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Vinculin binds to specific sites of mechanically unfolded talin rod domains to reinforce the coupling of the cell's exterior to its force generation machinery. Force-dependent vinculin–talin complexation and dissociation was previously observed as contraction or extension of the unfolded talin domains respectively using magnetic tweezers. However, the structural mechanism underlying vinculin recognition of unfolded vinculin binding sites (VBSs) in talin remains unknown. Using molecular dynamics simulations, we demonstrate that a VBS dynamically refolds under force, and that vinculin can recognize and bind to partially unfolded VBS states. Vinculin binding enables refolding of the mechanically strained VBS and stabilizes its folded α-helical conformation, providing resistance against mechanical stress. Together, these results provide an understanding of a recognition mechanism of proteins unfolded by force and insight into the initial moments of how vinculin binds unfolded talin rod domains during the assembly of this mechanosensing meshwork. Author summary: Cells attach to and interact with their surroundings, and sense mechanical signals from the microenvironment. Vinculin and talin are classical examples of mechanosensitive proteins that complex in a force-dependent manner. Talin domains unfold under mechanical loads exposing buried vinculin binding sites, namely single α-helices. Such mechanical stretching enables recognition by vinculin, however, the structural states of vinculin's binding site when under force and the complexation mechanisms are poorly understood. We used molecular dynamics simulations to study the behavior of a single vinculin binding helix under mechanical load and during the initial moments of the vinculin–talin binding process. Our results reveal the highly dynamic nature of a vinculin binding helix under mechanical load. Depending on the force magnitude, the helix frequently reforms to a partially folded conformation on the nanosecond to microsecond timescale. Crucially, vinculin recognizes such partially folded helices in talin and promotes their refolding against force. Our findings are beyond the resolution limits imposed by experimental single molecule methods, suggesting a model which may be useful as a general mechanism for protein-protein binding under mechanical load. [ABSTRACT FROM AUTHOR]

Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing. Here, we demonstrate that, in absence of α-catenin, β-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that β-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and β-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving β-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin. Adherens junctions mediate force transmission and connect the cytoskeleton of adjacent cells. This study demonstrates that force transmission can be achieved through direct and functional β-catenin/vinculin interaction in the absence of α-catenin. [ABSTRACT FROM AUTHOR]

Cardiac tissue engineering has emerged as a promising approach for restoring the functionality of damaged cardiac tissues following myocardial infarction. To effectively replicate the native anisotropic structure of cardiac tissues in vitro, this study focused on the fabrication of micropatterned gelatin methacryloyl hydrogels with varying geometric parameters. These substrates were evaluated for their ability to guide induced pluripotent stem cell-derived cardiomyocytes (CMs). The findings demonstrate that the mechanical properties of this hydrogel closely resemble those of native cardiac tissues, and it exhibits high fidelity in micropattern fabrication. Micropatterned hydrogel substrates lead to enhanced organization, maturation, and contraction of CMs. A microgroove with 20-μm-width and 20-μm-spacing was identified as the optimal configuration for maximizing the contact guidance effect, supported by analyses of nuclear orientation and F-actin organization. Furthermore, this specific micropattern design was found to promote CMs' maturation, as evidenced by increased expression of connexin 43 and vinculin, along with extended sarcomere length. It also enhanced CMs' contraction, resulting in larger contractile amplitudes and greater contractile motion anisotropy. In conclusion, these results underscore the significant benefits of optimizing micropatterned gelatin methacryloyl for improving CMs' organization, maturation, and contraction. This valuable insight paves the way for the development of highly organized and functionally mature cardiac tissues in vitro. [ABSTRACT FROM AUTHOR]

Focal adhesions (FAs) are mechanosensory structures that transform physical stimuli into chemical signals guiding cell migration. Comprehensive studies postulate correlation between the FA parameters and cell motility metrics for individual migrating cells. However, which properties of the FAs are critical for epithelial cell motility in a monolayer remains poorly elucidated. We used high-throughput microscopy to describe relationship between the FA parameters and cell migration in immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) with depleted or inhibited vinculin and focal adhesion kinase (FAK) FA proteins. To evaluate relationship between the FA morphology and cell migration, we used substrates with varying stiffness in the model of wound healing. Cells cultivated on fibronectin had the highest FA area values, migration rate, and upregulated expression of FAK and vinculin mRNAs, while the smallest FA area and slower migration rate to the wound were specific to cells cultivated on glass. Suppression of vinculin expression in both normal and tumor cells caused decrease of the FA size and fluorescence intensity but did not affect cell migration into the wound. In contrast, downregulation or inactivation of FAK did not affect the FA size but significantly slowed down the wound closure rate by both HaCaT and A549 cell lines. We also showed that the FAK knockdown results in the FA lifetime decrease for the cells cultivated both on glass and fibronectin. Our data indicate that the FA lifetime is the most important parameter defining migration of epithelial cells in a monolayer. The observed change in the cell migration rate in a monolayer caused by changes in expression/activation of FAK kinase makes FAK a promising target for anticancer therapy of lung carcinoma. [ABSTRACT FROM AUTHOR]

SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte‐specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins—paxillin, vinculin, and talin‐1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin‐1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin‐induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin‐1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin‐1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease. [ABSTRACT FROM AUTHOR]

The expression levels of certain genes or proteins may exhibit various features regardless of the tissue type. The housekeeping genes and reference proteins are often used during the normalization of expression studies. Herein, we aim to determine and validate the most stable reference gene and protein for a healthy human fibroblast cell line. This study used a finite fibroblast cell line derived from healthy human skin (CCD1079Sk). The samples between 25 and until senescence occurs at passage 55 were collected. Morphological changes during aging by cultivation were observed in each passage. Seven genes (ACTB, SDHA, GAPDH, RPLP0, VCL, TBP, 18S) were evaluated during cultivation and ranked according to their stability via the RefFinder interface. This study reports strong variations for both gene and protein expression studies and the morphology of the cells. Notably, during cultivation, the cell morphology showed an irreversible change starting from passage 43, before the known replicative lifespan of CCD1079Sk. The lack of an RPLP0 subunit after passage 39 may limit the cultivation range when protein expression is required. GAPDH is also an ideal reference gene and protein when studies include gene and protein expression. Or, as a second option, Vinculin may be the first choice when mRNA expression is required, and β-Actin is the best choice for reference protein when the studies include only protein expression. Plus, such as SDHA, RPLP0, and TBP should be avoided. These results will benefit further studies, including normalization for both gene, protein, and cell type.

