Adherens Junctions Research Articles

Porous honeycomb film membranes enhance endothelial barrier integrity in human vascular wall bilayer model compared to standard track-etched membranes.

In vitro vascular wall bilayer models for drug testing and disease modeling must emulate the physical and biological properties of healthy vascular tissue and its endothelial barrier function. Both endothelial cell (EC)-vascular smooth muscle cell (SMC) interaction across the internal elastic lamina (IEL) and blood vessel stiffness impact endothelial barrier integrity. Polymeric porous track-etched membranes (TEM) typically represent the IEL in laboratory vascular bilayer models. However, TEM stiffness exceeds that of diseased blood vessels, and the membrane pore architecture limits EC-SMC interaction. The mechanical properties of compliant honeycomb film (HCF) membranes better simulate the Young's modulus of healthy blood vessels, and HCFs are thinner (4 vs. 10 μm) and more porous (57 vs. 6.5%) than TEMs. We compared endothelial barrier integrity in vascular wall bilayer models with human ECs and SMCs statically cultured on opposite sides of HCFs and TEMs (5 μm pores) for up to 12 days. Highly segregated localization of tight junction (ZO-1) and adherens junction (VE-cadherin) proteins and quiescent F-actin cytoskeletons demonstrated superior and earlier maturation of interendothelial junctions. Quantifying barrier integrity based on transendothelial electrical resistance (TEER), membranes showed only minor but significant TEER differences despite enhanced junctional protein localization on HCF. Elongated ECs on HCF likely experienced greater paracellular diffusion than blocky ECs on TEM. Also, larger populations of plaques of connexin 43 subunit-containing gap junctions suggested enhanced EC-SMC communication across the more porous, thinner HCF. Compared with standard TEMs, engineered vascular wall bilayers cultured on HCFs better replicate physiologic endothelial barrier integrity.

Bi-allelic variations in CRB2, encoding the crumbs cell polarity complex component 2, lead to non-communicating hydrocephalus due to atresia of the aqueduct of sylvius and central canal of the medulla

Congenital hydrocephalus is a common condition caused by the accumulation of cerebrospinal fluid in the ventricular system. Four major genes are currently known to be causally involved in hydrocephalus, either isolated or as a common clinical feature: L1CAM, AP1S2, MPDZ and CCDC88C. Here, we report 3 cases from 2 families with congenital hydrocephalus due to bi-allelic variations in CRB2, a gene previously reported to cause nephrotic syndrome, variably associated with hydrocephalus. While 2 cases presented with renal cysts, one case presented with isolated hydrocephalus. Neurohistopathological analysis allowed us to demonstrate that, contrary to what was previously proposed, the pathological mechanisms underlying hydrocephalus secondary to CRB2 variations are not due to stenosis but to atresia of both Sylvius Aqueduct and central medullar canal. While CRB2 has been largely shown crucial for apico-basal polarity, immunolabelling experiments in our fetal cases showed normal localization and level of PAR complex components (PKCι and PKCζ) as well as of tight (ZO-1) and adherens (β-catenin and N-Cadherin) junction molecules indicating a priori normal apicobasal polarity and cell–cell adhesion of the ventricular epithelium suggesting another pathological mechanism. Interestingly, atresia but not stenosis of Sylvius aqueduct was also described in cases with variations in MPDZ and CCDC88C encoding proteins previously linked functionally to the Crumbs (CRB) polarity complex, and all 3 being more recently involved in apical constriction, a process crucial for the formation of the central medullar canal. Overall, our findings argue for a common mechanism of CRB2, MPDZ and CCDC88C variations that might lead to abnormal apical constriction of the ventricular cells of the neural tube that will form the ependymal cells lining the definitive central canal of the medulla. Our study thus highlights that hydrocephalus related to CRB2, MPDZ and CCDC88C constitutes a separate pathogenic group of congenital non-communicating hydrocephalus with atresia of both Sylvius aqueduct and central canal of the medulla.

