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European Journal of Cell Biology Jun 2024Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and...
Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and transducer by recruiting vinculin and transducing signals that influence cell behaviors. α-catenin/vinculin complex-mediated mechanotransduction regulates multiple pathways, such as Hippo pathway. However, their associations with the α-catenin-based tension sensors at cell junctions are still not fully addressed. Here, we uncovered the TRIP6/LATS1 complex co-localizes with α-catenin/vinculin at both bicellular junctions (BCJs) and tricellular junctions (TCJs). The localization of TRIP6/LATS1 complex to both TCJs and BCJs requires ROCK1 and α-catenin. Treatment by cytochalasin B, Y-27632 and blebbistatin all impaired the BCJ and TCJ junctional localization of TRIP6/LATS1, indicating that the junctional localization of TRIP6/LATS1 is mechanosensitive. The α-catenin/vinculin/TRIP6/LATS1 complex strongly localized to TCJs and exhibited a discontinuous button-like pattern on BCJs. Additionally, we developed and validated an α-catenin/vinculin BiFC-based mechanosensor that co-localizes with TRIP6/LATS1 at BCJs and TCJs. The mechanosensor exhibited a discontinuous distribution and motile signals at BCJs. Overall, our study revealed that TRIP6 and LATS1 are novel compositions of the tension sensor, together with the core complex of α-catenin/vinculin, at both the BCJs and TCJs.
Topics: alpha Catenin; Humans; Protein Serine-Threonine Kinases; Vinculin; Mechanotransduction, Cellular; Adaptor Proteins, Signal Transducing; Intercellular Junctions; HEK293 Cells; rho-Associated Kinases; Transcription Factors
PubMed: 38805800
DOI: 10.1016/j.ejcb.2024.151426 -
Cytoskeleton (Hoboken, N.J.) May 2024Focal adhesions serve as structural and signaling hubs, facilitating bidirectional communication at the cell-extracellular matrix interface. Paxillin and the related...
Focal adhesions serve as structural and signaling hubs, facilitating bidirectional communication at the cell-extracellular matrix interface. Paxillin and the related Hic-5 (TGFβ1i1) are adaptor/scaffold proteins that recruit numerous structural and regulatory proteins to focal adhesions, where they perform both overlapping and discrete functions. In this study, paxillin and Hic-5 were expressed in U2OS osteosarcoma cells as biotin ligase (BioID2) fusion proteins and used as bait proteins for proximity-dependent biotinylation in order to directly compare their respective interactomes. The fusion proteins localized to both focal adhesions and the centrosome, resulting in biotinylation of components of each of these structures. Biotinylated proteins were purified and analyzed by mass spectrometry. The list of proximity interactors for paxillin and Hic-5 comprised numerous shared core focal adhesion proteins that likely contribute to their similar functions in cell adhesion and migration, as well as proteins unique to paxillin and Hic-5 that have been previously localized to focal adhesions, the centrosome, or the nucleus. Western blotting confirmed biotinylation and enrichment of FAK and vinculin, known interactors of Hic-5 and paxillin, as well as several potentially unique proximity interactors of Hic-5 and paxillin, including septin 7 and ponsin, respectively. Further investigation into the functional relationship between the unique interactors and Hic-5 or paxillin may yield novel insights into their distinct roles in cell migration.
PubMed: 38801098
DOI: 10.1002/cm.21878 -
Frontiers in Oncology 2024Cancer metastasis is dependent on cell migration. Several mechanisms, including epithelial-to-mesenchymal transition (EMT) and actin fiber formation, could be involved...
BACKGROUND
Cancer metastasis is dependent on cell migration. Several mechanisms, including epithelial-to-mesenchymal transition (EMT) and actin fiber formation, could be involved in cancer cell migration. As a downstream effector of the Hippo signaling pathway, transcriptional coactivator with PDZ-binding motif (TAZ) is recognized as a key mediator of the metastatic ability of breast cancer cells. We aimed to examine whether TAZ affects the migration of breast cancer cells through the regulation of EMT or actin cytoskeleton.
METHODS
MCF-7 and MDA-MB-231 cells were treated with siRNA to attenuate TAZ abundance. Transwell migration assay and scratch wound healing assay were performed to study the effects of TAZ knockdown on cancer cell migration. Fluorescence microscopy was conducted to examine the vinculin and phalloidin. Semiquantitative immunoblotting and quantitative real-time PCR were performed to study the expression of small GTPases and kinases. Changes in the expression of genes associated with cell migration were examined through next-generation sequencing.
RESULTS
TAZ-siRNA treatment reduced TAZ abundance in MCF-7 and MDA-MB-231 breast cancer cells, which was associated with a significant decrease in cell migration. TAZ knockdown increased the expression of fibronectin, but it did not exhibit the typical pattern of EMT progression. TGF-β treatment in MDA-MB-231 cells resulted in a reduction in TAZ and an increase in fibronectin levels. However, it paradoxically promoted cell migration, suggesting that EMT is unlikely to be involved in the decreased migration of breast cancer cells in response to TAZ suppression. RhoA, a small Rho GTPase protein, was significantly reduced in response to TAZ knockdown. This caused a decrease in the expression of the Rho-dependent downstream pathway, i.e., LIM kinase 1 (LIMK1), phosphorylated LIMK1/2, and phosphorylated cofilin, leading to actin depolymerization. Furthermore, myosin light chain kinase (MLCK) and phosphorylated MLC2 were significantly decreased in MDA-MB-231 cells with TAZ knockdown, inhibiting the assembly of stress fibers and focal adhesions.