Gravity is a primary physical force that has a profound influence on the stability of the cell cytoskeleton. In our research, we investigated the influence of microgravity on altering the cytoskeletal pathways of glioblastoma cells. The highly infiltrative behavior of glioblastoma is supported by cytoskeletal dynamics and surface proteins that allow glioblastoma cells to avoid stable connections with the tissue environment and other cells. Glioblastoma cell line C6 was exposed to a microgravity environment for 24, 48, and 72 h by 3D-RPM, a laboratory instrument recognized to reproduce the effect of microgravity in cell cultures. The immunofluorescence for GFAP, vinculin, and Connexin-43 was investigated as signals related to cytoskeleton dynamics. The polymerization of GFAP and the expression of focal contact structured by vinculin were found to be altered, especially after 48 and 72 h of microgravity. Connexin-43, involved in several intracellular pathways that critically promote cell motility and invasion of glioma cells, was found to be largely reduced following microgravity exposure. In conclusion, microgravity, by reducing the expression of Connexin-43, alters the architecture of specific cytoskeletal elements such as GFAP and increases the focal contact, which can induce a reduction in glioma cell mobility, thereby inhibiting their aggressive metastatic behavior.

In cell–matrix adhesions, integrin receptors and associated proteins provide a dynamic coupling of the extracellular matrix (ECM) to the cytoskeleton. This allows bidirectional transmission of forces between the ECM and the cytoskeleton, which tunes intracellular signaling cascades that control survival, proliferation, differentiation, and motility. The quantitative relationships between recruitment of distinct cell–matrix adhesion proteins and local cellular traction forces are not known. Here, we applied quantitative super-resolution microscopy to cell–matrix adhesions formed on fibronectin-stamped elastomeric pillars and developed an approach to relate the number of talin, vinculin, paxillin, and focal adhesion kinase (FAK) molecules to the local cellular traction force. We find that FAK recruitment does not show an association with traction-force application, whereas a ∼60 pN force increase is associated with the recruitment of one talin, two vinculin, and two paxillin molecules on a substrate with an effective stiffness of 47 kPa. On a substrate with a fourfold lower effective stiffness, the stoichiometry of talin:vinculin:paxillin changes to 2:12:6 for the same ∼60 pN traction force. The relative change in force-related vinculin recruitment indicates a stiffness-dependent switch in vinculin function in cell–matrix adhesions. Our results reveal a substrate-stiffness-dependent modulation of the relationship between cellular traction-force and the molecular stoichiometry of cell–matrix adhesions. [ABSTRACT FROM AUTHOR]

The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding remain unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of the variants did not affect Vcn activation but reduced Vcn loading and altered exchange dynamics, consistent with the loss of directional catch bonding. Expression of Vcn variants perturbed the coordination of subcellular structures and cell migration, establishing key cellular functions for Vcn directional catch bonding. This study provides insights into the directional catch bonding of the linker protein vinculin by elucidating the molecular basis of mechanical reinforcement as well as unveiling its key role in subcellular organization and cellular processes. [ABSTRACT FROM AUTHOR]

The ability of cells to move in a mechanically coupled, coordinated manner, referred to as collective cell migration, is central to many developmental, physiological, and pathophysiological processes. Limited understanding of how mechanical forces and biochemical regulation interact to affect coupling has been a major obstacle to unravelling the underlying mechanisms. Focusing on the linker protein vinculin, we use a suite of Förster resonance energy transfer-based biosensors to probe its mechanical functions and biochemical regulation, revealing a switch that toggles vinculin between loadable and unloadable states. Perturbation of the switch causes covarying changes in cell speed and coordination, suggesting alteration of the friction within the system. Molecular scale modelling reveals that increasing levels of loadable vinculin increases friction, due to engagement of self-stabilizing catch bonds. Together, this work reveals a regulatory switch for controlling cell coupling and describes a paradigm for relating biochemical regulation, altered mechanical properties, and changes in cell behaviors. [ABSTRACT FROM AUTHOR]

Gravity is a primary physical force that has a profound influence on the stability of the cell cytoskeleton. In our research, we investigated the influence of microgravity on altering the cytoskeletal pathways of glioblastoma cells. The highly infiltrative behavior of glioblastoma is supported by cytoskeletal dynamics and surface proteins that allow glioblastoma cells to avoid stable connections with the tissue environment and other cells. Glioblastoma cell line C6 was exposed to a microgravity environment for 24, 48, and 72 h by 3D-RPM, a laboratory instrument recognized to reproduce the effect of microgravity in cell cultures. The immunofluorescence for GFAP, vinculin, and Connexin-43 was investigated as signals related to cytoskeleton dynamics. The polymerization of GFAP and the expression of focal contact structured by vinculin were found to be altered, especially after 48 and 72 h of microgravity. Connexin-43, involved in several intracellular pathways that critically promote cell motility and invasion of glioma cells, was found to be largely reduced following microgravity exposure. In conclusion, microgravity, by reducing the expression of Connexin-43, alters the architecture of specific cytoskeletal elements such as GFAP and increases the focal contact, which can induce a reduction in glioma cell mobility, thereby inhibiting their aggressive metastatic behavior. [ABSTRACT FROM AUTHOR]

