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Communications Biology Mar 2021The LIM domain-dependent localization of the adapter protein paxillin to β3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by...
The LIM domain-dependent localization of the adapter protein paxillin to β3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of β3 integrin at a putative paxillin binding site (β3) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to β3 integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of β3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the β3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of β3 integrin-FAs.
Topics: Animals; Binding Sites; Fibroblasts; Fluorescence Recovery After Photobleaching; Focal Adhesions; Integrin alphaVbeta3; Kinetics; Mice; Microscopy, Confocal; Microscopy, Fluorescence; NIH 3T3 Cells; Paxillin; Phenotype; Protein Binding; Protein Interaction Domains and Motifs; Protein Stability; Structure-Activity Relationship
PubMed: 33782527
DOI: 10.1038/s42003-021-01886-9 -
Investigative Ophthalmology & Visual... Jan 2024Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in...
PURPOSE
Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in scleral remodeling in myopia and its underlying mechanisms.
METHODS
A myopic cell model was induced using hypoxia. Following loss- and gain-of function experiments, the expression of the myofibroblast transdifferentiation-related and collagen production-related factors Forkhead box M1 (FOXM1), APOA1, and methyltransferase-like 3 (METTL3) in the myopic cell model was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The proliferation and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry, respectively. Chromatin immunoprecipitation (ChIP) was employed to examine FOXM1 enrichment in the METTL3 promoter, methylated RNA immunoprecipitation (Me-RIP) to examine the N6-methyladenosine (m6A) modification level of APOA1, and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to examine the binding between METTL3 and APOA1.
RESULTS
Hypoxia-induced human scleral fibroblasts (HSFs) had high APOA1 and FOXM1 expression and low METTL3 expression. FOXM1 knockdown elevated METTL3 expression and downregulated APOA1 expression. FOXM1 was enriched in METTL3 promoter. APOA1 or FOXM1 knockdown or METTL3 overexpression reversed the hypoxia-induced elevation in vinculin, paxillin, and α-smooth muscle actin (α-SMA) levels and apoptosis and the reduction in collagen, type I, alpha 1 (COL1A1) level and cell proliferation in HSFs. METTL3 or YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) knockdown or APOA1 overexpression reversed the impacts of FOXM1 knockdown on vinculin, paxillin, α-SMA, and COL1A1 expression and cell proliferation and apoptosis.
CONCLUSIONS
FOXM1 elevated the m6A methylation level of APOA1 by repressing METTL3 transcription and enhanced APOA1 mRNA stability and transcription by reducing the YTHDF2-recognized m6A methylated transcripts.
Topics: Humans; Apolipoprotein A-I; Paxillin; Vinculin; Myopia, Degenerative; Transcription Factors; Hypoxia; Methyltransferases; Forkhead Box Protein M1; RNA-Binding Proteins
PubMed: 38190128
DOI: 10.1167/iovs.65.1.19 -
Cell and Tissue Research Nov 2022ZINC40099027 (ZN27) is a specific focal adhesion kinase (FAK) activator that promotes murine mucosal wound closure after ischemic or NSAID-induced injury. Diverse...