Can biomarkers identified from the uterine fluid transcriptome be used to establish a noninvasive endometrial receptivity prediction tool? A proof-of-concept study

BackgroundEmbryo implantation in a receptive endometrium is crucial for successful pregnancy. Endometrial receptivity (ER) prediction tools based on endometrial transcriptome biomarkers by endometrial biopsy have been used to guide successful embryo implantation in in vitro fertilization (IVF) patients. However, no reliable noninvasive ER prediction method has been established, and one is greatly needed. We aimed to identify biomarkers from uterine fluid transcriptomic sequencing data for establishing noninvasive ER prediction tool and to evaluate its clinical application potential in patients undergoing IVF.MethodsThe non-invasive RNA-seq based endometrial receptivity test (nirsERT) was established by analyzing transcriptomic profile of 144 uterine fluid specimens (LH + 5, LH + 7, and LH + 9) at three different receptive status from 48 IVF patients with normal ER in combination with random forest algorithm. Subsequently, 22 IVF patients who underwent frozen-thaw blastocyst transfer were recruited and analyzed the correlation between the predicted results of nirsERT and pregnancy outcomes.ResultsA total of 864 ER-associated differentially expressed genes (DEGs) involved in biological processes associated with endometrium-embryo crosstalk, including protein binding, signal reception and transduction, biomacromolecule transport and cell-cell adherens junctions, were selected. Subsequently, a nirsERT model consisting of 87 markers and 3 hub genes was established using a random forest algorithm. 10-fold cross-validation resulted in a mean accuracy of 93.0%. A small cohort (n = 22) retrospective observation shows that 77.8% (14/18) of IVF patients predicted with a normal WOI had successful intrauterine pregnancies, while none of the 3 patients with a displaced WOI had successful pregnancies. One patient failed due to poor sequencing data quality.ConclusionsNirsERT based on uterine fluid transcriptome biomarkers can predict the WOI period relatively accurately and may serve as a noninvasive, reliable and same cycle test for ER in reproductive clinics.Trial registrationChinese Clinical Trial Registry: ChiCTR-DDD-17013375. Registered 14 November 2017, http://www.chictr.org.cn/index.aspx.

Membrane adhesion junctions regulate airway smooth muscle phenotype and function.

The local environment surrounding airway smooth muscle (ASM) cells has profound effects on the physiological and phenotypic properties of ASM tissues. ASM is continually subjected to the mechanical forces generated during breathing and to the constituents of its surrounding extracellular milieu. The smooth muscle cells within the airways continually modulate their properties to adapt to these changing environmental influences. Smooth muscle cells connect to the extracellular cell matrix (ECM) at membrane adhesion junctions that provide mechanical coupling between smooth muscle cells within the tissue. Membrane adhesion junctions also sense local environmental signals and transduce them to cytoplasmic and nuclear signaling pathways in the ASM cell. Adhesion junctions are composed of clusters of transmembrane integrin proteins that bind to ECM proteins outside the cell and to large multiprotein complexes in the submembranous cytoplasm. Physiological conditions and stimuli from the surrounding ECM are sensed by integrin proteins and transduced by submembranous adhesion complexes to signaling pathways to the cytoskeleton and nucleus. The transmission of information between the local environment of the cells and intracellular processes enables ASM cells to rapidly adapt their physiological properties to modulating influences in their extracellular environment: mechanical and physical forces that impinge on the cell, ECM constituents, local mediators, and metabolites. The structure and molecular organization of adhesion junction complexes and the actin cytoskeleton are dynamic and constantly changing in response to environmental influences. The ability of ASM to rapidly accommodate to the ever-changing conditions and fluctuating physical forces within its local environment is essential for its normal physiological function.

Dialogue between VE-Cadherin and Sphingosine 1 Phosphate Receptor1 (S1PR1) for Protecting Endothelial Functions.

