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Tert-butyl hydroperoxide (t-BuOOH) is an organic hydroperoxide widely used as a model compound to induce oxidative stress. It leads to a plethora of cellular damage, including lipid peroxidation, DNA double-strand breaks (DNA DSBs), and breakdown of the mitochondrial membrane potential (MMP). We could show in several cell lines that t-BuOOH induces ferroptosis, triggered by iron-dependent lipid peroxidation. We have further revealed that not only t-BuOOH-mediated ferroptosis, but also DNA DSBs and loss of MMP are prevented by cell–cell contacts. The underlying mechanisms are not known. Here, we show in murine fibroblasts and a human colon carcinoma cell line that t-BuOOH (50 or 100 µM, resp.) causes an increase in intracellular Ca2+, and that this increase is key to lipid peroxidation and ferroptosis, DNA DSB formation and dissipation of the MMP. We further demonstrate that cell–cell contacts prevent t-BuOOH-mediated raise in intracellular Ca2+. Hence, we provide novel insights into the mechanism of t-BuOOH-triggered cellular damage including ferroptosis and propose a model in which cell–cell contacts control intracellular Ca2+ levels to prevent lipid peroxidation, DNA DSB-formation and loss of MMP. Since Ca2+ is a central player of toxicity in response to oxidative stress and is involved in various cell death pathways, our observations suggest a broad protective function of cell–cell contacts against a variety of exogenous toxicants. [ABSTRACT FROM AUTHOR]

Meibomian gland dysfunction (MGD) is one of the main causes of dry eye disease. To better understand the physiological functions of human meibomian glands (MGs), the present study compared MGs with free sebaceous glands (SGs) and hair-associated SGs of humans using morphological, immunohistochemical, and liquid chromatography—mass spectrometry (LCMS)-based lipidomic approaches. Eyelids with MGs, nostrils, lips, and external auditory canals with free SGs, and scalp with hair-associated SGs of body donors were probed with antibodies against cytokeratins (CK) 1, 8, 10, and 14, stem cell markers keratin 15 and N-cadherin, cell–cell contact markers desmoglein 1 (Dsg1), desmocollin 3 (Dsc3), desmoplakin (Dp), plakoglobin (Pg), and E-cadherin, and the tight junction protein claudin 5. In addition, Oil Red O staining (ORO) was performed in cryosections. Secretions of MGs as well as of SGs of nostrils, external auditory canals, and scalps were collected from healthy volunteers, analyzed by LCMS, and the data were processed using various multivariate statistical analysis approaches. Serial sections of MGs, free SGs, and hair-associated SGs were 3D reconstructed and compared. CK1 was expressed differently in hair-associated SGs than in MGs and other free SGs. The expression levels of CK8, CK10, and CK14 in MGs were different from those in hair-associated SGs and other free SGs. KRT15 was expressed differently in hair-associated SGs, whereas N-cadherin was expressed equally in all types of glands. The cell–cell contact markers Dsg1, Dp, Dsc3, Pg, and E-cadherin revealed no differences. ORO staining showed that lipids in MGs were more highly dispersed and had larger lipid droplets than lipids in other free SGs. Hair-associated SGs had a smaller number of lipid droplets. LCMS revealed that the lipid composition of meibum was distinctively different from that of the sebum of the nostrils, external auditory canals, and scalp. The 3D reconstructions of the different glands revealed different morphologies of the SGs compared with MGs which are by far the largest type of glands. In humans, MGs differ in their morphology and secretory composition and show major differences from free and hair-associated SGs. The composition of meibum differs significantly from that of sebum from free SGs and from hair-associated SGs. Therefore, the MG can be considered as a highly specialized type of holocrine gland that exhibits all the histological characteristics of SGs, but is significantly different from them in terms of morphology and lipid composition. [ABSTRACT FROM AUTHOR]

Tumour cell detachment from the primary tumour is an early and crucial step of the metastatic cascade. At the single cell level, it was already shown that migrating melanoma cells establish both intra‐ and extracellular pH gradients and that the Na+/H+ exchanger NHE1 accumulates at the leading edges to strengthen cell‐matrix interactions. However, less is known about the role of NHE1 in collective cell migration and the specific pH microenvironment at tumour cell–cell contacts. We used MV3 melanoma cells transfected with a NHE1‐expressing vector or a control vector. NHE1 localization at cell–cell contacts was assessed via immunofluorescence imaging. Collective migration was analysed by live‐cell imaging. The NHE1 activity and the perimembranous pH were measured both intra‐ and extracellularly by ratiometric fluorescence microscopy. NHE1 clearly localizes at cell–cell contacts. Its overexpression further increases migratory speed and translocation in multidirectional pathway analyses. NHE1 overexpressing MV3 cells also move further away from their neighbouring cells during wound closure assays. pH measurements revealed that the NHE1 is highly active at cell–cell contacts of melanoma cells. NHE1‐mediated pH dynamics at such contact sites are more prominent in NHE1‐overexpressing melanoma cells. Our findings highlight the contribution of the NHE1 towards modulation and plasticity of melanoma cell–cell contacts. We propose that its localization and functional activity at cell–cell contacts promotes evasion of single melanoma cells from the primary tumour. [ABSTRACT FROM AUTHOR]

Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na+/K+-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.

Simple Summary: Connexins are proteins which comprise gap junctions in cells. These junctions can directly connect neighboring cells and the cell interior with the extracellular microenvironment and thus they act as tissue integrators. Alterations in connexin regulation can lead to unfavorable shifts in the tissue adhesive context thus eradicating the constraints of the normal tissue microenvironment, triggering (or enhancing) cell motility. This review tries to examine the role of connexins in orchestrating the tumor microenvironment and hence their role in malignancy. Today's research on the processes of carcinogenesis and the vital activity of tumor tissues implies more attention be paid to constituents of the tumor microenvironment and their interactions. These interactions between cells in the tumor microenvironment can be mediated via different types of protein junctions. Connexins are one of the major contributors to intercellular communication. They form the gap junctions responsible for the transfer of ions, metabolites, peptides, miRNA, etc., between neighboring tumor cells as well as between tumor and stromal cells. Connexin hemichannels mediate purinergic signaling and bidirectional molecular transport with the extracellular environment. Additionally, connexins have been reported to localize in tumor-derived exosomes and facilitate the release of their cargo. A large body of evidence implies that the role of connexins in cancer is multifaceted. The pro- or anti-tumorigenic properties of connexins are determined by their abundance, localization, and functionality as well as their channel assembly and non-channel functions. In this review, we have summarized the data on the contribution of connexins to the formation of the tumor microenvironment and to cancer initiation and progression. [ABSTRACT FROM AUTHOR]

