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Sample records for activated focal adhesion

  1. Lipid binding promotes oligomerization and focal adhesion activity of vinculin

    PubMed Central

    Chinthalapudi, Krishna; Rangarajan, Erumbi S.; Patil, Dipak N.; George, Eric M.; Brown, David T.

    2014-01-01

    Adherens junctions (AJs) and focal adhesion (FA) complexes are necessary for cell migration and morphogenesis, and for the development, growth, and survival of all metazoans. Vinculin is an essential regulator of both AJs and FAs, where it provides links to the actin cytoskeleton. Phosphatidylinositol 4,5-bisphosphate (PIP2) affects the functions of many targets, including vinculin. Here we report the crystal structure of vinculin in complex with PIP2, which revealed that PIP2 binding alters vinculin structure to direct higher-order oligomerization and suggests that PIP2 and F-actin binding to vinculin are mutually permissive. Forced expression of PIP2-binding–deficient mutants of vinculin in vinculin-null mouse embryonic fibroblasts revealed that PIP2 binding is necessary for maintaining optimal FAs, for organization of actin stress fibers, and for cell migration and spreading. Finally, photobleaching experiments indicated that PIP2 binding is required for the control of vinculin dynamics and turnover in FAs. Thus, through oligomerization, PIP2 directs a transient vinculin sequestration at FAs that is necessary for proper FA function. PMID:25488920

  2. Focal adhesions in osteoneogenesis

    PubMed Central

    Biggs, M.J.P; Dalby, M.J

    2010-01-01

    As materials technology and the field of tissue engineering advances, the role of cellular adhesive mechanisms, in particular the interactions with implantable devices, becomes more relevant in both research and clinical practice. A key tenet of medical device technology is to use the exquisite ability of biological systems to respond to the material surface or chemical stimuli in order to help develop next-generation biomaterials. The focus of this review is on recent studies and developments concerning focal adhesion formation in osteoneogenesis, with an emphasis on the influence of synthetic constructs on integrin mediated cellular adhesion and function. PMID:21287830

  3. In-situ coupling between kinase activities and protein dynamics within single focal adhesions

    PubMed Central

    Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

    2016-01-01

    The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells. PMID:27383747

  4. In-situ coupling between kinase activities and protein dynamics within single focal adhesions.

    PubMed

    Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

    2016-01-01

    The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells. PMID:27383747

  5. p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion

    PubMed Central

    George, Margaret D.; Wine, Robert N.; Lackford, Brad; Kissling, Grace E.; Akiyama, Steven K.; Olden, Kenneth; Roberts, John D.

    2014-01-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading and that this association can be regulated by factors in the tumor microenvironment. PMID:24219282

  6. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

    NASA Astrophysics Data System (ADS)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

    2014-07-01

    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  7. Molecular mechanism of vinculin activation and nano-scale spatial organization in focal adhesions

    PubMed Central

    Case, Lindsay B.; Baird, Michelle A.; Shtengel, Gleb; Campbell, Sharon L.; Hess, Harald F.; Davidson, Michael W.; Waterman, Clare M.

    2015-01-01

    Focal adhesions (FAs) link the extracellular matrix (ECM) to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signaling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin’s interactions are spatiotemporally organized within FA is unknown. Using interferometric photo-activation localization (iPALM) super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FA. Inactive vinculin localizes to the lower integrin signaling layer in FA by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FA where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FA at the nano-scale to regulate vinculin activation and function. PMID:26053221

  8. Novel anticancer agent, SQAP, binds to focal adhesion kinase and modulates its activity

    PubMed Central

    Izaguirre-Carbonell, Jesus; Kawakubo, Hirofumi; Murata, Hiroshi; Tanabe, Atsushi; Takeuchi, Toshifumi; Kusayanagi, Tomoe; Tsukuda, Senko; Hirakawa, Takeshi; Iwabata, Kazuki; Kanai, Yoshihiro; Ohta, Keisuke; Miura, Masahiko; Sakaguchi, Kengo; Matsunaga, Sachihiro; Sahara, Hiroeki; Kamisuki, Shinji; Sugawara, Fumio

    2015-01-01

    SQAP is a novel and promising anticancer agent that was obtained by structural modifications from a natural compound. SQAP inhibits angiogenesis in vivo resulting in increased hypoxia and reduced tumor volume. In this study, the mechanism by which SQAP modifies the tumor microenvironment was revealed through the application of a T7 phage display screening. This approach identified five SQAP-binding proteins including sterol carrier protein 2, multifunctional enzyme type 2, proteasomal ubiquitin receptor, UV excision repair protein and focal adhesion kinase (FAK). All the interactions were confirmed by surface plasmon resonance analysis. Since FAK plays an important role in cell turnover and angiogenesis, the influence of SQAP on FAK was the principal goal of this study. SQAP decreased FAK phosphorylation and cell migration in human umbilical vein endothelial cells and A549 cancer cells. These findings suggest that inhibition of FAK phosphorylation works as the mechanism for the anti-angiogenesis activity of SQAP. PMID:26456697

  9. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    EPA Science Inventory

    Prolactin-Induced Tyrosine Phosphorylation, Activation and Receptor
    Association of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells.
    Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental Protection
    Agency, MD-72, Research Triangle Park, NC 27711, and

  10. Numerically bridging lamellipodial and filopodial activity during cell spreading reveals a potentially novel trigger of focal adhesion maturation.

    PubMed

    Loosli, Y; Vianay, B; Luginbuehl, R; Snedeker, J G

    2012-05-01

    We present a novel approach to modeling cell spreading, and use it to reveal a potentially central mechanism regulating focal adhesion maturation in various cell phenotypes. Actin bundles that span neighboring focal complexes at the lamellipodium-lamellum interface were assumed to be loaded by intracellular forces in proportion to bundle length. We hypothesized that the length of an actin bundle (with the corresponding accumulated force at its adhesions) may thus regulate adhesion maturation to ensure cell mechanical stability and morphological integrity. We developed a model to test this hypothesis, implementing a "top-down" approach to simplify certain cellular processes while explicitly incorporating complexity of other key subcellular mechanisms. Filopodial and lamellipodial activities were treated as modular processes with functional spatiotemporal interactions coordinated by rules regarding focal adhesion turnover and actin bundle dynamics. This theoretical framework was able to robustly predict temporal evolution of cell area and cytoskeletal organization as reported from a wide range of cell spreading experiments using micropatterned substrates. We conclude that a geometric/temporal modeling framework can capture the key functional aspects of the rapid spreading phase and resultant cytoskeletal complexity. Hence the model is used to reveal mechanistic insight into basic cell behavior essential for spreading. It demonstrates that actin bundles spanning nascent focal adhesions such that they are aligned to the leading edge may accumulate centripetal endogenous forces along their length, and could thus trigger focal adhesion maturation in a force-length dependent fashion. We suggest that this mechanism could be a central "integrating" factor that effectively coordinates force-mediated adhesion maturation at the lamellipodium-lamellum interface. PMID:22453759

  11. Laminin α2-Mediated Focal Adhesion Kinase Activation Triggers Alport Glomerular Pathogenesis

    PubMed Central

    Delimont, Duane; Dufek, Brianna M.; Meehan, Daniel T.; Zallocchi, Marisa; Gratton, Michael Anne; Phillips, Grady; Cosgrove, Dominic

    2014-01-01

    It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages. PMID:24915008

  12. Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes.

    PubMed

    Goñi, Guillermina M; Epifano, Carolina; Boskovic, Jasminka; Camacho-Artacho, Marta; Zhou, Jing; Bronowska, Agnieszka; Martín, M Teresa; Eck, Michael J; Kremer, Leonor; Gräter, Frauke; Gervasio, Francesco Luigi; Perez-Moreno, Mirna; Lietha, Daniel

    2014-08-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase (NRTK) with key roles in integrating growth and cell matrix adhesion signals, and FAK is a major driver of invasion and metastasis in cancer. Cell adhesion via integrin receptors is well known to trigger FAK signaling, and many of the players involved are known; however, mechanistically, FAK activation is not understood. Here, using a multidisciplinary approach, including biochemical, biophysical, structural, computational, and cell biology approaches, we provide a detailed view of a multistep activation mechanism of FAK initiated by phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Interestingly, the mechanism differs from canonical NRTK activation and is tailored to the dual catalytic and scaffolding function of FAK. We find PI(4,5)P2 induces clustering of FAK on the lipid bilayer by binding a basic region in the regulatory 4.1, ezrin, radixin, moesin homology (FERM) domain. In these clusters, PI(4,5)P2 induces a partially open FAK conformation where the autophosphorylation site is exposed, facilitating efficient autophosphorylation and subsequent Src recruitment. However, PI(4,5)P2 does not release autoinhibitory interactions; rather, Src phosphorylation of the activation loop in FAK results in release of the FERM/kinase tether and full catalytic activation. We propose that PI(4,5)P2 and its generation in focal adhesions by the enzyme phosphatidylinositol 4-phosphate 5-kinase type Iγ are important in linking integrin signaling to FAK activation. PMID:25049397

  13. Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes

    PubMed Central

    Goñi, Guillermina M.; Epifano, Carolina; Boskovic, Jasminka; Camacho-Artacho, Marta; Zhou, Jing; Bronowska, Agnieszka; Martín, M. Teresa; Eck, Michael J.; Kremer, Leonor; Gräter, Frauke; Gervasio, Francesco Luigi; Perez-Moreno, Mirna; Lietha, Daniel

    2014-01-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase (NRTK) with key roles in integrating growth and cell matrix adhesion signals, and FAK is a major driver of invasion and metastasis in cancer. Cell adhesion via integrin receptors is well known to trigger FAK signaling, and many of the players involved are known; however, mechanistically, FAK activation is not understood. Here, using a multidisciplinary approach, including biochemical, biophysical, structural, computational, and cell biology approaches, we provide a detailed view of a multistep activation mechanism of FAK initiated by phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Interestingly, the mechanism differs from canonical NRTK activation and is tailored to the dual catalytic and scaffolding function of FAK. We find PI(4,5)P2 induces clustering of FAK on the lipid bilayer by binding a basic region in the regulatory 4.1, ezrin, radixin, moesin homology (FERM) domain. In these clusters, PI(4,5)P2 induces a partially open FAK conformation where the autophosphorylation site is exposed, facilitating efficient autophosphorylation and subsequent Src recruitment. However, PI(4,5)P2 does not release autoinhibitory interactions; rather, Src phosphorylation of the activation loop in FAK results in release of the FERM/kinase tether and full catalytic activation. We propose that PI(4,5)P2 and its generation in focal adhesions by the enzyme phosphatidylinositol 4-phosphate 5-kinase type Iγ are important in linking integrin signaling to FAK activation. PMID:25049397

  14. Focal Adhesion Kinase-Dependent Regulation of Adhesive Force Involves Vinculin Recruitment to Focal Adhesions

    PubMed Central

    Hanks, Steven K.; García, Andrés J.

    2016-01-01

    Background information Focal adhesion kinase (FAK), an essential non-receptor tyrosine kinase, plays pivotal roles in migratory responses, adhesive signaling, and mechanotransduction. FAK-dependent regulation of cell migration involves focal adhesion turnover dynamics as well as actin cytoskeleton polymerization and lamellipodia protrusion. Whereas roles for FAK in migratory and mechanosensing responses have been established, the contributions of FAK to the generation of adhesive forces are not well understood. Results Using FAK-null cells expressing wild-type and mutant FAK under an inducible tetracycline promoter, we analyzed the role of FAK in the generation of steady-state adhesive forces using micropatterned substrates and a hydrodynamic adhesion assay. FAK expression reduced steady-state strength by 30% compared to FAK-null cells. FAK expression reduced vinculin localization to focal adhesions by 35% independently from changes in integrin binding and localization of talin and paxillin. RNAi knockdown of vinculin abrogated the FAK-dependent differences in adhesive force. FAK-dependent changes in vinculin localization and adhesive force were confirmed in human primary fibroblasts with FAK knocked down by RNAi. The autophosphorylation Y397 and kinase domain Y576/Y577 sites were differentially required for FAK-mediated adhesive responses. Conclusions We demonstrate that FAK reduces steady-state adhesion strength by modulating vinculin recruitment to focal adhesions. These findings provide insights into the role of FAK in mechanical interactions between a cell and the extracellular matrix. PMID:19883375

  15. A kinetic model for RNA-interference of focal adhesions

    PubMed Central

    2013-01-01

    -interference of focal adhesions. Its predictions are in good agreement with known experimental results and can now guide the design of RNAi-experiments. In the future, it can be extended to include more components of the adhesome. It also could be extended by spatial aspects, for example by the differential activation of the Rac- and Rho-pathways in different parts of the cell. PMID:23311633

  16. Mechanism of Focal Adhesion Kinase Mechanosensing.

    PubMed

    Zhou, Jing; Aponte-Santamaría, Camilo; Sturm, Sebastian; Bullerjahn, Jakob Tómas; Bronowska, Agnieszka; Gräter, Frauke

    2015-11-01

    Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP2 binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP2 concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP2-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments. PMID:26544178

  17. Mechanism of Focal Adhesion Kinase Mechanosensing

    PubMed Central

    Sturm, Sebastian; Bullerjahn, Jakob Tómas; Bronowska, Agnieszka; Gräter, Frauke

    2015-01-01

    Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP2 binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP2 concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP2-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments. PMID:26544178

  18. Arsenic alters vascular smooth muscle cell focal adhesion complexes leading to activation of FAK-src mediated pathways

    SciTech Connect

    Pysher, Michele D. Chen, Qin M.; Vaillancourt, Richard R.

    2008-09-01

    Chronic exposure to arsenic has been linked to tumorigenesis, cardiovascular disease, hypertension, atherosclerosis, and peripheral vascular disease; however, the molecular mechanisms underlying its pathological effects remain elusive. In this study, we investigated arsenic-induced alteration of focal adhesion protein complexes in normal, primary vascular smooth muscle cells. We demonstrate that exposure to environmentally relevant concentrations of arsenic (50 ppb As{sup 3+}) can alter focal adhesion protein co-association leading to activation of downstream pathways. Co-associated proteins were identified and quantitated via co-immunoprecipitation, SDS-PAGE, and Western blot analysis followed by scanning densitometry. Activation of MAPK pathways in total cell lysates was evaluated using phosphor-specific antibodies. In our model, arsenic treatment caused a sustained increase in FAK-src association and activation, and induced the formation of unique signaling complexes (beginning after 3-hour As{sup 3+} exposure and continuing throughout the 12-hour time course studied). The effects of these alterations were manifested as chronic stimulation of downstream PAK, ERK and JNK pathways. Past studies have demonstrated that these pathways are involved in cellular survival, growth, proliferation, and migration in VSMCs.

  19. Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion

    PubMed Central

    Hoskin, Victoria; Szeto, Alvin; Ghaffari, Abdi; Greer, Peter A.; Côté, Graham P.; Elliott, Bruce E.

    2015-01-01

    Up-regulation of the cytoskeleton linker protein ezrin frequently occurs in aggressive cancer types and is closely linked with metastatic progression. However, the underlying molecular mechanisms detailing how ezrin is involved in the invasive and metastatic phenotype remain unclear. Here we report a novel function of ezrin in regulating focal adhesion (FA) and invadopodia dynamics, two key processes required for efficient invasion to occur. We show that depletion of ezrin expression in invasive breast cancer cells impairs both FA and invadopodia turnover. We also demonstrate that ezrin-depleted cells display reduced calpain-mediated cleavage of the FA and invadopodia-associated proteins talin, focal adhesion kinase (FAK), and cortactin and reduced calpain-1–specific membrane localization, suggesting a requirement for ezrin in maintaining proper localization and activity of calpain-1. Furthermore, we show that ezrin is required for cell directionality, early lung seeding, and distant organ colonization but not primary tumor growth. Collectively our results unveil a novel mechanism by which ezrin regulates breast cancer cell invasion and metastasis. PMID:26246600

  20. Activation of focal adhesion kinase through an interaction with β4 integrin contributes to tumorigenicity of colon cancer.

    PubMed

    Tai, Yu-Ling; Lai, I-Rue; Peng, Yu-Ju; Ding, Shih-Torng; Shen, Tang-Long

    2016-06-01

    High expression of either β4 integrin or focal adhesion kinase (FAK) has been reported in human colon cancer. However, it remains unclear how β4 integrin together with FAK contributes to the tumorigenicity of colon cancer. Here, we demonstrate that the co-overexpression of β4 integrin and FAK positively correlates with advanced stages of human colon cancer. Activated β4 integrin interacts with FAK and subsequently induces FAK phosphorylation at Tyr397. Furthermore, ablation of the β4 integrin/FAK complex and/or FAK activation impair colon cancer cell proliferation, anchorage-independent growth, and tumorigenicity. Our data indicate that the β4 integrin/FAK complex and subsequent FAK activation are essential regulators during the tumorigenicity of colon cancer, and we suggest an alternative strategy for colon cancer therapy. PMID:27178753

  1. Focal adhesion kinases in adhesion structures and disease.

    PubMed

    Eleniste, Pierre P; Bruzzaniti, Angela

    2012-01-01

    Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases. PMID:22888421

  2. Focal Adhesion Kinases in Adhesion Structures and Disease

    PubMed Central

    Eleniste, Pierre P.; Bruzzaniti, Angela

    2012-01-01

    Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases. PMID:22888421

  3. Focal adhesion kinase regulates the activity of the osmosensitive transcription factor TonEBP/NFAT5 under hypertonic conditions

    PubMed Central

    Neuhofer, Wolfgang; Küper, Christoph; Lichtnekert, Julia; Holzapfel, Konstantin; Rupanagudi, Khader V.; Fraek, Maria-Luisa; Bartels, Helmut; Beck, Franz-Xaver

    2014-01-01

    TonEBP/NFAT5 is a major regulator of the urinary concentrating process and is essential for the osmoadaptation of renal medullary cells. Focal adhesion kinase (FAK) is a mechanosensitive non-receptor protein tyrosine kinase expressed abundantly in the renal medulla. Since osmotic stress causes cell shrinkage, the present study investigated the contribution of FAK on TonEBP/NFAT5 activation. Osmotic stress induced time-dependent activation of FAK as evidenced by phosphorylation at Tyr-397, and furosemide reduces FAK Tyr-397 phosphorylation in the rat renal medulla. Both pharmacological inhibition of FAK and siRNA-mediated knockdown of FAK drastically reduced TonEBP/NFAT5 transcriptional activity and target gene expression in HEK293 cells. This effect was not mediated by impaired nuclear translocation or by reduced transactivating activity of TonEBP/NFAT5. However, TonEBP/NFAT5 abundance under hypertonic conditions was diminished by 50% by FAK inhibition or siRNA knockdown of FAK. FAK inhibition only marginally reduced transcription of the TonEBP/NFAT5 gene. Rather, TonEBP/NFAT5 mRNA stability was diminished significantly by FAK inhibition, which correlated with reduced reporter activity of the TonEBP/NFAT5 mRNA 3′ untranslated region (3′-UTR). In conclusion, FAK is a major regulator of TonEBP/NFAT5 activity by increasing its abundance via stabilization of the mRNA. This in turn, depends on the presence of the TonEBP/NFAT5 3′-UTR. PMID:24772088

  4. Insulin-induced tyrosine dephosphorylation of paxillin and focal adhesion kinase requires active phosphotyrosine phosphatase 1D.

    PubMed Central

    Ouwens, D M; Mikkers, H M; van der Zon, G C; Stein-Gerlach, M; Ullrich, A; Maassen, J A

    1996-01-01

    Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor beta-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(L-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D. PMID:8809054

  5. Focal adhesions, stress fibers and mechanical tension

    PubMed Central

    Burridge, Keith; Guilluy, Christophe

    2016-01-01

    Stress fibers and focal adhesions are complex protein arrays that produce, transmit and sense mechanical tension. Evidence accumulated over many years led to the conclusion that mechanical tension generated within stress fibers contributes to the assembly of both stress fibers themselves and their associated focal adhesions. However, several lines of evidence have recently been presented against this model. Here we discuss the evidence for and against the role of mechanical tension in driving the assembly of these structures. We also consider how their assembly is influenced by the rigidity of the substratum to which cells are adhering. Finally, we discuss the recently identified connections between stress fibers and the nucleus, and the roles that these may play, both in cell migration and regulating nuclear function. PMID:26519907

  6. Inhibition of focal adhesion kinase (FAK) activity prevents anchorage-independent ovarian carcinoma cell growth and tumor progression

    PubMed Central

    Ward, Kristy K.; Tancioni, Isabelle; Lawson, Christine; Miller, Nichol L.G.; Jean, Christine; Chen, Xiao Lei; Uryu, Sean; Kim, Josephine; Tarin, David; Stupack, Dwayne G.; Plaxe, Steven C.; Schlaepfer, David D.

    2013-01-01

    Recurrence and spread of ovarian cancer is the 5th leading cause of death for women in the United States. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase located on chromosome 8q24.3 (gene is Ptk2), a site commonly amplified in serous ovarian cancer. Elevated FAK mRNA levels in serous ovarian carcinoma are associated with decreased (logrank P = 0.0007, hazard ratio 1.43) patient overall survival, but how FAK functions in tumor progression remains undefined. We have isolated aggressive ovarian carcinoma cells termed ID8-IP after intraperitoneal (IP) growth of murine ID8 cells in C57Bl6 mice. Upon orthotopic implantation within the periovarian bursa space, ID8-IP cells exhibit greater tumor growth, local and distant metastasis, and elevated numbers of ascites-associated cells compared to parental ID8 cells. ID8-IP cells exhibit enhanced growth under non-adherent conditions with elevated FAK and c-Src tyrosine kinase activation compared to parental ID8 cells. In vitro, the small molecule FAK inhibitor (Pfizer, PF562,271, PF-271) at 0.1 uM selectively prevented anchorage-independent ID8-IP cell growth with the inhibition of FAK tyrosine (Y)397 but not c-Src Y416 phosphorylation. Oral PF-271 administration (30 mg/kg, twice daily) blocked FAK but not c-Src tyrosine phosphorylation in ID8-IP tumors. This was associated with decreased tumor size, prevention of peritoneal metastasis, reduced tumor-associated endothelial cell number, and increased tumor cell-associated apoptosis. FAK knockdown and re-expression assays showed that FAK activity selectively promoted anchorage-independent ID8-IP cell survival. These results support the continued evaluation of FAK inhibitors as a promising clinical treatment for ovarian cancer. PMID:23275034

  7. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    SciTech Connect

    Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew; Grawé, Jan; McKinney-Freeman, Shannon L.; Daley, George Q.; Welsh, Michael

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased

  8. Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization

    PubMed Central

    Datla, Srinivasa Raju; McGrail, Daniel J.; Vukelic, Sasa; Huff, Lauren P.; Lyle, Alicia N.; Pounkova, Lily; Lee, Minyoung; Seidel-Rogol, Bonnie; Khalil, Mazen K.; Hilenski, Lula L.; Terada, Lance S.; Dawson, Michelle R.; Lassègue, Bernard

    2014-01-01

    Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner. PMID:25063792

  9. Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization.

    PubMed

    Datla, Srinivasa Raju; McGrail, Daniel J; Vukelic, Sasa; Huff, Lauren P; Lyle, Alicia N; Pounkova, Lily; Lee, Minyoung; Seidel-Rogol, Bonnie; Khalil, Mazen K; Hilenski, Lula L; Terada, Lance S; Dawson, Michelle R; Lassègue, Bernard; Griendling, Kathy K

    2014-10-01

    Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner. PMID:25063792

  10. RAFTK, a Novel Member of the Focal Adhesion Kinase Family, Is Phosphorylated and Associates with Signaling Molecules upon Activation of Mature T Lymphocytes

    PubMed Central

    Ganju, Ramesh K.; Hatch, William C.; Avraham, Hava; Ona, Mel A.; Druker, Brian; Avraham, Shalom; Groopman, Jerome E.

    1997-01-01

    The related adhesion focal tyrosine kinase (RAFTK), a recently discovered member of the focal adhesion kinase family, has previously been reported to participate in signal transduction in neuronal cells, megakaryocytes, and B lymphocytes. We have found that RAFTK is constitutively expressed in human T cells and is rapidly phosphorylated upon the activation of the T cell receptor (TCR). This activation also results in an increase in the autophosphorylation and kinase activity of RAFTK. After its stimulation, there was an increase in the association of the src cytoplasmic tyrosine kinase Fyn and the adapter protein Grb2. This association was mediated through the SH2 domains of Fyn and Grb2. RAFTK also co-immunoprecipitates with the SH2 domain of Lck and with the cytoskeletal protein paxillin through its COOH-terminal proline-rich domain. The tyrosine phosphorylation of RAFTK after T cell receptor-mediated stimulation was reduced by the pretreatment of cells with cytochalasin D, suggesting the role of the cytoskeleton in this process. These observations indicate that RAFTK participates in T cell receptor signaling and may act to link signals from the cell surface to the cytoskeleton and thereby affect the host immune response. PMID:9091579

  11. Stability of focal adhesion enhanced by its inner force fluctuation

    NASA Astrophysics Data System (ADS)

    Mao, Zhi-Xiu; Chen, Xiao-Feng; Chen, Bin

    2015-08-01

    Cells actively sense and respond to mechanical signals from the extracellular matrix through focal adhesions. By representing a single focal adhesion as a cluster of slip bonds, it has been demonstrated that the cluster often became unstable under fluctuated forces. However, an unusual case was also reported, where the stability of the cluster might be substantially enhanced by a fluctuated force with a relatively low fluctuation frequency and high fluctuation amplitude. Such an observation cannot be explained by the conventional fracture theory of fatigue. Here, we intensively investigate this intriguing observation by carrying out systematic parametric studies. Our intensive simulation results indicate that stability enhancement of this kind is in fact quite robust, which can be affected by the stochastic features of a single bond and the profile of the fluctuated forces such as the average value of bond force. We then suggest that the fluctuation of traction force within a focal adhesion might enhance its stability in a certain way. Project supported by the National Natural Science Foundation of China (Grant No.*11372279).

  12. Neuronal polarity selection by topography-induced focal adhesion control.

    PubMed

    Ferrari, Aldo; Cecchini, Marco; Serresi, Michela; Faraci, Paolo; Pisignano, Dario; Beltram, Fabio

    2010-06-01

    Interaction between differentiating neurons and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. In previous works we demonstrated that PC12 cell interaction with nanogratings (alternating lines of ridges and grooves of submicron size) promotes bipolarity and alignment to the substrate topography. Here, we investigate the role of focal adhesions, cell contractility, and actin dynamics in this process. Exploiting nanoimprint lithography techniques and a cyclic olefin copolymer, we engineered biocompatible nanostructured substrates designed for high-resolution live-cell microscopy. Our results reveal that neuronal polarization and contact guidance are based on a geometrical constraint of focal adhesions resulting in an angular modulation of their maturation and persistence. We report on ROCK1/2-myosin-II pathway activity and demonstrate that ROCK-mediated contractility contributes to polarity selection during neuronal differentiation. Importantly, the selection process confined the generation of actin-supported membrane protrusions and the initiation of new neurites at the poles. Maintenance of the established polarity was independent from NGF stimulation. Altogether our results imply that focal adhesions and cell contractility stably link the topographical configuration of the extracellular environment to a corresponding neuronal polarity state. PMID:20304485

  13. Allosteric regulation of focal adhesion kinase by PIP₂ and ATP.

    PubMed

    Zhou, Jing; Bronowska, Agnieszka; Le Coq, Johanne; Lietha, Daniel; Gräter, Frauke

    2015-02-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains--the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP₂) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP₂. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP₂. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP₂ binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP₂ to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP₂ binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation. PMID:25650936

  14. Kindlin-2 phosphorylation by Src at Y193 enhances Src activity and is involved in Migfilin recruitment to the focal adhesions.

