Traction forces exerted through N-cadherin contacts

BACKGROUND INFORMATION: Mechanical forces play an important role in the organization, growth and function of living tissues. The ability of cells to transduce mechanical signals is governed by two types of microscale structures: focal adhesions, which link cells to the extracellular matrix, and adherens junctions, which link adjacent cells through cadherins. Although many studies have examined forces induced by focal adhesions, there is little known about the role of adherens junctions in force-regulation processes. The present study focuses on the determination of force transduction through cadherins at a single cell level. RESULTS: We characterized for the first time the distribution of forces developed by the cell through cadherin contacts. A N-cadherin (neural cadherin)-Fc chimaera, which mimicks the cell adhesion molecule N-cadherin, was immobilized on a muFSA (micro-force sensor array), comprising a dense array of vertical elastomer pillars, which were used both as a cell culture support for N-cadherin-expressing C2 myogenic cells and as detectors for force mapping. We coated the top of the pillars on which cells adhere and recruit adhesion complexes and F-actin. Individual pillar bending allowed the measurement of forces that mainly developed at the cell edge and directed toward their centre. Similar force distribution and amplitude were detected with an unrelated cell line of neuronal origin. Further comparison with forces applied by cells on pillars coated with fibronectin indicates that mechanical stresses transduced through both types of adhesions were comparable in distribution, orientation and amplitude. CONCLUSIONS: These results present a versatile method to measure and map forces exerted by cell-cell adhesion complexes. They show that cells transduce mechanical stress through cadherin contacts which are of the same order as magnitude of those previously characterized for focal adhesions. Altogether, they emphasize the mechanotransduction role of cytoskeleton-linked adhesion receptors of the cadherin family in tissue cohesion and reshaping.     

The vinculin C-terminal hairpin mediates F-actin bundle formation, focal adhesion, and cell mechanical properties

Vinculin is an essential and highly conserved cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples integrins or cadherins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility, and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) contains determinants necessary for binding and bundling of actin filaments. Actin binding to Vt has been proposed to induce formation of a Vt dimer that is necessary for cross-linking actin filaments. Results from this study provide additional support for actin-induced Vt self-association. Moreover, the actin-induced Vt dimer appears distinct from the dimer formed in the absence of actin. To better characterize the role of the Vt strap and carboxyl terminus (CT) in actin binding, Vt self-association, and actin bundling, we employed smaller amino-terminal (NT) and CT deletions that do not perturb the structural integrity of Vt. Although both NT and CT deletions retain actin binding, removal of the CT hairpin (1061-1066) selectively impairs actin bundling in vitro. Moreover, expression of vinculin lacking the CT hairpin in vinculin knock-out murine embryonic fibroblasts affects the number of focal adhesions formed, cell spreading as well as cellular stiffening in response to mechanical force.     

Integrating actin dynamics,mechanotransduction and integrin activation: The multiple functions of actin binding proteins in focal adhesions

Focal adhesions are clusters of integrin transmembrane receptors that mechanically couple the extracellular matrix to the actin cytoskeleton during cell migration. Focal adhesions sense and respond to variations in force transmission along a chain of protein-protein interactions linking successively actin filaments, actin binding proteins, integrins and the extracellular matrix to adapt cell-matrix adhesion to the composition and mechanical properties of the extracellular matrix. This review focuses on the molecular mechanisms by which actin binding proteins integrate actin dynamics, mechanotransduction and integrin activation to control force transmission in focal adhesions.     

The role of apical cell–cell junctions and associated cytoskeleton in mechanotransduction

Tissues of multicellular organisms are characterised by several types of specialised cell–cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton‐associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ.     

Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions.

The cell-cell adherens junction is a site for cadherin-mediated cell adhesion where actin filaments are densely associated with the plasma membrane through its well-developed plasmalemmal undercoat. Recent research has focused on the molecular linkage between cadherins and actin filaments in the undercoat of adherens junctions in order to understand the functions of these undercoat-constitutive proteins in the regulation and signal transduction of cadherin-based cell adhesion.     

Mapping the dynamics of shear stress-induced structural changes in endothelial cells

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.     

Vinculin, cadherin mechanotransduction and homeostasis of cell–cell junctions

Cell adhesion junctions characteristically arise from the cooperative integration of adhesion receptors, cell signalling pathways and the cytoskeleton. This is exemplified by cell–cell interactions mediated by classical cadherin adhesion receptors. These junctions are sites where cadherin adhesion systems functionally couple to the dynamic actin cytoskeleton, a process that entails physical interactions with many actin regulators and regulation by cell signalling pathways. Such integration implies a potential role for molecules that may stand at the interface between adhesion, signalling and the cytoskeleton. One such candidate is the cortical scaffolding protein, vinculin, which is a component of both cell–cell and cell–matrix adhesions. While its contribution to integrin-based adhesions has been extensively studied, less is known about how vinculin contributes to cell–cell adhesions. A major recent advance has come with the realisation that cadherin adhesions are active mechanical structures, where cadherin serves as part of a mechanotransduction pathway by which junctions sense and elicit cellular responses to mechanical stimuli. Vinculin has emerged as an important element in cadherin mechanotransduction, a perspective that illuminates its role in cell–cell interactions. We now review its role as a cortical scaffold and its role in cadherin mechanotransduction.     

A helping hand: How vinculin contributes to cell-matrix and cell-cell force transfer

Vinculin helps cells regulate and respond to mechanical forces. It is a scaffolding protein that tightly regulates its interactions with potential binding partners within adhesive structures—including focal adhesions that link the cell to the extracellular matrix and adherens junctions that link cells to each other—that physically connect the force-generating actin cytoskeleton (CSK) with the extracellular environment. This tight control of binding partner interaction—mediated by vinculin's autoinhibitory head–tail interaction—allows vinculin to rapidly interact and detach in response to changes in the dynamic forces applied through the cell. In doing so, vinculin modulates the structural composition of focal adhesions and the cell's ability to generate traction forces and adhesion strength. Recent evidence suggests that vinculin plays a similar role in regulating the fate and function of cell–cell junctions, further underscoring the importance of this protein. Using our lab's recent work as a starting point, this commentary explores several outstanding questions regarding the nature of vinculin activation and its function within focal adhesions and adherens junctions.     

Vinculin regulation of F-actin bundle formation

Vinculin is an essential cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples transmembrane proteins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) has the ability to both bind and bundle actin filaments. Binding to actin induces a conformational change in Vt believed to promote formation of a Vt dimer that is able to crosslink actin filaments. We have recently provided additional evidence for the actin-induced Vt dimer and have shown that the vinculin carboxyl (C)-terminal hairpin is critical for both the formation of the Vt dimer and for bundling F-actin. We have also demonstrated the importance of the C-terminal hairpin in cells as deletion of this region impacts both adhesion properties and force transduction. Intriguingly, we have identified bundling deficient variants of vinculin that show different cellular phenotypes. These results suggest additional role(s) for the C-terminal hairpin, distinct from its bundling function. In this commentary, we will expand on our previous findings and further investigate these actin bundling deficient vinculin variants.     

Identification of a novel intermediate filament-linked N-cadherin/gamma-catenin complex involved in the establishment of the cytoarchitecture of differentiated lens fiber cells

Tissue morphogenesis and maintenance of complex tissue architecture requires a variety of cell-cell junctions. Typically, cells adhere to one another through cadherin junctions, both adherens and desmosomal junctions, strengthened by association with cytoskeletal networks during development. Both β- and γ-catenins are reported to link classical cadherins to the actin cytoskeleton, but only γ-catenin binds to the desmosomal cadherins, which links them to intermediate filaments through its association with desmoplakin. Here we provide the first biochemical evidence that, in vivo, γ-catenin also mediates interactions between classical cadherins and the intermediate filament cytoskeleton, linked through desmoplakin. In the developing lens, which has no desmosomes, we discovered that vimentin became linked to N-cadherin complexes in a differentiation-state specific manner. This newly identified junctional complex was tissue specific but not unique to the lens. To determine whether in this junction N-cadherin was linked to vimentin through γ-catenin or β-catenin we developed an innovative “double” immunoprecipitation technique. This approach made possible, for the first time, the separation of N-cadherin/γ-catenin from N-cadherin/β-catenin complexes and the identification of multiple members of each of these isolated protein complexes. The study revealed that vimentin was associated exclusively with N-cadherin/γ-catenin junctions. Assembly of this novel class of cadherin junctions was coincident with establishment of the unique cytoarchitecture of lens fiber cells. In addition, γ-catenin had a distinctive localization to the vertices of these hexagonally shaped differentiating lens fiber cells, a region devoid of actin; while β-catenin co-localized with actin at lateral cell interfaces. We believe this novel vimentin-linked N-cadherin/γ-catenin junction provides the tensile strength necessary to establish and maintain structural integrity in tissues that lack desmosomes.     

