Dsg2 ectodomain organization increases throughout desmosome assembly.

Desmosomes are intercellular junctions that regulate mechanical integrity in epithelia and cardiac muscle. Dynamic desmosome remodeling is essential for wound healing and development, yet the mechanisms governing junction assembly remain elusive. While we and others have shown that cadherin ectodomains are highly organized, how this ordered architecture emerges during assembly is unknown. Using fluorescence polarization microscopy, we show that desmoglein 2 (Dsg2) ectodomain order gradually increases during 8 h of assembly, coinciding with increasing adhesive strength. In a scratch wound assay, we observed a similar increase in order in desmosomes assembling at the leading edge of migratory cells. Together, our findings indicate that cadherin organization is a hallmark of desmosome maturity and may play a role in conferring adhesive strength.

Age-progressive interplay of HSP-proteostasis, ECM-cell junctions and biomechanics ensures C. elegans astroglial architecture.

Tissue integrity is sensitive to temperature, tension, age, and is sustained throughout life by adaptive cell-autonomous or extrinsic mechanisms. Safeguarding the remarkably-complex architectures of neurons and glia ensures age-dependent integrity of functional circuits. Here, we report mechanisms sustaining the integrity of C. elegans CEPsh astrocyte-like glia. We combine large-scale genetics with manipulation of genes, cells, and their environment, quantitative imaging of cellular/ subcellular features, tissue material properties and extracellular matrix (ECM). We identify mutants with age-progressive, environment-dependent defects in glial architecture, consequent disruption of neuronal architecture, and abnormal aging. Functional loss of epithelial Hsp70/Hsc70-cochaperone BAG2 causes ECM disruption, altered tissue biomechanics, and hypersensitivity of glia to environmental temperature and mechanics. Glial-cell junctions ensure epithelia-ECM-CEPsh glia association. Modifying glial junctions or ECM mechanics safeguards glial integrity against disrupted BAG2-proteostasis. Overall, we present a finely-regulated interplay of proteostasis-ECM and cell junctions with conserved components that ensures age-progressive robustness of glial architecture.

Molecular and Cellular Characterization of Primary Endothelial Cells from a Familial Cavernomatosis Patient.

Cerebral cavernous malformation (CCM) or familial cavernomatosis is a rare, autosomal dominant, inherited disease characterized by the presence of vascular malformations consisting of blood vessels with an abnormal structure in the form of clusters. Based on the altered gene (CCM1/Krit1, CCM2, CCM3) and its origin (spontaneous or familial), different types of this disease can be found. In this work we have isolated and cultivated primary endothelial cells (ECs) from peripheral blood of a type 1 CCM patient. Differential functional and gene expression profiles of these cells were analyzed and compared to primary ECs from a healthy donor. The mutation of the familial index case consisted of a heterozygous point mutation in the position +1 splicing consensus between exons 15 and 16, causing failure in RNA processing and in the final protein. Furthermore, gene expression analysis by quantitative PCR revealed a decreased expression of genes involved in intercellular junction formation, angiogenesis, and vascular homeostasis. Cell biology analysis showed that CCM1 ECs were impaired in angiogenesis and cell migration. Taken together, the results obtained suggest that the alterations found in CCM1 ECs are already present in the heterozygous condition, suffering from vascular impairment and somewhat predisposed to vascular damage.

Afadin-nectin forces its way to the front.

Force transmission at cell-cell junctions critically regulates embryogenesis, tissue homeostasis, and diseases including cancer. The cadherin-catenin linkage has been considered the keystone of junctional force transmission, but new findings challenge this paradigm, arguing instead that the nectin-afadin linkage plays the more important role in mature junctions in the intestinal epithelium.

Transcriptional Levels of Intercellular Junction Proteins in an Alveolar Epithelial Cell Line Exposed to Irradiation or Bleomycin.

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.

Regulation of Epithelial and Endothelial Barriers by Molecular Chaperones.

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.

Tight junction membrane proteins regulate the mechanical resistance of the apical junctional complex.

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.

The Dilute domain in Canoe is not essential for linking cell junctions to the cytoskeleton but supports morphogenesis robustness.

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.

The zonula adherens matura redefines the apical junction of intestinal epithelia.

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.

Palmitoylation of proteolipid protein M6 promotes tricellular junction assembly in epithelia of Drosophila.

