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Journal of Cell Science Dec 2016Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. Here,...
Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. Here, we examine how altering vinculin activity perturbs integrin function within the context of Drosophila development. Whereas loss of vinculin produced relatively minor phenotypes, gain of vinculin activity, through a loss of head-tail autoinhibition, caused lethality. The minimal domain capable of inducing lethality is the talin-binding D1 domain, and this appears to require talin-binding activity, as lethality was suppressed by competition with single vinculin-binding sites from talin. Activated Drosophila vinculin triggered the formation of cytoplasmic adhesion complexes through the rod of talin, but independently of integrin. These complexes contain a subset of adhesion proteins but no longer link the membrane to actin. The negative effects of hyperactive vinculin were segregated into morphogenetic defects caused by its whole head domain and lethality caused by its D1 domain. These findings demonstrate the crucial importance of the tight control of the activity of vinculin.
Topics: Animals; Cell Adhesion; Cytoplasm; Drosophila melanogaster; Embryo, Nonmammalian; Integrins; Models, Biological; Muscles; Protein Aggregates; Protein Binding; Protein Domains; Vinculin
PubMed: 27737911
DOI: 10.1242/jcs.189878 -
Methods in Cell Biology 2018Productive chromosome movements require that a large multiprotein complex called the kinetochore assemble on sister centromeres. The kinetochore fulfills two critical...
Productive chromosome movements require that a large multiprotein complex called the kinetochore assemble on sister centromeres. The kinetochore fulfills two critical functions as (1) the physical linkage between chromosomes and spindle microtubules and (2) a mechanomolecular sensor that relays a spindle assembly checkpoint signal delaying anaphase onset until chromosomes are attached to spindle microtubules and bioriented. Given its central roles in such a vital process, the kinetochore is one of the most important force-transducing structures in cells; yet it has been technically challenging to measure kinetochore forces. Barriers to measuring cellular forces have begun to be broken by the development of fluorescence-based tension sensors. In this chapter, two methods will be described for measuring kinetochore forces in living cells and strategies for applying these sensors to other force-transducing processes and molecules will be discussed.
Topics: Animals; Biomechanical Phenomena; Biosensing Techniques; Cytological Techniques; Drosophila; Fluorescence Resonance Energy Transfer; Mitosis; Photobleaching; Talin; Vinculin
PubMed: 29804669
DOI: 10.1016/bs.mcb.2018.03.007 -
Journal of Cell Science Jul 2013Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface....
Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. Although it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force.
Topics: Biomechanical Phenomena; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Polarity; Cell Shape; Cytoskeleton; Fluorescence Resonance Energy Transfer; Focal Adhesions; Humans; Laser Therapy; Models, Biological; Protein Transport; Stress Fibers; Vinculin
PubMed: 23687380
DOI: 10.1242/jcs.119032 -
Biophysical Journal Aug 2010Vinculin activation plays a critical role in focal adhesion initiation and formation. In its native state, vinculin is in an autoinhibitory conformation in which domain...
Vinculin activation plays a critical role in focal adhesion initiation and formation. In its native state, vinculin is in an autoinhibitory conformation in which domain 1 prevents interaction of the vinculin tail domain with actin by steric hindrance. Once activated, vinculin is able to interact with both actin and talin. Several hypotheses have been put forth addressing the mechanisms of vinculin activation. One set of studies suggests that vinculin interaction with talin is sufficient to cause activation, whereas another set of studies suggests that a simultaneous interaction with several binding partners is necessary to achieve vinculin activation. Using molecular-dynamics (MD) simulations, we investigate the mechanisms of vinculin activation and suggest both a trajectory of conformational changes leading to vinculin activation, and key structural features that are likely involved in stabilizing the autoinhibited conformation. Assuming that the simultaneous interaction of vinculin with both actin and talin causes a stretching force on vinculin, and that vinculin activation results from a removal of steric hindrance blocking the actin-binding sites, we simulate with MD the stretching and activation of vinculin. The MD simulations are further confirmed by normal-mode analysis and simulation after residue modification. Taken together, the results of these simulations suggest that bending of the vinculin-binding-site region in vinculin away from the vinculin tail is the likely trajectory of vinculin activation.
