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Journal of Cell Science Mar 2021Hippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6 and LIMD1 have each been identified as being required for tension-dependent inhibition...
Hippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6 and LIMD1 have each been identified as being required for tension-dependent inhibition of the Hippo pathway LATS kinases and their recruitment to adherens junctions, but the relationship between TRIP6 and LIMD1 was unknown. Using siRNA-mediated gene knockdown, we show that TRIP6 is required for LIMD1 localization to adherens junctions, whereas LIMD1 is not required for TRIP6 localization. TRIP6, but not LIMD1, is also required for the recruitment of vinculin and VASP to adherens junctions. Knockdown of TRIP6 or vinculin, but not of LIMD1, also influences the localization of myosin and F-actin. In TRIP6 knockdown cells, actin stress fibers are lost apically but increased basally, and there is a corresponding increase in the recruitment of vinculin and VASP to basal focal adhesions. Our observations identify a role for TRIP6 in organizing F-actin and maintaining tension at adherens junctions that could account for its influence on LIMD1 and LATS. They also suggest that focal adhesions and adherens junctions compete for key proteins needed to maintain attachments to contractile F-actin.
Topics: Actin Cytoskeleton; Actins; Adherens Junctions; Cytoskeleton; Focal Adhesions; Vinculin
PubMed: 33558314
DOI: 10.1242/jcs.247866 -
Developmental Dynamics : An Official... Jul 2023Drosophila Singed (mammalian Fascin) is an actin-binding protein that is known mainly for bundling parallel actin filaments. Among many functions of Singed, it is...
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.
Topics: Animals; Actin Cytoskeleton; Actins; Cell Movement; Drosophila; Vinculin
PubMed: 36912821
DOI: 10.1002/dvdy.585 -
Scientific Reports Jul 2019Focal adhesions (FAs) are multiprotein structures that link the intracellular cytoskeleton to the extracellular matrix. They mediate cell adhesion and migration, crucial...
Focal adhesions (FAs) are multiprotein structures that link the intracellular cytoskeleton to the extracellular matrix. They mediate cell adhesion and migration, crucial to many (patho-) physiological processes. We examined in two cell types from different species the binding dynamics of functionally related FA protein pairs: paxillin and vinculin versus zyxin and VASP. In photobleaching experiments ~40% of paxillin and vinculin remained stably associated with a FA for over half an hour. Zyxin and VASP predominantly displayed more transient interactions. We show protein binding dynamics are influenced by FA location and orientation. In FAs located close to the edge of the adherent membrane paxillin, zyxin and VASP were more dynamic and had larger bound fractions. Zyxin and VASP were also more dynamic and had larger bound fractions at FAs perpendicular compared to parallel to this edge. Finally, we developed a photoconversion assay to specifically visualise stably bound proteins within subcellular structures and organelles. This revealed that while paxillin and vinculin are distributed evenly throughout FAs, their stably bound fractions form small clusters within the FA-complex. These clusters are more concentrated for paxillin than for vinculin and are mostly found at the proximal half of the FA where actin also enters.
Topics: Animals; Bone Neoplasms; Cell Adhesion Molecules; Cytoskeleton; Dogs; Extracellular Matrix; Focal Adhesions; Humans; Madin Darby Canine Kidney Cells; Microfilament Proteins; Osteosarcoma; Paxillin; Phosphoproteins; Tumor Cells, Cultured; Vinculin; Zyxin
PubMed: 31320676
DOI: 10.1038/s41598-019-46905-2 -
Vascular Biology (Bristol, England) Jan 2023Remodelling of cell-cell junctions is crucial for proper tissue development and barrier function. The cadherin-based adherens junctions anchor via β-catenin and...
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.
PubMed: 36260739
DOI: 10.1530/VB-22-0012 -
Cell Reports Nov 2023Focal adhesions (FAs) are dynamic protein assemblies that connect cytoskeletons to the extracellular matrix and are crucial for cell adhesion and migration. KANKs are...