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.

Campylobacter jejuni Surface-Bound Protease HtrA, but Not the Secreted Protease nor Protease in Shed Membrane Vesicles, Disrupts Epithelial Cell-to-Cell Junctions.

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.

ESCRT-III-dependent adhesive and mechanical changes are triggered by a mechanism detecting alteration of septate junction integrity in Drosophila epithelial cells.

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, ß-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 ß-Heavy Spectrin Karst, and ß-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.

Cleavage of cell junction proteins as a host invasion strategy in leptospirosis.

Infection and invasion are the prerequisites for developing the disease symptoms in a host. While the probable mechanism of host invasion and pathogenesis is known in many pathogens, very little information is available on Leptospira invasion/pathogenesis. For causing systemic infection Leptospira must transmigrate across epithelial barriers, which is the most critical and challenging step. Extracellular and membrane-bound proteases play a crucial role in the invasion process. An extensive search for the proteins experimentally proven to be involved in the invasion process through cell junction cleavage in other pathogens has resulted in identifying 26 proteins. The similarity searches on the Leptospira genome for counterparts of these 26 pathogenesis-related proteins identified at least 12 probable coding sequences. The proteins were either extracellular or membrane-bound with a proteolytic domain to cleave the cell junction proteins. This review will emphasize our current understanding of the pathogenic aspects of host cell junction-pathogenic protein interactions involved in the invasion process. Further, potential candidate proteins with cell junction cleavage properties that may be exploited in the diagnostic/therapeutic aspects of leptospirosis will also be discussed. KEY POINTS: • The review focussed on the cell junction cleavage proteins in bacterial pathogenesis • Cell junction disruptors from Leptospira genome are identified using bioinformatics • The review provides insights into the therapeutic/diagnostic interventions possible.

Assembly, dynamics and remodeling of epithelial cell junctions throughout development.

Cell junctions play key roles in epithelial integrity. During development, when epithelia undergo extensive morphogenesis, these junctions must be remodeled in order to maintain mechanochemical barriers and ensure the cohesion of the tissue. In this Review, we present a comprehensive and integrated description of junctional remodeling mechanisms in epithelial cells during development, from embryonic to adult epithelia. We largely focus on Drosophila, as quantitative analyses in this organism have provided a detailed characterization of the molecular mechanisms governing cell topologies, and discuss the conservation of these mechanisms across metazoans. We consider how changes at the molecular level translate to tissue-scale irreversible deformations, exploring the composition and assembly of cellular interfaces to unveil how junctions are remodeled to preserve tissue homeostasis during cell division, intercalation, invagination, ingression and extrusion.

Physiological cyclic stretching potentiates the cell-cell junctions in vascular endothelial layer formed on aligned fiber substrate.

In vascular tissue engineering, formation of stable endothelial cell-cell and cell-substrate adhesions is essential for maintaining long-term patency of the tissue-engineered vascular grafts (TEVGs). In this study, sheet-like aligned fibrous substrates of poly(l-lactide-co-caprolactone) (PLCL) were prepared by electrospinning to provide basement membrane-resembling structural support to endothelial cells (ECs). Cyclic stretching at physiological and pathological levels was then applied to human umbilical vein endothelial cells (HUVECs) cultured on chosen fibrous substrate using a force-loading device, from which effects of the cyclic stretching on cell-cell and cell-substrate adhesions were examined. It was found that applying uniaxial 1 Hz cyclic stretch at physiological levels (5 % and 10 % elongation) strengthened the cell-cell junctions, thus leading to improved structural integrity, functional expression and resistance to thrombin-induced damaging impacts in the formed endothelial layer. The cell-cell junctions were disrupted at pathological level (15 % elongation) cyclic stretching, which however facilitated the formation of focal adhesions (FAs) at cell-substrate interface. Mechanistically, the effects of cyclic stretching on endothelial cell-cell and cell-substrate adhesions were identified to be correlated with the RhoA/ROCK signaling pathway. Results from this study highlight the relevance between applying dynamic mechanical stimulation and maintaining the structural integrity of the formed endothelial layer, and implicate a necessity to implement appropriate dynamic mechanical training (i.e., preconditioning) to obtain tissue-engineered blood vessels with long-term patency post-implantation.

