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Metabolic syndrome often accompanies obesity and hyperglycemia and is associated with a breakdown in the integrity of the intestinal barrier and increased risk of systemic infection. Thaiss et al. found that mice with systemic infection of a Salmonella analog, Citrobacter rodentium , also exhibited hyperglycemia. Deletion of the glucose transporter GLUT2 altered sensitivity to chemically induced epithelial permeability and protected mice from pathogen invasion. The authors also found a correlation in humans between glycated hemoglobin (an indicator of hyperglycemia) and serum levels of pathogen recognition receptor ligands. Science , this issue p. 1376 High blood sugar levels cause epithelial reprogramming, compromising gut barrier integrity and increasing susceptibility to pathogens. Obesity, diabetes, and related manifestations are associated with an enhanced, but poorly understood, risk for mucosal infection and systemic inflammation. Here, we show in mouse models of obesity and diabetes that hyperglycemia drives intestinal barrier permeability, through GLUT2-dependent transcriptional reprogramming of intestinal epithelial cells and alteration of tight and adherence junction integrity. Consequently, hyperglycemia-mediated barrier disruption leads to systemic influx of microbial products and enhanced dissemination of enteric infection. Treatment of hyperglycemia, intestinal epithelial–specific GLUT2 deletion, or inhibition of glucose metabolism restores barrier function and bacterial containment. In humans, systemic influx of intestinal microbiome products correlates with individualized glycemic control, indicated by glycated hemoglobin levels. Together, our results mechanistically link hyperglycemia and intestinal barrier function with systemic infectious and inflammatory consequences of obesity and diabetes.

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC 50 -values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins αvβ3, αvβ5, αvβ6, αvβ8, α5β1, αIIbβ3, using homogenous ELISA-like solid phase binding assay.

Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2–Neuregulin and Hippo–YAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelial–mesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.Aharonov et al. use in vivo genetic approaches to show that ErBB2-mediated YAP activation initiates epithelial–mesenchymal transition-like processes and dedifferentiation of cardiomyocytes to drive heart regeneration.

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s −1 . This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The ‘allosteric vinculin mutant’ is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere. Vinculin binding to talin is a key event in focal adhesion dynamics; yet, how vinculin is activated to recruit actin remains unknown. Here, the authors use a multiscale approach to reveal that talin activates vinculin through an intricate allosteric mechanism tightly regulated by force.

Cardiomyocyte (CM) maturation in mammals is accompanied by a sharp decline in their proliferative and regenerative potential shortly after birth. In this study, we explored the role of the mechanical properties of the underlying matrix in the regulation of CM maturation. We show that rat and mouse neonatal CMs cultured on rigid surfaces exhibited increased myofibrillar organization, spread morphology, and reduced cell cycle activity. In contrast, compliant elastic matrices induced features of CM dedifferentiation, including a disorganized sarcomere network, rounding, and conspicuous cell-cycle re-entry. The rigid matrix facilitated nuclear division (karyokinesis) leading to binucleation, while compliant matrices promoted CM mitotic rounding and cell division (cytokinesis), associated with loss of differentiation markers. Moreover, the compliant matrix potentiated clonal expansion of CMs that involves multiple cell divisions. Thus, the compliant microenvironment facilitates CM dedifferentiation and proliferation via its effect on the organization of the myoskeleton. Our findings may be exploited to design new cardiac regenerative approaches. Heart muscle contracts and relaxes in a regular rhythm to pump blood around the body. Soon after birth, the cells that form our heart muscle stop multiplying. As we grow, these cells increase in size and their internal skeleton—called myoskeleton—becomes more complex, to withstand the demands of pumping more blood. However, because the cells can no longer divide, the body is unable to replace heart muscle cells that are damaged by a heart attack or other illness. This lack of ability to regenerate heart muscle is a major challenge for medicine. While researchers have documented many of the changes that occur in heart muscle cells (known as cardiomyocytes) after birth, it is not known exactly what triggers these changes. A network of proteins and other molecules—also known as a matrix—surrounds the cardiomyocytes and affects their behavior. Here, Yahalom-Ronen et al. investigated the degree to which the mechanical properties of this matrix affect the ability of cardiomyocytes to divide. In the experiments, cardiomyocytes from newborn rodents were grown on matrices with different rigidities. The cells grown on rigid matrices stopped dividing and became larger with a more robust myoskeleton. These cells also contained two nuclei, which indicates that these cells have become mature cardiomyoctyes. In contrast, heart cells grown on a softer matrix continued to multiply. These cells also began to lose some of the features that distinguish mature cardiomyocytes from the cardiomyocytes found in embryos. Next, Yahalom-Ronen et al. treated the cardiomyoctes with a drug that stops them from contracting, which led to increases in cell multiplication. Yahalom-Ronen et al.'s findings suggest that the stiffness of the matrix that surrounds heart muscle cells regulates their ability to divide and mature. In the future, these findings may pave the way towards the development of soft scaffolds that can stimulate the regeneration of adult human heart.

Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.

