Activation of vinculin induced by cholinergic stimulation regulates contraction of tracheal smooth muscle tissue

Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.     

The vinculin binding sites of talin and alpha-actinin are sufficient to activate vinculin

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.     

Structural basis for amplifying vinculin activation by talin

Talin interactions with vinculin are essential for focal adhesions. Curiously, talin contains three noncontiguous vinculin binding sites (VBS) that can bind individually to the vinculin head (Vh) domain. Here we report the crystal structure of the human Vh.VBS1 complex, a validated model of the Vh.VBS2 structure, and biochemical studies that demonstrate that all of talin VBSs activate vinculin by provoking helical bundle conversion of the Vh domain, which displaces the vinculin tail (Vt) domain. Thus, helical bundle conversion is a structurally conserved response in talin-vinculin interactions. Furthermore, talin VBSs bind to Vh in a mutually exclusive manner but do differ in their affinity for Vh and in their ability to displace Vt, suggesting that the strengths of these interactions could lead to differences in signaling outcome. These findings support a model in which talin binds to and activates multiple vinculin molecules to provoke rapid reorganization of the actin cytoskeleton.     

PIPKIγ regulates focal adhesion dynamics and colon cancer cell invasion

Focal adhesion assembly and disassembly are essential for cell migration and cancer invasion, but the detailed molecular mechanisms regulating these processes remain to be elucidated. Phosphatidylinositol phosphate kinase type Iγ (PIPKIγ) binds talin and is required for focal adhesion formation in EGF-stimulated cells, but its role in regulating focal adhesion dynamics and cancer invasion is poorly understood. We show here that overexpression of PIPKIγ promoted focal adhesion formation, whereas cells expressing either PIPKIγ^K188,200R or PIPKIγ^D316K, two kinase-dead mutants, had much fewer focal adhesions than those expressing WT PIPKIγ in CHO-K1 cells and HCT116 colon cancer cells. Furthermore, overexpression of PIPKIγ, but not PIPKIγ^K188,200R, resulted in an increase in both focal adhesion assembly and disassembly rates. Depletion of PIPKIγ by using shRNA strongly inhibited formation of focal adhesions in HCT116 cells. Overexpression of PIPKIγ^K188,200R or depletion of PIPKIγ reduced the strength of HCT116 cell adhesion to fibronection and inhibited the invasive capacities of HCT116 cells. PIPKIγ depletion reduced PIP₂ levels to ∼40% of control and PIP₃ to undetectable levels, and inhibited vinculin localizing to focal adhesions. Taken together, PIPKIγ positively regulates focal adhesion dynamics and cancer invasion, most probably through PIP₂-mediated vinculin activation.     

Purification and characterization of an 85 kDa talin-binding fragment of vinculin

Vinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin-binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125I-talin and the 125I-85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin-binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125I-talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment was about three-fold more effective at competing for 125I-85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin-talin interaction even though the talin-binding domain is localized in the 85 kDa fragment.     

Cell adhesion strengthening: contributions of adhesive area, integrin binding, and focal adhesion assembly

Mechanical interactions between a cell and its environment regulate migration, contractility, gene expression, and cell fate. We integrated micropatterned substrates to engineer adhesive area and a hydrodynamic assay to analyze fibroblast adhesion strengthening on fibronectin. Independently of cell spreading, integrin binding and focal adhesion assembly resulted in rapid sevenfold increases in adhesion strength to steady-state levels. Adhesive area strongly modulated adhesion strength, integrin binding, and vinculin and talin recruitment, exhibiting linear increases for small areas. However, above a threshold area, adhesion strength and focal adhesion assembly reached a saturation limit, whereas integrin binding transitioned from a uniform distribution to discrete complexes. Adhesion strength exhibited exponential increases with bound integrin numbers as well as vinculin and talin recruitment, and the relationship between adhesion strength and these biochemical events was accurately described by a simple mechanical model. Furthermore, adhesion strength was regulated by the position of an adhesive patch, comprised of bound integrins and cytoskeletal elements, which generated a constant 200-nN adhesive force. Unexpectedly, focal adhesion assembly, in particular vinculin recruitment, contributed only 30% of the adhesion strength. This work elucidates the roles of adhesive complex size and position in the generation of cell-extracellular matrix forces.     

Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin‐containing focal adhesions

Actin–myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30–40% higher than serum‐free cultures. Inhibition of myosin light chain kinase or Rho‐kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum‐induced enhancements in strengthening. Blebbistatin‐induced inhibition of myosin II reduced serum‐induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin‐null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin‐dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum‐induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK‐dependent, vinculin‐containing focal adhesion assembly. J. Cell. Physiol. 223:746–756, 2010. © 2010 Wiley‐Liss, Inc.     

