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Metastatic breast cancer cells disseminate to organs with a soft microenvironment. Whether and how the mechanical properties of the local tissue influence their response to treatment remains unclear. Here we found that a soft extracellular matrix empowers redox homeostasis. Cells cultured on a soft extracellular matrix display increased peri-mitochondrial F-actin, promoted by Spire1C and Arp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission. Changes in mitochondrial dynamics lead to increased production of mitochondrial reactive oxygen species and activate the NRF2 antioxidant transcriptional response, including increased cystine uptake and glutathione metabolism. This retrograde response endows cells with resistance to oxidative stress and reactive oxygen species-dependent chemotherapy drugs. This is relevant in a mouse model of metastatic breast cancer cells dormant in the lung soft tissue, where inhibition of DRP1 and NRF2 restored cisplatin sensitivity and prevented disseminated cancer-cell awakening. We propose that targeting this mitochondrial dynamics- and redox-based mechanotransduction pathway could open avenues to prevent metastatic relapse. Romani et al. report that cells on soft extracellular matrix have increased mitochondrial fission, with subsequent production of mitochondrial reactive oxygen species and NRF2, which may increase resistance to reactive oxygen species-dependent chemotherapy drugs in breast cancer cells in vitro and in mouse lungs.

Force transduction at cell-cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both sub-cellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell-cell junctions. At the multi-cellular scale, cell-cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell-cell adhesions. The intestines, liver, and skin are all examples of organs that perform specific functions. Organs are comprised of tissues, which are themselves made up of cells. Epithelial tissue is one of the four basic types of tissue found in animals, and it occurs in almost every organ in the body. For example, epithelial tissue makes up the outermost layer of the skin, and the lining of the lungs and the intestines; the cells in epithelial tissues are attached to one another via ‘adhesion molecules’. Organs and tissues need to be maintained throughout life in order for them to work properly. Epithelial cells in particular are very short-lived and must be constantly replaced. If epithelial tissue is cut or damaged in any way, the surrounding healthy epithelial cells must work together to repair the wound and restore the tissue's integrity. These processes require individual epithelial cells to communicate with one another. While chemical signals provide one means of cell-to-cell communication, cells also sense and respond to the physical presence of surrounding cells. In adults, organs and tissues generally do not change shape or size; as such there is a tightly balanced exchange of mechanical forces between the individual cells. Damage to the tissue causes a detectable change in these mechanical forces, which is sensed by nearby healthy epithelial cells and causes them to work towards healing the wound. While the importance of mechanical forces in maintaining tissue integrity is widely recognized, there were few tools to study these forces; this meant that mechanical communication through cell–cell adhesion sites was not well understood. Now Ng, Besser et al. describe the development and use of a new method for measuring and mapping the exchange of mechanical forces at cell–cell adhesion sites. Changes in the strength of the forces exchanged between cells could be measured across clusters of multiple cells or for specific parts of individual cells. Ng, Besser et al. found that when an epithelial cell in a cluster started to divide to form two new cells, the cell exerted less mechanical force on its neighboring cells. Ng, Besser et al. found that the forces exerted between cells were strongest when there was more of an adhesion molecule called E-cadherin in the cell surface membrane at the cell–cell adhesion sites. The opposite was also true, as these forces were weakest at cell–cell adhesion sites with fewer E-cadherin molecules. The new method and findings will now help to guide future studies into how mechanical forces are transmitted between living cells.

