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Current Biology : CB Oct 2022Filopodia are narrow cell extensions involved in various physiological processes. Integrins mediate filopodia adhesion and likely transmit adhesive force to regulate...
Filopodia are narrow cell extensions involved in various physiological processes. Integrins mediate filopodia adhesion and likely transmit adhesive force to regulate filopodia formation and functions, but the force is extremely weak to study and remains poorly understood. Using integrative tension sensor (ITS), we imaged filopodia adhesive force at the single molecular tension level and investigated the force dynamics and sources. Results show that filopodia integrin tension (FIT) is generated in discrete foci (force nodes) along single filopodia with a spacing of ∼1 μm. Inhibitions of actin polymerization or myosin II activity markedly reduced FIT signals in force nodes at filopodia tips and at filopodia bases, respectively, suggesting differential force sources of FIT in the distal force nodes and proximal ones in filopodia. Using two ITS constructs with different force thresholds for activation, we showed that the molecular force level of FIT is greater at filopodia bases than that at filopodia tips. We also tested the role of vinculin and myosin X in the FIT transmission. With vinculin knockout in cells, filopodia and associated force nodes were still formed normally, suggesting that vinculin is dispensable for the formation of filopodia and force nodes. However, vinculin is indeed required for the transmission of strong FIT (capable of rupturing DNA in a shear conformation), as the strong FIT vanished in filopodia with vinculin knockout. Co-imaging of FIT and myosin X shows no apparent co-localization, demonstrating that myosin X is not directly responsible for generating FIT, despite its prominent role in filopodium elongation.
Topics: Pseudopodia; Vinculin; Integrins; Actins; Adhesives; Myosins
PubMed: 36084647
DOI: 10.1016/j.cub.2022.08.040 -
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 -
PLoS Computational Biology Apr 2013Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin...
Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion.
Topics: Actin Cytoskeleton; Actins; Animals; Binding Sites; Chickens; Focal Adhesions; Humans; Molecular Dynamics Simulation; Protein Binding; Protein Structure, Tertiary; Vinculin
PubMed: 23633939
DOI: 10.1371/journal.pcbi.1002995 -
Molecular Biology of the Cell Sep 2022Vinculin is a protein found in both focal adhesions (FAs) and adherens junctions (AJs) which regulates actin connectivity to these structures. Many studies have...
Vinculin is a protein found in both focal adhesions (FAs) and adherens junctions (AJs) which regulates actin connectivity to these structures. Many studies have demonstrated that mechanical perturbations of cells result in enhanced recruitment of vinculin to FAs and/or AJs. Likewise, many other studies have shown "cross-talk" between FAs and AJs. Vinculin itself has been suggested to be a probable regulator of this adhesion cross-talk. In this study we used MDCK as a model system of epithelia, developing cell lines in which vinculin recruitment was reduced or enhanced at AJs. Careful analysis of these cells revealed that perturbing vinculin recruitment to AJs resulted in a reduction of detectable FAs. Interestingly the cross-talk between these two structures was not due to a limited pool of vinculin, as increasing expression of vinculin did not rescue FA formation. Instead, we demonstrate that vinculin translocation between AJs and FAs is necessary for actin cytoskeleton rearrangements that occur during cell migration, which is necessary for large, well-formed FAs. Last, we show using a wound assay that collective cell migration is similarly hindered when vinculin recruitment is reduced or enhanced at AJs, highlighting that vinculin translocation between each compartment is necessary for efficient collective migration.
Topics: Adherens Junctions; Catenins; Cell Adhesion; Focal Adhesions; Vinculin; alpha Catenin
PubMed: 35921161
DOI: 10.1091/mbc.E22-02-0071 -
Nature Cell Biology Jan 2017Multicellularity in animals requires dynamic maintenance of cell-cell contacts. Intercellularly ligated cadherins recruit numerous proteins to form supramolecular...
Multicellularity in animals requires dynamic maintenance of cell-cell contacts. Intercellularly ligated cadherins recruit numerous proteins to form supramolecular complexes that connect with the actin cytoskeleton and support force transmission. However, the molecular organization within such structures remains unknown. Here we mapped protein organization in cadherin-based adhesions by super-resolution microscopy, revealing a multi-compartment nanoscale architecture, with the plasma-membrane-proximal cadherin-catenin compartment segregated from the actin cytoskeletal compartment, bridged by an interface zone containing vinculin. Vinculin position is determined by α-catenin, and following activation, vinculin can extend ∼30 nm to bridge the cadherin-catenin and actin compartments, while modulating the nanoscale positions of the actin regulators zyxin and VASP. Vinculin conformational activation requires tension and tyrosine phosphorylation, regulated by Abl kinase and PTP1B phosphatase. Such modular architecture provides a structural framework for mechanical and biochemical signal integration by vinculin, which may differentially engage cadherin-catenin complexes with the actomyosin machinery to regulate cell adhesions.
