Apical Junctions Research Articles

Integrated analysis of the M2 macrophage-related signature associated with prognosis in ovarian cancer.

BackgroundM2 macrophages play an important role in cancer development. However, the underlying biological fator affecting M2 macrophages infiltration in ovarian cancer (OV) has not been elucidated.MethodsR software v 4.0.0 was used for all the analysis. The expression profile and clinical information of OV patients enrolled in this study were all downloaded from The Cancer Genome Atlas and Gene Expression Omnibus databases.ResultsThe CIBERSORT algorithm was used to quantify the M2 macrophage infiltration in OV tissue, which was found a risk factor for patients survival. Based on the limma package, a total of 196 DEGs were identified between OV patients with high and low M2 macrophage infiltration, which were defined as M2 macrophages related genes. Finally, the genes PTGFR, LILRA2 and KCNA1 were identified for prognosis model construction, which showed a great prediction efficiency in both training and validation cohorts (Training cohort, 1-year AUC = 0.661, 3-year AUC = 0.682, 8-year AUC = 0.846; Validation cohort, 1-year AUC = 0.642, 3-year AUC = 0.716, 5-year AUC = 0.741). Clinical correlation showed that the riskscore was associated with the worse clinical features. Pathway enrichment analysis showed that in high risk patients, the pathway of epithelial-mesenchymal transition (EMT), TNF-α signaling via NFKB, IL2/STAT5 signaling, apical junction, inflammatory response, KRAS signaling, myogenesis were activated. Moreover, we found that the PTGFR, LILRA2 and KCNA1 were all positively correlated with M2 macrophage infiltration and PTGFR was significantly associated with the pathway of autophagy regulation. Moreover, we found that the low risk patients might be more sensitive to cisplatin, while high risk patient might be more sensitive to axitinib, bexarotene, bortezomib, nilotinib, pazopanib.ConclusionsIn this study, we identified the genes associated with M2 macrophage infiltration and developed a model that could effectively predict the prognosis of OV patients.

Characterization of human nasal organoids from chronic rhinosinusitis patients.

ABSTRACTPatient-derived organoids grown in three-dimensional cultures provide an excellent platform for phenotypic high-throughput screening and drug-response research. Organoid technology has been applied to study stem cell biology and various human pathologies. This study investigates the characteristics and cellular morphology of organoids derived from primary human nasal epithelial cells (HNECs) of chronic rhinosinusitis (CRS) patients. Nasal organoids were cultured up to 20 days and morphological, cell composition and functional parameters were measured by immunofluorescence, RT-qPCR, western blot and FACS analysis. The results showed that nasal organoids expressed the stem cell marker leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and markers for apical junction genes, goblet cells and ciliated cells. Moreover, we were able to regrow and expand the nasal organoids well after freezing and thawing. This study provides an effective and feasible method for development of human nasal organoids, suitable for the phenotypic high-throughput screening and drug response research.

Combined effects of cadmium and ochratoxin A on intestinal barrier dysfunction in human Caco-2 cells and pig small intestinal epithelial cells.

Hazardous chemicals are commonly found in cereals and cereal-based products. However, most studies focus on the individual effects of these mycotoxins or metals, rather than their combined toxicity. The main objective of this study was to evaluate the combined effects of cadmium (Cd) and ochratoxin A (OTA) on intestinal barrier integrity using Caco-2 cells and pig small intestinal epithelial (PSI) cells as models of intestinal epithelial cells and to measure alterations in cell survival and barrier integrity. The combined effects on cell viability were assessed in terms of a combination of index values. These findings showed that co-exposure to Cd + OTA had synergistic effects on Caco-2 and PSI cells at 25%, 50%, and 75% inhibitory concentrations (IC25, IC50, and IC75, respectively) against cell viability. Individual Cd and OTA treatments had no effect, but combined Cd + OTA exposure resulted in synergistic down-regulation of paracellular apical junction complex proteins, such as claudin-1, occludin, and E-cadherin. The current findings indicate that the combined effects of OTA + Cd may have consequences at the gut level, which should not be underestimated when considering their risk to human health.

