Integrin Adhesion Complexes Research Articles

Talin Is Required Continuously for Cardiomyocyte Remodeling during Heart Growth in Drosophila.

Mechanotransduction of tension can govern the remodeling of cardiomyocytes during growth or cardiomyopathy. Tension is signaled through the integrin adhesion complexes found at muscle insertions and costameres but the relative importance of signalling during cardiomyocyte growth versus remodelling has not been assessed. Employing the Drosophila cardiomyocyte as a genetically amenable model, we depleted the levels of Talin, a central component of the integrin adhesion complex, at different stages of heart growth and remodeling. We demonstrate a continuous requirement for Talin during heart growth to maintain the one-to-one apposition of myofibril ends between cardiomyocytes. Retracted myofibrils cannot regenerate appositions to adjacent cells after restoration of normal Talin expression, and the resulting deficit reduces heart contraction and lifespan. Reduction of Talin during heart remodeling after hatching or during metamorphosis results in pervasive degeneration of cell contacts, myofibril length and number, for which restored Talin expression is insufficient for regeneration. Resultant dilated cardiomyopathy results in a fibrillating heart with poor rhythmicity. Cardiomyocytes have poor capacity to regenerate deficits in myofibril orientation and insertion, despite an ongoing capacity to remodel integrin based adhesions.

A defensin fragment regulates CHO-K1 cell spreading and induces renewal of fatty acid composition in cell membrane phospholipids.

A tetrapeptide defensin fragment has been shown to stimulate the spreading of CHOK1 cells. The tetrapeptide investigated had virtually no effect on the composition of cell membrane phospholipids but participated in the regulation of the renewal of fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Incubation of cells with the peptide resulted in a change in the composition of the unsaturated fatty acid residues in the phospholipids investigated: specifically, the content of monoenoic and/or dienoic acids increased and that of polyenoic acids decreased. The possible role of the peptide investigated (1) in the regulation of the functional activity of integrin receptors, and (2) in changes in the packing density of the phospholipid acyl chains in cell membrane microdomains, which affects the rates of integrin clustering and adhesion complex formation, is discussed.

The talin head domain reinforces integrin-mediated adhesion by promoting adhesion complex stability and clustering.

Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.

Suppressor Mutations Suggest a Surface on PAT-4 (Integrin-linked Kinase) That Interacts with UNC-112 (Kindlin)

Caenorhabditis elegans striated muscle cells attach to basement membrane and transmit the force of muscle contraction through integrin adhesion complexes. The cytoplasmic tail of β-integrin (PAT-3) is associated with a conserved four-protein complex that includes UNC-112 (kindlin), PAT-4 (integrin-linked kinase), PAT-6 (α-parvin/actopaxin), and UNC-97 (PINCH). The proper localization of UNC-112 to muscle integrin adhesion sites requires PAT-4. A recent report (Qadota, H., Moerman, D. G., and Benian, G. M. (2012) A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (kindlin) to integrin adhesion sites. J. Biol. Chem. 287, 28537-28551) suggests a possible molecular mechanism for this requirement: that UNC-112 exists in closed inactive and open active conformations, and conversion to the open active form is promoted by binding to PAT-4 (ILK). Previously, we also reported identification of a single missense mutation in UNC-112, D382V, which abolishes both binding to PAT-4 and normal localization to integrin adhesion sites in vivo. In this report, we describe isolation and characterization of PAT-4 missense mutations that permit binding with UNC-112 D382V and place nine affected residues on a homology model of PAT-4. These nine residues cluster in two regions on the surface of PAT-4, do not overlap the likely binding surface for PAT-6 (α-parvin), and therefore may reside along the interaction surface of PAT-4 for UNC-112 (kindlin). We also show that one of these PAT-4 mutations restores the ability of UNC-112 D382V to localize to integrin adhesions and participate in complex formation.

