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Cell Communication and Signaling : CCS Oct 2023Integrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile...
BACKGROUND
Integrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile stress could influence cell differentiation through integrin αVβ3.
METHODS
We inhibited the function of integrin αVβ3 of human mesenchymal stem cells by treating with c(RGDyk). Using cytochalasin D and verteporfin to inhibit polymerization of microfilament and function of nuclear Yes-associated protein (YAP), respectively. For each application, mesenchymal stem cells were loaded by cyclic tensile stress of 10% at 0.5 Hz for 2 h daily. Mesenchymal stem cells were harvested on day 7 post-treatment. Western blotting and quantitative RT-PCR were used to detect the expression of alkaline phosphatase (ALP), RUNX2, β-actin, integrin αVβ3, talin-1, vinculin, FAK, and nuclear YAP. Immunofluorescence staining detected vinculin, actin filaments, and YAP nuclear localization.
RESULTS
Cyclic tensile stress could increase the expression of ALP and RUNX2. Inhibition of integrin αVβ3 activation led to rearrangement of actin filaments and downregulated the expression of ALP, RUNX2 and promoted YAP nuclear localization. When microfilament polymerization was inhibited, ALP, RUNX2, and nuclear YAP nuclear localization decreased. Inhibition of YAP nuclear localization could reduce the expression of ALP and RUNX2.
CONCLUSIONS
Cyclic tensile stress promotes early osteogenesis of human mesenchymal stem cells via the integrin αVβ3-actin filaments axis. YAP nuclear localization participates in this process of human mesenchymal stem cells. Video Abstract.
Topics: Humans; Actin Cytoskeleton; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Integrin alphaVbeta3; Mesenchymal Stem Cells; Osteogenesis; Vinculin
PubMed: 37904190
DOI: 10.1186/s12964-022-01027-7 -
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 -
BioMed Research International 2020To investigate the physical properties of the modified microgroove (MG) and antibacterial nanocoated surfaces. In addition, the biological interactions of the modified...
OBJECTIVES
To investigate the physical properties of the modified microgroove (MG) and antibacterial nanocoated surfaces. In addition, the biological interactions of the modified surfaces with human gingival fibroblasts (HGFs) and the antibacterial activity of the surfaces against were studied.
METHODS
The titanium nitride (TiN) and silver (Ag) coatings were deposited onto the smooth and MG surfaces using magnetron sputtering. A smooth titanium surface (Ti-S) was used as the control. The physicochemical properties including surface morphology, roughness, and hydrophilicity were characterized using scanning electron microscopy, atomic force microscopy, and an optical contact angle analyzer. The "contact guidance" morphology was assessed using confocal laser scanning microscopy. Cell proliferation was analyzed using the Cell Counting Kit-8 assay. The expression level of the main focal adhesion-related structural protein vinculin was compared using quantitative reverse transcription PCR and Western blotting. The antibacterial activity against . was evaluated using the LIVE/DEAD BacLight™ Bacterial Viability Kit.
RESULTS
The Ag and TiN antibacterial nanocoatings were successfully deposited onto the smooth and MG surfaces using magnetron sputtering technology. TiN coating on a grooved surface (TiN-MG) resulted in less nanoroughness and greater surface hydrophilicity than Ag coating on a smooth surface (Ag-S), which was more hydrophobic. Cell proliferation and expression of vinculin were higher on the TiN-MG surface than on the Ag-coated surfaces. Ag-coated surfaces showed the strongest antibacterial activity, followed by TiN-coated surfaces.
CONCLUSION
Nano-Ag coating resulted in good antimicrobial activity; however, the biocompatibility was questionable. TiN nanocoating on an MG surface showed antibacterial properties with an optimal biocompatibility and maintained the "contact guidance" effects for HGFs.
Topics: Anti-Bacterial Agents; Cell Proliferation; Cells, Cultured; Coated Materials, Biocompatible; Equipment Design; Fibroblasts; Gingiva; Humans; Microbial Viability; Nanostructures; Porphyromonas gingivalis; Silver; Surface Properties; Titanium
PubMed: 32626766
DOI: 10.1155/2020/8387574 -
Frontiers in Cell and Developmental... 2021Protein localization in cells has been analyzed by fluorescent labeling using indirect immunofluorescence and fluorescent protein tagging. However, the relationships...
