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Matrix Biology : Journal of the... Nov 2017Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4...
Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4 knockdown was performed in order to address its role in endothelial cells (EC) behavior. Normal EC and shRNA-Syn4-EC cells were studied comparatively using complementary confocal, super-resolution and non-linear microscopic techniques. Confocal and super-resolution microscopy revealed that Syn4 knockdown alters the level and arrangement of essential proteins for focal adhesion, evidenced by the decoupling of vinculin from F-actin filaments. Furthermore, Syn4 knockdown alters the actin network leading to filopodial protrusions connected by VE-cadherin-rich junction. shRNA-Syn4-EC showed reduced adhesion and increased migration. Also, Syn4 silencing alters cell cycle as well as cell proliferation. Moreover, the ability of EC to form tube-like structures in matrigel is reduced when Syn4 is silenced. Together, the results suggest a mechanism in which Syndecan-4 acts as a central mediator that bridges fibronectin, integrin and intracellular components (actin and vinculin) and once silenced, the cytoskeleton protein network is disrupted. Ultimately, the results highlight Syn4 relevance for balanced cell behavior.
Topics: Actins; Animals; Carcinogenesis; Cells, Cultured; Endothelial Cells; Male; Mice, Inbred BALB C; Mice, SCID; Neoplasm Transplantation; Rabbits; Signal Transduction; Syndecan-4; Vinculin
PubMed: 28062282
DOI: 10.1016/j.matbio.2016.12.006 -
Nature Communications Dec 2015The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding...
The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs.
Topics: Actin Cytoskeleton; Actins; Actomyosin; Animals; Cell Polarity; Focal Adhesions; Mice; NIH 3T3 Cells; Protein Binding; Protein Structure, Tertiary; Talin; Vinculin
PubMed: 26634421
DOI: 10.1038/ncomms10038 -
Clinical Rheumatology Jun 2021Systemic sclerosis (SSc) is an autoimmune disorder and commonly presents with vascular system involvement and motility disorders in the gastrointestinal (GI) tract....
BACKGROUND
Systemic sclerosis (SSc) is an autoimmune disorder and commonly presents with vascular system involvement and motility disorders in the gastrointestinal (GI) tract. Vinculin is a cytoskeletal protein that plays major roles in cell-cell adhesion and is expressed in the neuromuscular apparatus of the gut. Antibodies to vinculin have been identified as a biomarker of irritable bowel syndrome (IBS). Our aim was to evaluate serum anti-vinculin antibodies in patients with SSc.
METHODS
Patients were recruited from two SSc centers: group I (GI-enriched group), University of Leeds, UK, and Group II (vascular predominant), University of California, Los Angeles. Serum samples of patients recruited from two SSc centres, Group I ( GI enriched group), University of Leeds, UK and Group II (Vascular predominant), University of California, Los Angeles) were collected. Samples from age- and sex-matched healthy volunteers (N = 88) were used as controls.
RESULTS
Group I (GI-enriched group, N = 83) patients were 58 [50-67] years old; 83% were females with a median body mass index (BMI) of 20.3 (21.2 ± 4.5) [18-23]. Group II (vascular-enriched group, N = 72) patients were 58 [50-67] years old; 80% were female, and BMI was 23.9 (21.3-26.9). More subjects in group I had prominent GI involvement (N = 55, 66%) than group II (12, 16%), p ˂ 0.0001. Anti-vinculin antibody levels in SSc group I (1.3 [0.9]) were significantly higher than in HC (0.7 [0.8]; p = 0.002). When pooled, circulating anti-vinculin levels in both SSc groups remained significantly higher than in the HC group (p = 0.02). Higher anti-vinculin levels were associated with higher GI-visual analogue scale (GI-VAS) scores and specifically with GI-VAS scores of ≥ 4 (p < 0.0001).
CONCLUSION
This study demonstrates that elevated anti-vinculin antibody levels are common in SSc and suggests a potential link between increased anti-vinculin levels and GI tract symptoms.
KEY POINTS
• Anti-vinculin antibodies are elevated in systemic sclerosis and are relatively common. • In these SSc patients, anti-vinculin antibodies are associated with higher levels of GI symptoms in SSc. • A potential link between anti-vinculin antibodies and vascular system involvement was shown.
