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Methods in Molecular Biology (Clifton,... 2020Adhesion is defined as a fundamental ability of the adherent cells isolated from the multicellular organisms to attach to an extracellular matrix or another cell, and it...
Adhesion is defined as a fundamental ability of the adherent cells isolated from the multicellular organisms to attach to an extracellular matrix or another cell, and it takes a key role in a wide variety of the important molecular mechanisms such as cell communication, regulation, differentiation, migration, wound healing, immune response, inflammation, embryonic development, and maintenance of tissues. Adhesion assays therefore provide information about not only the interactions between cell-cell and cell-extracellular matrix but also the other cellular events. In this chapter, it was desired to describe an easily applicable cell-extracellular matrix adhesion assay by explaining the purposes of each experimental step because it seems, to the best of our knowledge, that there is no a complete protocol that explains well the purposes and the related molecular mechanisms of the experimental steps though there are many well-written protocols for adhesion assays.
Topics: Cell Adhesion; Cell Communication; Cell Line; Extracellular Matrix; Humans; Keratinocytes
PubMed: 31201684
DOI: 10.1007/7651_2019_246 -
Digestive Diseases and Sciences Mar 2020Irritable bowel syndrome (IBS) is an extremely common and often very debilitating chronic functional gastrointestinal disorder. Despite its prevalence, significant... (Review)
Review
Irritable bowel syndrome (IBS) is an extremely common and often very debilitating chronic functional gastrointestinal disorder. Despite its prevalence, significant associated healthcare costs, and quality-of-life issues for affected individuals, our understanding of its etiology remained limited. However, it is now evident that microbial factors play key roles in IBS pathophysiology. Acute gastroenteritis following exposure to pathogens can precipitate the development of IBS, and studies have demonstrated changes in the gut microbiome in IBS patients. These changes may explain some of the symptoms of IBS, including visceral hypersensitivity, as gut microbes exert effects on the host immune system and gut barrier function, as well as the brain-gut axis. Microbial differences also appear to underlie the two main functional categories of IBS: diarrhea-predominant IBS (IBS-D) is associated with small intestinal bacterial overgrowth, which can be diagnosed by a positive hydrogen breath test, and constipation-predominant IBS (IBS-C) is associated with increased levels of methanogenic archaea, which can be diagnosed by a positive methane breath test. Mechanistically, the pathogens that cause gastroenteritis and trigger subsequent IBS development produce a common toxin, cytolethal distending toxin B (CdtB), and antibodies raised against CdtB cross-react with the cytoskeletal protein vinculin and impair gut motility, facilitating bacterial overgrowth. In contrast, methane gas slows intestinal contractility, which may facilitate the development of constipation. While antibiotics and dietary manipulations have been used to relieve IBS symptoms, with varying success, elucidating the specific mechanisms by which gut microbes exert their effects on the host may allow the development of targeted treatments that may successfully treat the underlying causes of IBS.
Topics: Anti-Bacterial Agents; Case-Control Studies; Constipation; Diarrhea; Gastrointestinal Microbiome; Humans; Irritable Bowel Syndrome; Randomized Controlled Trials as Topic
PubMed: 32026278
DOI: 10.1007/s10620-020-06109-5 -
American Journal of Physiology. Cell... Apr 2020This review analyzes data concerning patients with cardiomyopathies or skeletal myopathies associated with a variation in the intermediate filament (IF) synemin gene (),... (Review)
Review
This review analyzes data concerning patients with cardiomyopathies or skeletal myopathies associated with a variation in the intermediate filament (IF) synemin gene (), also referred to as desmuslin (). Molecular studies demonstrate that synemin copolymerizes with desmin and vimentin IF and interacts with vinculin, α-actinin, α-dystrobrevin, dystrophin, talin, and zyxin. It has been found that synemin is an A-kinase-anchoring protein (AKAP) that anchors protein kinase A (PKA) and modulates the PKA-dependent phosphorylation of several cytoskeletal substrates such as desmin. Because several IF proteins, including desmin, have been implicated in human genetic disorders such as dominant or recessive congenital and adult-onset myopathy, synemin becomes a significant candidate for cardiac and skeletal myopathies of unknown etiology. Because is a new candidate gene that displays numerous sequence polymorphisms, in this review, we summarize the genetic and clinical literature about mutations. Protein-changing variants (missense, frameshifts, nonsense) were further evaluated based on structural modifications and amino acid interactions. We present in silico modeling of helical salt-bridges between residues to evaluate the impact of the synemin networks crucial to interactions with cytoskeletal proteins. Finally, a discussion is featured regarding certain variants that may contribute to the disease state.