    PubMed

    Liu, Zhaoli; Lu, Danyu; Wang, Xiang; Wan, Junhu; Liu, Chang; Zhang, Hongquan

    2015-07-01

    Kindlin-2 regulates external to internal cell signaling by interaction with integrins in a process that involves the tyrosine kinase, Src. However, the underlying mechanisms remain elusive. Here we report that Src binds to and phosphorylates Kindlin-2 at Y193. Reciprocally, Kindlin-2-Y193 phosphorylation activates and maintains Src kinase activity. Kindlin-2-Y193 phosphorylation is also involved in its binding capacity with Migfilin and the recruitment of Migfilin to the focal adhesions. Functionally, we demonstrate that Kindlin-2-Y193 phosphorylation regulates Kindlin-2-mediated cell spreading and migration. These findings suggest that Src, Kindlin-2 and Migfilin together constitute a positive feedback loop that controls Src activity and regulates integrin-mediated cellular functions. PMID:26037143

  15. Assembly and mechanosensory function of focal adhesions: experiments and models.

    PubMed

    Bershadsky, Alexander D; Ballestrem, Christoph; Carramusa, Letizia; Zilberman, Yuliya; Gilquin, Benoit; Khochbin, Saadi; Alexandrova, Antonina Y; Verkhovsky, Alexander B; Shemesh, Tom; Kozlov, Michael M

    2006-04-01

    Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex

  16. Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration

    PubMed Central

    Wu, Jui-Chung; Chen, Yu-Chen; Kuo, Chih-Ting; Wenshin Yu, Helen; Chen, Yin-Quan; Chiou, Arthur; Kuo, Jean-Cheng

    2015-01-01

    Directed cell migration requires dynamical control of the protein complex within focal adhesions (FAs) and this control is regulated by signaling events involving tyrosine phosphorylation. We screened the SH2 domains present in tyrosine-specific kinases and phosphatases found within FAs, including SRC, SHP1 and SHP2, and examined whether these enzymes transiently target FAs via their SH2 domains. We found that the SRC_SH2 domain and the SHP2_N-SH2 domain are associated with FAs, but only the SRC_SH2 domain is able to be regulated by focal adhesion kinase (FAK). The FAK-dependent association of the SRC_SH2 domain is necessary and sufficient for SRC FA targeting. When the targeting of SRC into FAs is inhibited, there is significant suppression of SRC-mediated phosphorylation of paxillin and FAK; this results in an inhibition of FA formation and maturation and a reduction in cell migration. This study reveals an association between FAs and the SRC_SH2 domain as well as between FAs and the SHP2_N-SH2 domains. This supports the hypothesis that the FAK-regulated SRC_SH2 domain plays an important role in directing SRC into FAs and that this SRC-mediated FA signaling drives cell migration. PMID:26681405

  17. RhoJ interacts with the GIT-PIX complex and regulates focal adhesion disassembly.

    PubMed

    Wilson, Eleanor; Leszczynska, Katarzyna; Poulter, Natalie S; Edelmann, Francesca; Salisbury, Victoria A; Noy, Peter J; Bacon, Andrea; Rappoport, Joshua Z; Heath, John K; Bicknell, Roy; Heath, Victoria L

    2014-07-15

    RhoJ is a Rho GTPase expressed in endothelial cells and tumour cells, which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; small interfering RNA (siRNA)-mediated knockdown of RhoJ increased focal adhesion disassembly time, whereas expression of an active mutant (daRhoJ) decreased it. Furthermore, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast two-hybrid experiments, and this depended on the Spa homology domain of GIT1. GIT1, GIT2, β-PIX (also known as ARHGEF7) and RhoJ all colocalised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of both GIT1 and GIT2, or β-PIX phenocopying RhoJ knockdown. RhoJ-knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give new insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation. PMID:24928894

  18. In vivo epidermal migration requires focal adhesion targeting of ACF7.

    PubMed

    Yue, Jiping; Zhang, Yao; Liang, Wenguang G; Gou, Xuewen; Lee, Philbert; Liu, Han; Lyu, Wanqing; Tang, Wei-Jen; Chen, Shao-Yu; Yang, Feng; Liang, Hong; Wu, Xiaoyang

    2016-01-01

    Turnover of focal adhesions allows cell retraction, which is essential for cell migration. The mammalian spectraplakin protein, ACF7 (Actin-Crosslinking Factor 7), promotes focal adhesion dynamics by targeting of microtubule plus ends towards focal adhesions. However, it remains unclear how the activity of ACF7 is regulated spatiotemporally to achieve focal adhesion-specific guidance of microtubule. To explore the potential mechanisms, we resolve the crystal structure of ACF7's NT (amino-terminal) domain, which mediates F-actin interactions. Structural analysis leads to identification of a key tyrosine residue at the calponin homology (CH) domain of ACF7, whose phosphorylation by Src/FAK (focal adhesion kinase) complex is essential for F-actin binding of ACF7. Using skin epidermis as a model system, we further demonstrate that the phosphorylation of ACF7 plays an indispensable role in focal adhesion dynamics and epidermal migration in vitro and in vivo. Together, our findings provide critical insights into the molecular mechanisms underlying coordinated cytoskeletal dynamics during cell movement. PMID:27216888

  19. In vivo epidermal migration requires focal adhesion targeting of ACF7

    DOE PAGESBeta

    Yue, Jiping; Zhang, Yao; Liang, Wenguang G.; Gou, Xuewen; Lee, Philbert; Liu, Han; Lyu, Wanqing; Tang, Wei -Jen; Chen, Shao -Yu; Yang, Feng; et al

    2016-05-24

    Turnover of focal adhesions allows cell retraction, which is essential for cell migration. The mammalian spectraplakin protein, ACF7 (Actin-Crosslinking Factor 7), promotes focal adhesion dynamics by targeting of microtubule plus ends towards focal adhesions. However, it remains unclear how the activity of ACF7 is regulated spatiotemporally to achieve focal adhesion-specific guidance of microtubule. To explore the potential mechanisms, we resolve the crystal structure of ACF7's NT (amino-terminal) domain, which mediates F-actin interactions. Structural analysis leads to identification of a key tyrosine residue at the calponin homology (CH) domain of ACF7, whose phosphorylation by Src/FAK (focal adhesion kinase) complex is essentialmore » for F-actin binding of ACF7. Using skin epidermis as a model system, we further demonstrate that the phosphorylation of ACF7 plays an indispensable role in focal adhesion dynamics and epidermal migration in vitro and in vivo. Altogether, our findings provide critical insights into the molecular mechanisms underlying coordinated cytoskeletal dynamics during cell movement.« less

  20. A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis

    PubMed Central

    Daley, William P.; Kohn, Joshua M.; Larsen, Melinda

    2011-01-01

    Cleft formation is the initial step of branching morphogenesis in many organs. We previously demonstrated that ROCK 1 regulates a non-muscle myosin II-dependent mechanochemical checkpoint to transition initiated clefts to progressing clefts in developing submandibular salivary glands. Here, we report that ROCK-mediated integrin activation and subsequent formation of focal adhesion complexes comprise this mechanochemical checkpoint. Inhibition of ROCK1 and non-muscle myosin II activity decreased integrin β1 activation in the cleft region and interfered with localization and activation of focal adhesion complex proteins, such as focal adhesion kinase (FAK). Inhibition of FAK activity also prevented cleft progression, by disrupting recruitment of the focal adhesion proteins talin and vinculin and subsequent fibronectin assembly in the cleft region while decreasing ERK1/2 activation. These results demonstrate that inside-out integrin signaling leading to a localized recruitment of active FAK-containing focal adhesion protein complexes generates a mechanochemical checkpoint that facilitates progression of branching morphogenesis. PMID:22016182

  1. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

    PubMed Central

    Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

    2015-01-01

    Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. PMID:26703626

  2. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling

    PubMed Central

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2014-01-01

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. ST2L, the receptor for IL-33, is expressed on immune effector cells and lung epithelia, and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system, however its upstream internalization has not been studied. Here, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2LS446A) and phosphorylation site mutant (ST2LS442A) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Further, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. PMID:25472995

  3. A Small Physiological Electric Field Mediated Responses of Extravillous Trophoblasts Derived from HTR8/SVneo Cells: Involvement of Activation of Focal Adhesion Kinase Signaling

    PubMed Central

    Zhang, Juan; Ren, Rongmei; Luo, Xuefeng; Fan, Ping; Liu, Xinghui; Liang, Shanshan; Ma, Lei; Yu, Ping; Bai, Huai

    2014-01-01

    Moderate invasion of trophoblast cells into endometrium is essential for the placental development and normal pregnancy. Electric field (EF)-induced effects on cellular behaviors have been observed in many cell types. This study was to investigate the effect of physiological direct current EF (dc EF) on cellular responses such as elongation, orientation and motility of trophoblast cells. Immortalized first trimester extravillous trophoblast cells (HTR-8/SVneo) were exposed to the dc EF at physiological magnitude. Cell images were recorded and analyzed by image analyzer. Cell lysates were used to detect protein expression by Western blot. Cultured in the dc EFs the cells showed elongation, orientation and enhanced migration rate compared with non-EF stimulated cells at field strengths of 100 mV/mm to 200 mV/mm. EF exposure increased focal adhesion kinase (FAK) phosphorylation in a time-dependent manner and increased expression levels of MMP-2. Pharmacological inhibition of FAK impaired the EF-induced responses including motility and abrogated the elevation of MMP-2 expression. However, the expression levels of integrins like integrin α1, α5, αV and β1 were not affected by EF stimulation. Our results demonstrate the importance of FAK activation in migration/motility of trophobalst cells driven by EFs. In addition, it raises the feasibility of using applied EFs to promote placentation through effects on trophoblast cells. PMID:24643246

  4. Theory of the mechanical response of focal adhesions to shear flow

    NASA Astrophysics Data System (ADS)

    Biton, Y. Y.; Safran, S. A.

    2010-05-01

    The response of cells to shear flow is primarily determined by the asymmetry of the external forces and moments that are sensed by each member of a focal adhesion pair connected by a contractile stress fiber. In the theory presented here, we suggest a physical model in which each member of such a pair of focal adhesions is treated as an elastic body subject to both a myosin-activated contractile force and the shear stress induced by the external flow. The elastic response of a focal adhesion complex is much faster than the active cellular processes that determine the size of the associated focal adhesions and the direction of the complex relative to the imposed flow. Therefore, the complex attains its mechanical equilibrium configuration which may change because of the cellular activity. Our theory is based on the experimental observation that focal adhesions modulate their cross-sectional area in order to attain an optimal shear. Using this assumption, our elastic model shows that such a complex can passively change its orientation to align parallel to the direction of the flow.

  5. Cell morphology and focal adhesion location alters internal cell stress.

    PubMed

    Mullen, C A; Vaughan, T J; Voisin, M C; Brennan, M A; Layrolle, P; McNamara, L M

    2014-12-01

    Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues. PMID:25297316

  6. DFak56 is a novel Drosophila melanogaster focal adhesion kinase.

    PubMed

    Palmer, R H; Fessler, L I; Edeen, P T; Madigan, S J; McKeown, M; Hunter, T

    1999-12-10

    The mammalian focal adhesion kinase (FAK) family of nonreceptor protein-tyrosine kinases have been implicated in controlling a multitude of cellular responses to the engagement of cell surface integrins and G protein-coupled receptors. We describe here a Drosophila melanogaster FAK homologue, DFak56, which maps to band 56D on the right arm of the second chromosome. Full-length DFak56 cDNA encodes a phosphoprotein of 140 kDa, which shares strong sequence similarity not only with mammalian p125(FAK) but also with the more recently described mammalian Pyk2 (also known as CAKbeta, RAFTK, FAK2, and CADTK) FAK family member. DFak56 has intrinsic tyrosine kinase activity and is phosphorylated on tyrosine in vivo. As is the case for FAK, tyrosine phosphorylation of DFak56 is increased upon plating Drosophila embryo cells on extracellular matrix proteins. In situ hybridization and immunofluorescence staining analysis showed that DFak56 is ubiquitously expressed with particularly high levels within the developing central nervous system. We utilized the UAS-GAL4 expression system to express DFak56 and analyze its function in vivo. Overexpression of DFak56 in the wing imaginal disc results in wing blistering in adults, a phenotype also observed with both position-specific integrin loss of function and position-specific integrin overexpression. Our results imply a role for DFak56 in adhesion-dependent signaling pathways in vivo during D. melanogaster development. PMID:10585440

  7. Cell morphology and focal adhesion location alters internal cell stress

    PubMed Central

    Mullen, C. A.; Vaughan, T. J.; Voisin, M. C.; Brennan, M. A.; Layrolle, P.; McNamara, L. M.

    2014-01-01

    Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues. PMID:25297316

  8. Cadherin-11 localizes to focal adhesions and promotes cell–substrate adhesion

    PubMed Central

    Langhe, Rahul P.; Gudzenko, Tetyana; Bachmann, Michael; Becker, Sarah F.; Gonnermann, Carina; Winter, Claudia; Abbruzzese, Genevieve; Alfandari, Dominique; Kratzer, Marie-Claire; Franz, Clemens M.; Kashef, Jubin

    2016-01-01

    Cadherin receptors have a well-established role in cell–cell adhesion, cell polarization and differentiation. However, some cadherins also promote cell and tissue movement during embryonic development and tumour progression. In particular, cadherin-11 is upregulated during tumour and inflammatory cell invasion, but the mechanisms underlying cadherin-11 stimulated cell migration are still incompletely understood. Here, we show that cadherin-11 localizes to focal adhesions and promotes adhesion to fibronectin in Xenopus neural crest, a highly migratory embryonic cell population. Transfected cadherin-11 also localizes to focal adhesions in different mammalian cell lines, while endogenous cadherin-11 shows focal adhesion localization in primary human fibroblasts. In focal adhesions, cadherin-11 co-localizes with β1-integrin and paxillin and physically interacts with the fibronectin-binding proteoglycan syndecan-4. Adhesion to fibronectin mediated by cadherin-11/syndecan-4 complexes requires both the extracellular domain of syndecan-4, and the transmembrane and cytoplasmic domains of cadherin-11. These results reveal an unexpected role of a classical cadherin in cell–matrix adhesion during cell migration. PMID:26952325

  9. Nanog Increases Focal Adhesion Kinase (FAK) Promoter Activity and Expression and Directly Binds to FAK Protein to Be Phosphorylated*

    PubMed Central

    Ho, Baotran; Olson, Gretchen; Figel, Sheila; Gelman, Irwin; Cance, William G.; Golubovskaya, Vita M.

    2012-01-01

    Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis. PMID:22493428

  10. Nanog increases focal adhesion kinase (FAK) promoter activity and expression and directly binds to FAK protein to be phosphorylated.

    PubMed

    Ho, Baotran; Olson, Gretchen; Figel, Sheila; Gelman, Irwin; Cance, William G; Golubovskaya, Vita M

    2012-05-25

    Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis. PMID:22493428

  11. Concentrated growth factor promotes Schwann cell migration partly through the integrin β1-mediated activation of the focal adhesion kinase pathway.

    PubMed

    Qin, Jie; Wang, Lin; Zheng, Ling; Zhou, Xiaoyan; Zhang, Yidi; Yang, Tingting; Zhou, Yanmin

    2016-05-01

    Nerve injury is a serious complication associated with dental implant surgery. Following nerve injury, the migration of Schwann cells (SCs) supports nerve regeneration. Concentrated growth factor (CGF) belongs to a new generation of biomaterials that contain fibrin matrix, as well as a number of growth factors that affect the migration of various types of cells, including endothelial cells and cancer cells. To the very best of our knowledge, there are no available studies to date on the promoting effect of CGF on the migration of SCs. Thus, the aim of the present study was to characterize the structure of CGF and growth factor release, examine the effects of CGF on the migration of SCs, and to examine the role of integrin β1 and the focal adhesion kinase (FAK) pathway in the CGF-induced migration of SCs. For this purpose, CGF was prepared by centrifuging rat venous blood, which produced a fiber-like matrix capable of releasing transforming growth factor-β1 (TGF-β1) over a sustained period of time (at least 13 days). The soluble component of CGF was used to produce conditioned media to treat the SC cells in culture. The results demonstrated that CGF promoted the migration of SCs and increased the expression of integrin β1. These effects appeared to involve FAK phosphorylation, which occurred downstream of integrin β1 activation. The short-interfering RNA (siRNA)-mediated downregulation of integrin β1 expression did not block the ability of CGF to promote the migration of SCs. These data suggest that CGF promotes the migration of SCs partly through the integrin β1-mediated activation of the FAK pathway. PMID:26986804

  12. Focal Adhesion Kinase (FAK)-related Non-kinase Inhibits Myofibroblast Differentiation through Differential MAPK Activation in a FAK-dependent Manner*

    PubMed Central

    Ding, Qiang; Gladson, Candece L.; Wu, Hongju; Hayasaka, Haurko; Olman, Mitchell A.

    2008-01-01

    Transforming growth factor (TGF)-β1 induces fibroblast transdifferentiation to myofibroblasts, a process that requires the involvement of integrin-mediated signaling and focal adhesion kinase (FAK). FAK-related non-kinase (FRNK) is known for its role in inhibiting integrin-mediated cell migration; however, its role in myofibroblast differentiation has not been defined. Here, we report that FRNK abrogates TGF-β1-induced myofibroblast differentiation in vitro and in vivo. TGF-β1 can induce α-smooth muscle actin (α-SMA) expression in the presence or absence of FAK; however, TGF-β1-induced α-SMA expression is reduced (∼73%) in FAK-deficient fibroblasts. Although both ERK and p38 MAPK activation is required for maximal TGF-β1-induced α-SMA expression, ERK is the major signaling intermediate in cells that express FAK. In contrast, p38 MAPK is the dominant mediator of TGF-β1-induced α-SMA expression in FAK-deficient cells. FRNK overexpression blocks TGF-β1-induced ERK or p38 MAPK activation in the presence, and surprisingly, in the absence of FAK. The loss of FRNK was tested in vivo during experimentally induced pulmonary fibrosis in mice. FRNK knock-out mice have a greater increase in α-SMA-expressing cells in response to a pulmonary fibrotic stimulus in vivo, as compared with congenic wild type mice. This is the first time that FRNK loss has been shown to modify the pathobiology in any animal disease model. Together, the data demonstrate that FRNK negatively regulates myofibroblast differentiation in vitro and in vivo. These data further suggest that modulation FRNK expression may be a novel avenue for therapeutic intervention in tissue fibrosis. PMID:18669633

  13. Neuropilin-2 regulates α6β1 integrin in the formation of focal adhesions and signaling.

    PubMed

    Goel, Hira Lal; Pursell, Bryan; Standley, Clive; Fogarty, Kevin; Mercurio, Arthur M

    2012-01-15

    The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2(high) and NRP2(low) populations that differed in integrin expression and adhesion to laminin. Specifically, the NRP2(high) population adhered more avidly to laminin and expressed high levels of the α6β1 integrin than the NRP2(low) population. The NRP2(high) population formed numerous focal adhesions on laminin that were not seen in the NRP2(low) population. These results were substantiated using breast carcinoma cell lines that express NRP2 and α6β1 integrin. Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for adhesion on laminin. A specific interaction between NRP2 and α6β1 integrin was detected by co-immunoprecipitation. NRP2 is necessary for focal adhesion formation on laminin and for the association of α6β1 integrin with the cytoskeleton. NRP2 also facilitates α6β1-integrin-mediated activation of FAK and Src. Unexpectedly, we discovered that NRP2 is located in focal adhesions on laminin. The mechanism by which NRP2 regulates the interaction of α6β1 integrin with laminin to form focal adhesions involves PKC activation. Together, our data reveal a new VEGF-NRP2 signaling pathway that activates the α6β1 integrin and enables it to form focal adhesions and signal. This pathway is important in the pathogenesis of breast cancer. PMID:22302985

  14. Functional Characterization of KIN-32, the Caenorhabditis elegans Homolog of Focal Adhesion Kinase

    PubMed Central

    Cram, Erin J.; Fontanez, Kristina Marie; Schwarzbauer, Jean E.

    2014-01-01

    We have identified the single Caenorhabditis elegans focal adhesion kinase (FAK) homolog KIN-32, which has the signature FAK structure including an N-terminal Four.1-Ezrin-Radixin-Moesin (FERM) domain followed by a tyrosine kinase domain and a C-terminal domain with weak homology to the focal adhesion targeting domain. The functional requirements for KIN-32 were examined using RNA interference depletion experiments and analysis of a deletion allele, kin-32(ok166), in which a large segment of the FERM domain is missing. Our results show that reduced levels of expression or absence of the FERM domain do not affect viability, fertility, or anatomy in C. elegans. Expression of an analogous FERM deletion in mouse FAK showed kinase activity in vitro and supported normal focal adhesion localization in cell culture. Thus, the FERM domain of KIN-32, and possibly KIN-32 activity in general, appears to be dispensable for normal C. elegans physiology. PMID:18297732

  15. Integrin upregulation and localization to focal adhesion sites in pregnant human myometrium.

    PubMed

    Burkin, Heather R; Rice, Monica; Sarathy, Apurva; Thompson, Sara; Singer, Cherie A; Buxton, Iain L O

    2013-07-01

    Focal adhesions are integrin-rich microdomains that structurally link the cytoskeleton to the extracellular matrix and transmit mechanical signals. In the pregnant uterus, increases in integrin expression and activation are thought to be critical for the formation of the mechanical syncytium required for labor. The aim of this study was to determine which integrins are upregulated and localized to focal adhesions in pregnant human myometrium. We used quantitative polymerase chain reaction, Western blotting, and confocal microscopy to determine the expression levels and colocalization with focal adhesion proteins. We observed increases in several integrin transcripts in pregnant myometrium. At the protein level, integrins such as α5-integrin (ITGA5), ITGA7, ITGAV, and ITGB3 were significantly increased during pregnancy. The integrins ITGA3, ITGA5, ITGA7, and ITGB1 colocalized with focal adhesion proteins in term human myometrium. These data suggest that integrins α3β1, α5β1, and α7β1 are the most likely candidates to transmit mechanical signals from the extracellular matrix through focal adhesions in pregnant human myometrium. PMID:23298868

  16. Focal adhesion kinase is involved in mechanosensing during fibroblast migration

    NASA Technical Reports Server (NTRS)

    Wang, H. B.; Dembo, M.; Hanks, S. K.; Wang, Y.

    2001-01-01

    Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

  17. Microtubules regulate focal adhesion dynamics through MAP4K4.

    PubMed

    Yue, Jiping; Xie, Min; Gou, Xuewen; Lee, Philbert; Schneider, Michael D; Wu, Xiaoyang

    2014-12-01

    Disassembly of focal adhesions (FAs) allows cell retraction and integrin detachment from the extracellular matrix, processes critical for cell movement. Growth of microtubules (MTs) can promote FA turnover by serving as tracks to deliver proteins essential for FA disassembly. The molecular nature of this FA "disassembly factor," however, remains elusive. By quantitative proteomics, we identified mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) as an FA regulator that associates with MTs. Knockout of MAP4K4 stabilizes FAs and impairs cell migration. By exploring underlying mechanisms, we further show that MAP4K4 associates with ending binding 2 (EB2) and IQ motif and SEC7 domain-containing protein 1 (IQSEC1), a guanine nucleotide exchange factor specific for Arf6, whose activation promotes integrin internalization. Together, our findings provide critical insight into FA disassembly, suggesting that MTs can deliver MAP4K4 toward FAs through EB2, where MAP4K4 can, in turn, activate Arf6 via IQSEC1 and enhance FA dissolution. PMID:25490267

  18. Microtubules Regulate Focal Adhesion Dynamics through MAP4K4

    PubMed Central

    Yue, Jiping; Xie, Min; Gou, Xuewen; Lee, Philbert; Schneider, Michael D; Wu, Xiaoyang

    2014-01-01

    Disassembly of focal adhesions (FAs) allows cell retraction and integrin detachment from the ECM, processes critical for cell movement. Growth of MT (microtubule) can promote FA turnover by serving as tracks to deliver proteins essential for FA disassembly. The molecular nature of this FA “disassembly factor”, however, remains elusive. By quantitative proteomics, we identified MAP4K4 (mitogen-activated protein kinase kinase kinase kinase 4) as a FA regulator that associates with MTs. Conditional knockout (cKO) of MAP4K4 in skin stabilizes FAs and impairs epidermal migration. By exploring underlying mechanisms, we further show that MAP4K4 associates with EB2, a MT binding protein, and IQSEC1, a guanine nucleotide exchange factor (GEF) specific for Arf6, whose activation promotes integrin internalization. Together, our findings provide critical insights into FA disassembly, suggesting that MTs can deliver MAP4K4 toward FAs through EB2, where MAP4K4 can in turn activate Arf6 via IQSEC1 and enhance FA dissolution. PMID:25490267

  19. Focal Adhesion Kinase Inhibitors in Combination with Erlotinib Demonstrate Enhanced Anti-Tumor Activity in Non-Small Cell Lung Cancer

    PubMed Central

    Howe, Grant A.; Xiao, Bin; Zhao, Huijun; Al-Zahrani, Khalid N.; Hasim, Mohamed S.; Villeneuve, James; Sekhon, Harmanjatinder S.; Goss, Glenwood D.; Sabourin, Luc A.; Dimitroulakos, Jim; Addison, Christina L.