Predictors of myocardial recovery in heart failure (HF) are poorly understood. We explored if vinculin (VCL) variants are associated with myocardial recovery in dilated cardiomyopathy (DCM). Six infants with DCM with a VCL loss-of-function (LOF) variant were identified. Median age at diagnosis was 2 months, median LV ejection fraction was 24%, and median LV end-diastolic diameter z-score was 10.8. All patients received HF medications. Five patients (83%) showed normalization of LV function at a median age of 2.7 years. One patient progressed to end-stage HF requiring heart transplant. This case series identified a unique phenotype of HF with reduced ejection fraction at presentation that evolved to HF with recovered EF in over 80% of infant DCM cases with LOF VCL variants. These findings have prognostic implications for counseling and management of VCL-associated DCM and highlight a possible genetic basis for HF with recovered ejection fraction. [ABSTRACT FROM AUTHOR]

Abstract SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte‐specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins—paxillin, vinculin, and talin‐1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin‐1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin‐induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin‐1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin‐1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease.

Podosomes have been known for several decades as micron-sized, F-actin-rich structures that play a pivotal role in cell migration and invasion, as they are able to mediate both cell-matrix attachment as well as extracellular matrix degradation. Particularly in monocytic cells, podosomes have been shown to fulfill a variety of additional functions such as sensing of substrate rigidity and topography, or cell-cell fusion. Increasing evidence now points to the involvement of podosome-like structures also during phagocytosis by immune cells such as macrophages, dendritic cells, and neutrophils. Here, we compare the different cell models and experimental set ups where “phagocytic podosomes” have been described. We also discuss the composition and architecture of these structures, their potential involvement in mechanosensing and particle disruption, as well as the pros and cons for addressing them as bona fide podosomes.

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s−1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere. Vinculin binding to talin is a key event in focal adhesion dynamics; yet, how vinculin is activated to recruit actin remains unknown. Here, the authors use a multiscale approach to reveal that talin activates vinculin through an intricate allosteric mechanism tightly regulated by force. [ABSTRACT FROM AUTHOR]

Background: In the present study, we measured the serum vascular endothelial cadherin (VEC) and vinculin (Vcn) concentrations in patients with type B acute aortic dissection (TBAD) to evaluate their diagnostic value for this condition. Methods: A total of 100 patients with TBAD and 90 matched controls were included in the study. The serum concentrations of VEC and Vcn were measured using enzyme-linked immunosorbent assays. Results: The serum VEC and Vcn concentrations were significantly higher in participants with TBAD than in healthy controls. Compared with patients with acute myocardial infarction (AMI), the serum concentrations of VEC and Vcn in patients with TBAD were higher, and the Vcn showed significant difference, with statistical significance. Receiver operating characteristic analysis generated areas under the curves for VEC and Vcn that were diagnostic for TBAD (0.599 and 0.655, respectively). The optimal cut-off values were 3.975 ng/μL and 128.1 pg/mL, the sensitivities were 43.0% and 35.0%, and the specificities were 73.3% and 90.0%, respectively. In addition, the use of a combination of serum VEC and Vcn increased the AUC to 0.661, with a sensitivity of 33.0% and a specificity of 93.33%. A high serum Vcn concentration was associated with a higher risk of visceral malperfusion in participants with TBAD (odds ratio (OR) = 1.007, 95% confidence interval [CI]: 1.001–1.013, p = 0.014). In participants with refractory pain, the adjusted OR for the serum VEC concentration increased to 1.172 (95% CI: 1.010–1.361; p = 0.036), compared with participants without refractory pain. Conclusion: This study is the first to show the diagnostic value of serum VEC and Vcn for AAD and their relationships with the clinical characteristics of patients with TBAD. Thus, VEC and Vcn are potential serum markers of TBAD. [ABSTRACT FROM AUTHOR]

Increased intestinal and blood-brain barriers (BBB) permeability has been suggested to have a role in autism spectrum disorder (ASD). Claudin-5, claudin-11, occludin, β-catenin, vinculin, and paxillin are crucial components of these barriers. This study assessed concentrations of these molecules in preschool children with ASD. A total of 80 children with ASD and 40 controls aged 18–60 months were enrolled in this study. Serum levels of biochemical variables were determined using commercial enzyme-linked immunosorbent assay kits. Serum claudin-11, occludin, and β-catenin levels were significantly higher in the ASD group than in the control group. However, no significant difference for serum claudin-5, vinculin, and paxillin levels was detected between the groups. These findings suggest that claudin-11, occludin, and β-catenin may be involved in the pathogenesis of ASD. These proteins may affect the brain by causing dysregulation in intestinal or blood-brain barrier permeability or with other unknown mechanisms. [ABSTRACT FROM AUTHOR]

The present study was designed to clarify the activity of human gingival fibroblasts (HGFs) on the fibronectin (FN)-coated silanized microgroove titanium surface. The surface elemental composition of titanium discs was detected using XPS. HGFs' adhesion to the titanium discs was detected by immunofluorescence staining of vinculin. HGFs' number on the titanium discs was detected using the CCK8 assay. HGFs' secretion of type 1 collagen after five days of culturing was detected using ELISA and qPCR. HGFs could proliferate and spread well on the surface. The viability of HGFs in the experimental group was significantly more than in the control group. The HGFs in the experimental group significantly secreted more type 1 collagen than in the control group. Therefore, FNcoated can improve the morphology, viability, and type 1 collagen secretion of HGFs silanized microgroove titanium surface, which might ameliorate the efficacy of implants. [ABSTRACT FROM AUTHOR]

