N-cadherin association with lipid rafts regulates its dynamic assembly at cell-cell junctions in C2C12 myoblasts

Cadherins are homophilic cell-cell adhesion molecules implicated in cell growth, differentiation, and organization into tissues during embryonic development. They accumulate at cell-cell contact sites and act as adhesion-activated signaling receptors. Here, we show that the dynamic assembly of N-cadherin at cell-cell contacts involves lipid rafts. In C2C12 myoblasts, immunofluorescence and biochemical experiments demonstrate that N-cadherin present at cell-cell contacts is colocalized with lipid rafts. Disruption of lipid rafts leads to the inhibition of cell-cell adhesion and disorganization of N-cadherin-dependent cell-cell contacts without modifying the association of N-cadherin with catenins and its availability at the plasma membrane. Fluorescent recovery after photobleaching experiments demonstrate that at the dorsal plasma membrane, lipid rafts are not directly involved in the diffusional mobility of N-cadherin. In contrast, at cell-cell junctions N-cadherin association with lipid rafts allows its stabilization enabling the formation of a functional adhesive complex. We show that lipid rafts, as homophilic interaction and F-actin association, stabilize cadherin-dependent adhesive complexes. Homophilic interactions and F-actin association of N-cadherin are both required for its association to lipid rafts. We thus identify lipid rafts as new regulators of cadherin-mediated cell adhesion.     

Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction

OL-protocadherin (OL-pc) is a transmembrane protein belonging to the cadherin superfamily, which has been shown to accumulate at cell-cell contacts via its homophilic interaction, but its molecular roles remain elusive. In this study, we show that OL-pc bound Nck-associated protein 1 (Nap1), a protein that regulates WAVE-mediated actin assembly. In astrocytoma U251 cells not expressing OL-pc, Nap1 was localized only along the lamellipodia. However, exogenous expression of OL-pc in these cells recruited Nap1 as well as WAVE1 to cell-cell contact sites. Although OL-pc expression had no effect on the motility of solitary U251 cells, it accelerated their movement when they were in contact with one another, causing concomitant reorganization of F-actin and N-cadherin at cell junctions. OL-pc mutants lacking the Nap1-binding site exhibited no such effect. N-cadherin knockdown mimicked OL-pc expression in enhancing cell movement. These results suggest that OL-pc remodels the motility and adhesion machinery at cell junctions by recruiting the Nap1-WAVE1 complex to these sites and, in turn, promotes the migration of cells.     

Regulation of Homotypic Cell-Cell Adhesion by Branched N-Glycosylation of N-cadherin Extracellular EC2 and EC3 Domains

The effects of altering N-cadherin N-glycosylation on several cadherin-mediated cellular behaviors were investigated using small interfering RNA and site-directed mutagenesis. In HT1080 fibrosarcoma cells, small interfering RNA-directed knockdown of N-acetylglucosaminyltransferase V (GnT-V), a glycosyltransferase up-regulated by oncogene signaling, caused decreased expression of N-linked β(1,6)-branched glycans expressed on N-cadherin, resulting in enhanced N-cadherin-mediated cell-cell adhesion, but had no effect on N-cadherin expression on the cell surface. This effect on adhesion was accompanied by decreased cell migration and invasion, opposite of the effects observed when GnT-V was overexpressed in these cells (Guo, H. B., Lee, I., Kamar, M., and Pierce, M. (2003) J. Biol. Chem. 278, 52412–52424). A detailed study using site-directed mutagenesis demonstrated that three of the eight putative N-glycosylation sites in the N-cadherin sequence showed N-glycan expression. Moreover, all three of these sites, located in the extracellular domains EC2 and EC3, were shown by leucoagglutinating phytohemagglutinin binding to express at least some β(1,6)-branched glycans, products of GnT-V activity. Deletion of these sites had no effect on cadherin levels on the cell surface but led to increased stabilization of cell-cell contacts, cell-cell adhesion- mediated intracellular signaling, and reduced cell migration. We show for the first time that these deletions had little effect on formation of the N-cadherin-catenin complex but instead resulted in increased N-cadherin cis-dimerization. Branched N-glycan expression at three sites in the EC2 and -3 domains regulates N-cadherin-mediated cell-cell contact formation, outside-in signaling, and cell migration and is probably a significant contributor to the increase in the migratory/invasive phenotype of cancer cells that results when GnT-V activity is up-regulated by oncogene signaling.     