Integrin-mediated adhesion is regulated by multiple features of the adhesive surface, including its chemical composition, topography, and physical properties. In this study we investigated integrin lateral clustering, as a mechanism to control integrin functions, by characterizing the effect of nanoscale variations in the spacing between adhesive RGD ligands on cell spreading, migration, and focal adhesion dynamics. For this purpose, we used nanopatterned surfaces, containing RGD-biofunctionalized gold dots, surrounded by passivated gaps. By varying the spacing between the dots, we modulated the clustering of the associated integrins. We show that cell-surface attachment is not sensitive to pattern density, whereas the formation of stable focal adhesions and persistent spreading is. Thus cells plated on a 108-nm-spaced pattern exhibit delayed spreading with repeated protrusion-retraction cycles compared to cells growing on a 58-nm pattern. Cell motility on these surfaces is erratic and nonpersistent, leaving thin membrane tethers bound to the RGD pattern. Dynamic molecular profiling indicated that the adhesion sites formed with the 108-nm pattern undergo rapid turnover and contain reduced levels of zyxin. These findings indicate that a critical RGD density is essential for the establishment of mature and stable integrin adhesions, which, in turn, induce efficient cell spreading and formation of focal adhesions.

Focal adhesions (FAs) are large multi-protein complexes that act as transmembrane links between the extracellular matrix and the actin cytoskeleton. Recently, FAs were extensively characterized, yet the molecular mechanisms underlying their mechanical and signalling functions remain unresolved. To address this question, we isolated protein complexes containing different FA components, from chicken smooth muscle, and characterized their properties. Here we identified ‘hybrid complexes’ consisting of the actin-nucleating subunits of Arp2/3 and either vinculin or vinculin and α-actinin. We further show that suppression of p41-ARC, a central component of native Arp2/3, which is absent from the hybrid complexes, increases the levels of the Arp2/3-nucleating core in FA sites and stimulates FA growth and dynamics. In contrast, overexpression of p41-ARC adversely affects FAs. These results support the view that Arp2/3 can form modular ‘hybrid complexes’ containing an actin-nucleating ‘functional core’, and ‘anchoring domains’ (vinculin/p41-ARC) that regulate its subcellular localization. Arp2/3 is a seven-subunit actin-nucleating complex. Here, Chorev et al. identify a novel ‘hybrid complex’ consisting of the actin-nucleating three-subunit core of Arp2/3 and the focal adhesion adapter, vinculin, that localizes to, and modulates the growth of, focal adhesions.

Invadopodia are adhesive, actin-rich protrusions formed by metastatic cancer cells that degrade the extracellular matrix and facilitate invasion. They support the metastatic cascade by a spatially and temporally coordinated process whereby invading cells bind to the matrix, degrade it by specific metalloproteinases, and mechanically penetrate diverse tissue barriers by forming actin-rich extensions. However, despite the apparent involvement of invadopodia in the metastatic process, the molecular mechanisms that regulate invadopodia formation and function are still largely unclear. In this study, we have explored the involvement of the key Hippo pathway co-regulators, namely YAP, and TAZ, in invadopodia formation and matrix degradation. Toward that goal, we tested the effect of depletion of YAP, TAZ, or both on invadopodia formation and activity in multiple human cancer cell lines. We report that the knockdown of YAP and TAZ or their inhibition by verteporfin induces a significant elevation in matrix degradation and invadopodia formation in several cancer cell lines. Conversely, overexpression of these proteins strongly suppresses invadopodia formation and matrix degradation. Proteomic and transcriptomic profiling of MDA-MB-231 cells, following co-knockdown of YAP and TAZ, revealed a significant change in the levels of key invadopodia-associated proteins, including the crucial proteins Tks5 and MT1-MMP (MMP14). Collectively, our findings show that YAP and TAZ act as negative regulators of invadopodia formation in diverse cancer lines, most likely by reducing the levels of essential invadopodia components. Dissecting the molecular mechanisms of invadopodia formation in cancer invasion may eventually reveal novel targets for therapeutic applications against invasive cancer.

Little is known about the ultrastructure of focal adhesions, compared with their extensive molecular characterization. Cryo-electron tomography provides novel insights into the internal sub-structures at the interface between adhesions and the actin cytoskeleton. Focal adhesions are integrin-based multiprotein complexes, several micrometres in diameter, that mechanically link the extracellular matrix with the termini of actin bundles. The molecular diversity of focal adhesions and their role in cell migration and matrix sensing has been extensively studied, but their ultrastructural architecture is still unknown. We present the first three-dimensional structural reconstruction of focal adhesions using cryo-electron tomography. Our analyses reveal that the membrane–cytoskeleton interaction at focal adhesions is mediated through particles located at the cell membrane and attached to actin fibres. The particles have diameters of 25 ± 5 nm, and an average interspacing of approximately 45 nm. Treatment with the Rho-kinase inhibitor Y-27632 induces a rapid decrease in particle diameter, suggesting that they are highly mechanosensitive. Our findings clarify the internal architecture of focal adhesions at molecular resolution, and provide insights into their scaffolding and mechanosensory functions.