Mechanical stiffness is recognized as a key physical factor and directs cell function via a mechanotransduction process, from extracellular physical cues to intracellular signaling cascades that affect transcriptional activity. Cells continually receive mechanical signals from both the surrounding matrix and adjacent cells. However, how mechanical stiffness cue at cell–substrate interfaces coordinates cell–cell junctions in guiding mesenchymal stem cell behaviors is poorly understood. Here, polydimethylsiloxane substrates with different stiffnesses were used to study mechanosensation/transduction mechanisms in controlling odontogenic differentiation of dental papilla cells (DPCs). DPC phenotypes (morphology and differentiation) changed in response to the applied force derived from stiff substrates. Significantly, higher expression of paxillin on stiffer substrates promoted DPC dentinogenesis. Upon treatment with siRNA to knockdown paxillin, N-cadherin increased mainly in the cytomembrane at the area of cell–cell contacts, whereas β-catenin decreased in the nuclei. The result of a double luciferase reporter assay showed that stiffness promoted β-catenin binding to TCF, which could coactivate the target genes associated with odontogenic differentiation, as evidenced by bioinformatics analysis. Finally, we determined that the addition of a β-catenin inhibitor suppressed DPC mineralization in all the stiffness groups. Thus, our results indicated that a mechanotransduction process from cell–substrate interactions to cell–cell adhesions was required for DPC odontogenic differentiation under the stimulation of substrate stiffness. This finding suggests that stem cell fate specification under the stimulus of stiffness at the substrates is based on crosstalk between substrate interactions and adherens junctions, which provides an essential mechanism for cell-based tissue engineering. [ABSTRACT FROM AUTHOR]

Cell–cell junctions are pivotal for embryogenesis and tissue homeostasis but also play a major role in tumorigenesis, tumor invasion, and metastasis. E-cadherin (CDH1) and N-cadherin (CDH2) are two adherens junction's transmembrane glycoproteins with tissue-specific expression patterns in epithelial and neural/mesenchymal cells. Aberrant expression has been implicated in the process of epithelial–mesenchymal transition (EMT) in malignant tumors. We could hitherto demonstrate cis-E:N-cadherin heterodimer in endoderm-derived cells. Using immunoprecipitation in cultured cells of the line PLC as well as in human hepatocellular carcinoma (HCC)-lysates, we isolated E-N-cadherin heterodimers in a complex with the plaque proteins α- and β-catenin, plakoglobin, and vinculin. In confocal laser scanning microscopy, E-cadherin co-localized with N-cadherin at the basolateral membrane of normal hepatocytes, hepatocellular adenoma (HCA), and in most cases of HCC. In addition, we analyzed E- and N-cadherin expression via immunohistochemistry in a large cohort of 868 HCCs from 570 patients, 25 HCA, and respective non-neoplastic liver tissue, and correlated our results with multiple prognostic markers. While E- or N-cadherin were similarly expressed in tumor sites with vascular invasion or HCC metastases, HCC with vascular encapsulated tumor clusters (VETC) displayed slightly reduced E-cadherin, and slightly increased N-cadherin expression. Analyzing The Cancer Genome Atlas patient cohort, we found that reduced mRNA levels of CDH1, but not CDH2 were significantly associated with unfavorable prognosis; however, in multivariate analysis, CDH1 did not correlate with prognosis. In summary, E- and N-cadherin are specific markers for hepatocytes and derived HCA and HCC. E:N-cadherin heterodimers are constitutively expressed in the hepatocytic lineage and only slightly altered in malignant progression, thereby not complying with the concept of EMT. [ABSTRACT FROM AUTHOR]

Claudin-4, a protein with the structure of classic claudins most often found in cell-cell junctions, is frequently overexpressed in epithelial cancers where its localization has not been studied. In this study we aimed to find out where this membrane protein is localized in an ovarian tumor model, OVCAR3 cells, that express high levels of the protein. Immunohistochemical studies showed claudin-4 staining in a perinuclear region, at most plasma membranes and in cytoplasmic puncta. Native claudin-4 did not overlap with phosphorylated claudin-4, which was partially located in focal adhesions. Using claudin-4 BioID technology we confirmed that large amounts of claudin-4 are localized to the Golgi compartment, including in dispersed Golgi in cells where claudin-4 is partially knocked down and in dividing cells. Claudin-4 appears to be present in the vicinity of several types of cell-cell junctions, but there is no evidence that it forms tight junctions in these tumor cells. Both claudin-4, the Golgi marker GM130, and the plasma membrane receptor Notch2 were found in dispersed Golgi in dividing cells. This definition of the cellular architecture of claudin-4 should provide a framework for better understanding of the function of claudin-4 in tumor cells and its molecular interactions.

Simple Summary: The differential diagnosis of primary liver cancer versus liver metastases of an extrahepatic primary tumor may be challenging, especially in carcinoma of the pancreatobiliary phenotype, as in intrahepatic cholangiocarcinoma or metastases of ductal adenocarcinoma of the pancreas. Nevertheless, the distinction is of fundamental importance for therapy planning. In the frame of this study, we focus on the differential expression and localization of the cell–cell contact-proteins E-cadherin and N-cadherin, markers for cell differentiation in normal epithelia of the pancreaticobiliary tract as well as in derived tumors, and demonstrate that N-cadherin positivity distinguishes intrahepatic cholangiocarcinoma from liver metastases of ductal adenocarcinoma of the pancreas. We propose that N-cadherin-positivity together with other biliary markers may be used for this important histopathological differential diagnosis and may thus improve the accuracy of cholangiocarcinoma diagnosis. Carcinomas of the pancreatobiliary system confer an especially unfavorable prognosis. The differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and its subtypes versus liver metastasis of ductal adenocarcinoma of the pancreas (PDAC) is clinically important to allow the best possible therapy. We could previously show that E-cadherin and N-cadherin, transmembrane glycoproteins of adherens junctions, are characteristic features of hepatocytes and cholangiocytes. We therefore analyzed E-cadherin and N-cadherin in the embryonally related epithelia of the bile duct and pancreas, as well as in 312 iCCAs, 513 carcinomas of the extrahepatic bile ducts, 228 gallbladder carcinomas, 131 PDACs, and precursor lesions, with immunohistochemistry combined with image analysis, fluorescence microscopy, and immunoblots. In the physiological liver, N-cadherin colocalizes with E-cadherin in small intrahepatic bile ducts, whereas larger bile ducts and pancreatic ducts are positive for E-cadherin but contain decreasing amounts of N-cadherin. N-cadherin was highly expressed in most iCCAs, whereas in PDACs, N-cadherin was negative or only faintly expressed. E- and N-cadherin expression in tumors of the pancreaticobiliary tract recapitulate their expression in their normal tissue counterparts. N-cadherin is a helpful marker for the differential diagnosis between iCCA and PDAC, with a specificity of 96% and a sensitivity of 67% for small duct iCCAs and 50% for large duct iCCAs. [ABSTRACT FROM AUTHOR]

