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Kidney International Mar 2018Cell-matrix interactions and podocyte intercellular junctions are key for maintaining the glomerular filtration barrier. Vinculin, a cytoplasmic protein, couples actin...
Cell-matrix interactions and podocyte intercellular junctions are key for maintaining the glomerular filtration barrier. Vinculin, a cytoplasmic protein, couples actin filaments to integrin-mediated cell-matrix adhesions and to cadherin-based intercellular junctions. Here, we examined the role of vinculin in podocytes by the generation of a podocyte-specific knockout mouse. Mice lacking podocyte vinculin had increased albuminuria and foot process effacement following injury in vivo. Analysis of primary podocytes isolated from the mutant mice revealed defects in cell protrusions, altered focal adhesion size and signaling, as well as impaired cell migration. Furthermore, we found a marked mislocalization of the intercellular junction protein zonula occludens-1. In kidney sections from patients with focal segmental glomerulosclerosis, minimal change disease and membranous nephropathy, we observed dramatic differences in the expression levels and localization of vinculin. Thus, our results suggest that vinculin is necessary to maintain the integrity of the glomerular filtration barrier by modulating podocyte foot processes and stabilizing intercellular junctions.
Topics: Albuminuria; Animals; Cell Movement; Cell Surface Extensions; Cells, Cultured; Focal Adhesion Kinase 1; Focal Adhesions; Glomerulonephritis, Membranous; Glomerulosclerosis, Focal Segmental; Mechanotransduction, Cellular; Mice, Inbred C57BL; Mice, Knockout; Nephrosis, Lipoid; Phosphorylation; Podocytes; Vinculin; Zonula Occludens-1 Protein
PubMed: 29241625
DOI: 10.1016/j.kint.2017.09.021 -
Structure (London, England : 1993) Oct 2019Vinculin and its splice isoform metavinculin play key roles in regulating cellular morphology, motility, and force transduction. Vinculin is distinct from metavinculin...
Vinculin and its splice isoform metavinculin play key roles in regulating cellular morphology, motility, and force transduction. Vinculin is distinct from metavinculin in its ability to bundle filamentous actin (F-actin). To elucidate the molecular basis for these differences, we employed computational and experimental approaches. Results from these analyses suggest that the C terminus of both vinculin and metavinculin form stable interactions with the F-actin surface. However, the metavinculin tail (MVt) domain contains a 68 amino acid insert, with helix 1 (H1) sequestered into a globular subdomain, which protrudes from the F-actin surface and prevents actin bundling by sterically occluding actin filaments. Consistent with our model, deletion and selective point mutations within the MVt H1 disrupt this protruding structure, and facilitate actin bundling similar to vinculin tail (Vt) domain.
Topics: Actins; Alternative Splicing; Animals; Binding Sites; Cryoelectron Microscopy; Models, Molecular; Mutation; Protein Binding; Protein Domains; Protein Structure, Secondary; Vinculin
PubMed: 31422909
DOI: 10.1016/j.str.2019.07.013 -
FEBS Letters Feb 2022Lysophosphatidylcholine (LPC), the active metabolite of palmitate, triggers hepatocyte death by activating endoplasmic reticulum stress and JNK signalling-mediated...
Lysophosphatidylcholine (LPC), the active metabolite of palmitate, triggers hepatocyte death by activating endoplasmic reticulum stress and JNK signalling-mediated lipoapoptosis. However, LPC-induced cytotoxicity in hepatocytes is not well understood. Here, we found for the first time that LPC-induced cell rounding occurred prior to apoptosis. LPC-induced rounding of cells reduced both cell-extracellular matrix (ECM) adhesion and cell-cell junctions, which promoted detachment-induced apoptosis (defined as anoikis) in hepatocytes. Further study revealed that LPC altered cellular morphology and cell adhesion by inhibiting integrin and cadherin signalling-mediated microfilament polymerization. We also found that ECM supplementation and microfilament cytoskeletal stabilization inhibited LPC-induced hepatocyte death by attenuating anoikis. Our data indicate a novel cytotoxic process and signalling pathway induced by LPC.
Topics: Actin Cytoskeleton; Anoikis; Apoptosis Regulatory Proteins; Cadherins; Caspase 8; Cell Adhesion; Cell Line, Tumor; Endoplasmic Reticulum Stress; Extracellular Matrix; Gene Expression Regulation; Hep G2 Cells; Hepatocytes; Humans; Integrins; Intercellular Junctions; Lysophosphatidylcholines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Vinculin
PubMed: 35043979
DOI: 10.1002/1873-3468.14291 -
Experimental Eye Research Apr 2019Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell...
Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood. To examine the role of substrate compliancy on multi-layered epithelial sheet migration, we performed traction force and confocal microscopy to determine differences in traction forces and to examine focal adhesions on synthetic and biological substrates. The leading edges of corneal epithelial sheets undergo retraction or contraction prior to migration, and alterations in the sheet's stiffness are affected by the amount of force exerted by cells at the leading edge. On substrates of 30 kPa, cells exhibited greater and more rapid movement than on substrates of 8 kPa, which are similar to that of the corneal basement membrane. Vinculin and its phosphorylated residue Y1065 were prominent along the basal surface of migrating cells, while Y822 was prominent between neighboring cells along the leading edge. Vinculin localization was diffuse on a substrate where the basement membrane was removed. Furthermore, when cells were cultured on fibronectin-coated acrylamide substrates of 8 and 50 kPa and then wounded, there was an injury-induced phosphorylation of Y1065 and substrate dependent changes in the number and size of vinculin containing focal adhesions. These results demonstrate that changes in substrate stiffness affected traction forces and vinculin dynamics, which potentially could contribute to the delayed healing response associated with certain corneal pathologies.
