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Integrative Biology : Quantitative... Jun 2016Rigidity sensing is a critical determinant of cell fate and behavior but its molecular mechanisms are poorly understood. Focal adhesions (FAs) are complexes that anchor...
Rigidity sensing is a critical determinant of cell fate and behavior but its molecular mechanisms are poorly understood. Focal adhesions (FAs) are complexes that anchor cells to the matrix. Among their components, vinculin undergoes an auto-inhibitory head-tail interaction that regulates the recruitment of, and interactions with its partners in a force-dependent manner. It is unknown, however, whether this mechanism is involved in substrate rigidity sensing. Here, we use a range of quantitative fluorescence microscopies on live human Mesenchymal Stem Cells to address this question. We identify two distinct rigidity-sensing molecular modules in FAs, one of which involves vinculin and talin, is regulated by vinculin head-tail interaction, and targets cell morphology. Vinculin and talin are recruited independently in a rigidity-dependent manner to FAs where they directly interact in a rigidity-independent stoichiometry at a site proximal to talin head. Vinculin head-tail interaction is required on soft substrates to destabilize vinculin and talin in FAs, and to allow hMSCs branching. Another module involves paxillin and FAK, which soft substrates also destabilize, but independently of vinculin head-tail interaction. This multi-modularity may be key to allow a versatile response to complex biomechanical cues.
Topics: Cell Adhesion; Cell Size; Cells, Cultured; Cytoskeleton; Elastic Modulus; Focal Adhesions; Humans; Mechanotransduction, Cellular; Mesenchymal Stem Cells; Stress, Mechanical; Talin; Vinculin
PubMed: 27169142
DOI: 10.1039/c5ib00307e -
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 -
Journal of Cell Science Sep 2015The focal adhesion protein vinculin connects the actin cytoskeleton, through talin and integrins, with the extracellular matrix. Vinculin consists of a globular head and...
The focal adhesion protein vinculin connects the actin cytoskeleton, through talin and integrins, with the extracellular matrix. Vinculin consists of a globular head and tail domain, which undergo conformational changes from a closed auto-inhibited conformation in the cytoplasm to an open conformation in focal adhesions. Src-mediated phosphorylation has been suggested to regulate this conformational switch. To explore the role of phosphorylation in vinculin activation, we used knock-out mouse embryonic fibroblasts re-expressing different vinculin mutants in traction microscopy, magnetic tweezer microrheology, FRAP and actin-binding assays. Compared to cells expressing wild-type or constitutively active vinculin, we found reduced tractions, cytoskeletal stiffness, adhesion strength, and increased vinculin dynamics in cells expressing constitutively inactive vinculin or vinculin where Src-mediated phosphorylation was blocked by replacing tyrosine at position 100 and/or 1065 with a non-phosphorylatable phenylalanine residue. Replacing tyrosine residues with phospho-mimicking glutamic acid residues restored cellular tractions, stiffness and adhesion strength, as well as vinculin dynamics, and facilitated vinculin-actin binding. These data demonstrate that Src-mediated phosphorylation is necessary for vinculin activation, and that phosphorylation controls cytoskeletal mechanics by regulating force transmission between the actin cytoskeleton and focal adhesion proteins.
Topics: Animals; Cell Adhesion; Cytoskeleton; Energy Transfer; Fibroblasts; Integrins; Mice; Mice, Knockout; Phosphorylation; Point Mutation; Protein Structure, Secondary; Vinculin; src-Family Kinases
PubMed: 26240176
DOI: 10.1242/jcs.172031 -
Cell Adhesion and Communication Jan 1998Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical...
Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, gamma 227 and gamma 229, and a reconstituted gamma 229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins alpha- and beta-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines. BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculin-negative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted gamma 229 and gamma 227 cells that were formed in suspension during a two-hour static incubation at 37 degrees C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.
Topics: Actins; Animals; Cadherins; Carcinoma, Embryonal; Cell Adhesion; Cell Aggregation; Cytoskeletal Proteins; Cytoskeleton; Fluorescent Antibody Technique; Mice; Organoids; Rheology; Tensile Strength; Trans-Activators; Tumor Cells, Cultured; Vinculin; alpha Catenin; beta Catenin
PubMed: 9638341
DOI: 10.3109/15419069809005598 -
The Protein Journal Feb 2022To shed light onto the activation mechanism of vinculin, we carried out a detailed refinement of chicken vinculin and compared it to the human protein which is greater...
To shed light onto the activation mechanism of vinculin, we carried out a detailed refinement of chicken vinculin and compared it to the human protein which is greater than 95% identical. Refinement resulted in a complete and significantly improved model. This model includes important elements such as a pro-rich strap region (PRR) and C-terminus. The conformation of the PRR stabilized by its inter- and intra-molecular contacts shows a dynamic, but relatively stable motif that constitutes a docking platform for multiple molecules. The contact of the C-terminus with the PRR suggests that phosphorylation of Tyr1065 might control activation and membrane binding. Improved electron densities showed the presence of large solvent molecules such as phosphates/sulfates and a head-group of PIP2. The improved model allowed for a computational stability analysis to be performed by the program Corex/Best which located numerous hot-spots of increased and decreased stability. Proximity of the identified binding sites for regulatory partners involved in inducing or suppressing the activation of vinculin to the unstable elements sheds new light onto the activation pathway and differential activation. This stability analysis suggests that the activation pathway proceeds by unfurling of the super-bundle built from four bundles of helices without separation of the Vt region (840-1066) from the head. According to our mechanism, when activating proteins bind at the strap region a separation of N and C terminal bundles occurs, followed by unfurling of the super-bundle and flattening of the general shape of the molecule, which exposes the interaction sites for binding of auxiliary proteins.
