayer. In vivo, both MSC EVs and MSCs mitigated the loss of endothelial adherens junctions in the lung and small intestine, though only MSCs had a protective effect on epithelial tight junctions in the lung. Several plasma biomarkers including MMP8 and VEGF were elevated in LR- and EV-treated but not MSC-treated mice., Conclusions: In conclusion, MSC EVs could be a potential cell-free therapy targeting endotheliopathy after HS/T via preservation of the vascular endothelial barrier in multiple organs early after injury. Further research is needed to better understand the immunomodulatory effects of these products following HS/T and to move toward translating these therapies into clinical studies., (© 2024. The Author(s).)

Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca 2+ -binding sites in Scramb1 are essential for its activity raising the possibility that Ca 2+ signaling may control the assembly of SDs by impacting on Scramb1 activity., (© 2024. The Author(s).)

Background and Purpose: Oat β-glucan could ameliorate epidermal hyperplasia and accelerate epidermal barrier repair. Dectin-1 is one of the receptors of β-glucan and many biological functions of β-glucan are mediated by Dectin-1. Dectin-1 promotes wound healing through regulating the proliferation and migration of skin cells. Thus, this study aimed to investigate the role of oat β-glucan and Dectin-1 in epidermal barrier repair., Experimental Approach: To investigate the role of Dectin-1 in the epidermal barrier, indicators associated with the recovery of a damaged epidermal barrier, including histopathological changes, keratinization, proliferation, apoptosis, differentiation, cell-cell junctions and lipid content were compared between WT and Dectin-1 -/- mice. Further, the effect of oat β-glucan on the disruption of the epidermal barrier was also compared between WT and Dectin-1 -/- mice., Key Results: Dectin-1 deficiency resulted in delayed recovery and marked keratinization, as well as abnormal levels of keratinocyte differentiation, cell-cell junctions and lipid synthesis during the restoration of the epidermal barrier. Oat β-glucan significantly reduces epidermal hyperplasia, promotes epidermal differentiation, increases cell-cell junction expression, promotes lipid synthesis and ultimately accelerates the recovery of damaged epidermal barriers via Dectin-1. Oat β-glucan could promote CaS receptor expression and activate the PPAR-γ signalling pathway via Dectin-1., Conclusion and Implications: Oat β-glucan promote the recovery of damaged epidermal barriers through promoting epidermal differentiation, increasing the expression of cell-cell junctions and lipid synthesis through Dectin-1. Dectin-1 deficiency delay the recovery of epidermal barriers, which indicated that Dectin-1 may be a potential target in epidermal barrier repair., (© 2023 British Pharmacological Society.)

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1 flox/flox and/or ROCK2 flox/flox ;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, β-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/β-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and transducer by recruiting vinculin and transducing signals that influence cell behaviors. α-catenin/vinculin complex-mediated mechanotransduction regulates multiple pathways, such as Hippo pathway. However, their associations with the α-catenin-based tension sensors at cell junctions are still not fully addressed. Here, we uncovered the TRIP6/LATS1 complex co-localizes with α-catenin/vinculin at both bicellular junctions (BCJs) and tricellular junctions (TCJs). The localization of TRIP6/LATS1 complex to both TCJs and BCJs requires ROCK1 and α-catenin. Treatment by cytochalasin B, Y-27632 and blebbistatin all impaired the BCJ and TCJ junctional localization of TRIP6/LATS1, indicating that the junctional localization of TRIP6/LATS1 is mechanosensitive. The α-catenin/vinculin/TRIP6/LATS1 complex strongly localized to TCJs and exhibited a discontinuous button-like pattern on BCJs. Additionally, we developed and validated an α-catenin/vinculin BiFC-based mechanosensor that co-localizes with TRIP6/LATS1 at BCJs and TCJs. The mechanosensor exhibited a discontinuous distribution and motile signals at BCJs. Overall, our study revealed that TRIP6 and LATS1 are novel compositions of the tension sensor, together with the core complex of α-catenin/vinculin, at both the BCJs and TCJs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions fracture when claudin/JAM-A-deficient cells undergo spontaneous cell stretching. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A-deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells., (© 2024 Nguyen et al.)

