Formation Of Adherens Junctions Research Articles

Mechanotransductive N-cadherin binding induces differentiation in human neural stem cells

The neural stem cell niche is a complex microenvironment that includes cellular factors, secreted factors, and physical factors that impact stem cell behavior and development. Cellular interactions through cadherins, cell–cell binding proteins, have implications in embryonic development and mesenchymal stem cell differentiation. However, little is known about the influence of cadherins within the neural stem cell microenvironment and their effect on human stem cell maintenance and differentiation. Therefore, the purpose of this study was to develop synthetic substrates to examine the effect of cadherin mechanotransduction on human neural stem cells. Glass substrates were fabricated using silane, protein A, and recombinant N-cadherin; we used these substrates to examine the effect of N-cadherin binding on neural stem cell proliferation, cytoskeletal structure and morphology, Yes-associated protein-1 (YAP) translocation, and differentiation. Bound exogenous N-cadherin induced concentration-dependent increases in adherens junction formation, YAP translocation, and early expression of neurogenic differentiation markers. Strong F-actin ring structures were initiated by homophilic N-cadherin binding, eliciting neuronal differentiation of cells within 96 ​h without added soluble differentiation factors. Our findings show that active N-cadherin binding plays an important role for differentiation of human iPS-derived neural stem cells towards neurons, providing a new tool to differentiate cells in vitro.

8399 Adherens Junctions Regulate Aldosterone Production and Zona Glomerulosa Morphogenesis

Abstract Disclosure: M. Berber: None. N.A. Guagliardo: None. P.Q. Barrett: None. B. Haykir: None. V. Chortis: None. K.S. Borges: None. D.L. Carlone: None. D.T. Breault: None. Primary aldosteronism (PA) caused by the autonomous production of aldosterone by zona Glomerulosa (zG) cells of the adrenal gland is the most common cause of endocrine hypertension, which increases the risk of cardiovascular and renal disease, independent of blood pressure. Current treatments with mineralocorticoid receptor antagonists do not fully mitigate these risks. Thus, a better understanding of the mechanisms underlying PA pathogenesis is important for the development of new therapeutic strategies. zG cells are organized in multicellular rosette structures, which are the functional aldosterone-producing units. Rosettes are stabilized by intercellular adherens junctions (AJs) comprised of cadherins, beta-catenin (βCat) and alpha-catenin (αCat), which link to the actin non-muscle myosin II (NMII) network. Recent data implicate AJs and rosettes in aldosterone-producing cell clusters and an unbiased proteomic analysis of aldosterone-producing adenomas detected an upregulation of the GTPase RhoC, an important regulator of AJ stability.We hypothesized that AJs are potent modulators of aldosterone production and zG morphogenesis with important implications for PA. Using the NCI-H295R cell line with confocal microscopic and co-immunoprecipitation assays, we found that potassium (K+) and angiotensin II (AngII) stimulation markedly enhanced the formation and maintenance of AJs. In addition, treatment with inhibitors of Rho-associated kinase (ROCK) (Y-27632) and NMII (blebbistatin) largely abrogated the effects of K+ and AngII stimulation on AJs, suggesting the Rho-ROCK-NMII pathway regulates AJ stability in the zG. Moreover, disruption of AJs, using either blebbistatin or deletion of αCat using CRISPR, blunted the aldosterone response to K+ stimulation (100% and 50%, respectively). Furthermore, zG-specific deletion of αCat (αCat-LOF) in mice led to disruption of AJs, impaired rosette formation and decreased 24-hour urinary aldosterone excretion on standard (Control 5.6±1.7 ng/mg; αCat-LOF 2.9±0.7 ng/mg (aldosterone/creatinine)) or high K+ (Control: 34.7±13.7 ng/mg; αCat-LOF 19.4±9.5 ng/mg (aldosterone/creatinine)) diets. Finally, mice with zG-specific βcat-GOF demonstrated enhanced AJ stability, increased rosette size and zG hyperplasia, which was abrogated in zG-specific bigenic βCat-GOF::αCat-LOF mice, without affecting gene expression of canonical βcat targets. These data indicate that AJs modulate aldosterone production and zG morphology, revealing AJs as a novel molecular target for the treatment of high-risk patients with PA. Presentation: 6/3/2024

The Scribble-SGEF-Dlg1 complex regulates E-cadherin and ZO-1 stability, turnover and transcription in epithelial cells.

