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Vinculin

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David R Critchley - One of the best experts on this subject based on the ideXlab platform.

  • riam and Vinculin binding to talin are mutually exclusive and regulate adhesion assembly and turnover
    Journal of Biological Chemistry, 2013
    Co-Authors: Benjamin T. Goult, Christoph Ballestrem, Ricky Tsang, Thomas Zacharchenko, Neil Bate, Fiona Hey, Alexandre R Gingras, Paul R Elliott, Gordon C K Roberts, David R Critchley
    Abstract:

    Talin activates integrins, couples them to F-actin, and recruits Vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1–R13) organized into a compact cluster of four-helix bundles (R2–R4) within a linear chain of five-helix bundles. Nine of the bundles contain Vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that Vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, Vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas Vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit Vinculin to stabilize the complex.

  • the structure and regulation of Vinculin
    Trends in Cell Biology, 2006
    Co-Authors: Wolfgang H. Ziegler, Robert C Liddington, David R Critchley
    Abstract:

    Vinculin is a ubiquitously expressed actin-binding protein frequently used as a marker for both cell–cell and cell–extracellular matrix (focal adhesion) adherens-type junctions, but its function has remained elusive. Vinculin is made up of a globular head linked to a tail domain by a short proline-rich sequence, and an intramolecular interaction between the head and tail masks the numerous ligand-binding sites in the protein. Determination of the crystal structure of Vinculin has shed new light on the way that these ligand-binding sites are regulated. The picture that emerges is one in which Vinculin stabilizes focal adhesions and thereby suppresses cell migration, an effect that is relieved by transient changes in the local concentrations of inositol phospholipids. However, the finding that Vinculin modulates the signalling pathways involved in apoptosis suggests that additional roles for Vinculin remain to be discovered.

  • role of Vinculin in regulating focal adhesion turnover
    European Journal of Cell Biology, 2006
    Co-Authors: Ruth Saunders, Mark R. Holt, Andrey A Bobkov, Robert C Liddington, Lisa Jennings, Deborah H Sutton, Igor L Barsukov, Eileen A Adamson, Graham Dunn, David R Critchley
    Abstract:

    Abstract Although Vinculin (−/−) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than Vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in Vinculin null compared to wild-type cells. Expression of Vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in Vinculin activation by relieving an intramolecular association between the Vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the Vinculin/PIP2 interaction in FA dynamics, we used a Vinculin mutant lacking the C-terminal arm (residues 1053–1066) and referred to as the ΔC mutation. This mutation reduced PIP2 binding to a VtΔC polypeptide by >90% compared to wild type without affecting binding to Vh or F -actin. Interestingly, cells expressing the VinculinΔC mutant assembled remarkably stable FAs. The results suggest that Vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.

  • mapping and consensus sequence identification for multiple Vinculin binding sites within the talin rod
    Journal of Biological Chemistry, 2005
    Co-Authors: Alexandre R Gingras, David R Critchley, Wolfgang H. Ziegler, Igor L Barsukov, Gordon C K Roberts, Ronald Frank, Jonas Emsley
    Abstract:

    The interaction between the cytoskeletal proteins talin and Vinculin plays a key role in integrin-mediated cell adhesion and migration. Three Vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the α-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS α-helices bind to a hydrophobic cleft in the N-terminal Vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX- VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for Vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the Vinculin Vd1 domain reveals a subtly different mode of Vinculin binding.

  • a Vinculin binding domain from the talin rod unfolds to form a complex with the Vinculin head
    Structure, 2005
    Co-Authors: Ian J Fillingham, David R Critchley, Bipin Patel, Alexandre R Gingras, Gordon C K Roberts, Evangelos Papagrigoriou, Jonas Emsley, Igor L Barsukov
    Abstract:

    Summary The cytoskeletal protein talin plays a key role in activating integrins and in coupling them to the actin cytoskeleton. Its N-terminal globular head, which binds β integrins, is linked to an extended rod having a C-terminal actin binding site and several Vinculin binding sites (VBSs). The NMR structure of residues 755–889 of the rod (containing a VBS) is shown to be an amphipathic four-helix bundle with a left-handed topology. A talin peptide corresponding to the VBS binds the Vinculin head; the X-ray crystallographic structure of this complex shows that the residues which interact with Vinculin are buried in the hydrophobic core of the talin fragment. NMR shows that the interaction involves a major structural change in the talin fragment, including unfolding of one of its helices, making the VBS accessible to Vinculin. Interestingly, the talin 755–889 fragment binds more than one Vinculin head molecule, suggesting that the talin rod may contain additional as yet unrecognized VBSs.

Susan W Craig - One of the best experts on this subject based on the ideXlab platform.

  • how Vinculin regulates force transmission
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: David W Dumbauld, Susan W Craig, Christopher S Chen, Ted Tang Lee, Ankur Singh, Jan Scrimgeour, Charles A Gersbach, Evan A Zamir, Jennifer E Curtis, Andres J Garcia
    Abstract:

    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 (VH) and tail domains. VH 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; VH 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 VH, correlate with applied force, supporting a mechanosensitive model for Vinculin activation in which forces stabilize Vinculin’s active conformation to promote force transfer.

  • α catenin uses a novel mechanism to activate Vinculin
    Journal of Biological Chemistry, 2012
    Co-Authors: Xiao Peng, Susan W Craig, Jessica L Maiers, Dilshad Choudhury, Kris A Demali
    Abstract:

    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, α-catenin, a cytoplasmic component of the cadherin adhesion complex, activates Vinculin. Direct binding of α-catenin to Vinculin is critical for this event because a point mutant (α-catenin L344P) lacking high affinity binding does not activate Vinculin. Furthermore, unlike all known Vinculin activators, α-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 α-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.

  • Coincidence of actin filaments and talin is required to activate Vinculin.
    The Journal of biological chemistry, 2006
    Co-Authors: Hui Chen, Dilshad M. Choudhury, Susan W Craig
    Abstract:

    Vinculin regulates cell adhesion by strengthening contacts between extracellular matrix and the cytoskeleton. Binding of the integrin ligand, talin, to the head domain of Vinculin and F-actin to its tail domain is a potential mechanism for this function, but Vinculin is autoinhibited by intramolecular interactions between its head and tail domain and must be activated to bind talin and actin. Because autoinhibition of Vinculin occurs by synergism between two head and tail interfaces, one hypothesis is that activation could occur by two ligands that coordinately disrupt both interfaces. To test this idea we use a fluorescence resonance energy transfer probe that reports directly on activation of Vinculin. Neither talin rod, VBS3 (a talin peptide that mimics a postulated activated state of talin), nor F-actin alone can activate Vinculin. But in the presence of F-actin either talin rod or VBS3 induces dose-dependent activation of Vinculin. The activation data are supported by solution phase binding studies, which show that talin rod or VBS3 fails to bind Vinculin, whereas the same two ligands bind tightly to Vinculin head domain (K(d) approximately 100 nM). These data strongly support a combinatorial mechanism of Vinculin activation; moreover, they are inconsistent with a model in which talin or activated talin is sufficient to activate Vinculin. Combinatorial activation implies that at cell adhesion sites Vinculin is a coincidence detector awaiting simultaneous signals from talin and actin polymerization to unleash its scaffolding activity.

  • a conformational switch in Vinculin drives formation and dynamics of a talin Vinculin complex at focal adhesions
    Journal of Biological Chemistry, 2006
    Co-Authors: Daniel M Cohen, Brett Kutscher, Hui Chen, Douglas B Murphy, Susan W Craig
    Abstract:

    Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in Vinculin regulates formation and lifetime of the talin-Vinculin complex, a proposed mediator of integrin–cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-Vinculin complexes, whereas ectopic complexes did not form between talin and wild-type Vinculin. Moreover, reduction of HTI altered the dynamic assembly of Vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of Vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of Vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated Vinculin. Significantly, HTI-deficient Vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or α-actinin. These data show that talin conformation in cells permits Vinculin binding, whereas the autoinhibited conformation of Vinculin constitutes the barrier to complex formation. Down-regulation of HTI in Vinculin to Kd ∼ 10–7 is sufficient to induce talin binding, and HTI is essential to the dynamics of Vinculin and talin at focal adhesions. We therefore conclude that Vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-Vinculin complexes in cells.

