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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 -
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 -
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 -
Investigative Ophthalmology & Visual... Jan 2024Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in...
PURPOSE
Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in scleral remodeling in myopia and its underlying mechanisms.
METHODS
A myopic cell model was induced using hypoxia. Following loss- and gain-of function experiments, the expression of the myofibroblast transdifferentiation-related and collagen production-related factors Forkhead box M1 (FOXM1), APOA1, and methyltransferase-like 3 (METTL3) in the myopic cell model was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The proliferation and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry, respectively. Chromatin immunoprecipitation (ChIP) was employed to examine FOXM1 enrichment in the METTL3 promoter, methylated RNA immunoprecipitation (Me-RIP) to examine the N6-methyladenosine (m6A) modification level of APOA1, and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to examine the binding between METTL3 and APOA1.
RESULTS
Hypoxia-induced human scleral fibroblasts (HSFs) had high APOA1 and FOXM1 expression and low METTL3 expression. FOXM1 knockdown elevated METTL3 expression and downregulated APOA1 expression. FOXM1 was enriched in METTL3 promoter. APOA1 or FOXM1 knockdown or METTL3 overexpression reversed the hypoxia-induced elevation in vinculin, paxillin, and α-smooth muscle actin (α-SMA) levels and apoptosis and the reduction in collagen, type I, alpha 1 (COL1A1) level and cell proliferation in HSFs. METTL3 or YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) knockdown or APOA1 overexpression reversed the impacts of FOXM1 knockdown on vinculin, paxillin, α-SMA, and COL1A1 expression and cell proliferation and apoptosis.
CONCLUSIONS
FOXM1 elevated the m6A methylation level of APOA1 by repressing METTL3 transcription and enhanced APOA1 mRNA stability and transcription by reducing the YTHDF2-recognized m6A methylated transcripts.
Topics: Humans; Apolipoprotein A-I; Paxillin; Vinculin; Myopia, Degenerative; Transcription Factors; Hypoxia; Methyltransferases; Forkhead Box Protein M1; RNA-Binding Proteins
PubMed: 38190128
DOI: 10.1167/iovs.65.1.19 -
Molecular Cancer Dec 2014Loss of cell-cell adhesion is important for the development of cancer invasion and metastasis. Vinculin, a key adhesion-related protein, can affect metastasis and...
BACKGROUND
Loss of cell-cell adhesion is important for the development of cancer invasion and metastasis. Vinculin, a key adhesion-related protein, can affect metastasis and prognosis in several tumours. Here, we determined the biological roles of vinculin in the metastasis of colorectal cancer (CRC) and evaluated its clinical significance as a potential disease biomarker.
METHODS
The expression level of vinculin in CRC cell lines and tissues was measured using Real-Time PCR and western blotting. Moreover, vinculin function was analysed using Transwell assays and in vivo metastasis assays in gain- and loss-of-function experiments. Furthermore, the impact of vinculin together with membrane-bound β-catenin on the prognosis of 228 CRC patients was investigated by immunohistochemistry. Additionally, the expression of epithelial-mesenchymal transition (EMT) indicators was verified by immunohistochemistry in CRC tissues obtained from these patients.
RESULT
Vinculin expression was found to be significantly downregulated in highly metastatic CRC cell lines and metastatic tissues. Both in vitro and in vivo experiments showed that vinculin suppressed invasion, migration and metastasis in CRC cells and that this suppression could be attenuated by silencing β-catenin. Moreover, the expression of vinculin and membrane-bound β-catenin were positively correlated in CRC tissues, and lack of vinculin expression emerged as an independent prognostic factor in patients with CRC. Finally, the loss of vinculin and membrane-bound β-catenin was associated with node metastasis, organ metastasis and expression of EMT indicators.
CONCLUSION
Our results suggest that vinculin may play specific roles in the EMT and metastasis of CRC and that loss of vinculin could be used as a prognostic factor for CRC.
Topics: Animals; Caco-2 Cells; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Down-Regulation; Epithelial-Mesenchymal Transition; Female; HCT116 Cells; HT29 Cells; Humans; Mice; Mice, Nude; Middle Aged; Neoplasm Metastasis; Prognosis; Vinculin; beta Catenin
PubMed: 25496021
DOI: 10.1186/1476-4598-13-263 -
Atherosclerosis Dec 2016Focal adhesions (FA) play an important role in the tissue remodeling and in the maintenance of tissue integrity and homeostasis. Talin and vinculin proteins are among...