The endothelial cells (EC) of established blood vessels in adults remain extraordinarily quiescent in the sense that they are not actively proliferating, but they fulfill the necessary role to control the permeability of their monolayer that lines the interior of blood vessels. The cell-cell junctions between ECs in the endothelium comprise tight junctions and adherens homotypic junctions, which are ubiquitous along the vascular tree. Adherens junctions are adhesive intercellular contacts that are crucial for the organization of the EC monolayer and its maintenance and regulation of normal microvascular function. The molecular components and underlying signaling pathways that control the association of adherens junctions have been described in the last few years. In contrast, the role that dysfunction of these adherens junctions has in contributing to human vascular disease remains an important open issue. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator found at high concentrations in blood which has important roles in the control of the vascular permeability, cell recruitment, and clotting that follow inflammatory processes. This role of S1P is achieved through a signaling pathway mediated through a family of G protein-coupled receptors designated as S1PR1. This review highlights novel evidence for a direct linkage between S1PR1 signaling and the mediation of EC cohesive properties that are controlled by VE-cadherin.

In colon cancer cells fascin1 regulates adherens junction remodeling.

Adherens junctions (AJs) are a defining feature of all epithelial cells. They regulate epithelial tissue architecture and integrity, and their dysregulation is a key step in tumor metastasis. AJ remodeling is crucial for cancer progression, and it plays a key role in tumor cell survival, growth, and dissemination. Few studies have examined AJ remodeling in cancer cells consequently, it remains poorly understood and unleveraged in the treatment of metastatic carcinomas. Fascin1 is an actin-bundling protein that is absent from the normal epithelium but its expression in colon cancer is linked to metastasis and increased mortality. Here, we provide the molecular mechanism of AJ remodeling in colon cancer cells and identify for the first time, fascin1's function in AJ remodeling. We show that in colon cancer cells fascin1 remodels junctional actin and actomyosin contractility which makes AJs less stable but more dynamic. By remodeling AJs fascin1 drives mechanoactivation of WNT/β-catenin signaling and generates "collective plasticity" which influences the behavior of cells during cell migration. The impact of mechanical inputs on WNT/β-catenin activation in cancer cells remains poorly understood. Our findings highlight the role of AJ remodeling and mechanosensitive WNT/β-catenin signaling in the growth and dissemination of colorectal carcinomas.

Inflammation modulates intercellular adhesion and mechanotransduction in human epidermis via ROCK2

Aberrant mechanotransduction and compromised epithelial barrier function are associated with numerous human pathologies including inflammatory skin disorders. However, the cytoskeletal mechanisms regulating inflammatory responses in the epidermis are not well understood. Here we addressed this question by inducing a psoriatic phenotype in human keratinocytes and reconstructed human epidermis using a cytokine stimulation model. We show that the inflammation upregulates the Rho-myosin II pathway and destabilizes adherens junctions (AJs) promoting YAP nuclear entry. The integrity of cell-cell adhesion but not the myosin II contractility per se is the determinative factor for the YAP regulation in epidermal keratinocytes. The inflammation-induced disruption of AJs, increased paracellular permeability, and YAP nuclear translocation are regulated by ROCK2, independently from myosin II activation. Using a specific inhibitor KD025, we show that ROCK2 executes its effects via cytoskeletal and transcription-dependent mechanisms to shape the inflammatory response in the epidermis.

Simultaneous attenuation of hyperglycemic memory-induced retinal, pulmonary, and glomerular dysfunctions by proinsulin C-peptide in diabetes

BackgroundHyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic complications. Although coincident diabetic complications are reported, research on their development and treatment is limited. Thus, we investigated whether C-peptide can simultaneously inhibit HGM-induced retinal, pulmonary, and glomerular dysfunctions in diabetic mice supplemented with insulin.MethodsInsulin-treated diabetic mice were supplemented with human C-peptide by subcutaneous implantation of K9-C-peptide depots for 4 weeks, and reactive oxygen species (ROS) generation, transglutaminase (TGase) activity, and vascular leakage were examined in the retina, lung, and kidney.ResultsWe found hyperglycemia-induced persistent ROS generation and TGase activation after blood glucose normalization in the retina, lung, and kidney of insulin-supplemented diabetic mice. These pathological events were inhibited by systemic supplementation of human C-peptide via subcutaneous implantation of a thermosensitive biopolymer-conjugated C-peptide depot. ROS generation and TGase activation were in a vicious cycle after glucose normalization, and C-peptide suppressed the vicious cycle and subsequent endothelial permeability in human retinal endothelial cells. Moreover, C-peptide supplementation ameliorated HGM-induced retinal vascular leakage and neurodegeneration, pulmonary vascular leakage and fibrosis, and glomerular adherens junction disruption and vascular leakage.ConclusionsOverall, our findings demonstrate that C-peptide supplementation simultaneously attenuates vascular and neuronal dysfunctions in the retina, lung, and glomerulus of insulin-supplemented diabetic mice.