The relationship between the changes in endothelial cell–cell junctions and microvascular abnormalities in the progression of hypertrophic cardiomyopathy (HCM), as well as their potential as early biomarkers, remains unclear. Here, we analysed single‐nucleus RNA‐sequencing data from the left ventricles of 44 health donors and HCM patients. First, we observed that endothelial cell–cell junctions were significantly altered in HCM vascular endothelial cells (ECs), including tight junctions, gap junctions and adherens junctions, especially in capillary ECs. The proposed pseudo‐timing analysis predicted that endothelial cell–cell junctions abnormalities occurred in the early stages of HCM. Second, we verified that endothelial cell–cell junctions disorders occur at early stages of HCM disease progression in two time‐series single‐nucleus datasets of mice. The expression of eight cell–cell junction genes showed an initial increase in the early stage, followed by a slight decrease in the middle stage, and a sharp increase in the later stage. Subsequently, cell communication and transcription factor analysis were used to explore the underlying mechanisms. Furthermore, an early HCM prediction model was developed and independently validated using three mRNA datasets comprising 204 health individuals and HCM patients for the eight genes panel, the accuracy was 0.81 [0.63–0.98]. Finally, we validated this panel in HCM tissues. This study demonstrated in humans and mice that eight cell–cell junction genes were significantly elevated in the early stages of HCM and may be potential biomarkers for the early diagnosis of HCM.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 1010 cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632–treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell–cell junctions, and cell–extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell–cell and cell–ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.

Cell–cell adhesions are often subjected to mechanical strains of different rates and magnitudes in normal tissue function. However, the rate-dependent mechanical behavior of individual cell–cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This is particularly true under large strain conditions, which may potentially lead to cell–cell adhesion dissociation and ultimately tissue fracture. In this study, we designed and fabricated a single-cell adhesion micro tensile tester (SCAμTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell–cell adhesion. Straining the cytoskeleton–cell adhesion complex system reveals a passive shear-thinning viscoelastic behavior and a rate-dependent active stress-relaxation mechanism mediated by cytoskeleton growth. Under low strain rates, stress relaxation mediated by the cytoskeleton can effectively relax junctional stress buildup and prevent adhesion bond rupture. Cadherin bond dissociation also exhibits rate-dependent strengthening, in which increased strain rate results in elevated stress levels at which cadherin bonds fail. This bond dissociation becomes a synchronized catastrophic event that leads to junction fracture at high strain rates. Even at high strain rates, a single cell–cell junction displays a remarkable tensile strength to sustain a strain as much as 200% before complete junction rupture. Collectively, the platform and the biophysical understandings in this study are expected to build a foundation for the mechanistic investigation of the adaptive viscoelasticity of the cell–cell junction.

Maintenance of cell junctions plays a crucial role in the regulation of cellular functions including cell proliferation, permeability, and cell death. Disruption of cell junctions is implicated in a variety of human disorders, such as inflammatory diseases and cancers. Understanding molecular regulation of cell junctions is important for development of therapeutic strategies for intervention of human diseases. Ubiquitination is an important type of post-translational modification that primarily regulates endogenous protein stability, receptor internalization, enzyme activity, and protein-protein interactions. Ubiquitination is tightly regulated by ubiquitin E3 ligases and can be reversed by deubiquitinating enzymes. Recent studies have been focusing on investigating the effect of protein stability in the regulation of cell-cell junctions. Ubiquitination and degradation of cadherins, claudins, and their interacting proteins are implicated in epithelial and endothelial barrier disruption. Recent studies have revealed that ubiquitination is involved in regulation of Rho GTPases' biological activities. Taken together these studies, ubiquitination plays a critical role in modulating cell junctions and motility. In this review, we will discuss the effects of ubiquitination and deubiquitination on protein stability and expression of key proteins in the cell-cell junctions, including junction proteins, their interacting proteins, and small Rho GTPases. We provide an overview of protein stability in modulation of epithelial and endothelial barrier integrity and introduce potential future search directions to better understand the effects of ubiquitination on human disorders caused by dysfunction of cell junctions.