Drosophila integrin adhesion complexes are essential for hemocyte migration in vivo

SummaryCell migration is an important biological process which has been intensively studied in the past decades. Numerous techniques, mainly involving two-dimensional cell culture systems, have contributed to dissecting the essential mechanisms underlying this process. However, the development of three-dimensional cell culture and in vivo systems has shown some differences with what was previously believed to be well-established cell migration mechanisms, suggesting that two-dimensional cell motility would be a poor predictor of in vivo behaviour. Drosophila is a widely recognized model organism to study developmental and homeostatic processes and has been widely used to investigate cell migration. Here, we focus on the migration of small groups of pupal hemocytes that accumulate during larval stages in dorsal patches. We show that integrins, and other known nascent adhesion-related proteins such as Rhea and Fermitin 1, are crucial for this process and that their depletion does not affect polarization in response to environmental cues. We also present evidence for the importance of adhesion maturation-related proteins in hemocyte migration, namely Zyxin. Zyxin depletion in hemocytes leads to a significant increase of cell speed without affecting their response to a chemotactic cue. This is the first report of a systematic analysis using Drosophila melanogaster hemocytes to study adhesion-related proteins and their function in cell migration in vivo. Our data point to mechanisms of cell migration similar to those described in three-dimensional in vitro systems and other in vivo model organisms.

Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

SummaryPrecise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration.

CPNA-1, a copine domain protein, is located at integrin adhesion sites and is required for myofilament stability in Caenorhabditis elegans.

We identify cpna-1 (F31D5.3) as a novel essential muscle gene in the nematode Caenorhabditis elegans. Antibodies specific to copine domain protein atypical-1 (CPNA-1), as well as a yellow fluorescent protein translational fusion, are localized to integrin attachment sites (M-lines and dense bodies) in the body-wall muscle of C. elegans. CPNA-1 contains an N-terminal predicted transmembrane domain and a C-terminal copine domain and binds to the M-line/dense body protein PAT-6 (actopaxin) and the M-line proteins UNC-89 (obscurin), LIM-9 (FHL), SCPL-1 (SCP), and UNC-96. Proper CPNA-1 localization is dependent upon PAT-6 in embryonic and adult muscle. Nematodes lacking cpna-1 arrest elongation at the twofold stage of embryogenesis and display disruption of the myofilament lattice. The thick-filament component myosin heavy chain MYO-3 and the M-line component UNC-89 are initially localized properly in cpna-1-null embryos. However, in these embryos, when contraction begins, MYO-3 and UNC-89 become mislocalized into large foci and animals die. We propose that CPNA-1 acts as a linker between an integrin-associated protein, PAT-6, and membrane-distal components of integrin adhesion complexes in the muscle of C. elegans.

Directional cell migration in an extracellular pH gradient: A model study with an engineered cell line and primary microvascular endothelial cells

Extracellular pH (pHe) gradients are characteristic of tumor and wound environments. Cell migration in these environments is critical to tumor progression and wound healing. While it has been shown previously that cell migration can be modulated in conditions of spatially invariant acidic pHe due to acid-induced activation of cell surface integrin receptors, the effects of pHe gradients on cell migration remain unknown. Here, we investigate cell migration in an extracellular pHe gradient, using both model αvβ3 CHO-B2 cells and primary microvascular endothelial cells. For both cell types, we find that the mean cell position shifts toward the acidic end of the gradient over time, and that cells preferentially polarize toward the acidic end of the gradient during migration. We further demonstrate that cell membrane protrusion stability and actin–integrin adhesion complex formation are increased in acidic pHe, which could contribute to the preferential polarization toward acidic pHe that we observed for cells in pHe gradients. These results provide the first demonstration of preferential cell migration toward acid in a pHe gradient, with intriguing implications for directed cell migration in the tumor and wound healing environments.

Rebuttal from Gunst and Panettieri

Rebuttal from Gunst and Panettieri

Mechanical force regulates integrin turnover in Drosophila in vivo

Regulated assembly and disassembly, or turnover, of integrin-mediated cell-extracellular matrix (ECM) adhesions is essential for dynamic cell movements and long-term tissue maintenance. For example, in Drosophila, misregulation of integrin turnover disrupts muscle-tendon attachment at myotendinous junctions (MTJs). We demonstrate that mechanical force, which modulates integrin activity, also regulates integrin and intracellular adhesion complex (IAC) turnover in vivo. Using conditional mutants to alter the tensile force on MTJs, we found that the proportion of IAC components undergoing turnover inversely correlated with the force applied on MTJs. This effect was disrupted by point mutations in β-integrin that interfere with ECM-induced conformational changes and activation of β-integrin or integrin-mediated cytoplasmic signalling. These mutants also disrupted integrin dynamics at MTJs during larval development. Together, these data suggest that specific β-integrin-mediated signals regulate adhesion turnover in response to tension during tissue formation. We propose that integrin-ECM adhesive stability is continuously controlled by force in vivo through integrin-dependent auto-regulatory feedback mechanisms so that tissues can quickly adapt to and withstand mechanical stresses.