SGEF (also known as ARHGEF26), a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of both E-cadherin and ZO-1 (also known as TJP1) protein levels. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble-SGEF-Dlg1 complex. Our results show that the three members of the ternary complex are required to maintain the stability of the apical junctions, ZO-1 protein levels and tight junction (TJ) permeability. In contrast, only SGEF is necessary to regulate E-cadherin levels. The absence of SGEF destabilizes the E-cadherin-catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, β-catenin signaling and the transcriptional repressor Slug (also known as SNAI2).

HIF2α-dependent Dock4/Rac1-signaling regulates formation of adherens junctions and cell polarity in normoxia.

Hypoxia-inducible factors (HIF) 1 and 2 regulate similar but distinct sets of target genes. Although HIFs are best known for their roles in mediating the hypoxia response accumulating evidence suggests that under certain conditions HIFs, particularly HIF2, may function also under normoxic conditions. Here we report that HIF2α functions under normoxic conditions in kidney epithelial cells to regulate formation of adherens junctions. HIF2α expression was required to induce Dock4/Rac1/Pak1-signaling mediating stability and compaction of E-cadherin at nascent adherens junctions. Impaired adherens junction formation in HIF2α- or Dock4-deficient cells led to aberrant cyst morphogenesis in 3D kidney epithelial cell cultures. Taken together, we show that HIF2α functions in normoxia to regulate epithelial morphogenesis.

Abstract 5874: Phenotypic plasticity of TKI resistance reprograms cytoskeleton organization through cadherin-catenin and integrin pathways

Abstract The acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) restricts its clinical utility. Clinically, acquired resistance has been highly associated with epithelial-mesenchymal transition (EMT) traits. In this study, we characterized shared phenotypic plasticity features of TKI-resistant cells and aimed to find its key driver in three different cell lines harboring mutant EGFR. In vitro resistance cell lines were generated by dose-escalation exposure to osimertinib, a 3rd generation TKI, for more than 2 months. We employed canonical markers (E-cadherin, vimentin, pan-cytokeratin, and N-cadherin) along with additional markers (EpCAM and fibronectin) to define the EMT status of resistant cells. Moreover, cytoskeleton architecture (F-actin and Vimentin) was utilized to clarify the types of cell motility. Transcriptomics analysis illustrated enriched pathways at focal adhesion and adherens junction. Loss of adherens junction-related catenin (α-E-catenin, β-catenin, γ-catenin, p120 catenin) and aberrant activation of focal adhesion-related protein (p-FAK Y397 and Y576/57, p-Src Y416) confirmed the transcriptomics analysis of reduced contact inhibition and cell polarity. The resulting migration mode of resistant cells highly resembles ameboid movement. Mechanistically, we identified that YTHDF3, a metastasis-promoting regulator of mRNA methylation, destabilizes the mRNA of E-cadherin. Depletion of YTHDF3 in resistant cells reversed the cadherin-catenin pathway and distribution of vimentin and f-actin. In conclusion, resistant cells display ameboid-like movement resulting from loss of contact inhibition and aberrant activation of focal adhesion. Mechanistically, we identified that YTHDF3, a metastasis-promoting regulator of mRNA methylation, destabilizes the mRNA of E-cadherin. Depletion of YTHDF3 in resistant cells reversed the cadherin-catenin pathway and distribution of vimentin and f-actin. YTHDF3 serves as an RNA epitranscriptomic regulator that destabilizes CDH1 mRNA, thereby disrupting adherens junction formation and reducing integrin-mediated focal adhesion stabilization. Citation Format: Chun Ma, Jerry Chieh-Yu Chen, Mong-Lien Wang. Phenotypic plasticity of TKI resistance reprograms cytoskeleton organization through cadherin-catenin and integrin pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5874.

A phase II open-label study of enfortumab vedotin in patients with previously treated locally advanced, recurrent, or metastatic pancreatic adenocarcinoma (EPIC).