    2016-01-01

    Blockade of epidermal growth factor receptor (EGFR) activity has been a primary therapeutic target for non-small cell lung cancers (NSCLC). As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs), there is a need for additional therapeutic approaches in patients with wild-type EGFR. As a key component of downstream integrin signalling and known receptor cross-talk with EGFR, we hypothesized that targeting focal adhesion kinase (FAK) activity, which has also been shown to correlate with aggressive stage in NSCLC, would lead to enhanced activity of EGFR TKIs. As such, EGFR TKI-resistant NSCLC cells (A549, H1299, H1975) were treated with the EGFR TKI erlotinib and FAK inhibitors (PF-573,228 or PF-562,271) both as single agents and in combination. We determined cell viability, apoptosis and 3-dimensional growth in vitro and assessed tumor growth in vivo. Treatment of EGFR TKI-resistant NSCLC cells with FAK inhibitor alone effectively inhibited cell viability in all cell lines tested; however, its use in combination with the EGFR TKI erlotinib was more effective at reducing cell viability than either treatment alone when tested in both 2- and 3-dimensional assays in vitro, with enhanced benefit seen in A549 cells. This increased efficacy may be due in part to the observed inhibition of Akt phosphorylation when the drugs were used in combination, where again A549 cells demonstrated the most inhibition following treatment with the drug combination. Combining erlotinib with FAK inhibitor was also potent in vivo as evidenced by reduced tumor growth in the A549 mouse xenograft model. We further ascertained that the enhanced sensitivity was irrespective of the LKB1 mutational status. In summary, we demonstrate the effectiveness of combining erlotinib and FAK inhibitors for use in known EGFR wild-type, EGFR TKI resistant cells, with the potential that a subset of cell types, which includes A549, could be particularly

  20. Focal Adhesion Assembly Induces Phenotypic Changes and Dedifferentiation in Chondrocytes.

    PubMed

    Shin, Hyunjun; Lee, Mi Nam; Choung, Jin Seung; Kim, Sanghee; Choi, Byung Hyune; Noh, Minsoo; Shin, Jennifer H

    2016-08-01

    The expansion of autologous chondrocytes in vitro is used to generate sufficient populations for cell-based therapies. However, during monolayer culture, chondrocytes lose inherent characteristics and shift to fibroblast-like cells as passage number increase. Here, we investigated passage-dependent changes in cellular physiology, including cellular morphology, motility, and gene and protein expression, as well as the role of focal adhesion and cytoskeletal regulation in the dedifferentiation process. We found that the gene and protein expression levels of both the focal adhesion complex and small Rho GTPases are upregulated with increasing passage number and are closely linked to chondrocyte dedifferentiation. The inhibition of focal adhesion kinase (FAK) but not small Rho GTPases induced the loss of fibroblastic traits and the recovery of collagen type II, aggrecan, and SOX9 expression levels in dedifferentiated chondrocytes. Based on these findings, we propose a strategy to suppress chondrogenic dedifferentiation by inhibiting the identified FAK or Src pathways while maintaining the expansion capability of chondrocytes in a 2D environment. These results highlight a potential therapeutic target for the treatment of skeletal diseases and the generation of cartilage in tissue-engineering approaches. J. Cell. Physiol. 231: 1822-1831, 2016. © 2015 Wiley Periodicals, Inc. PMID:26661891

  1. Nanotopographical modification: a regulator of cellular function through focal adhesions

    PubMed Central

    Biggs, Manus Jonathan Paul; Richards, R. Geoff; Dalby, Matthew J.

    2010-01-01

    As materials technology and the field of biomedical engineering advances, the role of cellular mechanisms, in particular adhesive interactions with implantable devices, becomes more relevant in both research and clinical practice. A key tenet of medical device design has evolved from the exquisite ability of biological systems to respond to topographical features or chemical stimuli, a process that has led to the development of next-generation biomaterials for a wide variety of clinical disorders. In vitro studies have identified nanoscale features as potent modulators of cellular behavior through the onset of focal adhesion formation. The focus of this review is on the recent developments concerning the role of nanoscale structures on integrin-mediated adhesion and cellular function with an emphasis on the generation of medical constructs with regenerative applications. PMID:20138244

  2. Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

    SciTech Connect

    Klemm, Anna H.; Diez, Gerold; Alonso, Jose-Luis

    2009-02-13

    Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.

  3. TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility

    PubMed Central

    Cáceres, Mónica; Ortiz, Liliana; Recabarren, Tatiana; Romero, Anibal; Colombo, Alicia; Leiva-Salcedo, Elías; Varela, Diego; Rivas, José; Silva, Ian; Morales, Diego; Campusano, Camilo; Almarza, Oscar; Simon, Felipe; Toledo, Hector; Park, Kang-Sik; Trimmer, James S.; Cerda, Oscar

    2015-01-01

    Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility. PMID:26110647

  4. Focal adhesion molecules as potential target of lead toxicity in NRK-52E cell line.

    PubMed

    Giuliani, Roberta; Bettoni, Francesca; Leali, Daria; Morandini, Fausta; Apostoli, Pietro; Grigolato, Piergiovanni; Cesana, Bruno Mario; Aleo, Maria Francesca

    2005-11-01

    In this study, we investigated the influence of inorganic lead (Pb(II)), an environmental pollutant having nephrotoxic action, on the focal adhesion (FA) organization of a rat kidney epithelial cell line (NRK-52E). In particular, we evaluated the effects of the metal on the recruitment of paxillin, focal adhesion kinase, vinculin and cytoskeleton proteins at the FAs complexes. We provided evidences that, in proliferating NRK-52E cell cultures, low concentrations of Pb(II) affect the cell adhesive ability and stimulate the disassembly of FAs, thus inhibiting the integrin-activated signalling. These effects appeared to be strictly associated to the Pb-induced arrest of cell cycle at G0/G1 phase also proved in this cell line. PMID:16253243

  5. Mechanotransduction at focal adhesions: integrating cytoskeletal mechanics in migrating cells

    PubMed Central

    Kuo, Jean-Cheng

    2013-01-01

    Focal adhesions (FAs) are complex plasma membrane-associated macromolecular assemblies that serve to physically connect the actin cytoskeleton to integrins that engage with the surrounding extracellular matrix (ECM). FAs undergo maturation wherein they grow and change composition differentially to provide traction and to transduce the signals that drive cell migration, which is crucial to various biological processes, including development, wound healing and cancer metastasis. FA-related signalling networks dynamically modulate the strength of the linkage between integrin and actin and control the organization of the actin cytoskeleton. In this review, we have summarized a number of recent investigations exploring how FA composition is affected by the mechanical forces that transduce signalling networks to modulate cellular function and drive cell migration. Understanding the fundamental mechanisms of how force governs adhesion signalling provides insights that will allow the manipulation of cell migration and help to control migration-related human diseases. PMID:23551528

  6. Focal adhesion kinase overexpression and its impact on human osteosarcoma

    PubMed Central

    Chen, Yong; Yang, Aizhen; Chen, Hui; Zhang, Jian; Wu, Sujia; Shi, Xin; Wang, Chen; Sun, Xiaoliang

    2015-01-01

    Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P < 0.001). There were similar differences in metastasis-free survival between groups. The Cox proportional hazards analysis showed that the FAK expression profile was an independent indicator of both overall and metastasis-free survival. siRNA-based knockdown of FAK not only dramatically reduced the migration and invasion of MG63 and 143B cells, but also had a distinct effect on osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis. PMID:26393679

  7. Autocrine netrin function inhibits glioma cell motility and promotes focal adhesion formation.

    PubMed

    Jarjour, Andrew A; Durko, Margaret; Luk, Tamarah L; Marçal, Nathalie; Shekarabi, Masoud; Kennedy, Timothy E

    2011-01-01

    Deregulation of mechanisms that control cell motility plays a key role in tumor progression by promoting tumor cell dissemination. Secreted netrins and their receptors, Deleted in Colorectal Cancer (DCC), neogenin, and the UNC5 homologues, regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Netrin and netrin receptor expression have previously been shown to be disrupted in invasive tumors, including glioblastoma. We determined that the human glioblastoma cell lines U87, U343, and U373 all express neogenin, UNC5 homologues, and netrin-1 or netrin-3, but only U87 cells express DCC. Using transfilter migration assays, we demonstrate DCC-dependent chemoattractant migration of U87 cells up a gradient of netrin-1. In contrast, U343 and U373 cells, which do not express DCC, were neither attracted nor repelled. Ectopic expression of DCC by U343 and U373 cells resulted in these cells becoming competent to respond to a gradient of netrin-1 as a chemoattractant, and also slowed their rate of spontaneous migration. Here, in addition to netrins' well-characterized chemotropic activity, we demonstrate an autocrine function for netrin-1 and netrin-3 in U87 and U373 cells that slows migration. We provide evidence that netrins promote the maturation of focal complexes, structures associated with cell movement, into focal adhesions. Consistent with this, netrin, DCC, and UNC5 homologues were associated with focal adhesions, but not focal complexes. Disrupting netrin or DCC function did not alter cell proliferation or survival. Our findings provide evidence that DCC can slow cell migration, and that neogenin and UNC5 homologues are not sufficient to substitute for DCC function in these cells. Furthermore, we identify a role for netrins as autocrine inhibitors of cell motility that promote focal adhesion formation. These findings suggest that disruption of netrin signalling may disable a mechanism that normally restrains inappropriate cell migration. PMID

  8. α-Actinin-4 Enhances Colorectal Cancer Cell Invasion by Suppressing Focal Adhesion Maturation

    PubMed Central

    Yamada, Tesshi; Takenawa, Tadaomi

    2015-01-01

    α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion. PMID:25860875

  9. Arginine stimulates intestinal cell migration through a focal adhesion kinase dependent mechanism

    PubMed Central

    Rhoads, J M; Chen, W; Gookin, J; Wu, G Y; Fu, Q; Blikslager, A T; Rippe, R A; Argenzio, R A; Cance, W G; Weaver, E M; Romer, L H

    2004-01-01

    Background: l-Arginine is a nutritional supplement that may be useful for promoting intestinal repair. Arginine is metabolised by the oxidative deiminase pathway to form nitric oxide (NO) and by the arginase pathway to yield ornithine and polyamines. Aims: To determine if arginine stimulates restitution via activation of NO synthesis and/or polyamine synthesis. Methods: We determined the effects of arginine on cultured intestinal cell migration, NO production, polyamine levels, and activation of focal adhesion kinase, a key mediator of cell migration. Results: Arginine increased the rate of cell migration in a dose dependent biphasic manner, and was additive with bovine serum concentrate (BSC). Arginine and an NO donor activated focal adhesion kinase (a tyrosine kinase which localises to cell matrix contacts and mediates β1 integrin signalling) after wounding. Arginine stimulated cell migration was dependent on focal adhesion kinase (FAK) signalling, as demonstrated using adenovirus mediated transfection with a kinase negative mutant of FAK. Arginine stimulated migration was dependent on NO production and was blocked by NO synthase inhibitors. Arginine dependent migration required synthesis of polyamines but elevating extracellular arginine concentration above 0.4 mM did not enhance cellular polyamine levels. Conclusions: These results showed that l-arginine stimulates cell migration through NO and FAK dependent pathways and that combination therapy with arginine and BSC may enhance intestinal restitution via separate and convergent pathways. PMID:15016745

  10. Requirement of focal adhesion kinase in branching tubulogenesis.

    PubMed

    Wei, Wei-Chun; Kopec, Anna K; Tang, Ming-Jer

    2009-01-01

    We previously demonstrated that alpha3beta1 integrins are essential to hepatocyte growth factor (HGF)-independent branching tubulogenesis in Mardin-Darby Canine Kidney (MDCK) cells. However, the involvement of integrin downstream signaling molecules remains unclear. In the present study, we successfully isolated cell lines possessing different tubulogenic potentials from the MDCK cells; cyst clones (CA4, CA6) forming cystic structures when cultured in 0.3% type I collagen gel and mass clones (M610, M611, M612) forming aggregated masses. Cyst clones maintained cystic structure in 0.1% collagen gel, whereas mass clones spontaneously developed into tubules. Both clones exhibited various morphologies when cultured on a dish: cyst clones formed aggregated islands, while mass clones were more scattered and exhibited higher migration capacity. Among several focal adhesion machinery proteins examined, only the expression and phosphorylation level of focal adhesion kinase (FAK) in mass clones was higher than in cyst clones, while other proteins showed no obvious differences. However, overexpression of wild type FAK in CA6 cells did not facilitate branching tubule formation in 0.1% collagen gel. Targeted decrease in the expression level of FAK in M610 cells with the application of antisense cDNA resulted in a marked reduction of branching tubule formation in 0.1% collagen gel and showed a down-regulation of fibronectin assembly, which is known to promote tubulogenesis. In contrast, overexpression of wild type FAK in CA6 cells had no effect on fibronectin assembly. Taken together, our data demonstrates that FAK is required, but not sufficient for HGF-independent branching tubulogenesis in MDCK cells. PMID:19272169

  11. Crystal Structure of the FERM Domain of Focal Adhesion Kinase

    SciTech Connect

    Ceccarelli,D.; Song, H.; Poy, F.; Schaller, M.; Eck, M.

    2006-01-01

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.

  12. Cell locomotion and focal adhesions are regulated by substrate flexibility

    PubMed Central

    Pelham, Robert J.; Wang, Yu-li

    1997-01-01

    Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism. PMID:9391082

  13. Src Kinase Determines the Dynamic Exchange of the Docking Protein NEDD9 (Neural Precursor Cell Expressed Developmentally Down-regulated Gene 9) at Focal Adhesions*

    PubMed Central

    Bradbury, Peta; Bach, Cuc T.; Paul, Andre; O'Neill, Geraldine M.

    2014-01-01

    Dynamic exchange of molecules between the cytoplasm and integrin-based focal adhesions provides a rapid response system for modulating cell adhesion. Increased residency time of molecules that regulate adhesion turnover contributes to adhesion stability, ultimately determining migration speed across two-dimensional surfaces. In the present study we test the role of Src kinase in regulating dynamic exchange of the focal adhesion protein NEDD9/HEF1/Cas-L. Using either chemical inhibition or fibroblasts genetically null for Src together with fluorescence recovery after photobleaching (FRAP), we find that Src significantly reduces NEDD9 exchange at focal adhesions. Analysis of NEDD9 mutant constructs with the two major Src-interacting domains disabled revealed the greatest effects were due to the NEDD9 SH2 binding domain. This correlated with a significant change in two-dimensional migratory speed. Given the emerging role of NEDD9 as a regulator of focal adhesion stability, the time of NEDD9 association at the focal adhesions is key in modulating rates of migration and invasion. Our study suggests that Src kinase activity determines NEDD9 exchange at focal adhesions and may similarly modulate other focal adhesion-targeted Src substrates to regulate cell migration. PMID:25059660

  14. Formation of focal adhesion-stress fibre complexes coordinated by adhesive and non-adhesive surface domains.

    PubMed

    Zimerman, B; Arnold, M; Ulmer, J; Blümmel, J; Besser, A; Spatz, J P; Geiger, B

    2004-04-01

    Cell motility consists of repeating cycles of protrusion of a leading edge in the direction of migration, attachment of the advancing membrane to the matrix, and pulling of the trailing edge forward. In this dynamic process there is a major role for the cytoskeleton, which drives the protrusive events via polymerisation of actin in the lamellipodium, followed by actomyosin contractility. To study the transition of the actin cytoskeleton from a 'protrusive' to 'retractive' form, we have monitored the formation of focal adhesions and stress fibres during cell migration on a micro-patterned surface. This surface consisted of parallel arrays of 2 microm-wide, fibronectin-coated gold stripes, separated by non-adhesive (poly(ethylene glycol)-coated) glass areas with variable width, ranging from 4-12 microm. Monitoring the spreading of motile cells indicated that cell spreading was equally effective along and across the adhesive stripes, as long as the non-adhesive spaces between them did not exceed 6 microm. When the width of the PEG region was 8 microm or more, cells became highly polarised upon spreading, and failed to reach the neighboring adhesive stripes. It was also noted that as soon as the protruding lamella successfully crossed the PEG-coated area and reached an adhesive region, the organisation of actin in that area was transformed from a diffuse meshwork into a bundle, oriented perpendicularly to the stripes and anchored at its ends in focal adhesions. This transition depends on actomyosin-based contractility and is apparently triggered by the adhesion to the rigid fibronectin surface. PMID:16475844

  15. Talin determines the nanoscale architecture of focal adhesions

    PubMed Central

    Liu, Jaron; Wang, Yilin; Goh, Wah Ing; Goh, Honzhen; Baird, Michelle A.; Ruehland, Svenja; Teo, Shijia; Bate, Neil; Critchley, David R.; Davidson, Michael W.; Kanchanawong, Pakorn

    2015-01-01

    Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin–talin–actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites. PMID:26283369

  16. Talin determines the nanoscale architecture of focal adhesions.

    PubMed

    Liu, Jaron; Wang, Yilin; Goh, Wah Ing; Goh, Honzhen; Baird, Michelle A; Ruehland, Svenja; Teo, Shijia; Bate, Neil; Critchley, David R; Davidson, Michael W; Kanchanawong, Pakorn

    2015-09-01

    Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites. PMID:26283369

  17. Focal Adhesion of Osteoblastic Cells on Titanium Surface with Amine Functionalities Formed by Plasma Polymerization

    NASA Astrophysics Data System (ADS)

    Song, Heesang; Jung, Sang Chul; Kim, Byung Hoon

    2012-08-01

    To enhance the focal adhesion of osteoblastic cells on a titanium surface, plasma polymerized allyl amine (AAm) thin films were deposited by plasma polymerization. This plasma polymer functionalization of titanium is advantageous for osteoblastic focal adhesion formation. Such Ti surfaces are useful for the fabrication of titanium-based dental implants for enhancement of osseointegration.

  18. Persistent Focal Behavior and Physical Activity Performance

    ERIC Educational Resources Information Center

    Erfle, Stephen E.

    2014-01-01

    This article examines the proclivity and performance attributes of focal students across time and activities using data from 9,345 students. Three systematic focal behavior partitions are examined: Across activities, across time, and across activities and time. A student's performance is focal if it ends in 0 or 5 for push-ups and 0 for…

  19. The focal adhesion protein PINCH-1 associates with EPLIN at integrin adhesion sites

    PubMed Central

    Karaköse, Esra; Geiger, Tamar; Flynn, Kevin; Lorenz-Baath, Katrin; Zent, Roy; Mann, Matthias; Fässler, Reinhard

    2015-01-01

    ABSTRACT PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN. PMID:25609703

  20. The Focal Adhesion-Localized CdGAP Regulates Matrix Rigidity Sensing and Durotaxis

    PubMed Central

    Wormer, Duncan B.; Davis, Kevin A.; Henderson, James H.; Turner, Christopher E.

    2014-01-01

    Motile cells are capable of sensing the stiffness of the surrounding extracellular matrix through integrin-mediated focal adhesions and migrate towards regions of higher rigidity in a process known as durotaxis. Durotaxis plays an important role in normal development and disease progression, including tumor invasion and metastasis. However, the signaling mechanisms underlying focal adhesion-mediated rigidity sensing and durotaxis are poorly understood. Utilizing matrix-coated polydimethylsiloxane gels to manipulate substrate compliance, we show that cdGAP, an adhesion-localized Rac1 and Cdc42 specific GTPase activating protein, is necessary for U2OS osteosarcoma cells to coordinate cell shape changes and migration as a function of extracellular matrix stiffness. CdGAP regulated rigidity-dependent motility by controlling membrane protrusion and adhesion dynamics, as well as by modulating Rac1 activity. CdGAP was also found to be necessary for U2OS cell durotaxis. Taken together, these data identify cdGAP as an important component of an integrin-mediated signaling pathway that senses and responds to mechanical cues in the extracellular matrix in order to coordinate directed cell motility. PMID:24632816

  1. Osteogenic lineage restriction by osteoprogenitors cultured on nanometric grooved surfaces: the role of focal adhesion maturation.

    PubMed

    Cassidy, John W; Roberts, Jemma N; Smith, Carol-Anne; Robertson, Mary; White, Kate; Biggs, Manus J; Oreffo, Richard O C; Dalby, Matthew J

    2014-02-01

    The differentiation of progenitor cells is dependent on more than biochemical signalling. Topographical cues in natural bone extracellular matrix guide cellular differentiation through the formation of focal adhesions, contact guidance, cytoskeletal rearrangement and ultimately gene expression. Osteoarthritis and a number of bone disorders present as growing challenges for our society. Hence, there is a need for next generation implantable devices to substitute for, or guide, bone repair in vivo. Cellular responses to nanometric topographical cues need to be better understood in vitro in order to ensure the effective and efficient integration and performance of these orthopedic devices. In this study, the FDA-approved plastic polycaprolactone was embossed with nanometric grooves and the response of primary and immortalized osteoprogenitor cells observed. Nanometric groove dimensions were 240 nm or 540 nm deep and 12.5 μm wide. Cells cultured on test surfaces followed contact guidance along the length of groove edges, elongated along their major axis and showed nuclear distortion; they formed more focal complexes and lower proportions of mature adhesions relative to planar controls. Down-regulation of the osteoblast marker genes RUNX2 and BMPR2 in primary and immortalized cells was observed on grooved substrates. Down-regulation appeared to directly correlate with focal adhesion maturation, indicating the involvement of ERK 1/2 negative feedback pathways following integrin-mediated FAK activation. PMID:24252447

  2. NBR1 enables autophagy-dependent focal adhesion turnover

    PubMed Central

    Kenific, Candia M.; Stehbens, Samantha J.; Goldsmith, Juliet; Leidal, Andrew M.; Faure, Nathalie; Ye, Jordan; Wittmann, Torsten

    2016-01-01

    Autophagy is a catabolic pathway involving the sequestration of cellular contents into a double-membrane vesicle, the autophagosome. Although recent studies have demonstrated that autophagy supports cell migration, the underlying mechanisms remain unknown. Using live-cell imaging, we uncover that autophagy promotes optimal migratory rate and facilitates the dynamic assembly and disassembly of cell-matrix focal adhesions (FAs), which is essential for efficient motility. Additionally, our studies reveal that autophagosomes associate with FAs primarily during disassembly, suggesting autophagy locally facilitates the destabilization of cell-matrix contact sites. Furthermore, we identify the selective autophagy cargo receptor neighbor of BRCA1 (NBR1) as a key mediator of autophagy-dependent FA remodeling. NBR1 depletion impairs FA turnover and decreases targeting of autophagosomes to FAs, whereas ectopic expression of autophagy-competent, but not autophagy-defective, NBR1 enhances FA disassembly and reduces FA lifetime during migration. Our findings provide mechanistic insight into how autophagy promotes migration by revealing a requirement for NBR1-mediated selective autophagy in enabling FA disassembly in motile cells. PMID:26903539

  3. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    SciTech Connect

    Aanei, Carmen Mariana; Eloae, Florin Zugun; Flandrin-Gresta, Pascale; Tavernier, Emmanuelle; Carasevici, Eugen; Guyotat, Denis; Campos, Lydia

    2011-11-01

    Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90{alpha}/{beta} client protein, these results suggest the utility of HSP90{alpha}/{beta} inhibition as a target for adjuvant therapy for myelodysplasia.

  4. Highly Multiplexed Imaging Uncovers Changes in Compositional Noise within Assembling Focal Adhesions

    PubMed Central

    Harizanova, Jana; Fermin, Yessica; Malik-Sheriff, Rahuman S.; Wieczorek, Jakob; Ickstadt, Katja; Grecco, Hernán E.; Zamir, Eli

    2016-01-01

    Integrin adhesome proteins bind each other in alternative manners, forming within the cell diverse cell-matrix adhesion sites with distinct properties. An intriguing question is how such modular assembly of adhesion sites is achieved correctly solely by self-organization of their components. Here we address this question using high-throughput multiplexed imaging of eight proteins and two phosphorylation sites in a large number of single focal adhesions. We found that during the assembly of focal adhesions the variances of protein densities decrease while the correlations between them increase, suggesting reduction in the noise levels within these structures. These changes correlate independently with the area and internal density of focal adhesions, but not with their age or shape. Artificial neural network analysis indicates that a joint consideration of multiple components improves the predictability of paxillin and zyxin levels in internally dense focal adhesions. This suggests that paxillin and zyxin densities in focal adhesions are fine-tuned by integrating the levels of multiple other components, thus averaging-out stochastic fluctuations. Based on these results we propose that increase in internal protein densities facilitates noise suppression in focal adhesions, while noise suppression enables their stable growth and further density increase—hence forming a feedback loop giving rise to a quality-controlled assembly. PMID:27519053

  5. Highly Multiplexed Imaging Uncovers Changes in Compositional Noise within Assembling Focal Adhesions.

    PubMed

    Harizanova, Jana; Fermin, Yessica; Malik-Sheriff, Rahuman S; Wieczorek, Jakob; Ickstadt, Katja; Grecco, Hernán E; Zamir, Eli

    2016-01-01

    Integrin adhesome proteins bind each other in alternative manners, forming within the cell diverse cell-matrix adhesion sites with distinct properties. An intriguing question is how such modular assembly of adhesion sites is achieved correctly solely by self-organization of their components. Here we address this question using high-throughput multiplexed imaging of eight proteins and two phosphorylation sites in a large number of single focal adhesions. We found that during the assembly of focal adhesions the variances of protein densities decrease while the correlations between them increase, suggesting reduction in the noise levels within these structures. These changes correlate independently with the area and internal density of focal adhesions, but not with their age or shape. Artificial neural network analysis indicates that a joint consideration of multiple components improves the predictability of paxillin and zyxin levels in internally dense focal adhesions. This suggests that paxillin and zyxin densities in focal adhesions are fine-tuned by integrating the levels of multiple other components, thus averaging-out stochastic fluctuations. Based on these results we propose that increase in internal protein densities facilitates noise suppression in focal adhesions, while noise suppression enables their stable growth and further density increase-hence forming a feedback loop giving rise to a quality-controlled assembly. PMID:27519053

  6. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

    SciTech Connect

    Playford, Martin P.; Vadali, Kavita; Cai Xinming; Burridge, Keith; Schaller, Michael D.

    2008-10-15

    Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.

  7. Novel Phosphotidylinositol 4,5-Bisphosphate Binding Sites on Focal Adhesion Kinase.

    PubMed

    Feng, Jun; Mertz, Blake

    2015-01-01

    Focal adhesion kinase (FAK) is a protein tyrosine kinase that is ubiquitously expressed, recruited to focal adhesions, and engages in a variety of cellular signaling pathways. Diverse cellular responses, such as cell migration, proliferation, and survival, are regulated by FAK. Prior to activation, FAK adopts an autoinhibited conformation in which the FERM domain binds the kinase domain, blocking access to the activation loop and substrate binding site. Activation of FAK occurs through conformational change, and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2) are known to facilitate this process. PIP2 binding alters the autoinhibited conformation of the FERM and kinase domains and subsequently exposes the activation loop to phosphorylation. However, the detailed molecular mechanism of PIP2 binding and its role in FAK activation remain unclear. In this study, we conducted coarse-grained molecular dynamics simulations to investigate the binding of FAK to PIP2. Our simulations identified novel areas of basic residues in the kinase domain of FAK that potentially undergo transient binding to PIP2 through electrostatic attractions. Our investigation provides a molecular picture of PIP2-initiated FAK activation and introduces promising new pathways for future studies of FAK regulation. PMID:26186725

  8. Hormonal regulation of focal adhesions in bovine adrenocortical cells: induction of paxillin dephosphorylation by adrenocorticotropic hormone.