Bioinformatics Analysis Reveals a Shared Pathway for Common Forms of Adult Nephrotic Syndrome.

Focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), and minimal change disease (MCD) are common causes of nephrotic syndrome. Although triggers for these diseases differs, disease progression may share common molecular mechanisms. The aim of this study was to investigate the presence of molecular pathways that are dysregulated across these glomerular diseases. The gene expression dataset GSE200828 from the NEPTUNE Study was obtained from the GEO database. R and Python packages, Cytoscape software, and online tools (DAVID and STRING) were used to identify core genes and topologically relevant nodes and molecular pathways. Single cell RNA sequencing analysis was applied to identify expression patterns of core genes across kidney cell types in glomerular compartments. A total of 1087 differentially expressed genes (DEGs) were identified, including 691 up-regulated genes and 396 down-regulated genes, common in all three forms of nephrotic syndrome compared to kidney donor controls (FDR p<0.01). A multi-approach bioinformatics analysis narrowed down 28 genes similarly dysregulated across the three proteinuric glomerulopathies. The most topologically relevant nodes belonged to the adherens junction, focal adhesion, and cytoskeleton pathways, where zyxin covers all of those Gene Ontology terms. We report that dysregulation of cell adhesion complexes was present in the three most common forms of glomerular disease. Zyxin could be a biomarker in all three common forms of nephrotic syndrome. If further functional studies confirm its role in their development, zyxin could be a potential therapeutic target.

Junctional integrity and directional mobility of lymphatic endothelial cell monolayers are disrupted by saturated fatty acids.

The lymphatic circulation regulates transfer of tissue fluid and immune cells toward the venous circulation. While obesity impairs lymphatic vessel function, the contribution of lymphatic endothelial cells (LEC) to metabolic disease phenotypes is poorly understood. LEC of lymphatic microvessels are in direct contact with the interstitial fluid, whose composition changes during the development of obesity, markedly by increases in saturated fatty acids. Palmitate, the most prevalent saturated fatty acid in lymph and blood, is detrimental to metabolism and function of diverse tissues, but its impact on LEC function is relatively unknown. Here, palmitate (but not its unsaturated counterpart palmitoleate) destabilized adherens junctions in human microvascular LEC in culture, visualized as changes in VE-cadherin, α-catenin, and β-catenin localization. Detachment of these proteins from cortical actin filaments was associated with abundant actomyosin stress fibers. The effects were Rho-associated protein kinase (ROCK)- and myosin-dependent, as inhibition with Y27632 or blebbistatin, respectively, prevented stress fiber accumulation and preserved junctions. Without functional junctions, palmitate-treated LEC failed to directionally migrate to close wounds in two dimensions and failed to form endothelial tubes in three dimensions. A reorganization of the lymphatic endothelial actin cytoskeleton may contribute to lymphatic dysfunction in obesity and could be considered as a therapeutic target.

The immunolocalization of cadherins and beta-catenin in the cervix and vagina of cycling cows.