The Drosophila nervous system comprises a small number of well characterized glial cell classes. The outer surface of the central nervous system (CNS) is protected by a glial derived blood-brain barrier generated by perineurial and subperineurial glia. All neural stem cells and all neurons are engulfed by cortex glial cells. The inner neuropil region, that harbors all synapses and dendrites, is covered by ensheathing glia and infiltrated by astrocyte-like glial cells. All these glial cells show a tiled organization with an often remarkable plasticity where glial cells of one cell type invade the territory of the neighboring glial cell type upon its ablation. Here, we summarize the different glial tiling patterns and based on the different modes of cell-cell contacts we hypothesize that different molecular mechanisms underlie tiling of the different glial cell types. [ABSTRACT FROM AUTHOR]

The Drosophila nervous system comprises a small number of well characterized glial cell classes. The outer surface of the central nervous system (CNS) is protected by a glial derived blood-brain barrier generated by perineurial and subperineurial glia. All neural stem cells and all neurons are engulfed by cortex glial cells. The inner neuropil region, that harbors all synapses and dendrites, is covered by ensheathing glia and infiltrated by astrocyte-like glial cells. All these glial cells show a tiled organization with an often remarkable plasticity where glial cells of one cell type invade the territory of the neighboring glial cell type upon its ablation. Here, we summarize the different glial tiling patterns and based on the different modes of cell-cell contacts we hypothesize that different molecular mechanisms underlie tiling of the different glial cell types.

Today’s research on the processes of carcinogenesis and the vital activity of tumor tissues implies more attention be paid to constituents of the tumor microenvironment and their interactions. These interactions between cells in the tumor microenvironment can be mediated via different types of protein junctions. Connexins are one of the major contributors to intercellular communication. They form the gap junctions responsible for the transfer of ions, metabolites, peptides, miRNA, etc., between neighboring tumor cells as well as between tumor and stromal cells. Connexin hemichannels mediate purinergic signaling and bidirectional molecular transport with the extracellular environment. Additionally, connexins have been reported to localize in tumor-derived exosomes and facilitate the release of their cargo. A large body of evidence implies that the role of connexins in cancer is multifaceted. The pro- or anti-tumorigenic properties of connexins are determined by their abundance, localization, and functionality as well as their channel assembly and non-channel functions. In this review, we have summarized the data on the contribution of connexins to the formation of the tumor microenvironment and to cancer initiation and progression.

Cell–cell junctions are pivotal for embryogenesis and tissue homeostasis but also play a major role in tumorigenesis, tumor invasion, and metastasis. E-cadherin (CDH1) and N-cadherin (CDH2) are two adherens junction’s transmembrane glycoproteins with tissue-specific expression patterns in epithelial and neural/mesenchymal cells. Aberrant expression has been implicated in the process of epithelial–mesenchymal transition (EMT) in malignant tumors. We could hitherto demonstrate cis-E:N-cadherin heterodimer in endoderm-derived cells. Using immunoprecipitation in cultured cells of the line PLC as well as in human hepatocellular carcinoma (HCC)-lysates, we isolated E-N-cadherin heterodimers in a complex with the plaque proteins α- and β-catenin, plakoglobin, and vinculin. In confocal laser scanning microscopy, E-cadherin co-localized with N-cadherin at the basolateral membrane of normal hepatocytes, hepatocellular adenoma (HCA), and in most cases of HCC. In addition, we analyzed E- and N-cadherin expression via immunohistochemistry in a large cohort of 868 HCCs from 570 patients, 25 HCA, and respective non-neoplastic liver tissue, and correlated our results with multiple prognostic markers. While E- or N-cadherin were similarly expressed in tumor sites with vascular invasion or HCC metastases, HCC with vascular encapsulated tumor clusters (VETC) displayed slightly reduced E-cadherin, and slightly increased N-cadherin expression. Analyzing The Cancer Genome Atlas patient cohort, we found that reduced mRNA levels of CDH1, but not CDH2 were significantly associated with unfavorable prognosis; however, in multivariate analysis, CDH1 did not correlate with prognosis. In summary, E- and N-cadherin are specific markers for hepatocytes and derived HCA and HCC. E:N-cadherin heterodimers are constitutively expressed in the hepatocytic lineage and only slightly altered in malignant progression, thereby not complying with the concept of EMT.

Breast cancer (BC) is the leading cause of death from malignant neoplasms among women worldwide, and metastatic BC presents the biggest problems for treatment. Previously, it was shown that lower expression of ELOVL5 and IGFBP6 genes is associated with a higher risk of the formation of distant metastases in BC. In this work, we studied the change in phenotypical traits, as well as in the transcriptomic and proteomic profiles of BC cells as a result of the stable knockdown of ELOVL5 and IGFBP6 genes. The knockdown of ELOVL5 and IGFBP6 genes was found to lead to a strong increase in the expression of the matrix metalloproteinase (MMP) MMP1. These results were in good agreement with the correlation analysis of gene expression in tumor samples from patients and were additionally confirmed by zymography. The knockdown of ELOVL5 and IGFBP6 genes was also discovered to change the expression of a group of genes involved in the formation of intercellular contacts. In particular, the expression of the CDH11 gene was markedly reduced, which also complies with the correlation analysis. The spheroid formation assay showed that intercellular adhesion decreased as a result of the knockdown of the ELOVL5 and IGFBP6 genes. Thus, the obtained data indicate that malignant breast tumors with reduced expression of the ELOVL5 and IGFBP6 genes can metastasize with a higher probability due to a more efficient invasion of tumor cells.

