Cadherin-mediated Cell Adhesion Research Articles

A computational study for understanding the impact of p120-catenin on the cis-dimerization of cadherin.

A prototype of cross-membrane signal transduction is that extracellular binding of cell surface receptors to their ligands induces intracellular signalling cascades. However, much less is known about the process in the opposite direction, called inside-out signalling. Recent studies show that it plays a more important role in regulating the functions of many cell surface receptors than we used to think. In particular, in cadherin-mediated cell adhesion, recent experiments indicate that intracellular binding of the scaffold protein p120-catenin (p120ctn) can promote extracellular clustering of cadherin and alter its adhesive function. The underlying mechanism, however, is not well understood. To explore possible mechanisms, we designed a new multiscale simulation procedure. Using all-atom molecular dynamics simulations, we found that the conformational dynamics of the cadherin extracellular region can be altered by the intracellular binding of p120ctn. More intriguingly, by integrating all-atom simulation results into coarse-grained random sampling, we showed that the altered conformational dynamics of cadherin caused by the binding of p120ctn can increase the probability of lateral interactions between cadherins on the cell surface. These results suggest that p120ctn could allosterically regulate the cis-dimerization of cadherin through two mechanisms. First, p120ctn controls the extracellular conformational dynamics of cadherin. Second, p120ctn oligomerization can further promote cadherin clustering. Therefore, our study provides a mechanistic foundation for the inside-out signalling in cadherin-mediated cell adhesion, while the computational framework can be generally applied to other cross-membrane signal transduction systems.

CAMP: A master regulator of cadherin-mediated binding in endothelium, epithelium and myocardium.

Regulation of cadherin-mediated cell adhesion is crucial not only for maintaining tissue integrity and barrier function in the endothelium and epithelium but also for electromechanical coupling within the myocardium. Therefore, loss of cadherin-mediated adhesion causes various disorders, including vascular inflammation and desmosome-related diseases such as the autoimmune blistering skin dermatosis pemphigus and arrhythmogenic cardiomyopathy. Mechanisms regulating cadherin-mediated binding contribute to the pathogenesis of diseases and may also be used as therapeutic targets. Over the last 30 years, cyclic adenosine 3',5'-monophosphate (cAMP) has emerged as one of the master regulators of cell adhesion in endothelium and, more recently, also in epithelial cells as well as in cardiomyocytes. A broad spectrum of experimental models from vascular physiology and cell biology applied by different generations of researchers provided evidence that not only cadherins of endothelial adherens junctions (AJ) but also desmosomal contacts in keratinocytes and the cardiomyocyte intercalated discs are central targets in this scenario. The molecular mechanisms involve protein kinase A- and exchange protein directly activated by cAMP-mediated regulation of Rho family GTPases and S665 phosphorylation of the AJ and desmosome adaptor protein plakoglobin. In line with this, phosphodiesterase 4 inhibitors such as apremilast have been proposed as a therapeutic strategy to stabilize cadherin-mediated adhesion in pemphigus and may also be effective to treat other disorders where cadherin-mediated binding is compromised.

Deficiency of AP1 Complex Ap1g1 in Zebrafish Model Led to Perturbation of Neurodevelopment, Female and Male Fertility; New Insight to Understand Adaptinopathies.

In vertebrates, two homologous heterotetrameric AP1 complexes regulate the intracellular protein sorting via vesicles. AP-1 complexes are ubiquitously expressed and are composed of four different subunits: γ, β1, μ1 and σ1. Two different complexes are present in eukaryotic cells, AP1G1 (contains γ1 subunit) and AP1G2 (contains γ2 subunit); both are indispensable for development. One additional tissue-specific isoform exists for μ1A, the polarized epithelial cells specific to μ1B; two additional tissue-specific isoforms exist for σ1A: σ1B and σ1C. Both AP1 complexes fulfil specific functions at the trans-Golgi network and endosomes. The use of different animal models demonstrated their crucial role in the development of multicellular organisms and the specification of neuronal and epithelial cells. Ap1g1 (γ1) knockout mice cease development at the blastocyst stage, while Ap1m1 (μ1A) knockouts cease during mid-organogenesis. A growing number of human diseases have been associated with mutations in genes encoding for the subunits of adaptor protein complexes. Recently, a new class of neurocutaneous and neurometabolic disorders affecting intracellular vesicular traffic have been referred to as adaptinopathies. To better understand the functional role of AP1G1 in adaptinopathies, we generated a zebrafish ap1g1 knockout using CRISPR/Cas9 genome editing. Zebrafish ap1g1 knockout embryos cease their development at the blastula stage. Interestingly, heterozygous females and males have reduced fertility and showed morphological alterations in the brain, gonads and intestinal epithelium. An analysis of mRNA profiles of different marker proteins and altered tissue morphologies revealed dysregulated cadherin-mediated cell adhesion. These data demonstrate that the zebrafish model organism enables us to study the molecular details of adaptinopathies and thus also develop treatment strategies.