Cover Picture: Proteomics 7‐8'12

Over recent years, advances in proteomic technologies have led to the rapid generation of vast volumes of data regarding many clinical applications, acquired from cells through to patients. High‐throughput analysis of protein‐protein interactions, quantification of protein abundance, global analysis of posttranslational modifications and other approaches have addressed the complexity of cellular regulation in health and disease, which has opened the way to systems‐level clinical research. The dysregulation of cell adhesion plays a key role in many disease states. The image represents a systematic analysis of integrin adhesion complexes, which were isolated from erythroleukemia cells and analyzed bymass spectrometry (see Byron et al., Science Signaling 2011, 4, pt2). To understand how specific adhesion complexes may function, the composition of different complexes was compared using hierarchical clustering, and a protein‐protein interaction network model of the core receptor‐bound subcomplex was constructed. Data‐driven, global investigations such as this could pave the road to new therapeutic targets and personalized medicines.Cover image created by Adam Byron (University of Manchester,Manchester, UK); cover design by SCHULZ Grafik‐Design.

Proteomic analysis of α4β1 integrin adhesion complexes reveals α‐subunit‐dependent protein recruitment

Integrin adhesion receptors mediate cell–cell and cell–extracellular matrix interactions, which control cell morphology and migration, differentiation, and tissue integrity. Integrins recruit multimolecular adhesion complexes to their cytoplasmic domains, which provide structural and mechanosensitive signaling connections between the extracellular and intracellular milieux. The different functions of specific integrin heterodimers, such as α4β1 and α5β1, have been attributed to distinct signal transduction mechanisms that are initiated by selective recruitment of adhesion complex components to integrin cytoplasmic tails. Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0), and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS. Using hierarchical clustering and interaction network analyses, we detail the differential recruitment of proteins and highlight enrichment patterns of proteins to distinct adhesion complexes. We identify previously unreported components of integrin adhesion complexes and observe receptor-specific enrichment of molecules with previously reported links to cell migration and cell signaling processes. Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells. These datasets provide a resource for future studies of integrin receptor-specific signaling events.

Alternative cellular roles for proteins identified using proteomics

The recent report by Grigera et al. [1] provided interesting new data on the ability of the actin-binding protein cortactin to associate with regulator of chromosome condensation-2 (RCC2; also known as TD-60), a component of the mitotic machinery [2]. Owing to the localisation of cortactin to the equatorial plane of dividing cells, the authors inferred that its interaction with RCC2 was functionally important in mitosis. This may be the case, but the data also support an alternative interpretation that RCC2 may play a role in cortactin-mediated branched actin regulation during cell movement. Cortactin promotes actin nucleation and stabilises F-actin, and thus it regulates many cell motility processes, such as focal adhesion assembly, lamellipodial protrusion, vesicle transport and endocytosis [3], although the full gamut of cortactin function is incompletely understood. Cortactin strongly localises to the leading edge of migrating cells, where it is phosphorylated, and signalling to small guanosine triphosphatases (GTPases), such as Rac1, plays an important part in its function [4,5]. Engagement of integrin adhesion receptors leads to activation of small GTPases, which play a central role in the modulation of cell adhesion complexes [6,7]. Using a proteomic approach, we showed that specific integrin adhesion complexes contain RCC2 (Fig. 1) [8–10]. Moreover, we demonstrated that, through precise restriction of Rac1 and Arf6 GTPase activity, RCC2 controls directional cell motility [8,9,11]. Indeed, our findings suggest that, rather than acting as a putative guanine nucleotide exchange factor and activating Rac1, RCC2 limits the activation of Rac1. Fig. 1 Proteomic analysis of molecular interactions can reveal alternative cellular roles for proteins. RCC2 (red circle, or node, in interaction network) is a passenger protein with a role in cell division [2]. Proteomic and bioinformatic analyses of integrin ... Together with the work of Grigera et al. [1], our data indicate an intriguing overlap between molecules implicated in cell adhesion processes and RCC2 function. Thus, the data reported by Grigera et al. [1] do not exclude the possibility that RCC2 interacts with cortactin as a component of the cell migration machinery [8,9] or indeed of integrin adhesion-regulated mitosis [12,13]. A current lack of data reporting the colocalisation of RCC2 with cortactin prevents a complete understanding of the interaction between these molecules, but further study of their contributions to cell behaviour is warranted. The works discussed above exemplify the discovery of unexpected cellular roles for proteins; such examples continue to increase as more data-driven proteomic studies appear in the literature. The consequence, in the postgenomic era, is that a single gene cannot be correlated with a single protein function. Proteins may possess multifunctionality within a single polypeptide chain, known as “moonlighting” [14], or as different splice variants. Therefore, understanding the context of protein interactions is critical to gaining mechanistic insight into protein function. Many MS-based proteomic studies rely on analysis of cell or tissue lysates, in which cellular context (e.g. subcellular localisation or cell type-specific expression) has been eliminated. Furthermore, many analyses of proteomic data use protein interaction networks as a tool for data interrogation. Such analyses lever the wealth of knowledge about all reported protein interactions and can be incredibly informative, including by suggesting alternative functions for proteins [15], but these datasets should be interpreted in the context of their derivation and the methodological limitations. Reported interactions in large-scale interaction networks have usually been amalgamated from data from multiple conditions, cell types and subcellular locations. With a lack of cellular context, proteins can appear to be promiscuous binders, whereas, in a cell, they may bind to distinct partners under restricted conditions. Accordingly, to achieve mechanistic insight into protein function, the analysis of protein interaction networks should rarely be the final interpretation of MS data, but instead be the springboard for further experimentation. Overall, data-driven MS-based proteomic and bioinformatic analyses, within the framework of hypothesis-driven research, provide a valuable opportunity for the discovery of new cellular roles for proteins, and will continue to provide surprising results about biological systems.