Topics: Animals; Cytoskeleton; Heart; Humans; Intermediate Filament Proteins; Intermediate Filaments; Muscle Proteins; Muscular Diseases
PubMed: 32023076
DOI: 10.1152/ajpcell.00485.2019 -
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 -
Frontiers in Oncology 2022The onset and progression of cancer are strongly associated with the dissipation of adhesion forces between cancer cells, thus facilitating their incessant attachment...
The onset and progression of cancer are strongly associated with the dissipation of adhesion forces between cancer cells, thus facilitating their incessant attachment and detachment from the extracellular matrix (ECM) to move toward metastasis. During this process, cancer cells undergo mechanical stresses and respond to these stresses with membrane deformation while inducing protrusions to invade the surrounding tissues. Cellular response to mechanical forces is inherently related to the reorganization of the cytoskeleton, the dissipation of cell-cell junctions, and the adhesion to the surrounding ECM. Moreover, the role of focal adhesion proteins, and particularly the role of vinculin in cell attachment and detachment during migration, is critical, indicating the tight cell-ECM junctions, which favor or inhibit the metastatic cascade. The biomechanical analysis of these sequences of events may elucidate the tumor progression and the potential of cancer cells for migration and metastasis. In this work, we focused on the evaluation of the spreading rate and the estimation of the adhesion strength between breast cancer cells and ECM prior to and post-treatment with anti-tumor agents. Specifically, different tamoxifen concentrations were used for ER breast cancer cells, while even concentrations of trastuzumab and pertuzumab were used for HER2 cells. Analysis of cell stiffness indicated an increased elastic Young's modulus post-treatment in both MCF-7 and SKBR-3 cells. The results showed that the post-treatment spreading rate was significantly decreased in both types of breast cancer, suggesting a lower metastatic potential. Additionally, treated cells required greater adhesion forces to detach from the ECM, thus preventing detachment events of cancer cells from the ECM, and therefore, the probability of cell motility, migration, and metastasis was confined. Furthermore, post-detachment and post-treatment vinculin levels were increased, indicating tighter cell-ECM junctions, hence limiting the probability of cell detachment and, therefore, cell motility and migration.
PubMed: 36052248
DOI: 10.3389/fonc.2022.811508 -
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 -
APL Bioengineering Jun 2022Two meters of DNA in each of our cells must be protected against many types of damage. Mechanoprotection is increasingly understood to be conferred by the nuclear lamina... (Review)
Review
Two meters of DNA in each of our cells must be protected against many types of damage. Mechanoprotection is increasingly understood to be conferred by the nuclear lamina of intermediate filament proteins, but very different patterns of expression and regulation between different cells and tissues remain a challenge to comprehend and translate into applications. We begin with a tutorial style presentation of "tissue blueprints" of lamin expression including single-cell RNA sequencing in major public datasets. Lamin-A, C profiles appear strikingly similar to those for the mechanosensitive factors Vinculin, Yap1, and Piezo1, whereas datasets for lamin-B1 align with and predict regulation by the cell cycle transcription factor, FOXM1, and further predict poor survival across multiple cancers. Various experiments support the distinction between the lamin types and add mechanistic insight into the mechano-regulation of lamin-A, C by both matrix elasticity and externally imposed tissue strain. Both A- and B-type lamins, nonetheless, protect the nucleus from rupture and damage. Ultimately, for mechanically active tissue constructs and organoids as well as cell therapies, lamin levels require particular attention as they help minimize nuclear damage and defects in a cell cycle.