Introduction: Drosophila Singed (mammalian Fascin) is an actin‐binding protein that is known mainly for bundling parallel actin filaments. Among many functions of Singed, it is required for cell motility for both Drosophila and mammalian systems. Increased Fascin‐1 levels positively correlate with greater metastasis and poor prognosis in human cancer. Border cell cluster, forms and migrates during Drosophila egg chamber development, shows higher expression of Singed compared with other follicle cells. Interestingly, loss of singed in border cells does not lead to any effect other than delay. Result: In this work, we have screened many actin‐binding proteins in search of functional redundancy with Singed for border cell migration. We have found that Vinculin works with Singed to regulate border cell migration, albeit mildly. Although Vinculin is known for anchoring F‐actin to the membrane, knockdown of both singed and vinculin leads to a reduced level of F‐actin and changes in protrusion characteristics in border cells. We have also observed that they may act together to control microvilli length of brush border membrane vesicles and the shape of egg chambers in Drosophila. Conclusions: We may conclude that singed and vinculin work together to control F‐actin and these interactions are consistent across multiple platforms. Key Findings: Singed and vinculin show mild genetic interaction in Drosophila singed and vinculin regulate F‐actin in border cell singed and vinculin work together in border cell, brush border membrane vesicles and egg chamber shape. [ABSTRACT FROM AUTHOR]

Abdallah Essa,1,2 Enas Said Essa,3 Sara Mahmoud El-deeb,3 Hossam Eldin Mostafa Seleem,1 Muthana Al Sahlawi,2 Omar Ahmed Al-Omair,2 Somaia Shehab-Eldeen1,2 1Tropical Medicine Department, Faculty of Medicine, Menoufia University, Shebin Elkom, Egypt; 2Internal Medicine Department, College of Medicine, King Faisal University, Al-Ahsa, Kingdom of Saudi Arabia; 3Clinical Pathology Department, Faculty of Medicine, Menoufia University, Shebin Elkom, EgyptCorrespondence: Somaia Shehab-Eldeen, Tropical Medicine Department, Faculty of Medicine, Menoufia University, Yassen Abd Al Ghafar Street, Shebin Elkom, Menoufia Governorate, 32511, Egypt, Tel +201117251523, Email Somaia.shehab@med.menofia.edu.egBackground: The stiffness of the extracellular matrix (ECM) controls many cellular processes, such as migration and differentiation. Cells detect stiffness through adhesion structures termed focal adhesions (FAs). Vinculin, an actin-binding FA protein, plays a pivotal role in FA-mediated mechanotransduction.Aim: This study aimed to explore the role of vinculin in the development of HBV/HCV-induced hepatocellular carcinoma (HCC).Methods: Vinculin levels in a total number of 100 serum samples from patients with HBV/HCV-induced liver cirrhosis and HCC, as well as healthy controls, were analyzed using an enzyme-linked immunosorbent assay (ELISA).Results: In patients with HCC and liver cirrhosis, the serum vinculin levels were significantly greater than in controls (503.8± 242.2 and 728.4± 1044.8 vs 77.7± 36.1 respectively, p< 0.001). However, results showed no link between serum vinculin and the clinicopathological features of HCC.Conclusion: Patients with HBVor HCV-induced liver cirrhosis and HCC have significantly higher serum levels of vinculin than do controls. This might point to a potential role for vinculin in the development of HCC. More research into how this protein affects the development of HCC at the molecular level could lead to better clinical treatments and the development of new molecular therapies.Keywords: vinculin, HCC, extracellular matrix, liver cirrhosis, focal adhesion proteins

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 0.05). 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

Remodelling of cell–cell junctions is crucial for proper tissue development and barrier function. The cadherin-based adherens junctions anchor via β-catenin and α-catenin to the actomyosin cytoskeleton, together forming a junctional mechanotransduction complex. Tension-induced conformational changes in the mechanosensitive α-catenin protein induce junctional vinculin recruitment. In endothelial cells, vinculin protects the remodelling of VE–cadherin junctions. In this study, we have addressed the role of vinculin in endothelial barrier function in the developing vasculature. In vitro experiments, using endothelial cells in which α-catenin was replaced by a vinculin-binding-deficient mutant, showed that junctional recruitment of vinculin promotes endothelial barrier function. To assess the role of vinculin within blood vessels in vivo, we next investigated barrier function in the vasculature of vcl knockout zebrafish. In the absence of vinculin, sprouting angiogenesis and vessel perfusion still occurred. Intriguingly, the absence of vinculin made the blood vessels more permeable for 10 kDa dextran molecules but not for larger tracers. Taken together, our findings demonstrate that vinculin strengthens the endothelial barrier and prevents vascular leakage in developing vessels.

Vinculin is an actin-binding protein present at cell-matrix and cell-cell adhesions, which plays a critical role in bearing force experienced by cells and dissipating it onto the cytoskeleton. Recently, we identified a key tyrosine residue, Y822, whose phosphorylation plays a critical role in force transmission at cell-cell adhesions. The role of Y822 in human cancer remains unknown, even though Y822 is mutated to Y822C in uterine cancers. Here, we investigated the effect of this amino acid substitution and that of a phosphodeficient Y822F vinculin in cancer cells. We observed that the presence of the Y822C mutation led to cells that proliferate and migrate more rapidly and contained smaller focal adhesions when compared to cells with wild-type vinculin. In contrast, the presence of the Y822F mutation led to highly spread cells with larger focal adhesions and increased contractility. Furthermore, we provide evidence that Y822C vinculin forms a disulfide bond with paxillin, accounting for some of the elevated phosphorylated paxillin recruitment. Taken together, these data suggest that vinculin Y822 modulates the recruitment of ligands. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background & Aims: The aim of this study is to determine whether activated hepatic stellate cells (HSCs) may represent a prognostic marker of progressive liver fibrosis in chronic viral hepatitis C (VHC) before antiviral therapy. The possible correlation between HSCs immunohistochemical features, histopathological aspects and clinical data before therapy were also studied. Methods: This retrospective pilot study was conducted on 27 liver biopsies from VHC patients before antiviral therapy. HSCs's immunohistochemical analysis used the antibodies alpha-smooth muscle actin (α-SMA), glial fibrillary acidic protein (GFAP) and vinculin. We correlated immunopositive HSCs with HCV load, liver stiffness (LS), fibrosis stage and necro-inflammatory degree before treatment. Also, we assessed the association between liver fibrosis after therapy, the sustained virological response at 12 weeks after therapy (SVR 12) and the type of therapy. Results: HSCs were increased in VHC patients compared to controls, mainly in the intermediate and periportal lobular regions. α-SMA and vinculin HSCs correlated positively with fibrosis stage (p=0.044), (p=0.028). Furthermore, α-SMA and vinculin HSCs were associated with LS (p=0.027), (p=0.002) and viral load (p=0.021), (p=0.006), but not with necro-inflammation degree. GFAP HSCs inversely correlated with fibrosis stage (r= -0.475), LS (r= -0.422) and HCV load (r= -0.517), but positively with necro-inflammation degree (p=0.038). Liver fibrosis post therapy correlated positively with SVR12 (p<0.001) and the type of therapy (p=0.006) and SVR12 correlated positively with treatment's type (p=0.002). Conclusions: Activated HSCs may represent a marker of increased liver fibrosis in VHC. Different immunohistochemical markers can detect various HSCs subpopulations involved in the evolution of VHC and liver fibrosis. [ABSTRACT FROM AUTHOR]

Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins (p ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins (p < 0.001), whose differential expression was confirmed by ELISA (p = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR. [ABSTRACT FROM AUTHOR]

Talin-1 is the core mechanosensitive adapter protein linking integrins to the cytoskeleton. The TLN1 gene is comprised of 57 exons that encode the 2,541 amino acid TLN1 protein. TLN1 was previously considered to be expressed as a single isoform. However, through differential pre-mRNA splicing analysis, we discovered a cancer-enriched, non-annotated 51-nucleotide exon in TLN1 between exons 17 and 18, which we refer to as exon 17b. TLN1 is comprised of an N-terminal FERM domain, linked to 13 force-dependent switch domains, R1-R13. Inclusion of exon 17b introduces an in-frame insertion of 17 amino acids immediately after Gln665 in the region between R1 and R2 which lowers the force required to open the R1-R2 switches potentially altering downstream mechanotransduction. Biochemical analysis of this isoform revealed enhanced vinculin binding, and cells expressing this variant show altered adhesion dynamics and motility. Finally, we showed that the TGF-β/SMAD3 signaling pathway regulates this isoform switch. Future studies will need to consider the balance of these two TLN1 isoforms. [ABSTRACT FROM AUTHOR]

Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell-matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane-cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin-talin-F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein-protein and protein-lipid interfaces, including the membrane-binding and integrin affinityregulating F1 loop, as well as the interaction with lipid-anchored Rap1 (Rap1a and Rap1b in mammals) GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell-matrix attachment sites. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that thrives in the inflamed environment of the gingival crevice, and is strongly associated with periodontal disease. The host response to P. gingivalis requires TLR2, however P. gingivalis benefits from TLR2-driven signaling via activation of PI3K. We studied TLR2 protein-protein interactions induced in response to P. gingivalis, and identified an interaction between TLR2 and the cytoskeletal protein vinculin (VCL), confirmed using a split-ubiquitin system. Computational modeling predicted critical TLR2 residues governing the physical association with VCL, and mutagenesis of interface residues W684 and F719, abrogated the TLR2-VCL interaction. In macrophages, VCL knock-down led to increased cytokine production, and enhanced PI3K signaling in response to P. gingivalis infection, effects that correlated with increased intracellular bacterial survival. Mechanistically, VCL suppressed TLR2 activation of PI3K by associating with its substrate PIP2. P. gingivalis induction of TLR2-VCL led to PIP2 release from VCL, enabling PI3K activation via TLR2. These results highlight the complexity of TLR signaling, and the importance of discovering protein-protein interactions that contribute to the outcome of infection. Author summary: Porphyromonas gingivalis is an anaerobic bacterium strongly associated with periodontitis. Remarkably, P. gingivalis flourishes in the presence of inflammation and activated innate immune cells. P. gingivalis is sensed by TLR2, however, sensing leads to escape from macrophage bactericidal activity in a manner dependent on PI3K/Akt signaling. We used chemical cross-linking to identify induced interacting partners of TLR2 that contribute to bacterial immune escape, and found that infection induces TLR2 association with vinculin (VCL). Modeling the TLR2-VCL interaction highlighted critical amino acid residues of the TLR2 intracellular domain that were then experimentally validated by site-directed mutagenesis. We then confirmed that the induced TLR2-VCL interaction contributes to P. gingivalis immune evasion by enabling PI3K/Akt activation. Our results demonstrate the importance of induced TLR2 intracellular interacting partners in orchestrating downstream signaling that influences the outcome of the host-pathogen encounter. [ABSTRACT FROM AUTHOR]

