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Orthopaedic Surgery Feb 2023To explore the potential effect of three allogenic bone substitute configurations on the viability, adhesion, and spreading of osteoblasts in vitro.
OBJECTIVE
To explore the potential effect of three allogenic bone substitute configurations on the viability, adhesion, and spreading of osteoblasts in vitro.
METHODS
Freeze-dried cortical bone were ground and fractions were divided into three groups with different sizes and shapes, defined as bone fiber (0.1 mm × 0.1 mm × 3 mm), bone powder (0.45-0.9 mm), and bone granule group (3-6 mm). MC3T3-E1 cells were divided and co-cultured within groups to induce cell adhesion. The configuration of allogenic bone was captured by scanning electron microscopy and confocal laser scanning microscopy, and substrate roughness values were quantified. Cell adhesion rate was assessed using the hemocyte counting method, cell viability was determined by CCK-8 assay and live/dead staining, and cell morphology was visualized by Phalloidin and DAPI, and the mRNA expression of adhesion-related gene (vinculin) of different substitutes were determined with quantitative real-time polymerase chain reaction.
RESULTS
The roughness values of bone fiber, bone powder, and bone granule group were 1.878 μm (1.578-2.415 μm), 5.066 μm (3.891-6.162 μm), and 0.860 μm (0.801-1.452 μm), respectively (bone powder group compared with bone granule group, H = 18.015, P < 0.001). Similar OD values of all groups in CCK-8 assay indicated good biocompatibility of these substitutes (bone fiber, 0.201 ± 0.004; bone powder, 0.206 ± 0.008; bone granule group, 0.197 ± 0.006; and the control group, 0.202 ± 0.016, F = 0.7152, P > 0.05). In addition, representative cell adhesion rates at 24 h showed significantly lower cell adhesion rate in bone fiber group (20.3 ± 1.6%) compared to bone powder (29.3 ± 4.4%) and bone granule group (27.3 ± 3.2%) (F = 10.51,P = 0.009 and P = 0.034, respectively), but there was no significant difference between the latter two groups (P > 0.05). Interestingly, the expression of vinculin mRNA steadily decreased in a time-dependent manner. The vinculin expression reached its peak at 6 h in each group, and the vinculin levels in bone fiber, bone powder, and bone granule group were 2.119 ± 0.052, 3.842 ± 0.108, and 3.585 ± 0.068 times higher than those in the control group, respectively (F = 733.643, all P < 0.001). Meanwhile, there was a significant difference in the expression of target gene between bone powder and bone granule group (P = 0.006).
CONCLUSION
All allogenic bone substitutes presented an excellent cell viability. Moreover, bone powder and bone granule group were more likely to promote cell adhesion and spreading compared to bone fiber group.
Topics: Humans; Cell Adhesion; Bone Substitutes; Vinculin; Powders; Osteoblasts; RNA, Messenger; Hematopoietic Stem Cell Transplantation; Cell Proliferation
PubMed: 36453151
DOI: 10.1111/os.13395 -
Cellular and Molecular Life Sciences :... Feb 2014Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins,...
Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.
Topics: Amino Acid Motifs; Animals; Biomechanical Phenomena; Cell Adhesion; Cell Line; Crk-Associated Substrate Protein; Fibroblasts; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Mice; Peptides; Phosphorylation; Protein Binding; Protein Interaction Maps; Vinculin; src Homology Domains
PubMed: 23974298
DOI: 10.1007/s00018-013-1450-x -
International Journal of Molecular... Oct 2022α-catulin, together with vinculin and the α-catenins, belongs to the vinculin family of proteins, best known for their actin-filament binding properties and crucial... (Review)
Review
α-catulin, together with vinculin and the α-catenins, belongs to the vinculin family of proteins, best known for their actin-filament binding properties and crucial roles in cell-cell and cell-substrate adhesion. In the past few years, an array of binding partners for α-catulin have surfaced, which has shed new light on the possible functions of this protein. Despite all this information, the molecular basis of how α-catulin acts in cells and controls a wide variety of signals during morphogenesis, tissue homeostasis, and cancer progression remains elusive. This review aims to highlight recent discoveries on how α-catulin is involved in a broad range of diverse biological processes with an emphasis on cancer progression.
Topics: Actins; Catenins; Family; Homeostasis; Humans; Neoplasms; Vinculin; alpha Catenin
PubMed: 36233261
DOI: 10.3390/ijms231911962 -
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 -
Journal of Thrombosis and Haemostasis :... Oct 2010Summary.
UNLABELLED
Summary.
BACKGROUND
Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F-actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin's function in platelets is unknown.
OBJECTIVE
To determine whether vinculin is required for the functions of platelets and their major integrin, α(IIb) β(3) .
METHODS
The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4-Cre mice. Platelet and integrin functions were studied in vivo and ex vivo.