    PubMed Central

    Vilgrain, I; Chinn, A; Gaillard, I; Chambaz, E M; Feige, J J

    1998-01-01

    A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes. PMID:9601084

  9. Actin cap associated focal adhesions and their distinct role in cellular mechanosensing

    PubMed Central

    Kim, Dong-Hwee; Khatau, Shyam B.; Feng, Yunfeng; Walcott, Sam; Sun, Sean X.; Longmore, Gregory D.; Wirtz, Denis

    2012-01-01

    The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to biochemical stimuli. Actin cap associated focal adhesions (ACAFAs) dominate cell mechanosensing over a wide range of matrix stiffness, an ACAFA-specific function regulated by actomyosin contractility in the actin cap, while conventional focal adhesions are restrictively involved in mechanosensing for extremely soft substrates. These results establish the perinuclear actin cap and associated ACAFAs as major mediators of cellular mechanosensing and a critical element of the physical pathway that transduce mechanical cues all the way to the nucleus. PMID:22870384

  10. A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region

    PubMed Central

    Moroco, Jamie A.; Baumgartner, Matthew P.; Rust, Heather L.; Choi, Hwan Geun; Hur, Wooyoung; Gray, Nathanael S.; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the ‘DFG-out’ conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. PMID:25376742

  11. A new link between the retrograde actin flow and focal adhesions.

    PubMed

    Yamashiro, Sawako; Watanabe, Naoki

    2014-11-01

    The retrograde actin flow, continuous centripetal movement of the cell peripheral actin networks, is widely observed in adherent cells. The retrograde flow is believed to facilitate cell migration when linked to cell adhesion molecules. In this review, we summarize our current knowledge regarding the functional relationship between the retrograde actin flow and focal adhesions (FAs). We also introduce our recent study in which single-molecule speckle (SiMS) microscopy dissected the complex interactions between FAs and the local actin flow. FAs do not simply impede the actin flow, but actively attract and remodel the local actin network. Our findings provide a new insight into the mechanisms for protrusion and traction force generation at the cell leading edge. Furthermore, we discuss possible roles of the actin flow-FA interaction based on the accumulated knowledge and our SiMS study. PMID:25190817

  12. Nuclear transport of paxillin depends on focal adhesion dynamics and FAT domains

    PubMed Central

    Sathe, Aneesh R.; Shivashankar, G. V.; Sheetz, Michael P.

    2016-01-01

    ABSTRACT The nuclear transport of paxillin appears to be crucial for paxillin function but the mechanism of transport remains unclear. Here, we show that the nuclear transport of paxillin is regulated by focal adhesion turnover and the presence of FAT domains. Focal adhesion turnover was controlled using triangular or circular fibronectin islands. Circular islands caused higher focal adhesion turnover and increased the nuclear transport of paxillin relative to triangular islands. Mutating several residues of paxillin had no effect on its nuclear transport, suggesting that the process is controlled by multiple domains. Knocking out FAK (also known as PTK2) and vinculin caused an increase in nuclear paxillin. This could be reversed by rescue with wild-type FAK but not by FAK with a mutated FAT domain, which inhibits paxillin binding. Expressing just the FAT domain of FAK not only brought down nuclear levels of paxillin but also caused a large immobile fraction of paxillin to be present at focal adhesions, as demonstrated by fluorescence recovery after photobleaching (FRAP) studies. Taken together, focal adhesion turnover and FAT domains regulate the nuclear localization of paxillin, suggesting a possible role for transcriptional control, through paxillin, by focal adhesions. PMID:27068537

  13. Secretagogin affects insulin secretion in pancreatic β-cells by regulating actin dynamics and focal adhesion

    PubMed Central

    Yang, Seo-Yun; Lee, Jae-Jin; Lee, Jin-Hee; Lee, Kyungeun; Oh, Seung Hoon; Lim, Yu-Mi; Lee, Myung-Shik; Lee, Kong-Joo

    2016-01-01

    Secretagogin (SCGN), a Ca2+-binding protein having six EF-hands, is selectively expressed in pancreatic β-cells and neuroendocrine cells. Previous studies suggested that SCGN enhances insulin secretion by functioning as a Ca2+-sensor protein, but the underlying mechanism has not been elucidated. The present study explored the mechanism by which SCGN enhances glucose-induced insulin secretion in NIT-1 insulinoma cells. To determine whether SCGN influences the first or second phase of insulin secretion, we examined how SCGN affects the kinetics of insulin secretion in NIT-1 cells. We found that silencing SCGN suppressed the second phase of insulin secretion induced by glucose and H2O2, but not the first phase induced by KCl stimulation. Recruitment of insulin granules in the second phase of insulin secretion was significantly impaired by knocking down SCGN in NIT-1 cells. In addition, we found that SCGN interacts with the actin cytoskeleton in the plasma membrane and regulates actin remodelling in a glucose-dependent manner. Since actin dynamics are known to regulate focal adhesion, a critical step in the second phase of insulin secretion, we examined the effect of silencing SCGN on focal adhesion molecules, including FAK (focal adhesion kinase) and paxillin, and the cell survival molecules ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. We found that glucose- and H2O2-induced activation of FAK, paxillin, ERK1/2 and Akt was significantly blocked by silencing SCGN. We conclude that SCGN controls glucose-stimulated insulin secretion and thus may be useful in the therapy of Type 2 diabetes. PMID:27095850

  14. Secretagogin affects insulin secretion in pancreatic β-cells by regulating actin dynamics and focal adhesion.

    PubMed

    Yang, Seo-Yun; Lee, Jae-Jin; Lee, Jin-Hee; Lee, Kyungeun; Oh, Seung Hoon; Lim, Yu-Mi; Lee, Myung-Shik; Lee, Kong-Joo

    2016-06-15

    Secretagogin (SCGN), a Ca(2+)-binding protein having six EF-hands, is selectively expressed in pancreatic β-cells and neuroendocrine cells. Previous studies suggested that SCGN enhances insulin secretion by functioning as a Ca(2+)-sensor protein, but the underlying mechanism has not been elucidated. The present study explored the mechanism by which SCGN enhances glucose-induced insulin secretion in NIT-1 insulinoma cells. To determine whether SCGN influences the first or second phase of insulin secretion, we examined how SCGN affects the kinetics of insulin secretion in NIT-1 cells. We found that silencing SCGN suppressed the second phase of insulin secretion induced by glucose and H2O2, but not the first phase induced by KCl stimulation. Recruitment of insulin granules in the second phase of insulin secretion was significantly impaired by knocking down SCGN in NIT-1 cells. In addition, we found that SCGN interacts with the actin cytoskeleton in the plasma membrane and regulates actin remodelling in a glucose-dependent manner. Since actin dynamics are known to regulate focal adhesion, a critical step in the second phase of insulin secretion, we examined the effect of silencing SCGN on focal adhesion molecules, including FAK (focal adhesion kinase) and paxillin, and the cell survival molecules ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. We found that glucose- and H2O2-induced activation of FAK, paxillin, ERK1/2 and Akt was significantly blocked by silencing SCGN. We conclude that SCGN controls glucose-stimulated insulin secretion and thus may be useful in the therapy of Type 2 diabetes. PMID:27095850

  15. Reorganization of the actin cytoskeleton via transcriptional regulation of cytoskeletal/focal adhesion genes by myocardin-related transcription factors (MRTFs/MAL/MKLs)

    SciTech Connect

    Morita, Tsuyoshi; Mayanagi, Taira; Sobue, Kenji

    2007-10-01

    RhoA is a crucial regulator of stress fiber and focal adhesion formation through the activation of actin nucleation and polymerization. It also regulates the nuclear translocation of myocardin-related transcription factor-A and -B (MRTF-A/B, MAL or MKL 1/2), which are co-activators of serum response factor (SRF). In dominant-negative MRTF-A (DN-MRTF-A)-expressing NIH 3T3 cell lines, the expressions of several cytoskeletal/focal adhesion genes were down-regulated, and the formation of stress fiber and focal adhesion was severely diminished. MRTF-A/B-knockdown cells also exhibited such cytoskeletal defects. In reporter assays, both RhoA and MRTF-A enhanced promoter activities of these genes in a CArG-box-dependent manner, and DN-MRTF-A inhibited the RhoA-mediated activation of these promoters. In dominant-negative RhoA (RhoA-N19)-expressing NIH 3T3 cell lines, the nuclear translocation of MRTF-A/B was predominantly prevented, resulting in the reduced expression of cytoskeletal/focal adhesion proteins. Further, constitutive-active MRTF-A/B increased the expression of endogenous cytoskeletal/focal adhesion proteins, and thereby rescued the defective phenotype of stress fibers and focal adhesions in RhoA-N19 expressing cells. These results indicate that MRTF-A/B act as pivotal mediators of stress fiber and focal adhesion formation via the transcriptional regulation of a subset of cytoskeletal/focal adhesion genes.

  16. LIM-kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505.

    PubMed Central

    Amano, T; Tanabe, K; Eto, T; Narumiya, S; Mizuno, K

    2001-01-01

    LIM-kinase 1 and 2 (LIMK1 and LIMK2) phosphorylate cofilin and induce actin cytoskeletal reorganization. LIMK1 is activated by Rho-associated, coiled-coil-forming protein kinase (ROCK) and p21-activated kinase 1 (PAK1), but activation mechanisms and cellular functions of LIMK2 have remained to be determined. We report here that LIMK1 and LIMK2 phosphorylate both cofilin and actin-depolymerizing factor (ADF) specifically at Ser-3 and exhibit partially distinct substrate specificity when tested using site-directed cofilin mutants as substrates. We also show that LIMK2 is activated by ROCK by phosphorylation at Thr-505 within the activation loop. Wild-type LIMK2, but not its mutant (T505V) with replacement of Thr-505 by Val, was activated by ROCK in vitro and in vivo. LIMK2 mutants with replacement of Thr-505 by one or two Glu residues (T505E or T505EE) increased the kinase activity about 3.6-fold but were not further activated by ROCK. When expressed in HeLa cells, wild-type LIMK2, but not the T505V mutant, induced the formation of stress fibres, focal adhesions and membrane blebs. Furthermore, inhibitors of Rho and ROCK significantly suppressed LIMK2-induced stress fibres and membrane blebs. These results suggest that LIMK2 functions downstream of the Rho-ROCK signalling pathway and plays a role in reorganization of actin filaments and membrane structures, by phosphorylating cofilin/ADF proteins. PMID:11171090

  17. Tuning the material-cytoskeleton crosstalk via nanoconfinement of focal adhesions.

    PubMed

    Natale, Carlo F; Ventre, Maurizio; Netti, Paolo A

    2014-03-01

    Material features proved to exert a potent influence on cell behaviour in terms of adhesion, migration and differentiation. In particular, biophysical and biochemical signals on material surfaces are able to affect focal adhesion distribution and cytoskeletal assemblies, which are known to regulate signalling pathways that ultimately influence cell fate and functions. However, a general, unifying model that correlates cytoskeletal-generated forces with genetic events has yet to be developed. Therefore, it is crucial to gain a better insight into the material-cytoskeleton crosstalk in order to design and fabricate biomaterials able to govern cell fate more accurately. In this work, we demonstrate that confining focal adhesion distribution and growth dramatically alters the cytoskeleton's structures and dynamics, which in turn dictate cellular and nuclear shape and polarization. MC3T3 preosteoblasts were cultivated on nanograted polydimethylsiloxane substrates and a thorough quantification - in static and dynamic modes - of the morphological and structural features of focal adhesions and cytoskeleton was performed. Nanoengineered surfaces provided well-defined zones for focal adhesions to form and grow. Unique cytoskeletal structures spontaneously assembled when focal adhesions were confined and, in fact, they proved to be very effective in deforming the nuclei. The results here presented provide elements to engineer surfaces apt to guide and control cell behaviour through the material-cytoskeleton-nucleus axis. PMID:24388800

  18. Activation of the lutropin/choriogonadotropin receptor (LHR) in MA-10 cells leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) by a pathway that involves Src family kinases*

    PubMed Central

    Mizutani, Tetsuya; Shiraishi, Koji; Welsh, Toni; Ascoli, Mario

    2006-01-01

    We show that activation of the endogenous or recombinant LHR in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577 but it does not affect the phosphorylation of Tyr397, Tyr861 or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes and overexpression of either of these two tyrosine kinases enhances the LHR-mediate phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Gα subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways. PMID:16293639

  19. Crystal Structures of Free and Ligand-Bound Focal Adhesion Targeting Domain of Pyk2

    SciTech Connect

    Lulo, J.; Yuzawa, S; Schlessinger, J

    2009-01-01

    Focal adhesion targeting (FAT) domains target the non-receptor tyrosine kinases FAK and Pyk2 to cellular focal adhesion areas, where the signaling molecule paxillin is also located. Here, we report the crystal structures of the Pyk2 FAT domain alone or in complex with paxillin LD4 peptides. The overall structure of Pyk2-FAT is an antiparallel four-helix bundle with an up-down, up-down, right-handed topology. In the LD4-bound FAT complex, two paxillin LD4 peptides interact with two opposite sides of Pyk2-FAT, at the surfaces of the a1a4 and a2a3 helices of each FAT molecule. We also demonstrate that, while paxillin is phosphorylated by Pyk2, complex formation between Pyk2 and paxillin does not depend on Pyk2 tyrosine kinase activity. These experiments reveal the structural basis underlying the selectivity of paxillin LD4 binding to the Pyk2 FAT domain and provide insights about the molecular details which influence the different behavior of these two closely-related kinases.

  20. Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions

    SciTech Connect

    Lee, Chunsik; Dixelius, Johan; Thulin, Asa; Kawamura, Harukiyo; Claesson-Welsh, Lena; Olsson, Anna-Karin . E-mail: Anna-Karin.Olsson@genpat.uu.se

    2006-08-01

    Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures.

  1. Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation

    PubMed Central

    Lagares, David; Busnadiego, Oscar; García-Fernández, Rosa Ana; Kapoor, Mohit; Liu, Shangxi; Carter, David E.; Abraham, David; Shi-Wen, Xu; Carreira, Patricia; Fontaine T, Benjamin A; Shea, Barry S; Tager, Andrew M; Leask, Andrew; Lamas, Santiago; Rodríguez-Pascual, Fernando

    2011-01-01

    Objective Enhanced adhesive signaling including activation of the focal adhesion kinase (FAK) is a hallmark of fibroblasts from lung fibrosis patients, and FAK has been therefore hypothesized to be a key mediator of this disease. This study was undertaken to characterize the contribution of FAK to the development of pulmonary fibrosis both in vivo and in vitro. Methods FAK expression and activity were analyzed in lung tissue samples from lung fibrosis patients by immunohistochemistry. Mice orally treated with the FAK inhibitor, PF-562,271, or with siRNA-mediated silencing of FAK, were exposed to intratracheally instilled bleomycin to induce lung fibrosis, and the lungs were harvested for histological and biochemical analysis. Using endothelin-1 (ET-1) as stimulus, cell adhesion and contraction, as well as profibrotic gene expression were studied in fibroblasts isolated from wild type and FAK-deficient mouse embryos. ET-1-mediated FAK activation and gene expression were studied in primary mouse lung fibroblasts, as well as in wild type and integrin β1-deficient fibroblasts. Results Increased FAK expression and activity are upregulated in fibroblast foci and remodeled vessels in lung fibrosis patients. Pharmacological or siRNA-mediated targeting of FAK resulted in marked abrogation of bleomycin-induced lung fibrosis. Loss of FAK impaired the acquisition of a profibrotic phenotype in response to ET-1. Profibrotic gene expression leading to myofibroblast differentiation required cell adhesion, and was driven by Jun N-terminal kinase activation through integrin β1/FAK signaling. Conclusion These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases. PMID:22492165

  2. Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

    PubMed

    Marley, K; Maier, C S; Helfand, S C

    2012-09-01

    Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. PMID:22487216

  3. Retrograde Fluxes of Focal Adhesion Proteins in Response to Cell Migration and Mechanical Signals

    PubMed Central

    Guo, Wei-hui

    2007-01-01

    Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell. PMID:17804814

  4. High-Resolution Quantification of Focal Adhesion Spatiotemporal Dynamics in Living Cells

    PubMed Central

    Hahn, Klaus M.; Gomez, Shawn M.

    2011-01-01

    Focal adhesions (FAs) are macromolecular complexes that provide a linkage between the cell and its external environment. In a motile cell, focal adhesions change size and position to govern cell migration, through the dynamic processes of assembly and disassembly. To better understand the dynamic regulation of focal adhesions, we have developed an analysis system for the automated detection, tracking, and data extraction of these structures in living cells. This analysis system was used to quantify the dynamics of fluorescently tagged Paxillin and FAK in NIH 3T3 fibroblasts followed via Total Internal Reflection Fluorescence Microscopy (TIRF). High content time series included the size, shape, intensity, and position of every adhesion present in a living cell. These properties were followed over time, revealing adhesion lifetime and turnover rates, and segregation of properties into distinct zones. As a proof-of-concept, we show how a single point mutation in Paxillin at the Jun-kinase phosphorylation site Serine 178 changes FA size, distribution, and rate of assembly. This study provides a detailed, quantitative picture of FA spatiotemporal dynamics as well as a set of tools and methodologies for advancing our understanding of how focal adhesions are dynamically regulated in living cells. A full, open-source software implementation of this pipeline is provided at http://gomezlab.bme.unc.edu/tools. PMID:21779367

  5. Sub-micron lateral topography affects endothelial migration by modulation of focal adhesion dynamics.

    PubMed

    Antonini, S; Meucci, S; Jacchetti, E; Klingauf, M; Beltram, F; Poulikakos, D; Cecchini, M; Ferrari, A

    2015-06-01

    Through the interaction with topographical features, endothelial cells tune their ability to populate target substrates, both in vivo and in vitro. Basal textures interfere with the establishment and maturation of focal adhesions (FAs) thus inducing specific cell-polarization patterns and regulating a plethora of cell activities that govern the overall endothelial function. In this study, we analyze the effect of topographical features on FAs in primary human endothelial cells. Reported data demonstrate a functional link between FA dynamics and cell polarization and spreading on structured substrates presenting variable lateral feature size. Our results reveal that gratings with 2 µm lateral periodicity maximize contact guidance. The effect is linked to the dynamical state of FAs. We argue that these results are readily applicable to the rational design of active surfaces at the interface with the blood stream. PMID:26106866

  6. Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

    PubMed Central

    Bouchet, Benjamin P; Gough, Rosemarie E; Ammon, York-Christoph; van de Willige, Dieudonnée; Post, Harm; Jacquemet, Guillaume; Altelaar, AF Maarten; Heck, Albert JR; Goult, Benjamin T; Akhmanova, Anna

    2016-01-01

    The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules. DOI: http://dx.doi.org/10.7554/eLife.18124.001 PMID:27410476

  7. Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2.

    PubMed

    Rose, David M; Liu, Shouchun; Woodside, Darren G; Han, Jaewon; Schlaepfer, David D; Ginsberg, Mark H

    2003-06-15

    Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1). PMID:12794117

  8. Crystallization of the Focal Adhesion Kinase Targeting (FAT) Domain in a Primitive Orthorhombic Space Group

    SciTech Connect

    Magis,A.; Bailey, K.; Kurenova, E.; Hernandez Prada, J.; Cance, W.; Ostrov, D.

    2008-01-01

    X-ray diffraction data from the targeting (FAT) domain of focal adhesion kinase (FAK) were collected from a single crystal that diffracted to 1.99 Angstroms resolution and reduced to the primitive orthorhombic lattice. A single molecule was predicted to be present in the asymmetric unit based on the Matthews coefficient. The data were phased using molecular-replacement methods using an existing model of the FAK FAT domain. All structures of human focal adhesion kinase FAT domains solved to date have been solved in a C-centered orthorhombic space group.

  9. Transactivation of the epidermal growth factor receptor mediates muscarinic stimulation of focal adhesion kinase in intestinal epithelial cells.

    PubMed

    Calandrella, Sean O; Barrett, Kim E; Keely, Stephen J

    2005-04-01

    We have previously shown that the Gq protein coupled receptor (GqPCR) agonist, carbachol (CCh), transactivates and recruits epidermal growth factor receptor (EGFr)-dependent signaling mechanisms in intestinal epithelial cells. Increasing evidence suggests that GqPCR agonists can also recruit focal adhesion-dependent signaling pathways in some cell types. Therefore, the aim of the present study was to investigate if CCh stimulates activation of the focal adhesion-associated protein, focal adhesion kinase (FAK), in intestinal epithelia and, if so, to examine the signaling mechanisms involved. Experiments were carried out on monolayers of T84 cells grown on permeable supports. CCh rapidly induced tyrosine phosphorylation of FAK in T84 cells. This effect was accompanied by phosphorylation of another focal adhesion-associated protein, paxillin, and association of FAK with paxillin. CCh-stimulated FAK phosphorylation was inhibited by a chelator of intracellular Ca2+, BAPTA/AM (20 microM), and was mimicked by thapsigargin (2 microM), which mobilizes intracellular Ca2+ in a receptor-independent fashion. CCh also induced association of FAK with the EGFr and FAK phosphorylation was attenuated by an EGFr inhibitor, tyrphostin AG1478, and an inhibitor of Src family kinases, PP2. The actin cytoskeleton disruptor, cytochalasin D (20 microM), abolished FAK phosphorylation in response to CCh but did not alter CCh-induced EGFr or ERK MAPK activation. In summary, these data demonstrate that agonists of GqPCRs have the ability to induce FAK activation in intestinal epithelial cells. GqPCR-induced FAK activation is mediated by via a pathway involving transactivation of the EGFr and alterations in the actin cytoskeleton. PMID:15389641

  10. Teneurin-4 promotes cellular protrusion formation and neurite outgrowth through focal adhesion kinase signaling

    PubMed Central

    Suzuki, Nobuharu; Numakawa, Tadahiro; Chou, Joshua; de Vega, Susana; Mizuniwa, Chihiro; Sekimoto, Kaori; Adachi, Naoki; Kunugi, Hiroshi; Arikawa-Hirasawa, Eri; Yamada, Yoshihiko; Akazawa, Chihiro

    2014-01-01

    Teneurin-4 (Ten-4), a transmembrane protein, is highly expressed in the central nervous system; however, its cellular and molecular function in neuronal differentiation remains unknown. In this study, we aimed to elucidate the function of Ten-4 in neurite outgrowth. Ten-4 expression was induced during neurite outgrowth of the neuroblastoma cell line Neuro-2a. Ten-4 protein was localized at the neurite growth cones. Knockdown of Ten-4 expression in Neuro-2a cells decreased the formation of the filopodia-like protrusions and the length of individual neurites. Conversely, overexpression of Ten-4 promoted filopodia-like protrusion formation. In addition, knockdown and overexpression of Ten-4 reduced and elevated the activation of focal adhesion kinase (FAK) and Rho-family small GTPases, Cdc42 and Rac1, key molecules for the membranous protrusion formation downstream of FAK, respectively. Inhibition of the activation of FAK and neural Wiskott-Aldrich syndrome protein (N-WASP), which is a downstream regulator of FAK and Cdc42, blocked protrusion formation by Ten-4 overexpression. Further, Ten-4 colocalized with phosphorylated FAK in the filopodia-like protrusion regions. Together, our findings show that Ten-4 is a novel positive regulator of cellular protrusion formation and neurite outgrowth through the FAK signaling pathway.—Suzuki, N., Numakawa, T., Chou, J., de Vega, S., Mizuniwa, C., Sekimoto, K., Adachi, N., Kunugi, H., Arikawa-Hirasawa, E., Yamada, Y., Akazawa, C. Teneurin-4 promotes cellular protrusion formation and neurite outgrowth through focal adhesion kinase signaling. PMID:24344332

  11. Probing mechanical principles of focal contacts in cell–matrix adhesion with a coupled stochastic–elastic modelling framework

    PubMed Central

    Gao, Huajian; Qian, Jin; Chen, Bin

    2011-01-01

    Cell–matrix adhesion depends on the collective behaviours of clusters of receptor–ligand bonds called focal contacts between cell and extracellular matrix. While the behaviour of a single molecular bond is governed by statistical mechanics at the molecular scale, continuum mechanics should be valid at a larger scale. This paper presents an overview of a series of recent theoretical studies aimed at probing the basic mechanical principles of focal contacts in cell–matrix adhesion via stochastic–elastic models in which stochastic descriptions of molecular bonds and elastic descriptions of interfacial traction–separation are unified in a single modelling framework. The intention here is to illustrate these principles using simple analytical and numerical models. The aim of the discussions is to provide possible clues to the following questions: why does the size of focal adhesions (FAs) fall into a narrow range around the micrometre scale? How can cells sense and respond to substrates of varied stiffness via FAs? How do the magnitude and orientation of mechanical forces affect the binding dynamics of FAs? The effects of cluster size, cell–matrix elastic modulus, loading direction and cytoskeletal pretension on the lifetime of FA clusters have been investigated by theoretical arguments as well as Monte Carlo numerical simulations, with results showing that intermediate adhesion size, stiff substrate, cytoskeleton stiffening, low-angle pulling and moderate cytoskeletal pretension are factors that contribute to stable FAs. From a mechanistic point of view, these results provide possible explanations for a wide range of experimental observations and suggest multiple mechanisms by which cells can actively control adhesion and de-adhesion via cytoskeletal contractile machinery in response to mechanical properties of their surroundings. PMID:21632610

  12. Focal adhesion linker proteins expression of fibroblast related to adhesion in response to different transmucosal abutment surfaces

    PubMed Central

    Moon, Yeon-Hee; Yoon, Mi-Kyeong; Moon, Jung-Sun; Kang, Jee-Hae; Kim, Sun-Hun; Yang, Hong-Seo

    2013-01-01

    PURPOSE To evaluate adherence of human gingival fibroblasts (HGFs) to transmucosal abutment of dental implant with different surface conditions with time and to investigate the roles of focal adhesion linker proteins (FALPs) involved in HGFs adhesion to abutment surfaces. MATERIALS AND METHODS Morphologies of cultured HGFs on titanium and ceramic discs with different surface were observed by scanning electron microscopy. Biocompatibility and focal adhesion were evaluated by ultrasonic wave application and cell viability assay. FALPs expression levels were assessed by RT-PCR and western blot. RESULTS There seemed to be little difference in biocompatibility and adhesion strength of HGFs depending on the surface conditions and materials. In all experimental groups, the number of cells remaining on the disc surface after ultrasonic wave application increased more than 2 times at 3 days after seeding compared to 1-day cultured cells and this continued until 7 days of culture. FALPs expression levels, especially of vinculin and paxillin, also increased in 5-day cultured cells compared to 1-day cultured fibroblasts on the disc surface. CONCLUSION These results might suggest that the strength of adhesion of fibroblasts to transmucosal abutment surfaces increases with time and it seemed to be related to expressions of FALPs. PMID:24049577

  13. Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins

    PubMed Central

    Lopez-Sanchez, Inmaculada; Kalogriopoulos, Nicholas; Lo, I-Chung; Kabir, Firooz; Midde, Krishna K.; Wang, Honghui; Ghosh, Pradipta

    2015-01-01

    GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via β1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated β1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell–ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression. PMID:26446841

  14. Role of focal adhesion kinase in lung remodeling of endotoxemic rats.

    PubMed

    Petroni, Ricardo Costa; Teodoro, Walcy R; Guido, Maria Carolina; Barbeiro, Hermes Vieira; Abatepaulo, Fátima; Theobaldo, Mariana Cardillo; Biselli, Paolo Cesare; Soriano, Francisco Garcia

    2012-05-01

    Despite significant advances in the care of critically ill patients, acute lung injury continues to be a complex problem with high mortality. The present study was designed to characterize early lipopolysaccharide (LPS)-induced pulmonary injury and small interfering RNA targeting focal adhesion kinase (FAK) as a possible therapeutic tool in the septic lung remodeling process. Male Wistar rats were assigned into endotoxemic group and control group. Total collagen deposition was performed 8, 16, and 24 h after LPS injection. Focal adhesion kinase expression, interstitial and vascular collagen deposition, and pulmonary mechanics were analyzed at 24 h. Intravenous injection of small interfering RNA targeting FAK was used to silence expression of the kinase in pulmonary tissue. Focal adhesion kinase, total collagen deposition, and pulmonary mechanics showed increased in LPS group. Types I, III, and V collagen showed increase in pulmonary parenchyma, but only type V increased in vessels 24 h after LPS injection. Focal adhesion kinase silencing prevented lung remodeling in pulmonary parenchyma at 24 h. In conclusion, LPS induced a precocious and important lung remodeling. There was fibrotic response in the lung characterized by increased amount in total and specific-type collagen. These data may explain the frequent clinical presentation during sepsis of reduced lung compliance, oxygen diffusion, and pulmonary hypertension. The fact that FAK silencing was protective against lung collagen deposition underscores the therapeutic potential of FAK targeting by small interfering RNA. PMID:22293597

  15. The Regulation of Traction Force in Relation to Cell Shape and Focal Adhesions

    PubMed Central

    Rape, Andrew; Guo, Wei-hui; Wang, Yu-li

    2011-01-01

    Mechanical forces provide critical inputs for proper cellular functions. The interplay between the generation of, and response to, mechanical forces regulate such cellular processes as differentiation, proliferation, and migration. We postulate that adherent cells respond to a number of physical and topographical factors, including cell size and shape, by detecting the magnitude and/or distribution of traction forces under different conditions. To address this possibility we introduce a new simple method for precise micropatterning of hydrogels, and then apply the technique to systematically investigate the relationship between cell geometry, focal adhesions, and traction forces in cells with a series of spread areas and aspect ratios. Contrary to previous findings, we find that traction force is not determined primarily by the cell spreading area but by the distance from cell center to the perimeter. This distance in turn controls traction forces by regulating the size of focal adhesions, such that constraining the size of focal adhesions by micropatterning can override the effect of geometry. We propose that the responses of traction forces to center-periphery distance, possibly through a positive feedback mechanism that regulates focal adhesions, provide the cell with the information on its own shape and size. A similar positive feedback control may allow cells to respond to a variety of physical or topographical signals via a unified mechanism. PMID:21163521

  16. Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

    NASA Technical Reports Server (NTRS)

    Chicurel, M. E.; Singer, R. H.; Meyer, C. J.; Ingber, D. E.