The interface between the cellular actin network and diverse forms of integrin-mediated cell adhesions displays a unique capacity to serve as accurate chemical and mechanical sensors of the cell's microenvironment. Focal adhesion-like structures of diverse cell types, podosomes in osteoclasts, and invadopodia of invading cancer cells display distinct morphologies and apparent functions. Yet, all three share a similar composition and mode of coupling between a protrusive structure (the lamellipodium, the core actin bundle of the podosome, and the invadopodia protrusion, respectively), and a nearby adhesion site. Cytoskeletal or external forces, applied to the adhesion sites, trigger a cascade of unfolding and activation of key adhesome components (e.g., talin, vinculin, integrin), which in turn, trigger the assembly of adhesion sites and generation of adhesion-mediated signals that affect cell behavior and fate. The structural and molecular mechanisms underlying the dynamic crosstalk between the actin cytoskeleton and the adhesome network are discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Titanium alloys currently are the most used material for the manufacture of dental endosseous implants. However, in partially or totally edentulous patients, varying degrees of maxillary bone resorption usually occur, making the application of these devices difficult or even impossible. In these cases, a suitable alternative is offered by subperiosteal implants, whose use is undergoing a revival of interest following the introduction of novel, computer-assisted manufacturing techniques. Several procedures have been developed for the modification of titanium surfaces so to improve their biocompatibility and integration with bone. Information is, however, still incomplete as far as the most convenient surface modifications to apply with subperiosteal implants, in which an integration with soft mucosal tissues is just as important. Objectives: The present study aimed at evaluating whether different treatments of titanium surfaces can produce different effects on the viability, attachment, and differentiation of gingival fibroblasts, i.e., the cell type mainly involved in osteointegration as well as the healing of soft tissues injured by surgical procedures, in order to verify whether any of the treatments are preferable under these respects. Methodology: The human immortalized gingival fibroblast (CRL-4061 line) were cultured in the presence of titanium specimens previously treated with five different procedures for surface modification: (i) raw machined (Ti-1); (ii) electropolished (Ti-2); (iii) sand-blasted acid-etched (Ti-3); (iv) Al Ti Color™ proprietary procedure (Ti-4); and (v) anodized (Ti-5). At different times of incubation, viability and proliferation of cells, was determined along with the changes in the expression patterns of ECM-related genes involved in fibroblast attachment and differentiation: vinculin, fibronectin, collagen type I-alpha 1 chain, focal adhesion kinase, integrin β-1, and N-cadherin. Three different experiments were carried out for each experimental point. The release from fibroblasts of endothelin-1 was also analyzed as a marker of inflammatory response. The proliferation and migration of fibroblasts were evaluated by scratch tests. Results: None of the five types of titanium surface tested significantly affected the fibroblasts' viability and proliferation. The release of endothelin-1 was also not significantly affected by any of the specimens. On the other hand, all titanium specimens significantly stimulated the expression of ECM-related genes at varying degrees. The proliferation and migration abilities of fibroblasts were also significantly stimulated by all types of titanium surface, with a higher-to-lower efficiency in the order: Ti-3 > Ti-4 > Ti-5 > Ti-2 > Ti-1, thus identifying sandblasting acid-etching as the most convenient treatment. Conclusions: Our observations suggest that the titanium alloys used for manufacturing subperiosteal dental implants do not produce cytotoxic or proinflammatory effects on gingival fibroblasts, and that sandblasting acid-etching may be the surface treatment of choice as to stimulate the differentiation of gingival fibroblasts in the direction of attachment and migration, i.e., the features allegedly associated with a more efficient implant osteointegration, wound healing, and connective tissue seal formation. [ABSTRACT FROM AUTHOR]

The physicochemical properties of biomaterials influence cell adhesion, shape, and polarization of macrophages. In this study, we aimed to evaluate the polarization of macrophages in terms of the regulation of cell adhesion and how synthetic mimics for heparin and poly(sodium-4-styrenesulfonate) can regulate macrophage polarization by modulating cell shape, focal adhesion, cell traction force, and intracellular tension. Our initial findings showed that macrophages cultured with heparin-mimicking polymer-based hydrogel matrix showed reduced expression of cell adhesion markers such as integrins, vinculin, RhoA, and ROCK1/2 and reduced cell shape, elongation, cell-matrix traction force, and intracellular tension. Furthermore, we observed a significant decrease in cell adhesion in cells cultured on the hydrogel, resulting in the promotion of M1 polarization. These findings offer insights into the important roles of cell-matrix interactions in macrophage polarization and offer a platform for heparin-mimicking polymer-based hydrogel matrices to induce M1 polarization by inducing cell adhesion without classical activators. • M1 polarization is promoted by a heparin-mimicking polymer-based hydrogel matrix. • M1 macrophages polarized by LPS and heparin-mimicking polymer-based hydrogel matrix show reduced cell adhesion. • Heparin-mimicking polymer-based hydrogel matrix reduces cell adhesion and alters cell shape. • Heparin-mimicking polymer-based hydrogel matrix reduces traction force and intracellular tension of macrophages. [ABSTRACT FROM AUTHOR]

Background and aims Atherosclerosis preferentially develops in arterial branches and curvatures where vascular endothelium is exposed to disturbed flow. In this study, the effects of disturbed flow on the regulation of vascular endothelial phosphoproteins and their contribution to therapeutic application in atherogenesis were elucidated. Methods Porcine models, large-scale phosphoproteomics, transgenic mice, and clinical specimens were used to discover novel site-specific phosphorylation alterations induced by disturbed flow in endothelial cells (ECs). Results A large-scale phosphoproteomics analysis of native endothelium from disturbed (athero-susceptible) vs. pulsatile flow (athero-resistant) regions of porcine aortas led to the identification of a novel atherosclerosis-related phosphoprotein vinculin (VCL) with disturbed flow-induced phosphorylation at serine 721 (VCLS721p). The induction of VCLS721p was mediated by G-protein-coupled receptor kinase 2 (GRK2)S29p and resulted in an inactive form of VCL with a closed conformation, leading to the VE-cadherin/catenin complex disruption to enhance endothelial permeability and atherogenesis. The generation of novel apolipoprotein E-deficient (ApoE−/−) mice overexpressing S721-non-phosphorylatable VCL mutant in ECs confirmed the critical role of VCLS721p in promoting atherosclerosis. The administration of a GRK2 inhibitor to ApoE−/− mice suppressed plaque formation by inhibiting endothelial VCLS721p. Studies on clinical specimens from patients with coronary artery disease (CAD) revealed that endothelial VCLS721p is a critical clinicopathological biomarker for atherosclerosis progression and that serum VCLS721p level is a promising biomarker for CAD diagnosis. Conclusions The findings of this study indicate that endothelial VCLS721p is a valuable hemodynamic-based target for clinical assessment and treatment of vascular disorders resulting from atherosclerosis. [ABSTRACT FROM AUTHOR]