The adherens junctions (AJs) maintain the epithelial cell layers' structural integrity and barrier function. AJs also play a vital role in various biological and pathological processes. AJs perform these functions through the cadherin-catenin adhesion complex. This study investigated the presence, cell-specific localization, and temporal distribution of AJ components such as classical type I cadherins and beta-catenin in the cow cervix and vagina during the estrous cycle. Immunohistochemistry and Western blot analysis results demonstrated that beta-catenin and epithelial (E)-, neural (N)-, and placental (P)-cadherins are expressed in the cow cervix and vagina during the estrous cycle. These adhesion molecules were localized in the membrane and cytoplasm of the ciliated and non-ciliated cervical cells and the stratified vaginal epithelial cells. Positive immunostaining for P-, N-cadherin, and beta-catenin was also observed in the vascular endothelial cells of the cervical and vaginal stroma. Quantitative immunohistochemistry examinations revealed that in the cervical and vaginal epithelia, P-cadherin's optical density values (ODv) were the highest; in contrast, the N-cadherin ODv were the lowest. The ODv of P-cadherin and beta-catenin in the cervical epithelium and E-cadherin in the vagina were significantly higher in the luteal phase versus the follicular phase of the estrous cycle. Furthermore, the ODv of P-cadherin, N-cadherin, and beta-catenin in the cervix's central and peripheral epithelial regions were different during the estrous cycle. These findings indicate that classical cadherins and beta-catenin in the cervix and vagina exhibit cell- and tissue-specific expression patterns under the influence of estrogen and progesterone hormones during the estrous cycle.

Microcystin-LR accelerates follicular atresia in mice via JNK-mediated adherent junction damage of ovarian granulosa cells

Microcystin-LR (MC-LR), one of aquatic environmental contaminants with reproductive toxicity produced by cyanobacterial blooms, but its toxic effects and mechanisms on the ovary are not fully understood. Here, proteomic techniques and molecular biology experiments were performed to study the potential mechanism of MC-LR-caused ovarian toxicity. Results showed that protein expression profile of ovarian granulosa cells (KK-1) was changed by 17 μg/mL MC-LR exposure. Comparing with the control group, 118 upregulated proteins as well as 97 downregulated proteins were identified in MC-LR group. Function of differentially expressed proteins was found to be enriched in pathways related to adherent junction, such as cadherin binding, cell-cell junction, cell adhesion and focal adherens. Furthermore, in vitro experiments, MC-LR significantly downregulated the expression levels of proteins associated with adherent junction (β-catenin, N-cadherin, and α-catenin) as well as caused cytoskeletal disruption in KK-1 cells (P < 0.05), indicating that the adherent junction was damaged. Results of in vivo experiments have shown that after 14 days of acute MC-LR exposure (40 μg/kg), damaged adherent junction and an increased number of atretic follicles were observed in mouse ovaries. Moreover, MC-LR activated JNK, an upstream regulator of adherent junction proteins, in KK-1 cells and mouse ovarian tissues. In contrast, JNK inhibition alleviated MC-LR-induced adherent junction damage in vivo and in vitro, as well as the number of atretic follicles. Taken together, findings from the present study indicated that JNK is involved in MC-LR-induced granulosa cell adherent junction damage, which accelerated follicular atresia. Our study clarified a novel mechanism of MC-LR-caused ovarian toxicity, providing a theoretical foundation for protecting female reproductive health from environmental pollutants.

Resolving cadherin-mediated inter-cell contractile tension in multicellular migration.

Cell migration plays an important role in physiological processes of multicellular organisms and cancer metastasis. The change from coherent collective cell motion to individual incoherent motion is a key aspect of metastasis. This motivates this study of how stresses are generated, transmitted and coordinated during collective migration of cancer cells. We focus on the role of cadherins located at adherens junctions, transmembrane proteins that mediate cell-cell adhesion and harness the contractile force of the cell via the acto-myosin cytoskeleton. Cadherins are mechanosensitive and mechanoconductive, and are believed to play an important role in cell coordination during migration by enabling cells to collectively polarize and migrate along the axis of minimal intercellular shear stress. How such collective processes are coordinated over large multi-cellular assemblies has remained unclear, however, as mechanical stresses exerted at adherens junctions have not been accessible experimentally. To determine the impact of cadherins on carcinoma cell contractility, we used Traction Force Microscopy (TFM) and Bayesian Inversion Stress Microscopy (BISM) within fluorescently tagged cadherins clusters. GNE495 was used to modulate these cadherin-based adhesions. This showed an increase in the magnitudes and durations of traction forces, strain energy and shear stresses. A tracking algorithm was used to study the role of cadherins and contractility on cell migration, as we would expect higher contractility to correlate with higher displacements. Instead, cell movement tracked by their Mean Squared Displacement (MSD) decreased after cadherin disruption. This increase in contractile work but decrease in migration suggests that cadherin adhesion dynamics may be crucial in coordinating forces during migration, and loss of this coordination decreases the efficiency of collective migration. This provides valuable insight on tying the biophysics of cadherins to the biology of metastasis and a broader understanding of how intercellular stresses govern multicellular migration.