The blood-testis barrier (BTB) is formed adjacent to the seminiferous basement membrane. It is a distinct ultrastructure, partitioning testicular seminiferous epithelium into apical (adluminal) and basal compartments. It plays a vital role in developing and maturing spermatocytes into spermatozoa via reorganizing its structure. This enables the transportation of preleptotene spermatocytes across the BTB, from basal to adluminal compartments in the seminiferous tubules. Several bioactive peptides and biomolecules secreted by testicular cells regulate the BTB function and support spermatogenesis. These peptides activate various downstream signaling proteins and can also be the target themself, which could improve the diffusion of drugs across the BTB. The gap junction (GJ) and its coexisting junctions at the BTB maintain the immunological barrier integrity and can be the "gateway" during spermatocyte transition. These junctions are the possible route for toxicant entry, causing male reproductive dysfunction. Herein, we summarize the detailed mechanism of all the regulators playing an essential role in the maintenance of the BTB, which will help researchers to understand and find targets for drug delivery inside the testis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Podocytes serve as part of the renal filtration unit with slit diaphragms. Although the structure of slit diaphragms between two cells is well characterized, how the tricellular contact of podocytes is organized and how it changes in injured podocytes remains unknown. This study focused on a tricellular junction protein, angulin-3, and its localization in healthy podocytes, in developmental stages, and in pathologic conditions, using a newly established monoclonal antibody. Angulin-3 was confined at tricellular junctions of primordial podocytes, then transiently localized at bicellular junctions as foot process interdigitation developed and the intercellular junctions rearranged into slit diaphragm, and eventually distributed in a sparse punctate pattern on the foot processes of adult podocytes. In the rodent podocyte injury models, angulin-3 showed bicellular localization between the foot processes, and the localization turned from punctate to dashed linear pattern along the effaced foot processes with the progression of podocyte injury. Angulin-3 also accumulated between foot processes in a linear pattern in kidney biopsy samples of human nephrotic syndrome. Additionally, the line length of angulin-3 staining signal correlated with risk of relapse under glucocorticoid therapy in patients with minimal change nephrotic syndrome. This study proposes an image program to score the linearity of the accumulation pattern of angulin-3 to evaluate the relapse risk of patients with minimal change nephrotic syndrome., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tricellular junctions (TCJs) seal epithelial cell vertices and are essential for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka, also known as Bark), Gliotactin (Gli) and M6 organize occluding TCJs. Aka and M6 localize in an interdependent manner to vertices and act jointly to localize Gli, but how these proteins interact to assemble TCJs was not previously known. Here, we show that the proteolipid protein M6 physically interacts with Aka and with itself, and that M6 is palmitoylated on conserved juxta-membrane cysteine residues. This modification promotes vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed localization of M6 and Aka but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, whereas subsequent TCJ growth relies on different mechanisms that are independent of M6 palmitoylation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Robust linkage between adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The Drosophila multidomain protein Canoe and its mammalian homolog afadin are crucial for this, as in their absence many events of morphogenesis fail. To define the mechanism of action for Canoe, we are taking it apart. Canoe has five folded protein domains and a long intrinsically disordered region. The largest is the Dilute domain, which is shared by Canoe and myosin V. To define the roles of this domain in Canoe, we combined biochemical, genetic and cell biological assays. AlphaFold was used to predict its structure, providing similarities and contrasts with Myosin V. Biochemical data suggested one potential shared function - the ability to dimerize. We generated Canoe mutants with the Dilute domain deleted (CnoΔDIL). Surprisingly, they were viable and fertile. CnoΔDIL localized to adherens junctions and was enriched at junctions under tension. However, when its dose was reduced, CnoΔDIL did not provide fully wild-type function. Furthermore, canoeΔDIL mutants had defects in the orchestrated cell rearrangements of eye development. This reveals the robustness of junction-cytoskeletal connections during morphogenesis and highlights the power of natural selection to maintain protein structure., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Embryos repair wounds rapidly, with no inflammation or scarring. Embryonic wound healing is driven by the collective movement of the cells around the lesion. The cells adjacent to the wound polarize the cytoskeletal protein actin and the molecular motor non-muscle myosin II, which accumulate at the wound edge forming a supracellular cable around the wound. Adherens junction proteins, including E-cadherin, are internalized from the wound edge and localize to former tricellular junctions at the wound margin, in a process necessary for cytoskeletal polarity. We found that the cells adjacent to wounds in the Drosophila embryonic epidermis polarized Talin, a core component of cell-extracellular matrix (ECM) adhesions, which preferentially accumulated at the wound edge. Integrin knockdown and inhibition of integrin binding delayed wound closure and reduced actin polarization and dynamics around the wound. Additionally, disrupting integrins caused a defect in E-cadherin reinforcement at tricellular junctions along the wound edge, suggesting crosstalk between integrin-based and cadherin-based adhesions. Our results show that cell-ECM adhesion contributes to embryonic wound repair and reveal an interplay between cell-cell and cell-ECM adhesion in the collective cell movements that drive rapid wound healing., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction ( zonula occludens ), the zonula adherens ( ZA ), and the macula adherens . The prevailing dogma is that at the ZA , E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura . We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia., Competing Interests: Competing interests statement:The authors declare no competing interest.