ZINC40099027 (ZN27) is a specific focal adhesion kinase (FAK) activator that promotes murine mucosal wound closure after ischemic or NSAID-induced injury. Diverse motogenic pathways involve FAK, but the direct consequences of pure FAK activation have not been studied, and how ZN27-induced FAK activation stimulates wound closure remained unclear. We investigated signaling and focal adhesion (FA) turnover after FAK activation by ZN27 in Caco-2 cells, confirming key results in CCD841 cells. ZN27 increased Caco-2 FAK-Y-397, FAK-Y-576/7, paxillin-Y-118, and ERK 1/2 phosphorylation and decreased FAK-Y-925 phosphorylation, without altering FAK-Y-861, p38, Jnk, or Akt phosphorylation. ZN27 increased FAK-paxillin interaction while decreasing FAK-Grb2 association. ZN27 increased membrane-associated FAK-Y-397 and FAK-Y-576/7 phosphorylation and paxillin-Y-118 and ERK 1/2 phosphorylation but decreased FAK-Y-925 phosphorylation without altering Src or Grb2. Moreover, ZN27 increased the fluorescence intensity of GFP-FAK and pFAK-Y397 in FAs and increased the total number of FAs but reduced their size in GFP-FAK-transfected Caco-2 cells, consistent with increased FA turnover. In contrast, FAK-Y397F transfection prevented ZN27 effects on FAK size and number and FAK and pFAK fluorescent intensity in FAs. We confirmed the proposed FAK/paxillin/ERK pathway using PP2 and U0126 to block Src and MEK1/2 in Caco-2 and CCD841 cells. These results suggest that ZN27 promotes intestinal epithelial monolayer defect closure by stimulating autophosphorylation of FAK in the cytosol, distinct from classical models of FAK activation in the FA. Phosphorylated FAK translocates to the membrane, where its downstream substrates paxillin and ERK are phosphorylated, leading to FA turnover and human intestinal epithelial cell migration.
Topics: Humans; Mice; Animals; Paxillin; Focal Adhesion Protein-Tyrosine Kinases; Caco-2 Cells; Cytosol; MAP Kinase Signaling System; Focal Adhesion Kinase 1; Phosphorylation; Cell Movement
PubMed: 36001146
DOI: 10.1007/s00441-022-03674-1 -
PloS One 2016Paxillin family proteins regulate intracellular signaling downstream of extracellular matrix adhesion. Tissue expression patterns and cellular functions of Paxillin...
BACKGROUND
Paxillin family proteins regulate intracellular signaling downstream of extracellular matrix adhesion. Tissue expression patterns and cellular functions of Paxillin proteins during embryo development remain poorly understood. Additionally, the evolution of this gene family has not been thoroughly investigated.
RESULTS
This report characterizes the evolution and expression of a novel Paxillin gene, called Paxillin-b, in Teleosts. Alignments indicate that Teleost Paxillin-a and Paxillin-b proteins are highly homologous to each other and to human Paxillin. Phylogenetic and synteny analyses suggest that these genes originated from the duplication of an ancestral Paxillin gene that was in a common ancestor of Teleosts and Tetrapods. Analysis of the spatiotemporal expression profiles of Paxillin-a and Paxillin-b using zebrafish revealed both overlapping and distinct domains for Paxillin-a and Paxillin-b during embryo development. Localization of zebrafish Paxillin orthologs expressed in mammalian cells demonstrated that both proteins localize to focal adhesions, similar to mammalian Paxillin. This suggests these proteins regulate adhesion-dependent processes in their endogenous tissues.
CONCLUSION
Paxillin-a and Paxillin-b were generated by duplication in Teleosts. These genes likely play similar roles as Paxillin genes in other organisms. This work provides a framework for functional investigation of Paxillin family members during development using the zebrafish as an in vivo model system.
Topics: Animals; Evolution, Molecular; Fish Proteins; Fishes; Focal Adhesions; Gene Duplication; Gene Expression Regulation, Developmental; Humans; Paxillin; Phylogeny; Synteny; Zebrafish; Zebrafish Proteins
PubMed: 27806088
DOI: 10.1371/journal.pone.0165266 -
International Review of Cell and... 2020The paxillin family of proteins, including paxillin, Hic-5, and leupaxin, are focal adhesion adaptor/scaffolding proteins which localize to cell-matrix adhesions and are...