TPS719 Background: Nectin-4 is a type I transmembrane protein member of the nectin family involved in the formation and maintenance of adherens junctions. In contrast to its limited expression in normal tissues, aberrant overexpression of nectin-4 has been identified in various solid tumors, including in pancreatic adenocarcinoma. About 71% of human PDAC specimens stain positive and 13% stain strongly positive for nectin-4. Mechanistically, nectin-4 has been associated with various pro-oncogenic cellular processes, including the promotion of tumor cell proliferation and metastasis via WNT- βcatenin pathway activation. It interacts with ERBB2/HER2 and activates Rac1 (small G protein) to ultimately upregulate PI3K-AKT pathway signaling, leading to cell survival and proliferation. Enfortumab vedotin (EV) is an antibody-drug conjugate that delivers the microtubule-disrupting agent monomethyl auristatin E (MMAE) to cells expressing Nectin-4. EV is FDA approved as a single agent for the treatment of adult patients with locally advanced or metastatic urothelial cancer who have previously received a programmed death receptor-1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitor and platinum-containing chemotherapy based on the EV-301 Trial which demonstrated overall survival benefit. Taken together, the above data and the efficacy of EV seen in bladder cancer warrants the evaluation of EV in advanced pancreatic adenocarcinoma. Methods: Design: Open-label, phase II single-arm trial of EV (n=28) in previously treated locally advanced, recurrent, or metastatic pancreatic adenocarcinoma. Treatment schedule: EV 1.25 mg/kg administered IV on D1, D8, D15 every 28 days until disease progression or unacceptable toxicity. Imaging assessment will be done every 8 weeks. A biopsy prior to therapy and a biopsy on treatment (between days 15-21 of cycle 1) is required. Eligibility: Patients with previously treated, locally advanced, recurrent, or metastatic pancreatic adenocarcinoma. Inclusion criteria also include ECOG PS 0-1 and at least 1 line of prior therapy. Objectives: The primary objective is to determine anti-tumor activity by overall response rate using RECIST 1.1. Secondary objectives include safety, duration of response, disease control rate, progression-free survival and overall survival. Exploratory objectives include Nectin-4 expression (H-score) in tumor tissue and the relationship between the mutational profile of the tumor and response. Statistical Plan: A Simon’s two-stage Minimax design will be used. In the first stage, 18 pts will be accrued. If there is less than 1 response in these 18 pts, the study will be stopped for futility. Otherwise, 10 additional pts will be accrued for a total of 28 pts. The null hypothesis will be rejected if 3 or more responses are observed in 28 pts. Enrollment is currently ongoing. Clinical trial information: NCT05915351 .

CAMSAP3, a microtubule orientation regulator, plays a vital role in manifesting differentiation-dependent characteristics in keratinocytes

Microtubules constitute pivotal structural elements integral to cellular architecture and physiological functionality. Within the epidermis of the skin, microtubules undergo a noteworthy transition in orientation, shifting from centrosomal to non-centrosomal configurations during the processes of differentiation and stratification. This transition aligns with a discernible increase in the expression of CAMSAP3, a protein that binds to the minus end of microtubules, thereby regulating their orientation. In this study, we identified microtubule-bound CAMSAP3 within HaCaT keratinocytes, revealing an upregulation during the mitotic phase and accumulation at the intercellular bridge during cytokinesis. Building upon this observation, we scrutinized cellular responses upon a tetracycline/doxycycline-inducible CAMSAP3 expression in CAMSAP3-deficient HaCaT cells. Remarkably, CAMSAP3 deficiency induced shifts in microtubule orientation, resulting in cell cycle exit and delayed cytokinesis in a subset of the cells. Furthermore, our inquiry unveiled that CAMSAP3 deficiency adversely impacted the formation and stability of Adherens Junctions and Tight Junctions. In contrast, these perturbations were rectified upon the re-expression of CAMSAP3, underscoring the pivotal role of CAMSAP3 in manifesting differentiation-dependent characteristics in stratified keratinocytes. These observations emphasize the significance of CAMSAP3 in maintaining epidermal homeostasis.