The integrity and permeability of epithelial and endothelial barriers depend on the formation of tight junctions, adherens junctions, and a junction-associated cytoskeleton. The establishment of this junction-cytoskeletal module relies on the correct folding and oligomerization of its protein components. Molecular chaperones are known regulators of protein folding and complex formation in different cellular compartments. Mammalian cells possess an elaborate chaperone network consisting of several hundred chaperones and co-chaperones. Only a small part of this network has been linked, however, to the regulation of intercellular adhesions, and the systematic analysis of chaperone functions at epithelial and endothelial barriers is lacking. This review describes the functions and mechanisms of the chaperone-assisted regulation of intercellular junctions. The major focus of this review is on heat shock protein chaperones, their co-chaperones, and chaperonins since these molecules are the focus of the majority of the articles published on the chaperone-mediated control of tissue barriers. This review discusses the roles of chaperones in the regulation of the steady-state integrity of epithelial and vascular barriers as well as the disruption of these barriers by pathogenic factors and extracellular stressors. Since cytoskeletal coupling is essential for junctional integrity and remodeling, chaperone-assisted assembly of the actomyosin cytoskeleton is also discussed.

Barrier functions of proliferative epithelia are constantly challenged by mechanical and chemical constraints. How epithelia respond to and cope with disturbances of barrier functions to allow tissue integrity maintenance is poorly characterised. Cellular junctions play an important role in this process and intracellular traffic contribute to their homeostasis. Here, we reveal that, in Drosophila pupal notum , alteration of the bi- or tricellular septate junctions (SJs) triggers a mechanism with two prominent outcomes. On one hand, there is an increase in the levels of E-cadherin, F-actin, and non-muscle myosin II in the plane of adherens junctions. On the other hand, β-integrin/Vinculin-positive cell contacts are reinforced along the lateral and basal membranes. We found that the weakening of SJ integrity, caused by the depletion of bi- or tricellular SJ components, alters ESCRT-III/Vps32/Shrub distribution, reduces degradation and instead favours recycling of SJ components, an effect that extends to other recycled transmembrane protein cargoes including Crumbs, its effector β-Heavy Spectrin Karst, and β-integrin. We propose a mechanism by which epithelial cells, upon sensing alterations of the SJ, reroute the function of Shrub to adjust the balance of degradation/recycling of junctional cargoes and thereby compensate for barrier junction defects to maintain epithelial integrity., Competing Interests: TE, MJ, ED, RL No competing interests declared, (© 2024, Esmangart de Bournonville et al.)