Characterization of MET Exon 14 Skipping Alterations (in NSCLC) and Identification of Potential Therapeutic Targets Using Whole Transcriptome Sequencing

IntroductionGenomic alterations in the juxtamembrane exon 14 splice sites in NSCLC lead to increased MET stability and oncogenesis. We present the largest cohort study of MET Exon 14 (METex14) using whole transcriptome sequencing. MethodsA total of 21,582 NSCLC tumor samples underwent complete genomic profiling with next-generation sequencing of DNA (592 Gene Panel, NextSeq, whole exome sequencing, NovaSeq) and RNA (NovaSeq, whole transcriptome sequencing). Clinicopathologic information including programmed death-ligand 1 and tumor mutational burden were collected and RNA expression for mutation subtypes and MET amplification were quantified. Immunogenic signatures and potential pathways of invasion were characterized using single-sample gene set enrichment analysis and mRNA gene signatures. ResultsA total of 533tumors (2.47%) with METex14 were identified. The most common alterations were point mutations (49.5%) at donor splice sites. Most alterations translated to increased MET expression, with MET co-amplification resulting in synergistic increase in expression (q < 0.05). Common coalterations were amplifications of MDM2 (19.0% versus 1.8% wild-type [WT]), HMGA2 (13.2% versus 0.98% WT), and CDK4 (10.0% versus 1.5% WT) (q < 0.05). High programmed death-ligand 1 > 50% (52.5% versus 27.3% WT, q < 0.0001) and lower proportion of high tumor mutational burden (>10 mutations per megabase, 8.3% versus 36.7% WT, p < 0.0001) were associated with METex14, which were also enriched in both immunogenic signatures and immunosuppressive checkpoints. Pathways associated with METex14 included angiogenesis and apical junction pathways (q < 0.05). ConclusionsMETex14 splicing alterations and MET co-amplification translated to higher and synergistic MET expression at the transcriptomic level. High frequencies of MDM2 and CDK4 co-amplifications and association with multiple immunosuppressive checkpoints and angiogenic pathways provide insight into potential actionable targets for combination strategies in METex14 NSCLC.

Nanoarchitecture and molecular interactions of epithelial cell junction proteins revealed by super-resolution microscopy.

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin-matrix interactions. However, it is increasingly recognized that resident cell junction proteins can be found in varying locations and with previously unrecognized functions. Our study here presents the nanoarchitecture and nanocolocalization of cell junction proteins in culture and tissue by stochastic optical reconstruction microscopy (STORM). The Z-axial view of noncancerous MDCK-II and PZ-HPV-7 cell-cell junctions resolved β-catenin and p120ctn localizations to TJs and AJs, with p120ctn apical to β-catenin and colocalizing with TJ protein claudin-7. More basally, p120ctn and β-catenin become colocalized. This topography was lost in isogenic Ras-transformed MDCK cells and cancerous PC3 cells, where p120ctn becomes basally localized in relation to β-catenin. Claudin-7 gene conditional knockout (cKO) in mice also have altered polarity of p120ctn relative to β-catenin, like that seen in normal-to-cancer cell phenotypic transformation. Additionally, claudin-7 cKO resulted in redistribution and relocalization of other cell junction proteins, including claudin-1, zonula occludens-1, integrin α2, epithelial cell adhesion molecule, and focal adhesion kinase (FAK); specifically, integrin α2 and FAK were observed at the apical-lateral compartment. Our data show that STORM reveals regional cellular junction nanoarchitecture previously uncharacterized, providing new insight into potential trans-compartmental modulation of protein functions.