Genome-wide identification and spatiotemporal expression analysis of cadherin superfamily members in echinoderms

BackgroundCadherins are calcium-dependent transmembrane cell–cell adhesion proteins that are essential for metazoan development. They consist of three subfamilies: classical cadherins, which bind catenin, protocadherins, which contain 6–7 calcium-binding repeat domains, and atypical cadherins. Their functions include forming adherens junctions, establishing planar cell polarity (PCP), and regulating cell shape, proliferation, and migration. Because they are basal deuterostomes, echinoderms provide important insights into bilaterian evolution, but their only well-characterized cadherin is G-cadherin, a classical cadherin that is expressed by many embryonic epithelia. We aimed to better characterize echinoderm cadherins by conducting phylogenetic analyses and examining the spatiotemporal expression patterns of cadherin-encoding genes during Strongylocentrotus purpuratus development.ResultsOur phylogenetic analyses conducted on two echinoid, three asteroid, and one crinoid species identified ten echinoderm cadherins, including one deuterostome-specific ortholog, cadherin-23, and an echinoderm-specific atypical cadherin that possibly arose in an echinoid-asteroid ancestor. Catenin-binding domains in dachsous-2 orthologs were found to be a deuterostome-specific innovation that was selectively lost in mouse, while those in Fat4 orthologs appeared to be Ambulacraria-specific and were selectively lost in non-crinoid echinoderms. The identified suite of echinoderm cadherins lacks vertebrate-specific innovations but contains two proteins that are present in protostomes and absent from mouse. The spatiotemporal expression patterns of four embryonically expressed cadherins (fat atypical cadherins 1 and 4, dachsous-2, and protocadherin-9) were dynamic and mirrored the expression pattern of Frizzled 5/8, a non-canonical Wnt PCP pathway receptor protein essential for archenteron morphogenesis.ConclusionsThe echinoderm cadherin toolkit is more similar to that of an ancient bilaterian predating protostomes and deuterostomes than it is to the suite of cadherins found in extant vertebrates. However, it also appears that deuterostomes underwent several cadherin-related innovations. Based on their similar spatiotemporal expression patterns and orthologous relationships to PCP-related and tumor-suppressing proteins, we hypothesize that sea urchin cadherins may play a role in regulating the shape and growth of embryonic epithelia and organs. Future experiments will examine cadherin expression in non-echinoid echinoderms and explore the functions of cadherins during echinoderm development.

The Multi-Kinase Inhibitor RepSox Enforces Barrier Function in the Face of Both VEGF and Cytokines.

The therapeutic benefit provided by anti-vascular endothelial growth factor (VEGF) for patients with vision-threatening conditions such as diabetic retinopathy (DR) demonstrates the important role of VEGF in this affliction. Cytokines, which can be elevated in the vitreous of patients with DR, promote leakage of retinal blood vessels, and may also contribute to pathology, especially in those patients for whom anti-VEGF does not provide adequate benefit. In this in vitro study using primary human retinal endothelial cells, we compared anti-VEGF with the (transforming growth factor beta) TGFβ receptor inhibitor RepSox (RS) for their ability to enforce barrier function in the face of VEGF, cytokines, and the combination of both. RS was superior to anti-VEGF because it prevented permeability in response to VEGF, cytokines, and their combination, whereas anti-VEGF was effective against VEGF alone. The inhibitory effect of RS was associated with suppression of both agonist-induced pore formation and disorganization of adherens junctions. RS-mediated inhibition of the TGFβ pathway and increased expression of claudin-5 did not adequately explain how RS stabilized the endothelial cell barrier. Finally, RS not only prevented barrier relaxation, but also completely or partially reclosed a barrier relaxed with tumor necrosis factor α (TNF α) or VEGF, respectively. These studies demonstrate that RS stabilized the endothelial barrier in the face of both cytokines and VEGF, and thereby identify RS as a therapeutic that has the potential to overcome permeability driven by multiple agonists that play a role in the pathology of DR.