PD37-01 THE CADHERIN-CATENIN COMPLEX IS CRUCIAL FOR THE INITIAL STEP OF NEPHRON FORMATION

You have accessJournal of UrologyCME1 Apr 2023PD37-01 THE CADHERIN-CATENIN COMPLEX IS CRUCIAL FOR THE INITIAL STEP OF NEPHRON FORMATION Balint Der, Helena Bugacov, Muskaan Singh, and Andrew P. McMahon Balint DerBalint Der More articles by this author , Helena BugacovHelena Bugacov More articles by this author , Muskaan SinghMuskaan Singh More articles by this author , and Andrew P. McMahonAndrew P. McMahon More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003335.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Congenital anomalies of the kidney and urinary tract contribute to a significant share of pre- and neonatal developmental abnormalities. In nephron formation, canonical Wnt-signaling induces mesenchymal nephron progenitor cells (NPCs) to aggregate and epithelialize forming the renal vesicle (RV), the nephron precursor. β-catenin is essential for the inductive process though the mechanism of morphological transition is not well understood (Park et al., 2007). We hypothesized that ß-catenin also directs RV formation through its critical role in cadherin mediated cell adhesion, the potential redundancy amongst cadherin members confounding earlier genetic studies (Dahl et al., 2002). METHODS: Using a chemically defined culture medium replicating in vivo NPC signaling, we examined the requirement for multiple cadherin adhesion complex components in the canonical Wnt signaling induced aggregation of induced NPCs in vitro. RESULTS: Removal of extracellular calcium, an essential ion in cadherin complexes, resulted in reversible loss of cell-cell contacts, consistent with a role for cadherin adhesion in the aggregation process. In vitro knockout of β-catenin inhibited the induction and aggregation of NPCs replicating the findings of the in vivo loss-of-function mouse experiments. Interestingly, the deletion of α-catenin phenocopied the results of β-catenin knockout experiments blocking cell aggregation providing further evidence for the role of the cadherin-catenin complex in the aggregation. To identify the cadherin members of the adhesion complex, we screened in vitro bulk RNA-seq. dataset and validated the presence of four cadherin proteins by immunofluorescent staining. The quadruple removal of in vitro expressed cadherins replicated the previously characterized phenomenon of inhibited aggregation of the NPCs. CONCLUSIONS: The aggregation of NPCs in vitro is exclusively mediated via the cadherin-catenin complex which models the initial step of nephrogenesis in vivo. A mechanistic insight into NPC aggregation fits within the larger framework of how a healthy kidney is formed and might explain the genetic background of certain congenital kidney anomalies. Source of Funding: National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK054364 Cell Interaction in Development of the Mammalian Kidney) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e986 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Balint Der More articles by this author Helena Bugacov More articles by this author Muskaan Singh More articles by this author Andrew P. McMahon More articles by this author Expand All Advertisement PDF downloadLoading ...

Antagonism between wild-type and mutant β-catenin controls hepatoblastoma differentiation via fascin-1

