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Blood Advances Dec 2021Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbβ3. Here we show...
Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbβ3. Here we show that clot contraction is impaired by inhibitors of the platelet cytosolic protease calpain. We used subtiligase-mediated labeling of amino termini and mass spectrometry to identify proteolytically cleaved platelet proteins involved in clot contraction. Of 32 calpain-cleaved proteins after TRAP stimulation, 14 were cytoskeletal, most prominently talin and vinculin. A complex of talin and vinculin constitutes a mechanosensitive clutch connecting integrins bound to the extracellular matrix with the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain and a C-terminal rod domain organized into a series of 4- and 5-helix bundles. The bundles contain 11 vinculin binding sites (VBSs), each of which is an α-helix packed into a bundle interior and requiring structural rearrangement to initiate vinculin binding. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in α-helical regions in proximity to a VBS. There is evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force. Therefore, it is likely that calpain acts in the later stages of clot retraction through focal adhesion disassembly.
Topics: Binding Sites; Calpain; Fibrin; Humans; Talin; Thrombosis
PubMed: 34570183
DOI: 10.1182/bloodadvances.2021004582 -
Cells Apr 2024Mechanotransduction refers to the ability of cells to sense mechanical stimuli and convert them into biochemical signals. In this context, the key players are focal... (Review)
Review
Mechanotransduction refers to the ability of cells to sense mechanical stimuli and convert them into biochemical signals. In this context, the key players are focal adhesions (FAs): multiprotein complexes that link intracellular actin bundles and the extracellular matrix (ECM). FAs are involved in cellular adhesion, growth, differentiation, gene expression, migration, communication, force transmission, and contractility. Focal adhesion signaling molecules, including Focal Adhesion Kinase (FAK), integrins, vinculin, and paxillin, also play pivotal roles in cardiomyogenesis, impacting cell proliferation and heart tube looping. In fact, cardiomyocytes sense ECM stiffness through integrins, modulating signaling pathways like PI3K/AKT and Wnt/β-catenin. Moreover, FAK/Src complex activation mediates cardiac hypertrophic growth and survival signaling in response to mechanical loads. This review provides an overview of the molecular and mechanical mechanisms underlying the crosstalk between FAs and cardiac differentiation, as well as the role of FA-mediated mechanotransduction in guiding cardiac muscle responses to mechanical stimuli.
Topics: Mechanotransduction, Cellular; Focal Adhesions; Humans; Myocytes, Cardiac; Animals; Cell Differentiation; Extracellular Matrix
PubMed: 38667279
DOI: 10.3390/cells13080664 -
Molecular Biology of the Cell Sep 2022Increased tension on VE-cadherin (VE-cad) complexes activates adaptive cell stiffening and local cytoskeletal reinforcement--two key signatures of intercellular...
Increased tension on VE-cadherin (VE-cad) complexes activates adaptive cell stiffening and local cytoskeletal reinforcement--two key signatures of intercellular mechanotransduction. Here we demonstrate that tugging on VE-cad receptors initiates a cascade that results in downstream integrin activation. The formation of new integrin adhesions potentiates vinculin and actin recruitment to mechanically reinforce stressed cadherin adhesions. This cascade differs from documented antagonistic effects of integrins on intercellular junctions. We identify focal adhesion kinase, Abl kinase, and RhoA GTPase as key components of the positive feedback loop. Results further show that a consequence of integrin involvement is the sensitization of intercellular force transduction to the extracellular matrix (ECM) not by regulating junctional tension but by altering signal cascades that reinforce cell-cell adhesions. On type 1 collagen or fibronectin substrates, integrin subtypes α2β1 and α5β1, respectively, differentially control actin remodeling at VE-cad adhesions. Specifically, ECM-dependent differences in VE-cad force transduction mirror differences in the rigidity sensing mechanisms of α2β1 and α5β1 integrins. The findings verify the role of integrins in VE-cad force transduction and uncover a previously unappreciated mechanism by which the ECM impacts the mechanical reinforcement of interendothelial junctions.