Topics: Analysis of Variance; Biomechanical Phenomena; Cell Adhesion; Cell Movement; Cornea; Epithelial Cells; Epithelium; Humans; Limbus Corneae; Phosphorylation; Vinculin
PubMed: 30653966
DOI: 10.1016/j.exer.2019.01.014 -
Dental Materials Journal Jan 2023After periodontal tissue injury, reconstruct soft tissue sealing around the tooth surface is of fundamental importance to treat periodontitis. Among multiple cell types,...
After periodontal tissue injury, reconstruct soft tissue sealing around the tooth surface is of fundamental importance to treat periodontitis. Among multiple cell types, fibroblast plays a central role in reestablishing functional periodontium. To enhance fibroblast activity, a novel metal-organic framework-based nanoplatform is fabricated using mesoporous Prussian blue (MPB) nanoparticles to load baicalein (BA), named MPB-BA. Drug release test displayed sustained BA release of MPB-BA. Cell proliferation, transwell migration and wound healing tests revealed accelerated fibroblast proliferation and migration for the established MPB-BA nanoplatform. Moreover, vinculin immunofluorescence staining, western blot and quantitative real-time PCR analysis showed up-regulated vinculin protein and integrin α5 and integrin β1 gene expressions for MPB-BA, suggesting improved cell adhesion. In addition, hematoxylin and eosin (H&E) and Masson trichromatic staining suggested superior anti-inflammatory and collagen fiber reconstruction effects for MPB-BA in a rat experimental periodontitis model in vivo. Our study may provide a promising strategy for the treatment of periodontitis.
Topics: Rats; Animals; Vinculin; Metal-Organic Frameworks; Wound Healing; Fibroblasts; Periodontitis
PubMed: 36244739
DOI: 10.4012/dmj.2022-096 -
Shigella IpaA mediates actin bundling through diffusible vinculin oligomers with activation imprint.Cell Reports Apr 2023Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin...
Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domains that bind to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain, leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.
Topics: Bacterial Proteins; Antigens, Bacterial; Actins; Vinculin; Shigella; Protein Binding
PubMed: 37071535
DOI: 10.1016/j.celrep.2023.112405 -
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 -
PloS One 2019Vinculin (Vcn) is a ubiquitously expressed cytoskeletal protein that links transmembrane receptors to actin filaments, and plays a key role in regulating cell adhesion,...
Vinculin (Vcn) is a ubiquitously expressed cytoskeletal protein that links transmembrane receptors to actin filaments, and plays a key role in regulating cell adhesion, motility, and force transmission. Metavinculin (MVcn) is a Vcn splice isoform that contains an additional exon encoding a 68-residue insert within the actin binding tail domain. MVcn is selectively expressed at sub-stoichiometic amounts relative to Vcn in smooth and cardiac muscle cells. Mutations in the MVcn insert are linked to various cardiomyopathies. In vitro analysis has previously shown that while both proteins can engage filamentous (F)-actin, only Vcn can promote F-actin bundling. Moreover, we and others have shown that MVcn can negatively regulate Vcn-mediated F-actin bundling in vitro. To investigate functional differences between MVcn and Vcn, we stably expressed either Vcn or MVcn in Vcn-null mouse embryonic fibroblasts. While both MVcn and Vcn were observed at FAs, MVcn-expressing cells had larger but fewer focal adhesions per cell compared to Vcn-expressing cells. MVcn-expressing cells migrated faster and exhibited greater persistence compared to Vcn-expressing cells, even though Vcn-containing FAs assembled and disassembled faster. Magnetic tweezer measurements on Vcn-expressing cells show a typical cell stiffening phenotype in response to externally applied force; however, this was absent in Vcn-null and MVcn-expressing cells. Our findings that MVcn expression leads to larger but fewer FAs per cell, in conjunction with the inability of MVcn to bundle F-actin in vitro and rescue the cell stiffening response, are consistent with our previous findings of actin bundling deficient Vcn variants, suggesting that deficient actin-bundling may account for some of the differences between Vcn and MVcn.
Topics: Animals; Cell Line; Cell Movement; Focal Adhesions; Gene Expression Regulation; Mechanotransduction, Cellular; Mice; Models, Molecular; Protein Domains; Vinculin
PubMed: 31483833
DOI: 10.1371/journal.pone.0221962 -
International Journal of Molecular... May 2023Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events,...
Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins ( ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins ( < 0.001), whose differential expression was confirmed by ELISA ( = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR.
Topics: Humans; Crohn Disease; Tumor Necrosis Factor Inhibitors; Vinculin; Tumor Necrosis Factor-alpha; Antineoplastic Agents; Remission Induction; Infliximab
PubMed: 37240037
DOI: 10.3390/ijms24108695 -
Nature Cell Biology Jul 2015Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved...
Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin's interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.
Topics: Actins; Blotting, Western; Cell Line; Cell Line, Tumor; Fluorescence Resonance Energy Transfer; Focal Adhesions; Humans; Integrins; Luminescent Proteins; Microscopy, Fluorescence; Models, Molecular; Mutation; Nanostructures; Nanotechnology; Paxillin; Protein Binding; Protein Structure, Tertiary; RNA Interference; Talin; Vinculin
PubMed: 26053221
DOI: 10.1038/ncb3180