Breast cancer (BC) is the leading cause of death from malignant neoplasms among women worldwide, and metastatic BC presents the biggest problems for treatment. Previously, it was shown that lower expression of ELOVL5 and IGFBP6 genes is associated with a higher risk of the formation of distant metastases in BC. In this work, we studied the change in phenotypical traits, as well as in the transcriptomic and proteomic profiles of BC cells as a result of the stable knockdown of ELOVL5 and IGFBP6 genes. The knockdown of ELOVL5 and IGFBP6 genes was found to lead to a strong increase in the expression of the matrix metalloproteinase (MMP) MMP1. These results were in good agreement with the correlation analysis of gene expression in tumor samples from patients and were additionally confirmed by zymography. The knockdown of ELOVL5 and IGFBP6 genes was also discovered to change the expression of a group of genes involved in the formation of intercellular contacts. In particular, the expression of the CDH11 gene was markedly reduced, which also complies with the correlation analysis. The spheroid formation assay showed that intercellular adhesion decreased as a result of the knockdown of the ELOVL5 and IGFBP6 genes. Thus, the obtained data indicate that malignant breast tumors with reduced expression of the ELOVL5 and IGFBP6 genes can metastasize with a higher probability due to a more efficient invasion of tumor cells. [ABSTRACT FROM AUTHOR]

Cellular and molecular mechanisms of the peripheral immune system (e.g., macrophage and monocyte) in programming endotoxin tolerance (ET) have been well studied. However, regulatory mechanism in development of brain immune tolerance remains unclear. The inducible COX-2/PGE2 axis in microglia, the primary innate immune cells of the brain, is a pivotal feature in causing inflammation and neuronal injury, both in acute excitotoxic insults and chronic neurodegenerative diseases. This present study investigated the regulatory mechanism of PGE2 tolerance in microglia. Multiple reconstituted primary brain cells cultures, including neuron–glial (NG), mixed glial (MG), neuron-enriched, and microglia-enriched cultures, were performed and consequently applied to a treatment regimen for ET induction. Our results revealed that the levels of COX-2 mRNA and supernatant PGE2 in NG cultures, but not in microglia-enriched and MG cultures, were drastically reduced in response to the ET challenge, suggesting that the presence of neurons, rather than astroglia, is required for PGE2 tolerance in microglia. Furthermore, our data showed that neural contact, instead of its soluble factors, is sufficient for developing microglial PGE2 tolerance. Simultaneously, this finding determined how neurons regulated microglial PGE2 tolerance. Moreover, by inhibiting TLR4 activation and de novo protein synthesis by LPS-binding protein (LBP) manipulation and cycloheximide, our data showed that the TLR4 signal and de novo protein synthesis are necessary for microglia to develop PGE2 tolerance in NG cells under the ET challenge. Altogether, our findings demonstrated that neuron–microglia contacts are indispensable in emerging PGE2 tolerance through the regulation of TLR4-mediated de novo protein synthesis.

Stable cell–cell contacts underpin tissue architecture and organization. Quantification of junctions of mammalian epithelia requires laborious manual measurements that are a major roadblock for mechanistic studies. We designed Junction Mapper as an open access, semi-automated software that defines the status of adhesiveness via the simultaneous measurement of pre-defined parameters at cell–cell contacts. It identifies contacting interfaces and corners with minimal user input and quantifies length, area and intensity of junction markers. Its ability to measure fragmented junctions is unique. Importantly, junctions that considerably deviate from the contiguous staining and straight contact phenotype seen in epithelia are also successfully quantified (i.e. cardiomyocytes or endothelia). Distinct phenotypes of junction disruption can be clearly differentiated among various oncogenes, depletion of actin regulators or stimulation with other agents. Junction Mapper is thus a powerful, unbiased and highly applicable software for profiling cell–cell adhesion phenotypes and facilitate studies on junction dynamics in health and disease.

Ferroptosis is a regulated form of cell death characterized by iron dependency and increased lipid peroxidation. Initially assumed to be selectively induced in tumour cells, there is increasing evidence that ferroptosis plays an important role in pathophysiology and numerous cell types and tissues. Deregulated ferroptosis has been linked to human diseases, such as neurodegenerative diseases, cardiovascular disorders, and cancer. Along these lines, ferroptosis is a promising pathway to overcoming therapy resistance of cancer cells. It is therefore of utmost importance to understand the cellular signalling pathways and the molecular mechanisms underlying ferroptosis regulation, including context-specific effects mediated by the neighbouring cells through cell–cell contacts. Here, we give an overview on the molecular events and machinery linked to ferroptosis induction and commitment. We further summarize and discuss current knowledge about the role of cell–cell contacts, which differ in ferroptosis regulation between normal somatic cells and cancer cells. We present emerging concepts on the underlying mechanisms, address open questions, and discuss the possible impact of cell–cell contacts on exploiting ferroptosis in cancer therapy.

N-cadherin mediates cell–cell contacts in vascular smooth muscle cells (VSMCs), and regulates VSMC behaviours including migration and proliferation. Discoidin domain receptor 1 (DDR1) is a collagen binding receptor also implicated in these processes. Previous studies have shown that both N-cadherin and DDR1 are upregulated after vascular injury, but it is not known whether there is a relationship between the two molecules. In the current study we found that N-cadherin was mislocalised from cell–cell junctions in the absence of DDR1. This occurred in spite of the fact that there was no significant difference in total cell lysate levels of N-cadherin between DDR1+/+ and DDR1−/− VSMCs. Analysis of lipid raft fractions revealed decreased N-cadherin and associated junctional complex catenins in DDR1−/− compared to DDR1+/+ VSMCs. Treatment with cholesterol oxidase or methyl-β-cyclodextrin to disrupt lipid rafts removed N-cadherin and DDR1 from the raft fractions. Reciprocal co-immunoprecipitations suggested the association of DDR1 and N-cadherin. Importantly, transfection of DDR1−/− cells with full-length DDR1b rescued the formation of N-cadherin junctions. Together, these data reveal that N-cadherin cell–cell contacts in VSMCs are regulated through interactions with DDR1 and both molecules are located in lipid rafts.