Focal adhesions (FAs) are dynamic protein assemblies that connect cytoskeletons to the extracellular matrix and are crucial for cell adhesion and migration. KANKs are scaffold proteins that encircle FAs and act as key regulators of FA dynamics, but the molecular mechanism underlying their specified localization and functions remains poorly understood. Here, we determine the KANK1 structures in complex with talin and liprin-β, respectively. These structures, combined with our biochemical and cellular analyses, demonstrate how KANK1 scaffolds the FA core and associated proteins to modulate the FA shape in response to mechanical force. Additionally, we find that KANK1 undergoes liquid-liquid phase separation (LLPS), which is important for its localization at the FA edge and cytoskeleton connections to FAs. Our findings not only indicate the molecular basis of KANKs in bridging the core and periphery of FAs but also provide insights into the LLPS-mediated dynamic regulation of FA morphology.
Topics: Focal Adhesions; Protein Binding; Cell Adhesion; Cytoskeleton; Talin
PubMed: 37874676
DOI: 10.1016/j.celrep.2023.113321 -
Cell Stem Cell Nov 2020Matrix dynamics influence how individual cells develop into complex multicellular tissues. Here, we develop hydrogels with identical polymer components but different...
Matrix dynamics influence how individual cells develop into complex multicellular tissues. Here, we develop hydrogels with identical polymer components but different crosslinking capacities to enable the investigation of mechanisms underlying vascular morphogenesis. We show that dynamic (D) hydrogels increase the contractility of human endothelial colony-forming cells (hECFCs), promote the clustering of integrin β1, and promote the recruitment of vinculin, leading to the activation of focal adhesion kinase (FAK) and metalloproteinase expression. This leads to the robust assembly of vasculature and the deposition of new basement membrane. We also show that non-dynamic (N) hydrogels do not promote FAK signaling and that stiff D- and N-hydrogels are constrained for vascular morphogenesis. Furthermore, D-hydrogels promote hECFC microvessel formation and angiogenesis in vivo. Our results indicate that cell contractility mediates integrin signaling via inside-out signaling and emphasizes the importance of matrix dynamics in vascular tissue formation, thus informing future studies of vascularization and tissue engineering applications.
Topics: Endothelial Cells; Humans; Hydrogels; Morphogenesis; Signal Transduction; Tissue Engineering
PubMed: 32931729
DOI: 10.1016/j.stem.2020.08.005 -
Journal of Biochemical and Molecular... Sep 2020The influences of Vinculin on many cancers were blurry, including ovarian cancer. Thus, we concentrated on the efficient role of Vinculin in ovarian cancer and explored...
The influences of Vinculin on many cancers were blurry, including ovarian cancer. Thus, we concentrated on the efficient role of Vinculin in ovarian cancer and explored the potential mechanism(s). Expression of Vinculin in ovarian cancer tissues and cell lines was investigated by real-time polymerase chain reaction, immunohistochemistry, and Western blot. The Kaplan-Meier manner with the logrank was performed to assess overall survival. We further evaluated the relations between Vinculin expression and clinicopathological features of ovarian cancer. Moreover, Vinculin was overexpressed or silenced by respectively transfection with pcDNA-Vinculin or small interfering (si-Vinculin) into human ovarian cancer cell line Caov3 or human ovarian epithelial cell line (HOEpiC). Thereafter, cell viability, cell apoptosis, and migration were checked by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, flow cytometer, and scratch assay, respectively. Likewise, the apoptosis- and migration-related proteins were distinguished by Western blot. Compared to the nontumor tissues or HOEpiC cells, Vinculin was significantly lower expressed in the ovarian cancer tissues and cells. Furthermore, we found out that Vinculin was primarily distributed at the cell membrane and cytoplasm. Moreover, Vinculin was negatively associated with International Federation of Gynecology and Obstetrics stage, grade, and distant metastasis. Overexpression of Vinculin dramatically weakened cell viability and migration and stimulated apoptosis. Conversely, suppression of Vinculin showed opposite results. Vinculin presents unfavorable prediction in ovarian cancer and inhibits ovarian cancer proliferation and migration.
