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The Journal of Cell Biology Oct 2022The formation of healthy tissue involves continuous remodeling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM...
The formation of healthy tissue involves continuous remodeling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM adhesion sites (CMAs) and actomyosin-mediated forces, the underlying mechanisms remain unclear. Here, we examine how tensin3 contributes to the formation of fibrillar adhesions (FBs) and fibronectin fibrillogenesis. Using BioID mass spectrometry and a mitochondrial targeting assay, we establish that tensin3 associates with the mechanosensors such as talin and vinculin. We show that the talin R11 rod domain binds directly to a helical motif within the central intrinsically disordered region (IDR) of tensin3, whilst vinculin binds indirectly to tensin3 via talin. Using CRISPR knock-out cells in combination with defined tensin3 mutations, we show (i) that tensin3 is critical for the formation of α5β1-integrin FBs and for fibronectin fibrillogenesis, and (ii) the talin/tensin3 interaction drives this process, with vinculin acting to potentiate it.
Topics: Cell Adhesion; Extracellular Matrix; Fibronectins; Focal Adhesions; Integrins; Talin; Tensins; Vinculin
PubMed: 36074065
DOI: 10.1083/jcb.202107022 -
European Journal of Clinical... Dec 2021Distinct faecal microbiota profiles are reported to be associated with various subtypes of IBS. Circulating antibodies to cytolethal distending toxin B (CdtB) and...
BACKGROUND
Distinct faecal microbiota profiles are reported to be associated with various subtypes of IBS. Circulating antibodies to cytolethal distending toxin B (CdtB) and vinculin are proposed as biomarkers to identify post-infectious IBS. The aim of our study was to analyse serum levels of anti-CdtB and anti-vinculin antibodies in patients with different functional gastrointestinal disorders (FGID) and their correlation with the composition of faecal microbiome.
METHODS
The study cohort comprised 65 prospectively recruited individuals: 15 with diarrhoea-type-IBS (IBS-D), 13 with constipation-type-IBS (IBS-C), 15 with functional dyspepsia (FD) and 22 healthy controls. FGID subgroups were defined according to Rome III criteria. Serum levels of anti-CdtB and anti-vinculin antibodies were measured by ELISA. Faecal microbiome composition analysis and assessment of dysbiosis were performed by GA-map® Dysbiosis Test.
RESULTS
Positivity rate either for anti-CdtB or anti-vinculin antibodies was higher in the IBS-C group (76.9%) compared to IBS-D (40.0%), FD (60%) and healthy (63.6%) groups. Dysbiosis was more frequent in subjects positive for anti-CdtB antibodies and in IBS-C patients, who showed an increased amount of opportunistic/pro-inflammatory bacteria and reduced gut protective bacteria. IBS-C patients showed a high inter-individual variation of bacterial communities compared to other FGID subgroups and healthy individuals, whereas microbial profiles of patients with IBS-D and FD were overlapping with those of healthy controls. No bacteria markers showed significant differences between FGID subgroups and healthy controls.
CONCLUSION
Neither anti-CdtB/anti-vinculin antibodies nor faecal microbial profiles allowed to discriminate between specific FGID subgroups. Dysbiosis was more frequent in patients presenting with anti-CdtB antibodies and in IBS-C patients.
Topics: Adult; Aged; Antibodies, Bacterial; Autoantibodies; Bacterial Toxins; Case-Control Studies; Constipation; Cross Reactions; Diarrhea; Dysbiosis; Dyspepsia; Female; Gastrointestinal Diseases; Gastrointestinal Microbiome; Humans; Irritable Bowel Syndrome; Male; Middle Aged; Vinculin; Young Adult
PubMed: 34390492
DOI: 10.1111/eci.13666 -
FASEB Journal : Official Publication of... Aug 2022Mechanosensors control muscle integrity as demonstrated in mice. However, no information is available in human muscle about the distribution of mechanosensors and their... (Randomized Controlled Trial)
Randomized Controlled Trial
Skeletal muscle fiber type-specific expressions of mechanosensors integrin-linked kinase, talin, and vinculin and their modulation by loading and environmental conditions in humans.
Mechanosensors control muscle integrity as demonstrated in mice. However, no information is available in human muscle about the distribution of mechanosensors and their adaptations to mechanical loading and environmental conditions (hypoxia). Here, we hypothesized that mechanosensors show fiber-type-specific distributions and that loading and environmental conditions specifically regulate mechanosensors. We randomly subjected 28 healthy males to one of the following groups (n = 7 each) consisting of nine loading sessions within 3 weeks: normoxia moderate (NM), normoxia intensive (NI), hypoxia moderate (HM), and hypoxia intensive (HI). We took six biopsies: pre (T0), 4 h (T1), and 24 h (T2) after the third as well as 4 h (T3), 24 h (T4), and 72 h (T5) after the ninth training session. We analyzed subjects' maximal oxygen consumption (V̇O max), maximal power output (Pmax), muscle fiber types and cross-sectional areas (CSA), fiber-type-specific integrin-linked kinase (ILK) localizations as well as ILK, vinculin and talin protein and gene expressions in dependence on loading and environmental conditions. V̇O max increased upon NM and HM, Pmax upon all interventions. Fiber types did not change, whereas CSA increased upon NI and HI, but decreased upon HM. ILK showed a type 2-specific fiber type localization. ILK, vinculin, and talin protein and gene expressions differed depending on loading and environmental conditions. Our data demonstrate that mechanosensors show fiber type-specific distributions and that exercise intensities rather than environmental variables influence their profiles in human muscles. These data are the first of their kind in human muscle and indicate that mechanosensors manage the mechanosensing at a fiber-type-specific resolution and that the intensity of mechanical stimulation has a major impact.