β-catenin is a well-known effector of the Wnt pathway, and a key player in cadherin-mediated cell adhesion. Oncogenic mutations of β-catenin are very frequent in paediatric liver primary tumours. Those mutations are mostly heterozygous, which allows the co-expression of wild-type (WT) and mutated β-catenins in tumour cells. We investigated the interplay between WT and mutated β-catenins in liver tumour cells, and searched for new actors of the β-catenin pathway. Using an RNAi strategy in β-catenin-mutated hepatoblastoma (HB) cells, we dissociated the structural and transcriptional activities of β-catenin, which are carried mainly by WT and mutated proteins, respectively. Their impact was characterised using transcriptomic and functional analyses. We studied mice that develop liver tumours upon activation of β-catenin in hepatocytes (APCKO and β-cateninΔexon3 mice). We used transcriptomic data from mouse and human HB specimens, and used immunohistochemistry to analyse samples. We highlighted an antagonistic role of WT and mutated β-catenins with regard to hepatocyte differentiation, as attested by alterations in the expression of hepatocyte markers and the formation of bile canaliculi. We characterised fascin-1 as a transcriptional target of mutated β-catenin involved in tumour cell differentiation. Using mouse models, we found that fascin-1 is highly expressed in undifferentiated tumours. Finally, we found that fascin-1 is a specific marker of primitive cells including embryonal and blastemal cells in human HBs. Fascin-1 expression is linked to a loss of differentiation and polarity of hepatocytes. We present fascin-1 as a previously unrecognised factor in the modulation of hepatocyte differentiation associated with β-catenin pathway alteration in the liver, and as a new potential target in HB. The FSCN1 gene, encoding fascin-1, was reported to be a metastasis-related gene in various cancers. Herein, we uncover its expression in poor-prognosis hepatoblastomas, a paediatric liver cancer. We show that fascin-1 expression is driven by the mutated beta-catenin in liver tumour cells. We provide new insights on the impact of fascin-1 expression on tumour cell differentiation. We highlight fascin-1 as a marker of immature cells in mouse and human hepatoblastomas.

Defining domain-specific orientational order in the desmosomal cadherins

Desmosomes are large, macromolecular protein assemblies that mechanically couple the intermediate filament cytoskeleton to sites of cadherin-mediated cell adhesion, thereby providing structural integrity to tissues that routinely experience large forces. Proper desmosomal adhesion is necessary for the normal development and maintenance of vertebrate tissues, such as epithelia and cardiac muscle, while dysfunction can lead to severe disease of the heart and skin. Therefore, it is important to understand the relationship between desmosomal adhesion and the architecture of the molecules that form the adhesive interface, the desmosomal cadherins (DCs). However, desmosomes are embedded in two plasma membranes and are linked to the cytoskeletal networks of two cells, imposing extreme difficulty on traditional structural studies of DC architecture, which have yielded conflicting results. Consequently, the relationship between DC architecture and adhesive function remains unclear. To overcome these challenges, we utilized excitation-resolved fluorescence polarization microscopy to quantify the orientational order of the extracellular and intracellular domains of three DC isoforms: desmoglein 2, desmocollin 2, and desmoglein 3. We found that DC ectodomains were significantly more ordered than their cytoplasmic counterparts, indicating a drastic difference in DC architecture between opposing sides of the plasma membrane. This difference was conserved among all DCs tested, suggesting that it may be an important feature of desmosomal architecture. Moreover, our findings suggest that the organization of DC ectodomains is predominantly the result of extracellular adhesive interactions. We employed azimuthal orientation mapping to show that DC ectodomains are arranged with rotational symmetry about the membrane normal. Finally, we performed a series of mathematical simulations to test the feasibility of a recently proposed antiparallel arrangement of DC ectodomains, finding that it is supported by our experimental data. Importantly, the strategies employed here have the potential to elucidate molecular mechanisms for diseases that result from defective desmosome architecture.

Cadherin-mediated adhesion takes control.

The formation of a centralised apical membrane initiation site (AMIS) is a key event in epithelial cell polarisation. A recent study by Liang et al demonstrates that AMIS localisation relies on cadherin-mediated cell adhesion.

N-Cadherin Stabilizes β-Catenin and Promotes β-Catenin/TCF Transcriptional Activation and Cell Adhesion-Mediated Drug Resistance in Multiple Myeloma