PubMed: 32369671
DOI: 10.1002/jbt.22525 -
Current Eye Research Apr 2021RhoA signaling is important for the regulation of intraocular pressure through the trabecular meshwork (TM). However, the relationship between RhoA signaling and...
PURPOSE
RhoA signaling is important for the regulation of intraocular pressure through the trabecular meshwork (TM). However, the relationship between RhoA signaling and phagocytosis in TM cells is unclear. The purpose of this study was to investigate the effects of RhoA signaling on the phagocytosis of TM cells.
MATERIALS AND METHODS
TM cells were isolated from enucleated porcine eyes and treated with lysophosphatidic acid (LPA) or calpeptin to activate RhoA to determine phagocytic activity. To assess phagocytic activity, TM cells were incubated with pHrodo® Red bioparticles, and the fluorescence intensity was measured using a cell sorter. The phagocytic activity of RhoA knockdown TM cells was also assessed using small interfering RNA (siRNA). To resolve the effects of dexamethasone on phagocytosis, TM cells were treated with dexamethasone for 72 h. The immunocytochemistry of vinculin and F-actin were evaluated in LPA- and dexamethasone-treated TM cells.
RESULTS
RhoA activities after treatment with 10 µM LPA and 100 µM calpeptin were 1.38 ± 0.026-fold and 1.47 ± 0.070-fold higher, respectively, compared with the control. The phagocytic activity was reduced by LPA (0.67 ± 0.099) and calpeptin (0.57 ± 0.016), compared with the control. C3 transferase (Rho inhibitor) and Y-27632 (Rho-associated kinase inhibitor) prevented the effects of LPA on phagocytosis, and C3 partially inhibited the effects of calpeptin on phagocytosis. Knockdown of RhoA prevented the effect of LPA on phagocytosis. By immunostaining, LPA-induced stress fiber and focal adhesion formation was prevented by C3 and Y-27632 treatment. Moreover, RhoA knockdown prevented the effects of LPA on F-actin and focal adhesion. Dexamethasone treatment decreased phagocytic activity and increased stress fiber and focal adhesion. Y-27632 prevented the effects of dexamethasone on phagocytosis, and on stress fiber and focal adhesion fomation.
CONCLUSIONS
These results suggest that the RhoA signal pathway regulates the phagocytic activity of TM cells.
UNLABELLED
: TM: trabecular meshwork; LPA: lysophosphatidic acid; C3: C3 transferase; ROCK: Rho-associated kinase; siRNA: small interfering RNA.
Topics: Actins; Animals; Blotting, Western; Cell Survival; Cells, Cultured; Dexamethasone; Dipeptides; Glucocorticoids; Immunohistochemistry; Lysophospholipids; Phagocytosis; Signal Transduction; Staphylococcus aureus; Swine; Trabecular Meshwork; Vinculin; rhoA GTP-Binding Protein
PubMed: 32847411
DOI: 10.1080/02713683.2020.1815791 -
Journal of Cellular Biochemistry Aug 2019Vinculin is a highly conserved protein involved in cell proliferation, migration, and adhesion. However, the effects of vinculin on gastric cancer (GC) remain unclear....
