Cadherin Adhesion Research Articles

Cadherin adhesion complexes direct cell aggregation in the epithelial transition of Wnt-induced nephron progenitor cells.

In the developing mammalian kidney, nephron formation is initiated by a subset of nephron progenitor cells (NPCs). Wnt input activates a β-catenin (Ctnnb1)-driven, transcriptional nephrogenic program and the mesenchymal to epithelial transition (MET) of NPCs. Using an in vitro mouse NPC culture model, we observed that activation of the Wnt pathway results in the aggregation of induced NPCs, which is an initiating step in the MET program. Genetic removal showed aggregation was dependent on β-catenin. Modulating extracellular Ca2+ levels showed cell-cell contacts were Ca2+ dependent, suggesting a role for cadherin (Cdh)-directed cell adhesion. Molecular analysis identified Cdh2, Cdh4 and Cdh11 in NPCs, and the β-catenin directed upregulation of Cdh3 and Cdh4 accompanying the MET of induced NPCs. Mutational analysis of β-catenin supported a role for a Lef/Tcf-β-catenin-mediated transcriptional response in the cell aggregation process. Genetic removal of all four cadherins, and independent removal of α-catenin or of β-catenin-α-catenin interactions, abolished aggregation, but not the inductive response to Wnt pathway activation. These findings, and data in an accompanying article highlight the role of β-catenin in linking transcriptional programs to the morphogenesis of NPCs in mammalian nephrogenesis.

Multiphoton excited polymerized biomimetic models of collagen fiber morphology to study single cell and collective migration dynamics in pancreatic cancer

The respective roles of aligned collagen fiber morphology found in the extracellular matrix (ECM) of pancreatic cancer patients and cellular migration dynamics have been gaining attention because of their connection with increased aggressive phenotypes and poor prognosis. To better understand how collagen fiber morphology influences cell-matrix interactions associated with metastasis, we used Second Harmonic Generation (SHG) images from patient biopsies with Pancreatic ductal adenocarcinoma (PDAC) as models to fabricate collagen scaffolds to investigate processes associated with motility. Using the PDAC BxPC-3 metastatic cell line, we investigated single and collective cell dynamics on scaffolds of varying collagen alignment. Collective or clustered cells grown on the scaffolds with the highest collagen fiber alignment had increased E-cadherin expression and larger focal adhesion sites compared to single cells, consistent with metastatic behavior. Analysis of single cell motility revealed that the dynamics were characterized by random walk on all substrates. However, examining collective motility over different time points showed that the migration was super-diffusive and enhanced on highly aligned fibers, whereas it was hindered and sub-diffusive on un-patterned substrates. This was further supported by the more elongated morphology observed in collectively migrating cells on aligned collagen fibers. Overall, this approach allows the decoupling of single and collective cell behavior as a function of collagen alignment and shows the relative importance of collective cell behavior as well as fiber morphology in PDAC metastasis. We suggest these scaffolds can be used for further investigations of PDAC cell biology. Statement of significancePancreatic ductal adenocarcinoma (PDAC) has a high mortality rate, where aligned collagen has been associated with poor prognosis. Biomimetic models representing this architecture are needed to understand complex cellular interactions. The SHG image-based models based on stromal collagen from human biopsies afford the measurements of cell morphology, cadherin and focal adhesion expression as well as detailed motility dynamics. Using a metastatic cell line, we decoupled the roles of single cell and collective cell behavior as well as that arising from aligned collagen. Our data suggests that metastatic characteristics are enhanced by increased collagen alignment and that collective cell behavior is more relevant to metastatic processes. These scaffolds provide new insight in this disease and can be a platform for further experiments such as testing drug efficacy.

Vitamin C Improves the Inhibitory Effects of Oxaliplatin on HCT-116 Colorectal Cancer Growth and Progression Through Cellular Oxidant Function-associated Cadherin Molecules.

