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A431 cells show loss of E-cadherin staining as they convert to A431D cells. A431 cells were grown in 10⁻⁷ M dexamethasone for 2 wk, transferred to glass coverslips, and processed for immunofluorescence. Phase microscopy (A and C) depicts the altered morphology of the cells as they are converted to A431D cells. Normal-looking A431 cells surround the islands of A431D cells in A and in the bottom right hand corner of C. Arrows in A and B point to islands of A431D cells. Arrows in C and D point to borders between the A431 parent cells and the A431D cells. Immunofluorescence microscopy shows cell border staining for E-cadherin (B and D). Note the absence of staining in the cells that have converted to A431D cells and the decreased staining in cells that appear to be converting to A431D cells (arrowheads). Bar, 30 μm.
Source publication
Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immedia...
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Three proteins identified by quite different criteria in three different systems, the Drosophila segment polarity gene armadillo, the human desmosomal protein plakoglobin, and the Xenopus E-cadherin-associated protein beta-catenin, share amino acid sequence similarity. These findings raise questions about the relationship among the three molecules...
Plakoglobin (Pg) and desmoplakin (DP) are adapter proteins within the desmosome, providing a mechanical link between desmosomal cadherins as transmembrane adhesion molecules and the intermediate filament cytoskeleton. Because in the severe skin blistering disease pemphigus autoantibodies against desmosomal adhesion molecules induce loss of keratino...
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Citations
... Consequently, the presence of JUP in the CDH17/DSC1 complex should contribute to the stability and resistance to stress of CTC clusters, underscoring the significance of desmosomal proteins in CRC invasion and metastasis. It's also noteworthy that JUP (also known as γ-catenin) connects both, desmosomal and classical cadherins, playing an essential role in the assembly and interplay of AJs and desmosomes [3,38]. Therefore, CDH17 might participate in the assembly of AJs and desmosomes through its interaction with DSC1 and JUP, playing a similar function to E-cadherin in the formation of nascent desmosomes [24]. ...
Background
Cadherin-17 (CDH17), a marker of differentiation in intestinal cells, binds and activates α2β1 integrin to promote cell adhesion and proliferation in colorectal cancer (CRC) metastasis. Furthermore, CDH17 associates with p120- and β-catenin in a manner yet to be fully elucidated. In this report, we explored the molecular mediators involved in this association, their contribution to CRC dissemination and potential therapeutic implications.
Methods
Proteomic and confocal analyses were employed to identify and validate CDH17 interactors. Functional characterization involved the study of proliferation, migration, and invasion in cell lines representative of various phenotypes. Immunohistochemistry was conducted on CRC tissue microarrays (TMA). In vivo animal experiments were carried out for metastatic studies.
Results
We found that desmocollin-1 (DSC1), a desmosomal cadherin, interacts with CDH17 via its extracellular domain. DSC1 depletion led to increased or decreased invasion in CRC cells displaying epithelial or mesenchymal phenotype, respectively, in a process mediated by the association with p120-catenin. Down-regulation of DSC1 resulted in an increased expression of p120-catenin isoform 1 in epithelial cells or a shift in cellular location in mesenchymal cells. Opposite results were observed after forced expression of CDH17. DSC1 is highly expressed in budding cells at the leading edge of the tumor and associates with poor prognosis in the stem-like, mesenchymal CRC subtypes, while correlates with a more favorable prognosis in the less-aggressive subtypes. In vivo experiments demonstrated that DSC1 silencing reduced tumor growth, liver homing, and metastasis in CRC mesenchymal cells. Furthermore, a synthetic peptide derived from CDH17, containing the NLV motif, effectively inhibited invasion and liver homing in vivo, opening up new possibilities for the development of novel therapies focused on desmosomal cadherins.
Conclusions
These findings shed light on the multifaceted roles of CDH17, DSC1, and p120-catenin in CRC metastasis, offering insights into potential therapeutic interventions for targeting desmosomal cadherins in poorly-differentiated carcinomas.
... Both Crk and CrkL were shown to localise to desmosomes and to be critical for desmosome integrity, in particular for the localisation of PG to the desmosomal plaque in the epidermis of Crk/CrkL knock-out mice (Badu-Nkansah and Lechler, 2020). These proteins were over-represented in cell-cell adhesion-relevant terms "cadherin binding" and "molecular adaptor activity" and outline the proximity with potential signalling crosstalk to the neighbouring cadherinand nectin-associated adherens junctions (Barron et al., 2008;Lewis et al., 1997;Vasioukhin et al., 2000;Yoshida et al., 2010). A novel association appeared in Cluster 9 that hinted at some crosstalk of desmosomes with tight junctions. ...
