Vinculin Y822 is an important determinant of ligand binding.

Vinculin is an actin-binding protein present at cell-matrix and cell-cell adhesions, which plays a critical role in bearing force experienced by cells and dissipating it onto the cytoskeleton. Recently, we identified a key tyrosine residue, Y822, whose phosphorylation plays a critical role in force transmission at cell-cell adhesions. The role of Y822 in human cancer remains unknown, even though Y822 is mutated to Y822C in uterine cancers. Here, we investigated the effect of this amino acid substitution and that of a phosphodeficient Y822F vinculin in cancer cells. We observed that the presence of the Y822C mutation led to cells that proliferate and migrate more rapidly and contained smaller focal adhesions when compared to cells with wild-type vinculin. In contrast, the presence of the Y822F mutation led to highly spread cells with larger focal adhesions and increased contractility. Furthermore, we provide evidence that Y822C vinculin forms a disulfide bond with paxillin, accounting for some of the elevated phosphorylated paxillin recruitment. Taken together, these data suggest that vinculin Y822 modulates the recruitment of ligands.

Metabolic reprogramming in response to cell mechanics.

Much attention has been dedicated to understanding how cells sense and respond to mechanical forces. The types of forces cells experience as well as the repertoire of cell surface receptors that sense these forces have been identified. Key mechanisms for transmitting that force to the cell interior have also emerged. Yet, how cells process mechanical information and integrate it with other cellular events remains largely unexplored. Here we review the mechanisms underlying mechanotransduction at cell-cell and cell-matrix adhesions, and we summarize the current understanding of how cells integrate information from the distinct adhesion complexes with cell metabolism.

Fueling the cytoskeleton - links between cell metabolism and actin remodeling.

Attention has long focused on the actin cytoskeleton as a unit capable of organizing into ensembles that control cell shape, polarity, migration and the establishment of intercellular contacts that support tissue architecture. However, these investigations do not consider observations made over 40 years ago that the actin cytoskeleton directly binds metabolic enzymes, or emerging evidence suggesting that the rearrangement and assembly of the actin cytoskeleton is a major energetic drain. This Review examines recent studies probing how cells adjust their metabolism to provide the energy necessary for cytoskeletal remodeling that occurs during cell migration, epithelial to mesenchymal transitions, and the cellular response to external forces. These studies have revealed that mechanotransduction, cell migration, and epithelial to mesenchymal transitions are accompanied by alterations in glycolysis and oxidative phosphorylation. These metabolic changes provide energy to support the actin cytoskeletal rearrangements necessary to allow cells to assemble the branched actin networks required for cell movement and epithelial to mesenchymal transitions and the large actin bundles necessary for cells to withstand forces. In this Review, we discuss the emerging evidence suggesting that the regulation of these events is highly complex with metabolism affecting the actin cytoskeleton and vice versa.

The High Content of Fructose in Human Semen Competitively Inhibits Broad and Potent Antivirals That Target High-Mannose Glycans.

Semen is the primary transmission vehicle for various pathogenic viruses. Initial steps of transmission, including cell attachment and entry, likely occur in the presence of semen. However, the unstable nature of human seminal plasma and its toxic effects on cells in culture limit the ability to study in vitro virus infection and inhibition in this medium. We found that whole semen significantly reduces the potency of antibodies and microbicides that target glycans on the envelope glycoproteins (Envs) of HIV-1. The extraordinarily high concentration of the monosaccharide fructose in semen contributes significantly to the effect by competitively inhibiting the binding of ligands to α1,2-linked mannose residues on Env. Infection and inhibition in whole human seminal plasma are accurately mimicked by a stable synthetic simulant of seminal fluid that we formulated. Our findings indicate that, in addition to the protein content of biological secretions, their small-solute composition impacts the potency of antiviral microbicides and mucosal antibodies.IMPORTANCE Biological secretions allow viruses to spread between individuals. Each type of secretion has a unique composition of proteins, salts, and sugars, which can affect the infectivity potential of the virus and inhibition of this process. Here, we describe HIV-1 infection and inhibition in whole human seminal plasma and a synthetic simulant that we formulated. We discovered that the sugar fructose in semen decreases the activity of a broad and potent class of antiviral agents that target mannose sugars on the envelope protein of HIV-1. This effect of semen fructose likely reduces the efficacy of such inhibitors to prevent the sexual transmission of HIV-1. Our findings suggest that the preclinical evaluation of microbicides and vaccine-elicited antibodies will be improved by their in vitro assessment in synthetic formulations that simulate the effects of semen on HIV-1 infection and inhibition.

SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822.

The response of cells to mechanical inputs is a key determinant of cell behavior. In response to external forces, E-cadherin initiates signal transduction cascades that allow the cell to modulate its contractility to withstand the force. Much attention has focused on identifying the E-cadherin signaling pathways that promote contractility, but the negative regulators remain undefined. In this study, we identify SHP-2 as a force-activated phosphatase that negatively regulates E-cadherin force transmission by dephosphorylating vinculin Y822. To specifically probe a role for SHP-2 in E-cadherin mechanotransduction, we mutated vinculin so that it retains its phosphorylation but cannot be dephosphorylated. Cells expressing the mutant vinculin have increased contractility. This work provides a mechanism for inactivating E-cadherin mechanotransduction and provides a new method for specifically targeting the action of phosphatases in cells.

Interplay between tight junctions & adherens junctions.

Cell-cell adhesions are critical for the development and maintenance of tissues. Present at sites of cell-cell contact are the adherens junctions and tight junctions. The adherens junctions mediate cell-cell adhesion via the actions of nectins and cadherins. The tight junctions regulate passage of ions and small molecules between cells and establish cell polarity. Historically, the adherens and tight junctions have been thought of as discrete complexes. However, it is now clear that a high level of interdependency exists between the two junctional complexes. The adherens junctions and tight junctions are physically linked, by the zonula occludens proteins, and linked via signaling molecules including several polarity complexes and actin cytoskeletal modifiers. This review will first describe the individual components of both the adherens and tight junctions and then discuss the coupling of the two complexes with an emphasis on the signaling links and physical interactions between the two junctional complexes.

Vinculin in cell-cell and cell-matrix adhesions.

Vinculin was identified as a component of focal adhesions and adherens junctions nearly 40 years ago. Since that time, remarkable progress has been made in understanding its activation, regulation and function. Here we discuss the current understanding of the roles of vinculin in cell-cell and cell-matrix adhesions. Emphasis is placed on the how vinculin is recruited, activated and regulated. We also highlight the recent understanding of how vinculin responds to and transmits force at integrin- and cadherin-containing adhesion complexes to the cytoskeleton. Furthermore, we discuss roles of vinculin in binding to and rearranging the actin cytoskeleton.

Interferon regulatory factor 6 regulates keratinocyte migration.

Interferon regulatory factor 6 (Irf6) regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post-conception failed to close their wound compared with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell-cell and cell-matrix adhesions were investigated. We show that wild-type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPase-activating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration.

Vinculin-dependent actin bundling regulates cell migration and traction forces.

Vinculin binding to actin filaments is thought to be critical for force transduction within a cell, but direct experimental evidence to support this conclusion has been limited. In the present study, we found mutation (R1049E) of the vinculin tail impairs its ability to bind F-actin, stimulate actin polymerization, and bundle F-actin in vitro. Further, mutant (R1049E) vinculin expressing cells are altered in cell migration, which is accompanied by changes in cell adhesion, cell spreading and cell generation of traction forces, providing direct evidence for the critical role of vinculin in mechanotransduction at adhesion sites. Lastly, we discuss the viability of models detailing the F-actin-binding surface on vinculin in the context of our mutational analysis.

ZO-1 recruitment to α-catenin--a novel mechanism for coupling the assembly of tight junctions to adherens junctions.

The formation of a barrier between epithelial cells is a fundamental determinant of cellular homeostasis, protecting underlying cells against pathogens, dehydration and damage. Assembly of the tight junction barrier is dependent upon neighboring epithelial cells binding to one another and forming adherens junctions, but the mechanism for how these processes are linked is poorly understood. Using a knockdown and substitution system, we studied whether ZO-1 binding to α-catenin is required for coupling tight junction assembly to the formation of adherens junctions. We found that preventing ZO-1 binding to α-catenin did not appear to affect adherens junctions. Rather the assembly and maintenance of the epithelial barrier were disrupted. This disruption was accompanied by alterations in the mobility of ZO-1 and the organization of the actin cytoskeleton. Thus, our study identifies α-catenin binding to ZO-1 as a new mechanism for coupling the assembly of the epithelial barrier to cell-to-cell adhesion.

Force transmission at cell-cell and cell-matrix adhesions.

