Benzo(a)pyrene,but not 2,3,7,8-tetrachlorodibenzo-p-dioxin,alters cell adhesion proteins in human uterine RL95-2 cells

This study compared the effects of benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two aryl hydrocarbon receptor agonists, on cell attachment and adherens junction proteins in RL95-2 human uterine endometrial cells. Exposure to 10 microM BaP significantly decreased cell attachment to Matrigel, whereas 10 nM TCDD had no effect. Immunocytochemistry and Western immunoblot analysis showed that BaP, but not TCDD, produced a marked loss of plasma membrane epidermal growth factor receptor (EGF-R) localized along intercellular boundaries. BaP-treated cells exhibited significant decreases in beta-catenin and cadherin protein levels, while vinculin levels remained unchanged relative to control. In contrast, TCDD treatment had no effect on the levels of beta-catenin, cadherin, or vinculin. Further studies using the fluorescein labeled peptide phalloidin showed the presence of continuous subcortical actin filaments in control cells, whereas BaP-treated cells had subcortical actin aggregates. Thus, in contrast to TCDD, BaP produces a loss of cell attachment involving decreased localization of molecules important for cell-cell interactions in RL95-2 cells.     

Accumulation of talin in nodes at the edge of the lamellipodium and separate incorporation into adhesion plaques at focal contacts in fibroblasts

The focal contact forms beneath F-actin-rich ribs, or cytoplasmic precursors, present in the lamellipodia of fibroblasts. The basal part of the precursor is retained at the contact as the initial adhesion plaque. We have examined the distribution of talin in the lamellipodia and adhesion plaques of chicken embryo fibroblasts relative to the process of focal contact formation. Motility of single cells was recorded with differential interference contrast or interference reflection microscopy before fixation and fluorescent staining for talin, F-actin, and vinculin. Talin is present along the extreme edge of the lamellipodium, where it is further concentrated into a series of nodes. The nodes of talin are present at the tips of both larger and finer F-actin-rich ribs and at small structural nodes at the edge of the lamellipodium. We suggest that the talin in the nodes functions, via a cross-linking activity, in the convergence of actin filaments at the membrane during development of the ribs. Talin accumulates de novo in the adhesion plaque, independent of that at the tip of the precursor, in response to contact with the substrate. This second accumulation of talin at the focal contact starts before vinculin, consistent with a sequential binding of talin at the membrane and of vinculin to talin. The results imply that talin functions independently at two steps during formation of the focal contact: the development of the F-actin-rich precursor of the contact; and development of the contact-associated adhesion plaque, both involving organization of F-actin at the membrane.     

Isolation and partial characterization of human platelet vinculin

A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE-Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.     

Actin-independent association of vinculin with the cytoplasmic aspect of the plasma membrane in cell-contact areas

We investigated the mode of association of vinculin with areas of contact between the termini of microfilament bundles and the cell membrane in sites of focal contact with the substrate by selective removal of actin from these areas. Opened-up substrate-attached membranes of chick fibroblasts as well as detergent-permeabilized cells were treated with fragmin from Physarum in the presence of Ca+2. This treatment removed actin filaments from the cytoplasmic faces of the membranes, along with several actin-associated proteins (alpha-actinin, tropomyosin, myosin, and filamin). Vinculin distribution was not affected by treatment. Moreover, rhodamine- or fluorescein-conjugated vinculin, when added to these preparations, became specifically associated with the focal contacts regardless of whether the latter were pretreated with fragmin or not. We conclude that the association of vinculin with focal contacts is largely actin-independent. We discuss the implications of these findings in the molecular mechanisms of microfilament membrane association in areas of cell contact.     