Protein localization in cells has been analyzed by fluorescent labeling using indirect immunofluorescence and fluorescent protein tagging. However, the relationships between the localization of different proteins had not been analyzed using artificial intelligence. Here, we applied convolutional networks for the prediction of localization of the cytoskeletal proteins from the localization of the other proteins. Lamellipodia are one of the actin-dependent subcellular structures involved in cell migration and are mainly generated by the Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 2 (WAVE2) and the membrane remodeling I-BAR domain protein IRSp53. Focal adhesion is another actin-based structure that contains vinculin protein and promotes lamellipodia formation and cell migration. In contrast, microtubules are not directly related to actin filaments. The convolutional network was trained using images of actin filaments paired with WAVE2, IRSp53, vinculin, and microtubules. The generated images of WAVE2, IRSp53, and vinculin were highly similar to their real images. In contrast, the microtubule images generated from actin filament images were inferior without the generation of filamentous structures, suggesting that microscopic images of actin filaments provide more information about actin-related protein localization. Collectively, this study suggests that image translation by the convolutional network can predict the localization of functionally related proteins, and the convolutional network might be used to describe the relationships between the proteins by their localization.
PubMed: 34422790
DOI: 10.3389/fcell.2021.635231 -
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 -
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 -
Journal of Cell Science Dec 2019Podosomes are dynamic adhesion structures formed constitutively by macrophages, dendritic cells and osteoclasts and transiently in a wide variety of cells, such as... (Review)
Review
Podosomes are dynamic adhesion structures formed constitutively by macrophages, dendritic cells and osteoclasts and transiently in a wide variety of cells, such as endothelial cells and megakaryocytes. They mediate numerous functions, including cell-matrix adhesion, extracellular matrix degradation, mechanosensing and cell migration. Podosomes present as micron-sized F-actin cores surrounded by an adhesive ring of integrins and integrin-actin linkers, such as talin and vinculin. In this Review, we highlight recent research that has considerably advanced our understanding of the complex architecture-function relationship of podosomes by demonstrating that the podosome ring actually consists of discontinuous nano-clusters and that the actin network in between podosomes comprises two subsets of unbranched actin filaments, lateral and dorsal podosome-connecting filaments. These lateral and dorsal podosome-connecting filaments connect the core and ring of individual podosomes and adjacent podosomes, respectively. We also highlight recent insights into the podosome cap as a novel regulatory module of actomyosin-based contractility. We propose that these newly identified features are instrumental for the ability of podosomes to generate protrusion forces and to mechanically probe their environment. Furthermore, these new results point to an increasing complexity of podosome architecture and have led to our current view of podosomes as autonomous force generators that drive cell migration.
Topics: Animals; Cell Movement; Endothelial Cells; Humans; Megakaryocytes; Myosin Type II; Podosomes
PubMed: 31836688
DOI: 10.1242/jcs.236828 -
Frontiers in Immunology 2022Activation of the integrin phagocytic receptors CR3 (αβ, CD11b/CD18) and CR4 (αβ, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin...
Activation of the integrin phagocytic receptors CR3 (αβ, CD11b/CD18) and CR4 (αβ, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (α), ITGAX (α) and ITGB2 (β) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored α expression. In general, the expression of α was less responsive to jasplakinolide treatment than α, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αβ expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored α expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αβ and αβ during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.
Topics: Actins; Adaptor Proteins, Signal Transducing; Cell Adhesion Molecules; Co-Repressor Proteins; HL-60 Cells; Humans; Integrin alphaXbeta2; Integrins; Macrophage-1 Antigen; Membrane Proteins; Microfilament Proteins; Neutrophils; Phosphoproteins; RNA, Messenger; Serum Response Factor; Talin; Vinculin
PubMed: 36238292
DOI: 10.3389/fimmu.2022.951280 -
Biomaterials Advances Feb 2023Cells are not only anchored to the extracellular matrix via the focal adhesion complex, the focal adhesion complex also serves as a sensor for force transduction. How...
Cells are not only anchored to the extracellular matrix via the focal adhesion complex, the focal adhesion complex also serves as a sensor for force transduction. How tension influences the structure of focal adhesions is not well understood. Here, we analyse the effect of tension on the location of key focal adhesion proteins, namely vinculin, paxillin and actin. We use micropatterning on gold surfaces to manipulate the cell shape, to create focal adhesions at specific cell areas, and to perform metal-induced energy transfer (MIET) measurements on the patterned cells. MIET resolves the different protein locations with respect to the gold surface with nanometer accuracy. Further, we use drugs influencing the cellular motor protein myosin or mechanosensitive ion channels to get deeper insight into focal adhesions at different tension states. We show here that in particular actin is affected by the rationally tuned force balance. Blocking mechanosensitive ion channels has a particularly high influence on the actin and focal adhesion architecture, resulting in larger focal adhesions with elevated paxillin and vinculin and strongly lowered actin stress fibres. Our results can be explained by a balance of adhesion tension with cellular tension together with ion channel-controlled focal adhesion homeostasis, where high cellular tension leads to an elevation of vinculin and actin, while high adhesion tension lowers these proteins.
Topics: Focal Adhesions; Actins; Paxillin; Cytoskeleton; Vinculin; Cell Shape
PubMed: 36621197
DOI: 10.1016/j.bioadv.2022.213277 -
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