TNFα pathway blockade ameliorates toxic effects of FSGS plasma on podocyte cytoskeleton and β3 integrin activation

In the absence of mutant genes encoding components of the podocyte slit diaphragm, about 30-50 % of children with primary glucocorticoid-resistant focal segmental glomerulosclerosis (FSGS) develop recurrent proteinuria and slowly progressive FSGS lesions following renal transplantation. Recurrence of FSGS in the allograft strongly suggests a circulating factor that disturbs normal podocyte biology. To date, the nature of the circulating factor is unclear, and there is no cure for the recurrent form of FSGS (R-FSGS). Cultured differentiated human podocytes were exposed to the plasmapheresis effluent or blood plasma samples from pediatric patients with recurrent or primary FSGS; in some cases, podocytes were pre-incubated with specific antibodies to block the tumor necrosis factor-alpha (TNFα) signaling pathway. Integrity of focal adhesion complexes and actin cytoskeleton were investigated by immunofluorescent microscopy. Plasmapheresis effluent from an R-FSGS child or fresh plasma from two children with primary FSGS rapidly disturbed the cytoskeleton of normal human podocytes in vitro. Plasma from a child with R-FSGS also activated β3 integrin and dispersed focal adhesion complexes. The effects were reversed by pre-incubation with antibodies against TNFα or either of the two TNFα receptors. When our patient with R-FSGS became resistant to plasmapheresis, we initiated treatment with twice weekly etanercept injections and then infliximab. Within 3 weeks of regular anti-TNFα therapy, the patient achieved sustained partial remission of proteinuria, allowing us to wean her off plasmapheresis completely. We suggest that in some FSGS patients, disruption of the podocyte cytoskeleton and β3 integrin-mediated podocyte attachment are driven by the TNFα pathway.

β2 Integrin Adhesion Complexes Maintain the Integrity of HIV‐1 Assembly Compartments in Primary Macrophages

In human monocyte-derived macrophages (MDM), human immunodeficiency virus type 1 (HIV-1) assembly takes place primarily on complex intracellular plasma membrane domains connected to the cell surface by closely apposed membrane sheets or narrow channels. Some of the membranes associated with these compartments are decorated by thick (≈30 nm), electron-dense, cytoplasmic coats. Here we show by immunolabelling of ultrathin cryosections that the β2 integrin CD18, together with the αM and αX integrins (CD11b and CD11c), is clustered at these coated domains, and that the coats themselves contain the cytoskeletal linker proteins talin, vinculin and paxillin that connect the integrin complexes to the actin cytoskeleton. Intracellular plasma membrane-connected compartments (IPMC) with CD18-containing focal adhesion-like coats are also present in uninfected MDM. These compartments become more prominent as the cells mature in tissue culture and their appearance correlates with increased expression of CD18, CD11b/c and paxillin. Depletion of CD18 by RNA interference leads to parallel down-regulation of CD11b and CD11c, as well as of paxillin, and the disappearance of the adhesion-like coats. In addition, CD18 knockdown alters the appearance of virus-containing IPMC in HIV-infected MDM, indicating that the β2 integrin/focal adhesion-like coat structures are involved in the organization of these compartments.