Mechanotransduction in vivo by repeated talin stretch-relaxation events depends upon vinculin

Mechanotransduction is a critical function for cells, in terms of cell viability, shaping of tissues, and cellular behavior. In vitro, cellular level forces can stretch adhesion proteins that link extracellular matrix to the actin cytoskeleton exposing hidden binding sites. However, there is no evidence that in vivo forces produce significant in vivo stretching to cause domain unfolding. We now report that the adhesion protein, talin, is repeatedly stretched by 100-350 nm in vivo by myosin contraction of actin filaments. Using a functional EGFP-N-Talin1-C-mCherry to measure the length of single talin molecules, we observed that the C-terminal mCherry was normally displaced in the direction of actin flow by 90 to >250 nm from N-EGFP but only by 50-60 nm (talin's length in vitro) after myosin inhibition. Individual talin molecules transiently stretched and relaxed. Peripheral, multimolecular adhesions had green outside and red proximal edges. They also exhibited transient, myosin-dependent stretching of 50-350 nm for 6-16 s; however, expression of the talin-binding head of vinculin increased stretching to about 400 nm and suppressed dynamics. We suggest that rearward moving actin filaments bind, stretch, and release talin in multiple, stochastic stick-slip cycles and that multiple vinculin binding and release cycles integrate pulling on matrices into biochemical signals.     

Talin: an emerging focal point of adhesion dynamics

The adhesion protein talin and the phosphoinositide PIP2 are emerging as key modulators of adhesion dynamics. Recent genetic studies on talin demonstrate its physiological role in organizing adhesions, stabilizing integrin-actin linkages and mediating integrin signaling in vivo. Biophysical force measurements provide further evidence that it is required for the reinforcement of the extracellular matrix-integrin-actin connection. Knockdown data along with structural analyses establish a major role for talin in 'inside-out' integrin activation through its direct interaction with integrin cytoplasmic domains. A recently uncovered role for talin is the recruitment of a PIPKI gamma isoform to adhesions. This introduces a novel connection between talin and PIP2 generation. Finally, PIP2 also stimulates the transient, direct binding interaction of the Arp2/3 complex with vinculin and thus may couple adhesion to actin assembly.     

The conformational states of talin autoinhibition complex and its activation under forces

Talin is an integrin-binding protein located at focal adhesion site and serves as both an adapter and a force transmitter. Its integrin binding activity is regulated by the intramolecular autoinhibition interaction between its F3 and RS domains. Here, we used atomic force microscopy to measure the strength of talin autoinhibition complex. Our results suggest that the lifetime of talin autoinhibition complex shows weak catch bond behavior and does not change significantly at smaller forces, while it drops rapidly at larger forces (>10 pN). Moreover, besides the complex conformation revealed by crystal structure, our molecular dynamics (MD) simulations indicate the possible existence of another stable conformation. Further analysis indicates that forces may regulate the equilibrium of the two stable binding states and result in the non-exponential force dependence of the binding lifetime. Our findings reveal a negative regulation mechanism on talin activation and provide a new point of view on the function of talin in focal adhesion.     

Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides

Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P(2). This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P(2)-induced talin activation. Finally, sequestration of PI4,5P(2) by a specific pleckstrin homology domain confirms that PI4,5P(2) is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P(2) exposes the integrin-binding site on talin. We propose that PI4,5P(2)-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P(2) to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.     

The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.

Focal adhesion kinase (FAK) is a tyrosine kinase found in focal adhesions, intracellular signaling complexes that are formed following engagement of the extracellular matrix by integrins. The C-terminal 'focal adhesion targeting' (FAT) region is necessary and sufficient for localizing FAK to focal adhesions. We have determined the crystal structure of FAT and show that it forms a four-helix bundle that resembles those found in two other proteins involved in cell adhesion, alpha-catenin and vinculin. The binding of FAT to the focal adhesion protein, paxillin, requires the integrity of the helical bundle, whereas binding to another focal adhesion protein, talin, does not. We show by mutagenesis that paxillin binding involves two hydrophobic patches on opposite faces of the bundle and propose a model in which two LD motifs of paxillin adopt amphipathic helices that augment the hydrophobic core of FAT, creating a six-helix bundle.     

Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.     

