Cadherins In Adherens Junctions Research Articles

Somatic mutational landscapes of adherens junctions and their functional consequences in cutaneous melanoma development.

Cell-cell interaction in skin homeostasis is tightly controlled by adherens junctions (AJs). Alterations in such regulation lead to melanoma development. However, mutations in AJs and their functional consequences are still largely unknown.Methods: Cadherin mutations in skin cutaneous melanoma were identified using sequencing data from TCGA dataset, followed by cross-validation with data from non-TCGA cohorts. Mutations with significant occurrence were subjected to structural prediction using MODELLER and functional protein simulation using GROMACS software. Neo-antigen prediction was carried out using NetMHCpan tool. Cell-based fluorescence reporter assay was used to validate β-catenin activity in the presence of cadherin mutations. Clinical significance was analyzed using datasets from TCGA and other non-TCGA cohorts. Targeted gene exon sequencing and immunofluorescence staining on melanoma tissues were performed to confirm the in silico findings.Results: Highly frequent mutations in type-II classical cadherins were found in melanoma with one unique recurrent mutation (S524L) in the fifth domain of CDH6, which potentially destabilizes Ca2+-binding and cell-cell contacts. Mutational co-occurrence and physical dynamics analyses placed CDH6 at the center of the top-four mutated cadherins (core CDHs; all type-II), suggesting altered heterophilic interactions in melanoma development. Mutations in the intracellular domains significantly disturbed CDH6/β-catenin complex formation, resulting in β-catenin translocation into cytosol or nucleus and dysregulation of canonical Wnt/β-catenin signaling. Although mutations in core CDH genes correlated with advanced cancer stages and lymph node invasion, the overall and disease-free survival times in those patients were longer in patients with wild-type. Peptide/MHC-I binding affinity predictions confirmed overall increased neo-antigen potentials of mutated cadherins, which associated with T-lymphocyte infiltration and better clinical outcomes after immunotherapy.Conclusion: Changes in cell-cell communications by somatic mutations in AJ cadherins function as one of mechanisms to trigger melanoma development. Certain mutations in AJs may serve as potential neo-antigens which conversely benefit patients for longer survival times.

Cadherin flexibility provides a key difference between desmosomes and adherens junctions

Desmosomes and adherens junctions are intercellular adhesive structures essential for the development and integrity of vertebrate tissue, including the epidermis and heart. Their cell adhesion molecules are cadherins: type 1 cadherins in adherens junctions and desmosomal cadherins in desmosomes. A fundamental difference is that desmosomes have a highly ordered structure in their extracellular region and exhibit calcium-independent hyperadhesion, whereas adherens junctions appear to lack such ordered arrays, and their adhesion is always calcium-dependent. We present here the structure of the entire ectodomain of desmosomal cadherin desmoglein 2 (Dsg2), using a combination of small-angle X-ray scattering, electron microscopy, and solution-based biophysical techniques. This structure reveals that the ectodomain of Dsg2 is flexible even in the calcium-bound state and, on average, is shorter than the type 1 cadherin crystal structures. The Dsg2 structure has an excellent fit with the electron tomography reconstructions of human desmosomes. This fit suggests an arrangement in which desmosomal cadherins form trans interactions but are too far apart to interact in cis, in agreement with previously reported observations. Cadherin flexibility may be key to explaining the plasticity of desmosomes that maintain tissue integrity in their hyperadhesive form, but can adopt a weaker, calcium-dependent adhesion during wound healing and early development.

Correlative Light- and Electron Microscopy with chemical tags

Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.

Down-regulation of microRNAs of the miR-200 family and miR-205, and an altered expression of classic and desmosomal cadherins in spindle cell carcinoma of the head and neck—hallmark of epithelial-mesenchymal transition