The paxillin family of proteins, including paxillin, Hic-5, and leupaxin, are focal adhesion adaptor/scaffolding proteins which localize to cell-matrix adhesions and are important in cell adhesion and migration of both normal and cancer cells. Historically, the role of these proteins in regulating the actin cytoskeleton through focal adhesion-mediated signaling has been well documented. However, studies in recent years have revealed additional functions in modulating the microtubule and intermediate filament cytoskeletons to affect diverse processes including cell polarization, vesicle trafficking and mechanosignaling. Expression of paxillin family proteins in stromal cells is also important in regulating tumor cell migration and invasion through non-cell autonomous effects on the extracellular matrix. Both paxillin and Hic-5 can also influence gene expression through a variety of mechanisms, while their own expression is frequently dysregulated in various cancers. Accordingly, these proteins may serve as valuable targets for novel diagnostic and treatment approaches in cancer.
Topics: Focal Adhesions; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Neoplasm Invasiveness; Neoplasms; Paxillin; Signal Transduction
PubMed: 32859368
DOI: 10.1016/bs.ircmb.2020.05.003 -
Autophagy Mar 2021Cell migration is a highly dynamic and energy-intensive process that ensures the correct targeting of cells during embryonic and postnatal development. In recent work,...
Cell migration is a highly dynamic and energy-intensive process that ensures the correct targeting of cells during embryonic and postnatal development. In recent work, we highlighted the importance of macroautophagy/autophagy in regulating the dynamics of cell migration under baseline conditions and in response to a diverse set of molecular factors. Genetic suppression of autophagy-related genes induced longer stationary phases in migrating cells and cell stalling at the beginning of the migratory stream. We also showed that autophagy is required for recycling of the focal adhesion molecule PXN (paxillin), and is induced by energy levels of cells via AMPK activation. This recent study revealed the importance of autophagy in the maintenance of cell migration, and showed that the dynamic interplay between autophagy and energy levels is required to sustain neuronal migration and to cope with diverse micro-environmental factors.
Topics: AMP-Activated Protein Kinases; Adenosine Diphosphate; Adenosine Triphosphate; Autophagy; Cell Movement
PubMed: 33172335
DOI: 10.1080/15548627.2020.1848120 -
Genes To Cells : Devoted To Molecular &... Jul 2018The tyrosine kinase c-Src is frequently overexpressed and activated in a wide variety of human cancers. However, the molecular mechanisms responsible for the...
The tyrosine kinase c-Src is frequently overexpressed and activated in a wide variety of human cancers. However, the molecular mechanisms responsible for the upregulation of c-Src remain elusive. To examine whether microRNA-mediated c-Src upregulation promotes cancer progression, we screened miRNAs with complementarity to the 3'-UTR of c-Src mRNA. Among these miRNAs, down-regulation of miR-137 was tightly associated with c-Src-mediated tumor progression of human colon cancer cells/tissues. Re-expression of miR-137 in human colon cancer cells suppressed tumor growth and caused the disruption of focal contacts, suppression of cell adhesion, and invasion, although restoration of c-Src in miR-137-treated cells could not fully rescue the tumor-suppressive effect of miR-137. We found that miR-137 targets AKT2 and paxillin also and miR-137-mediated regulation of c-Src /AKT2 is crucial for controlling tumor growth, whereas that of c-Src/paxillin contributes to malignancy. miR-137 suppressed Src-related oncogenic signaling and changed the expression of miRNAs that are regulated by Src activation. miR-137 controls the expression of c-Src/AKT2/paxillin and synergistically suppresses Src oncogenic signaling evoked from focal adhesions. In various human cancers that harbor c-Src upregulation, the dysfunction of this novel mechanism would serve as a critical trigger for tumor progression.
PubMed: 29962093
DOI: 10.1111/gtc.12610 -
International Journal of Molecular... Nov 2023Metastasis is the leading cause of death in breast cancer patients due to the lack of effective therapies. Elevated levels of paxillin expression have been observed in...