Indirect correlative light and electron microscopy an integrated rule-based imaging approach with robust quantitative capabilities.

Although powerful, the utility of correlative light and electron microscopy (CLEM) is severely restricted by lack of experimental throughput and quantitative capability. We present indirect CLEM (iCLEM), a rule-based imaging (RUBI) pipeline for low-cost, high throughput assessment of multiscale structure. iCLEM correlates multiscale morphometric measurements independently obtained from each technique by exploiting structural landmarks identifiable via both light and electron microscopy, sidestepping CLEM's rate-limiting steps. Exploiting landmarks robust to perturbation enables comparison between health and disease. As proof of principle, we have used iCLEM to compile a cohesive and comprehensive quantitative description of cell-cell contact structures in cardiac muscle known as intercalated discs (ID). For this, we exploited ID components, such as gap junctions, adherens junctions and desmosomes, as structural landmarks. Confocal microscopy enabled assessments on organ through cellular scales, such as cell geometry, the sizes of IDs in different parts of the heart, and the distribution of ion transport proteins between IDs and other parts of heart muscle cells. Paired sets of transmission electron micrographs at different magnifications enable quantification of ultrastructure from micro through nanoscales ranging from the size and tortuosity of the whole ID to intermembrane spacing within its different nanodomains. Next, we assessed the spatial distribution of ion transport proteins within the ID, relative to structural landmarks and each other, using super-resolution imaging (STORM single molecule localization microscopy). Molecular organization was assessed by spatial analysis of fluorescence images and machine learning-based cluster analysis of single molecule localizations. These data enabled construction of 3D finite element models of IDs populated with ion transport proteins mimicking experimental observations. Integration of these models into physiological models is revealing previously unappreciated structure-function relationships underlying health and disease.

Mechanical regulation of vinculin conformations and interactions.

Vinculin, as an essential mechanosensitive protein, transmits intracellular tension initiated from the cell-cell and the cell-matrix interfaces, to modulate cellular and tissue functions. However, the tension-dependent conformations and interactions of vinculin are largely unknown. Vinculin provides the mechanical linkages between talin and actin at Focal adhesions, or alpha-catenin and actin at Adherens junctions. In drosophila, vinculin also functions at the muscle attachment sites with Cbl-associated proteins (CAPs) to regulate the cytoskeletal organization and membrane morphology. In this work, we combined single-molecule manipulation, AlphaFold2-based structural prediction, molecular dynamic (MD) simulation and theoretical modelling to understand the tension-dependent conformational changes of drosophila vinculin domains and its interaction with drosophila CAP isoform E (dCAP-E). We reveal that vinculin domains unfold in discrete steps, within 5-15 pN at a physiologically relevant tension loading rate. Based on the structural conformations predicted by AlphaFold2 and confirmed by MD simulation, a theoretical model is developed which predicts a tension-dependent binding affinity between the drosophila vinculin linker and dCAP-E within the physiologically relevant tension range. Together, the results suggest two roles of vinculin, as a tension buffering structure via tension-dependent unfolding and refolding of domains and as a mechanosensor via tension-dependent binding with its binding partners.

A systematic investigation of the cardiac intercalated disk in health and disease.