Regulation of intercellular adhesion strength in fibroblasts

The regulation of adherens junction formation in cells of mesenchymal lineage is of critical importance in tumorigenesis but is poorly characterized. As actin filaments are crucial components of adherens junction assembly, we studied the role of gelsolin, a calcium-dependent, actin severing protein, in the formation of N-cadherin-mediated intercellular adhesions. With a homotypic, donor-acceptor cell model and plates or beads coated with recombinant N-cadherin-Fc chimeric protein, we found that gelsolin spatially co-localizes to, and is transiently associated with, cadherin adhesion complexes. Fibroblasts from gelsolin-null mice exhibited marked reductions in kinetics and strengthening of N-cadherin-dependent junctions when compared with wild-type cells. Experiments with lanthanum chloride (250 microm) showed that adhesion strength was dependent on entry of calcium ions subsequent to N-cadherin ligation. Cadherin-associated gelsolin severing activity was required for localized actin assembly as determined by rhodamine actin monomer incorporation onto actin barbed ends at intercellular adhesion sites. Scanning electron microscopy showed that gelsolin was an important determinant of actin filament architecture of adherens junctions at nascent N-cadherin-mediated contacts. These data indicate that increased actin barbed end generation by the severing activity of gelsolin associated with N-cadherin regulates intercellular adhesion strength.     

Regulation of substrate adhesion dynamics during cell motility

The movement of a metazoan cell entails the regulated creation and turnover of adhesions with the surface on which it moves. Adhesion sites form as a result of signaling between the extracellular matrix on the outside and the actin cytoskeleton on the inside, and they are associated with specific assembles of actin filaments. Two broad categories of adhesion sites can be distinguished: (1) "focal complexes" associated with lamellipodia and filopodia that support protrusion and traction at the cell front; and (2) "focal adhesions" at the termini of stress fibre bundles that serve in longer term anchorage. Focal complexes are signaled via Rac1 or Cdc42 and can either turnover on a minute scale or differentiate, via intervention of the RhoA pathway, into longer-lived focal adhesions. All classes of adhesion sites depend on the stress in the actin cytoskeleton for their formation and maintenance. Different cell types use different adhesion strategies to move, in terms of the relative engagement of filopodia and lamellipodia in focal complex formation and protrusion and the extent of focal adhesion formation. These differences can be attributed to variations in the relative activities of Rho family members. However, the Rho GTPases alone are unable to signal asymmetry in the actin cytoskeleton, necessary for polarisation and movement. Polarisation requires the collaboration of the microtubule cytoskeleton. Changes in the polymerisation state of microtubules influences the activities of both Rac1 and RhoA and microtubules interact directly with adhesion foci and promote their turnover. Possible mechanisms of cross-talk between the microtubule and actin cytoskeletons in determining polarity are discussed.     

The key feature for early migratory processes: Dependence of adhesion,actin bundles,force generation and transmission on filopodia

Migration of cells is one of the most essential prerequisites to form higher organisms and depends on a strongly coordinated sequence of processes. Early migratory events include substrate sensing, adhesion formation, actin bundle assembly and force generation. While substrate sensing was ascribed to filopodia, all other processes were believed to depend mainly on lamellipodia of migrating cells. In this work we show for motile keratinocytes that all processes from substrate sensing to force generation strongly depend on filopodial focal complexes as well as on filopodial actin bundles. In a coordinated step by step process, filopodial focal complexes have to be tightly adhered to the substrate and to filopodial actin bundles to enlarge upon lamellipodial contact forming classical focal adhesions. Lamellipodial actin filaments attached to those focal adhesions originate from filopodia. Upon cell progression, the incorporation of filopodial actin bundles into the lamellipodium goes along with a complete change in actin cross-linker composition from filopodial fascin to lamellipodial α-actinin. α-Actinin in turn is replaced by myosin II and becomes incorporated directly behind the leading edge. Myosin II activity makes this class of actin bundles with their attached FAs the major source of force generation and transmission at the cell front. Furthermore, connection of FAs to force generating actin bundles leads to their stabilization and further enlargement. Consequently, adhesion sites formed independently of filopodia are not connected to detectable actin bundles, transmit weak forces to the substrate and disassemble within a few minutes without having been increased in size.Key words: filopodia, focal complexes, cell migration, focal adhesion, myosin II, force, actin flow, maturation     