We performed a comparative study of the effects of X-ray irradiation and bleomycin on the mRNA levels of E-cadherin and tight junction proteins (claudin-3, claudin-4, claudin-18, ZO-2, and occludin) in an alveolar epithelial cell line L2. Irradiation decreased claudin-4 levels and increased occludin levels, while the levels of other mRNAs remained unchanged. Bleomycin increased the expression levels of all proteins examined except claudin-3. Irradiation and bleomycin have different effects on the expression level of intercellular junction proteins, indicating different reactions triggered in alveolar epithelial cells and a great prospects of further comparative studies., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

PLEKHG4B is a Cdc42-targeting guanine-nucleotide exchange factor implicated in forming epithelial cell-cell junctions. Here we explored the mechanism regulating PLEKHG4B localization. PLEKHG4B localized to the basal membrane in normal Ca 2+ medium but accumulated at cell-cell junctions upon ionomycin treatment. Ionomycin-induced junctional localization of PLEKHG4B was suppressed upon disrupting its annexin-A2 (ANXA2)-binding ability. Thus, Ca 2+ influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca 2+ or BAPTA-AM (an intracellular Ca 2+ chelator) suppressed PLEKHG4B localization to the basal membrane. Mutations of the phosphoinositide-binding motif in the pleckstrin homology (PH) domain of PLEKHG4B or masking of membrane phosphatidylinositol-4,5-biphosphate [PI(4,5)P 2 ] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca 2+ levels and PI(4,5)P 2 binding of the PH domain. Activation of mechanosensitive ion channels (MSCs) promoted PLEKHG4B localization to cell-cell junctions, and their inhibition suppressed it. Moreover, similar to the PLEKHG4B knockdown phenotypes, inhibition of MSCs or treatment with BAPTA-AM disturbed the integrity of actin filaments at cell-cell junctions. Taken together, our results suggest that Ca 2+ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.

Fundamental functions of the intestinal epithelium include the digestion of food, absorption of nutrients, and its ability to act as the first barrier against intruding microbes. Campylobacter jejuni is a major zoonotic pathogen accounting for a substantial portion of bacterial foodborne illnesses. The germ colonizes the intestines of birds and is mainly transmitted to humans through the consumption of contaminated poultry meat. In the human gastrointestinal tract, the bacterium triggers campylobacteriosis that can progress to serious secondary disorders, including reactive arthritis, inflammatory bowel disease and Guillain-Barré syndrome. We recently discovered that C. jejuni serine protease HtrA disrupts intestinal epithelial barrier functions via cleavage of the tight and adherens junction components occludin, claudin-8 and E-cadherin. However, it is unknown whether epithelial damage is mediated by the secreted soluble enzyme, by HtrA contained in shed outer-membrane vesicles (OMVs) or by another mechanism that has yet to be identified. In the present study, we investigated whether soluble recombinant HtrA and/or purified OMVs induce junctional damage to polarized intestinal epithelial cells compared to live C. jejuni bacteria. By using electron and confocal immunofluorescence microscopy, we show that HtrA-expressing C. jejuni bacteria trigger efficient junctional cell damage, but not soluble purified HtrA or HtrA-containing OMVs, not even at high concentrations far exceeding physiological levels. Instead, we found that only bacteria with active protein biosynthesis effectively cleave junctional proteins, which is followed by paracellular transmigration of C. jejuni through the epithelial cell layer. These findings shed new light on the pathogenic activities of HtrA and virulence strategies of C. jejuni .