    1998-01-01

    The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

  17. MUC16 contributes to the metastasis of pancreatic ductal adenocarcinoma through focal adhesion mediated signaling mechanism

    PubMed Central

    Chugh, Seema; Rachagani, Satyanarayana; Lakshmanan, Imayavaramban; Gupta, Suprit; Seshacharyulu, Parthasarathy; Smith, Lynette M.; Ponnusamy, Moorthy P.; Batra, Surinder K.

    2016-01-01

    MUC16, a heavily glycosylated type-I transmembrane mucin is overexpressed in several cancers including pancreatic ductal adenocarcinoma (PDAC). Previously, we have shown that MUC16 is significantly overexpressed in human PDAC tissues. However, the functional consequences and its role in PDAC is poorly understood. Here, we show that MUC16 knockdown decreases PDAC cell proliferation, colony formation and migration in vitro. Also, MUC16 knockdown decreases the tumor formation and metastasis in orthotopic xenograft mouse model. Mechanistically, immunoprecipitation and immunofluorescence analyses confirms MUC16 interaction with galectin-3 and mesothelin in PDAC cells. Adhesion assay displayed decreased cell attachment of MUC16 knockdown cells with recombinant galectin-1 and galectin-3 protein. Further, CRISPR/Cas9-mediated MUC16 knockout cells show decreased tumor-associated carbohydrate antigens (T and Tn) in PDAC cells. Importantly, carbohydrate antigens were decreased in the region that corresponds to MUC16 and suggests for the decreased MUC16-galectin interactions. Co-immunoprecipitation also revealed a novel interaction between MUC16 and FAK in PDAC cells. Interestingly, we observed decreased expression of mesenchymal and increased expression of epithelial markers in MUC16-silenced cells. Additionally, MUC16 loss showed a decreased FAK-mediated Akt and ERK/MAPK activation. Altogether, these findings suggest that MUC16-focal adhesion signaling may play a critical role in facilitating PDAC growth and metastasis. PMID:27382435

  18. Expression of DFak56, a Drosophila homolog of vertebrate focal adhesion kinase, supports a role in cell migration in vivo

    PubMed Central

    Fox, George L.; Rebay, Ilaria; Hynes, Richard O.

    1999-01-01

    Focal adhesion kinase (FAK) is a highly conserved, cytoplasmic tyrosine kinase that has been implicated in promoting cell migration and transmission of antiapoptotic signals in vertebrate cells. In cultured cells, integrin engagement with the extracellular matrix promotes the recruitment of FAK to focal contacts and increases in its phosphotyrosine content and kinase activity, suggesting FAK is an intracellular mediator of integrin signaling. We have identified a Drosophila FAK homolog, DFak56, that is 33% identical to vertebrate FAK, with the highest degree of homology in domains critical for FAK function, including the kinase and focal adhesion targeting domains, and several protein–protein interaction motifs. Furthermore, when expressed in NIH 3T3 cells, DFak56 both localizes to focal contacts and displays the characteristic elevation of phosphotyrosine content in response to plating the cells on fibronectin. During embryogenesis, DFak56 is broadly expressed, and it becomes elevated in the gut and central nervous system at later stages. Consistent with a role in cell migration, we also observe that DFak56 is abundant in the border cells of developing egg chambers before the onset of, and during, their migration. PMID:10611323

  19. Displacement of p130Cas from focal adhesions links actomyosin contraction to cell migration.

    PubMed

    Machiyama, Hiroaki; Hirata, Hiroaki; Loh, Xia Kun; Kanchi, Madhu Mathi; Fujita, Hideaki; Tan, Song Hui; Kawauchi, Keiko; Sawada, Yasuhiro

    2014-08-15

    Cell adhesion complexes provide platforms where cell-generated forces are transmitted to the extracellular matrix (ECM). Tyrosine phosphorylation of focal adhesion proteins is crucial for cells to communicate with the extracellular environment. However, the mechanisms that transmit actin cytoskeletal motion to the extracellular environment to drive cell migration are poorly understood. We find that the movement of p130Cas (Cas, also known as BCAR1), a mechanosensor at focal adhesions, correlates with actin retrograde flow and depends upon actomyosin contraction and phosphorylation of the Cas substrate domain (CasSD). This indicates that CasSD phosphorylation underpins the physical link between Cas and the actin cytoskeleton. Fluorescence recovery after photobleaching (FRAP) experiments reveal that CasSD phosphorylation, as opposed to the association of Cas with Src, facilitates Cas displacement from adhesion complexes in migrating cells. Furthermore, the stabilization of Src-Cas binding and inhibition of myosin II, both of which sustain CasSD phosphorylation but mitigate Cas displacement from adhesion sites, retard cell migration. These results indicate that Cas promotes cell migration by linking actomyosin contractions to the adhesion complexes through a dynamic interaction with Src as well as through the phosphorylation-dependent association with the actin cytoskeleton. PMID:24928898

  20. A Chemomechanical Model of Matrix and Nuclear Rigidity Regulation of Focal Adhesion Size.

    PubMed

    Cao, Xuan; Lin, Yuan; Driscoll, Tristian P; Franco-Barraza, Janusz; Cukierman, Edna; Mauck, Robert L; Shenoy, Vivek B

    2015-11-01

    In this work, a chemomechanical model describing the growth dynamics of cell-matrix adhesion structures (i.e., focal adhesions (FAs)) is developed. We show that there are three regimes for FA evolution depending on their size. Specifically, nascent adhesions with initial lengths below a critical value that are yet to engage in actin fibers will dissolve, whereas bigger ones will grow into mature FAs with a steady state size. In adhesions where growth surpasses the steady state size, disassembly will occur until their sizes are reduced to the equilibrium state. This finding arises from the fact that polymerization of adhesion proteins is force-dependent. Under actomyosin contraction, individual integrin bonds within small FAs (i.e., nascent adhesions or focal complexes) must transmit higher loads while the phenomenon of stress concentration occurs at the edge of large adhesion patches. As such, an effective stiffness of the FA-extracellular matrix complex that is either too small or too large will be relatively low, resulting in a limited actomyosin pulling force developed at the edge that is insufficient to prevent disassembly. Furthermore, it is found that a stiffer extracellular matrix and/or nucleus, as well as a stronger chemomechanical feedback, will induce larger adhesions along with a higher level of contraction force. Interestingly, switching the extracellular side from an elastic half-space, corresponding to some widely used in vitro gel substrates, to a one-dimensional fiber (as in the case of cells anchoring to a fibrous scaffold in vivo) does not qualitative change these conclusions. Our model predictions are in good agreement with a variety of experimental observations obtained in this study as well as those reported in the literature. Furthermore, this new model, to our knowledge, provides a framework with which to understand how both intracellular and extracellular perturbations lead to changes in adhesion structure number and size. PMID:26536258

  1. β1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival

    PubMed Central

    Santos, Andrea Rachelle C.; Corredor, Raul G.; Obeso, Betty Albo; Trakhtenberg, Ephraim F.; Wang, Ying; Ponmattam, Jamie; Dvoriantchikova, Galina; Ivanov, Dmitry; Shestopalov, Valery I.; Goldberg, Jeffrey L.; Fini, Mary Elizabeth; Bajenaru, Michaela Livia

    2012-01-01

    Extracellular matrix (ECM) integrity in the central nervous system (CNS) is essential for neuronal homeostasis. Signals from the ECM are transmitted to neurons through integrins, a family of cell surface receptors that mediate cell attachment to ECM. We have previously established a causal link between the activation of the matrix metalloproteinase-9 (MMP-9), degradation of laminin in the ECM of retinal ganglion cells (RGCs), and RGC death in a mouse model of retinal ischemia-reperfusion injury (RIRI). Here we investigated the role of laminin-integrin signaling in RGC survival in vitro, and after ischemia in vivo. In purified primary rat RGCs, stimulation of the β1 integrin receptor with laminin, or agonist antibodies enhanced RGC survival in correlation with activation of β1 integrin’s major downstream regulator, focal adhesion kinase (FAK). Furthermore, β1 integrin binding and FAK activation were required for RGCs’ survival response to laminin. Finally, in vivo after RIRI, we observed an up-regulation of MMP-9, proteolytic degradation of laminin, decreased RGC expression of β1 integrin, FAK and Akt dephosphorylation, and reduced expression of the pro-survival molecule bcl-xL in the period preceding RGC apoptosis. RGC death was prevented, in the context of laminin degradation, by maintaining β1 integrin activation with agonist antibodies. Thus, disruption of homeostatic RGC-laminin interaction and signaling leads to cell death after retinal ischemia, and maintaining integrin activation may be a therapeutic approach to neuroprotection. PMID:23118988

  2. Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions

    PubMed Central

    Kaverina, Irina; Rottner, Klemens; Small, J. Victor

    1998-01-01

    By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a “cross talk” between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity. PMID:9660872

  3. Discovery of Clinical Candidate CEP-37440, a Selective Inhibitor of Focal Adhesion Kinase (FAK) and Anaplastic Lymphoma Kinase (ALK).

    PubMed

    Ott, Gregory R; Cheng, Mangeng; Learn, Keith S; Wagner, Jason; Gingrich, Diane E; Lisko, Joseph G; Curry, Matthew; Mesaros, Eugen F; Ghose, Arup K; Quail, Matthew R; Wan, Weihua; Lu, Lihui; Dobrzanski, Pawel; Albom, Mark S; Angeles, Thelma S; Wells-Knecht, Kevin; Huang, Zeqi; Aimone, Lisa D; Bruckheimer, Elizabeth; Anderson, Nathan; Friedman, Jay; Fernandez, Sandra V; Ator, Mark A; Ruggeri, Bruce A; Dorsey, Bruce D

    2016-08-25

    Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein. PMID:27527804

  4. Non-channel mechanosensors working at focal adhesion-stress fiber complex.

    PubMed

    Hirata, Hiroaki; Tatsumi, Hitoshi; Hayakawa, Kimihide; Sokabe, Masahiro

    2015-01-01

    Mechanosensitive ion channels (MSCs) have long been the only established molecular class of cell mechanosensors; however, in the last decade, a variety of non-channel type mechanosensor molecules have been identified. Many of them are focal adhesion-associated proteins that include integrin, talin, and actin. Mechanosensors must be non-soluble molecules firmly interacting with relatively rigid cellular structures such as membranes (in terms of lateral stiffness), cytoskeletons, and adhesion structures. The partner of MSCs is the membrane in which MSC proteins efficiently transduce changes in the membrane tension into conformational changes that lead to channel opening. By contrast, the integrin, talin, and actin filament form a linear complex of which both ends are typically anchored to the extracellular matrices via integrins. Upon cell deformation by forces, this structure turns out to be a portion that efficiently transduces the generated stress into conformational changes of composite molecules, leading to the activation of integrin (catch bond with extracellular matrices) and talin (unfolding to induce vinculin bindings). Importantly, this structure also serves as an "active" mechanosensor to detect substrate rigidity by pulling the substrate with contraction of actin stress fibers (SFs), which may induce talin unfolding and an activation of MSCs in the vicinity of integrins. A recent study demonstrates that the actin filament acts as a mechanosensor with unique characteristics; the filament behaves as a negative tension sensor in which increased torsional fluctuations by tension decrease accelerate ADF/cofilin binding, leading to filament disruption. Here, we review the latest progress in the study of those non-channel mechanosensors and discuss their activation mechanisms and physiological roles. PMID:24965068

  5. Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale.

    PubMed

    Dong, Jing-Ming; Tay, Felicia Pei-Ling; Swa, Hannah Lee-Foon; Gunaratne, Jayantha; Leung, Thomas; Burke, Brian; Manser, Ed

    2016-01-01

    Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the "adhesome." We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds β integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to β integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin β1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope-labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome. PMID:27303058

  6. Low-intensity pulsed ultrasound promotes chondrogenic progenitor cell migration via focal adhesion kinase pathway.

    PubMed

    Jang, Kee W; Ding, Lei; Seol, Dongrim; Lim, Tae-Hong; Buckwalter, Joseph A; Martin, James A

    2014-06-01

    Low-intensity pulsed ultrasound (LIPUS) has been studied frequently for its beneficial effects on the repair of injured articular cartilage. We hypothesized that these effects are due to stimulation of chondrogenic progenitor cell (CPC) migration toward injured areas of cartilage through focal adhesion kinase (FAK) activation. CPC chemotaxis in bluntly injured osteochondral explants was examined by confocal microscopy, and migratory activity of cultured CPCs was measured in transwell and monolayer scratch assays. FAK activation by LIPUS was analyzed in cultured CPCs by Western blot. LIPUS effects were compared with the effects of two known chemotactic factors: N-formyl-methionyl-leucyl-phenylalanine (fMLF) and high-mobility group box 1 (HMGB1) protein. LIPUS significantly enhanced CPC migration on explants and in cell culture assays. Phosphorylation of FAK at the kinase domain (Tyr 576/577) was maximized by 5 min of exposure to LIPUS at a dose of 27.5 mW/cm(2) and frequency of 3.5 MHz. Treatment with fMLF, but not HMBG1, enhanced FAK activation to a degree similar to that of LIPUS, but neither fMLF nor HMGB1 enhanced the LIPUS effect. LIPUS-induced CPC migration was blocked by suppressing FAK phosphorylation with a Src family kinase inhibitor that blocks FAK phosphorylation. Our results imply that LIPUS might be used to promote cartilage healing by inducing the migration of CPCs to injured sites, which could delay or prevent the onset of post-traumatic osteoarthritis. PMID:24612644

  7. Mechanically Induced Focal Adhesion Assembly Amplifies Anti-Adipogenic Pathways in Mesenchymal Stem Cells

    PubMed Central

    Sen, Buer; Guilluy, Christophe; Xie, Zhihui; Case, Natasha; Styner, Maya; Thomas, Jacob; Oguz, Ipek; Rubin, Clinton; Burridge, Keith; Rubin, Janet

    2013-01-01

    The fate of pluripotent mesenchymal stem cells (MSC) is determined through integration of chemical, spatial, and physical signals. The suppression of MSC adipogenesis by mechanical stimuli, which requires Akt-induced inhibition of glycogen synthase kinase 3β (GSK3β) with β-catenin activation, can be enhanced by repetitive dosing within a single day. Here, we demonstrate that reapplication of cyclic strain within a 24-hour period leads to amplification of both Akt activation and its subsequent inhibition of GSK3β, such that total cycle number can be reduced while still inhibiting adipogenesis. Amplification of Akt signaling is facilitated by a dynamic restructuring of the cell in response to mechanical signals, as evidenced by a transient increase in focal adhesion (FA) number and increased RhoA activity. Preventing FA assembly or development of tension blocks activation of Akt by mechanical signals, but not by insulin. This indicates that the FA infrastructure is essential to the physical, but not necessarily the chemical, sensitivity, and responsiveness of the cell. Exploiting the transient nature of cytoskeletal remodeling may represent a process to enhance cell responsiveness to mechanical input and ultimately define the fate of MSCs with a minimal input. PMID:21898699

  8. Focal adhesion kinase as a mechanotransducer during rapid brain growth of the chick embryo.

    PubMed

    Desmond, Mary E; Knepper, Janice E; DiBenedetto, Angela J; Malaugh, Elizabeth; Callejo, Sagrario; Carretero, Raquel; Alonso, Maria-Isabel; Gato, Angel

    2014-01-01

    Expansion of the hollow fluid-filled embryonic brain occurs by an increase in intraluminal pressure created by accumulation of cerebrospinal fluid (CSF). Experiments have shown a direct correlation between cavity pressure and cell proliferation within the neuroepithelium. These findings lead us to ask how mechanistically this might come about. Are there perhaps molecules on the luminal surface of the embryonic neuroepithelium, such as focal adhesion kinases (FAKs) known to respond to tension in other epithelial cells? Immunodetection using antibodies to total FAK and p-FAK was performed with subsequent confocal analysis of the pattern of their activation under normal intraluminal pressure and induced chronic pressure. Western analysis was also done to look at the amount of FAK expression, as well as its activation under these same conditions. Using immunolocalization, we have shown that FAK is present and activated on both apical and basolateral surfaces and within the cytoplasm of the neuroepithelial cells. This pattern changed profoundly when the neuroepithelium was under pressure. By Western blot, we have shown that FAK was upregulated and activated in the neuroepithelium of the embryos just after the neural tube becomes a closed pressurized system, with phosphorylation detected on the luminal instead of the basal surface, along with an increase in cell proliferation. Chronic hyper-pressure does not induce an increase in phosphorylation of FAK. In conclusion, here we show that neuroepithelial cells respond to intraluminal pressure via FAK phosphorylation on the luminal surface. PMID:24860993

  9. Targeting the Metastasis Suppressor, N-Myc Downstream Regulated Gene-1, with Novel Di-2-Pyridylketone Thiosemicarbazones: Suppression of Tumor Cell Migration and Cell-Collagen Adhesion by Inhibiting Focal Adhesion Kinase/Paxillin Signaling.

    PubMed

    Wangpu, Xiongzhi; Lu, Jiaoyang; Xi, Ruxing; Yue, Fei; Sahni, Sumit; Park, Kyung Chan; Menezes, Sharleen; Huang, Michael L H; Zheng, Minhua; Kovacevic, Zaklina; Richardson, Des R

    2016-05-01

    Metastasis is a complex process that is regulated by multiple signaling pathways, with the focal adhesion kinase (FAK)/paxillin pathway playing a major role in the formation of focal adhesions and cell motility. N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor in many solid tumor types, including prostate and colon cancer. Considering the antimetastatic effect of NDRG1 and the crucial involvement of the FAK/paxillin pathway in cellular migration and cell-matrix adhesion, we assessed the effects of NDRG1 on this important oncogenic pathway. In the present study, NDRG1 overexpression and silencing models of HT29 colon cancer and DU145 prostate cancer cells were used to examine the activation of FAK/paxillin signaling and the formation of focal adhesions. The expression of NDRG1 resulted in a marked and significant decrease in the activating phosphorylation of FAK and paxillin, whereas silencing of NDRG1 resulted in an opposite effect. The expression of NDRG1 also inhibited the formation of focal adhesions as well as cell migration and cell-collagen adhesion. Incubation of cells with novel thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, that upregulate NDRG1 also resulted in decreased phosphorylation of FAK and paxillin. The ability of these thiosemicarbazones to inhibit cell migration and metastasis could be mediated, at least in part, through the FAK/paxillin pathway. PMID:26895766

  10. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee.

    PubMed

    Advani, Alexander S; Chen, Annie Y; Babbitt, Courtney C

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors. PMID:26971204

  11. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee

    PubMed Central

    Advani, Alexander S.; Chen, Annie Y.; Babbitt, Courtney C.

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors. PMID:26971204

  12. Dipeptidyl peptidase 9 subcellular localization and a role in cell adhesion involving focal adhesion kinase and paxillin.

    PubMed

    Zhang, Hui; Chen, Yiqian; Wadham, Carol; McCaughan, Geoffrey W; Keane, Fiona M; Gorrell, Mark D

    2015-02-01

    Dipeptidyl peptidase 9 (DPP9) is a ubiquitously expressed member of the DPP4 gene and protease family. Deciphering the biological functions of DPP9 and its roles in pathogenesis has implicated DPP9 in tumor biology, the immune response, apoptosis, intracellular epidermal growth factor-dependent signaling and cell adhesion and migration. We investigated the intracellular distribution of DPP9 chimeric fluorescent proteins and consequent functions of DPP9. We showed that while some DPP9 is associated with mitochondria, the strongest co-localization was with microtubules. Under steady state conditions, DPP9 was not seen at the plasma membrane, but upon stimulation with either phorbol 12-myristate 13-acetate or epidermal growth factor, some DPP9 re-distributed towards the ruffling membrane. DPP9 was seen at the leading edge of the migrating cell and co-localized with the focal adhesion proteins, integrin-β1 and talin. DPP9 gene silencing and treatment with a DPP8/DPP9 specific inhibitor both reduced cell adhesion and migration. Expression of integrin-β1 and talin was decreased in DPP9-deficient and DPP9-enzyme-inactive cells. There was a concomitant decrease in the phosphorylation of focal adhesion kinase and paxillin, indicating that DPP9 knockdown or enzyme inhibition suppressed the associated adhesion signaling pathway, causing impaired cell movement. These novel findings provide mechanistic insights into the regulatory role of DPP9 in cell movement, and may thus implicate DPP9 in tissue and tumor growth and metastasis. PMID:25486458

  13. Role of focal adhesions and mechanical stresses in the formation and progression of the lamellipodium-lamellum interface [corrected].

    PubMed

    Shemesh, Tom; Verkhovsky, Alexander B; Svitkina, Tatyana M; Bershadsky, Alexander D; Kozlov, Michael M

    2009-09-01

    Actin network in the front part of a moving cell is organized into a lamellipodium and a lamellum. A distinct lamellipodium-lamellum interface is associated with focal adhesions and consists of a series of arclike segments linking neighboring focal adhesions in the front row. The interface advances by leaping onto new rows of focal adhesions maturating underneath the lamellipodium. We propose a mechanism of the lamellipodium-lamellum boundary generation, shape formation, and progression based on the elastic stresses generated in the lamellipodial actin gel by its friction against the focal adhesions. The crucial assumption of the model is that stretching stresses trigger actin gel disintegration. We compute the stress distribution throughout the actin gel and show that the gel-disintegrating stresses drive formation of a gel boundary passing through the row of focal adhesions. Our computations recover the lamellipodium-lamellum boundary shapes detected in cells and predict the mode of the boundary transition to the row of the newly maturing focal adhesions in agreement with the experimental observations. The model fully accounts for the current phenomenology of the lamellipodium-lamellum interface formation and advancing, and makes experimentally testable predictions on the dependence of these phenomena on the sizes of the focal adhesions, the character of the focal adhesion distribution on the substrate, and the velocity of the actin retrograde flow with respect to the focal adhesions. The phase diagram resulting from the model provides a background for quantitative classification of different cell types with respect to their ability to form a lamellipodium-lamellum interface. In addition, the model suggests a mechanism of nucleation of the dorsal and arclike actin bundles found in the lamellum. PMID:19720013

  14. Polychlorinated biphenyl quinone induces endothelial barrier dysregulation by setting the cross talk between VE-cadherin, focal adhesion, and MAPK signaling.

