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Molecular Biology of the Cell May 2024α-catenin (α-cat) displays force-dependent unfolding and binding to actin filaments through direct and indirect means, but features of adherens junction structure and...
α-catenin (α-cat) displays force-dependent unfolding and binding to actin filaments through direct and indirect means, but features of adherens junction structure and function most vulnerable to loss of these allosteric mechanisms have not been directly compared. By reconstituting an α-cat F-actin-binding domain unfolding mutant known to exhibit enhanced binding to actin (α-cat-H0-FABD) into α-cat knockout Madin Darby Canine Kidney (MDCK) cells, we show that partial loss of the α-cat catch bond mechanism (via an altered H0 α-helix) leads to stronger epithelial sheet integrity with greater colocalization between the α-cat-H0-FABD mutant and actin. α-cat-H0-FABD -expressing cells are less efficient at closing scratch-wounds, suggesting reduced capacity for more dynamic cell-cell coordination. Evidence that α-cat-H0-FABD is equally accessible to the conformationally sensitive α18 antibody epitope as WT α-cat and shows similar vinculin recruitment suggests this mutant engages lower tension cortical actin networks, as its M-domain is not persistently open. Conversely, α-cat-M-domain salt-bridge mutants with persistent recruitment of vinculin and phosphorylated myosin light chain show only intermediate monolayer adhesive strengths, but display less directionally coordinated and thereby slower migration speeds during wound-repair. These data show α-cat M- and FABD-unfolding mutants differentially impact cell-cell cohesion and migration properties, and suggest signals favoring α-cat-cortical actin interaction without persistent M-domain opening may improve epithelial monolayer strength through enhanced coupling to lower tension actin networks.
Topics: Dogs; Animals; alpha Catenin; Madin Darby Canine Kidney Cells; Actins; Cell Movement; Epithelial Cells; Actin Cytoskeleton; Protein Binding; Protein Domains; Mutation; Adherens Junctions; Protein Unfolding; Cell Adhesion; Vinculin
PubMed: 38507238
DOI: 10.1091/mbc.E23-01-0036 -
BioRxiv : the Preprint Server For... Mar 2024Cadherins are transmembrane adhesion receptors. Cadherin ectodomains form adhesive 2D clusters through cooperative and interactions, whereas its intracellular region...
Cadherins are transmembrane adhesion receptors. Cadherin ectodomains form adhesive 2D clusters through cooperative and interactions, whereas its intracellular region interacts with specific cytosolic proteins, termed catenins, to anchor the cadherin-catenin complex (CCC) to the actin cytoskeleton. How these two types of interactions are coordinated in the formation of specialized cell-cell adhesions, adherens junctions (AJ), remains unclear. We focus here on the role of the actin-binding domain of α-catenin (αABD) by showing that the interaction of αABD with actin generates actin-bound CCC oligomers (CCC/actin strands) incorporating up to six CCCs. The strands are primarily formed on the actin-rich cell protrusions. Once in cell-cell interface, the strands become involved in cadherin ectodomain clustering. Such combination of the extracellular and intracellular oligomerizations gives rise to the composite oligomers, CCC/actin clusters. To mature, these clusters then rearrange their actin filaments using several redundant pathways, two of which are characterized here: one depends on the α-catenin-associated protein, vinculin and the second one depends on the unstructured C-terminus of αABD. Thus, AJ assembly proceeds through spontaneous formation of CCC/actin clusters and their successive reorganization.
PubMed: 38496678
DOI: 10.1101/2024.03.04.583373 -
ACS Biomaterials Science & Engineering Apr 2024Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such...
Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or -(2-hydroxyethyl)piperazine--ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.
Topics: Collagen; Extracellular Matrix; Macrophages; Transcription Factors; Hydrogels; Cytokines
PubMed: 38467019
DOI: 10.1021/acsbiomaterials.3c01892 -
BioRxiv : the Preprint Server For... Feb 2024Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity...
Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity (increasing the signal-to-noise ratio) and robustness (signalling above a threshold rather than activation by a single receptor) of the signal compared to signalling by single receptors. Compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, if nanoclusters function as signalling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at sub-diffraction limited resolution of ~100nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in immobile population of integrin β3 organised as nanoclusters, which (ii) in turn serve to organise nanoclusters of associated key adhesome proteins- vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signalling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct dynamics dependent on function. (iv) long-lived nanoclusters function as signalling hubs- wherein phosphorylated FAK and paxillin formed stable nanoclusters in close proximity to immobile integrin nanoclusters which are disassembled in response to inactivation signal by phosphatase PTPN12 (v) signalling takes place in response to an external signal such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. Taken together, these results demonstrate that signalling downstream of transmembrane receptors is organised as hubs of signalling proteins (FAK, paxillin, vinculin) seeded by nanoclusters of the transmembrane receptor (integrin).
PubMed: 38464288
DOI: 10.1101/2024.02.25.581925 -
Research Square Feb 2024Curvature is a critical factor in cornea mechanobiology, but its impact on phenotypic alterations and extracellular matrix remodeling of cornea stroma remains unclear....
Curvature is a critical factor in cornea mechanobiology, but its impact on phenotypic alterations and extracellular matrix remodeling of cornea stroma remains unclear. In this work, we investigated how curvature influences the corneal stroma using a hydraulically controlled curvature array chip. The responses of stromal cells to low, medium, and high curvatures were observed by preparing three phenotypes of corneal stromal cells: corneal keratocytes, fibroblasts, and myofibroblasts. Keratocytes exhibited phenotypic alterations in response to curvature changes, notably including a decrease in ALDH3 expression and an increase in α-SMA expression. For focal adhesion, corneal fibroblast and myofibroblasts showed enhanced vinculin localization in response to curvature, while corneal keratocytes presented reduced vinculin expression. For cell alignment and ECM expression, most stromal cells under all curvatures showed a radially organized f-actin and collagen fibrils. Interestingly, for corneal fibroblast under medium curvature, we observed orthogonal cell alignment, which is linked to the unique hoop and meridional stress profiles of the curved surface. Furthermore, lumican expression was upregulated in corneal keratocytes, and keratocan expression was increased in corneal fibroblasts and myofibroblasts due to curvature. These results demonstrate that curvature influences both the phenotype of corneal stromal cells and the structural organization of corneal stroma tissue without any external stimuli. This curvature-dependent behavior of corneal stromal cells presents potential opportunities for creating therapeutic strategies for corneal shape dysfunctions.
PubMed: 38464213
DOI: 10.21203/rs.3.rs-3973873/v1 -
Clinical Proteomics Mar 2024Cardiac rupture (CR) is a rare but catastrophic mechanical complication of acute myocardial infarction (AMI) that seriously threatens human health. However, the reliable...
BACKGROUND
Cardiac rupture (CR) is a rare but catastrophic mechanical complication of acute myocardial infarction (AMI) that seriously threatens human health. However, the reliable biomarkers for clinical diagnosis and the underlying signaling pathways insights of CR has yet to be elucidated.
METHODS
In the present study, a quantitative approach with tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry was used to characterize the differential protein expression profiles of patients with CR. Plasma samples were collected from patients with CR (n = 37), patients with AMI (n = 47), and healthy controls (n = 47). Candidate proteins were selected for validation by multiple reaction monitoring (MRM) and enzyme-linked immunosorbent assay (ELISA).
RESULTS
In total, 1208 proteins were quantified and 958 differentially expressed proteins (DEPs) were identified. The difference in the expression levels of the DEPs was more noticeable between the CR and Con groups than between the AMI and Con groups. Bioinformatics analysis showed most of the DEPs to be involved in numerous crucial biological processes and signaling pathways, such as RNA transport, ribosome, proteasome, and protein processing in the endoplasmic reticulum, as well as necroptosis and leukocyte transendothelial migration, which might play essential roles in the complex pathological processes associated with CR. MRM analysis confirmed the accuracy of the proteomic analysis results. Four proteins i.e., C-reactive protein (CRP), heat shock protein beta-1 (HSPB1), vinculin (VINC) and growth/differentiation factor 15 (GDF15), were further validated via ELISA. By receiver operating characteristic (ROC) analysis, combinations of these four proteins distinguished CR patients from AMI patients with a high area under the curve (AUC) value (0.895, 95% CI, 0.802-0.988, p < 0.001).