During each heartbeat, action potential (AP) propagation at intercalated discs (ID), specialized structures that electrically (gap junctions [GJ]) and mechanically (adherens junctions [AJ], desmosomes [Des]) couple cardiomyocytes, coordinates the contraction of billions of cardiomyocytes. Electrogenic proteins underlying excitability (cardiac sodium channels [NaV1.5], inward-rectifier potassium channels [Kir2.1] and sodium-potassium ATPase [NKA]) enriched within ID nanodomains are emerging as vital machinery for AP propagation. ID disruption is a hallmark of arrhythmic disease, yet, IDs are omitted from physiological models due to lack of structural data. We used indirect correlative light and electron microscopy [iCLEM] to quantify ID ultrastructure and molecular organization in health and disease and link them with functional properties (conduction velocity, arrhythmia vulnerability). Additional studies in hearts treated with adhesion inhibitor peptides targeting different ID nanodomains helped bridge health with complex disease. In healthy mouse hearts, transmission electron microscopy uncovered smaller, more frequent junctions and closer intermembrane distance at peri-junctional nanodomains in atria vs. ventricles. Confocal microscopy revealed greater NaV1.5, Kir2.1, NKA expression with more preferential ID enrichment in atrial myocytes than ventricular. STORM single molecule localization microscopy, a super-resolution technique, identified chamber-specific differences in NaV1.5, Kir2.1 and NKA clustering and distribution relative to AJ, Des, GJ as well as each other. Adhesion inhibitor peptides Nadp1 (target: AJ), dadp1 (target: Des) and βadp1 (target: GJ-adjacent perinexi), but not corresponding inactive control peptides, acutely (60 minutes) disrupted their respective target nanodomains and dispersed NaV1.5 clusters from those sites. These structural impacts led to conduction slowing and reentrant arrhythmias with severity proportional to the amount of NaV1.5 present at the target site. These data parallel our previous observations in atrial fibrillation (mouse models and human patients) and highlight NaV1.5-rich ID nanodomains as potential targets for antiarrhythmic therapy.

Study of cell-cell adhesion formation and dynamics in cancer cells using a hybrid cell-lipid bilayer system.

The formation and regulation of connections between cells play a crucial role in maintaining tissue homeostasis and tumour repression. In many tissues, particular importance comes to adherens junctions that are formed by the binding of E-cadherin proteins at the surface of adjacent cells, followed by their clustering and recruitment of catenins and other adaptor proteins on the intracellular site linking the protein complex to the actomyosin cytoskeleton. This process depends on multiple parameters such as the mobility, density, type of cadherins, and the mechanical forces acting on the cadherin-catenin complexes and can change dramatically during developmental stages or in diseases such as cancer. To study the initial steps of adherens junction formation in a controlled environment in cancer cells, we have established a reconstituted system of cells adhering to supported lipid bilayers decorated with extracellular domains of E-cadherin proteins. This reconstituted system allowed us to capture the initiation and dynamics of cadherin interactions, the real-time localisation and clustering of adaptor proteins and the maturation of adherens junction in cancer cells over time using live cell imaging with confocal and total internal reflection microscopy. To understand the importance of the different parameters and to mimic the cell/tissue abnormalities occurring in cancer scenarios, we then perturb the controlled system by varying cadherin density, membrane mobility and inducing mechanical stress by confining the cell system over time. We find, for example, that reduced E-cadherin mobility leads to the formation of stable regions showing actin enrichment and that confinement increases the steady state adhesion zone. This approach provides new insights about cell surface dynamics during cell-cell adhesion and can improve our understanding of the role of cell mechanics and E-cadherin organisation patterns in cancer progression.

Systems biology approach to understand the interplay between Bacillus anthracis and human host genes that leads to CVDs

Humans infected with invasive Bacillus anthracis (B. anthracis) have a very poor prognosis and are at high risk for developing cardiovascular diseases (CVDs) and shock. Several bacterial elements probably have significant pathogenic roles in this pathogenic process of anthrax. In our current work, we have analysed the molecular level interactions between B. anthracis and human genes to understand the interplay during anthrax that leads to the CVDs. Our results have shown dense interactions between the functional partners in both host and the B. anthracis Gene interaction network (GIN). The functional enrichment analysis indicated that the clusters in the host GIN had genes related to hypoxia and autophagy in response to the lethal toxin; and genes related to adherens junction and actin cytoskeleton in response to edema toxin play a significant role in multiple stages of the disease. The B. anthracis genes BA_0530, guaA, polA, rpoB, ribD, secDF, metS, dinG and human genes ACTB, EGFR, EP300, CTNNB1, ESR1 have shown more than 50 direct interactions with the functional partners and hence they can be considered as hub genes in the network and they are observed to have important roles in CVDs. The outcome of our study will help to understand the molecular pathogenesis of CVDs in anthrax. The hub genes reported in the study can be considered potential drug targets and they can be exploited for new drug discovery.

