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Molecular & Cellular Proteomics : MCP Dec 2022Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by immune complex deposition in multiple organs. Despite the severe symptoms caused by it, the...
Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by immune complex deposition in multiple organs. Despite the severe symptoms caused by it, the underlying mechanisms of SLE, especially phosphorylation-dependent regulatory networks remain elusive. Herein, by combining high-throughput phosphoproteomics with bioinformatics approaches, we established the global phosphoproteome landscape of the peripheral blood mononuclear cells from a large number of SLE patients, including the remission stage (SLE_S), active stage (SLE_A), rheumatoid arthritis, and healthy controls, and thus a deep mechanistic insight into SLE signaling mechanism was yielded. Phosphorylation upregulation was preferentially in patients with SLE (SLE_S and SLE_A) compared with healthy controls and rheumatoid arthritis populations, resulting in an atypical enrichment in cell adhesion and migration signatures. Several specifically upregulated phosphosites were identified, and the leukocyte transendothelial migration pathway was enriched in the SLE_A group by expression pattern clustering analysis. Phosphosites identified by 4D-label-free quantification unveiled key kinases and kinase-regulated networks in SLE, then further validated by parallel reaction monitoring. Some of these validated phosphosites including vinculin S275, vinculin S579 and transforming growth factor beta-1-induced transcript 1 S68, primarily were phosphorylation of Actin Cytoskeleton -related proteins. Some predicted kinases including MAP3K7, TBK1, IKKβ, and GSK3β, were validated by Western blot using kinases phosphorylation sites-specific antibodies. Taken together, the study has yielded fundamental insights into the phosphosites, kinases, and kinase-regulated networks in SLE. The map of the global phosphoproteomics enables further understanding of this disease and will provide great help for seeking more potential therapeutic targets for SLE.
Topics: Humans; Vinculin; Leukocytes, Mononuclear; Lupus Erythematosus, Systemic; Protein Serine-Threonine Kinases; Arthritis, Rheumatoid
PubMed: 36309313
DOI: 10.1016/j.mcpro.2022.100434 -
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 -
Canadian Journal of Physiology and... Feb 2015Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin... (Review)
Review
Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) - p21-activated kinase (PAK) - PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist.
Topics: Actins; GTP Phosphohydrolases; Humans; Lung; Muscle Contraction; Muscle, Smooth; Signal Transduction; rhoA GTP-Binding Protein
PubMed: 25531582
DOI: 10.1139/cjpp-2014-0388 -
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 -
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 -
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 -
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 -
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