MicroRNAs are small, noncoding RNAs that regulate gene expression by posttranscriptional regulation of target genes. miR-200 family and miR-205 have been shown experimentally to regulate epithelial-mesenchymal transition. As epithelial-mesenchymal transition is the postulated pathogenetic mechanism in spindle cell carcinoma, we analyzed the expression of these microRNAs in spindle cell carcinoma of the head and neck in comparison to conventional squamous cell carcinoma of similar location and stage. We also analyzed the expression of classic and desmosomal cadherins, which are believed to be important targets during epithelial-mesenchymal transition. Forty-five cases of spindle cell carcinoma and 45 cases of squamous cell carcinoma of the head and neck were analyzed using real-time polymerase chain reaction for microRNAs, and immunohistochemistry for classic cadherins (E- and N-cadherins) and desmosomal cadherins. We found a significant down-regulation of the miR-200 family and miR-205, loss of desmosomal cadherins, and an altered expression of classic cadherins in spindle cell carcinoma in comparison to squamous cell carcinoma. Down-regulation of the miR-200 family and miR-205 strongly supports the postulated role of epithelial-mesenchymal transition in spindle cell carcinoma. These microRNAs act on transcription repressors that were also up-regulated in our cases of spindle cell carcinoma, both on mRNA and on protein levels. The result is not only an altered expression of classic cadherins in adherens junctions but also a complete loss of desmosomal cadherins.

Hyper-adhesion in desmosomes: its regulation in wound healing and possible relationship to cadherin crystal structure

The resistance of tissues to physical stress is dependent upon strong cell-cell adhesion in which desmosomes play a crucial role. We propose that desmosomes fulfil this function by adopting a more strongly adhesive state, hyper-adhesion, than other junctions. We show that the hyper-adhesive desmosomes in epidermis resist disruption by ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) and are thus independent of Ca2+. We propose that Ca2+ independence is the normal condition for tissue desmosomes. Ca2+ independence is associated with an organised arrangement of the intercellular adhesive material exemplified by a dense midline. When epidermis is wounded, desmosomes in the wound-edge epithelium lose hyper-adhesiveness and become Ca2+ dependent, i.e. readily dissociated by EGTA. Ca2+-dependent desmosomes lack a midline and show narrowing of the intercellular space. We suggest that this indicates a less-organised, weakly adhesive arrangement of the desmosomal cadherins, resembling classical cadherins in adherens junctions. Transition to Ca2+ dependence on wounding is accompanied by relocalisation of protein kinase C alpha to desmosomal plaques suggesting that an 'inside-out' transmembrane signal is responsible for changing desmosomal adhesiveness. We model hyper-adhesive desmosomes using the crystal packing observed for the ectodomain of C-cadherin and show how the regularity of this 3D array provides a possible explanation for Ca2+ independence.

Differential molecular interactions of β-catenin and plakoglobin in adhesion, signaling and cancer

Plakoglobin and β-catenin are homologous proteins functioning in cell adhesion and transactivation. Their activities are controlled by three types of interactions: those with cadherins in adherens junctions, linking them to the actin cytoskeleton; interactions in the nucleus, where they bind to transcription factors and stimulate gene expression; interactions of free cytoplasmic β-catenin with axin and adenomatous polyposis coli (APC) protein which target it for degradation. Studies in the past year have demonstrated the complex interplay between these three types of interactions and the different behavior of β-catenin and plakoglobin in their involvement in morphogenesis and tumorigenesis strongly suggesting that catenins play key roles in adhesion-mediated signaling.

Effect of hepatocyte growth factor on cadherin-mediated cell-cell adhesion.

Hepatocyte growth factor (HGF) is known to induce the dispersion of epithelial cells, as scatter factor. On the other hand, cadherins play a crucial role in connecting cells together. Two groups of cadherins are involved in epithelial cell adhesion, those locating in adherens junctions (AJ) and in desmosomes. Here, we examined the effect of HGF on the function of these cadherins in keratinocyte cell lines F and 308R, which expressed E- and P-cadherin in AJ (referred to as AJ cadherins) and desmoplakin in desmosomes. In the presence of HGF, these cells spread more extensively than in control cultures and their associations apparently loosened. However, they maintained cell-cell contacts where cadherins and desmoplakin concentrated, although the level of the concentration was reduced by HGF treatment. When antibodies to E- and P-cadherins were added to cultures of these cells without HGF, AJ cadherins were redistributed into non-junctional areas of the cells, but desmoplakin still localized at cell-cell boundaries. When HGF was added together with anti-AJ cadherin antibodies to the cultures, cell-cell contacts were now disrupted. In these cultures, not only AJ cadherins but also desmoplakin were lost at cell-cell contact sites, indicating that HGF can disrupt desmosomal cell-cell adhesion when AJ cadherins are inactive. These results suggest that, although HGF cannot block cadherin-mediated cell-cell adhesion when the entire cadherin system is intact, it might modulate the activities of cadherins, especially, of desmosomal cadherins.