Colorectal cancer (CRC) is strongly associated with altered cadherin adhesion molecules. Oxaliplatin is a standard treatment for CRC, yet high-doses have concerning side effects. In this study, the effects of oxaliplatin and the combination of oxaliplatin with vitamin C on HCT-116 CRC cell migration and invasion were studied through the roles of cellular oxidative stress associated with cadherin molecules. The cellular assays used in this research were MTT, DCFH-DA, immunofluorescence, and western blotting. Cancer progression was examined using wound healing and Boyden chamber techniques. The results indicate that hydrogen peroxide-induced cellular oxidative stress induced cancer cell migration and invasion. The combined treatment of oxaliplatin with a pro-oxidant concentration of vitamin C resulted in higher toxicity than treatment with oxaliplatin alone. However, treatment with the combination of oxaliplatin and antioxidant concentrations of vitamin C suppressed cancer migration and invasion. Furthermore, the combination treatment increased E-cadherin expression, whereas decreased that of N-cadherin. Treatment with the combination of oxaliplatin with vitamin C can inhibit CRC cell growth and decrease cancer cell migration and invasion, via oxidative stress and cadherins.

Abstract P2074: Site-specific Phosphorylation Of Vinculin Regulates N-cadherin Junction Assembly

Introduction: During physiological and pathological growth, cardiomyocytes adapt and remodel in response to changes in mechanical load. The cytoskeleton plays a central role in tissue remodeling as it both senses mechanical stress and mediates structural remodeling which affects the functional response within the cardiomyocyte. Abl kinase-dependent vinculin (VCL) phosphorylation at tyrosine (Y) residue 822 (pVCL-Y822) has been implicated in force-induced cytoskeletal remodeling and adhesion strengthening at cadherin junctions. However, the functional significance of VCL phosphorylation in the working myocardium remains unknown. Objective: To determine the role of vinculin-mediated mechanotransduction in cardiac development, homeostasis, and disease. Methods and Results: Using a phospho-specific antibody, we found that pVCL-Y822 correlates with dynamic junction remodeling in embryonic cardiomyocytes which declines in the postnatal heart when N-cadherin junctions assemble at the intercalated disc. Notably, pVCL-Y822 is upregulated in border zone cardiomyocytes following myocardial infarction. To investigate VCL-mediated mechanotransduction in the heart, CRISPR-mediated genome editing was used to mutate the tyrosine at position 822 to a non-phosphorylatable phenylalanine (F) in the mouse Vcl gene. Vcl Y822F mice were born in the expected Mendelian frequency and are viable. Blocking VCL phosphorylation causes a preferential reduction of mutant VCL protein at the myocyte termini together with the N-cadherin-catenin adhesion complex. Whereas, α5/β1 integrin and fibronectin are both increased along the lateral border of the Vcl Y822F postnatal cardiomyocytes. Vcl Y822F mice exhibit reduced cardiac function with age; however, cardiac remodeling is attenuated in the mutant hearts. Conclusion: This novel animal model demonstrates how blocking vinculin Y822 phosphorylation affects the balance between cadherin adhesions and integrin adhesions and thereby highlights the importance of understanding the crosstalk between the two adhesion systems and its implications for disease pathogenesis.

Adherens junction: the ensemble of specialized cadherin clusters.

The cell-cell connections in adherens junctions (AJs) are mediated by transmembrane receptors, type I cadherins (referred to here as cadherins). These cadherin-based connections (or trans bonds) are weak. To upregulate their strength, cadherins exploit avidity, the increased affinity of binding between cadherin clusters compared with isolated monomers. Formation of such clusters is a unique molecular process that is driven by a synergy of direct and indirect cis interactions between cadherins located at the same cell. In addition to their role in adhesion, cadherin clusters provide structural scaffolds for cytosolic proteins, which implicate cadherin into different cellular activities and signaling pathways. The cluster lifetime, which depends on the actin cytoskeleton, and on the mechanical forces it generates, determines the strength of AJs and their plasticity. The key aspects of cadherin adhesion, therefore, cannot be understood at the level of isolated cadherin molecules, but should be discussed in the context of cadherin clusters.

1422 Uncoupling desmosomal cadherin adhesion activates quiescent hair follicle stem cells and orchestrates self-organized regeneration through outside-in signaling

1422 Uncoupling desmosomal cadherin adhesion activates quiescent hair follicle stem cells and orchestrates self-organized regeneration through outside-in signaling

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 ...

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

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

Vinculin recruitment to α-catenin halts the differentiation and maturation of enterocyte progenitors to maintain homeostasis of the Drosophila intestine.

Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by peristalsis of its variable content. Using the Drosophila intestinal epithelium, we investigate the role of vinculin, one of the best characterised mechanoeffectors, which functions in both cadherin and integrin adhesion complexes. We discovered that vinculin regulates cell fate decisions, by preventing precocious activation and differentiation of intestinal progenitors into absorptive cells. It achieves this in concert with α-catenin at sites of cadherin adhesion, rather than as part of integrin function. Following asymmetric division of the stem cell into a stem cell and an enteroblast (EB), the two cells initially remain connected by adherens junctions, where vinculin is required, only on the EB side, to maintain the EB in a quiescent state and inhibit further divisions of the stem cell. By manipulating cell tension, we show that vinculin recruitment to adherens junction regulates EB activation and numbers. Consequently, removing vinculin results in an enlarged gut with improved resistance to starvation. Thus, mechanical regulation at the contact between stem cells and their progeny is used to control tissue cell number.

Extracellular matrix regulates force transduction at VE-cadherin junctions.

Increased tension on VE-cadherin (VE-cad) complexes activates adaptive cell stiffening and local cytoskeletal reinforcement--two key signatures of intercellular mechanotransduction. Here we demonstrate that tugging on VE-cad receptors initiates a cascade that results in downstream integrin activation. The formation of new integrin adhesions potentiates vinculin and actin recruitment to mechanically reinforce stressed cadherin adhesions. This cascade differs from documented antagonistic effects of integrins on intercellular junctions. We identify focal adhesion kinase, Abl kinase, and RhoA GTPase as key components of the positive feedback loop. Results further show that a consequence of integrin involvement is the sensitization of intercellular force transduction to the extracellular matrix (ECM) not by regulating junctional tension but by altering signal cascades that reinforce cell-cell adhesions. On type 1 collagen or fibronectin substrates, integrin subtypes α2β1 and α5β1, respectively, differentially control actin remodeling at VE-cad adhesions. Specifically, ECM-dependent differences in VE-cad force transduction mirror differences in the rigidity sensing mechanisms of α2β1 and α5β1 integrins. The findings verify the role of integrins in VE-cad force transduction and uncover a previously unappreciated mechanism by which the ECM impacts the mechanical reinforcement of interendothelial junctions.

Mind bomb 2 promotes cell migration and epithelial structure by regulating adhesion complexes and the actin cytoskeleton

Cell migration is essential in animal development and co-opted during metastasis and inflammatory diseases. Some cells migrate collectively, which requires them to balance epithelial characteristics such as stable cell-cell adhesions with features of motility like rapid turnover of adhesions and dynamic cytoskeletal structures. How this is regulated is not entirely clear but important to understand. While investigating Drosophila oogenesis, we found that the putative E3 ubiquitin ligase, Mind bomb 2 (Mib2), is required to promote epithelial stability and the collective cell migration of border cells. Through biochemical analysis, we identified components of Mib2 complexes, which include E-cadherin and α- and β-catenins, as well as actin regulators. We also found that three Mib2 interacting proteins, RhoGAP19D, Supervillin, and Myosin heavy chain-like, affect border cell migration. mib2 mutant main body follicle cells have drastically reduced E-cadherin-based adhesion complexes and diminished actin filaments. We conclude that Mib2 acts to stabilize E-cadherin-based adhesion complexes and promote a robust actin cytoskeletal network, which is important for maintenance of epithelial integrity. The interaction with cadherin adhesion complexes and other cytoskeletal regulators contribute to its role in collective cell migration. Since Mib2 is well conserved, it may have similar functional significance in other organisms.

Association between abnormal brain oscillations and cognitive performance in patients with bipolar disorder: Molecular mechanisms and clinical evidence.

Brain oscillations have gained great attention in neuroscience during recent decades as functional building blocks of cognitive-sensory processes. Research has shown that oscillations in "alpha," "beta," "gamma," "delta," and "theta" frequency windows are highly modified in brain pathology, including in patients with cognitive impairment like bipolar disorder (BD). The study of changes in brain oscillations can provide fundamental knowledge for exploring neurophysiological biomarkers in cognitive impairment. The present article reviews findings from the role and molecular basis of abnormal neural oscillation and synchronization in the symptoms of patients with BD. An overview of the results clearly demonstrates that, in cognitive-sensory processes, resting and evoked/event-related electroencephalogram (EEG) spectra in the delta, theta, alpha, beta, and gamma bands are abnormally changed in patients with BD showing psychotic features. Abnormal oscillations have been found to be associated with several neural dysfunctions and abnormalities contributing to BD, including abnormal GABAergic neurotransmission signaling, hippocampal cell discharge, abnormal hippocampal neurogenesis, impaired cadherin and synaptic contact-based cell adhesion processes, extended lateral ventricles, decreased prefrontal cortical gray matter, and decreased hippocampal volume. Mechanistically, impairment in calcium voltage-gated channel subunit alpha1 I, neurotrophic tyrosine receptor kinase proteins, genes involved in brain neurogenesis and synaptogenesis like WNT3 and ACTG2, genes involved in the cell adhesion process like CDH12 and DISC1, and gamma-aminobutyric acid (GABA) signaling have been reported as the main molecular contributors to the abnormalities in resting-state low-frequency oscillations in BD patients. Findings also showed the association of impaired synaptic connections and disrupted membrane potential with abnormal beta/gamma oscillatory activity in patients with BD. Of note, the synaptic GABA neurotransmitter has been found to be a fundamental requirement for the occurrence of long-distance synchronous gamma oscillations necessary for coordinating the activity of neural networks between various brain regions.