Introduction:Inappropriate activation of Wnt/β-catenin signaling plays a role in some cancers. β-catenin (β-cat) levels in the cell can be regulated by a cadherin-mediated sequestration into membrane-bound and free cytosolic pools, with the later translocating to the nucleus and driving TCF-mediated transcriptional activity following Wnt signal transduction. While sequencing has shown that MM lacks the mutations that typically lead to constitutive β-cat activation seen in other cancers, we and others have demonstrated that Wnt/β-catenin signaling is nonetheless activated in MM and can regulate MM growth. The mechanism driving β-cat stabilization and activation in MM is unclear. E- and N-cadherin (N-cad) expression is elevated in MM compared to plasma cells from healthy donors. We hypothesized that that cadherins can regulate Wnt/β-catenin signaling in MM.Materials and Methods:We detected different forms of β-cat expression in a panel of human MM cell lines (HMCLs) and CD138 PC from MM patients by several approaches. Cadherin gain- or loss-of-function MM models were produced by expressing wild-type N-cad in MMS1 and ARP1 (lack endogenous N-cadherin expression) using a lentiviral system to create stable cell lines (N-Cad/MMS1 and N-cad/ARP1) and empty vector control (EV/MMS1, and EV-ARP1). We knocked down N-cadherin in the JJN3 cell line expressing high level of endogenous N-cadherin using shRNA specific for N-cad (shNcad/JJN3) or scrambled control shRNA (shCont/JJN3) by lentiviral-mediated transfection. We used a TCF reporter system to evaluate β-cat transcriptional activity as previously described.Results:We surveyed 25 HMCLs and CD138-selected plasma cells from 72 newly diagnosed MM for active β-cat with an antibody that specifically recognizes the unphosphorylated active form of β-cat. Higher levels of cytosolic and/or nuclear β-cat protein were seen in 13 of 25 (52%) HMCLs and 36 of 72 (50%) primary MM PC. Correlation of β-cat protein levels with global mRNA expression levels in primary PC revealed significant correlation with only one gene, CDH2 (N-cad). Remarkably, those primary MM with high β-cat levels but low CDH2 levels expressed high levels of E-cadherin/CHD1 mRNA. This posed the question of whether CDH2 is a direct target of TCF/β-cat transcriptional activity or whether high levels of CDH2 lead to increased levels of β-cat protein via sequestration. Both CDH2 mRNA and protein were correlated with β-cat protein but not β-cat mRNA in 23/25 HMCLs. Co-immunoprecipitation revealed that N-cad and β-cat complexes could be identified in HMCLs and primary MM. Consistent with N-cad-mediated stabilization of β-cat both total and unphosphorylated β-cat levels and TCF activity were significantly elevated in N-cad/MMS1 and N-Cad/ARP1 cells relative to controls. In contrast, shRNA mediated knockdown of N-cad led to a loss of both N-cad and β-cat protein levels and TCF-dependent transcription activity relative to controls. These findings provide evidence that β-cat/TCF signaling can be regulated by N-cad in MM. CDH2 mRNA is significantly elevated in the MS and HY subgroups of MM. To search for a potential mechanism of CDH2 transcriptional regulation in MS MM, we compared TCF activity and β-cat protein levels in MS versus non-MS HMCLs. TCF activity and active β-cat were elevated in MS versus non-MS forms of MM and B-cell lymphoma lacking N-cadherin. To determine if MMSET is required to up-regulate N-cad expression, we depleted the full-length MMSET protein in KMS11 cells. The results revealed a dramatic loss of total and unphosphorylated β-cat protein, but not mRNA, and loss of both CDH2 mRNA and protein relative to controls. These data suggest that MMSET can regulate the transcription of the CDH2 gene. MMS1 and ARP1 cells stably expressing N-cad exhibited enhanced adhesion to bone marrow stromal cells and decreased sensitivity to bortezomib (Bzb). In contrast, blocking N-cadherin-mediated adhesion by CDH2 shRNA increased sensitivity to Bzb. These results suggests that N-cad/β-cat complexes can contribute to adhesion-mediated drug resistance in MM.Conclusion: Taken together, these findings establish that β-cat is stabilized by N-cadherin-, and likely E-cadherin-, adhesins junction formation in MM. This in turn leads to an increased pool of β-cat that can drive TCF transcriptional activation as well participate in cadherin-mediated cell adhesion and drug resistance. DisclosuresDavies: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau.

Urokinase-type plasminogen activator-mediated crosstalk between N-cadherin and β-catenin promotes wound healing.

Urokinase-type plasminogen activator (uPA; encoded by Plau) is a serine proteinase that, in the central nervous system, induces astrocytic activation. β-Catenin is a protein that links the cytoplasmic tail of cadherins to the actin cytoskeleton, thus securing the formation of cadherin-mediated cell adhesion complexes. Disruption of cell-cell contacts leads to the detachment of β-catenin from cadherins, and β-catenin is then degraded by the proteasome following its phosphorylation by GSK3β. Here, we show that astrocytes release uPA following a scratch injury, and that this uPA promotes wound healing via a plasminogen-independent mechanism. We found that uPA induces the detachment of β-catenin from the cytoplasmic tail of N-cadherin (NCAD; also known as CDH2) by triggering its phosphorylation at Tyr654. Surprisingly, this is not followed by degradation of β-catenin because uPA also induces the phosphorylation of the low density lipoprotein receptor-related protein 6 (LRP6) at Ser1490, which then blocks the kinase activity of GSK3β. Our work indicates that the ensuing cytoplasmic accumulation of β-catenin is followed by its nuclear translocation and β-catenin-triggered transcription of the receptor for uPA (Plaur), which in turn is required for uPA to induce astrocytic wound healing.