Summary: Lowe syndrome (LS) is a rare, X‐linked disorder characterised by numerous symptoms affecting the brain, the eyes, and the kidneys. It is caused by mutations in the oculocerebrorenal syndrome of Lowe (OCRL) protein, a 5‐phosphatase localised in different cellular compartments that dephosphorylates phosphatidylinositol‐4,5‐bisphosphate into phosphatidylinositol‐4‐monophosphate. Some patients with LS also have bleeding disorders, with normal to low platelet (PLT) count and impaired PLT function. However, the mechanism of PLT dysfunction in patients with LS is not completely understood. The main function of PLTs is to activate upon vessel wall injury and stop the bleeding by clot formation. PLT activation is accompanied by a shape change that is a result of massive cytoskeletal rearrangements. Here, we show that OCRL‐inhibited human PLTs do not fully spread, form mostly filopodia, and accumulate actin nodules. These nodules co‐localise with ARP2/3 subunit p34, vinculin, and sorting nexin 9. Furthermore, OCRL‐inhibited PLTs have a retained microtubular coil with high levels of acetylated tubulin. Also, myosin light chain phosphorylation is decreased upon OCRL inhibition, without impaired degranulation or integrin activation. Taken together, these results suggest that OCRL contributes to cytoskeletal rearrangements during PLT activation that could explain mild bleeding problems in patients with LS. [ABSTRACT FROM AUTHOR]

The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding α-catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of α-catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of α-catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that increased tension sensitivity of α-catenin affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive α-catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that the appropriate tension sensitivity of α-catenin is crucial for the coordinated round morphogenesis. Key words: α-catenin, vinculin, adherens junction, morphogenesis, mechanotransduction

Poly(ADP-ribose) (PAR) is a highly negatively charged polymer. PAR is synthesized by poly(ADP-ribose) polymerases (PARPs) and is involved in the assembly and stabilization of macromolecular complexes. Here, the presence and putative roles of poly(ADP-ribosyl)ation (PARylation) associated to adherens junctions (AJ) and the actin cytoskeleton in epithelial and Schwann cells, is reviewed. The hypothesis generated by analogy, stating that PAR is associated to AJ in other cell types, is postulated. According to this hypothesis, PAR associated to puncta adherentia in chemical synapses would participate in plasticity, learning and memory. In turn, PAR associated to fascia adherens in cardiomyocytes, would affect heart beating. PARP inhibitors are currently under development and clinical testing. Basic research in different tissues will probably influence their clinical uses. [ABSTRACT FROM AUTHOR]

Skin pigmentation is among the defenses against ultraviolet (UV) radiation. During formation of skin pigmentation, melanosomes that are transported to the cell membrane and released are internalized by keratinocytes. We here examined whether vinculin, the origin of actin fibers, is involved in this intracellular transport of melanosomes by using melanocytes with suppressed vinculin expression. Using fluorescence immunostaining, the migration of vinculin to the cell membrane due to exposure to 365-nm LED light was examined. The intracellular distribution of melanosomes after irradiation was weighted toward the pericellular region compared with non-irradiated cells. With the suppression of vinculin expression, the amount of extracellularly released melanin decreased. We conclude that the membrane migration of vinculin after UVA exposure is involved in the intracellular transport of melanosomes. [ABSTRACT FROM AUTHOR]

The assembly of a mature vascular network involves coordinated endothelial cell (EC) shape changes, including the process of EC elongation. How EC elongation is dynamically regulated in vivo is not fully understood. Here, we have generated a zebrafish mutant that is deficient for the integrin adaptor protein Talin 1 (Tln1). Using a new focal adhesion (FA) marker line expressing endothelial VinculinbeGFP, we demonstrate that EC FAs function dynamically and are lost in our tln1 mutants, allowing us to uncouple the primary roles of FAs in EC morphogenesis from the secondary effects that occur due to systemic vessel failure or loss of blood flow. Tln1 loss led to compromised F-actin rearrangements, perturbed EC elongation and disrupted cell-cell junction linearisation in vessel remodelling. Finally, chemical induction of actin polymerisation restored actin dynamics and EC elongation during vascular morphogenesis. Together, we identify that FAs are essential for EC elongation and junction linearisation in flow-pressured vessels and that they influence actin polymerisation in cellular morphogenesis. These observations can explain the severely compromised vessel beds and vascular leakage observed in mutant models that lack integrin signalling. [ABSTRACT FROM AUTHOR]

The Mercator projection map of the world provides a useful, but distorted, view of the relative scale of countries. Current cellular models suffer from a similar distortion. Here, we undertook an in-depth structural analysis of the molecular dimensions in the cell's computational machinery, the MeshCODE, that is assembled from a meshwork of binary switches in the scaffolding proteins talin and vinculin. Talin contains a series of force-dependent binary switches and each domain switching state introduces quantised step-changes in talin length on a micrometre scale. The average dendritic spine is 1 mm in diameter so this analysis identifies a plausible Gearbox-like mechanism for dynamic regulation of synaptic function, whereby the positioning of enzymes and substrates relative to each other, mechanically-encoded by the MeshCODE switch patterns, might control synaptic transmission. Based on biophysical rules and experimentally derived distances, this analysis yields a novel perspective on biological digital information. [ABSTRACT FROM AUTHOR]

To penetrate the zona pellucida before sperm-egg binding, sperm must undergo highly time-controlled capacitation and acrosome reaction in the female reproductive tract. Our previous study demonstrated that miR-21-5p is the most abundant miRNA in boar seminal plasma (SP)-derived extracellular vesicles (EVs) and can target Vinculin (VCL) gene, which may participate in boar sperm capacitation. Thus, this study aims to explore the potential role of miR-21-5p from SP-derived EVs in preventing sperm capacitation and its underlying mechanism. We observed that sperm could incorporate miR-21-5p from SP-derived EVs. The roles of SP-derived EVs miR-21-5p in sperm capacitation were then determined using gain- and loss-of-function analyses. In addition, the expression levels of miR-21-5p, VCL, and VCL protein in liquid-preserved boar sperm following transfection were determined using RT-qPCR and Western blotting. Our results revealed that miR-21-5p overexpression inhibited sperm capacitation and acrosome reaction. Similarly, miR-21-5p expression was significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. However, the protein level of VCL was also significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. Furthermore, immunofluorescence analysis showed that VCL protein mainly located in sperm head and sperm capacitation was inhibited after treating with VCL protein inhibitor (Chrysin). In conclusion, our study provides reasonable evidence that miR-21-5p expression in SP-derived EVs could prevent sperm capacitation via VCL inhibition. • Sperm could incorporate miR-21-5p from SP-derived EVs. • MiR-21-5p inhibited sperm capacitation and acrosome reaction. • MiR-21-5p was down regulated in boar sperm during sperm capacitation. • VCL protein was required for sperm capacitation. [ABSTRACT FROM AUTHOR]