All cells are subjected to mechanical forces throughout their lifetimes. These forces are sensed by cell surface adhesion receptors and trigger robust actin cytoskeletal rearrangements and growth of the associated adhesion complex to counter the applied force. In this review, we discuss how integrins and cadherins sense force and transmit these forces into the cell interior. We focus on the complement of proteins each adhesion complex recruits to bear the force and the signal transduction pathways activated to allow the cell to tune its contractility. A discussion of the similarities, differences, and crosstalk between cadherin- and integrin-mediated force transmission is also presented.

Bone marrow stromal antigen 2 expressed in cancer cells promotes mammary tumor growth and metastasis.

INTRODUCTION: Several innate immunity genes are overexpressed in human cancers and their roles remain controversial. Bone marrow stromal antigen 2 (BST-2) is one such gene whose role in cancer is not clear. BST-2 is a unique innate immunity gene with both antiviral and pro-tumor functions and therefore can serve as a paradigm for understanding the roles of other innate immunity genes in cancers. METHODS: Meta-analysis of tumors from breast cancer patients obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets were evaluated for levels of BST-2 expression and for tumor aggressiveness. In vivo, we examined the effect of knockdown of BST-2 in two different murine carcinoma cells on tumor growth, metastasis, and survival. In vitro, we assessed the effect of carcinoma cell BST-2 knockdown and/or overexpression on adhesion, anchorage-independent growth, migration, and invasion. RESULTS: BST-2 in breast tumors and mammary cancer cells is a strong predictor of tumor size, tumor aggressiveness, and host survival. In humans, BST-2 mRNA is elevated in metastatic and invasive breast tumors. In mice, orthotopic implantation of mammary tumor cells lacking BST-2 increased tumor latency, decreased primary tumor growth, reduced metastases to distal organs, and prolonged host survival. Furthermore, we found that the cellular basis for the role of BST-2 in promoting tumorigenesis include BST-2-directed enhancement in cancer cell adhesion, anchorage-independency, migration, and invasion. CONCLUSIONS: BST-2 contributes to the emergence of neoplasia and malignant progression of breast cancer. Thus, BST-2 may (1) serve as a biomarker for aggressive breast cancers, and (2) be a novel target for breast cancer therapeutics.

Mechanical activation of VE-cadherin stimulates AMPK to increase endothelial cell metabolism and vasodilation.

Endothelia cells respond to mechanical force by stimulating cellular signaling, but how these pathways are linked to elevations in cell metabolism and whether metabolism supports the mechanical response remains poorly understood. Here, we show that application of force to VE-cadherin stimulates liver kinase B1 (LKB1) to activate AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis. VE-cadherin stimulated AMPK increases eNOS activity and localization to the plasma membrane as well as reinforcement of the actin cytoskeleton and cadherin adhesion complex, and glucose uptake. We present evidence for the increase in metabolism being necessary to fortify the adhesion complex, actin cytoskeleton, and cellular alignment. Together these data extend the paradigm for how mechanotransduction and metabolism are linked to include a connection to vasodilation, thereby providing new insight into how diseases involving contractile, metabolic, and vasodilatory disturbances arise.

Functional analysis of a de novo ACTB mutation in a patient with atypical Baraitser-Winter syndrome.

Exome sequence analysis can be instrumental in identifying the genetic etiology behind atypical disease. We report a patient presenting with microcephaly, dysmorphic features, and intellectual disability with a tentative diagnosis of Dubowitz syndrome. Exome analysis was performed on the patient and both parents. A de novo missense variant was identified in ACTB, c.349G>A, p.E117K. Recent work in Baraitser-Winter syndrome has identified ACTB and ACTG1 mutations in a cohort of individuals, and we rediagnosed the patient with atypical Baraitser-Winter syndrome. We performed functional characterization of the variant actin and show that it alters cell adhesion and polymer formation supporting its role in disease. We present the clinical findings in the patient, comparison of this patient to other patients with ACTB/ACTG1 mutations, and results from actin functional studies that demonstrate novel functional attributes of this mutant protein.

α-Catenin uses a novel mechanism to activate vinculin.

Vinculin, an actin-binding protein, is emerging as an important regulator of adherens junctions. In focal-adhesions, vinculin is activated by simultaneous binding of talin to its head domain and actin filaments to its tail domain. Talin is not present in adherens junctions. Consequently, the identity of the ligand that activates vinculin in cell-cell junctions is not known. Here we show that in the presence of F-actin, α-catenin, a cytoplasmic component of the cadherin adhesion complex, activates vinculin. Direct binding of α-catenin to vinculin is critical for this event because a point mutant (α-catenin L344P) lacking high affinity binding does not activate vinculin. Furthermore, unlike all known vinculin activators, α-catenin binds to and activates vinculin independently of an A50I substitution in the vinculin head, a mutation that inhibits vinculin binding to talin and IpaA. Collectively, these data suggest that α-catenin employs a novel mechanism to activate vinculin and may explain how vinculin is differentially recruited and/or activated in cell-cell and cell-matrix adhesions.