Cell-cell interactions typically occur in a 3D context that is distinct from conventional 2D cell-substrate interactions in a Petri dish. Here, we describe a benchtop method to combine a 2D extracellular matrix surface with a 3D, vertical boundary functionalized with the extracellular domain of E-cadherin. The methodology is suitable for any biology laboratory without requiring advanced microfabrication equipment or training. Overall, this cell-mimetic interface uniquely recapitulates key aspects of cell-cell adhesion and can serve as a versatile, reductionist technique to study general cell-cell interactions in a 3D context., (© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The RhoGEF Trio is a large multi-domain protein and an activator of the small GTPases Rac1, RhoG, and RhoA. Although Trio has been implicated in many cellular mechanisms like leukocyte transendothelial migration, cell-cell junction stability, lamellipodia formation, axon outgrowth, and muscle fusion, it remains unclear how Trio is activated. Using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis of endothelial cells, we identified two serine residues (S1785/S1786) located in between the two exchange domains of Trio that were highly phosphorylated upon short thrombin treatment. Using phosphomimetic Trio S1785D/S1786D double mutants, we did not find an increase in Rac1/RhoG activity, indicating that the phosphorylation events do not increase Trio exchange activity. However, we found that the Trio mutants localized more strongly at cell-cell junctions and prevented junction destabilization upon thrombin treatment, judged by junction linearity. Our data suggest that serine phosphorylation of Trio potentiates the localization of Trio to junctional regions, resulting in locally promoting the exchange for Rac1 at junction regions and increasing endothelial cell-cell junction stability upon permeability-inducing reagents such as thrombin.

Various functions within our bodies require the generation and maintenance of compartments with distinct compositions, which in turn necessitate the formation of semipermeable cellular diffusion barriers. For example, the blood-brain barrier protects the brain by allowing only specific molecules to pass through. Another instance is the intestinal barrier, which allows the uptake of essential nutrients, while restricting the passage of pathogenic molecules and bacteria. Breakdown of such barriers causes various pathologies, such as brain or retinal edema, or diarrhoea. Epithelia and endothelia are the most common barrier-forming cells. Individual cells in such barriers are held together by cell-cell adhesion structures - also known as intercellular junctions - that are essential for barrier formation and maintenance. Here, we will focus on the structure and assembly of tight junctions (TJs) and their functions as barriers, but will refer to other adhesive structures crucial for barrier regulation such as adherens junctions (AJs) and focal adhesions to the extracellular matrix (ECM) (Figure 1A,B). We will also discuss additional functions of TJs in cell surface polarity and the regulation of gene expression, cell function, and cell behaviour., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hydrogen sulfide (H 2 S) promotes microangiogenesis and revascularization after ischemia. Neovascularization starts with the destruction of intercellular junctions and is accompanied by various endothelial cell angiogenic behaviors. Follistatin-like 1 (FSTL1) is a cardiovascular-protective myokine that works against ischemic injury. The present study examined whether FSTL1 was involved in H 2 S-induced angiogenesis and explored the underlying molecular mechanism. We observed that H 2 S accelerated blood perfusion after ischemia in the mouse hindlimb ischemia model. Western blot analysis showed that H 2 S stabilized FSTL1 transcript and increased FSTL1 and Human antigen R (HuR) levels in skeletal muscle. RNA-interference HuR significantly inhibited the H 2 S-promoted increase in FSTL1 levels. Exogenous FSTL1 promoted the wound-healing migration of human umbilical vein endothelial cells (HUVECs) and increased monolayer endothelial barrier permeability. Immunostaining showed that FSTL1 increased interendothelial gap formation and decreased VE-Cadherin, Occludin, Connexin-43, and Claudin-5 expression. In addition, FSTL1 significantly increased the phosphorylation of Src and VEGFR2. However, the Src inhibitor, not the VEGFR2 inhibitor, could block FSTL1-induced effects in angiogenesis. In conclusion, we demonstrated that H 2 S could upregulate the expression of FSTL1 by increasing the HuR levels in skeletal muscle, and paracrine FSTL1 could initiate angiogenesis by opening intercellular junctions via the Src signaling pathway. NEW & NOTEWORTHY The myocyte-derived paracrine protein FSTL1 acts on vascular endothelial cells and initiates the process of angiogenesis by opening the intercellular junction via activating Src kinase. H 2 S can significantly upregulate FSTL1 protein levels in skeletal muscles by increasing HuR expression.