Plakoglobin is a component of the filamentous subplasmalemmal coat of lens cells

The plasma membranes of the cells of the superficial layer of the eye lens and the lens fibres are in close intercellular contact, leaving an intermembrane space of approximately 20 nm or less throughout their entire length. This plasma membrane is underlaid by a filamentous, cytoplasmic web containing actin, proteins of the spectrin and band 4.1 families, alpha-actinin and vinculin. Using immunofluorescence microscopy and immunoblotting of gel electrophoretically separated proteins, we show that plakoglobin, the plaque protein common to desmosomal and nondesmosomal adhering junctions, is present in lens cells and is also a component of the subplasmalemmal coat of these cells. Plakoglobin also exists in the extended regions of intercellular contacts between cultured lenticular cells where it often colocalizes with vinculin but does not occur in other vinculin-rich plasma membrane regions such as the focal adhesions at the ventral cell surface. Plakoglobin associated with plasma membrane regions can also be identified in various other adhesive cultured cells, but it is not detected in cells and tissues that do not establish firm intercellular junctions such as erythrocytes, platelets, cultured myeloma cells and smooth muscle tissue. We conclude that plakoglobin occurs, at least in lens cells, throughout the entire subplasmalemmal coat, coexisting in this situation not only with vinculin but also with spectrin and 4.1 protein(s). This colocalization infers the presence of a distinct, complex type of membrane-skeleton assembly involving the actin filament-associated junctional plaque elements plakoglobin and vinculin together with actin-associated proteins of the spectrin and band 4.1 protein families.     

Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells

In this study, we examined the effects of shark cartilage extract on the attachment and spreading properties and the focal adhesion structure of cultured bovine pulmonary artery endothelial cells. Treatment with cartilage extract resulted in cell detachment from the substratum. Immunofluorescence staining of those treated cells that remained attached showed that, instead of being present in both central and peripheral focal adhesions as in control cells, both integrin alpha(v)beta(3) and vinculin were found only in peripheral focal adhesion and thinner actin filament bundles were seen. In addition to causing cell detachment, cartilage extract partially inhibited the initial adherence of the cells to the substratum in a dose-dependent manner. Integrin alpha(v)beta(3) and vinculin staining of these cells also showed a peripheral focal adhesion distribution pattern. Vitronectin induced cell spreading in the absence of serum, but was blocked by simultaneous incubation with cartilage extract, which was shown to inhibit both integrin alpha(v)beta(3) and vinculin recruitment to focal adhesion and the formation of stress fibers. Dot binding assays showed that these inhibitory effects on cell attachment and spreading were not due to direct binding of cartilage extract components to integrin alpha(v)beta(3) or vitronectin. Shark cartilage chondroitin sulfate had no inhibitory effect on either cell attachment or spreading of endothelial cells. These results show that the inhibitory effects of cartilage extract on cell attachment and spreading are mediated by modification of the organization of focal adhesion proteins.     

A plasma membrane integral sialoglycoprotein (Sgp 130) molecularly distinguishes nonjunctional dense plaque sites of microfilament attachment

An integral sialoglycoprotein with Mr approximately 130,000 (Sgp 130) and highest expression in adult chicken gizzard smooth muscle has been recently identified as an excellent candidate for classification as a plasma membrane protein natively associated (directly or indirectly) with actin microfilaments (Rogalski, A.A., and S.J. Singer, 1985, J. Cell Biol., 101:785-801). In this study, the relative in situ distributions of the Sgp 130 integral species (a designation that also includes non-smooth muscle molecular forms) and the peripheral protein, vinculin, have been simultaneously revealed for the first time in selected cultured cells and tissues abundant in microfilament-membrane attachment sites, particularly, smooth and cardiac muscle. Specific antibody probes against Sgp 130 (mouse mAb 30B6) and vinculin (affinity-purified rabbit antibody) were used in double indirect immunofluorescent and immunoelectron microscopic experiments. In contrast to the widespread distributions of vinculin at microfilament-membrane attachment sites, Sgp 130 has been shown to exhibit striking site-specific variation in its abundancy levels in the plasma membrane. Sgp 130 and vinculin were found coincidentally concentrated at focal contact sites in cultured chick embryo fibroblasts and endothelial cells, membrane dense plaques of smooth muscle, and sarcolemma dense plaque sites overlying the Z line in cardiac muscle. However, at the fascia adherens junctional sites of cardiac muscle where vinculin is sharply confined, Sgp 130 was immunologically undetectable in both intact and EGTA-uncoupled tissue. This latter result was confirmed with immunoblotting experiments using isolated forms of the fascia adherens. The double immunolabeling studies of this report establish Sgp 130 as a major integral protein component of nonjunctional membrane dense plaque structures and raise the possibility that the 130-kD integral sialoglycoprotein (Sgp 130) and vinculin assume stable transmembrane associations at these particular microfilament-membrane attachment sites. Nonjunctional dense plaques are further suggested to be a molecularly distinct class of plasma membrane structures rather than a subgroup of adherens junctions. Our data also support a hypothesis that Sgp 130 is involved in plasma membrane force coupling events but not in junctional-related cell-cell coupling.     