Mechanotransduction: Forcing a change in metabolism.

Epithelial and endothelial cells experience numerous mechanical cues throughout their lifetimes. Cells resist these forces by fortifying their cytoskeletal networks and adhesions. This reinforcement is energetically costly. Here we describe how these energetic demands are met. We focus on the response of epithelial and endothelial cells to mechanical cues, describe the energetic needs of epithelia and endothelia, and identify the mechanisms these cells employ to increase glycolysis, oxidative phosphorylation, and fatty acid metabolism. We discuss the similarities and differences in the responses of the two cell types.

Vinculin regulates cell-surface E-cadherin expression by binding to beta-catenin.

Vinculin was identified as a component of adherens junctions 30 years ago, yet its function there remains elusive. Deletion studies are consistent with the idea that vinculin is important for the organization of cell-cell junctions. However, this approach removes vinculin from both cell-matrix and cell-cell adhesions, making it impossible to distinguish its contribution at each site. To define the role of vinculin in cell-cell junctions, we established a powerful short hairpin-RNA-based knockdown/substitution model system that perturbs vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cadherin-dependent adhesion was reduced. These defects resulted from impaired E-cadherin cell-surface expression. We have investigated the mechanism for the effects of vinculin and found that the reduced surface E-cadherin expression could be rescued by introduction of vinculin, but not of a vinculin A50I substitution mutant that is defective for beta-catenin binding. These findings suggest that an interaction between beta-catenin and vinculin is crucial for stabilizing E-cadherin at the cell surface. This was confirmed by analyzing a beta-catenin mutant that fails to bind vinculin. Thus, our study identifies vinculin as a novel regulator of E-cadherin function and provides important new insight into the dynamic regulation of adherens junctions.

IRF6 Regulates the Delivery of E-Cadherin to the Plasma Membrane.

IRF6 is a transcription factor that is required for craniofacial development and epidermal morphogenesis. Specifically, Irf6-deficient mice lack the terminally differentiated epidermal layers, leading to an absence of barrier function. This phenotype also includes intraoral adhesions due to the absence of the oral periderm, leading to the mislocalization of E-cadherin and other cell‒cell adhesion proteins of the oral epithelium. However, the mechanisms by which IRF6 controls the localization of cell adhesion proteins are not understood. In this study, we show that in human and murine keratinocytes, loss of IRF6 leads to a breakdown of epidermal sheets after mechanical stress. This defect is due to a reduction of adhesion proteins at the plasma membrane. Dynamin inhibitors rescued the IRF6-dependent resistance of epidermal sheets to mechanical stress, but only inhibition of clathrin-mediated endocytosis rescued the localization of junctional proteins at the membrane. Our data show that E-cadherin recycling but not its endocytosis is affected by loss of IRF6. Overall, we demonstrate a role for IRF6 in the delivery of adhesion proteins to the cell membrane.

Integrin signaling to the actin cytoskeleton.

Integrin engagement stimulates the activity of numerous signaling molecules, including the Rho family of GTPases, tyrosine phosphatases, cAMP-dependent protein kinase and protein kinase C, and stimulates production of PtdIns(4,5)P2. Integrins promote actin assembly via the recruitment of molecules that directly activate the actin polymerization machinery or physically link it to sites of cell adhesion.

Dynamics of the Actin Cytoskeleton at Adhesion Complexes.

The shape of cells is altered to allow cells to adapt to their changing environments, including responding to internally generated and externally applied force. Force is sensed by cell surface adhesion proteins that are enriched in sites where cells bind to the extracellular matrix (focal adhesions) and neighboring cells (cell-cell or adherens junctions). Receptors at these adhesion sites stimulate intracellular signal transduction cascades that culminate in dramatic changes in the actin cytoskeleton. New actin filaments form, and/or new and existing filaments can be cleaved, branched, or bundled. Here, we discuss the actin cytoskeleton and its functions. We will examine the current understanding for how the actin cytoskeleton is tethered to adhesion sites. Finally, we will highlight recent studies describing how the actin cytoskeleton at these adhesion sites is remodeled in response to force.