O-239 CD147 expression in human embryos and its role during implantation

Abstract Study question Do human pre-implantation embryos express CD147, does it play a role during implantation, and can it be used as non-invasive marker for embryo selection? Summary answer CD147 is expressed throughout pre-implantation development. Blocking results in implantation failure in vitro, but CD147 secretion has no immediate link with implantation in vivo. What is known already Embryo selection remains a subjective process with room for improvement. Researchers focus on alternative techniques to reduce time to pregnancy. While preimplantation genetic testing and time-lapse studies increased our understanding, they failed to efficiently improve embryo selection. CD147, a cluster domain well studied in cancer, has been proven to positively influence implantation in mice. When the protein is blocked, the embryo develops to blastocyst but fails to implant. This study aims to visualize the expression of CD147 throughout human pre-implantation development and to understand its role during implantation in humans, using both an in vitro and in vivo model. Study design, size, duration CD147 expression was studied in oocytes (n = 13) and embryos (n = 30) between 3 and 7 days post-fertilisation (dpf) with immunohistochemistry. To investigate its function, 5dpf blastocysts (n = 45) were co-cultured with Ishikawa cells for two days in an in vitro attachment model with two distinct anti-CD147 antibodies (ab666 and ab119114). Attachment models containing embryos (n = 8) were stained using ActinGreen™ probes. Additionally, spent culture medium was collected in the IVF lab after fresh blastocyst transfer (n = 153, sequential medium). Participants/materials, setting, methods Fresh immature oocytes and vitrified/warmed embryos were obtained after patients’ informed consent, fixed in 4% paraformaldehyde and stained using ab666. The counterstained samples were visualized with confocal microscopy. For blocking experiments, embryos were incubated in ab666 or ab119114 (Abcam) and co-cultured in the attachment model. Attachment was measured after 24hrs by gentle pipetting of the medium. Attached embryos were stained for F-actin. CD147 concentrations in spent embryo culture medium were determined by ELISA (EH78RB, Thermo-Fisher-Scientific). Main results and the role of chance In immature oocytes, CD147 was present on the oolemma only. After fertilization, CD147 protein was restricted to the membrane of all blastomeres but fluorescence intensity decreased gradually during cleavage stages. After embryonic genome activation the expression increased again mainly on the membranes of the cells. After blastulation (5 dpf), the protein was expressed in all cells but mainly concentrated on the membranes. With the expansion of the blastocysts (6-7 dpf) CD147 became more abundant on the membranes of trophectoderm cells (TE), the signal being the strongest on the apical membranes and TE junctions. Blocking of CD147 binding in blastocysts was achieved with two anti-CD147 antibodies ab666 and ab119114 with respectively 52% and 70 % reduction of attachment on Ishikawa cells compared to non-treated control groups (n = 45, p &amp;lt; 0.05). Finally, secreted CD147 was found in culture medium of embryos 5 dpf. Most of the blastocysts (150/153) secreted CD147 with concentrations ranging from 40–229 pg/ml. No correlation with pregnancy outcome or morphology was noted, indicating that CD147 cannot be used as non-invasive marker for embryo selection. Although these results clearly highlight the importance of CD147 in human embryo implantation in vitro, more research is needed to investigate its role. Limitations, reasons for caution The experiments have been performed using an in vitro model for attachment, the impact of additional interfering factors in vivo cannot be ruled out. Wider implications of the findings Protein expression highlights the importance of CD147 in human blastocysts, especially for the attachment function of the apical TE membrane. Blocking CD147protein reduces implantation dramatically, proving its involvement in establishing a successful pregnancy. In addition, secretion of CD147 demonstrates its potential paracrine role during the interaction with the endometrium. Trial registration number not applicable