CONCLUSION
TAZ knockdown inhibits the migration of breast cancer cells by regulating the intracellular actin cytoskeletal organization. This is achieved, in part, by reducing the abundance of RhoA and Rho-dependent downstream kinase proteins, which results in actin depolymerization and the disassembly of stress fibers and focal adhesions.
PubMed: 38774409
DOI: 10.3389/fonc.2024.1376831 -
Archives of Biochemistry and Biophysics Jul 2024Biomechanical signals in the extracellular niche are considered promising for programming the lineage specification of stem cells. Recent studies have reported that...
Biomechanical signals in the extracellular niche are considered promising for programming the lineage specification of stem cells. Recent studies have reported that biomechanics, such as the microstructure of nanomaterials, can induce adipose-derived stem cells (ASCs) to differentiate into osteoblasts, mediating gene regulation at the epigenetic level. Therefore, in this study, transcriptome expression levels of histone demethylases in ASCs were screened after treatment with different matrix stiffnesses, and histone lysine demethylase 3B (KDM3B) was found to promote osteogenic differentiation of ASCs in response to matrix stiffness, indicating a positive modulatory effect on this biological process. ASCs exhibited widespread and polygonal shapes with a distinct bundle-like expression of vinculin parallel to the axial cytoskeleton along the cell margins on the stiff matrix rather than round shapes with a smeared and shorter expression on the soft matrix. Comparatively rigid polydimethylsiloxane material directed ASCs into an osteogenic phenotype in inductive culture media via the upregulation of osteocalcin, alkaline phosphatase, and runt-related transcription factor 2. Treatment with KDM3B-siRNA decreased the expression of osteogenic differentiation markers and impaired mitochondrial dynamics and mitochondrial membrane potential. These results illustrate the critical role of KDM3B in the biomechanics-induced osteogenic commitment of ASCs and provide new avenues for the further application of stem cells as potential therapeutics for bone regeneration.
Topics: Osteogenesis; Humans; Cell Differentiation; Jumonji Domain-Containing Histone Demethylases; Adipose Tissue; Stem Cells; Cells, Cultured; Extracellular Matrix; Dimethylpolysiloxanes
PubMed: 38768746
DOI: 10.1016/j.abb.2024.110028 -
Life Science Alliance Aug 2024Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in , encoding the first step in the synthesis...
Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in , encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that in mice there is a failure of the α7β1 integrin complex that is specific to affected muscle. We observed that in hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression of .
Topics: Animals; Mice; Vinculin; Mice, Knockout; Muscular Dystrophies; Integrins; Choline Kinase; Sarcolemma; Humans; Focal Adhesions; Cell Membrane; Actinin; Muscle, Skeletal; Phosphatidylinositol 4,5-Diphosphate; Actins; Disease Models, Animal
PubMed: 38749543
DOI: 10.26508/lsa.202301956 -
Cancers Apr 2024Colorectal tumorigenesis involves the development of aberrant crypt foci (ACF) or preneoplastic lesions, representing the earliest morphological lesion visible in colon...
Colorectal tumorigenesis involves the development of aberrant crypt foci (ACF) or preneoplastic lesions, representing the earliest morphological lesion visible in colon cancer. The purpose of this study was to determine changes in protein expression in carcinogen-induced ACF as they mature and transform into adenomas. Protein expression profiles of azoxymethane (AOM)-induced F344 rat colon ACF and adenomas were compared at four time points, 4 (control), 8, 16, and 24 weeks post AOM administration ( = 9/group), with time points correlating with induction and transformation events. At each time point, micro-dissected ACF and/or adenoma tissues were analyzed across multiple quantitative two-dimensional (2D-DIGE) gels using a Cy-dye labeling technique and a pooled internal standard to quantify expression changes with statistical confidence. Western blot and subsequent network pathway mapping were used to confirm and elucidate differentially expressed ( ≤ 0.05) proteins, including changes in vinculin (; = 0.007), scinderin (; = 0.02), and profilin (; = 0.01), By determining protein expression changes in ACF as they mature and transform into adenomas, a "baseline" of altered regulatory proteins associated with adenocarcinoma development in this model has been elucidated. These data will enable future studies aimed at biomarker identification and understanding the molecular biology of intestinal tumorigenesis and adenocarcinoma maturation under varying intestinal conditions.
PubMed: 38730628
DOI: 10.3390/cancers16091678 -
Cells May 2024Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several...
Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of β-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-β-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.