A comprehensive analysis of different types of databases reveals that CDH1 mRNA and E-cadherin protein are not downregulated in most carcinoma tissues and carcinoma cell lines

BackgroundThe CDH1 gene codes for the epithelial-cadherin (E-cad) protein, which is embedded in the plasma membrane of epithelial cells to form adherens junctions. E-cad is known to be essential for maintaining the integrity of epithelial tissues, and the loss of E-cad has been widely considered a hallmark of metastatic cancers enabling carcinoma cells to acquire the ability to migrate and invade nearby tissues. However, this conclusion has come under scrutiny.MethodsTo assess how CDH1 and E-cad expression changes during cancer progression, we analyzed multiple large transcriptomics, proteomics, and immunohistochemistry datasets on clinical cancer samples and cancer cell lines to determine the CDH1 mRNA and E-cad protein expression profiles in tumor and normal cells.ResultsIn contrast to the textbook knowledge of the loss of E-cad during tumor progression and metastasis, the levels of CDH1 mRNA and E-cad protein are either upregulated or remain unchanged in most carcinoma cells compared to normal cells. In addition, the CDH1 mRNA upregulation occurs in the early stages of tumor development and the levels remain elevated as tumors progress to later stages across most carcinoma types. Furthermore, E-cad protein levels are not downregulated in most metastatic tumor cells compared to primary tumor cells. The CDH1 mRNA and E-cad protein levels are positively correlated, and the CDH1 mRNA levels are positively correlated to cancer patient’s survival. We have discussed potential mechanisms underlying the observed expression changes in CDH1 and E-cad during tumor progression.ConclusionsCDH1 mRNA and E-cadherin protein are not downregulated in most tumor tissues and cell lines derived from commonly occurring carcinomas. The role of E-cad in tumor progression and metastasis may have previously been oversimplified. CDH1 mRNA levels may serve as a reliable biomarker for the diagnosis of some tumors (such as colon and endometrial carcinomas) due to the marked upregulation of CDH1 mRNA in the early stages of tumor development of these carcinomas.

THY1 (CD90) Maintains the Adherens Junctions in Nasopharyngeal Carcinoma via Inhibition of SRC Activation

We had previously shown that THY1 (CD90) is a tumor suppressor in nasopharyngeal carcinoma (NPC) and that its down-regulation and loss of expression are associated with tumor metastasis, yet the mechanism leading to such effects remains unknown. In this study we show that tumor invasion could be suppressed by THY1 via adherens junction formation in a few NPC cell lines, and knockdown of THY1 would disrupt this cell-cell adhesion phenotype. Mechanistically, the activity of the SRC family kinase (SFK) member, SRC, and canonical Wnt signaling were dramatically reduced when THY1 was constitutively expressed. Previous studies by others have found that high levels of SRC activity in NPCs are associated with EMT and a poor prognosis. We hypothesized that THY1 can suppress tumor invasion in NPC via inhibition of SRC. By gene silencing of SRC, we found that the in vitro NPC cell invasion was significantly reduced and adherens junctions were restored. Through proteomic analysis, we identified that platelet-derived growth factor receptor β (PDGF-Rβ) and protein tyrosine phosphatase nonreceptor type 22 (PTPN22) are novel and potential binding partners of THY1, which were subsequently verified by co-immunoprecipitation (co-IP) analysis. The ligand of PDGF-Rβ (PDGF-BB) could highly induce SRC activation and NPC cell invasion, which could be almost completely suppressed by THY1 expression. On the other hand, the PTPN22 siRNA could enhance both the SRC activities and the cell invasion and could also disrupt the adherens junctions in the THY1-expressing NPC cells; the original THY1-induced phenotypes were reverted when the PTPN22 expression was reduced. Together, our results identified that PTPN22 is essential for THY1 to suppress cell invasion and SRC activity, maintain tight adherens junctions, and prevent NPC metastasis. These results suggested that PDGF-Rβ and SRC can be used as drug targets for suppressing NPC metastasis. Indeed, our in vivo assay using the SRC inhibitor KX2-391, clearly showed that inhibition of SRC signaling can prevent the metastasis of NPC, indicating that targeting SRC can be a promising approach to control the NPC progression.

PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing

Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.