Tight junctions (TJs) are the most apical components of junctional complexes in epithelial and endothelial cells. Barrier function is one of the major functions of TJ, which restricts the ions and small water-soluble molecules from passing through the paracellular pathway. Adherens junctions (AJs) play an important role in cell-cell adhesion and cell signaling. Gap junctions (GJs) are intercellular channels regulating electrical and metabolic signals between cells. It is well known that TJ integral membrane proteins, such as claudins and occludins, are the molecular building blocks responsible for TJ barrier function. However, recent studies demonstrate that proteins of other junctional complexes can influence and regulate TJ barrier function. Therefore, the crosstalk between different cell junctions represents a common means to modulate cellular activities. In this review, we will discuss the interactions among TJ, AJ, and GJ by focusing on how AJ and GJ proteins regulate TJ barrier function in different biological systems.

Tissue cells in epithelial or endothelial monolayers are connected through cell-cell junctions, which are stabilized by transmembrane E-cadherin bonds and intracellular actin filaments. These bonds and junctions play a crucial role in maintaining the barrier function of epithelia and endothelia and are believed to transmit forces between cells. Additionally, E-cadherin bonds can impact the shape of cell-cell junctions. In this study, we develop a continuum mechanical model of the cell-cell junction by explicitly incorporating the cell membrane, distributions of E-cadherin bonds, cytoplasmic fluid pressure, and F-actin dynamics. The static force-balanced version of the model is able to analyze the influences of cell cortical tension, actin dynamics, and cytoplasmic pressure on the junction shape and E-cadherin bonds. Furthermore, an extended model that incorporates fluid flow, across the cell boundary as well as around the cell, is also examined. This model can couple cell-shape changes with cell cortical tension and fluid flow, and predicts the additional effect of fluid motion on cell-cell junction mechanics. Taken together, our models serve as an intermediate link between molecular-scale models of cell-junction molecules and cell-scale models of tissue and epithelia., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Critical for the maintenance of epidermal integrity and function are attachments between intermediate filaments (IF) and intercellular junctions called desmosomes. The desmosomal cytoplasmic plaque protein desmoplakin (DP) is essential for anchoring IF to the junction. DP-IF interactions are regulated by a phospho-regulatory motif within the DP C-terminus controlling keratinocyte intercellular adhesion. Here we identify the protein phosphatase 2A (PP2A)-B55α holoenzyme as the major serine/threonine phosphatase regulating DP's C-terminus and consequent intercellular adhesion. Using a combination of chemical and genetic approaches, we show that the PP2A-B55α holoenzyme interacts with DP at intercellular membranes in 2D- and 3D- epidermal models and human skin samples. Our experiments demonstrate that PP2A-B55α regulates the phosphorylation status of junctional DP and is required for maintaining strong desmosome-mediated intercellular adhesion. These data identify PP2A-B55α as part of a regulatory module capable of tuning intercellular adhesion strength and a candidate disease target in desmosome-related disorders of the skin and heart., (© 2023. Springer Nature Limited.)

One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. We focus on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. We have combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Our bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When we looked over shorter evolutionary time scales, we identified multiple conserved motifs. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. We next identified the lesions in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including the conserved motifs in the IDR, are critical for protein function. These data provide the foundation for further analysis of IDR function., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gurley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

As biologics used in the clinic outpace the number of new small molecule drugs, an important challenge for their efficacy and widespread use has emerged, namely tissue penetrance. Macromolecular drugs - bulky, high-molecular weight, hydrophilic agents - exhibit low permeability across biological barriers. Epithelial and endothelial layers, for example within the gastrointestinal tract or at the blood-brain barrier, present the most significant obstacle to drug transport. Within epithelium, two subcellular structures are responsible for limiting absorption: cell membranes and intercellular tight junctions. Previously considered impenetrable to macromolecular drugs, tight junctions control paracellular flux and dictate drug transport between cells. Recent work, however, has shown tight junctions to be dynamic, anisotropic structures that can be targeted for delivery. This review aims to summarize new approaches for targeting tight junctions, both directly and indirectly, and to highlight how manipulation of tight junction interactions may help usher in a new era of precision drug delivery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