CONCLUSIONS
Our study highlights the value of comprehensive proteomic characterization for identifying plasma proteome changes in patients with CR. This pilot study could serve as a valid foundation and initiation point for elucidation of the mechanisms of CR, which might aid in identifying effective diagnostic biomarkers in the future.
PubMed: 38429673
DOI: 10.1186/s12014-024-09474-9 -
Cytoskeleton (Hoboken, N.J.) Mar 2024A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitment of the LIM domain proteins TRIP6 and LIMD1 to adherens junctions....
The ability of the LIMD1 and TRIP6 LIM domains to bind strained f-actin is critical for their tension dependent localization to adherens junctions and association with the Hippo pathway kinase LATS1.
A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitment of the LIM domain proteins TRIP6 and LIMD1 to adherens junctions. Mechanical tension also triggers TRIP6 and LIMD1 to bind and inhibit the Hippo pathway kinase LATS1. How TRIP6 and LIMD1 are recruited to adherens junctions in response to tension is not clear, but previous studies suggested that they could be regulated by the known mechanosensory proteins α-catenin and vinculin at adherens junctions. We found that the three LIM domains of TRIP6 and LIMD1 are necessary and sufficient for tension-dependent localization to adherens junctions. The LIM domains of TRIP6, LIMD1, and certain other LIM domain proteins have been shown to bind to actin networks under strain/tension. Consistent with this, we show that TRIP6 and LIMD1 colocalize with the ends of actin fibers at adherens junctions. Point mutations in a key conserved residue in each LIM domain that are predicted to impair binding to f-actin under strain inhibits TRIP6 and LIMD1 localization to adherens junctions and their ability to bind to and recruit LATS1 to adherens junctions. Together these results show that the ability of TRIP6 and LIMD1 to bind to strained actin underlies their ability to localize to adherens junctions and regulate LATS1 in response to mechanical tension.
PubMed: 38426816
DOI: 10.1002/cm.21847 -
Cell Communication and Signaling : CCS Feb 2024Tubulointerstitial kidney disease associated microenvironmental dysregulation, like acidification, inflammation and fibrosis, affects tubule cells and fibroblasts....
BACKGROUND
Tubulointerstitial kidney disease associated microenvironmental dysregulation, like acidification, inflammation and fibrosis, affects tubule cells and fibroblasts. Micromilieu homeostasis influences intracellular signaling and intercellular crosstalk. Cell-cell communication in turn modulates the interstitial microenvironment. We assessed the impact of acidosis on inflammatory and fibrotic responses in proximal tubule cells and fibroblasts as a function of cellular crosstalk. Furthermore, cellular signaling pathways involved were identified.
METHODS
HK-2 (human proximal tubule) and CCD-1092Sk (human fibroblasts), in mono and coculture, were exposed to acidic or control media for 3 or 48 h. Protein expression of inflammation markers (TNF, TGF-ß and COX-2), dedifferentiation markers (N-cadherin, vinculin, ß-catenin and vimentin), fibrosis markers (collagen III and fibronectin) and phospho- as well as total MAPK levels were determined by western blot. Secreted collagen III and fibronectin were measured by ELISA. The impact of MAPK activation was assessed by pharmacological intervention. In addition, necrosis, apoptosis and epithelial permeability were determined.
RESULTS
Independent of culture conditions, acidosis caused a decrease of COX-2, vimentin and fibronectin expression in proximal tubule cells. Only in monoculture, ß-Catenin expression decreased and collagen III expression increased in tubule cells during acidosis. By contrast, in coculture collagen III protein expression of tubule cells was reduced. In fibroblasts acidosis led to an increase of TNF, COX-2, vimentin, vinculin, N-cadherin protein expression and a decrease of TGF-ß expression exclusively in coculture. In monoculture, expression of COX-2 and fibronectin was reduced. Collagen III expression of fibroblasts was reduced by acidosis independent of culture conditions. In coculture, acidosis enhanced phosphorylation of ERK1/2, JNK1/2 and p38 transiently in proximal tubule cells. In fibroblasts, acidosis enhanced phosphorylation of p38 in a sustained and very strong manner. ERK1/2 and JNK1/2 were not affected in fibroblasts. Inhibition of JNK1/2 and p38 under coculture conditions reduced acidosis-induced changes in fibroblasts significantly.