The Hippo pathway is an evolutionarily conserved pathway that serves to promote cell death and differentiation while inhibiting cellular proliferation across species. The downstream effectors of this pathway, yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), are considered vital in promoting the output of the Hippo pathway, with activation of upstream kinases negatively regulating YAP/TAZ activity. The upstream regulation of the Hippo pathway is not entirely understood on a molecular level. However, several studies have shown that numerous cellular and non-cellular mechanisms such as cell polarity, contact inhibition, soluble factors, mechanical forces, and metabolism can convey external stimuli to the intracellular kinase cascade, promoting the activation of key components of the Hippo pathway and therefore regulating the subcellular localisation and protein activity of YAP/TAZ. This review will summarise what we have learnt about the role of intercellular junction-associated proteins in the activation of this pathway, including adherens junctions and tight junctions, and in particular our latest findings about the desmosomal components, including desmoglein-3 (DSG3), in the regulation of YAP signalling, phosphorylation, and subcellular translocation.

When migrating in vivo, cells are exposed to numerous conflicting signals: chemokines, repellents, extracellular matrix, growth factors. The roles of several of these molecules have been studied individually in vitro or in vivo, but we have yet to understand how cells integrate them. To start addressing this question, we used the cephalic neural crest as a model system and looked at the roles of its best examples of positive and negative signals: stromal-cell derived factor 1 (Sdf1/Cxcl12) and class3-Semaphorins. Here we show that Sdf1 and Sema3A antagonistically control cell-matrix adhesion via opposite effects on Rac1 activity at the single cell level. Directional migration at the population level emerges as a result of global Semaphorin-dependent confinement and broad activation of adhesion by Sdf1 in the context of a biased Fibronectin distribution. These results indicate that uneven in vivo topology renders the need for precise distribution of secreted signals mostly dispensable. Migrating cells encounter multiple signals such as extracellular matrix (ECM) and chemokinetic factors but how these integrate in vivo is unclear. Here, the authors report that overall control of cell-ECM adhesion by Sema3A and Sdf1 can be converted into directional migration by a biased ECM network.

Transmembrane proteins of adherens and tight junctions are known targets for viruses and bacterial toxins. The coronavirus receptor ACE2 has been localized at the apical surface of epithelial cells, but it is not clear whether ACE2 is localized at apical Cell—Cell junctions and whether it associates with junctional proteins. Here we explored the expression and localization of ACE2 and its association with transmembrane and tight junction proteins in epithelial tissues and cultured cells by data mining, immunoblotting, immunofluorescence microscopy, and co-immunoprecipitation experiments. ACE2 mRNA is abundant in epithelial tissues, where its expression correlates with the expression of the tight junction proteins cingulin and occludin. In cultured epithelial cells ACE2 mRNA is upregulated upon differentiation and ACE2 protein is widely expressed and co-immunoprecipitates with the transmembrane proteins ADAM17 and CD9. We show by immunofluorescence microscopy that ACE2 colocalizes with ADAM17 and CD9 and the tight junction protein cingulin at apical junctions of intestinal (Caco-2), mammary (Eph4) and kidney (mCCD) epithelial cells. These observations identify ACE2, ADAM17 and CD9 as new epithelial junctional transmembrane proteins and suggest that the cytokine-enhanced endocytic internalization of junction-associated protein complexes comprising ACE2 may promote coronavirus entry.

This review is dedicated to E-cadherin, a calcium-dependent cell-cell adhesion molecule with pivotal roles in epithelial cell behavior, tissue formation, and suppression of cancer. As founder member of the cadherin superfamily, it has been extensively investigated. We summarize the structure and regulation of the E-cadherin gene and transcript. Models for E-cadherin-catenin complexes and cell junctions are presented. The structure of the E-cadherin protein is discussed in view of the diverse functions of this remarkable protein. Homophilic and heterophilic adhesion are compared, including the role of E-cadherin as a receptor for pathogens. The complex post-translational processing of E-cadherin is reviewed, as well as the many signaling activities. The role of E-cadherin in embryonic development and morphogenesis is discussed for several animal models. Finally, we review the multiple mechanisms that disrupt E-cadherin function in cancer: inactivating somatic and germline mutations, epigenetic silencing by DNA methylation and epithelial to mesenchymal transition-inducing transcription factors, and dysregulated protein processing.