β-CATENIN is an evolutionarily conserved multifunctional molecule that maintains cell adhesion as a cell junction protein to safeguard the integrity of the mammalian blood-testes barrier, and also regulates cell proliferation and apoptosis as a key signaling molecule in the WNT/β-CATENIN signaling pathway. In the crustacean Eriocheir sinensis, Es-β-CATENIN has been shown to be involved in spermatogenesis, but the testes of E. sinensis have large and well-defined structural differences from those of mammals, and the impact of Es-β-CATENIN in them is still unknown. In the present study, we found that Es-β-CATENIN, Es-α-CATENIN and Es-ZO-1 interact differently in the testes of the crab compared to mammals. In addition, defective Es-β-CATENIN resulted in increased Es-α-CATENIN protein expression levels, distorted and deformed F-ACTIN, and disturbed localization of Es-α-CATENIN and Es-ZO-1, leading to loss of hemolymph-testes barrier integrity and impaired sperm release. In addition to this, we also performed the first molecular cloning and bioinformatics analysis of Es-AXIN in the WNT/β-CATENIN pathway to exclude the effect of the WNT/β-CATENIN pathway on the cytoskeleton. In conclusion, Es-β-CATENIN participates in maintaining the hemolymph-testes barrier in the spermatogenesis of E. sinensis., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bortezomib (BTZ), a chemotherapeutic drug used to treat multiple myeloma, induces life-threatening side effects, including severe pulmonary toxicity. However, the mechanisms underlying these effects remain unclear. The objectives of this study were to (1) investigate whether BTZ influences vascular permeability and (2) clarify the effect of BTZ on the expression of molecules associated with cell-cell junctions using human pulmonary microvascular endothelial cells in vitro. Clinically relevant concentrations of BTZ induced limited cytotoxicity and increased the permeability of human pulmonary microvascular endothelial cell monolayers. BTZ decreased the protein expression of claudin-5, occludin, and VE-cadherin but not that of ZO-1 and β-catenin. Additionally, BTZ decreased the mRNA expression of claudin-5, occludin, ZO-1, VE-cadherin, and β-catenin. Our results suggest that BTZ increases the vascular permeability of the pulmonary microvascular endothelium by downregulating cell-cell junction molecules, particularly claudin-5, occludin, and VE-cadherin.

Although perineurium has an important role in maintenance of the blood-nerve barrier, understanding of perineurial cell-cell junctions is insufficient. The aim of this study was to analyze the expression of junctional cadherin 5 associated (JCAD) and epidermal growth factor receptor (EGFR) in the perineurium of the human inferior alveolar nerve (IAN) and investigate their roles in perineurial cell-cell junctions using cultured human perineurial cells (HPNCs). In human IAN, JCAD was strongly expressed in endoneurial microvessels. JCAD and EGFR were expressed at various intensities in the perineurium. In HPNCs, JCAD was clearly expressed at cell-cell junctions. EGFR inhibitor AG1478 treatment changed cell morphology and the ratio of JCAD-positive cell-cell contacts of HPNCs. Therefore, JCAD and EGFR may have a role in the regulation of perineurial cell-cell junctions.

Multipotent epithelial progenitor cells can be expanded from human embryonic lungs as organoids and maintained in a self-renewing state using a defined medium. The organoid cells are columnar, resembling the cell morphology of the developing lung tip epithelium in vivo. Cell shape dynamics and fate are tightly coordinated during development. We therefore used the organoid system to identify signalling pathways that maintain the columnar shape of human lung tip progenitors. We found that EGF, FGF7 and FGF10 have distinct functions in lung tip progenitors. FGF7 activates MAPK/ERK and PI3K/AKT signalling, and is sufficient to promote columnar cell shape in primary tip progenitors. Inhibitor experiments show that MAPK/ERK and PI3K/AKT signalling are key downstream pathways, regulating cell proliferation, columnar cell shape and cell junctions. We identified integrin signalling as a key pathway downstream of MAPK/ERK in the tip progenitors; disrupting integrin alters polarity, cell adhesion and tight junction assembly. By contrast, stimulation with FGF10 or EGF alone is not sufficient to maintain organoid columnar cell shape. This study employs organoids to provide insight into the cellular mechanisms regulating human lung development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)