Topics: Actins; Animals; Binding Sites; Chickens; Humans; Protein Binding; Protein Conformation; Vinculin
PubMed: 35006498
DOI: 10.1007/s10930-022-10040-1 -
The Journal of Biological Chemistry Apr 2010The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin...
The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into three-dimensional collagen matrices. Cell motility was investigated in vinculin knockout and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knockout cells were 2-fold more motile on two-dimensional collagen-coated substrates compared with wild-type cells, but 3-fold less invasive in 2.4 mg/ml three-dimensional collagen matrices. Vinculin knockout cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced two-dimensional motility but does not explain their reduced three-dimensional invasiveness. Importantly, vinculin-expressing cells adhered more strongly to collagen and generated 3-fold higher traction forces compared with vinculin knockout cells. Moreover, vinculin-expressing cells were able to migrate into dense (5.8 mg/ml) three-dimensional collagen matrices that were impenetrable for vinculin knockout cells. These findings suggest that vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.
Topics: Animals; Cell Adhesion; Cell Movement; Cells, Cultured; Collagen; Cytoskeleton; Embryo, Mammalian; Extracellular Matrix; Fibroblasts; Mice; Mice, Knockout; Vinculin
PubMed: 20181946
DOI: 10.1074/jbc.M109.087171 -
Nature Communications Dec 2023The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to...
The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding remain unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of the variants did not affect Vcn activation but reduced Vcn loading and altered exchange dynamics, consistent with the loss of directional catch bonding. Expression of Vcn variants perturbed the coordination of subcellular structures and cell migration, establishing key cellular functions for Vcn directional catch bonding.
Topics: Actins; Vinculin; Actin Cytoskeleton; Cell Movement; Protein Binding
PubMed: 38097542
DOI: 10.1038/s41467-023-43779-x -
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... Jan 2023Cells of the cardiovascular system are physiologically exposed to a variety of mechanical forces fundamental for both cardiac development and functions. In this context,...
Cells of the cardiovascular system are physiologically exposed to a variety of mechanical forces fundamental for both cardiac development and functions. In this context, forces generated by actomyosin networks and those transmitted through focal adhesion (FA) complexes represent the key regulators of cellular behaviors in terms of cytoskeleton dynamism, cell adhesion, migration, differentiation, and tissue organization. In this study, we investigated the involvement of FAs on cardiomyocyte differentiation. In particular, vinculin and focal adhesion kinase (FAK) family, which are known to be involved in cardiac differentiation, were studied. Results revealed that differentiation conditions induce an upregulation of both FAK-Tyr397 and vinculin, resulting also in the translocation to the cell membrane. Moreover, the role of mechanical stress in contractile phenotype expression was investigated by applying a uniaxial mechanical stretching (5% substrate deformation, 1 Hz frequency). Morphological evaluation revealed that the cell shape showed a spindle shape and reoriented following the stretching direction. Substrate deformation resulted also in modification of the length and the number of vinculin-positive FAs. We can, therefore, suggest that mechanotransductive pathways, activated through FAs, are highly involved in cardiomyocyte differentiation, thus confirming their role during cytoskeleton rearrangement and cardiac myofilament maturation.
Topics: Focal Adhesions; Vinculin; Cell Adhesion; Cell Membrane; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesion Kinase 1; Cell Differentiation
PubMed: 36768766
DOI: 10.3390/ijms24032444 -
Cells Jun 2022The focal adhesion protein Vinculin (VCL) is ascribed to various cytoplasmic functions; however, its nuclear role has so far been ambiguous. We observed that VCL...
The focal adhesion protein Vinculin (VCL) is ascribed to various cytoplasmic functions; however, its nuclear role has so far been ambiguous. We observed that VCL localizes to the nuclei of mouse primary spermatocytes undergoing first meiotic division. Specifically, VCL localizes along the meiosis-specific structure synaptonemal complex (SC) during prophase I and the centromeric regions, where it remains until metaphase I. To study the role of VCL in meiotic division, we prepared a conditional knock-out mouse (VCL). We found that the VCL male mice were semi-fertile, with a decreased number of offspring compared to wild-type animals. This study of events in late prophase I indicated premature splitting of homologous chromosomes, accompanied by an untimely loss of SCP1. This caused erroneous kinetochore formation, followed by failure of the meiotic spindle assembly and metaphase I arrest. To assess the mechanism of VCL involvement in meiosis, we searched for its possible interacting partners. A mass spectrometry approach identified several putative interactors which belong to the ubiquitin-proteasome pathway (UPS). The depletion of VLC leads to the dysregulation of a key subunit of the proteasome complex in the meiotic nuclei and an altered nuclear SUMOylation level. Taken together, we show for the first time the presence of VCL in the nucleus of spermatocytes and its involvement in proper meiotic progress. It also suggests the direction for future studies regarding the role of VCL in spermatogenesis through regulation of UPS.
Topics: Animals; Centromere; Focal Adhesions; Male; Mice; Proteasome Endopeptidase Complex; Spermatogenesis; Vinculin
PubMed: 35805097
DOI: 10.3390/cells11132013