Anti-infectious pneumonia target discovery and molecular mechanism study of Jingfang Granules

This study aimed to identify the direct pharmacological targets of Jingfang Granules in treating infectious pneumonia via &quot;target fishing&quot; strategy. Moreover, the molecular mechanism of Jingfang Granules in treating infectious pneumonia was also investigated based on target-related pharmacological signaling pathways. First, the Jingfang Granules extract-bound magnetic nanoparticles were prepared, which were incubated with lipopolysaccharide(LPS)-induced mouse pneumonia tissue lysates. The captured proteins were analyzed by high-resolution mass spectrometry(HRMS), and the target groups with specific binding to the Jingfang Granules extract were screened out. Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis was used to identify the target protein-associated signaling pathways. On this basis, the LPS-induced mouse model of infectious pneumonia was established. The possible biological functions of target proteins were verified by hematoxylin-eosin(HE) staining and immunohistochemical assay. A total of 186 Jingfang Granules-specific binding proteins were identified from lung tissues. KEGG pathway enrichment analysis showed that the target protein-associated signaling pathways mainly included Salmonella infection, vascular and pulmonary epithelial adherens junction, ribosomal viral replication, viral endocytosis, and fatty acid degradation. The target functions of Jingfang Granules were related to pulmonary inflammation and immunity, pulmonary energy metabolism, pulmonary microcirculation, and viral infection. Based on the in vivo inflammation model, Jingfang Granules significantly improved the alveolar structure of the LPS-induced mouse model of infectious pneumonia and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6). Meanwhile, Jingfang Gra-nules significantly up-regulated the expressions of key proteins of mitochondrial function COX Ⅳ and ATP, microcirculation-related proteins CD31 and Occludin, and proteins associated with viral infection DDX21 and DDX3. These results suggest that Jingfang Gra-nules can inhibit lung inflammation, improve lung energy metabolism and pulmonary microcirculation, resist virus infection, thus playing a protective role in the lung. This study systematically explains the molecular mechanism of Jingfang Granules in the treatment of respiratory inflammation from the perspective of target-signaling pathway-pharmacological efficacy, thereby providing key information for clinical rational use of Jingfang Granules and expanding potential pharmacological application.

Illuminating dynamic structural states in hiPSC through endogenous gene tagging.

The Allen Institute for Cell Science aims to understand the principles by which human induced pluripotent stem cells (hiPSCs) establish and maintain robust dynamic localization of cellular structures, and how they transition between states during differentiation and disease. As a first step [JG1], we have created a collection of endogenously tagged hiPSC lines to illuminate cell organization. The tagged proteins include notable membrane-bound and membrane-less cellular organelles, signaling complexes, phase transition markers, transcription factors, and structural markers specific to differentiated cells. To date, the Allen Cell Collection consists of 54 single- or dual-edited lines that have undergone extensive quality control testing to ensure genomic, cell biological, and stem cell integrity. Here, we highlight our gene-editing and quality control workflow for mono- and biallelic editing of expressed or silent genes that are expressed specifically during differentiation and function as reporters of cellular state. Furthermore, we underscore our efforts to increase efficiency of the current workflow by utilizing Adeno-Associated Virus (AAV) to deliver donor DNAs and multiplexing transfection strategies for gene tagging at multiple loci. Our most recently released lines are CTCF-mEGFP and PCNA-mEGFP, for dynamic visualization of chromatin domains and replication foci, G3BP1-mEGFP to label stress granules, mono- and bi-allelic mEGFP-E-cadherin to visualize adherens junctions and DCP1a-mEGFP for P-bodies. We now have gene [RG2] [JG3]-edited cell populations for N- and VE-cadherin ready for single cell sorting. Our overarching goal is to make these tools openly available to the cell biology community to help accelerate biomedical research. In addition to our cell lines, the donor plasmids, segmented 3D images of about 200,000 cells from our lines, image analysis, visualization tools, integrated cell models and biological findings are all openly available to the research community (www.allencell.org).