RESULTS
Vinculin was undetectable in platelets from Vcl fl/fl Cre(+) mice, as determined by immunoblotting and fluorescence microscopy. Vinculin-deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on α(IIb) β(3) with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin-deficient platelets displayed normal agonist-induced fibrinogen binding to α(IIb) β(3) , aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin-deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin-deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild-type littermates in response to carotid artery injury with FeCl(3) .
CONCLUSION
Despite promoting membrane cytoskeleton integrity when mechanical force is applied to α(IIb) β(3) , vinculin is not required for the traditional functions of α(IIb) β(3) or the platelet actin cytoskeleton.
Topics: Actins; Animals; Blood Platelets; Cell Lineage; Collagen; Cytoskeleton; Fibrinogen; Gene Deletion; Megakaryocytes; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Vinculin
PubMed: 20670372
DOI: 10.1111/j.1538-7836.2010.03998.x -
European Journal of Cell Biology 2011Vinculin, discovered in 1979 (Geiger, 1979), is an adapter protein with binding sites for more than 15 proteins. Biochemical and structural analyses have contributed to... (Review)
Review
Vinculin, discovered in 1979 (Geiger, 1979), is an adapter protein with binding sites for more than 15 proteins. Biochemical and structural analyses have contributed to detailed knowledge about potential binding partners and the understanding of how their binding may be regulated. Despite all this information the molecular basis of how vinculin acts in cells and controls a wide variety of signals remains elusive. This review aims to highlight recent discoveries with an emphasis on how vinculin is involved in the coordination of a network of signals.
Topics: Animals; Cell Adhesion; Cell Growth Processes; Humans; Mice; Mice, Knockout; Protein Binding; Signal Transduction; Vinculin
PubMed: 20655620
DOI: 10.1016/j.ejcb.2010.06.007 -
The Journal of Biological Chemistry Mar 2013Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13...
Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1-R13) organized into a compact cluster of four-helix bundles (R2-R4) within a linear chain of five-helix bundles. Nine of the bundles contain vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit vinculin to stabilize the complex.
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Crystallography, X-Ray; Focal Adhesions; Human Umbilical Vein Endothelial Cells; Humans; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Mice; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; Protein Structure, Secondary; Talin; Vinculin
PubMed: 23389036
DOI: 10.1074/jbc.M112.438119 -
ELife Oct 2022Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by...
Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by peristalsis of its variable content. Using the intestinal epithelium, we investigate the role of vinculin, one of the best characterised mechanoeffectors, which functions in both cadherin and integrin adhesion complexes. We discovered that vinculin regulates cell fate decisions, by preventing precocious activation and differentiation of intestinal progenitors into absorptive cells. It achieves this in concert with α-catenin at sites of cadherin adhesion, rather than as part of integrin function. Following asymmetric division of the stem cell into a stem cell and an enteroblast (EB), the two cells initially remain connected by adherens junctions, where vinculin is required, only on the EB side, to maintain the EB in a quiescent state and inhibit further divisions of the stem cell. By manipulating cell tension, we show that vinculin recruitment to adherens junction regulates EB activation and numbers. Consequently, removing vinculin results in an enlarged gut with improved resistance to starvation. Thus, mechanical regulation at the contact between stem cells and their progeny is used to control tissue cell number.
Topics: Animals; alpha Catenin; Vinculin; Drosophila; Enterocytes; Actins; Cadherins; Integrins; Homeostasis
PubMed: 36269226
DOI: 10.7554/eLife.72836 -
Open Biology Jun 2013Mesenchymal cell migration is important for embryogenesis and tissue regeneration. In addition, it has been implicated in pathological conditions such as the... (Review)
Review
Mesenchymal cell migration is important for embryogenesis and tissue regeneration. In addition, it has been implicated in pathological conditions such as the dissemination of cancer cells. A characteristic of mesenchymal-migrating cells is the presence of actin stress fibres, which are thought to mediate myosin II-based contractility in close cooperation with associated focal adhesions. Myosin II-based contractility regulates various cellular activities, which occur in a spatial and temporal manner to achieve directional cell migration. These myosin II-based activities involve the maturation of integrin-based adhesions, generation of traction forces, establishment of the front-to-back polarity axis, retraction of the trailing edge, extracellular matrix remodelling and mechanotransduction. Growing evidence suggests that actin stress fibre subtypes, namely dorsal stress fibres, transverse arcs and ventral stress fibres, could provide this spatial and temporal myosin II-based activity. Consistent with their functional differences, recent studies have demonstrated that the molecular composition of actin stress fibre subtypes differ significantly. This present review focuses on the current view of the molecular composition of actin stress fibre subtypes and how these fibre subtypes regulate mesenchymal cell migration.
Topics: Actins; Cell Movement; Extracellular Matrix; Humans; Mesenchymal Stem Cells; Myosin Type II; Stress Fibers; Vinculin
PubMed: 23782578
DOI: 10.1098/rsob.130001 -
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