Topics: Humans; Hypoxia; Male; Muscle Fibers, Skeletal; Muscle, Skeletal; Protein Serine-Threonine Kinases; Talin; Vinculin
PubMed: 35867073
DOI: 10.1096/fj.202101377RR -
Journal of Cell Science Jun 2023Vinculin is an actin-binding protein present at cell-matrix and cell-cell adhesions, which plays a critical role in bearing force experienced by cells and dissipating it...
Vinculin is an actin-binding protein present at cell-matrix and cell-cell adhesions, which plays a critical role in bearing force experienced by cells and dissipating it onto the cytoskeleton. Recently, we identified a key tyrosine residue, Y822, whose phosphorylation plays a critical role in force transmission at cell-cell adhesions. The role of Y822 in human cancer remains unknown, even though Y822 is mutated to Y822C in uterine cancers. Here, we investigated the effect of this amino acid substitution and that of a phosphodeficient Y822F vinculin in cancer cells. We observed that the presence of the Y822C mutation led to cells that proliferate and migrate more rapidly and contained smaller focal adhesions when compared to cells with wild-type vinculin. In contrast, the presence of the Y822F mutation led to highly spread cells with larger focal adhesions and increased contractility. Furthermore, we provide evidence that Y822C vinculin forms a disulfide bond with paxillin, accounting for some of the elevated phosphorylated paxillin recruitment. Taken together, these data suggest that vinculin Y822 modulates the recruitment of ligands.
Topics: Humans; Vinculin; Paxillin; Ligands; Cell Adhesion; Cell Communication; Focal Adhesions
PubMed: 37248996
DOI: 10.1242/jcs.260104 -
ELife Nov 2020Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling...
Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.
Topics: Actin Cytoskeleton; Actin-Related Protein 2-3 Complex; Actins; Animals; Cell Adhesion; Cryoelectron Microscopy; Electron Microscope Tomography; Extracellular Matrix; Humans; Integrins; Microscopy, Confocal; Sf9 Cells; Talin; Vinculin
PubMed: 33185186
DOI: 10.7554/eLife.53990 -
Cell Communication and Signaling : CCS Oct 2023Integrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile...
BACKGROUND
Integrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile stress could influence cell differentiation through integrin αVβ3.
METHODS
We inhibited the function of integrin αVβ3 of human mesenchymal stem cells by treating with c(RGDyk). Using cytochalasin D and verteporfin to inhibit polymerization of microfilament and function of nuclear Yes-associated protein (YAP), respectively. For each application, mesenchymal stem cells were loaded by cyclic tensile stress of 10% at 0.5 Hz for 2 h daily. Mesenchymal stem cells were harvested on day 7 post-treatment. Western blotting and quantitative RT-PCR were used to detect the expression of alkaline phosphatase (ALP), RUNX2, β-actin, integrin αVβ3, talin-1, vinculin, FAK, and nuclear YAP. Immunofluorescence staining detected vinculin, actin filaments, and YAP nuclear localization.
RESULTS
Cyclic tensile stress could increase the expression of ALP and RUNX2. Inhibition of integrin αVβ3 activation led to rearrangement of actin filaments and downregulated the expression of ALP, RUNX2 and promoted YAP nuclear localization. When microfilament polymerization was inhibited, ALP, RUNX2, and nuclear YAP nuclear localization decreased. Inhibition of YAP nuclear localization could reduce the expression of ALP and RUNX2.
CONCLUSIONS
Cyclic tensile stress promotes early osteogenesis of human mesenchymal stem cells via the integrin αVβ3-actin filaments axis. YAP nuclear localization participates in this process of human mesenchymal stem cells. Video Abstract.
Topics: Humans; Actin Cytoskeleton; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Integrin alphaVbeta3; Mesenchymal Stem Cells; Osteogenesis; Vinculin
PubMed: 37904190
DOI: 10.1186/s12964-022-01027-7 -
ELife Mar 2021Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated...
Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated NA formation, we find these molecules concurrently recruited to the subclass of NAs maturing to focal adhesions. After the initial recruitment under minimal load, vinculin accumulates in maturing NAs at a ~ fivefold higher rate than in non-maturing NAs, and is accompanied by a faster traction force increase. We identify the R8 domain in talin, which exposes a vinculin-binding-site (VBS) in the absence of load, as required for NA maturation. Disruption of R8 domain function reduces load-free vinculin binding to talin, and reduces the rate of additional vinculin recruitment. Taken together, these data show that the concurrent recruitment of talin and vinculin prior to mechanical engagement with integrins is essential for the traction-mediated unfolding of talin, exposure of additional VBSs, further recruitment of vinculin, and ultimately, NA maturation.
Topics: Animals; Binding Sites; CHO Cells; Cricetinae; Cricetulus; Focal Adhesions; Mice; Protein Binding; Talin; Vinculin
PubMed: 33783351
DOI: 10.7554/eLife.66151 -
Nature Communications Apr 2022Mechanical loading generally weakens adhesive structures and eventually leads to their rupture. However, biological systems can adapt to loads by strengthening...
Mechanical loading generally weakens adhesive structures and eventually leads to their rupture. However, biological systems can adapt to loads by strengthening adhesions, which is essential for maintaining the integrity of tissue and whole organisms. Inspired by cellular focal adhesions, we suggest here a generic, molecular mechanism that allows adhesion systems to harness applied loads for self-stabilization through adhesion growth. The mechanism is based on conformation changes of adhesion molecules that are dynamically exchanged with a reservoir. Tangential loading drives the occupation of some states out of equilibrium, which, for thermodynamic reasons, leads to association of further molecules with the cluster. Self-stabilization robustly increases adhesion lifetimes in broad parameter ranges. Unlike for catch-bonds, bond rupture rates can increase monotonically with force. The self-stabilization principle can be realized in many ways in complex adhesion-state networks; we show how it naturally occurs in cellular adhesions involving the adaptor proteins talin and vinculin.
Topics: Cell Adhesion; Focal Adhesions; Mechanical Phenomena; Talin; Vinculin
PubMed: 35459276
DOI: 10.1038/s41467-022-29823-2 -
The Journal of Biological Chemistry 2021α-Catenin binds directly to β-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation....
α-Catenin binds directly to β-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation. Actomyosin-generated force stretches the middle (M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. It is not known whether the intramolecular interactions that regulate epithelial (αE)-catenin binding are conserved across the α-catenin family. Here, we describe the biochemical properties of testes (αT)-catenin, an α-catenin isoform critical for cardiac function and how intramolecular interactions regulate vinculin-binding autoinhibition. Isothermal titration calorimetry showed that αT-catenin binds the β-catenin-N-cadherin complex with a similar low nanomolar affinity to that of αE-catenin. Limited proteolysis revealed that the αT-catenin M-region adopts a more open conformation than αE-catenin. The αT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated αT-catenin M-region is not autoinhibited and thereby distinct from αE-catenin. However, the αT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates the M-region binding to vinculin. In cells, αT-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results show that the αT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of αT-catenin allow it to function as a scaffold for building specific adhesion complexes.
Topics: Actin Cytoskeleton; Binding Sites; Myocardium; Protein Binding; Proteolysis; Vinculin; alpha Catenin
PubMed: 33771561
DOI: 10.1016/j.jbc.2021.100582 -
Biophysical Journal Apr 2018Cell migration is a complex process, requiring coordination of many subcellular processes including membrane protrusion, adhesion, and contractility. For efficient cell...
Cell migration is a complex process, requiring coordination of many subcellular processes including membrane protrusion, adhesion, and contractility. For efficient cell migration, cells must concurrently control both transmission of large forces through adhesion structures and translocation of the cell body via adhesion turnover. Although mechanical regulation of protein dynamics has been proposed to play a major role in force transmission during cell migration, the key proteins and their exact roles are not completely understood. Vinculin is an adhesion protein that mediates force-sensitive processes, such as adhesion assembly under cytoskeletal load. Here, we elucidate the mechanical regulation of vinculin dynamics. Specifically, we paired measurements of vinculin loads using a Förster resonance energy transfer-based tension sensor and vinculin dynamics using fluorescence recovery after photobleaching to measure force-sensitive protein dynamics in living cells. We find that vinculin adopts a variety of mechanical states at adhesions, and the relationship between vinculin load and vinculin dynamics can be altered by the inhibition of vinculin binding to talin or actin or reduction of cytoskeletal contractility. Furthermore, the force-stabilized state of vinculin required for the stabilization of membrane protrusions is unnecessary for random migration, but is required for directional migration along a substrate-bound cue. These data show that the force-sensitive dynamics of vinculin impact force transmission and enable the mechanical integration of subcellular processes. These results suggest that the regulation of force-sensitive protein dynamics may have an underappreciated role in many cellular processes.
Topics: Actomyosin; Animals; Biomechanical Phenomena; Cell Line; Cell Movement; Cell Survival; Focal Adhesions; Mechanical Phenomena; Mice; Talin; Vinculin; rho-Associated Kinases
PubMed: 29642037
DOI: 10.1016/j.bpj.2018.02.019