Topics: Molecular Dynamics Simulation; Protein Structure, Secondary; Reproducibility of Results; Vinculin
PubMed: 20712990
DOI: 10.1016/j.bpj.2010.05.024 -
The Journal of Biological Chemistry Jul 2011Internalization of Shigella into host epithelial cells, where the bacteria replicates and spreads to neighboring cells, requires a type 3 secretion system (T3SS)...
Internalization of Shigella into host epithelial cells, where the bacteria replicates and spreads to neighboring cells, requires a type 3 secretion system (T3SS) effector coined IpaA. IpaA binds directly to and activates the cytoskeletal protein vinculin after injection in the host cell cytosol, and this was previously thought to be directed by two amphipathic α-helical vinculin-binding sites (VBS) found in the C-terminal tail domain of IpaA. Here, we report a third VBS, IpaA-VBS3, that is located N-terminal to the other two VBSs of IpaA and show that one IpaA molecule can bind up to three vinculin molecules. Biochemical in vitro Shigella invasion assays and the 1.6 Å crystal structure of the vinculin·IpaA-VBS3 complex showed that IpaA-VBS3 is functionally redundant with the other two IpaA-VBSs in cell invasion and in activating the latent F-actin binding functions of vinculin. Multiple VBSs in IpaA are reminiscent of talin, which harbors 11 VBSs. However, most of the talin VBSs have low affinity and are buried in helix bundles, whereas all three of the VBSs of IpaA are high affinity, readily available, and in close proximity to each other in the IpaA structure. Although deletion of IpaA-VBS3 has no detectable effects on Shigella invasion of epithelial cells, deletion of all three VBSs impaired bacterial invasion to levels found in an ipaA null mutant strain. Thus, IpaA-directed mimicry of talin in activating vinculin occurs through three high affinity VBSs that are essential for Shigella pathogenesis.
Topics: Antigens, Bacterial; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Dysentery, Bacillary; HeLa Cells; Humans; Molecular Mimicry; Protein Structure, Secondary; Protein Structure, Tertiary; Shigella flexneri; Talin; Vinculin
PubMed: 21525010
DOI: 10.1074/jbc.M110.184283 -
Scientific Reports Feb 2017Sudden unexplained nocturnal death syndrome (SUNDS) remains an autopsy negative disorder with unclear etiology. Vinculin (VCL) was linked to sudden arrhythmia death in...
Sudden unexplained nocturnal death syndrome (SUNDS) remains an autopsy negative disorder with unclear etiology. Vinculin (VCL) was linked to sudden arrhythmia death in VCL knockout mice prior to the appearance of cardiomyopathy. We hypothesized VCL mutations underlie risk for SUNDS. A rare heterozygous variant VCL-M94I was found in a SUNDS victim who suffered sudden nocturnal tachypnea and lacked pathogenic variants in known arrhythmia-causing genes. VCL was identified to interact with SCN5A in vitro/vivo. The VCL-M94I was co-expressed with the cardiac sodium channel in HEK293 cells and also overexpressed in induced pluripotent stem cells derived cardiomyocytes (iPSCs-CM). In HEK293 cells with pH 7.4, VCL-M94I caused ~30% decrease in peak sodium current (I) amplitude compared to WT; under acidotic conditions (pH 7.0) typically found with hypoxia during sleep apnea, M94I resulted in 37% reduction in peak I compared to WT and the combination of VCL-M94I and pH 7.0 decreased peak I by ~56% compared to WT at pH 7.4. In iPSCs-CM, similar effects of M94I on reduction of peak I were observed. This study initially shows both physical and functional interaction between VCL and cardiac sodium channel, and suggests an important role for respiratory acidosis in triggering the fatal arrhythmia underlying SUNDS.