Shigella interactions with the actin cytoskeleton in the absence of Ena/VASP family proteins

Shigella move through the cytosol of infected cells by assembly of a propulsive actin tail at one end of the bacterium. Vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins, is important in cellular actin dynamics and is present on intracellular Shigella. VASP binds both profilin, an actin monomer-binding protein, and vinculin, a component of intercellular contacts that also binds the Shigella actin assembly protein IcsA. It has been postulated that VASP might serve as a linker between vinculin and profilin on intracellular Shigella, thereby delivering profilin to the Shigella actin assembly machinery. We show that Shigella actin-based motility is unaltered in cells that are deficient for the Ena/VASP family of proteins. In these cells, Shigella form normal-appearing actin tails and move at rates that are comparable to the rates of bacterial movement in Ena/VASP-deficient cells complemented with the Ena/VASP family member Mena. Finally, whereas vinculin can bind the Arp2/3 complex, we show that Arp2/3 recruitment to Shigella is not correlated with vinculin recruitment, indicating that the role of vinculin in Shigella motility is not recruitment of Arp2/3. Thus, although VASP is recruited to the surface of intracellular Shigella, it is not essential for Shigella actin-based motility.     

IpaA targets beta1 integrins and rho to promote actin cytoskeleton rearrangements necessary for Shigella entry

Shigella invasion into the colonic epithelium involves many steps including the formation of large membrane protrusions by the epithelial cells that facilitate bacterial engulfment. IpaA, a Shigella protein secreted into target cells upon cell contact induces a loss of actin stress fibers in cells and promotes the reorganization of actin at the site of entry. The mechanism for this is not known but is thought to involve recruitment of the focal adhesion protein vinculin to IpaA. Here we have examined the mechanism for the effects of IpaA on the actin cytoskeleton. We show that IpaA-induced loss of actin stress fibers and cell rounding do not require vinculin expression or an intact vinculin binding site on IpaA. Rather, we find that cells expressing IpaA exhibited elevated Rho activity and increased myosin light chain phosphorylation. In addition, IpaA decreases integrin affinity for extracellular matrix ligands by interfering with talin recruitment to the integrin cytoplasmic tail. The combination of these two effects, namely weakened adhesion and increased contractility, account for the loss of actin stress fibers and cell rounding observed in cells exposed to IpaA.     

Vinculin

Vinculin is clearly a key element in the transmembrane assemblages that link cells to each other or to the substrate. However, despite all the studies that have been done on the protein, we still do not know its function within these assemblages. The bulk of the biochemical and cell biological evidence suggests that, in some unknown way, its presence in the junctions may be involved in the stable association of actin with the membrane, yet vinculin by itself does not appear to interact with actin. In the future, identification of additional junctional molecules that interconnect actin and vinculin may resolve this dilemma. Alternatively, studies with vinculin that is phosphorylated or acylated may yield clues to its function. Perhaps the complexity of the protein composition of microfilament-containing junctions suggests that protein assemblages rather than individual proteins provide novel functions. As new proteins belonging to these junctions are discovered, it will be important to assess their interaction with already known components such as vinculin and to ask if the protein combination has a particular function.     

Fibroblast adhesion to RGDS shows novel features compared with fibronectin

As previously shown by others, the fibroblast attachment and spreading activity of fibronectin is mimicked by a short peptide (RGDS or longer) from the cell binding domain. Normal rat kidney fibroblasts showed similar attachment kinetics on either peptide GRGDSC or bovine plasma fibronectin and binding to either substratum was inhibited by peptide alone. We now demonstrate, however, considerable differences in biological activity between peptide and fibronectin. In particular, cells developed novel adhesion structures on peptide-coated substrata. Interference reflection microscopy showed a predominance of small round dark grey/black patches of adherent membrane ("spots") with relatively few focal adhesions, which occurred only at the outermost cell margins in contrast to their distribution in cells spread on fibronectin. The spots were resistant to detergent extraction and stained less strongly or not at all for vinculin. Electron microscopy in vertical thin section showed that the ventral surface of the cell was characterized by "point-contacts", corresponding in size to the spot structures seen by interference reflection microscopy, and which were only occasionally associated with microfilaments. Cells also required a higher substratum loading of peptide than fibronectin to promote spreading and proceeded to spread less rapidly and to a lesser extent, developing very few and extremely fine actin cables.     

Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion

Cell migration involves many steps, including membrane protrusion and the development of new adhesions. Here we have investigated whether there is a link between actin polymerization and integrin engagement. In response to signals that trigger membrane protrusion, the actin-related protein (Arp)2/3 complex transiently binds to vinculin, an integrin-associated protein. The interaction is regulated, requiring phosphatidylinositol-4,5-bisphosphate and Rac1 activation, and is sufficient to recruit the Arp2/3 complex to new sites of integrin aggregation. Binding of the Arp2/3 complex to vinculin is direct and does not depend on the ability of vinculin to associate with actin. We have mapped the binding site for the Arp2/3 complex to the hinge region of vinculin, and a point mutation in this region selectively blocks binding to the Arp2/3 complex. Compared with WT vinculin, expression of this mutant in vinculin-null cells results in diminished lamellipodial protrusion and spreading on fibronectin. The recruitment of the Arp2/3 complex to vinculin may be one mechanism through which actin polymerization and membrane protrusion are coupled to integrin-mediated adhesion.     

Vasodilator-stimulated Phosphoprotein (VASP) Regulates Actin Polymerization and Contraction in Airway Smooth Muscle by a Vinculin-dependent Mechanism

Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser¹⁵⁷ phosphorylation by different kinases. Inhibition of VASP Ser¹⁵⁷ phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser¹⁵⁷ mediates its localization at the membrane, but that VASP Ser¹⁵⁷ phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM.     

Vinculin in relation to stress fibers in spread platelets.

To investigate the function of vinculin in blood platelets, we studied its localization in relation to other cytoskeletal proteins as well as its state of phosphorylation in platelets allowed to spread on fibrinogen-coated surfaces. By 5 minutes after loading the platelets onto the surfaces the 47 and 20 kDa polypeptides became phosphorylated, indicating activation. By 30 minutes, platelets formed small, typical bundles of fibers which stained brilliantly with rhodamine phalloidin. Myosin and tropomyosin, detected with specific antibodies, were localized in periodic arrays along these bundles. By indirect immunofluorescence, a discrete patch of vinculin was observed at each end of every actin-containing bundle. Vinculin phosphorylation was not detected in immunoprecipitates protected against phosphatases. Interference reflection images showed that regions of close binding to the substratum (adhesion plaques) closely matched the vinculin staining sites. Talin appeared diffusely localized. It could be shown to be present in the plaques when platelets were stabilized with ZnCl2 by the method of Geiger and then sonicated to remove some of the surface membrane. Localizations of vinculin and myosin were unaltered by this treatment. Talin phosphorylation or proteolysis could not account for vinculin translocation. We conclude that platelets, in response to an appropriate physiological surface, form typical actin bundles with vinculin at the termination of each bundle, in close relation to adhesion plaques. The signal for this translocation does not appear to depend on phosphorylation of vinculin or on phosphorylation or proteolysis of talin. Our findings support the conclusion that in platelets, as in nucleated cells, vinculin serves as at least part of the connection between bundled actin fibers and the extracellular matrix. Such a connection seems required for platelets' known ability to exert tension on surfaces.     

Effect of phosphatidyl-L-serine and vinculin on actin polymerization

The effects of phosphatidyl-L-serine (PS) and/or vinculin on actin polymerization are examined by spectrophotometry, viscometry and electrophoresis. Actin polymerization is inhibited by PS alone and stimulated by PS and vinculin. The results suggest that actin does not directly adhere to cell membrane and that vinculin is a protein which is involved in structures connecting actin microfilaments to cell membranes.     

