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Biomaterials Jul 2024Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal...
Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.
Topics: Talin; Vinculin; Humans; Cell Nucleus; YAP-Signaling Proteins; Mechanotransduction, Cellular; Adaptor Proteins, Signal Transducing; Transcription Factors; Mesenchymal Stem Cells; Animals; Focal Adhesions; Mice; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Protein Binding
PubMed: 38547833
DOI: 10.1016/j.biomaterials.2024.122542 -
The FEBS Journal Jun 2022Focal adhesions (FA) are large macromolecular assemblies relevant for various cellular and pathological events such as migration, polarization, and metastatic cancer... (Review)
Review
Focal adhesions (FA) are large macromolecular assemblies relevant for various cellular and pathological events such as migration, polarization, and metastatic cancer formation. At FA sites at the migrating periphery of a cell, hundreds of players gather and form a network to respond to extra cellular stimuli transmitted by the integrin receptor, the most upstream component within a cell, initiating the FA signaling pathway. Numerous cellular experiments have been performed to understand the FA architecture and functions; however, their intricate network formation hampers unraveling the precise molecular actions of individual players. Here, in vitro bottom-up reconstitution presents an advantageous approach for elucidating the FA machinery and the hierarchical crosstalk of involved cellular players.
Topics: Actins; Cell Adhesion; Focal Adhesions; Integrins; Talin; Vinculin
PubMed: 33999507
DOI: 10.1111/febs.16023 -
American Journal of Respiratory and... Oct 2022The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. To relate functional abnormalities in...
The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated β1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.
Topics: Animals; Antiviral Agents; Elafin; Endogenous Retroviruses; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Integrins; Leukocyte Elastase; Mice; Neutrophils; Proteomics; Pulmonary Arterial Hypertension; Vinculin
PubMed: 35696338
DOI: 10.1164/rccm.202102-0446OC -
An ensemble of cadherin-catenin-vinculin complex employs vinculin as the major F-actin binding mode.Biophysical Journal Jun 2023The cell-cell adhesion cadherin-catenin complexes recruit vinculin to the adherens junction (AJ) to modulate the mechanical couplings between neighboring cells. However,...
The cell-cell adhesion cadherin-catenin complexes recruit vinculin to the adherens junction (AJ) to modulate the mechanical couplings between neighboring cells. However, it is unclear how vinculin influences the AJ structure and function. Here, we identified two patches of salt bridges that lock vinculin in the head-tail autoinhibited conformation and reconstituted the full-length vinculin activation mimetics bound to the cadherin-catenin complex. The cadherin-catenin-vinculin complex contains multiple disordered linkers and is highly dynamic, which poses a challenge for structural studies. We determined the ensemble conformation of this complex using small-angle x-ray and selective deuteration/contrast variation small-angle neutron scattering. In the complex, both α-catenin and vinculin adopt an ensemble of flexible conformations, but vinculin has fully open conformations with the vinculin head and actin-binding tail domains well separated from each other. F-actin binding experiments show that the cadherin-catenin-vinculin complex binds and bundles F-actin. However, when the vinculin actin-binding domain is removed from the complex, only a minor fraction of the complex binds to F-actin. The results show that the dynamic cadherin-catenin-vinculin complex employs vinculin as the primary F-actin binding mode to strengthen AJ-cytoskeleton interactions.