Ferroptosis is a recently discovered pathway of regulated necrosis dependent on iron and lipid peroxidation. It has gained broad attention since it is a promising approach to overcome resistance to apoptosis in cancer chemotherapy. We have recently identified tertiary-butyl hydroperoxide (t-BuOOH) as a novel inducer of ferroptosis. t-BuOOH is a widely used compound to induce oxidative stress in vitro. t-BuOOH induces lipid peroxidation and consequently ferroptosis in murine and human cell lines. t-BuOOH additionally results in a loss of mitochondrial membrane potential, formation of DNA double-strand breaks, and replication block. Here, we specifically address the question whether cell–cell contacts regulate t-BuOOH-induced ferroptosis and cellular damage. To this end, murine NIH3T3 or human HaCaT cells were seeded to confluence, but below their saturation density to allow the establishment of cell–cell contacts without inducing quiescence. Cells were then treated with t-BuOOH (50 or 200 µM, respectively). We revealed that cell–cell contacts reduce basal and t-BuOOH-triggered lipid peroxidation and consequently block ferroptosis. Similar results were obtained with the specific ferroptosis inducer erastin. Cell–cell contacts further protect against t-BuOOH-induced loss of mitochondrial membrane potential, and formation of DNA double-strand breaks. Interestingly, cell–cell contacts failed to prevent t-BuOOH-mediated replication block or formation of the oxidative base lesion 8-oxo-dG. Since evidence of protection against cell death was both (i) observed after treatment with hydrogen peroxide, methyl methanesulfonate or UV-C, and (ii) seen in several cell lines, we conclude that protection by cell–cell contacts is a widespread phenomenon. The impact of cell–cell contacts on toxicity might have important implications in cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Tumor resistance to therapy is associated with the 3D organization and peculiarities of the tumor microenvironment, of which intercellular adhesion is a key participant. In this work, the abundance of contact proteins was compared in SKOV-3 and SKOV-3.ip human ovarian adenocarcinoma cell lines, cultivated in monolayers, tumor spheroids and collagen hydrogels. Three-dimensional models were characterized by extremely low expression of basic molecules of adherens junctions E-cadherin and demonstrated a simultaneous decrease in desmosomal protein desmoglein-2, gap junction protein connexin-43 and tight junction proteins occludin and ZO-1. The reduction in the level of contact proteins was most pronounced in collagen hydrogel, accompanied by significantly increased resistance to treatment with doxorubicin and targeted anticancer toxin DARPin-LoPE. Thus, we suggest that 3D models of ovarian cancer, especially matrix-based models, tend to recapitulate tumor microenvironment and treatment responsiveness to a greater extent than monolayer culture, so they can be used as a highly relevant platform for drug efficiency evaluation.

Abstract: NTPDase2, a member of the CD39/NTPDase family, is an ecto‐nucleotidase anchored to the plasma membrane by two transmembrane domains, with a catalytic site facing the extracellular space and preferentially hydrolyzing nucleoside triphosphates. While NTPDase2 is expressed in many cell types, its unique functionality, mobility and dynamics at the cell membrane remain unexplored. We therefore constructed a recombinant NTPDase2 linked to the yellow fluorescent protein (EYFP) to investigate its dynamics by confocal microscopy. The present study shows that the expression of EYFP‐NTPDase2 in different cell lines does not affect its proliferation, migration and adhesion to extracellular matrices (ECM). Moreover, in human embryonic kidney cells 293 (HEK293) grown on collagen type I and fibronectin, EYFP‐NTPDase2 fluorescence is greater in free plasma membrane regions than in cell–cell contacts, in comparison with cells grown on other substrates. Differences in the time required for fluorescence recovery after photobleaching (FRAP) in free membrane regions and cell–cell contacts indicate that the mobility of EYFP‐NTPDase2 depends on the matrix to which the cells are attached. © 2018 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

The tumorvirus human T-cell lymphotropic virus type 1 (HTLV-1), a member of the delta-retrovirus family, is transmitted via cell-containing body fluids such as blood products, semen, and breast milk. In vivo, HTLV-1 preferentially infects CD4+ T-cells, and to a lesser extent, CD8+ T-cells, dendritic cells, and monocytes. Efficient infection of CD4+ T-cells requires cell-cell contacts while cell-free virus transmission is inefficient. Two types of cell-cell contacts have been described to be critical for HTLV-1 transmission, tight junctions and cellular conduits. Further, two non-exclusive mechanisms of virus transmission at cell-cell contacts have been proposed: (1) polarized budding of HTLV-1 into synaptic clefts; and (2) cell surface transfer of viral biofilms at virological synapses. In contrast to CD4+ T-cells, dendritic cells can be infected cell-free and, to a greater extent, via viral biofilms in vitro. Cell-to-cell transmission of HTLV-1 requires a coordinated action of steps in the virus infectious cycle with events in the cell-cell adhesion process; therefore, virus propagation from cell-to-cell depends on specific interactions between cellular and viral proteins. Here, we review the molecular mechanisms of HTLV-1 transmission with a focus on the HTLV-1-encoded proteins Tax and p8, their impact on host cell factors mediating cell-cell contacts, cytoskeletal remodeling, and thus, virus propagation. [ABSTRACT FROM AUTHOR]

Cadherin-based cell–cell adhesions are a primary determinant of tissue structure. For several decades, it had been thought that the primary function of these ubiquitous structures was to resist external mechanical loads. Here we review recent evidence that cadherins also couple together the force-generating actomyosin cytoskeletons of neighbouring cells, serve as potent regulators of the actomyosin cytoskeleton, and activate diverse signalling pathways in response to applied load. In considering the force sensitivity of the molecular-scale processes that mediate these events, we propose a dynamic picture of the force-sensitive processes in cell–cell contacts. This quantitative and physical understanding of the mechanobiology of cadherin cell–cell junctions will aid endeavours to study the fundamental processes mediating the development and maintenance of tissue structure. [ABSTRACT FROM AUTHOR]

Cell-cell contact-mediated Notch signaling is essential for mesenchymal stem cell (MSC) chondrogenesis during development. However, subsequent deactivation of Notch signaling is also required to allow for stem cell chondrogenic progression. Recent literature has shown that Notch signaling can also influence Wnt/β-catenin signaling, critical for MSC differentiation, through perturbations in cell-cell contacts. Traditionally, abundant cell-cell contacts, consistent with development, are emulated in vitro using pellet cultures for chondrogenesis. However, cells are often encapsulated within biomaterials-based scaffolds, such as hydrogels, to improve therapeutic cell localization in vivo. To explore the role of Notch and Wnt/β-catenin signaling in the context of hydrogel-encapsulated MSC chondrogenesis, we compared signaling and differentiation capacity of MSCs in both hydrogels and traditional pellet cultures. We demonstrate that encapsulation within poly(ethylene glycol) hydrogels reduces cell-cell contacts, and both Notch (7.5-fold) and Wnt/β-catenin (84.7-fold) pathway activation. Finally, we demonstrate that following establishment of cell-cell contacts and transient Notch signaling in pellet cultures, followed by Notch signaling deactivation, resulted in a 1.5-fold increase in MSC chondrogenesis. Taken together, these findings support that cellular condensation, and establishment of initial cell-cell contacts is critical for MSC chondrogenesis, and this process is inhibited by hydrogel encapsulation. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 1291-1302, 2015. [ABSTRACT FROM AUTHOR]