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 Drosophila 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. [ABSTRACT FROM AUTHOR]

Rationale: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. Objectives: To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Methods: 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. Measurements and Main Results: 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. Conclusions: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Denaturation of collagen inhibits adhesion and migration of HUVECs, while assembly favors these behaviors. • Expression of vinculin and F-actin are positively correlated with adhesion and migration behaviors. Cell adhesion and migration behaviors are widely involved in physiological processes, and can be mediated by various factors. Collagen is one representative constituents of extracellular matrix, possessing advantages including excellent biocompatibility, low immunogenicity, and ease to be engineered, which makes collagen be promising biomaterial for regulating cell behaviors. Furthermore, the biofunction of collagen is closely correlated with its configuration. Whereas, current studies on collagen-based biomaterials for regulating cell adhesion and migration are rare, let alone the exploration of the ralationship between the configurations and these two behaviors. (Human umbilical vein endothelial cells, HUVEC) plays significant roles in wounds healing and cancer progression. Therefore, we aimed at disclosing the relationships between the adhesion/migration capability and the collagen origin, structure, and molecular state. In summary, results clearly revealed that denaturation disfavors the adhesion/migration of HUVEC cells, while assembly promotes. The expression levels of F-actin and vinculin, together with the quantity of filopodia were positively correlated with the two processes. Finnaly, our study may provide directions for the further designing of collagen-based biomaterials in the treatment of these kinds of severe diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: In the present study, we measured the serum vascular endothelial cadherin (VEC) and vinculin (Vcn) concentrations in patients with type B acute aortic dissection (TBAD) to evaluate their diagnostic value for this condition. Methods: A total of 100 patients with TBAD and 90 matched controls were included in the study. The serum concentrations of VEC and Vcn were measured using enzyme-linked immunosorbent assays. Results: The serum VEC and Vcn concentrations were significantly higher in participants with TBAD than in healthy controls. Compared with patients with acute myocardial infarction (AMI), the serum concentrations of VEC and Vcn in patients with TBAD were higher, and the Vcn showed significant difference, with statistical significance. Receiver operating characteristic analysis generated areas under the curves for VEC and Vcn that were diagnostic for TBAD (0.599 and 0.655, respectively). The optimal cut-off values were 3.975 ng/μL and 128.1 pg/mL, the sensitivities were 43.0% and 35.0%, and the specificities were 73.3% and 90.0%, respectively. In addition, the use of a combination of serum VEC and Vcn increased the AUC to 0.661, with a sensitivity of 33.0% and a specificity of 93.33%. A high serum Vcn concentration was associated with a higher risk of visceral malperfusion in participants with TBAD (odds ratio (OR) = 1.007, 95% confidence interval [CI]: 1.001–1.013, p = 0.014). In participants with refractory pain, the adjusted OR for the serum VEC concentration increased to 1.172 (95% CI: 1.010–1.361; p = 0.036), compared with participants without refractory pain. Conclusion: This study is the first to show the diagnostic value of serum VEC and Vcn for AAD and their relationships with the clinical characteristics of patients with TBAD. Thus, VEC and Vcn are potential serum markers of TBAD.

The Mercator projection map of the world provides a useful, but distorted, view of the relative scale of countries. Current cellular models suffer from a similar distortion. Here, we undertook an in-depth structural analysis of the molecular dimensions in the cell’s computational machinery, the MeshCODE, that is assembled from a meshwork of binary switches in the scaffolding proteins talin and vinculin. Talin contains a series of force-dependent binary switches and each domain switching state introduces quantised step-changes in talin length on a micrometre scale. The average dendritic spine is 1 μm in diameter so this analysis identifies a plausible Gearbox-like mechanism for dynamic regulation of synaptic function, whereby the positioning of enzymes and substrates relative to each other, mechanically-encoded by the MeshCODE switch patterns, might control synaptic transmission. Based on biophysical rules and experimentally derived distances, this analysis yields a novel perspective on biological digital information.

Introduction: Cellular homeostasis with its environment is bidirectional and dynamic in nature. Extracellular matrix plays an important role in retinoblastoma tumor cell's response to chemotherapeutic drugs. The present study was designed to investigate the effect of mechanotransduction along with the chemotherapeutic effect on RB cells grown on three-dimensional matrices. Methods: Matrigels of varying stiffness (low and high) were prepared and characterized. Drug sensitivity of RB cells on various matrigel stiffness was measured using cytotoxicity (MTT) assay and IC50 value was found for carboplatin and etoposide. Further, the effect on the proliferation and migration of RB cells in various stiffness matrigels was investigated. Findings: The results showed that drug sensitivity decreased with an increase in matrix stiffness when compared to RB cells grown in suspension. In higher stiffness, the drug sensitivity decreases with an increase in cell proliferation. Data from a gene expression study revealed that Vinculin was variably regulated between metastatic and non-metastatic tumor cells, and upregulated RhoA was altered in different stiffness gradient matrigel during drug treatment. Interpretation: These results enhance the understanding of the fundamental molecules responding to drug sensitivity in a stiffness-altering environment. Additionally, it provides a new avenue for understanding how RB cells respond to chemotherapeutic intervention.