... Desmosomes also become Ca 2+ -dependent upon epithelial wounding (Kimura et al., 2007) and during wound healing, in areas where keratinocytes become activated for migration that requires motility and desmosome turnover. At this time desmosomes are less stable than during hyperadhesion (Bartle et al., 2020;Fülle et al., 2021;Kimura et al., 2007;Wallis et al., 2000) and both assembly and internalisation require crosstalk with the actomyosin machinery (Fülle et al., 2021;Lewis et al., 1997;Vasioukhin et al., 2001). Such crosstalk is reflected particularly in group of prey hits of PG* that were enriched in Ca 2+ -dependent conditions (cluster 11 in Fig. 4 A; Fig. S3 B and C; Fig. S4) and were mostly linked to actin associated terms ("actin-dependent AT-Pase activity", "actin filament binding", "actin binding" and "microfilament motor activity") ( Another large fraction of Dsc2a* proximal interactors (cluster 5 in Fig. 4 A and Fig. S4) fall into similar categories as actin regulatory proteins and contain the Rho GT-Pases and related proteins, such as Cdc42 effector protein 4 (CDC42EP4), RAS related (RRAS) and p21 (RAC1) activated kinase 2 (PAK2). ...
... This compartmentalisation of junctions is clearly indicated in our findings where the AJ proteins nectin 2 and 3 and α-catenin are close to Dsc2a when desmosomes are Ca 2+ -dependent (Box 1. in Fig. 6 B), but become spatially more distinct as desmosomes mature to hyper-adhesion (Box 1. in Fig. 6 C). These proteins, however, retain proximity to PG, possibly reflecting the capacity of PG to locate at adherens junctions as well as desmosomes (Lewis et al., 1997). This highlights the fact that both PG and Pkp2 can reside in non-desmosomal locations. ...
Desmosomes are multiprotein adhesion complexes that link intermediate filaments to the plasma membrane, ensuring the mechanical integrity of cells across tissues, but how they participate in the wider signalling network to exert their full function is unclear. To investigate this we carried out multiplexed protein proximity mapping using biotinylation (BioID). The combined interactomes of the essential desmosomal proteins desmocollin 2a, plakoglobin and plakophilin 2a (Pkp2a) in Madin-Darby canine kidney epithelial cells were mapped and their differences and commonalities characterised as desmosome matured from Ca ²⁺ -dependence to the mature, Ca ²⁺ -independent, hyper-adhesive state, which predominates in tissues. Results suggest that individual desmosomal proteins have distinct roles in connecting to cellular signalling pathways and that these roles alter substantially when cells change their adhesion state. The data provide further support for a dualistic concept of desmosomes in which the properties of Pkp2a differ from those of the other, more stable proteins. This body of data provides an invaluable resource for analysis of desmosome function.
... 192,220 Analogously to TJs, the development of desmosomes depends on a prior formation of AJs. 232 According to Lewis et al. (1997), Pg, which is the only common component of both, desmosomes and AJs, plays a crucial role in the desmosomal-AJ crosstalk. 232 Only the co-expression of E-cadherin and Pg stimulated the subsequent assembly of desmosomes. ...
... 192,220 Analogously to TJs, the development of desmosomes depends on a prior formation of AJs. 232 According to Lewis et al. (1997), Pg, which is the only common component of both, desmosomes and AJs, plays a crucial role in the desmosomal-AJ crosstalk. 232 Only the co-expression of E-cadherin and Pg stimulated the subsequent assembly of desmosomes. ...
... 232 According to Lewis et al. (1997), Pg, which is the only common component of both, desmosomes and AJs, plays a crucial role in the desmosomal-AJ crosstalk. 232 Only the co-expression of E-cadherin and Pg stimulated the subsequent assembly of desmosomes. 232 In addition, for the full maturation of desmosomes, the presence of both Dsg and Dsc is essential. ...