Topics: Adolescent; Adult; Animals; Brugada Syndrome; Female; Genotype; Humans; Induced Pluripotent Stem Cells; Male; Membrane Potentials; Mice; Mice, Knockout; Middle Aged; Myocardium; Myocytes, Cardiac; NAV1.5 Voltage-Gated Sodium Channel; Polymorphism, Genetic; Protein Binding; Vinculin; Young Adult
PubMed: 28218286
DOI: 10.1038/srep42953 -
Biochemistry Nov 2008Vinculin is a highly conserved cytoskeletal protein that localizes to sites of cell adhesion. The tail domain of vinculin (Vt) forms tight autoinhibitory interactions...
Vinculin is a highly conserved cytoskeletal protein that localizes to sites of cell adhesion. The tail domain of vinculin (Vt) forms tight autoinhibitory interactions with the head domain and down-regulates vinculin function by obscuring ligand binding sites. Ligand binding is required for both vinculin activation and function, and one of vinculin's primary roles as a cell adhesion protein involves its ability to link the Actin cytoskeleton to the cell membrane. Vt can bind F-Actin and phosphoinositol 4,5-bisphosphate, and association with these ligands has been reported to cause a conformational change in Vt. Moreover, a single histidine residue, H906, was reported to be critical for both a pH dependent conformational change and pH dependent self-association. In this study, we investigate the role of pH on Vt structure and self-association. In contrast to earlier observations, our studies do not support a significant alteration in Vt conformation over this pH range. Moreover, while we identify a site of Vt dimerization, similar to that observed previously by X-ray crystallography, the weak K(d) (approximately 300 microM) determined for Vt self-association does not differ significantly between pH 5.5 and pH 7.5.
Topics: Animals; Chickens; Histidine; Hydrogen-Ion Concentration; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Protons; Vinculin
PubMed: 18980387
DOI: 10.1021/bi801764a -
Cellular Signalling Jun 2016Endothelial cell (EC) barrier disruption induced by edemagenic agonists such as thrombin is a result of increased actomyosin contraction and enforcement of focal...
Endothelial cell (EC) barrier disruption induced by edemagenic agonists such as thrombin is a result of increased actomyosin contraction and enforcement of focal adhesions (FA) anchoring contracting stress fibers, which leads to cell retraction and force-induced disruption of cell junctions. In turn, EC barrier enhancement by oxidized phospholipids (OxPAPC) and other agonists is a result of increased tethering forces due to enforcement of the peripheral actin rim and enhancement of cell-cell adherens junction (AJ) complexes promoting EC barrier integrity. This study tested participation of the mechanosensitive adaptor, vinculin, which couples FA and AJ to actin cytoskeleton, in control of the EC permeability response to barrier disruptive (thrombin) and barrier enhancing (OxPAPC) stimulation. OxPAPC and thrombin induced different patterns of FA remodeling. Knockdown of vinculin attenuated both, OxPAPC-induced decrease and thrombin-induced increase in EC permeability. Thrombin stimulated the vinculin association with FA protein talin and suppressed the interaction with AJ protein, VE-cadherin. In contrast, OxPAPC stimulated the vinculin association with VE-cadherin. Thrombin and OxPAPC induced different levels of myosin light chain (MLC) phosphorylation and caused different patterns of intracellular phospho-MLC distribution. Thrombin-induced talin-vinculin and OxPAPC-induced VE-cadherin-vinculin association were abolished by myosin inhibitor blebbistatin. Expression of the vinculin mutant unable to interact with actin attenuated EC permeability changes and MLC phosphorylation caused by both, thrombin and OxPAPC. These data suggest that the specific vinculin interaction with FA or AJ in different contexts of agonist stimulation is defined by development of regional actyomyosin-based tension and participates in both, the barrier-disruptive and barrier-enhancing endothelial responses.