Talin1 Regulates Integrin Turnover To Promote Embryonic Epithelial Morphogenesis

Talin is a cytoskeletal protein that binds to integrin β cytoplasmic tails and regulates integrin activation. Talin1 ablation in mice disrupts gastrulation and causes embryonic lethality. However, the role of talin in mammalian epithelial morphogenesis is poorly understood. Here we demonstrate that embryoid bodies (EBs) differentiated from talin1-null embryonic stem cells are defective in integrin adhesion complex assembly, epiblast elongation, and lineage differentiation. These defects are accompanied by a significant reduction in integrin β1 protein levels due to accelerated degradation through an MG-132-sensitive proteasomal pathway. Overexpression of integrin β1 or MG-132 treatment in mutant EBs largely rescues the phenotype. In addition, epiblast cells isolated from talin1-null EBs exhibit impaired cell spreading and focal adhesion formation. Transfection of the mutant cells with green fluorescent protein (GFP)-tagged wild-type but not mutant talin1 that is defective in integrin binding normalizes integrin β1 protein levels and restores focal adhesion formation. Significantly, cell adhesion and spreading are also improved by overexpression of integrin β1. All together, these results suggest that talin1 binding to integrin promotes epiblast adhesion and morphogenesis in part by preventing integrin β1 degradation.

Mechanical stimuli and IL-13 interact at integrin adhesion complexes to regulate expression of smooth muscle myosin heavy chain in airway smooth muscle tissue

Airway smooth muscle phenotype may be modulated in response to external stimuli under physiological and pathophysiological conditions. The effect of mechanical forces on airway smooth muscle phenotype were evaluated in vitro by suspending weights of 0.5 or 1 g from the ends of canine tracheal smooth muscle tissues, incubating the weighted tissues for 6 h, and then measuring the expression of the phenotypic marker protein, smooth muscle myosin heavy chain (SmMHC). Incubation of the tissues at a high load significantly increased expression of SmMHC compared with incubation at low load. Incubation of the tissues at a high load also decreased activation of PKB/Akt, as indicated by its phosphorylation at Ser 473. Inhibition of Akt or phosphatidylinositol-3,4,5 triphosphate-kinase increased SmMHC expression in tissues at low load but did not affect SmMHC expression at high load. IL-13 induced a significant increase in Akt activation and suppressed the expression of SmMHC protein at both low and high loads. The role of integrin signaling in mechanotransduction was evaluated by expressing a PINCH (LIM1-2) fragment in the muscle tissues that prevents the membrane localization of the integrin-binding IPP complex (ILK/PINCH/α-parvin), and also by expressing an inactive integrin-linked kinase mutant (ILK S343A) that inhibits endogenous ILK activity. Both mutants inhibited Akt activation and increased expression of SmMHC protein at low load but had no effect at high load. These results suggest that mechanical stress and IL-13 both act through an integrin-mediated signaling pathway to oppositely regulate the expression of phenotypic marker proteins in intact airway smooth muscle tissues. The stimulatory effects of mechanical stress on contractile protein expression oppose the suppression of contractile protein expression mediated by IL-13; thus the imposition of mechanical strain may inhibit changes in airway smooth muscle phenotype induced by inflammatory mediators.

Quantitative proteomics of the integrin adhesome show a myosin II‐dependent recruitment of LIM domain proteins

A characteristic of integrins is their ability to transfer chemical and mechanical signals across the plasma membrane. Force generated by myosin II makes cells able to sense substrate stiffness and induce maturation of nascent adhesions into focal adhesions. In this paper, we present a comprehensive proteomic analysis of nascent and mature adhesions. The purification of integrin adhesion complexes combined with quantitative mass spectrometry enabled the identification and quantification of known and new adhesion-associated proteins. Furthermore, blocking adhesion maturation with the myosin II inhibitor blebbistatin markedly impaired the recruitment of LIM domain proteins to integrin adhesion sites. This suggests a common recruitment mechanism for a whole class of adhesion-associated proteins, involving myosin II and the zinc-finger-type LIM domain.

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.