This thesis aimed the development of a correlated device which combines FluidFM® with Fluorescence Microscopy (FL) (FL-FluidFM®) and enables the simultaneous quantification of adhesion forces and fluorescent visualization of mature cells. The implementation of a PIFOC was crucial to achieve a high-resolution as well as a stable but dynamic focus level. The functionality of SCFS after hardware modification was verified by comparing two force-curves, both showing the typical force progression and measured with the optimized and conventional hardware, respectively. Then, the integration of FL was examined by detaching fluorescently labeled REF52 cells. The fluorescence illumination of the cytoskeleton showed the expected characteristic force profile and no evidence of interference effects. Afterwards a corresponding correlative data analysis was addressed including manual force step fitting, the identification of visualized cellular unbinding, and a time-dependent correlation. This procedure revealed a link between the area of cytoskeletal unbinding and force-jumps. This was followed by a comparison of the detachment characteristics of intercellular connected HUVECs and individual REF52 cells. HUVECs showed maximum detachment forces in the same order of magnitude as the ones of single REF52 cells. This contrasted with the expected strong cohesiveness of endothelial cells and indicated a lack of cell-cell contact formation. The latter was confirmed by a comparison of HUVECs, primary HBMVECs, and immortalized EA.hy926 cells fluorescently labeled for two marker proteins of intercellular junctions. This unveiled that both the previous cultivation duration and the cell type have a major impact on the development of intercellular junctions. In summary, the correlative FL FluidFM® represents a powerful novel approach, which enables a truly contemporaneous performance and, thus, has the potential to reveal new insights into the mechanobiological properties of cell adhesion.
... While not considered an essential component of AJ per se (Aktary et al., 2017), Lewis et al. showed that γ-catenin plays a vital role in the stability of these adhesive complexes as epithelial cells lacking both γ-catenin and E-cadherin were unable to form AJs. Reintroduction of E-cadherin only, which facilitated the formation of AJs, was insufficient to stabilize the AJs. However, reintroduction of both γ-catenin and E-cadherin resulted in AJ stabilization and facilitated the organization of desmosomal junctions (Aktary et al., 2017;Lewis et al., 1997). Interestingly γ-catenin is also found in desmosomal junctions where it binds to desmosomal cadherins Desmocollin and Desmoglein. ...
The function and structure of the mammalian epithelial cell layer is maintained by distinct intercellular adhesion complexes including adherens junctions (AJs), tight junctions, and desmosomes. The AJ is most integral for stabilizing cell-cell adhesion and conserving the structural integrity of epithelial tissues. AJs are comprised of the transmembrane protein E-cadherin and cytoplasmic catenin cofactors (α, β, γ, and p120-catenin). One organ where malfunction of AJ is a major contributor to disease states is the mammalian intestine. In the intestine, cell-cell adhesion complexes work synergistically to maintain structural integrity and homeostasis of the epithelium and prevent its malfunction. Consequently, when AJ integrity is compromised in the intestinal epithelium, the ensuing homeostatic disruption leads to diseases such as inflammatory bowel disease and colorectal carcinoma. In addition to their function at the plasma membrane, protein components of AJs also have nuclear functions and are thus implicated in regulating gene expression and intracellular signaling. Within the nucleus, AJ proteins have been shown to interact with transcription factors such as TCF/LEF and Kaiso ( ZBTB33 ), which converge on the canonical Wnt signaling pathway. The multifaceted nature of AJ proteins highlights their complexity in modulating homeostasis and emphasizes the importance of their subcellular localization and expression in the mammalian intestine. In this review, we summarize the nuclear roles of AJ proteins in intestinal tissues; their interactions with transcription factors and how this leads to crosstalk with canonical Wnt signaling; and how nuclear AJ proteins are implicated in intestinal homeostasis and disease.
... We further evaluated the membranous expression of β-catenin, the key mediator of canonical Wnt signalling (Wodarz and Nusse 1998) and a component of a protein complex that constitutes adherens junctions (Lewis et al., 1997). β-catenin expression was robust in elongated and polarised cells lining the nasopharyngeal duct, especially in apical parts of epithelial cells ( Figure 10Q; Figure 11O). ...
Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5), a Wnt pathway member, has been previously recognised as a stem cell marker in numerous epithelial tissues. In this study, we used Lgr5-EGFP-CreERT2 mice to analyse the distribution of LGR5-positive cells during craniofacial development. LGR5 expressing cells were primarily located in the mesenchyme adjacent to the craniofacial epithelial structures undergoing folding, such as the nasopharyngeal duct, lingual groove, and vomeronasal organ. To follow the fate of LGR5-positive cells, we performed lineage tracing using an inducible Cre knock-in allele in combination with Rosa26-tdTomato reporter mice. The slight expansion of LGR5-positive cells was found around the vomeronasal organ, in the nasal cavity, and around the epithelium in the lingual groove. However, most LGR5 expressing cells remained in their original location, possibly supporting their signalling function for adjacent epithelium rather than exerting their role as progenitor cells for the craniofacial structures. Moreover, Lgr5 knockout mice displayed distinct defects in LGR5-positive areas, especially in the reduction of the nasopharyngeal duct, the alteration of the palatal shelves shape, abnormal epithelial folding in the lingual groove area, and the disruption of salivary gland development. The latter defect manifested as an atypical number and localisation of the glandular ducts. The gene expression of several Wnt pathway members (Rspo1-3, Axin2) was altered in Lgr5-deficient animals. However, the difference was not found in sorted EGFP-positive cells obtained from Lgr5 +/+ and Lgr5 −/− animals. Expression profiling of LGR5-positive cells revealed the expression of several markers of mesenchymal cells, antagonists, as well as agonists, of Wnt signalling, and molecules associated with the basal membrane. Therefore, LGR5-positive cells in the craniofacial area represent a very specific population of mesenchymal cells adjacent to the epithelium undergoing folding or groove formation. Our results indicate a possible novel role of LGR5 in the regulation of morphogenetic processes during the formation of complex epithelial structures in the craniofacial areas, a role which is not related to the stem cell properties of LGR5-positive cells as was previously defined for various epithelial tissues.