NCAD and ITGB1 Coordinately Regulate Growth Plate Cartilage Architecture

Spatial and temporal regulation of chondrocyte maturation in the growth plate drives long bone growth. A notable feature of growth plate cartilage is the progression of progenitor resting chondrocytes into clonal columns of transit‐amplifying proliferative chondrocytes. Following division of proliferative chondrocytes, daughter cell pairs undergo a unique 90‐degree rearrangement to form columns. Our previous studies using a murine fluorescent membrane reporter system and time‐lapse imaging along with chemical inhibition of cell adhesion suggested a primary requirement for cadherin adhesion at the daughter cell interface in column formation. Studies designed to test the force generating mechanism at the cadherin dependent daughter cell interface produced a paradoxical result with activated myosin motor proteins enriched at the cell‐matrix interface, an observation that suggests integrin adhesion molecules might drive cell rearrangement. The purpose of the current work was to determine the relative contributions of cadherin and integrin mediated cell adhesion in chondrocyte column formation. As a first step, we used live cell imaging to compare the daughter cell rearrangement phenotype in growth plate explants from embryos containing a cartilage‐specific deletion of N‐cadherin (NCAD) or integrin β1 (ITGB1). Deletion of either gene produces similar disruption of column formation, even though the mutant phenotypes show differences in cell shape, rearrangement dynamics, and kinetics of daughter cell separation following rearrangement. The apparent requirement for both NCAD and ITGB1 suggested potential reciprocal regulatory interaction between the two adhesion surfaces. To test this hypothesis, we analyzed phenotypes of an allelic series. We found that chondrocytes mutant for ITGB1 show defects in cell rearrangement and column formation (n=37 embryos) regardless of the NCAD genotype as expected from single mutant studies. Surprisingly, chondrocytes mutant for NCAD and heterozygous for ITGB1 are phenotypically wildtype with organized columns and normal rearrangement (n=13 embryos). These results support a model for which ITGB1, and not NCAD, is the driver of chondrocyte rearrangement and suggest that failure to rearrange in NCAD mutants results from increased ITGB1 function. Thus, rearrangement requires a narrow range of cell‐matrix adhesion strength and NCAD potentially acts as a brake by restricting ITGB1 activity in non‐rearranging resting chondrocytes. Current studies aim to confirm these genetic findings using gene expression analysis and an innovative quantitative adhesion assay as well as identify upstream regulators that control strength of antagonism between the cell adhesion interfaces.

Mechanical transmission enables EMT cancer cells to drive epithelial cancer cell migration to guide tumor spheroid disaggregation.

Cell phenotype heterogeneity within tumor tissue, especially which due to the emergence of epithelial-mesenchymal transition (EMT) in cancer cells, is associated with cancer invasion and metastasis. However, our understanding of the cellular mechanism(s) underlying the cooperation between EMT cell and epithelial cancer cell migration remains incomplete. Herein, heterotypic tumor spheroids containing both epithelial and EMT cancer cells were generated in vitro. We observed that EMT cells dominated the peripheral region of the self-organized heterotypic tumor spheroid. Furthermore, our results demonstrated that EMT cells could serve as leader cells to improve the collective migration efficiency of epithelial cancer cells and promote dispersion and invasion of the tumor spheroids, which was regulated by the force transition between EMT cells and epithelial cancer cells. Mechanistically, our data further suggest that force transmission is mediated by heterophilic N-cadherin/E-cadherin adhesion complexes between EMT and epithelial cancer cells. Impairment of N-cadherin/E-cadherin adhesion complex formation abrogated the ability of EMT cells to guide epithelial cancer cell migration and blocked the dispersion of tumor spheroids. Together, our data provide new insight into the mechanical interaction between epithelial and EMT cancer cells through heterophilic cadherin adhesion, which enables cooperative tumor cell migration, highlighting the role of EMT cells in tumor invasion.