    PubMed

    Zhang, Pu; Feng, Shan; Bai, Huiyuan; Zeng, Panying; Chen, Feng; Wu, Chengxiang; Peng, Yi; Zhang, Qin; Zhang, Qiuyao; Ye, Qichao; Xue, Qiang; Xu, Xiaoyu; Song, Erqun; Song, Yang

    2015-05-15

    Environmental hazardous material polychlorinated biphenyl (PCB) exposure is associated with vascular endothelial dysfunction, which may increase the risk of cardiovascular diseases and cancer metastasis. Our previous studies illustrated the cytotoxic, antiproliferative, and genotoxic effects of a synthetic, quinone-type, highly reactive metabolite of PCB, 2,3,5-trichloro-6-phenyl-[1,4]benzoquinone (PCB29-pQ). Here, we used it as the model compound to investigate its effects on vascular endothelial integrity and permeability. We demonstrated that noncytotoxic doses of PCB29-pQ induced vascular endothelial (VE)-cadherin junction disassembly by increasing the phosphorylation of VE-cadherin at Y658. We also found that focal adhesion assembly was required for PCB29-pQ-induced junction breakdown. Focal adhesion site-associated actin stress fibers may serve as holding points for cytoskeletal tension to regulate the cellular contractility. PCB29-pQ exposure promoted the association of actin stress fibers with paxillin-containing focal adhesion sites and enlarged the size/number of focal adhesions. In addition, PCB29-pQ treatment induced phosphorylation of paxillin at Y118. By using pharmacological inhibition, we further demonstrated that p38 activation was necessary for paxillin phosphorylation, whereas extracellular signal-regulated kinases-1/2 activation regulated VE-cadherin phosphorylation. In conclusion, these results indicated that PCB29-pQ stimulates endothelial hyperpermeability by mediating VE-cadherin disassembly, junction breakdown, and focal adhesion formation. Intervention strategies targeting focal adhesion and MAPK signaling could be used as therapeutic approaches for preventing adverse cardiovascular health effects induced by environmental toxicants such as PCBs. PMID:25770237

  15. Image Analysis for the Quantitative Comparison of Stress Fibers and Focal Adhesions

    PubMed Central

    Elosegui-Artola, Alberto; Jorge-Peñas, Alvaro; Moreno-Arotzena, Oihana; Oregi, Amaia; Lasa, Marta; García-Aznar, José Manuel; De Juan-Pardo, Elena M.; Aldabe, Rafael

    2014-01-01

    Actin stress fibers (SFs) detect and transmit forces to the extracellular matrix through focal adhesions (FAs), and molecules in this pathway determine cellular behavior. Here, we designed two different computational tools to quantify actin SFs and the distribution of actin cytoskeletal proteins within a normalized cellular morphology. Moreover, a systematic cell response comparison between the control cells and those with impaired actin cytoskeleton polymerization was performed to demonstrate the reliability of the tools. Indeed, a variety of proteins that were present within the string beginning at the focal adhesions (vinculin) up to the actin SFs contraction (non-muscle myosin II (NMMII)) were analyzed. Finally, the software used allows for the quantification of the SFs based on the relative positions of FAs. Therefore, it provides a better insight into the cell mechanics and broadens the knowledge of the nature of SFs. PMID:25269086

  16. Mechanotransduction through fibronectin-integrin focal adhesion in microvascular smooth muscle cells: is calcium essential?

    PubMed Central

    Li, Zhaohui; Meininger, Gerald A.

    2012-01-01

    It is believed that increased transmural pressure exerts force on vascular smooth muscle cells (VSMCs) and triggers Ca2+ signaling as an initiating event responsible for the arteriolar myogenic response. However, the mechanisms linking the pressure increase to Ca2+ signaling are unclear. We have shown previously using atomic force microscopy (AFM) that mechanical force induces a VSMC contractile response when applied to single fibronectin (FN; Sun Z, Martinez-Lemus LA, Hill MA, Meininger GA. Am J Physiol Cell Physiol 295; C268–C278, 2008) focal adhesion sites. This current study seeks to determine whether application of force to single focal adhesions can cause a change in VSMC Ca2+. Experiments were performed in low passage (p3∼10) as well as in freshly isolated skeletal muscle arteriole VSMCs. AFM-attached microbeads (5 μm) were coated with FN or collagen type I (CN-I) or type IV (CN-IV) and placed on a VSMC for 20 min, resulting in formation of a focal adhesion between the cell and the microbead. In low passage VSMCs, mechanically pulling on the FN-coated beads (800∼3000 pN) did not induce a Ca2+ increase but did cause a contractile response. In freshly isolated VSMCs, application of an FN or CN-I-coated bead onto the cell surface induced global Ca2+ increases. However, these Ca2+ increases were not correlated with the application of AFM pulling force to the bead or with the VSMC contractile responses to FN-coupled pulling. Chelating cytosolic Ca2+ using BAPTA loading had no negative effect on the focal adhesion-related contractile response in both freshly isolated and low passage VSMCs, while the Rho-kinase inhibitor Y27632 abolished the micromyogenic response in both cases. These observations suggest that, in freshly isolated and cultured VSMCs, application of mechanical force to a focal adhesion does not invoke an acute global Ca2+ increase. On the other hand, our data support a role for Rho-linked signaling mechanism involved in mechanotransduction

  17. Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins.

    PubMed

    Freeman, N L; Zurawski, D V; Chowrashi, P; Ayoob, J C; Huang, L; Mittal, B; Sanger, J M; Sanger, J W

    2000-12-01

    When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli. PMID:11093251

  18. Micrometer scale spacings between fibronectin nanodots regulate cell morphology and focal adhesions

    NASA Astrophysics Data System (ADS)

    Horzum, Utku; Ozdil, Berrin; Pesen-Okvur, Devrim

    2014-04-01

    Cell adhesion to extracellular matrix is an important process for both health and disease states. Surface protein patterns that are topographically flat, and do not introduce other chemical, topographical or rigidity related functionality and, more importantly, that mimic the organization of the in vivo extracellular matrix are desired. Previous work showed that vinculin and cytoskeletal organization are modulated by size and shape of surface nanopatterns. However, quantitative analysis on cell morphology and focal adhesions as a function of micrometer scale spacings of FN nanopatterns was absent. Here, electron beam lithography was used to pattern fibronectin nanodots with micrometer scale spacings on a K-casein background on indium tin oxide coated glass which, unlike silicon, is transparent and thus suitable for many light microscopy techniques. Exposure times were significantly reduced using the line exposure mode with micrometer scale step sizes. Micrometer scale spacings of 2, 4 and 8 μm between fibronectin nanodots proved to modulate cell adhesion through modification of cell area, focal adhesion number, size and circularity. Overall, cell behavior was shown to shift at the apparent threshold of 4 μm spacing. The findings presented here offer exciting new opportunities for cell biology research.

  19. Focal adhesions control cleavage furrow shape and spindle tilt during mitosis

    PubMed Central

    Taneja, Nilay; Fenix, Aidan M.; Rathbun, Lindsay; Millis, Bryan A.; Tyska, Matthew J.; Hehnly, Heidi; Burnette, Dylan T.

    2016-01-01

    The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells—MDCK—within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis. PMID:27432211

  20. Focal adhesion kinase (FAK) phosphorylation is not required for genistein-induced FAK-beta-1-integrin complex formation.

    PubMed

    Liu, Y; Kyle, E; Lieberman, R; Crowell, J; Kellof, G; Bergan, R C

    2000-01-01

    It has previously been shown that changes in the activity of focal adhesion kinase (FAK), and its binding to beta-1-integrin, accompany genistein-induced adhesion of prostate cells. Consumption of genistein world wide is associated with a lower incidence of metastatic prostate cancer. Early human clinical trials of genistein are under way to evaluate genistein's potential causal role in this regard. Though an important cell adhesion-associated signaling molecule, FAK's role in regulating prostate cell adhesion was not clear. Elucidation of this process would provide important information relating to both biology and potential clinical endpoints. It was hypothesized that FAK activation and complex formation are temporally related in prostate cells, and can thus be separated. Significant activation of FAK was demonstrated when cells adhered to fibronectin, as compared to poly-L-lysine, thus demonstrating that beta-1-integrin plays a significant role in activating FAK. Neither FAK activation, nor FAK-integrin complex formation, required beta-1-integrin ligand. However, disruption of the cellular cytoskeleton by cytochalasin D prevented FAK activation, but did not block genistein-induced complex formation. In the face of a disrupted cytoskeleton, signaling through FAK could not be restored through either integrin cross linking, or re-establishment of tensile forces via attachment to solid matrix. These studies demonstrate that FAK-beta-1-integrin complex formation does not require FAK activation, suggesting that it is an early event in prostate cell adhesion. An intact cytoskeleton is necessary for FAK activation. The functional importance of beta-1-integrin in prostate cells is demonstrated. Current findings support plans to test genistein in prostate cancer. PMID:11315093

  1. Diamagnetic levitation causes changes in the morphology, cytoskeleton, and focal adhesion proteins expression in osteocytes.

    PubMed

    Qian, A R; Wang, L; Gao, X; Zhang, W; Hu, L F; Han, J; Li, J B; Di, S M; Shang, Peng

    2012-01-01

    Diamagnetic levitation technology is a novel simulated weightless technique and has recently been applied in life-science research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely, μg (diamagnetic levitation), 1g, and 2g for diamagnetic materials. In this study, the effects of LG-HMF on the activity, morphology, and cytoskeleton (actin filament, microtubules, and vimentin intermediate filaments) in osteocyte - like cell line MLO-Y4 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, hematoxylin-eosin (HE) staining, and laser scanning confocal microscopy (LSCM), respectively. The changes induced by LG-HMF in distribution and expression of focal adhesion (FA) proteins, including vinculin, paxillin, and talin in MLO-Y4 were determined by LSCM and Western blotting. The results showed that LG-HMF produced by superconducting magnet had no lethal effects on MLO-Y4. Compared to control, diamagnetic levitation (μg) affected MLO-Y4 morphology, nucleus size, cytoskeleton architecture, and FA proteins distribution and expression. The study indicates that osteocytes are sensitive to altered gravity and FA proteins (vinculin, paxillin, and talin) may be involved in osteocyte mechanosensation. The diamagnetic levitation may be a novel ground-based space-gravity simulator and can be used for biological experiment at cellular level. PMID:21216704

  2. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    PubMed

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells. PMID:23387972

  3. LRRK2 G2019S mutation attenuates microglial motility by inhibiting focal adhesion kinase

    PubMed Central

    Choi, Insup; Kim, Beomsue; Byun, Ji-Won; Baik, Sung Hoon; Huh, Yun Hyun; Kim, Jong-Hyeon; Mook-Jung, Inhee; Song, Woo Keun; Shin, Joo-Ho; Seo, Hyemyung; Suh, Young Ho; Jou, Ilo; Park, Sang Myun; Kang, Ho Chul; Joe, Eun-Hye

    2015-01-01

    In response to brain injury, microglia rapidly extend processes that isolate lesion sites and protect the brain from further injury. Here we report that microglia carrying a pathogenic mutation in the Parkinson's disease (PD)-associated gene, G2019S-LRRK2 (GS-Tg microglia), show retarded ADP-induced motility and delayed isolation of injury, compared with non-Tg microglia. Conversely, LRRK2 knockdown microglia are highly motile compared with control cells. In our functional assays, LRRK2 binds to focal adhesion kinase (FAK) and phosphorylates its Thr–X–Arg/Lys (TXR/K) motif(s), eventually attenuating FAK activity marked by decreased pY397 phosphorylation (pY397). GS-LRRK2 decreases the levels of pY397 in the brain, microglia and HEK cells. In addition, treatment with an inhibitor of LRRK2 kinase restores pY397 levels, decreased pTXR levels and rescued motility of GS-Tg microglia. These results collectively suggest that G2019S mutation of LRRK2 may contribute to the development of PD by inhibiting microglial response to brain injury. PMID:26365310

  4. Raver1 Interactions with Vinculin and RNA Suggest a Feed-Forward Pathway in Directing mRNA to Focal Adhesions

    SciTech Connect

    Lee, Jun Hyuck; Rangarajan, Erumbi S.; Yogesha, S.D.; Izard, Tina; Scripps

    2009-09-11

    The translational machinery of the cell relocalizes to focal adhesions following the activation of integrin receptors. This response allows for rapid, local production of components needed for adhesion complex assembly and signaling. Vinculin links focal adhesions to the actin cytoskeleton following its activation by integrin signaling, which severs intramolecular interactions of vinculin's head and tail (Vt) domains. Our vinculin:raver1 crystal structures and binding studies show that activated Vt selectively interacts with one of the three RNA recognition motifs of raver1, that the vinculin:raver1 complex binds to F-actin, and that raver1 binds selectively to RNA, including a sequence found in vinculin mRNA. Further, mutation of residues that mediate interaction of raver1 with vinculin abolish their colocalization in cells. These findings suggest a feed-forward model where vinculin activation at focal adhesions provides a scaffold for recruitment of raver1 and its mRNA cargo to facilitate the production of components of adhesion complexes.

  5. Comparative Dynamics of Retrograde Actin Flow and Focal Adhesions: Formation of Nascent Adhesions Triggers Transition from Fast to Slow Flow

    PubMed Central

    Alexandrova, Antonina Y.; Arnold, Katya; Schaub, Sébastien; Vasiliev, Jury M.; Meister, Jean-Jacques; Bershadsky, Alexander D.; Verkhovsky, Alexander B.

    2008-01-01

    Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base. PMID:18800171

  6. Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow.

    PubMed

    Alexandrova, Antonina Y; Arnold, Katya; Schaub, Sébastien; Vasiliev, Jury M; Meister, Jean-Jacques; Bershadsky, Alexander D; Verkhovsky, Alexander B

    2008-01-01

    Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base. PMID:18800171

  7. Integrin activation and focal complex formation in cardiac hypertrophy

    NASA Technical Reports Server (NTRS)

    Laser, M.; Willey, C. D.; Jiang, W.; Cooper, G. 4th; Menick, D. R.; Zile, M. R.; Kuppuswamy, D.

    2000-01-01

    Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

  8. LOW-INTENSITY PULSED ULTRASOUND PROMOTES CHONDROGENIC PROGENITOR CELL MIGRATION VIA FOCAL ADHESION KINASE PATHWAY

    PubMed Central

    Jang, Kee W.; Ding, Lei; Seol, Dongrim; Lim, Tae-hong; Buckwalter, Joseph A.; Martin, James A.

    2014-01-01

    Low-intensity pulsed ultrasound (LIPUS) has been frequently studied for its beneficial effects on the repair of injured articular cartilage. Here, we hypothesized that these effects are due to stimulation of chondrogenic progenitor cell (CPC) migration toward injured areas in cartilage through focal adhesion kinase (FAK) activation. CPC chemotaxis in bluntly impacted osteochondral explants was examined by confocal microscopy and migratory activity of cultured CPCs was measured in trans-well and monolayer scratch assays. FAK activation by LIPUS was analyzed in cultured CPCs by western blot. LIPUS effects were compared with the effects of two known chemotactic factors; formylated-methionine peptides (fMLF), and high-mobility group box 1 (HMGB1) protein. LIPUS significantly enhanced CPC migration on explants and in cell culture assays. Phosphorylation of FAK at the kinase domain (Tyr 576/577) was maximized by 5 minute exposure to LIPUS at a dose of 27.5 mW/cm2 and at a frequency of 3.5 MHz. Treatment with fMLF, but not HMBG1 enhanced FAK activation to a degree similar to LIPUS, but neither fMLF nor HMGB1 enhanced the LIPUS effect. LIPUS-induced CPC migration was blocked by suppressing FAK phosphorylation with a Src family kinases (SFKs) inhibitor that blocks FAK phosphorylation. Our results imply that LIPUS might be utilized to promote cartilage healing by inducing the migration of CPCs to injured sites, which could delay or prevent the onset of post-traumatic osteoarthritis (PTOA). PMID:24612644

  9. Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity.

    PubMed

    Kumar, Abhishek; Ouyang, Mingxing; Van den Dries, Koen; McGhee, Ewan James; Tanaka, Keiichiro; Anderson, Marie D; Groisman, Alexander; Goult, Benjamin T; Anderson, Kurt I; Schwartz, Martin A

    2016-05-01

    Integrin-dependent adhesions are mechanosensitive structures in which talin mediates a linkage to actin filaments either directly or indirectly by recruiting vinculin. Here, we report the development and validation of a talin tension sensor. We find that talin in focal adhesions is under tension, which is higher in peripheral than central adhesions. Tension on talin is increased by vinculin and depends mainly on actin-binding site 2 (ABS2) within the middle of the rod domain, rather than ABS3 at the far C terminus. Unlike vinculin, talin is under lower tension on soft substrates. The difference between central and peripheral adhesions requires ABS3 but not vinculin or ABS2. However, differential stiffness sensing by talin requires ABS2 but not vinculin or ABS3. These results indicate that central versus peripheral adhesions must be organized and regulated differently, and that ABS2 and ABS3 have distinct functions in spatial variations and stiffness sensing. Overall, these results shed new light on talin function and constrain models for cellular mechanosensing. PMID:27161398

  10. Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly

    PubMed Central

    Skau, Colleen T.; Plotnikov, Sergey V.; Doyle, Andrew D.; Waterman, Clare M.

    2015-01-01

    Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF–FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM. PMID:25918420

  11. Structured illumination microscopy reveals focal adhesions are composed of linear subunits.

    PubMed

    Hu, Shiqiong; Tee, Yee-Han; Kabla, Alexandre; Zaidel-Bar, Ronen; Bershadsky, Alexander; Hersen, Pascal

    2015-05-01

    The ability to mechanically interact with the extracellular matrix is a fundamental feature of adherent eukaryotic cells. Cell-matrix adhesion in many cell types is mediated by protein complexes called focal adhesions (FAs). Recent progress in super resolution microscopy revealed FAs possess an internal organization, yet such methods do not enable observation of the formation and dynamics of their internal structure in living cells. Here, we combine structured illumination microscopy (SIM) with total internal reflection fluorescence microscopy (TIRF) to show that the proteins inside FA patches are distributed along elongated subunits, typically 300 ± 100 nm wide, separated by 400 ± 100 nm, and individually connected to actin cables. We further show that the formation and dynamics of these linear subunits are intimately linked to radial actin fiber formation and actomyosin contractility. We found FA growth to be the result of nucleation of new linear subunits and their coordinated elongation. Taken together, this study reveals that the basic units of mature focal adhesion are 300-nm-wide elongated, dynamic structures. We anticipate this ultrastructure to be relevant to investigation of the function of FAs and their behavior in response to mechanical stress. PMID:26012525

  12. Identification of methyl violet 2B as a novel blocker of focal adhesion kinase signaling pathway in cancer cells

    SciTech Connect

    Kim, Hwan; Kim, Nam Doo; Lee, Jiyeon; Han, Gyoonhee; Sim, Taebo

    2013-07-26

    Highlights: •FAK signaling cascade in cancer cells is profoundly inhibited by methyl violet 2B. •Methyl violet 2B identified by virtual screening is a novel allosteric FAK inhibitor. •Methyl violet 2B possesses extremely high kinase selectivity. •Methyl violet 2B suppresses strongly the proliferation of cancer cells. •Methyl violet 2B inhibits focal adhesion, invasion and migration of cancer cells. -- Abstract: The focal adhesion kinase (FAK) signaling cascade in cancer cells was profoundly inhibited by methyl violet 2B identified with the structure-based virtual screening. Methyl violet 2B was shown to be a non-competitive inhibitor of full-length FAK enzyme vs. ATP. It turned out that methyl violet 2B possesses extremely high kinase selectivity in biochemical kinase profiling using a large panel of kinases. Anti-proliferative activity measurement against several different cancer cells and Western blot analysis showed that this substance is capable of suppressing significantly the proliferation of cancer cells and is able to strongly block FAK/AKT/MAPK signaling pathways in a dose dependent manner at low nanomolar concentration. Especially, phosphorylation of Tyr925-FAK that is required for full activation of FAK was nearly completely suppressed even with 1 nM of methyl violet 2B in A375P cancer cells. To the best of our knowledge, it has never been reported that methyl violet possesses anti-cancer effects. Moreover, methyl violet 2B significantly inhibited FER kinase phosphorylation that activates FAK in cell. In addition, methyl violet 2B was found to induce cell apoptosis and to exhibit strong inhibitory effects on the focal adhesion, invasion, and migration of A375P cancer cells at low nanomolar concentrations. Taken together, these results show that methyl violet 2B is a novel, potent and selective blocker of FAK signaling cascade, which displays strong anti-proliferative activities against a variety of human cancer cells and suppresses adhesion

  13. Inhibition of smooth muscle force generation by focal adhesion kinase inhibitors in the hyperplastic human prostate.

    PubMed

    Kunit, Thomas; Gratzke, Christian; Schreiber, Andrea; Strittmatter, Frank; Waidelich, Raphaela; Rutz, Beata; Loidl, Wolfgang; Andersson, Karl-Erik; Stief, Christian G; Hennenberg, Martin

    2014-10-01

    Smooth muscle contraction may be critical for lower urinary tract symptoms (LUTS) in patients with benign prostate hyperplasia and requires stable anchorage of the cytoskeleton to the cell membrane. These connections are regulated by focal adhesion kinase (FAK). Here, we addressed the involvement of FAK in the regulation of smooth muscle contraction in hyperplastic human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Expression of FAK and focal adhesion proteins was assessed by Western blot analysis and immunohistochemical stainings. Effects of the FAK inhibitors PF-573228 and Y-11 on contraction of prostate strips were examined in the organ bath. Expression of FAK and focal adhesion proteins (integrin-5α, paxilin, and c-Src) was detected by Western blot analysis in prostate samples. By double immunofluorescence staining with calponin and pan-cytokeratin, expression of FAK was observed in stromal and epithelial cells. Immunoreactivity for FAK colocalized with integrin-5α, paxilin, talin, and c-Src. Stimulation of prostate tissues with the α1-adrenergic agonist phenylephrine increased the phosphorylation state of FAK at Tyr³⁹⁷ and Tyr⁹²⁵ with different kinetics, which was blocked by the α1-adrenoceptor antagonist tamsulosin. Norepinephrine and phenylephrine induced concentration-dependent contractions of prostate strips. Both FAK inhibitors PF-573228 and Y-11 significantly inhibited norepinephrine- and phenylephrine-induced contractions. Finally, PF-573228 and Y-11 inhibited contractions induced by electric field stimulation, which was significant at the highest frequency. In conclusion, α1-adrenergic smooth muscle contraction or its regulation involves FAK in the human prostate. Consequently, FAK may be involved in the pathophysiology of LUTS and in current or future LUTS therapies. PMID:25056351

  14. Structural Basis for Paxillin Binding and Focal Adhesion Targeting of β-Parvin*

    PubMed Central

    Stiegler, Amy L.; Draheim, Kyle M.; Li, Xiaofeng; Chayen, Naomi E.; Calderwood, David A.; Boggon, Titus J.

    2012-01-01

    β-Parvin is a cytoplasmic adaptor protein that localizes to focal adhesions where it interacts with integrin-linked kinase and is involved in linking integrin receptors to the cytoskeleton. It has been reported that despite high sequence similarity to α-parvin, β-parvin does not bind paxillin, suggesting distinct interactions and cellular functions for these two closely related parvins. Here, we reveal that β-parvin binds directly and specifically to leucine-aspartic acid repeat (LD) motifs in paxillin via its C-terminal calponin homology (CH2) domain. We present the co-crystal structure of β-parvin CH2 domain in complex with paxillin LD1 motif to 2.9 Å resolution and find that the interaction is similar to that previously observed between α-parvin and paxillin LD1. We also present crystal structures of unbound β-parvin CH2 domain at 2.1 Å and 2.0 Å resolution that show significant conformational flexibility in the N-terminal α-helix, suggesting an induced fit upon paxillin binding. We find that β-parvin has specificity for the LD1, LD2, and LD4 motifs of paxillin, with KD values determined to 27, 42, and 73 μm, respectively, by surface plasmon resonance. Furthermore, we show that proper localization of β-parvin to focal adhesions requires both the paxillin and integrin-linked kinase binding sites and that paxillin is important for early targeting of β-parvin. These studies provide the first molecular details of β-parvin binding to paxillin and help define the requirements for β-parvin localization to focal adhesions. PMID:22869380

  15. Disrupting the scaffold to improve focal adhesion kinase-targeted cancer therapeutics.

    PubMed

    Cance, William G; Kurenova, Elena; Marlowe, Timothy; Golubovskaya, Vita

    2013-03-26

    Focal adhesion kinase (FAK) is emerging as a promising cancer target because it is highly expressed at both the transcriptional and translational level in cancer and is involved in many aspects of tumor growth, invasion, and metastasis. Existing FAK-based therapeutics focus on inhibiting the kinase's catalytic function and not the large scaffold it creates that includes many oncogenic receptor tyrosine kinases and tumor suppressor proteins. Targeting the FAK scaffold is a feasible and promising approach for developing highly specific therapeutics that disrupt FAK signaling pathways in cancer. PMID:23532331

  16. Visualizing the interior architecture of focal adhesions with high-resolution traction maps.

    PubMed

    Morimatsu, Masatoshi; Mekhdjian, Armen H; Chang, Alice C; Tan, Steven J; Dunn, Alexander R

    2015-04-01

    Focal adhesions (FAs) are micron-sized protein assemblies that coordinate cell adhesion, migration, and mechanotransduction. How the many proteins within FAs are organized into force sensing and transmitting structures is poorly understood. We combined fluorescent molecular tension sensors with super-resolution light microscopy to visualize traction forces within FAs with <100 nm spatial resolution. We find that αvβ3 integrin selectively localizes to high force regions. Paxillin, which is not generally considered to play a direct role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or α-actinin, proteins with hypothesized roles as force transducers. These observations suggest that αvβ3 integrin and paxillin may play important roles in mechanotransduction. PMID:25730141

  17. New partners and phosphorylation sites of focal adhesion kinase identified by mass spectrometry.

    PubMed

    Masdeu, Maria del Mar; Armendáriz, Beatriz G; Soriano, Eduardo; Ureña, Jesús Mariano; Burgaya, Ferran

    2016-07-01

    The regulation of focal adhesion kinase (FAK) involves phosphorylation and multiple interactions with other signaling proteins. Some of these pathways are relevant for nervous system functions such as branching, axonal guidance, and plasticity. In this study, we screened mouse brain to identify FAK-interactive proteins and phosphorylatable residues as a first step to address the neuronal functions of this kinase. Using mass spectrometry analysis, we identified new phosphorylated sites (Thr 952, Thr 1048, and Ser 1049), which lie in the FAT domain; and putative new partners for FAK, which include cytoskeletal proteins such as drebrin and MAP 6, adhesion regulators such as neurabin-2 and plakophilin 1, and synapse-associated proteins such as SynGAP and a NMDA receptor subunit. Our findings support the participation of brain-localized FAK in neuronal plasticity. PMID:27033120

  18. Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-Mediated Contractility via Activation of Focal Adhesion Kinase and Extracellular Regulated Kinase 1/2 in Cerebral Arteries from Rat.