Cadherins in development and cancer

Proper embryonic development is guaranteed under conditions of regulated cell-cell and cell-matrix adhesion. The cells of an embryo have to be able to distinguish their neighbours as being alike or different. Cadherins, single-pass transmembrane, Ca(2+)-dependent adhesion molecules that mainly interact in a homophilic manner, are major contributors to cell-cell adhesion. Cadherins play pivotal roles in important morphogenetic and differentiation processes during development, and in maintaining tissue integrity and homeostasis. Changes in cadherin expression throughout development enable differentiation and the formation of various organs. In addition to these functions, cadherins have strong implications in tumourigenesis, since frequently tumour cells show deregulated cadherin expression and inappropriate switching among family members. In this review, I focus on E- and N-cadherin, giving an overview of their structure, cellular function, importance during development, role in cancer, and of the complexity of Ecadherin gene regulation.     

IQGAP1 and calmodulin modulate E-cadherin function

Ca(2+)-dependent cell-cell adhesion is mediated by the cadherin family of transmembrane proteins. Adhesion is achieved by homophilic interaction of the extracellular domains of cadherins on adjacent cells, with the cytoplasmic regions serving to couple the complex to the cytoskeleton. IQGAP1, a novel RasGAP-related protein that interacts with the cytoskeleton, binds to actin, members of the Rho family, and E-cadherin. Calmodulin binds to IQGAP1 and regulates its association with Cdc42 and actin. Here we demonstrate competition between calmodulin and E-cadherin for binding to IQGAP1 both in vitro and in a normal cellular milieu. Immunocytochemical analysis in MCF-7 (E-cadherin positive) and MDA-MB-231 (E-cadherin negative) epithelial cells revealed that E-cadherin is required for accumulation of IQGAP1 at cell-cell junctions. The cell-permeable calmodulin antagonist CGS9343B significantly increased IQGAP1 at areas of MCF-7 cell-cell contact, with a concomitant decrease in the amount of E-cadherin at cell-cell junctions. Analysis of E-cadherin function revealed that CGS9343B significantly decreased homophilic E-cadherin adhesion. On the basis of these data, we propose that disruption of the binding of calmodulin to IQGAP1 enhances the association of IQGAP1 with components of the cadherin-catenin complex at cell-cell junctions, resulting in impaired E-cadherin function.     

N-cadherin cell-cell adhesion complexes are regulated by fibronectin matrix assembly

Fibronectin is a principal component of the extracellular matrix. Soluble fibronectin molecules are assembled into the extracellular matrix as insoluble, fibrillar strands via a cell-dependent process. In turn, the interaction of cells with the extracellular matrix form of fibronectin stimulates cell functions critical for tissue repair. Cross-talk between cell-cell and cell-extracellular matrix adhesion complexes is essential for the organization of cells into complex, functional tissue during embryonic development and tissue remodeling. Here, we demonstrate that fibronectin matrix assembly affects the organization, composition, and function of N-cadherin-based adherens junctions. Using fibronectin-null mouse embryonic myofibroblasts, we identified a novel quaternary complex composed of N-cadherin, β-catenin, tensin, and actin that exists in the absence of a fibronectin matrix. In the absence of fibronectin, homophilic N-cadherin ligation recruited both tensin and α5β1 integrins into nascent cell-cell adhesions. Initiation of fibronectin matrix assembly disrupted the association of tensin and actin with N-cadherin, released α5β1 integrins and tensin from cell-cell contacts, stimulated N-cadherin reorganization into thin cellular protrusions, and decreased N-cadherin adhesion. Fibronectin matrix assembly has been shown to recruit α5β1 integrins and tensin into fibrillar adhesions. Taken together, these studies suggest that tensin serves as a common cytoskeletal link for integrin- and cadherin-based adhesions and that the translocation of α5β1 integrins from cell-cell contacts into fibrillar adhesions during fibronectin matrix assembly is a novel mechanism by which cell-cell and cell-matrix adhesions are coordinated.     