Topics: Aged; Autoimmunity; Female; Humans; Irritable Bowel Syndrome; Los Angeles; Male; Middle Aged; Scleroderma, Systemic; Vinculin
PubMed: 33231773
DOI: 10.1007/s10067-020-05479-5 -
Cell Reports Apr 2022Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving...
Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving vascular integrity. Here, we analyze the dynamics of endothelial adherens junctions during lumen formation in angiogenic sprouts in vivo. Live imaging in zebrafish reveals that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Junctional fingers are positively regulated by blood pressure, whereas flow inhibition prevents their formation. Using fluorescent reporters, we show that junctional fingers contain the mechanotransduction protein vinculin. Furthermore, genetic deletion of vinculin prevents finger formation, a junctional defect that could be rescued by transient endothelial expression of vinculin. Our findings suggest a mechanism whereby lumen expansion leads to an increase in junctional tension, triggering recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells employ force-dependent junctional remodeling to counteract external forces in order to maintain vascular integrity during sprouting angiogenesis.
Topics: Adherens Junctions; Animals; Cadherins; Endothelial Cells; Intercellular Junctions; Mechanotransduction, Cellular; Neovascularization, Physiologic; Vinculin; Zebrafish; Zebrafish Proteins
PubMed: 35417696
DOI: 10.1016/j.celrep.2022.110658 -
The Protein Journal Feb 2022To shed light onto the activation mechanism of vinculin, we carried out a detailed refinement of chicken vinculin and compared it to the human protein which is greater...
To shed light onto the activation mechanism of vinculin, we carried out a detailed refinement of chicken vinculin and compared it to the human protein which is greater than 95% identical. Refinement resulted in a complete and significantly improved model. This model includes important elements such as a pro-rich strap region (PRR) and C-terminus. The conformation of the PRR stabilized by its inter- and intra-molecular contacts shows a dynamic, but relatively stable motif that constitutes a docking platform for multiple molecules. The contact of the C-terminus with the PRR suggests that phosphorylation of Tyr1065 might control activation and membrane binding. Improved electron densities showed the presence of large solvent molecules such as phosphates/sulfates and a head-group of PIP2. The improved model allowed for a computational stability analysis to be performed by the program Corex/Best which located numerous hot-spots of increased and decreased stability. Proximity of the identified binding sites for regulatory partners involved in inducing or suppressing the activation of vinculin to the unstable elements sheds new light onto the activation pathway and differential activation. This stability analysis suggests that the activation pathway proceeds by unfurling of the super-bundle built from four bundles of helices without separation of the Vt region (840-1066) from the head. According to our mechanism, when activating proteins bind at the strap region a separation of N and C terminal bundles occurs, followed by unfurling of the super-bundle and flattening of the general shape of the molecule, which exposes the interaction sites for binding of auxiliary proteins.
Topics: Actins; Animals; Binding Sites; Chickens; Humans; Protein Binding; Protein Conformation; Vinculin
PubMed: 35006498
DOI: 10.1007/s10930-022-10040-1 -
Nature Communications Jan 2017Circadian clocks drive ∼24 h rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock...
Circadian clocks drive ∼24 h rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock complexes. However, little is known about how cell-intrinsic circadian clocks sense and respond to their microenvironment. Here, we reveal that the breast epithelial clock is regulated by the mechano-chemical stiffness of the cellular microenvironment in primary cell culture. Moreover, the mammary clock is controlled by the periductal extracellular matrix in vivo, which contributes to a dampened circadian rhythm during ageing. Mechanistically, the tension sensing cell-matrix adhesion molecule, vinculin, and the Rho/ROCK pathway, which transduces signals provided by extracellular stiffness into cells, regulate the activity of the core circadian clock complex. We also show that genetic perturbation, or age-associated disruption of self-sustained clocks, compromises the self-renewal capacity of mammary epithelia. Thus, circadian clocks are mechano-sensitive, providing a potential mechanism to explain how ageing influences their amplitude and function.