Vinculin is a highly conserved protein involved in cell proliferation, migration, and adhesion. However, the effects of vinculin on gastric cancer (GC) remain unclear. Therefore, we aimed to explore the functional role of vinculin in GC, as well as its underlying mechanism. Expression of vinculin in patients with GC was analyzed by real-time polymerase chain reaction, Western blot analysis, and immunohistochemistry. Overall survival was evaluated by the Kaplan-Meier method with the log-rank test. The relationship between vinculin and clinicopathological characteristics of patients with GC was further identified. In addition, we assessed the expression of vinculin in GC cell lines. Besides, vinculin was suppressed or overexpressed by transfection with small interfering (si-vinculin) or pcDNA-vinculin and then cell viability, cell apoptosis, and/or migration was respectively examined by the 3-(4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide assay, flow cytometer, and scratch assay, respectively. Moreover, the cell cycle- and apoptosis-related proteins were detected by Western blot analysis. The expression of vinculin was significantly increased in the GC tissues and cells compared with the nontumor tissues or cells. Vinculin protein positive staining was mainly located in the cell membrane and cytoplasm. Moreover, vinculin was significantly associated with Tumor Node Metastasis (TNM) and poor differentiation. Patients with high vinculin levels had significantly worse overall survival than those with low levels. Suppression of vinculin significantly decreased cell viability and migration and promoted cell apoptosis. However, overexpression of vinculin statistically increased cell viability but had no effects on cell apoptosis. Vinculin promotes GC proliferation and migration and predicts poor prognosis in patients with GC.
Topics: Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Prognosis; RNA, Messenger; Stomach Neoplasms; Vinculin
PubMed: 30989694
DOI: 10.1002/jcb.28686 -
Orthopaedic Surgery Feb 2023To explore the potential effect of three allogenic bone substitute configurations on the viability, adhesion, and spreading of osteoblasts in vitro.
OBJECTIVE
To explore the potential effect of three allogenic bone substitute configurations on the viability, adhesion, and spreading of osteoblasts in vitro.
METHODS
Freeze-dried cortical bone were ground and fractions were divided into three groups with different sizes and shapes, defined as bone fiber (0.1 mm × 0.1 mm × 3 mm), bone powder (0.45-0.9 mm), and bone granule group (3-6 mm). MC3T3-E1 cells were divided and co-cultured within groups to induce cell adhesion. The configuration of allogenic bone was captured by scanning electron microscopy and confocal laser scanning microscopy, and substrate roughness values were quantified. Cell adhesion rate was assessed using the hemocyte counting method, cell viability was determined by CCK-8 assay and live/dead staining, and cell morphology was visualized by Phalloidin and DAPI, and the mRNA expression of adhesion-related gene (vinculin) of different substitutes were determined with quantitative real-time polymerase chain reaction.
RESULTS
The roughness values of bone fiber, bone powder, and bone granule group were 1.878 μm (1.578-2.415 μm), 5.066 μm (3.891-6.162 μm), and 0.860 μm (0.801-1.452 μm), respectively (bone powder group compared with bone granule group, H = 18.015, P < 0.001). Similar OD values of all groups in CCK-8 assay indicated good biocompatibility of these substitutes (bone fiber, 0.201 ± 0.004; bone powder, 0.206 ± 0.008; bone granule group, 0.197 ± 0.006; and the control group, 0.202 ± 0.016, F = 0.7152, P > 0.05). In addition, representative cell adhesion rates at 24 h showed significantly lower cell adhesion rate in bone fiber group (20.3 ± 1.6%) compared to bone powder (29.3 ± 4.4%) and bone granule group (27.3 ± 3.2%) (F = 10.51,P = 0.009 and P = 0.034, respectively), but there was no significant difference between the latter two groups (P > 0.05). Interestingly, the expression of vinculin mRNA steadily decreased in a time-dependent manner. The vinculin expression reached its peak at 6 h in each group, and the vinculin levels in bone fiber, bone powder, and bone granule group were 2.119 ± 0.052, 3.842 ± 0.108, and 3.585 ± 0.068 times higher than those in the control group, respectively (F = 733.643, all P < 0.001). Meanwhile, there was a significant difference in the expression of target gene between bone powder and bone granule group (P = 0.006).
CONCLUSION
All allogenic bone substitutes presented an excellent cell viability. Moreover, bone powder and bone granule group were more likely to promote cell adhesion and spreading compared to bone fiber group.
Topics: Humans; Cell Adhesion; Bone Substitutes; Vinculin; Powders; Osteoblasts; RNA, Messenger; Hematopoietic Stem Cell Transplantation; Cell Proliferation
PubMed: 36453151
DOI: 10.1111/os.13395