PubMed: 35719698
DOI: 10.1063/5.0080392 -
Frontiers in Molecular Biosciences 2019The advent of cell-cell and cell-extracellular adhesion enabled cells to interact in a coherent manner, forming larger structures and giving rise to the development of... (Review)
Review
The advent of cell-cell and cell-extracellular adhesion enabled cells to interact in a coherent manner, forming larger structures and giving rise to the development of tissues, organs and complex multicellular life forms. The development of such organisms required tight regulation of dynamic adhesive structures by signaling pathways that coordinate cell attachment. Integrin-mediated adhesion to the extracellular matrix provides cells with support, survival signals and context-dependent cues that enable cells to run different cellular programs. One mysterious aspect of the process is how hundreds of proteins assemble seemingly spontaneously onto the activated integrin. An emerging concept is that adhesion assembly is regulated by autoinhibition of key proteins, a highly dynamic event that is modulated by a variety of signaling events. By enabling precise control of the activation state of proteins, autoinhibition enables localization of inactive proteins and the formation of pre-complexes. In response to the correct signals, these proteins become active and interact with other proteins, ultimately leading to development of cell-matrix junctions. Autoinhibition of key components of such adhesion complexes-including core components integrin, talin, vinculin, and FAK and important peripheral regulators such as RIAM, Src, and DLC1-leads to a view that the majority of proteins involved in complex assembly might be regulated by intramolecular interactions. Autoinhibition is relieved via multiple different signals including post-translation modification and proteolysis. More recently, mechanical forces have been shown to stabilize and increase the lifetimes of active conformations, identifying autoinhibition as a means of encoding mechanosensitivity. The complexity and scope for nuanced adhesion dynamics facilitated via autoinhibition provides numerous points of regulation. In this review, we discuss what is known about this mode of regulation and how it leads to rapid and tightly controlled assembly and disassembly of cell-matrix adhesion.
PubMed: 31921890
DOI: 10.3389/fmolb.2019.00144 -
Clinical Rheumatology Nov 2022Systemic sclerosis (SSc) is an autoimmune disorder that causes vasculopathy and scarring, most commonly in the lungs and skin, but it can also affect other organs....
INTRODUCTION
Systemic sclerosis (SSc) is an autoimmune disorder that causes vasculopathy and scarring, most commonly in the lungs and skin, but it can also affect other organs. Endothelial vinculin plays a critical role in angiogenesis regulation. Therefore, vinculin overexpression in SSc may give rise to anti-vinculin antibodies, which may contribute to the development of SSc vasculopathy. The current research aims to (1) determine whether anti-vinculin autoantibodies play a significant role in the diagnosis of SSc and (2) compare anti-vinculin serum levels between two scleroderma patient populations, namely, pulmonary artery hypertension (PAH)-predominant and interstitial pulmonary fibrosis (IPF)-predominant groups.
METHODS
This research included 140 participants categorized into three groups: group I-patients with PAH-predominant; group II-patients with ILD-predominant; group III-the control group. Anti-vinculin antibodies were detected in serum samples collected from all participants using ELISA. All subjects underwent high-resolution computed tomography (CT), diffusing capacity for carbon monoxide, and pulmonary function tests.
RESULTS
Patients in group I (PAH-predominant group, N = 35) were 41.3 [± 11.4] years old, with 80% being women. Patients in group II (ILD-predominant group, N = 35) were 41.0 [± 11.5] years old. The SSc group showed significantly higher anti-vinculin antibody levels than the control group (P < 0.001). The PAH-predominant group demonstrated significantly higher anti-vinculin antibody levels and anti-vinculin positivity than the ILD-predominant group.
CONCLUSION
Anti-vinculin antibodies in the blood appear to be diagnostic biomarkers for scleroderma. Furthermore, they shed light on some novel perspectives on the pathophysiology of specific lung fibrotic changes. Key Points • This study included two groups of systemic sclerosis patients (PAH-predominant group, ILD-predominant group) as well as a control group to investigate the significance of anti-vinculin antibodies in such cases. • Our results have demonstrated that anti-vinculin antibodies can play a significant role in diagnosing and monitoring systemic sclerosis disease.
Topics: Autoantibodies; Biomarkers; Carbon Monoxide; Egypt; Female; Humans; Lung Diseases, Interstitial; Male; Scleroderma, Systemic; Vascular Diseases
PubMed: 35876914
DOI: 10.1007/s10067-022-06301-0 -
Clinical Rheumatology Mar 2021Gastrointestinal (GI) disease is common in systemic sclerosis, and novel biomarkers are needed to identify and monitor these patients. Dr. Suliman and colleagues...
Gastrointestinal (GI) disease is common in systemic sclerosis, and novel biomarkers are needed to identify and monitor these patients. Dr. Suliman and colleagues identify anti-vinculin autoantibodies in a subset of systemic sclerosis patients which associate with GI symptom severity, although more work is needed to determine their clinical utility.
Topics: Autoantibodies; Biomarkers; Humans; Scleroderma, Systemic
PubMed: 33411141
DOI: 10.1007/s10067-020-05533-2