BACKGROUND AND AIMS
Focal adhesions (FA) play an important role in the tissue remodeling and in the maintenance of tissue integrity and homeostasis. Talin and vinculin proteins are among the major constituents of FAs contributing to cellular well-being and intercellular communication.
METHODS
Microarray analysis (MA) and qRT-PCR low-density array were implemented to analyze talin-1, talin-2, meta-vinculin and vinculin gene expression in circulating blood and arterial plaque.
RESULTS
All analyzed genes were significantly and consistently downregulated in plaques (carotid, abdominal aortic and femoral regions) compared to left internal thoracic artery (LITA) control. The use of LITA samples as controls for arterial plaque samples was validated using immunohistochemistry by comparing LITA samples with healthy arterial samples from a cadaver. Even though the differences in expression levels between stable and unstable plaques were not statistically significant, we observed further negative tendency in the expression in unstable atherosclerotic plaques. The confocal tissue imaging revealed gradient of talin-1 expression in plaque with reduction close to the vessel lumen. Similar gradient was observed for talin-2 expression in LITA controls but was not detected in plaques. This suggests that impaired tissue mechanostability affects the tissue remodeling and healing capabilities leading to development of unstable plaques.
CONCLUSIONS
The central role of talin and vinculin in cell adhesions suggests that the disintegration of the tissue in atherosclerosis could be partially driven by downregulation of these genes, leading to loosening of cell-ECM interactions and remodeling of the tissue.
Topics: Aged; Aged, 80 and over; Aorta, Abdominal; Aortic Diseases; Carotid Arteries; Carotid Artery Diseases; Case-Control Studies; Cell-Matrix Junctions; Down-Regulation; Female; Femoral Artery; Finland; Fluorescent Antibody Technique; Humans; Male; Microscopy, Confocal; Middle Aged; Peripheral Arterial Disease; Plaque, Atherosclerotic; Polymerase Chain Reaction; RNA, Messenger; Talin; Vascular Remodeling; Vinculin
PubMed: 27816808
DOI: 10.1016/j.atherosclerosis.2016.10.031 -
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 -
PLoS Computational Biology Oct 2023Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which...
Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.
Topics: Focal Adhesions; Vinculin; Actins; Integrins; Extracellular Matrix; Cell Adhesion; Talin
PubMed: 37801464
DOI: 10.1371/journal.pcbi.1011500 -
Anatomical Record (Hoboken, N.J. : 2007) Sep 2014Smooth muscle (SM) tissue is a complex organization of multiple cell types and is regulated by numerous signaling molecules (neurotransmitters, hormones, cytokines,... (Review)
Review
Smooth muscle (SM) tissue is a complex organization of multiple cell types and is regulated by numerous signaling molecules (neurotransmitters, hormones, cytokines, etc.). SM contractile function can be regulated via expression and distribution of the contractile and cytoskeletal proteins, and activation of any of the second messenger pathways that regulate them. Spatial-temporal changes in the contractile, cytoskeletal or regulatory components of SM cells (SMCs) have been proposed to alter SM contractile activity. Ca(2+) sensitization/desensitization can occur as a result of changes at any of these levels, and specific pathways have been identified at all of these levels. Understanding when and how proteins can translocate within the cytoplasm, or to-and-from the plasmalemma and the cytoplasm to alter contractile activity is critical. Numerous studies have reported translocation of proteins associated with the adherens junction and G protein-coupled receptor activation pathways in isolated SMC systems. Specific examples of translocation of vinculin to and from the adherens junction and protein kinase C (PKC) and 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) to and from the plasmalemma in isolated SMC systems but not in intact SM tissues are discussed. Using both isolated SMC systems and SM tissues in parallel to pursue these studies will advance our understanding of both the role and mechanism of these pathways as well as their possible significance for Ca(2+) sensitization in intact SM tissues and organ systems.
Topics: Adherens Junctions; Animals; Calcium; Excitation Contraction Coupling; Humans; Intracellular Signaling Peptides and Proteins; Muscle Contraction; Muscle Proteins; Muscle, Smooth; Myocytes, Smooth Muscle; Phosphoprotein Phosphatases; Protein Kinase C; Protein Transport; Receptors, G-Protein-Coupled; Signal Transduction; Time Factors; Vinculin
PubMed: 25125185
DOI: 10.1002/ar.22970