Topics: Actin Cytoskeleton; Actins; Animals; Biomarkers; Biomechanical Phenomena; Cadherins; Cell Adhesion; Cell Membrane; Cytoskeletal Proteins; Dogs; Intercellular Junctions; Interferometry; Madin Darby Canine Kidney Cells; Mice; Microscopy; Nanoparticles; Phosphorylation; Signal Transduction; Vinculin; alpha Catenin
PubMed: 27992406
DOI: 10.1038/ncb3456 -
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 -
PloS One 2012Cell migration is of paramount importance to organism development and maintenance as well as multiple pathological processes, including cancer metastasis. The RhoGTPases...
Cell migration is of paramount importance to organism development and maintenance as well as multiple pathological processes, including cancer metastasis. The RhoGTPases Rac1 and RhoA are indispensable for cell migration as they regulate cell protrusion, cell-extracellular matrix (ECM) interactions and force transduction. However, the consequences of their activity at a molecular level within the cell remain undetermined. Using a combination of FRET, FRAP and biochemical analyses we show that the interactions between the focal adhesion proteins vinculin and paxillin, as well as the closely related family member Hic-5 are spatially and reciprocally regulated by the activity of Rac1 and RhoA. Vinculin in its active conformation interacts with either paxillin or Hic-5 in adhesions in response to Rac1 and RhoA activation respectively, while inactive vinculin interacts with paxillin in the membrane following Rac1 inhibition. Additionally, Rac1 specifically regulates the dynamics of paxillin as well as its binding partner and F-actin interacting protein actopaxin (α-parvin) in adhesions. Furthermore, FRET analysis of protein:protein interactions within cell adhesions formed in 3D matrices revealed that, in contrast to 2D systems vinculin interacts preferentially with Hic-5. This study provides new insight into the complexity of cell-ECM adhesions in both 2D and 3D matrices by providing the first description of RhoGTPase-coordinated protein:protein interactions in a cellular microenvironment. These data identify discrete roles for paxillin and Hic-5 in Rac1 and RhoA-dependent cell adhesion formation and maturation; processes essential for productive cell migration.
Topics: 3T3 Cells; Animals; Cell Adhesion; Cell Movement; Cells, Cultured; Cytoskeletal Proteins; DNA-Binding Proteins; Focal Adhesions; LIM Domain Proteins; Mice; Paxillin; Signal Transduction; Vinculin; rac1 GTP-Binding Protein; rhoA GTP-Binding Protein
PubMed: 22629471
DOI: 10.1371/journal.pone.0037990 -
Proceedings of the National Academy of... Jun 2013Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct...
Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculin-null cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (V(H)) and tail domains. V(H) enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with VH, implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; V(H) has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or V(H), correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.
Topics: Actins; Animals; Blotting, Western; Cells, Cultured; Cytoskeleton; Embryo, Mammalian; Extracellular Matrix; Fibroblasts; Fluorescence Recovery After Photobleaching; Focal Adhesions; Green Fluorescent Proteins; Integrins; Mice; Mice, Knockout; Microscopy, Fluorescence; Models, Biological; Protein Binding; Stress, Mechanical; Talin; Vinculin
PubMed: 23716647
DOI: 10.1073/pnas.1216209110 -
Journal of Cell Science Mar 2021Hippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6 and LIMD1 have each been identified as being required for tension-dependent inhibition...
Hippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6 and LIMD1 have each been identified as being required for tension-dependent inhibition of the Hippo pathway LATS kinases and their recruitment to adherens junctions, but the relationship between TRIP6 and LIMD1 was unknown. Using siRNA-mediated gene knockdown, we show that TRIP6 is required for LIMD1 localization to adherens junctions, whereas LIMD1 is not required for TRIP6 localization. TRIP6, but not LIMD1, is also required for the recruitment of vinculin and VASP to adherens junctions. Knockdown of TRIP6 or vinculin, but not of LIMD1, also influences the localization of myosin and F-actin. In TRIP6 knockdown cells, actin stress fibers are lost apically but increased basally, and there is a corresponding increase in the recruitment of vinculin and VASP to basal focal adhesions. Our observations identify a role for TRIP6 in organizing F-actin and maintaining tension at adherens junctions that could account for its influence on LIMD1 and LATS. They also suggest that focal adhesions and adherens junctions compete for key proteins needed to maintain attachments to contractile F-actin.
Topics: Actin Cytoskeleton; Actins; Adherens Junctions; Cytoskeleton; Focal Adhesions; Vinculin
PubMed: 33558314
DOI: 10.1242/jcs.247866 -
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