Topics: Cadherins; Actins; Vinculin; alpha Catenin; Protein Binding; Actin Cytoskeleton; Cell Adhesion
PubMed: 37147801
DOI: 10.1016/j.bpj.2023.04.026 -
International Journal of Molecular... May 2022The mechanical signals within the extracellular matrix (ECM) regulate cell growth, proliferation and differentiation, and integrins function as the hub between the ECM... (Review)
Review
The mechanical signals within the extracellular matrix (ECM) regulate cell growth, proliferation and differentiation, and integrins function as the hub between the ECM and cellular actin. Focal adhesions (FAs) are multi‑protein, integrin‑containing complexes, acting as tension‑sensing anchoring points that bond cells to the extracellular microenvironment. Talin‑1 serves as the central protein of FAs that participates in the activation of integrins and connects them with the actin cytoskeleton. As a cytoplasmic protein, Talin‑1 consists of a globular head domain and a long rod comprised of a series of α‑helical bundles. The unique structure of the Talin‑1 rod domain permits folding and unfolding in response to the mechanical stress, revealing various binding sites. Thus, conformation changes of the Talin‑1 rod domain enable the cell to convert mechanical signals into chemical through multiple signaling pathways. The present review discusses the binding partners of Talin‑1, their interactions, effects on the cellular processes, and their possible roles in diseases.
Topics: Cell Adhesion; Focal Adhesions; Integrins; Mechanotransduction, Cellular; Protein Binding; Talin
PubMed: 35266014
DOI: 10.3892/ijmm.2022.5116 -
Science Advances Jan 2022E-cadherin is a major cell-cell adhesion molecule involved in mechanotransduction at cell-cell contacts in tissues. Because epithelial cells respond to rigidity and...
E-cadherin is a major cell-cell adhesion molecule involved in mechanotransduction at cell-cell contacts in tissues. Because epithelial cells respond to rigidity and tension in tissue through E-cadherin, there must be active processes that test and respond to the mechanical properties of these adhesive contacts. Using submicrometer, E-cadherin-coated polydimethylsiloxane pillars, we find that cells generate local contractions between E-cadherin adhesions and pull to a constant distance for a constant duration, irrespective of pillar rigidity. These cadherin contractions require nonmuscle myosin IIB, tropomyosin 2.1, α-catenin, and binding of vinculin to α-catenin. Cells spread to different areas on soft and rigid surfaces with contractions, but spread equally on soft and rigid without. We further observe that cadherin contractions enable cells to test myosin IIA-mediated tension of neighboring cells and sort out myosin IIA-depleted cells. Thus, we suggest that epithelial cells test and respond to the mechanical characteristics of neighboring cells through cadherin contractions.
PubMed: 35089785
DOI: 10.1126/sciadv.abk0387 -
Journal of Molecular Recognition : JMR Jun 2023Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix....
Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.
Topics: Animals; Mice; Cell Adhesion; Fibroblasts; Focal Adhesions; Integrins; Talin; Vinculin
PubMed: 36987702
DOI: 10.1002/jmr.3012 -
European Journal of Clinical... Jan 2022
Topics: Antibodies; Bacterial Toxins; Gastrointestinal Diseases; Gastrointestinal Microbiome; Humans; Irritable Bowel Syndrome; Vinculin
PubMed: 34738648
DOI: 10.1111/eci.13702 -
Journal of Dental Research Sep 2023Teeth consist of 3 mineralized tissues: enamel, dentin, and cementum. Tooth malformation, the most common craniofacial anomaly, arises from complex genetic and...
Teeth consist of 3 mineralized tissues: enamel, dentin, and cementum. Tooth malformation, the most common craniofacial anomaly, arises from complex genetic and environmental factors affecting enamel structure, size, shape, and tooth eruption. Hyaluronic acid (HA), a primary extracellular matrix component, contributes to structural and physiological functions in periodontal tissue. Transmembrane protein 2 (TMEM2), a novel cell surface hyaluronidase, has been shown to play a critical role during embryogenesis. In this study, we demonstrate messenger RNA expression in inner enamel epithelium and presecretory, secretory, and mature ameloblasts. knock-in reporter mice reveal TMEM2 protein localization at the apical and basal ends of secretory ameloblasts. Micro-computed tomography analysis of epithelial-specific conditional knockout (-) mice shows a significant reduction in enamel layer thickness and severe enamel deficiency. Enamel matrix protein expression was remarkably downregulated in - mice. Scanning electron microscopy of enamel from - mice revealed an irregular enamel prism structure, while the microhardness and density of enamel were significantly reduced, indicating impaired ameloblast differentiation and enamel matrix mineralization. Histological evaluation indicated weak adhesion between cells and the basement membrane in - mice. The reduced and irregular expressions of vinculin and integrin β1 suggest that deficiency attenuated focal adhesion formation. In addition, abnormal HA accumulation in the ameloblast layer and weak claudin 1 immunoreactivity in - mice indicate impaired tight junction gate function. Irregular actin filament assembly was also observed at the apical and basal ends of secretory ameloblasts. Last, we demonstrated that -deficient mHAT9d mouse ameloblasts exhibit defective adhesion to HA-containing substrates in vitro. Collectively, our data highlight the importance of TMEM2 in adhesion to HA-rich extracellular matrix, cell-to-cell adhesion, ameloblast differentiation, and enamel matrix mineralization.
Topics: Mice; Animals; Dental Enamel Hypoplasia; X-Ray Microtomography; Dental Enamel; Ameloblasts; Amelogenesis; Mice, Knockout; Membrane Proteins
PubMed: 37449307
DOI: 10.1177/00220345231182355 -
Cancers Sep 2023Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is...
Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is associated with a worse prognosis and shortened disease-free survival. Therefore, anti-HER2 therapies have been developed, such as monoclonal antibodies (trastuzumab, Tz), antibody-drug conjugates (ado-trastuzumab emtansine, T-DM1), and pharmacological inhibitors of tyrosine kinase activity (lapatinib, Lp). Although Tz, the standard treatment, has significantly improved the prognosis of patients, resistance still affects a significant population of women and is currently a major challenge in clinical oncology. Therefore, this study aims to identify potential biomarkers to predict disease progression (prognostic markers) and the efficacy of Tz treatment (predictive markers) in patients with HER2+ BC. We hypothesize that proteins involved in cell motility are implicated in Tz-resistance. We aim to identify alterations in Tz-resistant cells to guide more efficient oncologic decisions. By bioinformatics, we selected candidate proteins and determined how their expression, localization, and the process they modulate were affected by anti-HER2 treatments. Next, using HER2+ BC patients' data, we assessed these proteins as prognostic and predictive biomarkers. Finally, using Tz-resistant cells, we evaluated their roles in Tz response. We identified deregulated genes associated with cell motility in Tz/T-DM1-resistant vs. -sensitive cells. We showed that Tz, T-DM1, and Lp decrease cell viability, and their effect is enhanced in combinations. We determined synergism between Tz/T-DM1 and Lp, making possible a dose reduction of each drug to achieve the same therapeutic effect. We found that combinations (Tz/T-DM1 + Lp) efficiently inhibit cell adhesion and migration. Furthermore, we demonstrated the induction of FAK nuclear and cortactin peri-nuclear localization after T-DM1, Lp, and Tz/T-DM1 + Lp treatments. In parallel, we observed that combined treatments downregulate proteins essential for metastatic dissemination, such as SRC, FAK, and paxillin. We found that low vinculin (VCL) and cortactin (CTTN) mRNA expression predicts favorable survival rates and has diagnostic value to discriminate between Tz-sensible and Tz-resistant HER2+ BC patients. Finally, we confirmed that vinculin and cortactin are overexpressed in Tz-resistance cells, SKBR3-RTz. Moreover, we found that Tz plus FAK/paxillin/cortactin-silencing reduced cell adhesion/migration capacity in Tz-sensitive and -resistant cells. In conclusion, we demonstrate that combined therapies are encouraging since low doses of Tz/T-DM1 + Lp inhibit metastatic processes by downregulating critical protein expression and affecting its subcellular localization. We propose that vinculin and cortactin might contribute to Tz-sensibility/resistance in BC cells. Finally, we identify potential prognostic and predictive biomarkers that are promising for personalized BC management that would allow efficient patient selection in order to mitigate resistance and maximize the safety and efficacy of anti-HER2 therapies.
PubMed: 37686651
DOI: 10.3390/cancers15174374