Cadmium aggravates the blood-brain barrier disruption via inhibition of the Wnt7A/β-catenin signaling axis

Cadmium (Cd) is a non-biodegradable widespread environmental pollutant, which can cross the blood-brain barrier (BBB) and cause cerebral toxicity. However, the effect of Cd on the BBB is still unclear. In this study, a total of 80 (1-day-old) Hy-Line white variety chicks (20 chickens/group) were selected and randomly divided into four (4) groups: the control group (Con group) (fed with a basic diet, n = 20), the Cd 35 group (basic diet with 35 mg/kg CdCl2, n = 20), the Cd 70 group (basic diet with 70 mg/kg CdCl2, n = 20) and the Cd 140 group (basic diet with 140 mg/kg CdCl2, n = 20), and fed for 90 days. The pathological changes, factors associated with the BBB, oxidation level and the levels of Wingless-type MMTV integration site family, member 7 A (Wnt7A)/Wnt receptor Frizzled 4 (FZD4)/β-catenin signaling axis-related proteins in brain tissue were detected. Cd exposure induced capillary damage and neuronal swelling, degeneration and loss of neurons. Gene Set Enrichment Analysis (GSEA) showed the weakened Wnt/β-catenin signaling axis. The protein expression of the Wnt7A, FZD4, and β-catenin was decreased by Cd expusure. Inflammation generation and BBB dysfunction were induced by Cd, as manifested by impaired tight junctions (TJs) and adherens junctions (AJs) formation. These findings underscore that Cd induced BBB dysfunction via disturbing Wnt7A/FZD4/β-catenin signaling axis.

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.

Comprehensive analysis of LAMC1 expression and prognostic value in kidney renal papillary cell carcinoma and clear cell carcinoma.

Background: Laminin subunit gamma 1 (LAMC1) protein is associated with tumor cell invasion and metastasis. However, its role in kidney cancer remains unclear. In this work, we sought to probe the expression as well as its carcinogenic mechanisms of LAMC1 in kidney renal papillary cell carcinoma (KIRP) and kidney renal clear cell carcinoma (KIRC). Methods: Public databases including TIMER, Oncomine, UALCAN, TISIDB, TCGA, Kaplan–Meier plotter, UCSC Xena, cBioPortal, SurvivalMeth, KEGG, GeneMANIA, Metascape, GSCALite and GDSC were adopted, and the expression, clinical pathological correlation, prognostic signatures, dominant factors influencing LAMC1 expression, DNA methylation levels, gene mutations, copy number variations, functional networks, and drug sensitivity were analyzed. Expression of LAMC1 protein in clinical KIRP and KIRC was validated using tissue array. Results: LAMC1 expression in KIRP and KIRC were significantly higher than those in normal tissues. High LAMC1 expression indicated poor overall survival in KIRP patients and better overall survival in KIRC patients. Through the univariate and multivariate Cox analysis, we found that high LAMC1 expression was a potential independent marker for poor prognosis in KIRP, however it implied a better prognosis in KIRC by univariate Cox analysis. In addition, the LAMC1 expression in KIRP and KIRC was negatively correlated with methylation levels of LAMC1 DNA. Interestingly, LAMC1 expression was positively correlated with the infiltration of CD8+ T cells, dendritic cells and neutrophils in KIRP; however, it was positively correlated with the infiltration of CD4+ T cells, macrophages and neutrophils but negatively correlated with B cells in KIRC. Moreover, high level of CD8+ T cells is beneficial for KIRC prognosis but opposite for KIRP. LAMC1 may participate in signaling pathways involved in formation of adherens junction and basement membrane in KIRP and KIRC, and the high expression of LAMC1 is resistant to most drugs or small molecules of the Genomics of Drug Sensitivity in Cancer database. Conclusion: Enhanced LAMC1 expression suggests a poor prognosis in KIRP while a better prognosis in KIRC, and these opposite prognostic signatures of LAMC1 may be related to different immune microenvironments.