Abstract 3834: Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma

Abstract Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) have a dismal survival rate. The effects of the extracellular matrix (ECM) on cancer progression have been long studied, but the roles of specific integrins in the process of HNSCC metastasis remain to be dissected. This study aims to determine how HNSCC cells affect the production of laminin-binding integrins, and how these integrins participate in the ECM interactions necessary for a metastatic phenotype. Experimental design: Our laboratory has produced syngeneic mouse SCC cell lines, P029 and A223, derived from Keratin15+ stem cells with Smad4 loss and KrasG12D mutation. In syngeneic recipients, SCCs derived from P029 cells transplanted to the flank mouse skin produced spontaneous metastases to the lung while SCCs derived from A223 cells did not form metastases. Having the same genotype, these cell lines serve as models to examine cancer cell interactions with the ECM and resulting effects on invasion and metastasis. Bulk RNA sequencing (RNAseq) was performed to compare cultured metastatic P029 cells versus non-metastatic A223 cells and identify differentially expressed genes that regulate SCC cells and ECM interactions. Immunoassays and functional invasion assays were performed to evaluate ECM-cancer cell signaling and influence on cancer cell invasion in these two cell lines. Results: RNAseq analysis revealed that, relative to A223 cells, P029 cells have increased levels of integrins, the main mechanoreceptors for numerous ECM ligands and matrix proteins. Laminin-binding integrins, including integrins α4 and β6, were greater at the RNA and protein levels. Additionally, P029 cells expressed higher levels of fibronectin and laminin coding genes compared to non-metastatic A223 cells. Correlatively, high expression of these matrix proteins is associated with worse patient survival. Additionally, P029 SCC cells displayed differential gene expression of tight and apical junctions, PI3K signaling components, and regulators of cytoskeletal remodeling. Furthermore, ELISA and western blot analysis revealed that P029 has aberrant TGFβ-Smad signaling as indicated by the elevated release of TGFβ-1 protein and higher levels of phosphorylated Smad2 and Smad3 relative to non-metastatic A223. Treating P029 cells with TGFβ-1 significantly increased their motility and invasion. Conversely, migration and motility of P029 cells were radically reduced by the TGFβ inhibitor galunisertib. Additionally, the mouse cytokine array revealed that mouse plasma bearing P029 tumors had a greater circulation of CXCL16, MMP-9, proliferin, and serpin E1. These proteins are associated with ECM remodeling and metastasis in HNSCC. Conclusions: SCC cells with metastatic properties upregulate integrins which bind to non-collagen matrix proteins and have elevated invasive and migratory capacity contributed from activated TGFβ signaling. Citation Format: John Aleman, Khoa A. Nguyen, Yao Ke, Christian D. Young, Xiao-Jing Wang. Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3834.

Abstract 5969: Intra-tumoral coagulation is significantly associated with EMT, angiogenesis, and worse patient survival in gastric cancer

Abstract Background: Bleeding is one of the most commonly observed clinical signs of gastrointestinal cancer. Coagulation regulates inflammation and angiogenesis in cancer, and is associated with tumor development and metastasis. To date, there has been no measures to quantify the state of intra-tumoral coagulation. We hypothesized that intra-tumoral coagulation as quantified using coagulation score is associated with angiogenesis and worse prognosis in gastric cancer. Methods: The coagulation score was determined by the Gene set variation analysis (GSVA) algorithm as GSVA score of the “HALLMARK_COAGULATION” in the MSigDB hallmark gene sets collection. Transcriptome data of 807 gastric cancer patients of TCGA_STAD and GSE84437 cohorts were used for the analysis. Results: We found that high coagulation score was significantly associated with high AJCC T-category (p = 0.018), lymph node metastasis status (p = 0.036), AJCC stage (p = 0.006) in the TCGA cohort. The association of the score with staging were validated by GSE84437 cohort. Further, patients with high score were associated with worse disease-specific survival and overall survival (OS) (p = 0.019 and 0.011, respectively) in the TCGA cohort, and OS in GSE84437 cohort (p = 0.012). Although the coagulation score was not associated with cell proliferation-related nor immune-related signaling, gastric cancer with high coagulation score highly enriched epithelial mesenchymal transition (EMT), myogenesis, apical junction, transforming growth factor (TGF)-β signaling, and angiogenesis consistently in both cohorts (all false discovery rate&amp;lt;0.25). The coagulation score strongly correlated with angiogenesis score, and high coagulation was significantly associated with high infiltration of angiogenesis-related vascular cells, including endothelial cells, microvascular endothelial cells, lymphatic endothelial cells, and pericytes, calculated by xCell algorithm, as well as high expression of angiogenesis-related genes, including Endothelial cell marker, and vascular stability related genes. Conclusion: Intra-tumoral coagulation assessed by coagulation score is associated with clinical outcomes of gastric cancer patients. Citation Format: Masanori Oshi, Joy Sarkar, Rongrong Wu, Li Yan, Ryusei Matsuyama, Itaru Endo, Kazuaki Takabe. Intra-tumoral coagulation is significantly associated with EMT, angiogenesis, and worse patient survival in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5969.

Characterization of MET exon 14 skipping alterations (METex14) in non–small cell lung cancer (NSCLC) using whole transcriptome sequencing (WTS).