Topics: Cadherins; Cell Adhesion; Cell Membrane Permeability; Cells, Cultured; Cytoskeleton; Endothelial Cells; Endothelium, Vascular; Focal Adhesions; Humans; Myosin Light Chains; Phospholipids; Talin; Thrombin; Vinculin
PubMed: 26923917
DOI: 10.1016/j.cellsig.2016.02.015 -
Biophysical Journal Oct 2021Vinculin plays a key role during the first phase of focal adhesion formation and interacts with the plasma membrane through specific binding of its tail domain to the...
Vinculin plays a key role during the first phase of focal adhesion formation and interacts with the plasma membrane through specific binding of its tail domain to the lipid phosphatidylinositol 4,5-bisphosphate (PIP). Our understanding of the PIP-vinculin interaction has been hampered by contradictory biochemical and structural data. Here, we used a multiscale molecular dynamics simulation approach, in which unbiased coarse-grained molecular dynamics were used to generate starting structures for subsequent microsecond-long all-atom simulations. This allowed us to map the interaction of the vinculin tail with PIP-enriched membranes in atomistic detail. In agreement with experimental data, we have shown that membrane binding is sterically incompatible with the intramolecular interaction between vinculin's head and tail domain. Our simulations further confirmed biochemical and structural results, which identified two positively charged surfaces, the basic collar and the basic ladder, as the main PIP interaction sites. By introducing a valency-disaggregated binding network analysis, we were able to map the protein-lipid interactions in unprecedented detail. In contrast to the basic collar, in which PIP is specifically recognized by an up to hexavalent binding pocket, the basic ladder forms a series of low-valency binding sites. Importantly, many of these PIP binding residues are also involved in maintaining vinculin in a closed, autoinhibited conformation. These findings led us to propose a molecular mechanism for the coupling between vinculin activation and membrane binding. Finally, our refined binding site suggests an allosteric relationship between PIP and F-actin binding that disfavors simultaneous interaction with both ligands, despite nonoverlapping binding sites.
Topics: Actin Cytoskeleton; Actins; Binding Sites; Molecular Dynamics Simulation; Protein Binding; Vinculin
PubMed: 34411575
DOI: 10.1016/j.bpj.2021.08.018 -
Investigative Ophthalmology & Visual... Oct 2014To identify plasma protein biomarkers for age-related macular degeneration (AMD) using a large-scale quantitative proteomic discovery procedure.
PURPOSE
To identify plasma protein biomarkers for age-related macular degeneration (AMD) using a large-scale quantitative proteomic discovery procedure.
METHODS
Plasma proteomes from 20 exudative AMD patients and 20 healthy control patients were comparatively profiled by four-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins existing at statistically different levels were validated by enzyme-linked immunosorbent assay (ELISA) and Western blotting in 233 case-controlled samples. Newly discovered plasma biomarkers were further confirmed using in vivo and in vitro experiments.
RESULTS
Out of 320 proteins identified, vinculin, protein S100A9, triosephosphate isomerase, protein S100A8, protein Z-dependent protease inhibitor, C-X-C motif chemokine 7, and tenascin X showed significantly differential expression in AMD patient plasma compared to control plasma. Among these, the area under the curve (AUC) for vinculin was 0.871 for discriminating between exudative AMD and controls (n = 201) and 0.879 for discriminating between AMD and controls (n = 233). A proteogenomic combination model using vinculin and two known risk genotypes in ARMS2 and CFH genes additionally provided excellent discrimination of AMD from controls (AUC = 0.916). The plasma level of vinculin was not associated with any confounding clinical variables, such as age, smoking, and other comorbidities. Additionally, vinculin was strongly expressed in retinal pigment epithelial cells of human eyes, and its expression was elevated when exposed to oxidative stress in vitro.
CONCLUSIONS
Vinculin was identified as a potential plasma biomarker for AMD. The early detection of AMD using novel plasma biomarkers with genetic modeling may enable timely treatment and vision preservation in the elderly.
Topics: Aged; Biomarkers; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Humans; Macula Lutea; Macular Degeneration; Male; Proteomics; Retrospective Studies; Tandem Mass Spectrometry; Vinculin
PubMed: 25298412
DOI: 10.1167/iovs.14-15168