Topics: Animals; Actin Cytoskeleton; Actinin; Adherens Junctions; alpha Catenin; beta Catenin; Cell Adhesion; Cell Line, Tumor; Cell Movement; Epithelial Cells; Epithelial-Mesenchymal Transition; Integrins; Phosphorylation; Vinculin; Zyxin; Rats
PubMed: 38727316
DOI: 10.3390/cells13090780 -
APL Bioengineering Jun 2024Cardiac tissue engineering has emerged as a promising approach for restoring the functionality of damaged cardiac tissues following myocardial infarction. To effectively...
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 , 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-m-width and 20-m-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 .
PubMed: 38699629
DOI: 10.1063/5.0182585 -
Toxicology in Vitro : An International... Jun 2024Silver nanoparticles (AgNPs) are increasingly incorporated in diverse products to confer antimicrobial properties. They are released into the environment during...
Silver nanoparticles (AgNPs) are increasingly incorporated in diverse products to confer antimicrobial properties. They are released into the environment during manufacture, after disposal, and from the products during use. Because AgNPs bioaccumulate in brain, it is important to understand how they interact with neural cell physiology. We found that the focal adhesion (FA)-associated protein cadherin aggregated in a dose-dependent response to AgNP exposure in differentiating cultured B35 neuroblastoma cells. These aggregates tended to colocalize with F-actin inclusions that form in response to AgNP and also contain β-catenin. However, using hyperspectral microscopy, we demonstrate that these multi-protein aggregates did not colocalize with the AgNPs themselves. Furthermore, expression and organization of the FA protein vinculin did not change in cells exposed to AgNP. Our findings suggest that AgNPs activate an intermediate mechanism which leads to formation of aggregates via specific protein-protein interactions. Finally, we detail the changes in hyperspectral profiles of AgNPs during different stages of cell culture and immunocytochemistry processing. AgNPs in citrate-stabilized solution present mostly blue with some rainbow spectra and these are maintained upon mounting in Prolong Gold. Exposure to tissue culture medium results in a uniform green spectral shift that is not further altered by fixation and protein block steps of immunocytochemistry.
Topics: Metal Nanoparticles; Silver; Cadherins; Cell Line, Tumor; Animals; Protein Aggregates; Vinculin
PubMed: 38692336
DOI: 10.1016/j.tiv.2024.105837 -
Biomedicine & Pharmacotherapy =... Jun 2024DIREN is a SHE ethnic medicine with stasis-resolving, hemostasis, clearing heat, and removing toxin effects. It is clinically used in the treatment of gastrointestinal...
BACKGROUND
DIREN is a SHE ethnic medicine with stasis-resolving, hemostasis, clearing heat, and removing toxin effects. It is clinically used in the treatment of gastrointestinal bleeding, such as ulcerative colitis (UC).
AIM OF THE STUDY
Fibrosis is one of the pathological changes in the progression of UC, which can make it challenging to respond to a treatment. We aimed to illuminate the role of DIREN in DSS-induced UC and tried to unveil its related mechanisms from two perspectives: intestinal inflammation and collagen deposition.
MATERIALS AND METHODS
A 2.5 % dextran sulfate sodium (DSS) water solution was used to induce colitis in mice. The therapeutic effect of DIREN was assessed using the disease activity index, histopathological score, and colon length. Masson and Sirius Red staining was used to observe the fibrosis in the colon. Apoptosis of colonic epithelial cells was observed by TUNEL immunofluorescence staining. RNA-seq observed differential genes and enrichment pathways. Immunohistochemistry and RT-qPCR were used to detect the expression of molecules related to fibrosis and focal adhesion signaling in colon tissue.
RESULTS
The administration of DIREN resulted in a reduction of disease activity index (DAI) in mice with UC while simultaneously promoting an increase in colon length. DIREN mitigated the loss of goblet cells in the colon of UC mice and maintained the integrity of the intestinal mucosa barrier. Masson staining revealed a reduction in colonic fibrosis with DIREN treatment, while Sirius red staining demonstrated a decrease in collagen Ⅰ deposition. DIREN reduced apoptosis of colonic epithelial cells and the expression of genes, such as CDH2, ITGA1, and TGF-β2. Additionally, the results of GSEA analysis of colon tissue transcriptome showed that the differentially expressed genes were enriched in the focal adhesion pathway. DIREN was found to downregulate the protein expression of BAX, N-cadherin, β-catenin, Integrin A1, and Vinculin while upregulating the protein expression of BCL2. Additionally, it led to the co-expression of N-cadherin and α-SMA.
CONCLUSION
DIREN exerts a protective effect against DSS-induced UC by ameliorating colonic fibrosis via regulation of focal adhesion and the WNT/β-catenin signaling pathway, thereby inhibiting fibroblast migration and reducing collagen secretion.
Topics: Animals; Dextran Sulfate; Mice; Wnt Signaling Pathway; Collagen; Focal Adhesions; Colon; Mice, Inbred C57BL; Male; Colitis; Apoptosis; Fibrosis; Colitis, Ulcerative; Disease Models, Animal; beta Catenin
PubMed: 38678963
DOI: 10.1016/j.biopha.2024.116671