Desmosomes represent adhesive, spot-like intercellular junctions that in association with intermediate filaments mechanically link neighboring cells and stabilize tissue architecture. In addition to this structural function, desmosomes also act as signaling platforms involved in the regulation of cell proliferation, differentiation, migration, morphogenesis, and apoptosis. Thus, deregulation of desmosomal proteins has to be considered to contribute to tumorigenesis. Proteolytic fragmentation and downregulation of desmosomal cadherins and plaque proteins by transcriptional or epigenetic mechanisms were observed in different cancer entities suggesting a tumor-suppressive role. However, discrepant data in the literature indicate that context-dependent differences based on alternative intracellular, signal transduction lead to altered outcome. Here, modulation of Wnt/β-catenin signaling by plakoglobin or desmoplakin and of epidermal growth factor receptor signaling appears to be of special relevance. This review summarizes current evidence on how desmosomal proteins participate in carcinogenesis, and depicts the molecular mechanisms involved. [ABSTRACT FROM AUTHOR]

The tumorvirus human T-cell lymphotropic virus type 1 (HTLV-1), a member of the delta-retrovirus family, is transmitted via cell-containing body fluids such as blood products, semen, and breast milk. In vivo, HTLV-1 preferentially infects CD4+ T-cells, and to a lesser extent, CD8+ T-cells, dendritic cells, and monocytes. Efficient infection of CD4+ T-cells requires cell-cell contacts while cell-free virus transmission is inefficient. Two types of cell-cell contacts have been described to be critical for HTLV-1 transmission, tight junctions and cellular conduits. Further, two non-exclusive mechanisms of virus transmission at cell-cell contacts have been proposed: (1) polarized budding of HTLV-1 into synaptic clefts; and (2) cell surface transfer of viral biofilms at virological synapses. In contrast to CD4+ T-cells, dendritic cells can be infected cell-free and, to a greater extent, via viral biofilms in vitro. Cell-to-cell transmission of HTLV-1 requires a coordinated action of steps in the virus infectious cycle with events in the cell-cell adhesion process; therefore, virus propagation from cell-to-cell depends on specific interactions between cellular and viral proteins. Here, we review the molecular mechanisms of HTLV-1 transmission with a focus on the HTLV-1-encoded proteins Tax and p8, their impact on host cell factors mediating cell-cell contacts, cytoskeletal remodeling, and thus, virus propagation.

Background: Vinflunine (VFL) is a microtubule-targeting drug that suppresses microtubule dynamics, showing anti-metastatic properties both in vitro and in living cancer cells. An increasing body of evidence underlines the influence of the microtubules dynamics on the cadherin-dependent cell-cell adhesions. E-cadherin is a marker of epithelial-to-mesenchymal transition (EMT) and a tumour suppressor; its reduced levels in carcinoma are associated with poor prognosis. In this report, we investigate the role of VFL on cell-cell adhesions in bladder epithelial tumour cells. Methods: Human bladder epithelial tumour cell lines HT1376, 5637, SW780, T24 and UMUC3 were used to analyse cadherin-dependent cell-cell adhesions under VFL treatment. VFL effect on growth inhibition was measured by using a MTT colorimetric cell viability assay. Western blot, immunofluorescence and transmission electron microscopy analyses were performed to assess the roles of VFL effect on cell-cell adhesions, epithelial-to-mesenchymal markers and apoptosis. The role of the proteasome in controlling cell-cell adhesion was studied using the proteasome inhibitor MG132. Results: We show that VFL induces cell death in bladder cancer cells and activates epithelial differentiation of the remaining living cells, leading to an increase of E-cadherin-dependent cell-cell adhesion and a reduction of mesenchymal markers, such as N-cadherin or vimentin. Moreover, while E-cadherin is increased, the levels of Hakai, an E3 ubiquitin-ligase for E-cadherin, were significantly reduced in presence of VFL. In 5637, this reduction on Hakai expression was blocked by MG132 proteasome inhibitor, indicating that the proteasome pathway could be one of the molecular mechanisms involved in its degradation. Conclusions: Our findings underscore a critical function for VFL in cell-cell adhesions of epithelial bladder tumour cells, suggesting a novel molecular mechanism by which VFL may impact upon EMT and metastasis. [ABSTRACT FROM AUTHOR]

Background The role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance, both for the enhanced understanding of fundamental cancer biology as well as for improving molecular diagnostics and therapeutics. The aim of this study was to establish an in vitro model based on a co-culture of healthy human fibroblasts (HFs) and human osteosarcoma cells (MG-63s) to simulate the microenvironment including tumor and healthy cells. Methods The HFs and MG-63s were in vitro co-cultured for a period of time ranging from 24 h to 7 days. Cell morphology and organization were studied using phase contrast microscopy while the expression of Human Cartilage Glycoprotein 39 (YKL-40), Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloprotease 1 (MMP1) was investigated by Real Time PCR and Western Blotting. Results The results showed a characteristic disposition of tumor and healthy co-cultured cells in columns which are not visible in tumor and healthy cells grown singularly. The expression of YKL-40, VEGF and MMP1 significantly changed in co-cultured cells compared to HFs and MG-63s separately cultured. Conclusions We concluded that the tumor microenvironment has an influence on the protein expression of the healthy surrounding tissues and the process of tumorigenicity. [ABSTRACT FROM AUTHOR]

Cellular and molecular mechanisms of the peripheral immune system (e.g., macrophage and monocyte) in programming endotoxin tolerance (ET) have been well studied. However, regulatory mechanism in development of brain immune tolerance remains unclear. The inducible COX-2/PGE2 axis in microglia, the primary innate immune cells of the brain, is a pivotal feature in causing inflammation and neuronal injury, both in acute excitotoxic insults and chronic neurodegenerative diseases. This present study investigated the regulatory mechanism of PGE2 tolerance in microglia. Multiple reconstituted primary brain cells cultures, including neuron–glial (NG), mixed glial (MG), neuron-enriched, and microglia-enriched cultures, were performed and consequently applied to a treatment regimen for ET induction. Our results revealed that the levels of COX-2 mRNA and supernatant PGE2 in NG cultures, but not in microglia-enriched and MG cultures, were drastically reduced in response to the ET challenge, suggesting that the presence of neurons, rather than astroglia, is required for PGE2 tolerance in microglia. Furthermore, our data showed that neural contact, instead of its soluble factors, is sufficient for developing microglial PGE2 tolerance. Simultaneously, this finding determined how neurons regulated microglial PGE2 tolerance. Moreover, by inhibiting TLR4 activation and de novo protein synthesis by LPS-binding protein (LBP) manipulation and cycloheximide, our data showed that the TLR4 signal and de novo protein synthesis are necessary for microglia to develop PGE2 tolerance in NG cells under the ET challenge. Altogether, our findings demonstrated that neuron–microglia contacts are indispensable in emerging PGE2 tolerance through the regulation of TLR4-mediated de novo protein synthesis. [ABSTRACT FROM AUTHOR]