The key feature for early migratory processes

Migration of cells is one of the most essential prerequisites to form higher organisms and depends on a strongly coordinated sequence of processes. Early migratory events include substrate sensing, adhesion formation, actin bundle assembly and force generation. While substrate sensing was ascribed to filopodia, all other processes were believed to depend mainly on lamellipodia of migrating cells. In this work we show for motile keratinocytes that all processes from substrate sensing to force generation strongly depend on filopodial focal complexes as well as on filopodial actin bundles. In a coordinated step by step process filopodial focal complexes have to be tightly adhered to the substrate and to filopodial actin bundles to enlarge upon lamellipodial contact forming classical focal adhesions. Lamellipodial actin filaments attached to those focal adhesions originate from filopodia. Upon cell progression, the incorporation of filopodial actin bundles into the lamellipodium goes along with a complete change in actin cross-linker composition from filopodial fascin to lamellipodial α-actinin. α-Actinin in turn is replaced by myosin II and becomes incorporated directly behind the leading edge. Myosin II activity makes this class of actin bundles with their attached FAs the major source of force generation and transmission at the cell front. Furthermore, connection of FAs to force generating actin bundles leads to their stabilization and further enlargement. Consequently, adhesion sites formed independently of filopodia are not connected to detectable actin bundles, transmit weak forces to the substrate and disassemble within a few minutes without having been increased in size.     

A helping hand: How vinculin contributes to cell-matrix and cell-cell force transfer

Vinculin helps cells regulate and respond to mechanical forces. It is a scaffolding protein that tightly regulates its interactions with potential binding partners within adhesive structures—including focal adhesions that link the cell to the extracellular matrix and adherens junctions that link cells to each other—that physically connect the force-generating actin cytoskeleton (CSK) with the extracellular environment. This tight control of binding partner interaction—mediated by vinculin''s autoinhibitory head–tail interaction—allows vinculin to rapidly interact and detach in response to changes in the dynamic forces applied through the cell. In doing so, vinculin modulates the structural composition of focal adhesions and the cell''s ability to generate traction forces and adhesion strength. Recent evidence suggests that vinculin plays a similar role in regulating the fate and function of cell–cell junctions, further underscoring the importance of this protein. Using our lab''s recent work as a starting point, this commentary explores several outstanding questions regarding the nature of vinculin activation and its function within focal adhesions and adherens junctions.     

Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner

Cell surface receptors integrate chemical and mechanical cues to regulate a wide range of biological processes. Integrin complexes are the mechanotransducers between the extracellular matrix and the actomyosin cytoskeleton. By analogy, cadherin complexes may function as mechanosensors at cell–cell junctions, but this capacity of cadherins has not been directly demonstrated. Furthermore, the molecular composition of the link between E-cadherin and actin, which is needed to sustain such a function, is unresolved. In this study, we describe nanomechanical measurements demonstrating that E-cadherin complexes are functional mechanosensors that transmit force between F-actin and E-cadherin. Imaging experiments reveal that intercellular forces coincide with vinculin accumulation at actin-anchored cadherin adhesions, and nanomechanical measurements show that vinculin potentiates the E-cadherin mechanosensory response. These investigations directly demonstrate the mechanosensory capacity of the E-cadherin complex and identify a novel function for vinculin at cell–cell junctions. These findings have implications for barrier function, morphogenesis, cell migration, and invasion and may extend to all soft tissues in which classical cadherins regulate cell–cell adhesion.     