Identification of a novel de novo mutation in the CTNNB1 gene in an Iranian patient with intellectual disability.

CTNNB1 encodes for the β-catenin protein, a component of the cadherin adhesion complex, which regulates cell-cell adhesion and gene expression in the canonical Wnt signaling pathway. Mutations in CTNNB1 have been reported to be associated with cancer and mental disorders. Recently, loss-of-function mutations in CTNNB1 have been observed in patients with intellectual disability and some other clinical manifestations including motor and language delays, microcephaly, and mild visual defects. We report an 8-year-old Iranian girl with intellectual disability, hypotonia, impaired vision such as vitreomacular adhesion, motor delay, and speech delay. A novel, de novo nonsense mutation (c.1014G > A; p.Trp338Ter) in exon 7 of the CTNNB1 (NM_001904) gene was detected and confirmed by whole-exome sequencing and Sanger sequencing, respectively. This study helps to expand the growing list of loss-of-function mutations known in the CTNNB1 gene.

Mechanical disruption of E-cadherin complexes with epidermal growth factor receptor actuates growth factor–dependent signaling

Increased intercellular tension is associated with enhanced cell proliferation and tissue growth. Here, we present evidence for a force-transduction mechanism that links mechanical perturbations of epithelial (E)-cadherin (CDH1) receptors to the force-dependent activation of epidermal growth factor receptor (EGFR, ERBB1)-a key regulator of cell proliferation. Here, coimmunoprecipitation studies first show that E-cadherin and EGFR form complexes at the plasma membrane that are disrupted by either epidermal growth factor (EGF) or increased tension on homophilic E-cadherin bonds. Although force on E-cadherin bonds disrupts the complex in the absence of EGF, soluble EGF is required to mechanically activate EGFR at cadherin adhesions. Fully quantified spectral imaging fluorescence resonance energy transfer further revealed that E-cadherin and EGFR directly associate to form a heterotrimeric complex of two cadherins and one EGFR protein. Together, these results support a model in which the tugging forces on homophilic E-cadherin bonds trigger force-activated signaling by releasing EGFR monomers to dimerize, bind EGF ligand, and signal. These findings reveal the initial steps in E-cadherin-mediated force transduction that directly link intercellular force fluctuations to the activation of growth regulatory signaling cascades.

Abstract P383: 3D-collagen Matrix Enhanced Integrins And Matrix Metalloproteinases Expression In Cardiac Mesenchymal Cells

Introduction: Use of cardiac mesenchymal cells (CMCs) has been shown to improve cardiac function following myocardial infarction. Main drawback in cardiac cell therapy is the major loss of injected cells within few hours. Increase the retention of these injected cells could increase their efficacy, where cardiac patches with various cell types showed better outcome. Among, collagen patch plays lead role as a cell-laden matrix in cardiac tissue engineering. Creating a detailed understanding of how collagen matrix changes the cellular phenotype could provide seminal insights to regeneration therapy. Hypothesis: Growing CMCs in three dimensional (3D) collagen matrix could alter the expression of extracellular matrix (ECM) and adhesion molecules, which may enhance their efficacy. Methods: The bovine type I collagen was chemically modified and solubilized in culture medium with photo-initiator. The mouse CMCs were isolated and resuspended in collagen solution, printed using 3D bioprinter and UV-crosslinked to form 3D-CMC construct. The 3D-CMC construct was submerged in growth medium and cultured for 48h and analyzed for the expression of ECM and adhesion molecules (n=5/group). CMCs cultured in regular plastic tissue culture dish was used as control. Results: RT profiler array showed changes in the ECM and adhesion molecules expression, specifically certain integrins and matrix metalloproteinases (MMPs) in CMCs cultured 3D collagen construct compared to 2D monolayer. Subsequent qRT-PCR analysis revealed significant (p<0.01) upregulation of integrins such as Itga2 (2.96±0.13), Itgb1 (3.18±0.2) and Itgb3 (2.4±0.27) and MMPs such as MMP13 (37.2±3.36), MMP9 (5.23±1.06) and MMP3 (7.14±2.07). Western blot analysis further confirmed significant elevation of these integrins and matrix metalloproteinases at protein level. Collagen encapsulation did not alter the expression of N-cadherin in CMCs, which is a potential mesenchymal cadherin adhesion molecule. Conclusion: Integrin αβ heterodimers transduce signals that facilitate cell homing, migration, survival and differentiation. Similarly, MMPs plays vital role in cell migration and proliferation. Our results demonstrate that the 3D-collagen Niche enhances the expression of certain integrins and MMPs in CMCs. This suggest that the efficacy of CMCs could be magnified by providing 3D architecture with collagen matrix and further in vivo experiments would reveal functional benefits from CMCs for clinical use.