9122 Background: Multiple DNA alterations in exon 14 splice sites have been identified in NSCLC and result in skipping of the juxtamembrane domain Cbl-E3 ubiquitin ligase binding region, leading to increased MET stability and oncogenesis. The effects of these alterations on transcriptome-level have not been fully characterized. We present the largest cohort study of METex14 using WTS and identify key cellular pathways associated with invasion and metastases in METex14. Methods: 17,666 NSCLC tumor samples underwent genomic profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq, whole exome sequencing, NovaSeq) and RNA (NovaSeq, WTS). METex14 was captured via WTS. ssGSEA analysis was used to evaluate pathway enrichment. Wilcoxon, Fisher’s exact were used for statistical significance (p without and q values with multiple comparison correction). Results: 440 patients (2.5%) with METex14 were identified. METex14 patients were of older age, female gender, and enriched in sarcomatoid histology (Table 1). The most common alterations were point mutations (51.5%) and deletions (17.3%) at donor splice sites. Splice site alterations except point mutations at splice acceptor site translated to increased mRNA expression compared to wild-type MET (WT). MET amplification translated to higher mRNA expression compared to METex14 and WT with synergistic expression when co-altered with METex14 (q&lt;0.05). The most common co-alterations were amplifications of MDM2 (18.5% vs. 1.8% WT), HMGA2 (13.7% vs 0.9% WT), and CDK4 (10.4% vs 1.4% WT) (q &lt; 0.05). METex14 were mutually exclusive to mutations in KRAS and EGFR. High PD-L1 (22c3) &gt; 50% (53% vs. 27.6% WT, q&lt;0.001) and lower TMB (4 mut/Mb vs. 7 mut/Mb WT, p&lt;0.001) were observed with METex14 and pathways associated with skipping variants included IFNγ signaling, angiogenesis, and apical junction pathways on univariate analysis (q&lt;0.05). Conclusions: We present the largest WTS analysis of METex14. Splicing alterations and MET co-amplification translated to higher and synergistic MET expression at transcriptome level, respectively. Association with upregulated angiogenic and apical junction pathways support preclinical observation of vascular and cytoskeletal remodeling as potential mechanisms of invasion and metastases in MET ex14 NSCLC.[Table: see text]

Cigarette Smoke Extract Disturbs Mitochondria-Regulated Airway Epithelial Cell Responses to Pneumococci.

Mitochondrial functionality is crucial for the execution of physiologic functions of metabolically active cells in the respiratory tract including airway epithelial cells (AECs). Cigarette smoke is known to impair mitochondrial function in AECs. However, the potential contribution of mitochondrial dysfunction in AECs to airway infection and airway epithelial barrier dysfunction is unknown. In this study, we used an in vitro model based on AECs exposed to cigarette smoke extract (CSE) followed by an infection with Streptococcus pneumoniae (Sp). The levels of oxidative stress as an indicator of mitochondrial stress were quantified upon CSE and Sp treatment. In addition, expression of proteins associated with mitophagy, mitochondrial content, and biogenesis as well as mitochondrial fission and fusion was quantified. Transcriptional AEC profiling was performed to identify the potential changes in innate immune pathways and correlate them with indices of mitochondrial function. We observed that CSE exposure substantially altered mitochondrial function in AECs by suppressing mitochondrial complex protein levels, reducing mitochondrial membrane potential and increasing mitochondrial stress and mitophagy. Moreover, CSE-induced mitochondrial dysfunction correlated with reduced enrichment of genes involved in apical junctions and innate immune responses to Sp, particularly type I interferon responses. Together, our results demonstrated that CSE-induced mitochondrial dysfunction may contribute to impaired innate immune responses to Sp.

Crumbs complex-directed apical membrane dynamics in epithelial cell ingression.

Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cells lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. Here, we show that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. Our findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer.

Lmo7 recruits myosin II heavy chain to regulate actomyosin contractility and apical domain size in Xenopus ectoderm.

Apical constriction, or a reduction in size of the apical domain, underlies many morphogenetic events during development. Actomyosin complexes play an essential role in apical constriction; however, the detailed analysis of molecular mechanisms is still pending. Here, we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is primarily localized at apical junctions and promotes the formation of the dense circumferential actomyosin belt. Strikingly, Lmo7 binds non-muscle myosin II (NMII) and recruits it to apical junctions and the apical cortex. This NMII recruitment is essential for Lmo7-mediated apical constriction. Lmo7 knockdown decreases NMIIA localization at apical junctions and delays neural tube closure in Xenopus embryos. Our findings suggest that Lmo7 serves as a scaffold that regulates actomyosin contractility and apical domain size.