    PubMed

    Spray, Stine; Rasmussen, Marianne N P; Skovsted, Gry F; Warfvinge, Karin; Sheykhzade, Majid; Edvinsson, Lars

    2016-07-01

    Cerebral ischaemia results in enhanced endothelin B (ETB ) receptor-mediated contraction and receptor protein expression in the affected cerebrovascular smooth muscle cells (SMC). Organ culture of cerebral arteries is a method to induce similar alterations in ETB receptor expression. We suggest that rapid and sustained reduction in wall tension/stretch is a possible trigger mechanism for this vascular remodelling. Isolated rat middle cerebral artery (MCA) segments were incubated in a wire myograph with or without mechanical stretch, prior to assessment of their contractile response to the selective ETB receptor agonist sarafotoxin 6c. The involvement of extracellular regulated kinase (ERK) 1/2 and focal adhesion kinase (FAK) was studied by their specific inhibitors U0126 and PF-228, respectively. Compared with their stretched counterparts, unstretched MCA segments showed a significantly increased ETB receptor-mediated contractile response after 12 hr of incubation, which was attenuated by either U0126 or PF-228. The functionally increased ETB -mediated contractility could be attributed to two different mechanisms: (i) a difference in ETB receptor localization from primarily endothelial expression to SMC expression and (ii) an increased calcium sensitivity of the SMCs due to an increased expression of the calcium channel transient receptor potential canonical 1. Collectively, our results present a possible mechanism linking lack of vessel wall stretch/tension to changes in ETB receptor-mediated contractility via triggering of an early mechanosensitive signalling pathway involving ERK1/2 and FAK signalling. A mechanism likely to be an initiating factor for the increased ETB receptor-mediated contractility found after cerebral ischaemia. PMID:26781487

  19. KSHV-TK is a tyrosine kinase that disrupts focal adhesions and induces Rho-mediated cell contraction

    PubMed Central

    Gill, Michael B; Turner, Rachel; Stevenson, Philip G; Way, Michael

    2015-01-01

    Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV-TK, in contrast to HSV1-TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto-phosphorylation of tyrosines 65, 85 and 120 in the N-terminus of KSHV-TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3-Kinase, respectively. The interaction of Crk with KSHV-TK leads to tyrosine phoshorylation of this cellular adaptor. Auto-phosphorylation of KSHV-TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA-ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV-TK has no effect on focal adhesion integrity or cell morphology. Our observations demonstrate that by acting as a tyrosine kinase, KSHV-TK modulates signalling and cell morphology. PMID:25471072

  20. Phosphoproteomic profiling identifies focal adhesion kinase as a mediator of docetaxel resistance in castrate-resistant prostate cancer.

    PubMed

    Lee, Brian Y; Hochgräfe, Falko; Lin, Hui-Ming; Castillo, Lesley; Wu, Jianmin; Raftery, Mark J; Martin Shreeve, S; Horvath, Lisa G; Daly, Roger J

    2014-01-01

    Docetaxel remains the standard-of-care for men diagnosed with metastatic castrate-resistant prostate cancer (CRPC). However, only approximately 50% of patients benefit from treatment and all develop docetaxel-resistant disease. Here, we characterize global perturbations in tyrosine kinase signaling associated with docetaxel resistance and thereby develop a potential therapeutic strategy to reverse this phenotype. Using quantitative mass spectrometry-based phosphoproteomics, we identified that metastatic docetaxel-resistant prostate cancer cell lines (DU145-Rx and PC3-Rx) exhibit increased phosphorylation of focal adhesion kinase (FAK) on Y397 and Y576, in comparison with parental controls (DU145 and PC3, respectively). Bioinformatic analyses identified perturbations in pathways regulating focal adhesions and the actin cytoskeleton and in protein-protein interaction networks related to these pathways in docetaxel-resistant cells. Treatment with the FAK tyrosine kinase inhibitor (TKI) PF-00562271 reduced FAK phosphorylation in the resistant cells, but did not affect cell viability or Akt phosphorylation. Docetaxel administration reduced FAK and Akt phosphorylation, whereas cotreatment with PF-00562271 and docetaxel resulted in an additive attenuation of FAK and Akt phosphorylation and overcame the chemoresistant phenotype. The enhanced efficacy of cotreatment was due to increased autophagic cell death, rather than apoptosis. These data strongly support that enhanced FAK activation mediates chemoresistance in CRPC, and identify a potential clinical niche for FAK TKIs, where coadministration with docetaxel may be used in patients with CRPC to overcome chemoresistance. PMID:24194567

  1. Focal adhesion kinase regulation in stem cell alignment and spreading on nanofibers.

    PubMed

    Andalib, Mohammad Nahid; Lee, Jeong Soon; Ha, Ligyeom; Dzenis, Yuris; Lim, Jung Yul

    2016-05-13

    While electrospun nanofibers have demonstrated the potential for novel tissue engineering scaffolds, very little is known about the molecular mechanism of how cells sense and adapt to nanofibers. Here, we revealed the role of focal adhesion kinase (FAK), one of the key molecular sensors in the focal adhesion complex, in regulating mesenchymal stem cell (MSC) shaping on nanofibers. We produced uniaxially aligned and randomly distributed nanofibers from poly(l-lactic acid) to have the same diameters (about 130 nm) and evaluated MSC behavior on these nanofibers comparing with that on flat PLLA control. C3H10T1/2 murine MSCs exhibited upregulations in FAK expression and phosphorylation (pY397) on nanofibrous cultures as assessed by immunoblotting, and this trend was even greater on aligned nanofibers. MSCs showed significantly elongated and well-spread morphologies on aligned and random nanofibers, respectively. In the presence of FAK silencing via small hairpin RNA (shRNA), cell elongation length in the aligned nanofiber direction (cell major axis length) was significantly decreased, while cells still showed preferred orientation along the aligned nanofibers. On random nanofibers, MSCs with FAK-shRNA showed impaired cell spreading resulting in smaller cell area and higher circularity. Our study provides new data on how MSCs shape their morphologies on aligned and random nanofibrous cultures potentially via FAK-mediated mechanism. PMID:27040763

  2. Specific induction of pp125 focal adhesion kinase in human breast cancer

    PubMed Central

    Watermann, D O; Gabriel, B; Jäger, M; Orlowska-Volk, M; Hasenburg, A; zur Hausen, A; Gitsch, G; Stickeler, E

    2005-01-01

    The pp125 focal adhesion kinase (FAK) is involved in integrin-mediated cell signalling and overexpressed in a variety of solid tumours. Focal adhesion kinase expression has been correlated to invasion and metastasis, but the data on breast cancer are inconclusive. We analysed FAK mRNA, protein levels and expression patterns in primary breast cancer and normal breast tissue. FAK expression on the functional protein level and mRNA was determined in 55 matched pairs of breast cancer and corresponding normal tissue by Western blot, immunohistochemistry and RT–PCR. Using a score ranging from 0 to +5 for Western blots, we determined in normal breast tissue a score of 1.51±0.84 (mean±standard deviation), which was strongly induced to 2.91 (±1.22) in breast cancers (P<0.001). Overall, 45 out of 55 tissue pairs (81.8%) showed this upregulation of FAK protein in tumours in comparison to normal tissue. Immunohistochemistry confirmed these findings with a significant higher score for tumours vs physiological tissue (1.0±0.63 vs 2.27±0.91; P=0.001). Interestingly, no overall significant difference in the mRNA levels (P=0.359) was observed. In conclusion, expression levels of the FAK protein are specifically upregulated in breast cancer in comparison to matched normal breast tissue supporting its pivotal role in neoplastic signal transduction and representing a potential marker for malignant transformation. PMID:16136050

  3. Regulation of brain tumor dispersal by NKCC1 through a novel role in focal adhesion regulation.

    PubMed

    Garzon-Muvdi, Tomas; Schiapparelli, Paula; ap Rhys, Colette; Guerrero-Cazares, Hugo; Smith, Christopher; Kim, Deok-Ho; Kone, Lyonell; Farber, Harrison; Lee, Danielle Y; An, Steven S; Levchenko, Andre; Quiñones-Hinojosa, Alfredo

    2012-01-01

    Glioblastoma (GB) is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1) can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF), which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1) through the regulation of focal adhesion dynamics and cell contractility and (2) through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms. PMID:22570591

  4. Selected Contribution: Skeletal muscle focal adhesion kinase, paxillin, and serum response factor are loading dependent

    NASA Technical Reports Server (NTRS)

    Gordon, S. E.; Fluck, M.; Booth, F. W.

    2001-01-01

    This investigation examined the effect of mechanical loading state on focal adhesion kinase (FAK), paxillin, and serum response factor (SRF) in rat skeletal muscle. We found that FAK concentration and tyrosine phosphorylation, paxillin concentration, and SRF concentration are all lower in the lesser load-bearing fast-twitch plantaris and gastrocnemius muscles compared with the greater load-bearing slow-twitch soleus muscle. Of these three muscles, 7 days of mechanical unloading via tail suspension elicited a decrease in FAK tyrosine phosphorylation only in the soleus muscle and decreases in FAK and paxillin concentrations only in the plantaris and gastrocnemius muscles. Unloading decreased SRF concentration in all three muscles. Mechanical overloading (via bilateral gastrocnemius ablation) for 1 or 8 days increased FAK and paxillin concentrations in the soleus and plantaris muscles. Additionally, whereas FAK tyrosine phosphorylation and SRF concentration were increased by < or =1 day of overloading in the soleus muscle, these increases did not occur until somewhere between 1 and 8 days of overloading in the plantaris muscle. These data indicate that, in the skeletal muscles of rats, the focal adhesion complex proteins FAK and paxillin and the transcription factor SRF are generally modulated in association with the mechanical loading state of the muscle. However, the somewhat different patterns of adaptation of these proteins to altered loading in slow- vs. fast-twitch skeletal muscles indicate that the mechanisms and time course of adaptation may partly depend on the prior loading state of the muscle.

  5. The role of focal adhesion kinase in the regulation of cellular mechanical properties

    NASA Astrophysics Data System (ADS)

    Mierke, Claudia Tanja

    2013-12-01

    The regulation of mechanical properties is necessary for cell invasion into connective tissue or intra- and extravasation through the endothelium of blood or lymph vessels. Cell invasion is important for the regulation of many healthy processes such as immune response reactions and wound healing. In addition, cell invasion plays a role in disease-related processes such as tumor metastasis and autoimmune responses. Until now the role of focal adhesion kinase (FAK) in regulating mechanical properties of cells and its impact on cell invasion efficiency is still not well known. Thus, this review focuses on mechanical properties regulated by FAK in comparison to the mechano-regulating protein vinculin. Moreover, it points out the connection between cancer cell invasion and metastasis and FAK by showing that FAK regulates cellular mechanical properties required for cellular motility. Furthermore, it sheds light on the indirect interaction of FAK with vinculin by binding to paxillin, which then impairs the binding of paxillin to vinculin. In addition, this review emphasizes whether FAK fulfills regulatory functions similar to vinculin. In particular, it discusses the differences and the similarities between FAK and vinculin in regulating the biomechanical properties of cells. Finally, this paper highlights that both focal adhesion proteins, vinculin and FAK, synergize their functions to regulate the mechanical properties of cells such as stiffness and contractile forces. Subsequently, these mechanical properties determine cellular invasiveness into tissues and provide a source sink for future drug developments to inhibit excessive cell invasion and hence, metastases formation.

  6. Effects of PPARgamma agonists on cell survival and focal adhesions in a Chinese thyroid carcinoma cell line.

    PubMed

    Chen, Ying; Wang, Seu-Mei; Wu, Jiahn-Chun; Huang, Shih-Horng

    2006-07-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists cause cell death in several types of cancer cells. The aim of this study was to examine the effects of two PPARgamma agonists, ciglitazone and 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2), on the survival of thyroid carcinoma CGTH W-2 cells. Both ciglitazone and 15dPGJ2 decreased cell viability in a time- and dose-dependent manner. Cell death was mainly due to apoptosis, with a minor contribution from necrosis. Increased levels of active caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and cytosolic cytochrome-c were noted. In addition, ciglitazone and 15dPGJ2 induced detachment of CGTH W-2 cells from the culture substratum. Both the protein levels and immunostaining signals of focal adhesion (FA) proteins, including vinculin, integrin beta1, focal adhesion kinase (FAK), and paxillin were decreased after PPARgamma agonist treatment. Meanwhile, reduced phosphorylation of FAK and paxillin was noted. Furthermore, PPARgamma agonists induced expression of protein tyrosine phosphatase-PEST (PTP-PEST), and of phosphatase and tensin homologue deleted on chromosome ten (PTEN). The upregulation of these phosphatases might contribute to the dephosphorylation of FAK and paxillin, since pre-treatment with orthovanadate prevented PPARgamma agonist-induced dephosphorylation of FAK and paxillin. Perturbation of CGTH W-2 cells with anti-integrin beta1 antibodies induced FA disruption and apoptosis in the same cells, thus the downregulation of integrin beta1 by PPARgamma agonists resulted in FA disassembly and might induce apoptosis via anoikis. Our results suggested the presence of crosstalk between apoptosis and integrin-FA signaling. Moreover, upregulation and activation of PTEN was correlated with reduced phosphorylation of Akt, and this consequence disfavored cell survival. In conclusion, PPARgamma agonists induced apoptosis of thyroid carcinoma cells via the cytochrome-c caspase 3 and PTEN

  7. Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

    SciTech Connect

    Sakwe, Amos M.; Koumangoye, Rainelli; Guillory, Bobby; Ochieng, Josiah

    2011-04-01

    The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell-cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell-cell and/or cell-ECM contacts and anchorage-independent cell proliferation.

  8. Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

    PubMed Central

    Yu, Haiyang; Lui, Yuan Siang; Xiong, Sijing; Leong, Wen Shing; Wen, Feng; Nurkahfianto, Himawan; Rana, Sravendra; Leong, David Tai; Ng, Kee Woei

    2013-01-01

    We report the establishment of a novel platform to induce myogenic differentiation of human mesenchymal stem cells (hMSCs) via focal adhesion (FA) modulation, giving insights into the role of FA on stem cell differentiation. Micropatterning of collagen type I on a polyacrylamide gel with a stiffness of 10.2 kPa efficiently modulated elongated FA. This elongated FA profile preferentially recruited the β3 integrin cluster and induced specific myogenic differentiation at both transcription and translation levels with expression of myosin heavy chain and α-sarcomeric actin. This was initiated with elongation of FA complexes that triggered the RhoA downstream signaling toward a myogenic lineage commitment. This study also illustrates how one could partially control myogenic differentiation outcomes of similar-shaped hMSCs by modulating FA morphology and distribution. This technology increases our toolkit choice for controlled differentiation in muscle engineering. PMID:22765653

  9. Haematopoietic focal adhesion kinase deficiency alters haematopoietic homeostasis to drive tumour metastasis.

    PubMed

    Batista, Silvia; Maniati, Eleni; Reynolds, Louise E; Tavora, Bernardo; Lees, Delphine M; Fernandez, Isabelle; Elia, George; Casanovas, Oriol; Lo Celso, Cristina; Hagemann, Thorsten; Hodivala-Dilke, Kairbaan

    2014-01-01

    Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis. PMID:25270220

  10. Targeting the C-terminal focal adhesion kinase scaffold in pancreatic cancer.

    PubMed

    Gogate, Priyanka N; Kurenova, Elena V; Ethirajan, Manivannan; Liao, Jianqun; Yemma, Michael; Sen, Arindam; Pandey, Ravindra K; Cance, William G

    2014-10-28

    Preliminary studies in our laboratory have demonstrated the importance of both the NH2 and COOH terminus scaffolding functions of focal adhesion kinase (FAK). Here, we describe a new small molecule inhibitor, C10, that targets the FAK C-terminus scaffold. C10 showed marked selectivity for cells overexpressing VEGFR3 when tested in isogenic cell lines, MCF7 and MCF7-VEGFR3. C10 preferentially inhibited pancreatic tumor growth in vivo in cells with high FAK-Y925 and VEGFR3 expression. Treatment with C10 led to a significant inhibition in endothelial cell proliferation and tumor endothelial and lymphatic vessel density and decrease in interstitial fluid pressure. These results highlight the underlying importance of targeting the FAK scaffold to treat human cancers. PMID:25067788

  11. Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction

    PubMed Central

    Liu, Guofa; Beggs, Hilary; Jürgensen, Claudia; Park, Hwan-Tae; Tang, Hao; Gorski, Jessica; Jones, Kevin R; Reichardt, Louis F; Wu, Jane; Rao, Yi

    2008-01-01

    Although netrins are an important family of neuronal guidance proteins, intracellular mechanisms that mediate netrin function are not well understood. Here we show that netrin-1 induces tyrosine phosphorylation of proteins including focal adhesion kinase (FAK) and the Src family kinase Fyn. Blockers of Src family kinases inhibited FAK phosphorylation and axon outgrowth and attraction by netrin. Dominant-negative FAK and Fyn mutants inhibited the attractive turning response to netrin. Axon outgrowth and attraction induced by netrin-1 were significantly reduced in neurons lacking the FAK gene. Our results show the biochemical and functional links between netrin, a prototypical neuronal guidance cue, and FAK, a central player in intracellular signaling that is crucial for cell migration. PMID:15494732

  12. Focal Adhesion Kinase Is Involved in Rabies Virus Infection through Its Interaction with Viral Phosphoprotein P

    PubMed Central

    Fouquet, Baptiste; Nikolic, Jovan; Larrous, Florence; Bourhy, Hervé; Wirblich, Christoph

    2014-01-01

    ABSTRACT The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function. PMID:25410852

  13. Plectin-containing, centrally localized focal adhesions exert traction forces in primary lung epithelial cells

    PubMed Central

    Eisenberg, Jessica L.; Beaumont, Kristin G.; Takawira, Desire; Hopkinson, Susan B.; Mrksich, Milan; Budinger, G. R. Scott; Jones, Jonathan C. R.

    2013-01-01

    Summary Receptor clustering upon cell attachment to the substrate induces assembly of cytoplasmic protein complexes termed focal adhesions (FAs), which connect, albeit indirectly, the extracellular matrix to the cytoskeleton. A subset of cultured primary alveolar epithelial cells (AEC) display a unique pattern of vinculin/paxillin/talin-rich FAs in two concentric circles when cultured on glass and micropatterned substrates: one ring of FAs located at the cell periphery (pFAs), and another FA ring located centrally in the cell (cFAs). Unusually, cFAs associate with an aster-like actin array as well as keratin bundles. Moreover, cFAs show rapid paxillin turnover rates following fluorescence recovery after photobleaching and exert traction forces similar to those generated by FAs at the cell periphery. The plakin protein plectin localizes to cFAs and is normally absent from pFAs, whereas tensin, a marker of mature/fibrillar adhesions, is found in both cFAs and pFAs. In primary AEC in which plectin expression is depleted, cFAs are largely absent, with an attendant reorganization of both the keratin and actin cytoskeletons. We suggest that the mechanical environment in the lung gives rise to the assembly of unconventional FAs in AEC. These FAs not only show a distinctive arrangement, but also possess unique compositional and functional properties. PMID:23750011

  14. The simulation of stress fibre and focal adhesion development in cells on patterned substrates

    PubMed Central

    Pathak, Amit; Deshpande, Vikram S; McMeeking, Robert M; Evans, Anthony G

    2007-01-01

    The remodelling of the cytoskeleton and focal adhesion (FA) distributions for cells on substrates with micro-patterned ligand patches is investigated using a bio-chemo-mechanical model. We investigate the effect of ligand pattern shape on the cytoskeletal arrangements and FA distributions for cells having approximately the same area. The cytoskeleton model accounts for the dynamic rearrangement of the actin/myosin stress fibres. It entails the highly nonlinear interactions between signalling, the kinetics of tension-dependent stress-fibre formation/dissolution and stress-dependent contractility. This model is coupled with another model that governs FA formation and accounts for the mechano-sensitivity of the adhesions from thermodynamic considerations. This coupled modelling scheme is shown to capture a variety of key experimental observations including: (i) the formation of high concentrations of stress fibres and FAs at the periphery of circular and triangular, convex-shaped ligand patterns; (ii) the development of high FA concentrations along the edges of the V-, T-, Y- and U-shaped concave ligand patterns; and (iii) the formation of highly aligned stress fibres along the non-adhered edges of cells on the concave ligand patterns. When appropriately calibrated, the model also accurately predicts the radii of curvature of the non-adhered edges of cells on the concave-shaped ligand patterns. PMID:17939980

  15. Inhibition of Focal Adhesion Kinase and Src Increases Detachment and Apoptosis in Human Neuroblastoma Cell Lines

    PubMed Central

    Beierle, Elizabeth A.; Ma, Xiaojie; Trujillo, Angelica; Kurenova, Elena V.; Cance, William G.; Golubovskaya, Vita M.

    2010-01-01

    Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that is overexpressed in a number of human tumors including neuroblastoma, and regulates both cellular adhesion and survival. We have studied the effects of FAK inhibition upon neuroblastoma using adenovirus-containing FAK-CD (AdFAK-CD). Utilizing an isogenic MYCN+ / MYCN− neuroblastoma cell line, we found that the MYCN+ cells are more sensitive to FAK inhibition with AdFAK-CD than their MYCN negative counterparts. In addition, we have shown that phosphorylation of Src is increased in the untreated isogenic MYCN− neuroblastoma cells, and that the decreased sensitivity of the MYCN− neuroblastoma cells to FAK inhibition with AdFAK-CD is abrogated by the addition of the Src family kinase inhibitor, PP2. The results of the current study suggest that both FAK and Src play a role in protecting neuroblastoma cells from apoptosis, and that dual inhibition of these kinases may be important when designing therapeutic interventions for this tumor. PMID:19885861

  16. Association between tensin 1 and p130Cas at focal adhesions links actin inward flux to cell migration

    PubMed Central

    Zhao, Zhihai; Tan, Song Hui; Machiyama, Hiroaki; Kawauchi, Keiko; Araki, Keigo; Hirata, Hiroaki; Sawada, Yasuhiro

    2016-01-01

    ABSTRACT Cell migration is a highly dynamic process that plays pivotal roles in both physiological and pathological processes. We have previously reported that p130Cas supports cell migration through the binding to Src as well as phosphorylation-dependent association with actin retrograde flow at focal adhesions. However, it remains elusive how phosphorylated Cas interacts with actin cytoskeletons. We observe that the actin-binding protein, tensin 1, co-localizes with Cas, but not with its phosphorylation-defective mutant, at focal adhesions in leading regions of migrating cells. While a truncation mutant of tensin 1 that lacks the phosphotyrosine-binding PTB and SH2 domains (tensin 1-SH2PTB) poorly co-localizes or co-immunoprecitates with Cas, bacterially expressed recombinant tensin 1-SH2PTB protein binds to Cas in vitro in a Cas phosphorylation-dependent manner. Furthermore, exogenous expression of tensin 1-SH2PTB, which is devoid of the actin-interacting motifs, interferes with the Cas-driven cell migration, slows down the inward flux of Cas molecules, and impedes the displacement of Cas molecules from focal adhesions. Taken together, our results show that tensin 1 links inwardly moving actin cytoskeletons to phosphorylated Cas at focal adhesions, thereby driving cell migration. PMID:27029899

  17. Label-free proteomics uncovers energy metabolism and focal adhesion regulations responsive for endometrium receptivity.

    PubMed

    Chen, Qian; Zhang, Aijun; Yu, Feng; Gao, Jing; Liu, Yue; Yu, Chengli; Zhou, Hu; Xu, Chen

    2015-04-01

    The menstrual cycle of the female uterus leads to periodic changes of the endometrium. These changes are important for developing the endometrial receptivity and for achieving competency of embryo implantation. However, the molecular events underlying the endometrial receptivity process remain poorly understood. Here we applied an LC-MS-based label-free quantitative proteomic approach to compare the endometrial tissues in the midsecretory (receptive) phase with the endometrial tissues in the proliferative phase from age-matched woman (n = 6/group). The proteomes of endometrial tissues were extracted using an SDS-based detergent, digested by the filter-aided sample preparation procedures, and subsequently analyzed by nano-LC-MS/MS (Orbitrap XL) with a 4 h gradient. Reliable protein expression profiles were reproducibly obtained from the endometrial tissues in the receptive and proliferative phases. A total of 2138 protein groups were quantified under highly stringent criteria with a false discovery rate of <1% for peptide and protein groups. Among these proteins, 317 proteins had differences in expression that were statistically significant between the receptive and proliferative phases. Direct protein-protein interaction network analyses of these significantly changed proteins showed that the up-regulation of creatine kinase B-type (CKB) in the receptive phase may be related to endometrium receptivity. The interaction network also showed that proteins related to cell-cell adhesion were down-regulated. Moreover, the results from KEGG pathway analyses are consistent with the protein-protein interaction results. The proteins, including alpha-actinin (ACTN), extracellular matrix proteins, integrin alpha-V, and so on, that are involved in the focal adhesion pathway were down-regulated in the receptive phase compared with the proliferative phase, which may facilitate the implantation of the fertilized ovum. Selected proteins were validated by Western blot analysis and

  18. Adhesive curing through low-voltage activation

    PubMed Central

    Ping, Jianfeng; Gao, Feng; Chen, Jian Lin; Webster, Richard D.; Steele, Terry W. J.

    2015-01-01

    Instant curing adhesives typically fall within three categories, being activated by either light (photocuring), heat (thermocuring) or chemical means. These curing strategies limit applications to specific substrates and can only be activated under certain conditions. Here we present the development of an instant curing adhesive through low-voltage activation. The electrocuring adhesive is synthesized by grafting carbene precursors on polyamidoamine dendrimers and dissolving in aqueous solvents to form viscous gels. The electrocuring adhesives are activated at −2 V versus Ag/AgCl, allowing tunable crosslinking within the dendrimer matrix and on both electrode surfaces. As the applied voltage discontinued, crosslinking immediately terminated. Thus, crosslinking initiation and propagation are observed to be voltage and time dependent, enabling tuning of both material properties and adhesive strength. The electrocuring adhesive has immediate implications in manufacturing and development of implantable bioadhesives. PMID:26282730

  19. New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales.

    PubMed

    Yamashiro, Sawako; Mizuno, Hiroaki; Smith, Matthew B; Ryan, Gillian L; Kiuchi, Tai; Vavylonis, Dimitrios; Watanabe, Naoki

    2014-04-01

    Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein-actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8-8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network. PMID:24501425

  20. Epidermal Growth Factor-Induced Tumor Cell Invasion and Metastasis Initiated by Dephosphorylation and Downregulation of Focal Adhesion Kinase

    PubMed Central

    Lu, Zhimin; Jiang, Guoqiang; Blume-Jensen, Peter; Hunter, Tony

    2001-01-01

    Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis. PMID:11359909

  1. Role of c-Src and focal adhesion kinase in progression and metastasis of estrogen receptor-positive breast cancer

    SciTech Connect

    Planas-Silva, Maricarmen D. . E-mail: mcplanas@psu.edu; Bruggeman, Richard D.; Grenko, Ronald T.; Stanley Smith, J.

    2006-03-03

    The non-receptor tyrosine kinases c-Src and focal adhesion kinase (Fak) mediate signal transduction pathways that regulate cell proliferation, survival, invasion, and metastasis. Here, we investigated whether c-Src and Fak are activated during progression of hormone-dependent breast cancer. Maximally active c-Src was overexpressed in a subset of tamoxifen-resistant variants and in metastases of recurrent hormone-treated breast cancer. Active Fak was also frequently observed in these tumors. We also show that estrogen receptor (ER) can bind to Fak and that estrogen can modulate Fak autophosphorylation supporting a cross-talk between these two pathways. Inhibition of c-Src activity blocked proliferation of all tamoxifen-resistant variants, suggesting that inhibitors of c-Src-Fak activity may delay or prevent progression and metastasis of ER-positive tumors. These studies also raise the possibility that fully active forms of c-Src and Fak in breast tumors may be biomarkers to predict tamoxifen resistance and/or risk of recurrence in ER-positive breast cancer.