CONCLUSIONS
Our data show that the crosstalk between proximal tubule cells and fibroblasts is crucial for acidosis-induced dedifferentiation of fibroblasts into an inflammatory phenotype. This dedifferentiation is at least in part mediated by p38 and JNK1/2. Thus, cell-cell communication is essential for the pathophysiological impact of tubulointerstitial acidosis.
Topics: Humans; Acidosis; Cadherins; Catenins; Collagen; Cyclooxygenase 2; Fibroblasts; Fibronectins; Fibrosis; Inflammation; p38 Mitogen-Activated Protein Kinases; Vimentin; Vinculin; Mitogen-Activated Protein Kinase 14
PubMed: 38395872
DOI: 10.1186/s12964-024-01527-8 -
Theranostics 2024Osteosarcoma (OS), a common malignant bone tumor, calls for the investigation of novel treatment strategies. Low-intensity vibration (LIV) presents itself as a...
Osteosarcoma (OS), a common malignant bone tumor, calls for the investigation of novel treatment strategies. Low-intensity vibration (LIV) presents itself as a promising option, given its potential to enhance bone health and decrease cancer susceptibility. This research delves into the effects of LIV on OS cells and mesenchymal stem cells (MSCs), with a primary focus on generating induced tumor-suppressing cells (iTSCs) and tumor-suppressive conditioned medium (CM). To ascertain the influence of vibration frequency, we employed numerical simulations and conducted experiments to determine the most effective LIV conditions. Subsequently, we generated iTSCs and CM through LIV exposure and assessed the impact of CM on OS cells. We also explored the underlying mechanisms of the tumor-suppressive effects of LIV-treated MSC CM, with a specific focus on vinculin (VCL). We employed cytokine array, RNA sequencing, and Western blot techniques to investigate alterations in cytokine profiles, transcriptomes, and tumor suppressor proteins. Numerical simulations validated LIV frequencies within the 10-100 Hz range. LIV induced notable morphological changes in OS cells and MSCs, confirming its dual role in inhibiting OS cell progression and promoting MSC conversion into iTSCs. Upregulated VCL expression enhanced MSC responsiveness to LIV, significantly bolstering CM's efficacy. Notably, we identified tumor suppressor proteins in LIV-treated CM, including procollagen C endopeptidase enhancer (PCOLCE), histone H4 (H4), peptidylprolyl isomerase B (PPIB), and aldolase A (ALDOA). Consistently, cytokine levels decreased significantly in LIV-treated mouse femurs, and oncogenic transcript levels were downregulated in LIV-treated OS cells. Moreover, our study demonstrated that combining LIV-treated MSC CM with chemotherapy drugs yielded additive anti-tumor effects. LIV effectively impeded the progression of OS cells and facilitated the transformation of MSCs into iTSCs. Notably, iTSC-derived CM demonstrated robust anti-tumor properties and the augmentation of MSC responsiveness to LIV via VCL. Furthermore, the enrichment of tumor suppressor proteins within LIV-treated MSC CM and the reduction of cytokines within LIV-treated isolated bone underscore the pivotal tumor-suppressive role of LIV within the bone tumor microenvironment.
Topics: Animals; Mice; Vibration; Mesenchymal Stem Cells; Osteosarcoma; Cytokines; Bone Neoplasms; Tumor Suppressor Proteins; Tumor Microenvironment
PubMed: 38389836
DOI: 10.7150/thno.90945 -
Cellular & Molecular Biology Letters Feb 2024The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to...
BACKGROUND
The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2.
METHODS
Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-Glo Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays.
RESULTS
We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin.
CONCLUSIONS
This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.
Topics: Animals; Female; Humans; Breast Neoplasms; Cell Proliferation; Monomeric GTP-Binding Proteins; ras Proteins; Signal Transduction
PubMed: 38383288
DOI: 10.1186/s11658-024-00546-0