Abstract A24: Cell adhesion molecule (CAM)-related downregulated by oncogenes (CDON) promotes ovarian cancer adhesion and survival

Abstract Ovarian carcinoma (OC) broadly describes epithelial tumors involving the ovary and represents the most lethal gynecologic malignancy. The most frequently diagnosed OCs are high-grade serous carcinomas (HGSC), and the majority of these cancers are diagnosed at late stage, stage III or IV (51% and 29% respectively), with widespread disease dissemination. In addition, a significant number of patients develop malignant ascites. A key feature of tumor cells in present in ascites is the capacity to form multicellular aggregates that promote tumor cell survival and continued disease growth and spread. Cell adhesion molecule (CAM)-related downregulated by oncogenes (CDON) is a cell surface glycoprotein with established roles in normal development and associated with some tumor types. CDON signaling occurs via ligand-dependent mechanisms as a hedgehog co-receptor and ligand-independent mechanisms via interactions with cadherins. Little is known about the role of CDON in ovarian cancer, but given the importance of cadherin-mediated cell adhesion in disease dissemination, we asked whether CDON plays a role in HGSC growth and progression. We find that CDON is expressed in ovarian carcinoma (OC) cells, and protein levels are elevated by expression of oncogenes or by growth under nonadherent conditions. Depletion of CDON expression revealed that it plays an important role in HGSC cells regardless of growth conditions, resulting in decreased growth (proliferation) in cells grown under nonadherent conditions as multicellular aggregates or under adherent conditions as 2D monolayers. Alterations in signaling related to both cell adhesion and survival occur upon CDON downregulation, including decreased cadherin protein expression, integrin expression, and focal adhesion kinase activation. Depletion of CDON in OC cells results in significant tumor growth inhibition in xenograft and allograft models, suggesting that CDON plays an important role in promoting ovarian tumor growth. Treatment of OC spheroids with novel anti-CDON antibodies results in collapse of 3D cellular structures and induction of apoptosis, supporting a key role for CDON in OC cellular adhesion in tumor cell survival. These data indicate that CDON may play an essential role in OC spread since altered cell-cell adhesion and survival as multicellular aggregates is crucial for disease dissemination in patients. Taken together, our results nominate CDON as an important protein promoting HGSC growth and progression and as a novel therapeutic target for the treatment of this disease. Citation Format: Valerie L. Sodi, Shane W. O'Brien, Chao Wu, Roland L. Dunbrack, Tiffiney R. Hartman, Alana M. O'Reilly, Denise C. Connolly. Cell adhesion molecule (CAM)-related downregulated by oncogenes (CDON) promotes ovarian cancer adhesion and survival [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A24.

Lanthanides compete with calcium for binding to cadherins and inhibit cadherin-mediated cell adhesion.

Lanthanides are rare-earth metals with a broad range of applications in biological research and medicine. In addition to their unique magnetic and spectroscopic properties, lanthanides are also effective mimics of calcium and can stimulate or inhibit the function of calcium-binding proteins. Cadherins are a large family of calcium-binding proteins that facilitate cell adhesion and play key roles in embryo development, tissue homeostasis and tumour metastasis. However, whether lanthanides can bind cadherins and functionally replace calcium binding has not been comprehensively explored. In this study, we investigated the effect of lanthanide binding on cadherin structure and function using terbium, which is a commonly used lanthanide for protein spectroscopy and a proposed anti-cancer agent. We demonstrate that terbium can compete with calcium for binding to calcium-binding sites in cadherins. Terbium binding to cadherins abolished their cell adhesive activity and rendered cadherins sensitive to proteolysis by trypsin. Molecular dynamics simulations indicate that replacement of calcium by terbium results in structural rearrangements and increases the flexibility of the cadherin ectodomain. These changes in structure and dynamics are likely to underlie the inability of lanthanide-bound cadherins to support cell adhesion. Taken together, our findings further knowledge on lanthanide interactions with calcium-binding proteins and provide new insight into the influence of metal chemistry on cadherin structure, dynamics and function.