  • spatial distribution and functional significance of activated Vinculin in living cells
    Journal of Cell Biology, 2005
    Co-Authors: Hui Chen, Daniel M Cohen, Noriyuki Kioka, Dilshad M. Choudhury, Susan W Craig
    Abstract:

    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 β 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.

Tina Izard - One of the best experts on this subject based on the ideXlab platform.

  • lipid binding promotes oligomerization and focal adhesion activity of Vinculin
    Journal of Cell Biology, 2014
    Co-Authors: Krishna Chinthalapudi, Erumbi S Rangarajan, Dipak N Patil, Eric M George, David T Brown, Tina Izard
    Abstract:

    Adherens junctions (AJs) and focal adhesion (FA) complexes are necessary for cell migration and morphogenesis, and for the development, growth, and survival of all metazoans. Vinculin is an essential regulator of both AJs and FAs, where it provides links to the actin cytoskeleton. Phosphatidylinositol 4,5-bisphosphate (PIP2) affects the functions of many targets, including Vinculin. Here we report the crystal structure of Vinculin in complex with PIP2, which revealed that PIP2 binding alters Vinculin structure to direct higher-order oligomerization and suggests that PIP2 and F-actin binding to Vinculin are mutually permissive. Forced expression of PIP2-binding–deficient mutants of Vinculin in Vinculin-null mouse embryonic fibroblasts revealed that PIP2 binding is necessary for maintaining optimal FAs, for organization of actin stress fibers, and for cell migration and spreading. Finally, photobleaching experiments indicated that PIP2 binding is required for the control of Vinculin dynamics and turnover in FAs. Thus, through oligomerization, PIP2 directs a transient Vinculin sequestration at FAs that is necessary for proper FA function.

  • the cytoskeletal protein α catenin unfurls upon binding to Vinculin
    Journal of Biological Chemistry, 2012
    Co-Authors: Erumbi S Rangarajan, Tina Izard
    Abstract:

    Adherens junctions (AJs) are essential for cell-cell contacts, morphogenesis, and the development of all higher eukaryotes. AJs are formed by calcium-dependent homotypic interactions of the ectodomains of single membrane-pass cadherin family receptors. These homotypic interactions in turn promote binding of the intracellular cytoplasmic tail domains of cadherin receptors with β-catenin, a multifunctional protein that plays roles in both transcription and AJs. The cadherin receptor-β-catenin complex binds to the cytoskeletal protein α-catenin, which is essential for both the formation and the stabilization of these junctions. Precisely how α-catenin contributes to the formation and stabilization of AJs is hotly debated, although the latter is thought to involve its interactions with the cytoskeletal protein Vinculin. Here we report the crystal structure of the Vinculin binding domain (VBD) of α-catenin in complex with the Vinculin head domain (Vh1). This structure reveals that α-catenin is in a unique unfurled mode allowing dimer formation when bound to Vinculin. Finally, binding studies suggest that Vinculin must be in an activated state to bind to α-catenin and that this interaction is stabilized by the formation of a ternary α-catenin-Vinculin-F-actin complex, which can be formed via the F-actin binding domain of either protein. We propose a feed-forward model whereby α-catenin-Vinculin interactions promote their binding to the actin cytoskeleton to stabilize AJs.