A transmembrane relationship between fibronectin and vinculin (130 kd protein): Serum modulation in normal and transformed hamster fibroblasts

Using electron microscopy, we had previously demonstrated a very close transmembrane relationship between actin microfilaments and fibronectin fibrils, termed the fibronexus. Since vinculin, a recently discovered intracellular protein, is localized at the membrane-insertion regions of actin fibers, we studied its possible relationship to fibronectin and the fibronexus. Using double-label immunofluorescence microscopy, we have observed that the distributions of vinculin and fibronectin are strikingly coincident in normal Nil 8 hamster fibroblasts arrested in the G1 phase of the cell cycle, and in HSV-transformed Nil hamster cells treated with purified fibronectin after culturing in 0.3% serum. Extensively spread Nil 8 cells have numerous vinculin-positive focal patches, which are localized either directly over or in tandem with fibronectin fibers at the ventral surface. However, fibronectin and vinculin do not exhibit this relationship in Nil 8 cells grown in 5% serum. These vinculin patches closely resemble the vinculin plaques that Geiger found to be dark under interference-reflection microscopy, suggesting that fibronectin is associated with substrate-adhesion plaques in arrested cells. Fibronectin treatment of the HSV-transformed Nil cells cultured in a low concentration of serum results in the formation of ventral microprocesses, exhibiting an extraordinary congruence of vinculin and fibronectin staining. In addition, these cells bind matrix-like arrangements of fibronectin on their dorsal surface at sites of cell-cell interaction that are vinculin-negative. These results imply that two distinct types of fibronexuses may exist: a ventral substrate-adhesive nexus consisting of fibronectin, vinculin and actin, and a dorsal association between actin and intercellular fibronectin matrix fibers. Transmembrane vinculin-fibronectin associations are evidently sensitive to the growth state of the cell.     

纽带蛋白及其与微丝和膜结合的研究

 用落球粘度计测量低剪切粘度的方法研究了纽带蛋白-肌动蛋白的相互作用。与以前关于纽带蛋白可使肌动蛋白粘度降低并推测它抑制微丝间相互作用的报道不同,10—50μg/ml的纽带蛋白引起肌动蛋白细丝溶液粘度略有增加。电镜观察表明,由于纽带蛋白的作用,肌动蛋白丝聚集成束。 远紫外圆二色谱显示,在pH7.0下,220和207nm处呈双负峰,190nm处有一正峰。按三波长法计算,其α螺旋和β折叠含量分别占41％和22％。研究了pH,磷脂和去垢剂对其构象的影响。疏水性结合的和水溶性的纽带蛋白相比,存在少量构象差别。联系这一蛋白处在细胞骨架与质膜相联系的特殊位置,推测在体内,纽带蛋白与脂结合后也可能发生类似的构象变化。     

Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading

To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor- mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.     

Interaction of fibronectin-coated beads with attached and spread fibroblasts : Binding, phagocytosis, and cytoskeletal reorganization

After 15 min incubations, binding of 0.8-, 6-, and 16-microns fibronectin-coated latex beads occurred primarily at the margins of chick embryo fibroblasts that previously were attached and spread on fibronectin-coated glass coverslips. Extensive phagocytosis of the smallest beads and some phagocytosis of the larger beads occurred within 2 h. Following binding of the 16-micron beads, there were no changes in overall cell shape or in the distribution of several cytoskeletal proteins. There was, however, a local accumulation of actin and alpha-actinin patches adjacent to the sites where the beads were bound. The formation of alpha-actinin patches could be detected with 6- or 16-microns beads shortly after initial bead binding to the cells, but a similar reorganization of alpha-actinin in response to the binding of 0.8-micron beads was not detected. The patches of alpha-actinin appeared to be associated with membrane ruffles, since such structures were observed by scanning electron microscopy (SEM) to be sites of cell interaction with 6- but not 0.8-micron beads. Also, two other cytoskeletal proteins normally absent from membrane ruffles, tropomyosin and vinculin, were not detected at the sites of cell-bead interaction. No reorganization of vinculin at the cell-bead interaction sites was observed even when the 16-microns beads remained bound at the cell surfaces for up to 6 h. Nevertheless, prominent vinculin plaques were observed at the marginal attachment sites on the ventral cell surfaces. Consequently, formation of mature focal adhesions may be restricted to linear regions of cell-substratum interaction.     

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.