Single-cell adhesion tests against functionalized microspheres arrayed on AFM cantilevers confirm heterophilic E- and N-cadherin binding

We assess the cross-reactivity of both cellular as well as recombinant E- and N-cadherins using functionalized bead arrays assembled on atomic-force-microscope cantilevers. This new approach builds upon and enhances the utility of a recently developed force probe that integrates a custom-built, horizontal atomic force microscope with micropipette manipulation. It enables us to test multiple biomolecular interactions of the same cell in a swift sequential or cyclic manner and thus to resolve subtle differences between individual interactions that otherwise would be obscured by cell-cell baseline variability. For each cell, we contrast heterophilic E:N-cadherin binding with the respective homophilic bonds and with a suitable control. Clarifying previous literature reports, we establish that specific bonds between E- and N-cadherins form readily, albeit less frequently than homophilic bonds of either cadherin. We support this assessment with a rough estimate of the ratio of on-rate constants of E/N-cadherin binding.     

Axonin-1/TAG-1 mediates cell-cell adhesion by a cis-assisted trans-interaction.

The neural cell adhesion molecule axonin-1/TAG-1 mediates cell-cell interactions via homophilic and heterophilic contacts. It consists of six Ig and four fibronectin type III domains anchored to the membrane by glycosylphosphatidylinositol. The recently solved crystal structure indicates a module composed of the four N-terminal Ig domains as the contact site between trans-interacting axonin-1 molecules from apposed membranes. Here, we have tested domain-specific monoclonal antibodies for their capacity to interfere with homophilic binding in a cell aggregation assay. The results confirmed the existence of a binding region within the N-terminal Ig domains and identified a second region contributing to homophilic binding on the third and fourth fibronectin domains near the C terminus. The perturbation of each region alone resulted in a complete loss of cell aggregation, suggesting that axonin-1-mediated cell-cell contact results from a cooperative action of two homophilic binding regions. The data support that axonin-1-mediated cell-cell contact is formed by cis-assisted trans-binding. The N-terminal binding regions of axonin-1 establish a linear zipper-like string of trans-interacting axonin-1 molecules alternately provided by the two apposed membranes. The C-terminal binding regions strengthen the cell-cell contact by enhancing the expansion of the linear string into a two-dimensional array via cis-interactions. Cis-assisted trans-binding may be a basic binding mechanism common to many cell adhesion molecules.     

Role of N-cadherin in bone formation

Cell-cell adhesion mediated by cadherins is essential for the function of bone forming cells during osteogenesis. Here, the evidence that N-cadherin is an important regulator of osteoblast differentiation and osteogenesis is reviewed. Osteoblasts express a limited number of cadherins, including the classic N-cadherin. The expression profile of N-cadherin in osteoblasts during bone formation in vivo and in vitro suggests a role of this molecule in osteogenesis. Functional studies using neutralizing antibodies or antisense oligonucleotides indicate that N-cadherin is involved in the control the expression of osteoblast marker gene expression and differentiation. Cleavage of N-cadherin during osteoblast apoptosis also suggests a role of N-cadherin-mediated-cell-cell adhesion in osteoblast survival. Hormonal and local factors that regulate osteoblast function also regulate N-cadherin expression and subsequent cell-cell adhesion associated with osteoblast differentiation or survival. Signaling mechanisms involved in N-cadherin-mediated cell-cell adhesion and osteoblast gene expression have also been identified. Alterations of N-cadherin expression are associated with abnormal osteoblast differentiation and osteogenesis in pathological conditions. These findings indicate that N-cadherin plays a role in normal and pathological bone formation and provide some insight into the process involved in N-cadherin-mediated cell-cell adhesion and differentiation in osteoblasts.     

Biogenesis of N-Cadherin-dependent Cell-Cell Contacts in Living Fibroblasts Is a Microtubule-dependent Kinesin-driven Mechanism

Cadherin-mediated cell-cell adhesion is a dynamic process that is regulated during embryonic development, cell migration, and differentiation. Different cadherins are expressed in specific tissues consistent with their roles in cell type recognition. In this study, we examine the formation of N-cadherin-dependent cell-cell contacts in fibroblasts and myoblasts. In contrast to E-cadherin, both endogenous and ectopically expressed N-cadherin shuttles between an intracellular and a plasma membrane pool. Initial formation of N-cadherin-dependent cell-cell contacts results from the recruitment of the intracellular pool of N-cadherin to the plasma membrane. N-cadherin also localizes to the Golgi apparatus and both secretory and endocytotic vesicles. We demonstrate that the intracellular pool of N-cadherin is tightly associated with the microtubule (MT) network and that junction formation requires MTs. In addition, localization of N-cadherin to the cortex is dependent on an intact F-actin cytoskeleton. We show that N-cadherin transport requires the MT network as well as the activity of the MT-associated motor kinesin. In conclusion, we propose that N-cadherin distribution is a regulated process promoted by cell-cell contact formation, which controls the biogenesis and turnover of the junctions through the MT network.     

A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined by amino acids that flank the HAV motif

The classical cadherins (e.g. N-, E-, and P- cadherin) are well established homophilic adhesion molecules; however, the mechanism that governs cadherin specificity remains contentious. The classical cadherins contain an evolutionarily conserved His-Ala-Val (HAV) sequence, and linear peptides harboring this motif are capable of inhibiting a variety of cadherin-dependent processes. We now demonstrate that short cyclic HAV peptides can inhibit N-cadherin function. Interestingly, the nature of the amino acids that flank the HAV motif determine both the activity and specificity of the peptides. For example, when the HAV motif is flanked by a single aspartic acid, which mimics the natural HAVD sequence of N-cadherin, the peptide becomes a much more effective inhibitor of N-cadherin function. In contrast, when the HAV motif is flanked by a single serine, which mimics the natural HAVS sequence of E-cadherin, it loses its ability to inhibit the N-cadherin response. Our results demonstrate that subtle changes in the amino acids that flank the HAV motif can account for cadherin specificity and that small cyclic peptides can inhibit cadherin function. An emerging role for cadherins in a number of pathological processes suggests that the cyclic peptides reported in this study might be developed as therapeutic agents.     

Protein tyrosine phosphatase PTPRT as a regulator of synaptic formation and neuronal development

PTPRT/RPTPρ is the most recently isolated member of the type IIB receptor-type protein tyrosine phosphatase family and its expression is restricted to the nervous system. PTPRT plays a critical role in regulation of synaptic formation and neuronal development. When PTPRT was overexpressed in hippocampal neurons, synaptic formation and dendritic arborization were induced. On the other hand, knockdown of PTPRT decreased neuronal transmission and attenuated neuronal development. PTPRT strengthened neuronal synapses by forming homophilic trans dimers with each other and heterophilic cis complexes with neuronal adhesion molecules. Fyn tyrosine kinase regulated PTPRT activity through phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT. Phosphorylation induced homophilic cis dimerization of PTPRT and resulted in the inhibition of phosphatase activity. BCR-Rac1 GAP and Syntaxin-binding protein were found as new endogenous substrates of PTPRT in rat brain. PTPRT induced polymerization of actin cytoskeleton that determined the morphologies of dendrites and spines by inhibiting BCR-Rac1 GAP activity. Additionally, PTPRT appeared to regulate neurotransmitter release through reinforcement of interactions between Syntaxin-binding protein and Syntaxin, a SNARE protein. In conclusion, PTPRT regulates synaptic function and neuronal development through interactions with neuronal adhesion molecules and the dephosphorylation of synaptic molecules. [BMB Reports 2015; 48(5): 249-255]     

SH2B1β regulates N-cadherin levels, cell-cell adhesion and nerve growth factor-induced neurite initiation

Little is known regarding the role of inter-cellular interaction during neuronal differentiation. Homophilic N-cadherin engagement between cells contributes to neuronal migration. However, its function in neurite initiation is not clear. In this study, we provide the first evidence that the adaptor protein SH2B1β regulated N-cadherin levels and neurite initiation. Overexpression of SH2B1β reduces N-cadherin levels and increased phosphotyrosine 654 β-catenin, leading to increased nerve growth factor-induced neurite initiation in PC12 cells, an established model for neuronal differentiation. In contrast, overexpression of the dominant-negative mutant SH2B1β(R555E) increases N-cadherin expression, cell-cell aggregation, and reduces neurite initiation. Moreover, SH2B1β binds directly or indirectly to N-cadherin indicative of its involvement in regulating the levels of N-cadherin. Taken together, these findings provide significant new insights into how N-cadherin-mediated inter-cellular interactions may influence neurite initiation and how SH2B1β may regulate these processes.     

Galectin-3 Protein Regulates Mobility of N-cadherin and GM1 Ganglioside at Cell-Cell Junctions of Mammary Carcinoma Cells

Galectin-3 binding to cell surface glycoproteins, including branched N-glycans generated by N-acetylglucosaminyltransferase V (Mgat5) activity, forms a multivalent, heterogeneous, and dynamic lattice. This lattice has been shown to regulate integrin and receptor tyrosine kinase signaling promoting tumor cell migration. N-cadherin is a homotypic cell-cell adhesion receptor commonly overexpressed in tumor cells that contributes to cell motility. Here we show that galectin-3 and N-cadherin interact and colocalize with the lipid raft marker GM1 ganglioside in cell-cell junctions of mammary epithelial cancer cells. Disruption of the lattice by deletion of Mgat5, siRNA depletion of galectin-3, or competitive inhibition with lactose stabilizes cell-cell junctions. It also reduces, in a p120-catenin-dependent manner, the dynamic pool of junctional N-cadherin. Proteomic analysis of detergent-resistant membranes (DRMs) revealed that the galectin lattice opposes entry of many proteins into DRM rafts. N-cadherin and catenins are present in DRMs; however, their DRM distribution is not significantly affected by lattice disruption. Galectin lattice integrity increases the mobile fraction of the raft marker, GM1 ganglioside binding cholera toxin B subunit Ctb, at cell-cell contacts in a p120-catenin-independent manner, but does not affect the mobility of either Ctb-labeled GM1 or GFP-coupled N-cadherin in nonjunctional regions. Our results suggest that the galectin lattice independently enhances lateral molecular diffusion by direct interaction with specific glycoconjugates within the adherens junction. By promoting exchange between raft and non-raft microdomains as well as molecular dynamics within junction-specific raft microdomains, the lattice may enhance turnover of N-cadherin and other glycoconjugates that determine junctional stability and rates of cell migration.     

N-cadherin-associated proteins in chicken muscle

The development and functional activity of the heart depends on the regulated interaction of cardiac cells. This is in part mediated by cell-cell adhesion molecules such as N-cadherin. N-cadherin belongs to a family of Ca+(+)-dependent, transmembrane, adhesion glycoproteins that promote cell-cell adhesion by molecular self-association extracellularly, and interact intracellularly with the cytoskeleton through highly conserved carboxy-terminal domains. In this paper we show that embryonic chicken cardiac myocytes grown in vitro display Ca+(+)-dependent adhesion and express N-cadherin. When immunoprecipitated from detergent extracts of embryonic chicken cardiac and skeletal muscle cultures, N-cadherin associates with proteins immunologically unrelated to itself. The associated proteins are similar in molecular weight to proteins that coimmunoprecipatate with E-cadherin from human epithelial cells. We postulate that the coimmunoprecipitating proteins are involved in linking the cadherins to the cytoskeleton.     

Molecular mechanisms of cell-cell interaction in Dictyostelium discoideum

During development of the cellular slime mold Dictyostelium discoideum, cells migrate in response to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr80,000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell-cell adhesion. One of them has a Mr of 150,000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell-cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.     

N-cadherin activation substitutes for the cell contact control in cell cycle arrest and myogenic differentiation: involvement of p120 and beta-catenin

N-cadherin is expressed throughout skeletal myogenesis and has been proposed to be involved in the differentiation program of myogenic precursors. Here, we further characterize the N-cadherin involvement and its mechanism of action at the onset of differentiation, through controlled N-cadherin activation by plating isolated C2 myoblasts on surfaces coated with a chimeric Ncad-Fc homophilic ligand (N-cadherin ectodomain fused to the immunoglobulin G Fc fragment). We show that N-cadherin activation substitutes for the cell density in myogenic differentiation by promoting myogenin and troponin T expression. In addition, N-cadherin adhesion participates to the associated cell cycle arrest through the nuclear accumulation of cyclin-dependent kinase inhibitors p21 and p27. Mouse primary myoblast cultures exhibited similar responses to N-cadherin as C2 cells. RNA interference knockdowns of the N-cadherin-associated cytoplasmic proteins p120 and beta-catenin produced opposite effects on the differentiation pathway. p120 silencing resulted in a decreased myogenic differentiation, associated with a reduction in cadherin-catenin content, which may explain its action on myogenic differentiation. beta-Catenin silencing led to a stimulatory effect on myogenin expression, without any effect on cell cycle. Our results demonstrate that N-cadherin adhesion may account for cell-cell contact-dependent cell cycle arrest and differentiation of myogenic cells, involving regulation through p120 and beta-catenins.