Changes in transplanted sarcoma 45 tissue in outbred albino rats with tumor regression under the effect of magnetite nanoparticles (magnetic fluid) were studied by light and electron microscopy. The ultrastructure and cell death types in regressing tumors and signs of cell-cell interactions with participation of macrophages, lymphocytes, neutrophils, and degranulating mast cells were described. Some possible mechanisms of a pronounced antitumor activity of magnetite nanoparticles were discussed. [ABSTRACT FROM AUTHOR]

Highlights: [•] FAK induces a mesenchymal/epithelial switch affecting cell–cell cohesion. [•] FAK influences the macroscopic cell colony spreading in two and three dimensions. [•] FAK affects cellular velocity fields, persistence, and correlation length. [ABSTRACT FROM AUTHOR]

Background: During nephron induction, morphogenetic molecules are reciprocally exchanged between epithelial and mesenchymal stem/progenitor cells within the renal stem/progenitor cell niche. That these molecules remain concentrated, it is assumed that both cell populations stand in close contact to each other. However, recently published data illustrate that epithelial and mesenchymal cells are separated by an astonishingly wide interstitial interface. Methods: To gain deeper morphological insights into the spatial distribution of mesenchymal and epithelial stem/progenitor cells, the embryonic zone of neonatal rabbit kidney was fixed either with glutaraldehyde (GA) or in a combination with cupromeronic blue, ruthenium red or tannic acid. Transmission electron microscopy was then performed on exactly orientated sections. Results: Conventional fixation with GA illustrates that epithelial and mesenchymal stem/progenitor cells are separated by a bright but inconspicuously looking interstitial interface. In contrast, fixation of specimens in GA containing cupromeronic blue, ruthenium red or tannic acid elucidates that part of the interstitial interface exhibits a special extracellular matrix extending like woven strands between mesenchymal and epithelial stem/progenitor cells. In parallel, filigree projections from mesenchymal stem/progenitor cells cross the interstitial interface to penetrate the basal lamina of epithelial cells. Fusion of the plasma membranes cannot be observed. Instead, touching mesenchymal cell projections form a cone at the contact site with tunneling nanotubes. Conclusions: The results demonstrate that the contact between mesenchymal and epithelial stem/progenitor cells does not form accidentally but physiologically and appears to belong to a suspected system involved in the exchange of morphogenetic information. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Vascular integrity is fundamental to the formation of mature blood vessels and depends on a functional, quiescent endothelial monolayer. However, how endothelial cells enter and maintain quiescence in the presence of angiogenic factors is still poorly understood. Here we identify the fibroblast growth factor (FGF) antagonist Sprouty2 (Spry2) as a key player in mediating endothelial quiescence and barrier integrity in mouse aortic endothelial cells (MAECs): Spry2 knockout MAECs show spindle-like shapes and are incapable of forming a functional, impermeable endothelial monolayer in the presence of FGF2. Whereas dense wild type cells exhibit contact inhibition and stop to proliferate, Spry2 knockout MAECs remain responsive to FGF2 and continue to proliferate even at high cell densities. Importantly, the anti-proliferative effect of Spry2 is absent in sparsely plated cells. This cell density-dependent Spry2 function correlates with highly increased Spry2 expression in confluent wild type MAECs. Spry2 protein expression is barely detectable in single cells but steadily increases in cells growing to high cell densities, with hypoxia being one contributing factor. At confluence, Spry2 expression correlates with intact cell-cell contacts, whereas disruption of cell-cell contacts by EGTA, TNFα and thrombin decreases Spry2 protein expression. In confluent cells, high Spry2 levels correlate with decreased extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. In contrast, dense Spry2 knockout MAECs exhibit enhanced signaling by Erk1/2. Moreover, inhibiting Erk1/2 activity in Spry2 knockout cells restores wild type cobblestone monolayer morphology. This study thus reveals a novel Spry2 function, which mediates endothelial contact inhibition and barrier integrity. [ABSTRACT FROM AUTHOR]

The formation and stability of epithelial adhesive systems, such as adherens junctions, desmosomes and tight junctions, rely on a number of cellular processes that ensure a dynamic interaction with the cortical cytoskeleton, and appropriate delivery and turnover of receptors at the surface. Unique signalling pathways must be coordinated to allow the coexistence of distinct adhesive systems at discrete subdomains along junctions and the specific properties they confer to epithelial cells. Rho, Rac and Cdc42 are members of the Rho small GTPase family, and are well-known regulators of cell-cell adhesion. The spatio-temporal control of small GTPase activation drives specific intracellular processes to enable the hierarchical assembly, morphology and maturation of cell-cell contacts. Here, we discuss the small GTPase regulators that control the precise amplitude and duration of the levels of active Rho at cell-cell contacts, and the mechanisms that tailor the output of Rho signalling to a particular cellular event. Interestingly, the functional interaction is reciprocal; Rho regulators drive the maturation of cell-cell contacts, whereas junctions can also modulate the localisation and activity of Rho regulators to operate in diverse processes in the epithelial differentiation programme. [ABSTRACT FROM AUTHOR]

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphological and migration characteristics of epithelial cells of IAR1162 and IAR1170 clones derived from a mixed culture of N-RasV12 oncogene-transformed IAR-2 cell line. It was found that the oncogenic RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, assembled E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the oncogenic RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of cell motility in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the motility of transformed epithelial cells and plays an important role in their collective migration. [ABSTRACT FROM AUTHOR]