Actin-dependent membrane association of a Drosophila epithelial APC protein and its effect on junctional Armadillo

BACKGROUND: The adenomatous polyposis coli (APC) protein is an important tumour suppressor in the colon. It promotes the destabilisation of free cytoplasmic beta-catenin (the vertebrate homologue of the Drosophila protein Armadillo), a critical effector of the Wnt signalling pathway. The beta-catenin protein is also a component of adherens junctions, linking these to the actin cytoskeleton. In Drosophila epithelial cells, the ubiquitous form of APC, known as E-APC, is associated with adherens junctions. This association appears to be necessary for E-APC to function in destabilising Armadillo. RESULTS: Using actin-depolymerising drugs, we established that an intact actin cytoskeleton is required for the association of E-APC with adherens junctions in the Drosophila embryo. From an analysis of profilin mutants, whose actin cytoskeleton is disrupted, we found that E-APC also requires actin filaments to associate with adhesive cell membranes in the ovary. Notably, conditions that delocalised E-APC from membranes, including a mutation in E-APC itself, caused partial detachment of Armadillo from adhesive membranes. CONCLUSIONS: Actin filaments are continuously required for E-APC to be associated with junctional membranes. These filaments may serve as tracks for E-APC to reach the adherens junctions. The failure of E-APC to do so appears to affect the integrity of junctional complexes.     

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.     

Dissecting the roles of ROCK isoforms in stress-induced cell detachment

The homologous Rho kinases, ROCK1 and ROCK2, are involved in stress fiber assembly and cell adhesion and are assumed to be functionally redundant. Using mouse embryonic fibroblasts (MEFs) derived from ROCK1^−/− and ROCK2^−/− mice, we have recently reported that they play different roles in regulating doxorubicin-induced stress fiber disassembly and cell detachment: ROCK1 is involved in destabilizing the actin cytoskeleton and cell detachment, whereas ROCK2 is required for stabilizing the actin cytoskeleton and cell adhesion. Here, we present additional insights into the roles of ROCK1 and ROCK2 in regulating stress-induced impairment of cell-matrix and cell-cell adhesion. In response to doxorubicin, ROCK1^−/− MEFs showed significant preservation of both focal adhesions and adherens junctions, while ROCK2^−/− MEFs exhibited impaired focal adhesions but preserved adherens junctions compared with the wild-type MEFs. Additionally, inhibition of focal adhesion or adherens junction formations by chemical inhibitors abolished the anti-detachment effects of ROCK1 deletion. Finally, ROCK1^−/− MEFs, but not ROCK2^−/− MEFs, also exhibited preserved central stress fibers and reduced cell detachment in response to serum starvation. These results add new insights into a novel mechanism underlying the anti-detachment effects of ROCK1 deletion mediated by reduced peripheral actomyosin contraction and increased actin stabilization to promote cell-cell and cell-matrix adhesion. Our studies further support the differential roles of ROCK isoforms in regulating stress-induced loss of central stress fibers and focal adhesions as well as cell detachment.     

Adherens junctions remain dynamic

One of the four principal categories of cell-cell junctions that hold together and shape distinct tissues and organs in vertebrates, adherens junctions (AJs) form cell-cell contacts that connect transmembrane proteins with cytoskeletal actin filaments to provide architectural strength, aid in morphogenesis, and help to maintain proper tissue homeostasis. The classical organization of AJs, consisting of transmembrane cadherins and cytoplasmically attached β-catenins and α-catenins assembled together into a multiprotein complex, was once thought obligatory to craft a robust and stable connection to actin-based cytoskeletal elements, but this architecture has since been challenged and questioned to exist. In a stimulating paper published in a recent issue of BMC Biology, Millán et al. provide convincing evidence that in confluent vascular endothelial cells a novel dynamic vascular endothelial (VE)-cadherin-based AJ type exists that interacts with and physically connects prominent bundles of tension-mediating actin filaments, stress fibers, between neighboring cells. Stress fibers were known previously to link to integrin-based focal adhesion complexes but not to cell-cell adhesion mediating AJs. These new findings, together with previous results support the concept that different AJ subtypes, sharing the same transmembrane cadherin types, can assemble in various configurations to either increase barrier function and promote physical cell-cell adhesion, or to lessen cell-cell adhesion and promote cell separation and migration.