Anomalous cell migration triggers a switch to deviation from the undifferentiated state in colonies of human induced pluripotent stems on feeder layers

Understanding the fundamental mechanisms that trigger deviation from the undifferentiated state of human induced pluripotent stem cells (hiPSCs) provides key strategies to maintain their undifferentiated state during cell expansion. We assessed deviation from the undifferentiated state in hiPSC colonies by measuring cell migration rates in colonies with deviation that were targeted by the end of culture, in a backward manner. Analyses of migration rates of single cells in colonies with deviation demonstrated that the distribution of migration rates at the region with occurrence of deviated cells had a broad or narrow range compared with those at the regions of undifferentiated cells. It was found that deviated cells in hiPSC colonies accidentally occurred consequent to the appearance of relatively fast or slow migrating cells at the peripheral or central region of colonies, reflecting disorders owing to cell migration anomalies in the hiPSC colony. Fluorescence microscopy for F-actin, paxillin, and E-cadherin clarified the localization of integrin-mediated and cadherin-mediated adhesions, introducing the concept that the occurrence and pattern of deviation in a colony were responsive to changes of cell migration in that colony. Furthermore, a major component of the nuclear lamina, laminA/C displayed a rim at the nuclear periphery in the regions with occurrence of deviated cells, concomitant with the actin cytoskeleton associated with integrin- and cadherin-mediated cell adhesion. These results showed that an anomaly of cell migration in hiPSC colonies led to the accidental appearance of deviated cells therein through alternation of the nuclear lamina and imbalance between cell-cell and cell-substrate interactions.

Abstract 596: Disruption of Cholesterol Metabolism and Substrate Stiffness Coordinate to Regulate Cellular Biomechanics and Induce Cytoskeletal Remodeling in Vascular Smooth Muscle Cells

Cholesterol is the main culprit contributing to the development of atherosclerosis by inducing the endothelial dysfunction, triggering the inflammatory response in the blood vessel wall, and deposition in the foam cell at the atherosclerotic plaque. However, growing evidence suggests that the role cholesterol plays during the process of atherogenesis is not only triggering the inflammation, but also plays an important role as a regulator of cellular mechanics, cell spreading, and cell migration, which is the critical step in atherogenesis. In addition, the mechanical environment of the VSMCs in blood vessel undergoes progressive stiffening along with aging, which may potentially affect the development of atherosclerosis. In this study, our main hypothesis is that membrane cholesterol affects the VSMCs migration during the progression of atherosclerosis by regulating the integrin- and cadherin-mediated cell adhesion and this effect highly depends on the mechanical properties of extracellular matrix (ECM). VSMCs used were enzymatically isolated from the descending thoracic aorta of male Sprague-Dawley rats. Cell mechanics was measured using an atomic force microscope (AFM). F-actin cytoskeleton was visualized with confocal microscopy and AFM imaging. 1 μM fluvastatin was used to reduce the endogenous cellular cholesterol. Our results showed that fluvastatin treatment dramatically decreased cell E-modulus, but, significantly increased the average rupture force of collagen I-, fibronectin-, and N-cadherin-mediated cell adhesion. In addition, the confocal microscopy and AFM imaging studies showed that the fluvastatin treatment significantly disrupted the actin stress fiber organization. Interestingly, the effect of fluvastatin on the biomechanics and the architecture of cytoskeleton of VSMCs decreased on the soft substrate. These results indicate that cholesterol and ECM stiffness play the roles synergistically in biomechanics, cytoskeleton architecture, and thus in migration of VSMCs. The knowledge that we obtained in this project will lead to a novel therapeutic strategy to prevent the VSMCs migration and then formation of atherosclerotic plaque by regulating VSMCs biomechanics.

β-Catenin in the Adult Visual Cortex Regulates NMDA-Receptor Function and Visual Responses.

The formation, plasticity and maintenance of synaptic connections is regulated by molecular and electrical signals. β-Catenin is an important protein in these events and regulates cadherin-mediated cell adhesion and the recruitment of pre- and postsynaptic proteins in an activity-dependent fashion. Mutations in the β-catenin gene can cause cognitive disability and autism, with life-long consequences. Understanding its synaptic function may thus be relevant for the treatment of these disorders. So far, β-catenin's function has been studied predominantly in cell culture and during development but knowledge on its function in adulthood is limited. Here, we show that ablating β-catenin in excitatory neurons of the adult visual cortex does not cause the same synaptic deficits previously observed during development. Instead, it reduces NMDA-receptor currents and impairs visual processing. We conclude that β-catenin remains important for adult cortical function but through different mechanisms than during development.