Summary: The role of airway wall remodelling in bronchial asthma is well established. Myofibroblasts, the cells displaying features intermediate between fibroblasts and smooth muscle cells, are involved in this process but the mechanism of myofibroblasts activation in the onset of the disease remains obscure. Myofibroblasts can differentiate from various cell types, including resident fibroblasts, and the fibroblasts to myofibroblasts transition (FMT) can be reproduced in vitro. We aimed to investigate the process of FMT in human bronchial fibroblasts (HBF) derived from non-asthmatic (n = 7) and asthmatic (n = 7) subjects. We also tested whether cell–cell contacts affect FMT by using N-cadherin blocking antibody. HBF plated in low or high cell density were treated with TGF-β1 up to one week to induce FMT. The percentage of myofibroblsts was counted and expression of α−smooth muscle actin was evaluated by cytoimmunofluorescence, flow cytometry and immunobloting. We demonstrated that the intensity of FMT induced by TGF-β1 in vitro was strongly enhanced in asthmatic as compared to non-asthmatic HBF populations. This process was facilitated by low cell plating density in both groups of cultures. Furthermore, we proved that neither HBF-conditioned medium nor growth arrest in G0/G1 phase of cell cycle could stop the TGF-β1-induced FMT in asthmatic cell populations. However, even in sparse asthmatic HBF, the blocking of N-cadherin resulted in the inhibition of FMT. Our findings show for the first time that the initial absence or an induced loss of cell–cell adhesions in asthmatic HBF populations is important for the completion of FMT. [ABSTRACT FROM AUTHOR]

Abstract: Liprin-α1 to liprin-α4 constitute a family of cytoplasmic proteins, which have been found in various multiprotein complexes. For liprin-α1 roles in synapse formation and cell spreading were described but other liprin family members are not well characterized. We show here that liprin-α4 is upregulated in human clear cell renal cell carcinomas (RCC) as compared to normal kidney tissue. Liprin-α4 expression is downregulated by the von Hippel-Lindau tumor suppressor (VHL) and upregulated by hypoxia in RCC cell lines. The liprin-α4 gene promoter is directly activated by binding of the hypoxia-inducible factor 1α (HIF-1α) to HRE consensus binding sites as shown by reporter assays and chromatin immunoprecipitations. RNAi mediated knockdown of liprin-α4 leads to reduced E-cadherin and β-catenin levels at cell junctions and to dissociation of epithelial cell contacts. Our data describe for the first time liprin-α4 as a hypoxia-induced gene potentially involved in cell–cell adhesion. [ABSTRACT FROM AUTHOR]

A number of factors contribute to the control of stem cell fate. In particular, the evidence for how physical forces control the stem cell differentiation program is now accruing. In this review, the authors discuss the types of physical forces: mechanical forces, cell shape, extracellular matrix geometry/properties, and cell-cell contacts and morphogenic factors, which evidence suggests play a role in influencing stem cell fate. [ABSTRACT FROM AUTHOR]

Abstract: EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPβ and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell–cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell–cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain. [Copyright &y& Elsevier]

Abstract: Emerging evidence suggests that endothelial-to-mesenchymal transition (EndoMT) is an important contributor to cardiovascular diseases and to vascular development and pathologies as well as in cancer progression. As in epithelial–mesenchymal transition (EMT), EndoMT may involve several regulated steps: disassembly of adherence junctions or loss of cell–cell contacts, cytoskeletal reorganization, proteases, cytokines and growth factor synthesis and secretion, extracellular matrix remodeling, membrane receptor expression, cell detachment and cell migration and differentiation. Loss of cell–cell contacts is a necessary and sufficient step in the progression of EndoMT. In endothelial cells, adherence junctions are composed of transmembrane adhesive proteins belonging to the cadherin family, with the VE-cadherin being the most important. This protein interacts with β-catenin, which links cadherin to the actin cytoskeleton. Tyrosine phosphorylation of both VE-cadherin and β-catenin is considered an important mechanism associated to the disassembly of adherence junctions or loss of cell–cell contacts. Insulin-like growth factor receptor I (IGFIR) is a transmembrane tyrosine kinase that has been involved in the alterations of cell–cell contacts and in the expression of some genes during cancer development and progression. Here, it is hypothesized that IGFIR autophosphorylation may initiate a signaling pathway that would lead to the loss of cell–cell contacts or adherence junctions, remarkable remodeling of the cytoskeleton, increased cell motility, and finally to the progressive transition towards a mesenchymal phenotype. Data supporting this hypothesis are presented here. [Copyright &y& Elsevier]

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gramnegative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria ( Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated [1], but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes. [ABSTRACT FROM AUTHOR]

Claudins are the critical transmembrane proteins in tight junctions. Claudin-5, for instance, prevents paracellular permeation of small molecules. However, the molecular interaction mechanism is unknown. Hence, the claudin-claudin interaction and tight junction strand formation were investigated using systematic single mutations. Claudin-5 mutants transfected into tight junction-free cells demonstrated that the extracellular loop 2 is involved in strand formation via trans-interaction, but not via polymerization, along the plasma membrane of one cell. Three phenotypes were obtained: the tight junction type (wild-type-like trans- and cis-interaction; the disjunction type (blocked trans-interaction); the intracellular type (disturbed folding). Combining site-directed mutagenesis, live-cell imaging-, electron microscopy-, and molecular modeling data led to an antiparallel homodimer homology model of the loop. These data for the first time explain how two claudins hold onto each other and constrict the paracellular space. The intermolecular interface includes aromatic (F147, Y148, Y158) and hydrophilic (Q156, E159) residues. The aromatic residues form a strong binding core between two loops from opposing cells. Since nearly all these residues are conserved in most claudins, our findings are of general relevance for all classical claudins. On the basis of the data we have established a novel molecular concept for tight junction formation. [ABSTRACT FROM AUTHOR]

Abstract: Cell–cell contacts play important roles in the homeostasis of normal epithelium and in the steps of metastasis of tumor cells, although signaling mechanisms to regulate cell–cell contacts are unclear. In this study, we observed that phenotype of no cell–cell contacts in rat intestinal epithelial cell subline (RIE1-Sca) correlated with increased Erk1/2 signaling activity, compared to that of parental RIE1 cells growing in colonies. Furthermore, cell–cell contacts between RIE1-Sca cells were reformed by treatment with a specific MEK inhibitor (U0126), with translocation of ZO1 and β-catenin to cell–cell contacts, without changes of their expression levels. U0126 treatment also increased EGFR phosphorylation in a ligand-independent manner. Pretreatment with EGFR kinase inhibitor abolished U0126 treatment-mediated EGFR phosphorylation, and expression of dominant negative H-Ras N17 allowed EGFR phosphorylation and cell–cell contacts even without U0126 treatment. Furthermore, the expression of a nonphosphorylatable EGFR Y5F mutant abolished U0126-mediated cell–cell contacts. U0126 treatment also caused less efficient wound healing by keeping monolayer integrity intact, compared to control untreated cells. This U0126-mediated reduction in wound healing was further altered either by pretreatment of EGFR kinase inhibitor or expression of H-Ras N17 or EGFR Y5F. Taken together, this study supports a unique mechanism of cell–cell contact formation through MEK/Erks inhibition-mediated EGFR phosphorylation. [Copyright &y& Elsevier]