... The most striking finding was the reorganization of E-cadherin, a key marker of epithelial cells [24], by 18 hpi. E-cadherin is crucial in the establishment and maintenance of the cellular junction complex as a whole [25,26], and aberrant expression of E-cadherin is a hallmark of epithelial dysregulation [7]. Additionally, we observed an increase in vimentin staining correlating to the increase in vimentin mRNA. ...
Human astroviruses (HAstV), positive sense single-stranded RNA viruses, are one of the leading causes of diarrhea worldwide. Despite their high prevalence, the cellular mechanisms of astrovirus pathogenesis remain ill-defined. Previous studies showed HAstV increased epithelial barrier permeability by causing a re-localization of the tight junction protein, occludin. In these studies, we demonstrate that HAstV replication induces epithelial-mesenchymal transition (EMT), by upregulating the transcription of EMT-related genes within 8 hours post-infection (hpi), followed by the loss of cell-cell contacts and disruption of polarity by 24 hpi. While multiple classical HAstV serotypes, including clinical isolates, induce EMT, the non-classical genotype HAstV-VA1 and two strains of reovirus are incapable of inducing EMT. Unlike the re-localization of tight junction proteins, HAstV-induced EMT requires productive replication and is dependent transforming growth factor-β (TGF-β) activity. Finally, inhibiting TGF-β signaling and EMT reduces viral replication, highlighting its importance in the viral life cycle. This finding puts classical strains of HAstV-1 in an exclusive group of non-oncogenic viruses triggering EMT.
... The DJs are formed at the basal side of the AJC and play a role in the mechanical connections between neighboring cells (Garrod & Chidgey, 2008). The DJ formation is also dependent on the AJ formation (Lewis et al., 1997). ...
Multilayered proliferation in an adherent culture as well as proliferation in a suspension culture is a characteristic feature of cancer cells. We previously showed using T47D human mammary cancer cells that nectin‐4, upregulated in many cancer cells, cis‐interacts with ErbB2 and its trastuzumab‐resistant splice variants, p95‐ErbB2 and ErbB2ΔEx16, and enhances DNA synthesis mainly through the PI3K‐AKT pathway in an adherent culture. We showed here that only the combination of nectin‐4 and p95‐ErbB2, but not that of nectin‐4 and ErbB2 or that of nectin‐4 and ErbB2ΔEx16, cooperatively enhanced multilayered T47D cell proliferation through the Hippo pathway‐mediated SOX2 gene expression in an adherent culture. T47D cells expressed the components of the apical junctional complex (AJC) consisting of adherens junctions (AJs) and tight junctions and cell polarity molecules, but not the AJ component afadin. The AJC and apicobasal polarity were disorganized in T47D cells in a monolayer and T47D cells stably expressing both nectin‐4 and p95‐ErbB2 in multilayers. These results indicate that nectin‐4 and p95‐ErbB2 play a stimulatory role in multilayered proliferation in an adherent culture.
... They share a high degree of sequence similarity, and both plakoglobin and β-catenin contain 12 Armadillo repeats that allow for interactions with a wide variety of targets and results in overlapping functions (13). Both plakoglobin and β -catenin are required for cell-cell adhesion, contributing to the formation of adherens junctions that confer structural integrity to epithelial and non-epithelial cells (14)(15)(16); however, they can also have divergent functions, as only plakoglobin is present in desmosomes where it participates in cell-cell adhesion and provide resistance to mechanical stress (17). Plakoglobin may also compete with β -catenin for binding to TCF7L2, the transcriptional coactivator of the Wnt signalling pathway and inhibit β -catenin-dependent transcriptional activity in a context-. ...