How adherens junctions move cells during collective migration.

In this review, we consider how the association between adherens junctions and the actomyosin cytoskeleton influences collective cell movement. We focus on recent findings which reveal different ways for adherens junctions to promote the locomotion of cells within tissues: through lamellipodia and junctional contraction. These contributions reflect how classic cadherins establish sites of cortical actin assembly and how adherens junctions couple to contractile actomyosin, respectively. The diverse interplay between cadherin adhesion and the cytoskeleton thus provides different ways for adherens junctions to support epithelial locomotion.

Cadherin puncta are interdigitated dynamic actin protrusions necessary for stable cadherin adhesion

Cadherins harness the actin cytoskeleton to build cohesive sheets of cells using paradoxically weak bonds, but the molecular mechanisms are poorly understood. In one popular model, actin organizes cadherins into large, micrometer-sized clusters known as puncta. Myosin is thought to pull on these puncta to generate strong adhesion. Here, however, we show that cadherin puncta are actually interdigitated actin microspikes generated by actin polymerization mediated by three factors (Arp2/3, EVL, and CRMP-1). The convoluted membranes in these regions give the impression of cadherin clustering by fluorescence microscopy, but the ratio of cadherin to membrane is constant. Nevertheless, these interlocking fingers of membrane are important for adhesion because perturbing their formation disrupts cell adhesion. In contrast, blocking myosin-dependent contractility does not disrupt either the interdigitated microspikes or lateral membrane adhesion. "Puncta" are zones of strong cell-cell adhesion not due to cadherin clustering but that occur because the interdigitated microspikes expand the surface area available for adhesive bond formation and increase the asperity of the cell surface to promote friction between cells.

091 MARCH family E3 ubiquitin ligases selectively target cadherin family proteins for degradation

Cadherins play a central role in the assembly and adhesive function of adherens junctions (AJ). Adhesion and signaling functions of cadherins regulate epidermal and vascular barrier function, development, and tumor metastasis. Our group previously showed that a viral ubiquitin ligase K5, which is encoded in Human Herpesvirus 8 genome and involved in Kaposi Sarcoma, causes endocytosis and degradation of VE-cadherin (VE-cad). K5 is homologous to endogenous human membrane associated RING-CH-type ubiquitin ligase (MARCH) proteins. However, the contribution of endogenous MARCH family proteins to cadherin regulation is unclear. To assess the effect of MARCH family protein expression on epithelial and endothelial cadherins, MARCH proteins were expressed in epithelial and endothelial cells and the effect on E-cadherin (E-cad), VE-cad, and N-cadherin (N-cad) was monitored by western blot and immunofluorescence microscopy. MARCH1 had no discernable impact on E-cad, VE-cad or N-cad. MARCH8 downregulated E-cad and slightly downregulated VE-cad but not N-cad. In contrast, MARCH2, MARCH3 and MARCH4 transfection markedly downregulated VE-cad. Interestingly, MARCH2 and MARCH3 did not downregulate E-cad and N-cad, but MARCH4 downregulated E-cad, VE-cad and N-cad. These results revealed that different MARCH proteins have different cadherin specificities. To determine the amino-acid sequences within VE-cad that dictate this specificity, we generated VE-cad/N-cad chimeras by swapping the cadherin transmembrane and juxta-membrane domains (VE-cad-NcadTMDJMD, N-cad-VEcadTMDJMD). Interestingly, MARCH2 downregulated N-cad-VEcadTMDJMD but not VE-cad-NcadTMDJMD. These results indicate that MARCH proteins exhibit selectivity for different cadherins and that the amino acid sequence within the transmembrane and juxta-membrane domains is essential for this selectivity. These findings suggest that MARCH proteins may differentially regulate cadherins in epithelial and endothelial cells to modulate cell migration and barrier function during development and inflammation.