Topics: Aging; Amides; Animals; Breast; Breast Diseases; CLOCK Proteins; Cell Self Renewal; Cellular Microenvironment; Circadian Clocks; Circadian Rhythm; Epithelial Cells; Epithelium; Extracellular Matrix; Female; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Primary Cell Culture; Pyridines; RNA, Small Interfering; Signal Transduction; Spheroids, Cellular; Tissue Culture Techniques; Vinculin; rho-Associated Kinases
PubMed: 28134247
DOI: 10.1038/ncomms14287 -
European Heart Journal Jan 2023
Topics: Humans; Phosphorylation; Vinculin; Cardiovascular Diseases; Endothelial Cells; Permeability; Atherosclerosis
PubMed: 36514951
DOI: 10.1093/eurheartj/ehac704 -
ELife Oct 2022Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by...
Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by peristalsis of its variable content. Using the intestinal epithelium, we investigate the role of vinculin, one of the best characterised mechanoeffectors, which functions in both cadherin and integrin adhesion complexes. We discovered that vinculin regulates cell fate decisions, by preventing precocious activation and differentiation of intestinal progenitors into absorptive cells. It achieves this in concert with α-catenin at sites of cadherin adhesion, rather than as part of integrin function. Following asymmetric division of the stem cell into a stem cell and an enteroblast (EB), the two cells initially remain connected by adherens junctions, where vinculin is required, only on the EB side, to maintain the EB in a quiescent state and inhibit further divisions of the stem cell. By manipulating cell tension, we show that vinculin recruitment to adherens junction regulates EB activation and numbers. Consequently, removing vinculin results in an enlarged gut with improved resistance to starvation. Thus, mechanical regulation at the contact between stem cells and their progeny is used to control tissue cell number.
Topics: Animals; alpha Catenin; Vinculin; Drosophila; Enterocytes; Actins; Cadherins; Integrins; Homeostasis
PubMed: 36269226
DOI: 10.7554/eLife.72836 -
Scientific Reports Oct 2019Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and...
Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through αE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin upon binding to the E-cadherin•β-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in αE-catenin when bound to F-actin. Comparisons of wild-type αE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal β-catenin-binding and the middle (M) vinculin-binding domain of αE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length αE-catenin, and identify the M domain as a key transmitter of conformational changes.
Topics: Actins; Allosteric Regulation; Animals; Biomechanical Phenomena; Cysteine; Mice; Models, Molecular; Mutant Proteins; Protein Binding; Protein Conformation; Solutions; Sulfhydryl Compounds; Vinculin; alpha Catenin; beta Catenin
PubMed: 31653927
DOI: 10.1038/s41598-019-51816-3 -
Journal of Cell Science Sep 2015The focal adhesion protein vinculin connects the actin cytoskeleton, through talin and integrins, with the extracellular matrix. Vinculin consists of a globular head and...
The focal adhesion protein vinculin connects the actin cytoskeleton, through talin and integrins, with the extracellular matrix. Vinculin consists of a globular head and tail domain, which undergo conformational changes from a closed auto-inhibited conformation in the cytoplasm to an open conformation in focal adhesions. Src-mediated phosphorylation has been suggested to regulate this conformational switch. To explore the role of phosphorylation in vinculin activation, we used knock-out mouse embryonic fibroblasts re-expressing different vinculin mutants in traction microscopy, magnetic tweezer microrheology, FRAP and actin-binding assays. Compared to cells expressing wild-type or constitutively active vinculin, we found reduced tractions, cytoskeletal stiffness, adhesion strength, and increased vinculin dynamics in cells expressing constitutively inactive vinculin or vinculin where Src-mediated phosphorylation was blocked by replacing tyrosine at position 100 and/or 1065 with a non-phosphorylatable phenylalanine residue. Replacing tyrosine residues with phospho-mimicking glutamic acid residues restored cellular tractions, stiffness and adhesion strength, as well as vinculin dynamics, and facilitated vinculin-actin binding. These data demonstrate that Src-mediated phosphorylation is necessary for vinculin activation, and that phosphorylation controls cytoskeletal mechanics by regulating force transmission between the actin cytoskeleton and focal adhesion proteins.
Topics: Animals; Cell Adhesion; Cytoskeleton; Energy Transfer; Fibroblasts; Integrins; Mice; Mice, Knockout; Phosphorylation; Point Mutation; Protein Structure, Secondary; Vinculin; src-Family Kinases
PubMed: 26240176
DOI: 10.1242/jcs.172031