Topics: Actins; Antigens, CD; Cadherins; Cell Adhesion; Extracellular Matrix; Integrins; Intercellular Junctions; Mechanotransduction, Cellular
PubMed: 35653290
DOI: 10.1091/mbc.E22-03-0075 -
Small (Weinheim An Der Bergstrasse,... Aug 2020Grooved topography and inherent cell contact guidance has shown promising results regarding cell proliferation, morphology, and lineage-specific differentiation. Yet...
Grooved topography and inherent cell contact guidance has shown promising results regarding cell proliferation, morphology, and lineage-specific differentiation. Yet these approaches are limited to 2D applications. Sandwich-culture conditions are developed to bridge the gap between 2D and 3D culture, enabling both ventral and dorsal cell surface stimulation. The effect of grooved surface topography is accessed on cell orientation and elongation in a highly controlled manner, with simultaneous and independent stimuli on two cell sides. Nanogrooved and non-nanogrooved substrates are assembled into quasi-3D systems with variable relative orientations. A plethora of sandwich-culture conditions are created by seeding cells on lower, upper, or both substrates. Software image analysis demonstrates that F-actin of cells acquires the orientation of the substrate on which cells are initially seeded, independently from the orientation of the second top substrate. Contrasting cell morphologies are observed, with a higher elongation for nanogrooved 2D substrates than nanogrooved sandwich-culture conditions. Correlated with an increased pFAK activity and vinculin staining for sandwich-culture conditions, these results point to an enhanced cell surface stimulation versus control conditions. The pivotal role of initial cell-biomaterial contact on cellular alignment is highlighted, providing important insights for tissue engineering strategies aiming to guide cellular response through mechanotransduction approaches.
Topics: Cell Differentiation; Cell Proliferation; Mechanotransduction, Cellular; Surface Properties; Tissue Engineering
PubMed: 32603002
DOI: 10.1002/smll.202001975 -
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 -
Journal of Molecular Endocrinology Jan 2023Progesterone causes vascular smooth muscle cell relaxation through membrane progesterone receptors (mPRs), which are members of the progestin and adipoQ receptor (PAQR)...
Progesterone causes vascular smooth muscle cell relaxation through membrane progesterone receptors (mPRs), which are members of the progestin and adipoQ receptor (PAQR) family, and nuclear PRs (nPRs). However, beneficial vascular effects of progesterone in preventing pre-atherosclerosis and the involvement of mPRs and nPRs remain unclear. The results show short- to long-term treatments with 100 nM progesterone (P4) and specific agonists for mPRs, OD 02-0, and nPRs, R5020, inhibited pre-atherosclerotic events in human umbilical vein endothelial cells (HUVECs), decreasing focal adhesion (FA) by monocytes, FA signaling, HUVEC migration and invasion, and vinculin expression. Progesterone and OD 02-0, but not R5020, inhibited phosphorylation of Src and focal adhesion kinase, critical kinases of FA signaling, within 20 min and migration and invasion of HUVECs and monocyte adhesion after 3 h. These inhibitory P4 and 02-0 effects were attenuated with MAP kinase and Pi3k inhibitors, indicating involvement of these kinases in this mPR-mediated action. However, after 16 h, OD 02-0 was no longer effective in inhibiting FA signaling, while both progesterone and R5020 decreased the activity of the two kinases. Knockdown of receptor expression with siRNA confirmed that mPRα mediates short-term and nPR long-term inhibitory effects of progesterone on FA signaling. Thus, progesterone inhibition of FA signaling and pre-atherosclerosis is coordinated through mPRα and nPRs.
Topics: Humans; Focal Adhesions; Progesterone; Endothelial Cells; Phosphatidylinositol 3-Kinases
PubMed: 36099054
DOI: 10.1530/JME-22-0073 -
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 -
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