Role of moesin and its phosphorylation in VE-cadherin expression and distribution in endothelial adherens junctions

Background and aimVascular endothelial cadherin (VE-cadherin) is an important element of adherens junctions (AJs) between endothelial cells. Its expression and proper distribution are critical for AJ formation and vascular integrity. Our previous studies have demonstrated that moesin phosphorylation mediated the hyper-permeability in endothelial monolayer and microvessels. However, the role of moesin and its phosphorylation in VE-cadherin expression and distribution is not clear. Methods and resultsIn vivo, expression of VE-cadherin was significantly reduced in retina and other various tissues in moesin knock out mice (Msn-/Y). In vitro, by regulating moesin expression with siRNA and adenovirus transfection, we verified that moesin has an effect on VE-cadherin expression in HUVECs, while transcription factor KLF4 may participate in this process. In addition, treatment of advanced glycation end products (AGEs) induced abnormal distribution of VE-cadherin in retinal microvessels from C57BL/6 wild type mice, and in vitro studies indicated that moesin Thr558 phosphorylation had a critical role in AGE-induced VE-cadherin internalization from cytomembrane to cytoplasm. Further investigation demonstrated that the inhibition of F-actin polymerization with cytochalasin D could abolish AGE- and Thr558 phosphor-moesin-mediated VE-cadherin internalization. ConclusionThis study suggests that moesin regulates VE-cadherin expression through KLF4 and the state of moesin phosphorylation at Thr558 affects the integrity of VE-cadherin-based AJs. Thr558 phosphor-moesin mediates AGE-induced VE-cadherin internalization through cytoskeleton reassembling.

Contractility and Calcium Transient Maturation in the Human iPSC-Derived Cardiac Microfibers.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are considered immature in the sarcomere organization, contractile machinery, calcium transient, and transcriptome profile, which prevent them from further applications in modeling and studying cardiac development and disease. To improve the maturity of hiPSC-CMs, here, we engineered the hiPSC-CMs into cardiac microfibers (iCMFs) by a stencil-based micropatterning method, which enables the hiPSC-CMs to be aligned in an end-to-end connection for prolonged culture on the hydrogel of physiological stiffness. A series of characterization approaches were performed to evaluate the maturation in iCMFs on both structural and functional levels, including immunohistochemistry, calcium transient, reverse-transcription quantitative PCR, cardiac contractility, and electrical pacing analysis. Our results demonstrate an improved cardiac maturation of hiPSC-CMs in iCMFs compared to micropatterned or random single hiPSC-CMs and hiPSC-CMs in a random cluster at the same cell number of iCMFs. We found an increased sarcomere length, better regularity and alignment of sarcomeres, enhanced contractility, matured calcium transient, and T-tubule formation and improved adherens junction and gap junction formation. The hiPSC-CMs in iCMFs showed a robust calcium cycling in response to the programmed and continuous electrical pacing from 0.5 to 7 Hz. Moreover, we generated the iCMFs with hiPSC-CMs with mutations in myosin-binding protein C (MYBPC3) to have a proof-of-concept of iCMFs in modeling cardiac hypertrophic phenotype. These findings suggest that the multipatterned iCMF connection of hiPSC-CMs boosts the cardiac maturation structurally and functionally, which will reveal the full potential of the application of hiPSC-CM models in disease modeling of cardiomyopathy and cardiac regenerative medicine.

Unique and redundant functions of cytoplasmic actins and nonmuscle myosin II isoforms at epithelial junctions.

The integrity and functions of epithelial barriers depend on the formation of adherens junctions (AJs) and tight junctions (TJs). A characteristic feature of AJs and TJs is their association with the cortical cytoskeleton composed of actin filaments and nonmuscle myosin II (NM-II) motors. Mechanical forces generated by the actomyosin cytoskeleton are essential for junctional assembly, stability, and remodeling. Epithelial cells express two different actin proteins and three NM-II isoforms, all known to be associated with AJs and TJs. Despite their structural similarity, different actin and NM-II isoforms have distinct biochemical properties, cellular distribution, and functions. The diversity of epithelial actins and myosin motors could be essential for the regulation of different steps of junctional formation, maturation, and disassembly. This review focuses on the roles of actin and NM-II isoforms in controlling the integrity and barrier properties of various epithelia. We discuss the effects of the depletion of individual actin isoforms and NM-II motors on the assembly and barrier function of AJs and TJs in model epithelial monolayers in vitro. We also describe the functional consequences of either total or tissue-specific gene knockout of different actins and NM-II motors, with a focus on the development and integrity of different epithelia in vivo.

