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PloS One 2017The formation of multicellular tissues during development is governed by mechanical forces that drive cell shape and tissue architecture. Protein complexes at sites of...
The formation of multicellular tissues during development is governed by mechanical forces that drive cell shape and tissue architecture. Protein complexes at sites of adhesion to the extracellular matrix (ECM) and cell neighbors, not only transmit these mechanical forces, but also allow cells to respond to changes in force by inducing biochemical feedback pathways. Such force-induced signaling processes are termed mechanotransduction. Vinculin is a central protein in mechanotransduction that in both integrin-mediated cell-ECM and cadherin-mediated cell-cell adhesions mediates force-induced cytoskeletal remodeling and adhesion strengthening. Vinculin was found to be important for the integrity and remodeling of epithelial tissues in cell culture models and could therefore be expected to be of broad importance in epithelial morphogenesis in vivo. Besides a function in mouse heart development, however, the importance of vinculin in morphogenesis of other vertebrate tissues has remained unclear. To investigate this further, we knocked out vinculin functioning in zebrafish, which contain two fully functional isoforms designated as vinculin A and vinculin B that both show high sequence conservation with higher vertebrates. Using TALEN and CRISPR-Cas gene editing technology we generated vinculin-deficient zebrafish. While single vinculin A mutants are viable and able to reproduce, additional loss of zygotic vinculin B was lethal after embryonic stages. Remarkably, vinculin-deficient embryos do not show major developmental defects, apart from mild pericardial edemas. These results lead to the conclusion that vinculin is not of broad importance for the development and morphogenesis of zebrafish tissues.
Topics: Animals; Cell Adhesion; Dogs; Embryonic Development; Extracellular Matrix; Gene Expression Regulation, Developmental; Gene Knockout Techniques; HEK293 Cells; Humans; Madin Darby Canine Kidney Cells; Mechanotransduction, Cellular; Vinculin; Zebrafish; Zebrafish Proteins
PubMed: 28767718
DOI: 10.1371/journal.pone.0182278 -
Scientific Reports Feb 2020Neutrophils are innate immune effector cells that traffic from the circulation to extravascular sites of inflammation. β2 integrins are important mediators of the...
Neutrophils are innate immune effector cells that traffic from the circulation to extravascular sites of inflammation. β2 integrins are important mediators of the processes involved in neutrophil recruitment. Although neutrophils express the cytoskeletal protein vinculin, they do not form mature focal adhesions. Here, we characterize the role of vinculin in β2 integrin-dependent neutrophil adhesion, migration, mechanosensing, and recruitment. We observe that knockout of vinculin attenuates, but does not completely abrogate, neutrophil adhesion, spreading, and crawling under static conditions. However, we also found that vinculin deficiency does not affect these behaviors in the presence of forces from fluid flow. In addition, we identify a role for vinculin in mechanosensing, as vinculin-deficient neutrophils exhibit attenuated spreading on stiff, but not soft, substrates. Consistent with these findings, we observe that in vivo neutrophil recruitment into the inflamed peritoneum of mice remains intact in the absence of vinculin. Together, these data suggest that while vinculin regulates some aspects of neutrophil adhesion and spreading, it may be dispensable for β2 integrin-dependent neutrophil recruitment in vivo.
Topics: Animals; CD18 Antigens; Cell Adhesion; Cells, Cultured; Mechanotransduction, Cellular; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Neutrophils; Vinculin
PubMed: 32034208
DOI: 10.1038/s41598-020-58882-y -
Biophysical Journal Jan 2023Focal adhesions (FAs) transmit force and mediate mechanotransduction between cells and the matrix. Previous studies revealed that integrin-transmitted force is critical...