  • raver1 interactions with Vinculin and rna suggest a feed forward pathway in directing mrna to focal adhesions
    Structure, 2009
    Co-Authors: Jun Hyuck Lee, Erumbi S Rangarajan, Sollepura D Yogesha, Tina Izard
    Abstract:

    The translational machinery of the cell relocalizes to focal adhesions following the activation of integrin receptors. This response allows for rapid, local production of components needed for adhesion complex assembly and signaling. Vinculin links focal adhesions to the actin cytoskeleton following its activation by integrin signaling, which severs intramolecular interactions of Vinculin's head and tail (Vt) domains. Our Vinculin:raver1 crystal structures and binding studies show that activated Vt selectively interacts with one of the three RNA recognition motifs of raver1, that the Vinculin:raver1 complex binds to F-actin, and that raver1 binds selectively to RNA, including a sequence found in Vinculin mRNA. Further, mutation of residues that mediate interaction of raver1 with Vinculin abolish their colocalization in cells. These findings suggest a feed-forward model where Vinculin activation at focal adhesions provides a scaffold for recruitment of raver1 and its mRNA cargo to facilitate the production of components of adhesion complexes.

  • Vinculin binding in its closed conformation by a helix addition mechanism
    The EMBO Journal, 2007
    Co-Authors: Guy Tran Van Nhieu, Tina Izard
    Abstract:

    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, α-actinin, and the invasin IpaA of Shigella flexneri sever Vinculin's head–tail interaction by inserting an α-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 α-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.

  • the Vinculin binding sites of talin and α actinin are sufficient to activate Vinculin
    Journal of Biological Chemistry, 2006
    Co-Authors: Philippe R J Bois, Brendan P Ohara, Daniel Nietlispach, John Kirkpatrick, Tina Izard
    Abstract:

    Abstract 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 α-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 α-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 α-actinin and disassembling focal adhesions. In their native (inactive) states the Vinculin binding sites of talin and α-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 α-actinin, by provoking structural changes that allow their Vinculin binding sites to swing out, which are then sufficient to bind to and activate Vinculin.

Kris A Demali - One of the best experts on this subject based on the ideXlab platform.

  • Vinculin in cell cell and cell matrix adhesions
    Cellular and Molecular Life Sciences, 2017
    Co-Authors: Jennifer L Bays, Kris A Demali
    Abstract:

    Vinculin was identified as a component of focal adhesions and adherens junctions nearly 40 years ago. Since that time, remarkable progress has been made in understanding its activation, regulation and function. Here we discuss the current understanding of the roles of Vinculin in cell–cell and cell–matrix adhesions. Emphasis is placed on the how Vinculin is recruited, activated and regulated. We also highlight the recent understanding of how Vinculin responds to and transmits force at integrin- and cadherin-containing adhesion complexes to the cytoskeleton. Furthermore, we discuss roles of Vinculin in binding to and rearranging the actin cytoskeleton.

  • Vinculin phosphorylation differentially regulates mechanotransduction at cell cell and cell matrix adhesions
    Journal of Cell Biology, 2014
    Co-Authors: Jennifer Bays, Keith Burridge, Xiao Peng, Catlin E Tolbert, Christophe Guilluy, Ashley E Angell, Richard Superfine, Kris A Demali
    Abstract:

    Cells experience mechanical forces throughout their lifetimes. Vinculin is critical for transmitting these forces, yet how it achieves its distinct functions at cell–cell and cell–matrix adhesions remains unanswered. Here, we show Vinculin is phosphorylated at Y822 in cell–cell, but not cell–matrix, adhesions. Phosphorylation at Y822 was elevated when forces were applied to E-cadherin and was required for Vinculin to integrate into the cadherin complex. The mutation Y822F ablated these activities and prevented cells from stiffening in response to forces on E-cadherin. In contrast, Y822 phosphorylation was not required for Vinculin functions in cell–matrix adhesions, including integrin-induced cell stiffening. Finally, forces applied to E-cadherin activated Abelson (Abl) tyrosine kinase to phosphorylate Vinculin; Abl inhibition mimicked the loss of Vinculin phosphorylation. These data reveal an unexpected regulatory mechanism in which Vinculin Y822 phosphorylation determines whether cadherins transmit force and provides a paradigm for how a shared component of adhesions can produce biologically distinct functions.