Constructing modular and universal single molecule tension sensor using protein G to study mechano-sensitive receptors.

Recently a variety of molecular force sensors have been developed to study cellular forces acting through single mechano-sensitive receptors. A common strategy adopted is to attach ligand molecules on a surface through engineered molecular tethers which report cell-exerted tension on receptor-ligand bonds. This approach generally requires chemical conjugation of the ligand to the force reporting tether which can be time-consuming and labor-intensive. Moreover, ligand-tether conjugation can severely reduce the activity of protein ligands. To address this problem, we developed a Protein G (ProG)-based force sensor in which force-reporting tethers are conjugated to ProG instead of ligands. A recombinant ligand fused with IgG-Fc is conveniently assembled with the force sensor through ProG:Fc binding, therefore avoiding ligand conjugation and purification processes. Using this approach, we determined that molecular tension on E-cadherin is lower than dsDNA unzipping force (nominal value: 12 pN) during initial cadherin-mediated cell adhesion, followed by an escalation to forces higher than 43 pN (nominal value). This approach is highly modular and potentially universal as we demonstrate using two additional receptor-ligand interactions, P-selectin & PSGL-1 and Notch & DLL1.

Fluorescence-based gene reporter plasmid to track canonical Wnt signaling in ENS inflammation.

In several gut inflammatory or cancer diseases, cell-cell interactions are compromised, and an increased cytoplasmic expression of β-catenin is observed. Over the last decade, numerous studies provided compelling experimental evidence that the loss of cadherin-mediated cell adhesion can promote β-catenin release and signaling without any specific activation of the canonical Wnt pathway. In the present work, we took advantage of the ability of lipofectamine-like reagent to cause a synchronous dissociation of adherent junctions in cells isolated from the rat enteric nervous system (ENS) for obtaining an in vitro model of deregulated β-catenin signaling. Under these experimental conditions, a green fluorescent protein Wnt reporter plasmid called ΔTop_EGFP3a was successfully tested to screen β-catenin stabilization at resting and primed conditions with exogenous Wnt3a or lipopolysaccharide (LPS). ΔTop_EGFP3a provided a reliable and strong fluorescent signal that was easily measurable and at the same time highly sensitive to modulations of Wnt signaling following Wnt3a and LPS stimulation. The reporter gene was useful to demonstrate that Wnt3a exerts a protective activity in the ENS from overstimulated Wnt signaling by promoting a downregulation of the total β-catenin level. Based on this evidence, the use of ΔTop_EGFP3a reporter plasmid could represent a more reliable tool for the investigation of Wnt and cross-talking pathways in ENS inflammation.

Distinct adhesion-independent functions of β-catenin control stage-specific sensory neurogenesis and proliferation.

Backgroundβ-catenin plays a central role in multiple developmental processes. However, it has been difficult to study its pleiotropic effects, because of the dual capacity of β-catenin to coordinate cadherin-dependent cell adhesion and to act as a component of Wnt signal transduction. To distinguish between the divergent functions of β-catenin during peripheral nervous system development, we made use of a mutant allele of β-catenin that can mediate adhesion but not Wnt-induced TCF transcriptional activation. This allele was combined with various conditional inactivation approaches.ResultsWe show that of all peripheral nervous system structures, only sensory dorsal root ganglia require β-catenin for proper formation and growth. Surprisingly, however, dorsal root ganglia development is independent of cadherin-mediated cell adhesion. Rather, both progenitor cell proliferation and fate specification are controlled by β-catenin signaling. These can be divided into temporally sequential processes, each of which depends on a different function of β-catenin.ConclusionsWhile early stage proliferation and specific Neurog2- and Krox20-dependent waves of neuronal subtype specification involve activation of TCF transcription, late stage progenitor proliferation and Neurog1-marked sensory neurogenesis are regulated by a function of β-catenin independent of TCF activation and adhesion. Thus, switching modes of β-catenin function are associated with consecutive cell fate specification and stage-specific progenitor proliferation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0134-4) contains supplementary material, which is available to authorized users.