Focal adhesions (FAs) transmit force and mediate mechanotransduction between cells and the matrix. Previous studies revealed that integrin-transmitted force is critical to regulate FA formation. As vinculin is a prominent FA protein implicated in integrin tension transmission, this work studies the relation among integrin tensions (force), vinculin (protein), and FA formation (structure) by integrin tension manipulation, force visualization and vinculin knockout (KO). Two DNA-based integrin tension tools are adopted: tension gauge tether (TGT) and integrative tension sensor (ITS), with TGT restricting integrin tensions under a designed T (tension tolerance) value and ITS visualizing integrin tensions above the T value by fluorescence. Results show that large FAs (area >1 μm) were formed on the TGT surface with T of 54 pN but not on those with lower T values. Time-series analysis of FA formation shows that focal complexes (area <0.5 μm) appeared on all TGT surfaces 20 min after cell plating, but only matured to large FAs on TGT with T of 54 pN. Next, we tested FA formation in vinculin KO cells on TGT surfaces. Surprisingly, the T value of TGT required for large FA formation is drastically decreased to 23 pN. To explore the cause, we visualized integrin tensions in both wild-type and vinculin KO cells using ITS. The results showed that integrin tensions in FAs of wild-type cells frequently activate ITS with T of 54 pN. With vinculin KO, however, integrin tensions in FAs became lower and unable to activate 54 pN ITS. Force signal intensities of integrin tensions reported by 33 and 43 pN ITS were also significantly reduced with vinculin KO, suggesting that vinculin is essential to transmit high-level integrin tensions and involved in transmitting intermediate-level integrin tensions in FAs. However, the high-level integrin tensions transmitted by vinculin are not required by FA formation.
Topics: Integrins; Focal Adhesions; Cell Adhesion; Vinculin; Mechanotransduction, Cellular
PubMed: 36352785
DOI: 10.1016/j.bpj.2022.11.013 -
The Journal of Cell Biology Jan 2020Talin, vinculin, and paxillin are core components of the dynamic link between integrins and actomyosin. Here, we study the mechanisms that mediate their activation and...
Talin, vinculin, and paxillin are core components of the dynamic link between integrins and actomyosin. Here, we study the mechanisms that mediate their activation and association using a mitochondrial-targeting assay, structure-based mutants, and advanced microscopy. As expected, full-length vinculin and talin are autoinhibited and do not interact with each other. However, contrary to previous models that propose a critical role for forces driving talin-vinculin association, our data show that force-independent relief of autoinhibition is sufficient to mediate their tight interaction. We also found that paxillin can bind to both talin and vinculin when either is inactive. Further experiments demonstrated that adhesions containing paxillin and vinculin can form without talin following integrin activation. However, these are largely deficient in exerting traction forces to the matrix. Our observations lead to a model whereby paxillin contributes to talin and vinculin recruitment into nascent adhesions. Activation of the talin-vinculin axis subsequently leads to the engagement with the traction force machinery and focal adhesion maturation.
Topics: Actin Cytoskeleton; Animals; Cells, Cultured; Fibroblasts; Focal Adhesions; Mice; Mice, Inbred C57BL; Mice, Knockout; Paxillin; Protein Binding; Stress, Mechanical; Talin; Vinculin
PubMed: 31816055
DOI: 10.1083/jcb.201903134 -
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 -
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 -
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 -
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 -
Journal of Cell Science Jan 2009Cells govern tissue shape by exerting highly regulated forces at sites of matrix adhesion. As the major force-bearing adhesion-receptor protein, integrins have a central... (Review)
Review
Cells govern tissue shape by exerting highly regulated forces at sites of matrix adhesion. As the major force-bearing adhesion-receptor protein, integrins have a central role in how cells sense and respond to the mechanics of their surroundings. Recent studies have shown that a key aspect of mechanotransduction is the cycle by which integrins bind to the matrix at the leading cell edge, attach to the cytoskeleton, transduce mechanical force, aggregate in the plasma membrane as part of increasingly strengthened adhesion complexes, unbind and, ultimately, are recycled. This mechanical cycle enables the transition from early complexes to larger, more stable adhesions that can then rapidly release. Within this mechanical cycle, integrins themselves exhibit intramolecular conformational change that regulates their binding affinity and may also be dependent upon force. How the cell integrates these dynamic elements into a rigidity response is not clear. Here, we focus on the steps in the integrin mechanical cycle that are sensitive to force and closely linked to integrin function, such as the lateral alignment of integrin aggregates and related adhesion components.
Topics: Animals; Cell Adhesion; Cell Movement; Cytoskeleton; Humans; Integrins; Mechanotransduction, Cellular; Phosphorylation; Stress, Mechanical; Syndecans; Talin; Vinculin
PubMed: 19118210
DOI: 10.1242/jcs.042127 -
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