Identification and Characterization of Novel Macromolecular Assemblies Responsible for Cell Adhesion

Cadherins and nectins are cell surface glycoproteins that function as cell adhesion molecules (CAMs) to drive the adhesion of cells and thereby organize them into higher‐ordered structures during early development. Homophilic associations among the cadherins and homophilic as well as heterophilic associations among the nectins lead to the formation of adherens junction that drives cell‐to‐cell adhesion. However, several studies suggest for the existence of complex cross‐talk among these CAMs, which remains to be explored in greater detail. Based on SPR‐based protein‐protein interaction studies followed by structure‐guided mutagenesis and complementary biophysical analyses, we observed specific heterophilic associations among cadherins and nectin family members mediated through their membrane distal ectodomains. In particular, N‐cadherin was found to interact with nectin‐2, E‐cadherin to interact with nectin‐4, and E‐cadherin and N‐cadherin were also found to interact with each other. In conclusion to these observations, the biophysical and biochemical studies imply for direct heterophilic recognition and crosstalk among nectins and cadherins and the results obtained elaborate how these novel macromolecular assemblies can also be a part of adherens junction to drive physiologically relevant cell‐cell adhesion.

Heterocellular N-cadherin junctions enable nontransformed cells to inhibit the growth of adjacent transformed cells

BackgroundThe Src tyrosine kinase phosphorylates effector proteins to induce expression of the podoplanin (PDPN) receptor in order to promote tumor progression. However, nontransformed cells can normalize the growth and morphology of neighboring transformed cells. Transformed cells must escape this process, called “contact normalization”, to become invasive and malignant. Contact normalization requires junctional communication between transformed and nontransformed cells. However, specific junctions that mediate this process have not been defined. This study aimed to identify junctional proteins required for contact normalization.MethodsSrc transformed cells and oral squamous cell carcinoma cells were cultured with nontransformed cells. Formation of heterocellular adherens junctions between transformed and nontransformed cells was visualized by fluorescent microscopy. CRISPR technology was used to produce cadherin deficient and cadherin competent nontransformed cells to determine the requirement for adherens junctions during contact normalization. Contact normalization of transformed cells cultured with cadherin deficient or cadherin competent nontransformed cells was analyzed by growth assays, immunofluorescence, western blotting, and RNA-seq. In addition, Src transformed cells expressing PDPN under a constitutively active exogenous promoter were used to examine the ability of PDPN to override contact normalization.ResultsWe found that N-cadherin (N-Cdh) appeared to mediate contact normalization. Cadherin competent cells that expressed N-Cdh inhibited the growth of neighboring transformed cells in culture, while cadherin deficient cells failed to inhibit the growth of these cells. Results from RNA-seq analysis indicate that about 10% of the transcripts affected by contact normalization relied on cadherin mediated communication, and this set of genes includes PDPN. In contrast, cadherin deficient cells failed to inhibit PDPN expression or normalize the growth of adjacent transformed cells. These data indicate that nontransformed cells formed heterocellular cadherin junctions to inhibit PDPN expression in adjacent transformed cells. Moreover, we found that PDPN enabled transformed cells to override the effects of contact normalization in the face of continued N-Cdh expression. Cadherin competent cells failed to normalize the growth of transformed cells expressing PDPN under a constitutively active exogenous promoter.ConclusionsNontransformed cells form cadherin junctions with adjacent transformed cells to decrease PDPN expression in order to inhibit tumor cell proliferation.Plain English SummaryCancer begins when a single cell acquires changes that enables them to form tumors. During these beginning stages of cancer development, normal cells surround and directly contact the cancer cell to prevent tumor formation and inhibit cancer progression. This process is called contact normalization. Cancer cells must break free from contact normalization to progress into a malignant cancer. Contact normalization is a widespread and powerful process; however, not much is known about the mechanisms involved in this process. This work identifies proteins required to form contacts between normal cells and cancer cells, and explores pathways by which cancer cells override contact normalization to progress into malignant cancers.Graphical abstract1_HLfVkYUa-1fjeHgGEFH3Video