Integrin-based adhesions promote cell-cell junction and cytoskeletal remodelling to drive embryonic wound healing.

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.

The effect of Par3 on the cellular junctions and biological functions of odontoblast-lineage cells.

Perfect intercellular junctions are key for odontoblast barrier function. However, whether Partitioning defective-3 (Par3) is expressed in odontoblasts and its potential effects on odontoblast junctions are unknown. Herein, we investigated the effect of Par3 on cellular junctions and the biological behavior of odontoblast-lineage cells (OLCs). Whole-transcriptome sequencing was used to analyze the effects of Par3 on OLCs and the underlying molecular mechanism. Par3 was detected under physiological and inflammatory conditions in OLCs. To investigate the regulatory effect of Par3 on junctions between mouse OLCs, the effects of Par3 downregulation on the proliferation, migration, cycle and apoptosis of OLCs were detected by 5-ethyl-2'-deoxyuridine (EdU) and Transwell assays and flow cytometry. Western blotting and alizarin red S and alkaline phosphatase (ALP) staining were used to observe the effect of Par3 downregulation on OLC mineralization. Whole-transcriptome sequencing was used to investigate the biological role of Par3 in OLCs and potential molecular mechanisms. Par3 was located along the odontoblast layer in the rat pulp tissue and in the cytoplasm of OLCs. Par3 expression was downregulated under inflammatory conditions. The OLC junctions were discontinuous, and total Zona occluden-1 (ZO-1) expression and expression of ZO-1 at the membrane in OLCs were reduced after Par3 silencing (P < 0.05). Expression of a junction-related protein (ZO-1) was downregulated after the downregulation of Par3 (P < 0.05), and ZO-1 moved from the cell membrane to the cytoplasm. OLC proliferation and migration were enhanced, but apoptosis and mineralization were inhibited in shPar3-transfected cells (P < 0.05). Sequencing identified 2996 differentially expressed genes (DEGs), which were mainly enriched in the response to stimuli and binding. Downregulation of Par3 could overactivate the PI3k-AKT pathway by promoting AKT phosphorylation (P < 0.05). Downregulation of Par3 may disrupt junctions between OLCs by affecting ZO-1 expression and distribution and promote OLC proliferation and migration but inhibit OLC mineralization. Par3 may interact with 14-3-3 proteins for PI3K-AKT pathway activation to affect OLC junctions and function.

Calcium influx promotes PLEKHG4B localization to cell-cell junctions and regulates the integrity of junctional actin filaments.

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 Ca2+ 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, Ca2+ influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca2+ or BAPTA-AM (an intracellular Ca2+ 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)P2] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca2+ levels and PI(4,5)P2 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 Ca2+ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.

Porcine circovirus type 2 and Glaesserella parasuis serotype 4 co-infection activates Snail1 to disrupt the intercellular junctions and facilitate bacteria translocation across the tracheal epithelium.

Clinically, Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality. However, the mechanism of PCV2 and G. parasuis serotype 4 (GPS4) co-infection is still not fully understood. In this study, swine tracheal epithelial cells (STEC) were used as a barrier model, and our results showed that PCV2 infection increased the adhesion of GPS4 to STEC, while decreasing the levels of ZO-1, Occludin and increasing tracheal epithelial permeability, and ultimately facilitated GPS4 translocation. Snail1 is a transcriptional repressor, and has been known to induce epithelial-to-mesenchymal transition (EMT) during development or in cancer metastasis. Importantly, we found that Snail1, as a transcriptional repressor, was crucial in destroying the tracheal epithelial barrier induced by PCV2, GPS4, PCV2 and GPS4 coinfection. For the first time, we found that PCV2, GPS4, PCV2 and GPS4 coinfection cross-activates TGF-ß and p38/MAPK signaling pathways to upregulate the expression of Snail1, down-regulate the levels of ZO-1 and Occludin, and thus disrupt the integrity of tracheal epithelial barrier then promoting GPS4 translocation. Finally, PCV2 and GPS4 co-infection also can activate TGF-ß and p38/MAPK signaling pathways in vivo and upregulate Snail1, ultimately down-regulating the expression of ZO-1 and Occludin. Our study elucidates how PCV2 infection promotes GPS4 to breach the tracheal epithelial barrier and aggravate clinical manifestations.