  • α catenin uses a novel mechanism to activate Vinculin
    Journal of Biological Chemistry, 2012
    Co-Authors: Xiao Peng, Susan W Craig, Jessica L Maiers, Dilshad Choudhury, Kris A Demali
    Abstract:

    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, α-catenin, a cytoplasmic component of the cadherin adhesion complex, activates Vinculin. Direct binding of α-catenin to Vinculin is critical for this event because a point mutant (α-catenin L344P) lacking high affinity binding does not activate Vinculin. Furthermore, unlike all known Vinculin activators, α-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 α-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.

  • new insights into Vinculin function and regulation
    International Review of Cell and Molecular Biology, 2011
    Co-Authors: Xiao Peng, Jessica L Maiers, Elke S Nelson, Kris A Demali
    Abstract:

    Vinculin is a cytoplasmic actin-binding protein enriched in focal adhesions and adherens junctions that is essential for embryonic development. Much is now known regarding the role of Vinculin in governing cell-matrix adhesion. In the past decade that the crystal structure of Vinculin and the molecular details for how Vinculin regulates adhesion events have emerged. The recent data suggests a critical function for Vinculin in regulating integrin clustering, force generation, and strength of adhesion. In addition to an important role in cell-matrix adhesion, Vinculin is also emerging as a regulator of apoptosis, Shigella entry into host cells, and cadherin-based cell-cell adhesion. A close inspection of this work reveals that there are similarities between Vinculin's role in focal adhesions and these processes and also some intriguing differences.

  • Vinculin regulates cell surface e cadherin expression by binding to β catenin
    Journal of Cell Science, 2010
    Co-Authors: Xiao Peng, Laura E Cuff, Cort D Lawton, Kris A Demali
    Abstract:

    Vinculin was identified as a component of adherens junctions 30 years ago, yet its function there remains elusive. Deletion studies are consistent with the idea that Vinculin is important for the organization of cell-cell junctions. However, this approach removes Vinculin from both cell-matrix and cell-cell adhesions, making it impossible to distinguish its contribution at each site. To define the role of Vinculin in cell-cell junctions, we established a powerful short hairpin-RNA-based knockdown/substitution model system that perturbs Vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cadherin-dependent adhesion was reduced. These defects resulted from impaired E-cadherin cell-surface expression. We have investigated the mechanism for the effects of Vinculin and found that the reduced surface E-cadherin expression could be rescued by introduction of Vinculin, but not of a Vinculin A50I substitution mutant that is defective for β-catenin binding. These findings suggest that an interaction between β-catenin and Vinculin is crucial for stabilizing E-cadherin at the cell surface. This was confirmed by analyzing a β-catenin mutant that fails to bind Vinculin. Thus, our study identifies Vinculin as a novel regulator of E-cadherin function and provides important new insight into the dynamic regulation of adherens junctions.

Susan J Gunst - One of the best experts on this subject based on the ideXlab platform.

  • Vinculin phosphorylation at tyr1065 regulates Vinculin conformation and tension development in airway smooth muscle tissues
    Journal of Biological Chemistry, 2014
    Co-Authors: Youliang Huang, Richard O Day, Susan J Gunst
    Abstract:

    Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of Vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr1065 on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of Vinculin phosphorylation at Tyr1065 in regulating the conformation and function of Vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr1065 (Vinculin Y1065F and Vinculin Y1065E) and Vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr1065 phosphorylation affects smooth muscle contraction and the conformation and cellular functions of Vinculin. The results show that Vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr1065 phosphorylation regulates the conformation and scaffolding activity of the Vinculin molecule. We conclude that the phosphorylation of Vinculin at tyrosine 1065 provides a mechanism for regulating the function of Vinculin in airway smooth muscle in response to contractile stimulation.

  • activation of Vinculin induced by cholinergic stimulation regulates contraction of tracheal smooth muscle tissue
    Journal of Biological Chemistry, 2011
    Co-Authors: Youliang Huang, Wenwu Zhang, Susan J Gunst
    Abstract:

    Abstract 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.