The scaffold protein 14-3-3ζ is an established regulator of adipogenesis and postnatal adiposity. We and others have demonstrated that the 14-3-3ζ interactome to be diverse and dynamic, and it can be examined to identify novel regulators of physiological processes, including adipogenesis. In the present study, we sought to determine if factors that influence adipogenesis could be identified in the 14-3-3ζ interactome found in white adipose tissue of lean or obese TAP-tagged-14-3-3ζ overexpressing mice. Using mass spectrometry, changes in the abundance of novel, as well as established, adipogenic factors within the 14-3-3ζ interactome could detected. One novel candidate is plakoglobin, the homolog of the known adipogenic inhibitor β-catenin, and herein, we report that plakoglobin is involved in adipocyte differentiation. Plakoglobin is expressed in murine 3T3-L1 cells and is primarily localized to the nucleus, where its abundance decreases during adipogenesis. Ectopic overexpression and siRNA-mediated depletion of plakoglobin had dual effects on inhibiting adipogenesis and reducing PPARγ2 expression. Plakoglobin depletion in human adipose-derived stem cells also impaired adipogenesis and reduced lipid accumulation post-differentiation. Transcriptional assays indicated that plakoglobin does not participate in Wnt/β-catenin signaling, as its depletion did not affect Wnt3a-mediated SUPERTOPFlash activity. Taken together, our results establish plakoglobin as a novel regulator of adipogenesis in vitro and highlights the ability of using the 14-3-3ζ interactome to discover undiscovered pro-obesogenic factors.
... The parental A-431D line does not express E-cadherin, but it overexpresses EGFR (36). We also used MCF-10A cells-an extensively used human breast epithelial line that expresses normal levels of EGFR and E-cadherin. ...
... Studies of E-cadherin/EGFR interactions in mechanically stretched epithelial cells used A-431D epidermoid carcinoma cells that were engineered to stably express the full-length human E-cadherin (A-431D Ecad ) with a C-terminal green fluorescent protein (GFP) (deposited by Dr. Jennifer Stow, Addgene plasmid, 28009) (71). The A-431D line (from Prof. Keith Johnson, University of Nebraska) over expresses EGFR but does not express endogenous classical cadherins (36). Cell stretching studies were also done with MCF10A cells, which express typical levels of E-cadherin and EGFR. ...
Significance
Force transduction at interepithelial junctions involves E-cadherin–mediated activation of epidermal growth factor receptor (EGFR) signaling, which modulates local cytoskeletal remodeling and cell proliferation. Findings show that E-cadherin and EGFR form a heteroreceptor complex at the membrane. Increased tension on E-cadherin bonds disrupts the complex in the absence of epidermal growth factor (EGF), but the mechanical activation of EGFR signaling requires soluble EGF. Fully quantified spectral imaging fluorescence resonance energy transfer measurements further revealed that E-cadherin and EGFR form a heterotrimeric complex at the plasma membrane, comprising two E-cadherins bound to an EGFR monomer. These results suggest that tugging forces on E-cadherin adhesions activate force transduction cascades, by releasing EGFR monomers from the complex, to enable EGFR to homodimerize, bind EGF, and signal.
... The SNAP-tag allows fast and highly specific binding of any molecule functionalized with its ligand benzylguanine. 32 The expression of SNAP-E-cadherin in A431D cells, which lack endogenous expression of E-cadherin, 33 was successful but did not facilitate cell−cell adhesion ( Figure S2A). We designed a 45 base pair long DNA linker consisting of identical benzylguanine-tagged anchor strands, which covalently bind to the SNAP-tag and hybridize with two complementary linker strands. ...
The binding strength between epithelial cells is crucial for tissue integrity, signal transduction and collective cell dynamics. However, there is no experimental approach to precisely modulate cell–cell adhesion strength at the cellular and molecular level. Here, we establish DNA nanotechnology as a tool to control cell–cell adhesion of epithelial cells. We designed a DNA-E-cadherin hybrid system consisting of complementary DNA strands covalently bound to a truncated E-cadherin with a modified extracellular domain. DNA sequence design allows to tune the DNA-E-cadherin hybrid molecular binding strength, while retaining its cytosolic interactions and downstream signaling capabilities. The DNA-E-cadherin hybrid facilitates strong and reversible cell–cell adhesion in E-cadherin deficient cells by forming mechanotransducive adherens junctions. We assess the direct influence of cell–cell adhesion strength on intracellular signaling and collective cell dynamics. This highlights the scope of DNA nanotechnology as a precision technology to study and engineer cell collectives.