Clinical Correlation of Bladder Electron Microscopic Characteristics in Patients with Detrusor Underactivity of Various Etiologies.

This study aimed to investigate the ultrastructural characteristics of the bladder of patients with detrusor underactivity (DU) of various etiologies. Twenty-five patients with DU and control subjects underwent urodynamic testing and transmission electron microscopic examination of bladder specimens. The epithelium, lamina propria, and muscle layers were analyzed separately. The DU bladders exhibited total epithelial denudation (52%). In the bladders with remaining epithelium, apical cell uroplakins (44.4%) and tight junction complexes (77.8%) were also noted. The lamina propria was characterized by loose extracellular connective tissue (48%) and a lack of nerve terminals (76%). Smooth muscle shrinkage and a loss of their regular spindle shape (91.6%) were also noted in the detrusor layer. Patients with DU with intact epithelial cell layers had significantly larger void volumes and maximal flow rates than those with mild or severe epithelial denudation. Patients with remaining nerve terminals in lamina propria had a stronger first sensation of filling and smaller residual urine volume than those without nerve terminals. The proportion of ultrastructural defects of the bladder was not significantly different among patients with DU of various etiologies and treatment outcomes. DU bladders were characterized by ultrastructural defects in the entire bladder, and the defects were correlated to clinical parameters.

Actomyosin fibers DApPLE epithelial apical junctions.

Epithelial cell morphology is essential for cellular homeostasis, but the mechanisms by which cell shape is established remain unclear. In this study, Marivin et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202111002) identify DAPLE as a linker between polarity complexes and the actomyosin network at apical junctions. By recruiting CD2P and activating Gαβγ-mediated RhoA signaling, DAPLE ensures proper cell shape and function.

Synaptopodin stress fiber and contractomere at the epithelial junction.

The apical junction of epithelial cells can generate force to control cell geometry and perform contractile processes while maintaining barrier function and adhesion. Yet, the structural basis for force generation at the apical junction is not fully understood. Here, we describe two synaptopodin-dependent actomyosin structures that are spatially, temporally, and structurally distinct. The first structure is formed by the retrograde flow of synaptopodin initiated at the apical junction, creating a sarcomeric stress fiber that lies parallel to the apical junction. Contraction of the apical stress fiber is associated with either clustering of membrane components or shortening of junctional length. Upon junction maturation, apical stress fibers are disassembled. In mature epithelial monolayer, a motorized "contractomere" capable of "walking the junction" is formed at the junctional vertex. Actomyosin activities at the contractomere produce a compressive force evident by actin filament buckling and measurement with a new α-actinin-4 force sensor. The motility of contractomeres can adjust junctional length and change cell packing geometry during cell extrusion and intercellular movement. We propose a model of epithelial homeostasis that utilizes contractomere motility to support junction rearrangement while preserving the permeability barrier.

DAPLE orchestrates apical actomyosin assembly from junctional polarity complexes.

Establishment of apicobasal polarity and the organization of the cytoskeleton must operate coordinately to ensure proper epithelial cell shape and function. However, the precise molecular mechanisms by which polarity complexes directly instruct the cytoskeletal machinery to determine cell shape are poorly understood. Here, we define a mechanism by which the PAR polarity complex (PAR3-PAR6-aPKC) at apical cell junctions leads to efficient assembly of the apical actomyosin network to maintain epithelial cell morphology. We found that the PAR polarity complex recruits the protein DAPLE to apical cell junctions, which in turn triggers a two-pronged mechanism that converges upon assembly of apical actomyosin. More specifically, DAPLE directly recruits the actin-stabilizing protein CD2AP to apical junctions and, concomitantly, activates heterotrimeric G protein signaling in a GPCR-independent manner to favor RhoA-myosin activation. These observations establish DAPLE as a direct molecular link between junctional polarity complexes and the formation of apical cytoskeletal assemblies that support epithelial cell shape.

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair.