Metastasis is the leading cause of death in breast cancer patients due to the lack of effective therapies. Elevated levels of paxillin expression have been observed in various cancer types, with tyrosine phosphorylation shown to play a critical role in driving cancer cell migration. However, the specific impact of the distinct tyrosine phosphorylation events of paxillin in the progression of breast cancer remains to be fully elucidated. Here, we found that paxillin overexpression in breast cancer tissue is associated with a patient's poor prognosis. Paxillin knockdown inhibited the migration and invasion of breast cancer cells. Furthermore, the phosphorylation of paxillin tyrosine residue 31 (Tyr31) was significantly increased upon the TGF-β1-induced migration and invasion of breast cancer cells. Inhibiting Fyn activity or silencing Fyn decreases paxillin Tyr31 phosphorylation. The wild-type and constitutively active Fyn directly phosphorylate paxillin Tyr31 in an in vitro system, indicating that Fyn directly phosphorylates paxillin Tyr31. Additionally, the non-phosphorylatable mutant of paxillin at Tyr31 reduces actin stress fiber formation, migration, and invasion of breast cancer cells. Taken together, our results provide direct evidence that Fyn-mediated paxillin Tyr31 phosphorylation is required for breast cancer migration and invasion, suggesting that targeting paxillin Tyr31 phosphorylation could be a potential therapeutic strategy for mitigating breast cancer metastasis.
Topics: Female; Humans; Breast Neoplasms; Cell Movement; Paxillin; Phosphorylation; Tyrosine
PubMed: 37958964
DOI: 10.3390/ijms242115980 -
Biomaterials Advances Feb 2023Cells are not only anchored to the extracellular matrix via the focal adhesion complex, the focal adhesion complex also serves as a sensor for force transduction. How...
Cells are not only anchored to the extracellular matrix via the focal adhesion complex, the focal adhesion complex also serves as a sensor for force transduction. How tension influences the structure of focal adhesions is not well understood. Here, we analyse the effect of tension on the location of key focal adhesion proteins, namely vinculin, paxillin and actin. We use micropatterning on gold surfaces to manipulate the cell shape, to create focal adhesions at specific cell areas, and to perform metal-induced energy transfer (MIET) measurements on the patterned cells. MIET resolves the different protein locations with respect to the gold surface with nanometer accuracy. Further, we use drugs influencing the cellular motor protein myosin or mechanosensitive ion channels to get deeper insight into focal adhesions at different tension states. We show here that in particular actin is affected by the rationally tuned force balance. Blocking mechanosensitive ion channels has a particularly high influence on the actin and focal adhesion architecture, resulting in larger focal adhesions with elevated paxillin and vinculin and strongly lowered actin stress fibres. Our results can be explained by a balance of adhesion tension with cellular tension together with ion channel-controlled focal adhesion homeostasis, where high cellular tension leads to an elevation of vinculin and actin, while high adhesion tension lowers these proteins.
Topics: Focal Adhesions; Actins; Paxillin; Cytoskeleton; Vinculin; Cell Shape
PubMed: 36621197
DOI: 10.1016/j.bioadv.2022.213277 -
Genes Sep 2021Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents and accounts for approximately 2% of soft tissue sarcomas in adults. It is... (Review)
Review
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents and accounts for approximately 2% of soft tissue sarcomas in adults. It is subcategorized into distinct subtypes based on histological features and fusion status (). Despite advances in our understanding of the pathobiological and molecular landscape of RMS, the prognosis of these tumors has not significantly improved in recent years. Developing a better understanding of genetic abnormalities and risk stratification beyond the fusion status are crucial to developing better therapeutic strategies. Herein, we aim to highlight the genetic pathways/abnormalities involved, specifically in fusion-negative RMS, assess the currently available model systems to study RMS pathogenesis, and discuss available prognostic factors as well as their importance for risk stratification to achieve optimal therapeutic management.
Topics: Animals; Cell Line, Tumor; Epigenesis, Genetic; Gene Fusion; Humans; Mice; Models, Biological; Paxillin; Prognosis; Rhabdomyosarcoma; Signal Transduction
PubMed: 34680895
DOI: 10.3390/genes12101500