Computational Modeling of the Interplay between Cadherin-Mediated Cell Adhesion and Wnt Signaling Pathway

Wnt signaling and cadherin-mediated adhesion have been implicated in both processes of embryonic development and the progression of carcinomas. Recent experimental studies revealed that Wnt signaling and cadherin-mediated cell adhesion have close crosstalk with each other. A comprehensive model that investigates the dynamic balance of β-catenins in Wnt signaling and cell adhesion will improve our understanding to embryonic development and carcinomas. We constructed a network model to evaluate the dynamic interplay between adhesion and Wnt signaling. The network is decomposed into three interdependent modules: the cell adhesion, the degradation circle and the transcriptional regulation. In the cell adhesion module, we consider the effect of cadherin’s lateral clustering. We found adhesion negatively contributes to Wnt signaling through competition for cytoplasmic β-catenins. In the network of degradation circle, we incorporated features from various existing models. Our simulations reproduced the most recent experimental phenomena with semi-quantitative accuracy. Finally, in the transcriptional regulation module, we developed a function selection strategy to analyze the outcomes of genetic feedback loops in modulating the gene expression of Wnt targets. The specific cellular phenomena such as cadherin switch and Axin oscillation were archived and their biological insights were discussed. Our model provides the theoretical basis of how spatial organization regulates the dynamics of cellular signaling pathways. We suggest that cell adhesion affects Wnt signaling in both negative and positive ways. Cadherins can inhibit Wnt signaling not only in a way as a stoichiometric binding partner of β-catenins that sequesters them from signaling, but also in a way through their clustering to impacts the rate at which β-catenins are involved in the destruction loop. Additionally, cadherin clustering increases the phosphorylation rate of β-catenins and promotes its signaling in nucleus.

Covalent and Density-Controlled Surface Immobilization of E-Cadherin for Adhesion Force Spectroscopy

E-cadherin is a key cell-cell adhesion molecule but the impact of receptor density and the precise contribution of individual cadherin ectodomains in promoting cell adhesion are only incompletely understood. Investigating these mechanisms would benefit from artificial adhesion substrates carrying different cadherin ectodomains at defined surface density. We therefore developed a quantitative E-cadherin surface immobilization protocol based on the SNAP-tag technique. Extracellular (EC) fragments of E-cadherin fused to the SNAP-tag were covalently bound to self-assembled monolayers (SAM) of thiols carrying benzylguanine (BG) head groups. The adhesive functionality of the different E-cadherin surfaces was then assessed using cell spreading assays and single-cell (SCSF) and single-molecule (SMSF) force spectroscopy. We demonstrate that an E-cadherin construct containing only the first and second outmost EC domain (E1-2) is not sufficient for mediating cell adhesion and yields only low single cadherin-cadherin adhesion forces. In contrast, a construct containing all five EC domains (E1-5) efficiently promotes cell spreading and generates strong single cadherin and cell adhesion forces. By varying the concentration of BG head groups within the SAM we determined a lateral distance of 5–11 nm for optimal E-cadherin functionality. Integrating the results from SCMS and SMSF experiments furthermore demonstrated that the dissolution of E-cadherin adhesion contacts involves a sequential unbinding of individual cadherin receptors rather than the sudden rupture of larger cadherin receptor clusters. Our method of covalent, oriented and density-controlled E-cadherin immobilization thus provides a novel and versatile platform to study molecular mechanisms underlying cadherin-mediated cell adhesion under defined experimental conditions.

Microtubules CLASP to Adherens Junctions in epidermal progenitor cells

Cadherin-mediated cell adhesion at Adherens Junctions (AJs) and its dynamic connections with the microtubule (MT) cytoskeleton are important regulators of cellular architecture. However, the functional relevance of these interactions and the molecular players involved in different cellular contexts and cellular compartments are still not completely understood. Here, we comment on our recent findings showing that the MT plus-end binding protein CLASP2 interacts with the AJ component p120-catenin (p120) specifically in progenitor epidermal cells. Absence of either protein leads to alterations in MT dynamics and AJ functionality. These findings represent a novel mechanism of MT targeting to AJs that may be relevant for the maintenance of proper epidermal progenitor cell homeostasis. We also discuss the potential implication of other MT binding proteins previously associated to AJs in the wider context of epithelial tissues. We hypothesize the existence of adaptation mechanisms that regulate the formation and stability of AJs in different cellular contexts to allow the dynamic behavior of these complexes during tissue homeostasis and remodeling.