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell‐matrix adhesions. Although biochemical mechanisms that regulate the activity of non‐muscle myosin II (NM‐II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC‐45A is a cytoskeletal chaperone that is essential for proper folding of NM‐II heavy chains and myofilament assembly. We found abundant expression of UNC‐45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC‐45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9‐mediated knock‐out of UNC‐45A in HT‐29cf8 and SK‐CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC‐45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti‐migratory effects of UNC‐45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC‐45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC‐45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

LMBR1L regulates the proliferation and migration of endothelial cells through Norrin/β-catenin signaling.

Precise Norrin and β-catenin (Norrin/β-catenin; encoded by NDP and CTNNB1, respectively) signaling is critical for proper angiogenesis. Dysregulation of this signaling leads to various diseases, of which retinal exudative vitreoretinopathy is the most prevalent. Here, we used a global knockout mouse model to show that limb development membrane protein 1 like (LMBR1L), a transmembrane protein of unknown function in angiogenesis, is essential for retinal vascular development. In vitro experiments revealed that LMBR1L depletion results in aberrant activation of the Norrin/β-catenin signaling pathway via decreased ubiquitylation of FZD4 and increased Norrin co-receptor LRP5 and p-GSK3β-Ser9 expression levels, which cause accumulation of β-catenin. Moreover, inhibition of LMBR1L in human retinal microvascular endothelial cells (HRECs) caused increased proliferation ability and defective cell migration, which might have occurred as a result of upregulated expression levels of the apical junction components. Treatment with p-GSK3β-Ser9 inhibitor AR-A014418 restored the phenotypes in LMBR1L-null HRECs, which further demonstrated the important regulatory role of LMBR1L in the Norrin/β-catenin signaling pathway. Taken together, our data reveal an essential role for LMBR1L in angiogenesis. This article has an associated First Person interview with the first author of the paper.

Microbial Metabolite Regulation of Epithelial Cell-Cell Interactions and Barrier Function.

Epithelial cells that line tissues such as the intestine serve as the primary barrier to the outside world. Epithelia provide selective permeability in the presence of a large constellation of microbes, termed the microbiota. Recent studies have revealed that the symbiotic relationship between the healthy host and the microbiota includes the regulation of cell–cell interactions at the level of epithelial tight junctions. The most recent findings have identified multiple microbial-derived metabolites that influence intracellular signaling pathways which elicit activities at the epithelial apical junction complex. Here, we review recent findings that place microbiota-derived metabolites as primary regulators of epithelial cell–cell interactions and ultimately mucosal permeability in health and disease.

The relation between age and airway epithelial barrier function

BackgroundThe prevalence of age-associated diseases, such as chronic obstructive pulmonary disease (COPD), is increasing as the average life expectancy increases around the world. We previously identified a gene signature for ageing in the human lung which included genes involved in apical and tight junction assembly, suggesting a role for airway epithelial barrier dysfunction with ageing.AimTo investigate the association between genes involved in epithelial barrier function and age both in silico and in vitro in the airway epithelium.MethodsWe curated a gene signature of 274 genes for epithelial barrier function and tested the association with age in two independent cohorts of bronchial brushings from healthy individuals with no respiratory disease, using linear regression analysis (FDR < 0.05). Protein–protein interactions were identified using STRING©. The barrier function of primary bronchial epithelial cells at air–liquid interface and CRISPR–Cas9-induced knock-down of target genes in human bronchial 16HBE14o-cells was assessed using Trans epithelial resistance (TER) measurement and Electric cell-surface impedance sensing (ECIS) respectively.ResultsIn bronchial brushings, we found 55 genes involved in barrier function to be significantly associated with age (FDR < 0.05). EPCAM was most significantly associated with increasing age and TRPV4 with decreasing age. Protein interaction analysis identified CDH1, that was negatively associated with higher age, as potential key regulator of age-related epithelial barrier function changes. In vitro, barrier function was lower in bronchial epithelial cells from subjects > 45 years of age and significantly reduced in CDH1-deficient 16HBE14o-cells.ConclusionThe significant association between genes involved in epithelial barrier function and age, supported by functional studies in vitro, suggest a role for epithelial barrier dysfunction in age-related airway disease.