Vinculin Force-Sensitive Dynamics at Focal Adhesions Enable Effective Directed Cell Migration

Cell migration is a complex process, requiring coordination of many subcellular processes including membrane protrusion, adhesion, and contractility. For efficient cell migration, cells must concurrently control both transmission of large forces through adhesion structures and translocation of the cell body via adhesion turnover. Although mechanical regulation of protein dynamics has been proposed to play a major role in force transmission during cell migration, the key proteins and their exact roles are not completely understood. Vinculin is an adhesion protein that mediates force-sensitive processes, such as adhesion assembly under cytoskeletal load. Here, we elucidate the mechanical regulation of vinculin dynamics. Specifically, we paired measurements of vinculin loads using a Förster resonance energy transfer-based tension sensor and vinculin dynamics using fluorescence recovery after photobleaching to measure force-sensitive protein dynamics in living cells. We find that vinculin adopts a variety of mechanical states at adhesions, and the relationship between vinculin load and vinculin dynamics can be altered by the inhibition of vinculin binding to talin or actin or reduction of cytoskeletal contractility. Furthermore, the force-stabilized state of vinculin required for the stabilization of membrane protrusions is unnecessary for random migration, but is required for directional migration along a substrate-bound cue. These data show that the force-sensitive dynamics of vinculin impact force transmission and enable the mechanical integration of subcellular processes. These results suggest that the regulation of force-sensitive protein dynamics may have an underappreciated role in many cellular processes.     

Molecular dynamics study of talin-vinculin binding

Cells can sense mechanical force in regulating focal adhesion assembly. One vivid example is the force-induced recruitment of vinculin to reinforce initial contacts between a cell and the extracellular matrix. Crystal structures of the unbound proteins and bound complex between the vinculin head subdomain (Vh1) and the talin vinculin binding site 1 (VBS1) indicate that vinculin undergoes a conformational change upon binding to talin. However, the molecular basis for this event and the precise nature of the binding pathway remain elusive. In this article, molecular dynamics is used to investigate the binding mechanism of Vh1 and VBS1 under minimal constraints to facilitate binding. One simulation demonstrates binding of the two molecules in the complete absence of external force. VBS1 makes early hydrophobic contact with Vh1 by positioning the critical hydrophobic residues (L608, L615, and L622) in the groove formed by helices 1 and 2 of Vh1. The solvent-exposed hydrophobic residues (V619 and L623) then gradually penetrate the hydrophobic core of Vh1, thus further separating helix 1 from helix 2. These critical residues are highly conserved as large hydrophobic side groups in other vinculin binding sites; studies also have demonstrated that these residues are essential in Vh1-VBS1 binding. Similar binding mechanisms are also demonstrated in separate molecular dynamics simulations of Vh1 binding to other vinculin binding sites both in talin and α-actinin.     

Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin-binding site (VBS), folds into a five-helix bundle whereas talin 656–789 is a four-helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482–655 fold. We demonstrate that the talin 482–655 five-helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.     

Polyphosphoinositides inhibit the interaction of vinculin with actin filaments.

Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin's head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P2) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P2 on the actin and talin-binding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P2.     

FAK promotes recruitment of talin to nascent adhesions to control cell motility

Cell migration is a dynamic process that involves the continuous formation, maturation, and turnover of matrix-cell adhesion sites. New (nascent) adhesions form at the protruding cell edge in a tension-independent manner and are comprised of integrin receptors, signaling, and cytoskeletal-associated proteins. Integrins recruit focal adhesion kinase (FAK) and the cytoskeletal protein talin to nascent adhesions. Canonical models support a role for talin in mediating FAK localization and activation at adhesions. Here, alternatively, we show that FAK promotes talin recruitment to nascent adhesions occurring independently of talin binding to β1 integrins. The direct binding site for talin on FAK was identified, and a point mutation in FAK (E1015A) prevented talin association and talin localization to nascent adhesions but did not alter integrin-mediated FAK recruitment and activation at adhesions. Moreover, FAK E1015A inhibited cell motility and proteolytic talin cleavage needed for efficient adhesion dynamics. These results support an alternative linkage for FAK-talin interactions within nascent adhesions essential for the control of cell migration.     

MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts

Herein, we define how MEKK1, a MAPK kinase kinase, regulates cell migration. MEKK1 is associated with actin fibers and focal adhesions, localizing MEKK1 to sites critical in the control of cell adhesion and migration. EGF-induced ERK1/2 activation and chemotaxis are inhibited in MEKK1-/- fibroblasts. MEKK1 deficiency causes loss of vinculin in focal adhesions of migrating cells, increased cell adhesion and impeded rear-end detachment. MEKK1 is required for activation of the cysteine protease calpain and cleavage of spectrin and talin, proteins linking focal adhesions to the cytoskeleton. Inhibition of ERK1/2 or calpain, but not of JNK, mimics MEKK1 deficiency. Therefore, MEKK1 regulates calpain-mediated substratum release of migrating fibroblasts.     

Spatial distribution and functional significance of activated vinculin in living cells

Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin beta by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics.