How force might activate talin's vinculin binding sites: SMD reveals a structural mechanism.
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ABSTRACT:
SUBMITTER: Hytonen VP
PROVIDER: S-EPMC2242828 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Vinculin is a universal adaptor protein that transiently reinforces the mechanical stability of adhesion complexes. It stabilizes mechanical connections that cells establish between the actomyosin cytoskeleton and the extracellular matrix via integrins or to neighboring cells via cadherins, yet little is known regarding its mechanical design. Vinculin binding sites (VBSs) from different nonhomologous actin-binding proteins use conserved helical motifs to associate with the vinculin head domain. We studied the mechanical stability of such complexes by pulling VBS peptides derived from talin, α-actinin, and Shigella IpaA out of the vinculin head domain. Experimental data from atomic force microscopy single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations both revealed greater mechanical stability of the complex for shear-like than for zipper-like pulling configurations. This suggests that reinforcement occurs along preferential force directions, thus stabilizing those cytoskeletal filament architectures that result in shear-like pulling geometries. Large force-induced conformational changes in the vinculin head domain, as well as protein-specific fine-tuning of the VBS sequence, including sequence inversion, allow for an even more nuanced force response.
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Project description: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, ?-catenin, a cytoplasmic component of the cadherin adhesion complex, activates vinculin. Direct binding of ?-catenin to vinculin is critical for this event because a point mutant (?-catenin L344P) lacking high affinity binding does not activate vinculin. Furthermore, unlike all known vinculin activators, ?-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 ?-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.
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Project description:Focal adhesions are dynamic constructs at the leading edge of migrating cells, linking them to the extracellular matrix and enabling force sensing and transmission. The lifecycle of a focal adhesion is a highly coordinated process involving spatial and temporal variations of protein composition, interaction, and cellular tension. The assembly of focal adhesions requires the recruitment and activation of vinculin. Vinculin is present in the cytoplasm in an autoinhibited conformation in which its tail is held pincerlike by its head domains, further stabilized by two high-affinity head-tail interfaces. Vinculin has binding sites for talin and F-actin, but effective binding requires vinculin activation to release its head-tail associations. In migrating cells, it has been shown that the locations of vinculin activation coincide with areas of high cellular tension, and that the highest recorded tensions across vinculin are associated with adhesion assembly. Here, we use a structure-based model to investigate vinculin activation by talin modulated by tensile force generated by transient associations with F-actin. We show that vinculin activation may proceed from an intermediate state stabilized by partial talin-vinculin association. There is a low-force regime and a high-force regime where vinculin activation is dominated by two different pathways with distinct responses to force. Specifically, at zero or low forces, vinculin activation requires substantial destabilization of the main head-tail interface, which is rigid and undergoes very limited fluctuations, despite the other being relatively flexible. This pathway is not significantly affected by force; instead, higher forces favor an alternative pathway, which seeks to release the vinculin tail from its pincerlike head domains before destabilizing the head-tail interfaces. This pathway has a force-sensitive activation barrier and is significantly accelerated by force. Experimental data of vinculin during various stages of the focal adhesion lifecycle are consistent with the proposed force-regulated activation pathway.
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Project description:Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculin-null cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (V(H)) and tail domains. V(H) enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with VH, implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; V(H) has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or V(H), correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.
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Project description:αT (Testes)-catenin, a critical factor regulating cell-cell adhesion in the heart, directly couples the cadherin-catenin complex to the actin cytoskeleton at the intercalated disk (ICD), a unique cell-cell junction that couples cardiomyocytes. Loss of αT-catenin in mice reduces plakophilin2 and connexin 43 recruitment to the ICD. Since αT-catenin is subjected to mechanical stretch during actomyosin contraction in cardiomyocytes, its activity could be regulated by mechanical force. To provide insight in how force regulates αT-catenin function, we investigated the mechanical stability of the putative, force-sensing middle (M) domain of αT-catenin and determined how force impacts vinculin binding to αT-catenin. We show that 1) physiological levels of force, <15 pN, are sufficient to unfold the three M domains; 2) the M1 domain that harbors the vinculin-binding site is unfolded at ∼6 pN; and 3) unfolding of the M1 domain is necessary for high-affinity vinculin binding. In addition, we quantified the binding kinetics and affinity of vinculin to the mechanically exposed binding site in M1 and observed that αT-catenin binds vinculin with low nanomolar affinity. These results provide important new insights into the mechanosensing properties of αT-catenin and how αT-catenin regulates cell-cell adhesion at the cardiomyocyte ICD.
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Project description:Vinculin links integrin receptors to the actin cytoskeleton by binding to talin. Vinculin is held in an inactive, closed-clamp conformation through hydrophobic interactions between its head and tail domains, and vinculin activation has long been thought to be dependent upon severing the head-tail interaction. Talin, alpha-actinin, and the invasin IpaA of Shigella flexneri sever vinculin's head-tail interaction by inserting an alpha-helix into vinculin's N-terminal four-helical bundle, provoking extensive conformational changes by a helical bundle conversion mechanism; these alterations in vinculin structure displace its tail domain, allowing vinculin to bind to its other partners. IpaA harbors two juxtaposed alpha-helical vinculin-binding sites (VBS) in its C-terminus. Here, we report that the lower affinity VBS of IpaA can also bind to the adjacent C-terminal four-helical bundle of vinculin's head domain through a helix addition mechanism. These hydrophobic interactions do not alter the conformation of this helical bundle, and the architecture of the complex suggests that IpaA can simultaneously interact with both of the four-helical bundle domains of vinculin's N-terminus to stabilize vinculin-IpaA interactions.
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Project description:This study utilized a Förster resonance energy transfer (FRET)-based molecular tension sensor and live cell imaging to evaluate the effect of osteocytes, a mechanosensitive bone cell, on the migratory behavior of tumor cells. Two cell lines derived from MDA-MB-231 breast cancer cells were transfected with the vinculin tension sensor to quantitatively evaluate the force in focal adhesions of the tumor cell. Tumor cells treated with MLO-A5 osteocyte-conditioned media (CM) decreased the tensile forces in their focal adhesions and decreased their migratory potential. Tumor cells treated with media derived from MLO-A5 cells exposed to fluid flow-driven shear stress (FFCM) increased the tensile forces and increased migratory potential. Focal adhesion tension in tumor cells was also affected by distance from MLO-A5 cells when the two cells were co-cultured, where tumor cells close to MLO-A5 cells exhibited lower tension and decreased cell motility. Overall, this study demonstrates that focal adhesion tension is involved in altered migratory potential of tumor cells, and tumor-osteocyte interactions decrease the tension and motility of tumor cells.
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Project description:The shaping of a multicellular body and repair of adult tissues require fine--tuning of cell adhesion, cell mechanics, and intercellular transmission of mechanical load. Adherens junctions (AJs) are the major intercellular junctions by which cells sense and exert mechanical force on each other. However, how AJs adapt to mechanical stress and how this adaptation contributes to cell-cell cohesion and eventually to tissue-scale dynamics and mechanics remains largely unknown. Here, by analyzing the tension-dependent recruitment of vinculin, ?-catenin, and F-actin as a function of stiffness, as well as the dynamics of GFP-tagged wild-type and mutated ?-catenins, altered for their binding capability to vinculin, we demonstrate that the force-dependent binding of vinculin stabilizes ?-catenin and is responsible for AJ adaptation to force. Challenging cadherin complexes mechanical coupling with magnetic tweezers, and cell-cell cohesion during collective cell movements, further highlight that tension-dependent adaptation of AJs regulates cell-cell contact dynamics and coordinated collective cell migration. Altogether, these data demonstrate that the force-dependent ?-catenin/vinculin interaction, manipulated here by mutagenesis and mechanical control, is a core regulator of AJ mechanics and long-range cell-cell interactions.
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Project description:Actinomycin D (ActD) is a small molecule with strong antibiotic and anticancer activity. However, its biologically relevant DNA-binding mechanism has never been resolved, with some studies suggesting that the primary binding mode is intercalation, and others suggesting that single-stranded DNA binding is most important. To resolve this controversy, we develop a method to quantify ActD's equilibrium and kinetic DNA-binding properties as a function of stretching force applied to a single DNA molecule. We find that destabilization of double stranded DNA (dsDNA) by force exponentially facilitates the extremely slow ActD-dsDNA on and off rates, with a much stronger effect on association, resulting in overall enhancement of equilibrium ActD binding. While we find the preferred ActD-DNA-binding mode to be to two DNA strands, major duplex deformations appear to be a pre-requisite for ActD binding. These results provide quantitative support for a model in which the biologically active mode of ActD binding is to pre-melted dsDNA, as found in transcription bubbles. DNA in transcriptionally hyperactive cancer cells will therefore likely efficiently and rapidly bind low ActD concentrations (≈ 10 nM), essentially locking ActD within dsDNA due to its slow dissociation, blocking RNA synthesis and leading to cell death.
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Project description:Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.
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