  2. Kinetic Mechanism and Rate-Limiting Steps of Focal Adhesion Kinase-1

    SciTech Connect

    Schneck, Jessica L.; Briand, Jacques; Chen, Stephanie; Lehr, Ruth; McDevitt, Patrick; Zhao, Baoguang; Smallwood, Angela; Concha, Nestor; Oza, Khyati; Kirkpatrick, Robert; Yan, Kang; Villa, James P.; Meek, Thomas D.; Thrall, Sara H.

    2010-12-07

    Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH{sub 2}, FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k{sub cat} (0.052 {+-} 0.001 s{sup -1}), K{sub MgATP} (1.2 {+-} 0.1 {micro}M), K{sub iMgATP} (1.3 {+-} 0.2 {micro}M), K{sub FAK-tide} (5.6 {+-} 0.4 {micro}M), and K{sub iFAK-tide} (6.1 {+-} 1.1 {micro}M) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the {beta}{gamma}-bridge:{beta}-nonbridge positional oxygen exchange of [{gamma}-{sup 18}O{sub 4}]ATP in the presence of 1 mM [{gamma}-{sup 18}O{sub 4}]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k{sub x}/k{sub cat} = 0.14 {+-} 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k{sub cat}/K{sub FAK-tide}, while k{sub cat} and k{sub cat}/K{sub MgATP} were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on k{sub cat}/K{sub m} for MgATP but not on k{sub cat}/K{sub m} for FAK-tide. From the positional isotope exchange, viscosity, and

  3. Vasoconstrictor-induced endocytic recycling regulates focal adhesion protein localization and function in vascular smooth muscle

    PubMed Central

    Poythress, Ransom H.; Gallant, Cynthia; Vetterkind, Susanne

    2013-01-01

    Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle (VSM) cells. However, it is often assumed that FAs in nonmigratory, differentiated VSM (dVSM) cells embedded in the wall of healthy blood vessels are stable structures. Recent work has demonstrated agonist-induced actin polymerization and Src-dependent FA phosphorylation in dVSM cells, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the α-agonist phenylephrine. Vasodilator-stimulated phosphoprotein and zyxin displayed the largest redistributions, while β-integrin and FA kinase showed undetectable redistribution. Vinculin, metavinculin, Src, Crk-associated substrate, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and Duolink proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and early endosomal antigen 1. Endosomal disruption with the small-molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B, and colchicine, indicating that the redistribution is actin- and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus these studies are consistent with the concept that FAs in dVSM cells, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor. PMID:23703522

  4. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes

    PubMed Central

    Bellance, Catherine; Khan, Junaid A.; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-01-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(–) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl­amino)­phenyl-17β-hydroxy-17-(1-propynyl)­estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes. PMID:23485561

  5. Two distinct actin networks mediate traction oscillations to confer mechanosensitivity of focal adhesions

    NASA Astrophysics Data System (ADS)

    Wu, Zhanghan; Plotnikov, Sergey; Waterman, Clare; Liu, Jian

    Cells sense the mechanical stiffness of their extracellular matrix (ECM) by exerting traction force through focal adhesions (FAs), which are integrin-based protein assemblies. Strikingly, FA-mediated traction forces oscillate in time and space and govern durotaxis - the tendency of most cell types to migrate toward stiffer ECM. The underlying mechanism of this intriguing oscillation of FA traction force is unknown. Combing theory and experiment, we develop a model of FA growth, which integrates coordinated contributions of a branched actin network and stress fibers in the process. We show that retrograde flux of branched actin network contributes to a traction peak near the FA distal tip and that stress fiber-mediated actomyosin Contractility generates a second traction peak near the FA center. Formin-mediated stress fiber elongation negatively feeds back with actomyosin Contractility, resulting in the central traction peak oscillation. This underpins observed spatio-temporal patterns of the FA traction, and broadens the ECM stiffness range, over which FAs could accurately adapt with traction force generation. Our findings shed light on the fundamental mechanism of FA mechanosensing and hence durotaxis.

  6. Rac1 inactivation by lethal toxin from Clostridium sordellii modifies focal adhesions upstream of actin depolymerization.

    PubMed

    Geny, Blandine; Grassart, Alexandre; Manich, Maria; Chicanne, Gaëtan; Payrastre, Bernard; Sauvonnet, Nathalie; Popoff, Michel R

    2010-02-01

    Inactivation of different small GTPases upon their glucosylation by lethal toxin from Clostridium sordellii strain IP82 (LT-82) is already known to lead to cell rounding, adherens junction (AJ) disorganization and actin depolymerization. In the present work, we observed that LT-82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Among the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing Rac1(V12). Rac1 inactivation by LT-82 modifies interactions between proteins from AJ and FA complexes as shown by pull-down assays. We showed that in Triton X-100-insoluble membrane proteins from these complexes, namely E-cadherin, beta-catenin, p120-catenin and talin, are decreased upon LT-82 intoxication, a treatment that also induces a rapid decrease in cell phosphoinositide content. Therefore, we proposed that Rac inactivation by LT-82 alters phosphoinositide metabolism leading to FA and AJ complex disorganization and actin depolymerization. PMID:19840028

  7. Paxillin and Focal Adhesion Kinase (FAK) Regulate Cardiac Contractility in the Zebrafish Heart

    PubMed Central

    Hirth, Sofia; Bühler, Anja; Bührdel, John B.; Rudeck, Steven; Dahme, Tillman; Rottbauer, Wolfgang; Just, Steffen

    2016-01-01

    An orchestrated interplay of adaptor and signaling proteins at mechano-sensitive sites is essential to maintain cardiac contractility and when defective leads to heart failure. We recently showed that Integrin-linked Kinase (ILK), ß-Parvin and PINCH form the IPP-complex to grant tuned Protein Kinase B (PKB) signaling in the heart. Loss of one of the IPP-complex components results in destabilization of the whole complex, defective PKB signaling and finally heart failure. Two components of IPP, ILK and ß-Parvin directly bind to Paxillin; however, the impact of this direct interaction on the maintenance of heart function is not known yet. Here, we show that targeted gene inactivation of Paxillin results in progressive decrease of cardiac contractility and heart failure in zebrafish without affecting IPP-complex stability and PKB phosphorylation. However, we found that Paxillin deficiency leads to the destabilization of its known binding partner Focal Adhesion Kinase (FAK) and vice versa resulting in degradation of Vinculin and thereby heart failure. Our findings highlight an essential role of Paxillin and FAK in controlling cardiac contractility via the recruitment of Vinculin to mechano-sensitive sites in cardiomyocytes. PMID:26954676

  8. Dasatinib affects focal adhesion and myosin regulation to inhibit matrix contraction by Müller cells.

    PubMed

    Tsukahara, Rintaro; Umazume, Kazuhiko; Yamakawa, Naoyuki; McDonald, Kevin; Kaplan, Henry J; Tamiya, Shigeo

    2015-10-01

    Epiretinal membrane (ERM) contraction is associated with a variety of ocular diseases that cause macular dysfunction. Trans-differentiated Müller cells have been identified in ERMs, and have been implicated to be involved in the contractile process. In this study, we tested the effect of dasatinib, an FDA-approved tyrosine kinase inhibitor, on matrix contraction caused by Müller cells, and examined molecular mechanism of action. Type I collagen matrix contraction assays were used to examine the effect of drugs on matrix contraction by trans-differentiated Müller cells. Fluophore-conjugated phalloidin was used for the detection of actin cytoskeleton, and Western-blot analyses were carried out to examine protein expression and phosphorylation status. Dasatinib inhibited collagen matrix contraction by trans-differentiated Müller cells that was associated with decreased cell spreading and reduction of actomyosin stress fibers. Concomitantly, dasatinib-treated Müller cells had reduced phosphorylation of Src family kinase, paxillin, as well as myosin II light chain. Specific inhibitors of Rho/ROCK and myosin II confirmed the critical role played by this pathway in Müller cell contraction. Our data demonstrate that dasatinib significantly reduced matrix contraction by Müller cells via inhibition of focal adhesion, as well as actomyosin contraction. PMID:26240967

  9. Targeting Focal Adhesion Kinase Suppresses the Malignant Phenotype in Rhabdomyosarcoma Cells.

    PubMed

    Waters, Alicia M; Stafman, Laura L; Garner, Evan F; Mruthyunjayappa, Smitha; Stewart, Jerry E; Mroczek-Musulman, Elizabeth; Beierle, Elizabeth A

    2016-08-01

    Despite the tremendous advances in the treatment of childhood solid tumors, rhabdomyosarcoma (RMS) continues to provide a therapeutic challenge. Children with metastatic or relapsed disease have a disease-free survival rate under 30%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumorigenesis. Signaling pathways both upstream and downstream to FAK have been found to be important in sarcoma tumorigenesis, leading us to hypothesize that FAK would be present in RMS and would impact cellular survival. In the current study, we showed that FAK was present and phosphorylated in pediatric alveolar and embryonal RMS tumor specimens and cell lines. We also examined the effects of FAK inhibition upon two RMS cell lines utilizing parallel approaches including RNAi and small molecule inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Furthermore, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse RMS xenograft model. The findings from this study will help to further our understanding of the regulation of tumorigenesis in RMS and may provide desperately needed novel therapeutic strategies for these difficult-to-treat tumors. PMID:27567948

  10. The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins.

    PubMed Central

    Reinhard, M; Giehl, K; Abel, K; Haffner, C; Jarchau, T; Hoppe, V; Jockusch, B M; Walter, U

    1995-01-01

    Profilins are small proteins that form complexes with G-actin and phosphoinositides and are therefore considered to link the microfilament system to signal transduction pathways. In addition, they bind to poly-L-proline, but the biological significance of this interaction is not yet known. The recent molecular cloning of the vasodilator-stimulated phosphoprotein (VASP), an established in vivo substrate of cAMP- and cGMP-dependent protein kinases, revealed the presence of a proline-rich domain which prompted us to investigate a possible interaction with profilins. VASP is a microfilament and focal adhesion associated protein which is also concentrated in highly dynamic regions of the cell cortex. Here, we demonstrate that VASP is a natural proline-rich profilin ligand. Human platelet VASP bound directly to purified profilins from human platelets, calf thymus and birch pollen. Moreover, VASP and a novel protein were specifically extracted from total cell lysates by profilin affinity chromatography and subsequently eluted either with poly-L-proline or a peptide corresponding to a proline-rich VASP motif. Finally, the subcellular distributions of VASP and profilin suggest that both proteins also interact within living cells. Our data support the hypothesis that profilin and VASP act in concert to convey signal transduction to actin filament formation. Images PMID:7737110

  11. High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.

    PubMed

    Kroll, Torsten; Schmidt, David; Schwanitz, Georg; Ahmad, Mubashir; Hamann, Jana; Schlosser, Corinne; Lin, Yu-Chieh; Böhm, Konrad J; Tuckermann, Jan; Ploubidou, Aspasia

    2016-01-01

    High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc. PMID:27367288

  12. Focal adhesion kinase autophosphorylation inhibition decreases colon cancer cell growth and enhances the efficacy of chemotherapy.

    PubMed

    Heffler, Melissa; Golubovskaya, Vita M; Dunn, Kelli M Bullard; Cance, William

    2013-08-01

    Focal adhesion kinase (FAK) increasingly has been implicated in cancer growth and progression. 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) is a small molecule FAK inhibitor that blocks the Y397 autophosphorylation site. FAK inhibitor, Y15 decreased Y397 FAK in different colon cancer cells lines in a dose-dependent manner. In addition, Y15 decreased phosphorylated Src in SW480 and SW620 cells. Y15 decreased cell viability, increased detachment, and increased apoptosis in SW480 and SW620 cells in vitro. Combination of FAK inhibitor Y15 and Src inhibitor PP2 decreased colon cancer cell viability more effectively than each agent alone. In addition, when combined with 5-FU, oxaliplatin or 5-FU and oxaliplatin, colon cancer viability was decreased further, demonstrating that dual and triple therapy synergistically inhibits cell viability. In vivo, Y15 decreased subcutaneous SW620 tumor growth by 28%. Combination of oral Y15 with 5-FU/or oxaliplatin decreased tumor growth by 48% more effectively than each inhibitor alone. Finally, tumors treated with Y15 expressed less Y397 phosphorylation, Src phosphorylation and had greater apoptosis than controls. Thus, the small molecule FAK inhibitor, Y15, inhibits cell growth in vitro and in vivo and enhances the efficacy of chemotherapy, demonstrating that it can be an effective therapeutic inhibitor for treating colon cancer. PMID:23792569

  13. Cbl-b promotes cell detachment via ubiquitination of focal adhesion kinase

    PubMed Central

    Fan, Yibo; Qu, Xiujuan; Ma, Yanju; Liu, Yunpeng; Hu, Xuejun

    2016-01-01

    Cancer cell detachment from the primary tumor site represents the first stage of metastasis. Previous studies have identified that cell detachment is triggered by cytoskeletal disruption, which may induce a wide variety of cellular changes. Focal adhesion kinase (FAK) exhibits crucial cellular functions, including regulation of the cytoskeleton. These observations have provided exciting insights into the effect of FAK in cell detachment; however, the involvement of FAK in cell detachment remains controversial. The aim of the present study was to evaluate the effect of FAK and its function in the process of cell detachment. The results revealed that FAK expression was downregulated following trypsin treatment in human gastric, lung, colon and breast cancer cell lines, as well as a human gastric epithelial cell line. Knockdown of FAK enhanced cell detachment in gastric cancer MGC803 cells, indicating that FAK inhibits cell detachment. Further investigation revealed that trypsin induced monoubiquitination of FAK. In addition, the lysosome inhibitor, NH4Cl, decreased trypsin-induced degradation of FAK. Casitas B-lineage lymphoma-b (Cbl-b), an E3 ubiquitin ligase, was involved in this process, which interacted with FAK, as demonstrated by co-precipitation experiments, and promoted trypsin-induced ubiquitin-lysosome degradation of FAK. These results indicate that Cbl-b promotes cell detachment via ubiquitination of FAK. These findings provide novel insights regarding the effect of FAK and Cbl-b in the process of cancer cell detachment.

  14. Expression, purification and crystallization of a BH domain from the GTPase regulatory protein associated with focal adhesion kinase.

    PubMed

    Sheffield, P J; Derewenda, U; Taylor, J; Parsons, T J; Derewenda, Z S

    1999-01-01

    Signaling by small GTPases is down-regulated by GTPase activating proteins (GAPs) which enhance the rate of GTP hydrolysis. The activity of GAPs specific for Rho GTPases resides in the BH domain, many homologues of which are found in any mammalian genome. One of them was identified in the GTPase regulator associated with focal-adhesion kinase (GRAF). It shares approximately 20% sequence identity with p50RhoGAP. This GAP activates RhoA and Cdc42Hs, but not Rac. In order to dissect the molecular basis of this specificity, a 231-residue-long fragment corresponding to the BH domain of GRAF has been expressed, purified and crystallized. Trigonal crystals, of space group P3(1)21 or P3(2)21, with unit-cell dimensions a = b = 63.5, c = 90.38 A were grown from solutions of PEG 6000. Data to 2.15 A were collected from a flash-frozen sample on an R-AXIS IV imaging-plate detector mounted on a rotating anode X-ray generator. PMID:10232922

  15. Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase)

    PubMed Central

    Neerathilingam, Muniasamy; Bairy, Sneha G.; Mysore, Sumukh

    2016-01-01

    Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1–313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies. PMID:26928467

  16. PRL-3 engages the focal adhesion pathway in triple-negative breast cancer cells to alter actin structure and substrate adhesion properties critical for cell migration and invasion.

    PubMed

    Gari, Hamid H; DeGala, Gregory D; Ray, Rahul; Lucia, M Scott; Lambert, James R

    2016-10-01

    Triple-negative breast cancers (TNBCs) are among the most aggressive cancers characterized by a high propensity to invade, metastasize and relapse. We previously reported that the TNBC-specific inhibitor, AMPI-109, significantly impairs the ability of TNBC cells to migrate and invade by reducing levels of the metastasis-promoting phosphatase, PRL-3. Here, we examined the mechanisms by which AMPI-109 and loss of PRL-3 impede cell migration and invasion. AMPI-109 treatment or knock down of PRL-3 expression were associated with deactivation of Src and ERK signaling and concomitant downregulation of RhoA and Rac1/2/3 GTPase protein levels. These cellular changes led to rearranged filamentous actin networks necessary for cell migration and invasion. Conversely, overexpression of PRL-3 promoted TNBC cell invasion by upregulating matrix metalloproteinase 10, which resulted in increased TNBC cell adherence to, and degradation of, the major basement membrane component laminin. Our data demonstrate that PRL-3 engages the focal adhesion pathway in TNBC cells as a key mechanism for promoting TNBC cell migration and invasion. Collectively, these data suggest that blocking PRL-3 activity may be an effective method for reducing the metastatic potential of TNBC cells. PMID:27452906

  17. Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP.

    PubMed Central

    Haffner, C; Jarchau, T; Reinhard, M; Hoppe, J; Lohmann, S M; Walter, U

    1995-01-01

    The vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL-60 and canine MDCK cells, is organized into three distinct domains. A central proline-rich domain contains a GPPPPP motif as a single copy and as a 3-fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide-dependent protein kinases. A C-terminal domain contains a repetitive mixed-charge cluster which is predicted to form an alpha-helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over-expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F-actin-containing cell surface protrusions, whereas truncated VASP lacking the C-terminal domain was no longer concentrated at focal adhesions. These data indicate that the C-terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin-based cytoskeleton, including the cytoskeleton-membrane interface, may be able to exchange signals between these networks. Images PMID:7828592

  18. Simulated Microgravity Alters Actin Cytoskeleton and Integrin-Mediated Focal Adhesions of Cultured Human Mesenchymal Stromal Cells

    NASA Astrophysics Data System (ADS)

    Gershovich, P. M.; Gershovic, J. G.; Buravkova, L. B.

    2008-06-01

    Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG) (3, 4). One potential mechanism for cytoskeletal gravisensitivity is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, α-actinin, and actin filaments (4, 5). Integrins produce signals essential for proper cellular function, survival and differentiation. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinostat (Dutch Space, Netherlands). Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. The increasing of cells number with abnormal F-actin was observed after subsequent terms of 3D-clinorotation (6, 24, 48, 120 hours). Randomization of gravity vector altered dimensional structure of stress fibers and resulted in remodeling of actin fibers inside the cells. In addition, we observed vinculin redistribution inside the cells after 6 hours and prolonged terms of clinorotation. Tubulin fibers in a contrast with F-actin and vinculin didn't show any reorganization even after long 3Dclinorotation (120 hours). The expression of integrin α2 increased 1,5-6-fold in clinorotated hMSCs. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with involvement of some integrin subtypes.

  19. Ankyrin repeat domain 28 (ANKRD28), a novel binding partner of DOCK180, promotes cell migration by regulating focal adhesion formation.

    PubMed

    Tachibana, Mitsuhiro; Kiyokawa, Etsuko; Hara, Shigeo; Iemura, Shun-Ichiro; Natsume, Tohru; Manabe, Toshiaki; Matsuda, Michiyuki

    2009-03-10

    DOCK180 is a guanine exchange factor of Rac1 originally identified as a protein bound to an SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130(Cas), and recruits the Crk-p130(Cas) complex to focal adhesions. To understand the role of DOCK180 in cell adhesion and migration, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and identified ANKRD28, a protein that contains twenty-six ankyrin domain repeats. Knockdown of ANKRD28 by RNA interference reduced the velocity of migration of HeLa cells, suggesting that this protein plays a physiologic role in the DOCK180-Rac1 signaling pathway. Furthermore, knockdown of ANKRD28 was found to alter the distribution of focal adhesion proteins such as Crk, paxillin, and p130(Cas). On the other hand, expression of ANKRD28, p130(Cas), Crk, and DOCK180 induced hyper-phosphorylation of p130(Cas), and impaired detachment of the cell membrane during migration. Consequently, cells expressing ANKRD28 exhibited multiple long cellular processes. ANKRD28 associated with DOCK180 in an SH3-dependent manner and competed with ELMO, another protein bound to the SH3 domain of DOCK180. In striking contrast to ANKRD28, overexpression of ELMO induced extensive lamellipodial protrusion around the entire circumference. These data suggest that ANKRD28 specifies the localization and the activity of the DOCK180-Rac1 pathway. PMID:19118547

  20. Molecular mechanisms underlying the force-dependent regulation of actin-to-ECM linkage at the focal adhesions.

    PubMed

    Hirata, Hiroaki; Sokabe, Masahiro; Lim, Chwee Teck

    2014-01-01

    The linkage of the actin cytoskeleton to extracellular matrices (ECMs) at focal adhesions provides a physical path for cells to exert traction forces on substrates during cellular processes such as migration and morphogenesis. Mechanical strength of the actin-to-ECM linkage increases in response to forces loaded at this linkage. This is achieved by local accumulations of actin filaments, as well as linker proteins connecting actins to integrins, at force-bearing adhesion sites, which leads to an increase in the number of molecular bonds between the actin cytoskeleton- and ECM-bound integrins. Zyxin-dependent actin polymerization and filamin-mediated actin bundling are seemingly involved in the force-dependent actin accumulation. Each actin-integrin link is primarily mediated by the linker protein talin, which is strengthened by another linker protein vinculin connecting the actin filaments to talin in a force-dependent manner. This eliminates slippage between the actin cytoskeleton and talin (clutch mechanism), thus playing a crucial role in creating cell membrane protrusions mediated by actin polymerization. Finally, each integrin-ECM bond is also strengthened when a force is loaded on it, which ensures force transmission at focal adhesions, contributing to stable cell-substrate adhesion in cell migration. PMID:25081617

  1. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

    PubMed Central

    Schaller, M D; Hildebrand, J D; Shannon, J D; Fox, J W; Vines, R R; Parsons, J T

    1994-01-01

    The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins. Images PMID:7509446

  2. Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    SciTech Connect

    Xiong, Xiangyang; Wang, Yao; Liu, Chengmei; Lu, Quqin; Liu, Tao; Chen, Guoan; Rao, Hai; Luo, Shiwen

    2014-08-01

    Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton.

  3. The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions

    PubMed Central

    Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M.; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel

    2015-01-01

    In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature. PMID:26169353

  4. The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

    PubMed

    Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel; Søgaard-Andersen, Lotte; Mignot, Tâm

    2015-07-20

    In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature. PMID:26169353

  5. Mutation of Drosophila Focal Adhesion Kinase Induces Bang-Sensitive Behavior and Disrupts Glial Function, Axonal Conduction and Synaptic Transmission

    PubMed Central

    Ueda, Atsushi; Grabbe, Caroline; Lee, Jihye; Lee, Jisue; Palmer, Ruth H.; Wu, Chun-Fang

    2009-01-01

    The role of the conserved Focal Adhesion Kinase (FAK) family of protein tyrosine kinases (PTKs) in the development and physiological functions of the CNS has long been an area of interest among neuroscientists. In this report, we observe that Drosophila mutants lacking Fak56 exhibit a decreased life span, accompanied by a bang-sensitive phenotype, which is characterised by sensitivity to mechanical and high-frequency electrical stimulation. Fak56 mutant animals display lower thresholds and higher rates of seizures in response to electroconvulsive stimuli, and direct measurements of action potential conduction in larval segmental nerves demonstrate a slowed propagation speed and failure during high-frequency nerve stimulation. In addition, neuromuscular junctions in Fak56 mutant animals display transmission blockade during high-frequency activity as a result of action potential failure. Endogenous Fak56 protein is abundant in glial cells ensheathing the axon bundles, and structural alterations of segmental nerve bundles can be observed in mutants. Manipulation of Fak56 function specifically in glial cells also disrupts action potential conduction and neurotransmission, suggesting a glial component in the Fak56 bang-sensitive phenotype. Furthermore, we show that increased intracellular calcium levels result in the dephosphorylation of endogenous Fak56 protein in Drosophila cell lines, in parallel with our observations of highly variable synaptic potentials at a higher Ca2+ level in Fak56 mutant larvae. Together these findings suggest that modulation of Fak56 function is important for action potential propagation and Ca2+-regulated neuromuscular transmission in vivo. PMID:18540882

  6. A small molecule focal adhesion kinase (FAK) inhibitor, targeting Y397 site: 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide effectively inhibits FAK autophosphorylation activity and decreases cancer cell viability, clonogenicity and tumor growth in vivo.

    PubMed

    Golubovskaya, Vita M; Figel, Sheila; Ho, Baotran T; Johnson, Christopher P; Yemma, Michael; Huang, Grace; Zheng, Min; Nyberg, Carl; Magis, Andrew; Ostrov, David A; Gelman, Irwin H; Cance, William G

    2012-05-01

    Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in most solid types of tumors and plays an important role in the survival signaling. Recently, we have developed a novel computer modeling combined with a functional assay approach to target the main autophosphorylation site of FAK (Y397). Using these approaches, we identified 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide, called Y11, a small molecule inhibitor targeting Y397 site of FAK. Y11 significantly and specifically decreased FAK autophosphorylation, directly bound to the N-terminal domain of FAK. In addition, Y11 decreased Y397-FAK autophosphorylation, inhibited viability and clonogenicity of colon SW620 and breast BT474 cancer cells and increased detachment and apoptosis in vitro. Moreover, Y11 significantly decreased tumor growth in the colon cancer cell mouse xenograft model. Finally, tumors from the Y11-treated mice demonstrated decreased Y397-FAK autophosphorylation and activation of poly (ADP ribose) polymerase and caspase-3. Thus, targeting the major autophosphorylation site of FAK with Y11 inhibitor is critical for development of cancer therapeutics and carcinogenesis field. PMID:22402131

  7. Plekhh2, a novel podocyte protein downregulated in human focal segmental glomerulosclerosis, is involved in matrix adhesion and actin dynamics.

    PubMed

    Perisic, Ljubica; Lal, Mark; Hulkko, Jenny; Hultenby, Kjell; Önfelt, Björn; Sun, Ying; Dunér, Fredrik; Patrakka, Jaakko; Betsholtz, Christer; Uhlen, Mathias; Brismar, Hjalmar; Tryggvason, Karl; Wernerson, Annika; Pikkarainen, Timo

    2012-11-01

    Pleckstrin homology domain-containing, family H (with MyTH4 domain), member 2 (Plekhh2) is a 1491-residue intracellular protein highly enriched in renal glomerular podocytes for which no function has been ascribed. Analysis of renal biopsies from patients with focal segmental glomerulosclerosis revealed a significant reduction in total podocyte Plekhh2 expression compared to controls. Sequence analysis indicated a putative α-helical coiled-coil segment as the only recognizable domain within the N-terminal half of the polypeptide, while the C-terminal half contains two PH, a MyTH4, and a FERM domain. We identified a phosphatidylinositol-3-phosphate consensus-binding site in the PH1 domain required for Plekhh2 localization to peripheral regions of cell lamellipodia. The N-terminal half of Plekkh2 is not necessary for lamellipodial targeting but mediates self-association. Yeast two-hybrid screening showed that Plekhh2 directly interacts through its FERM domain with the focal adhesion protein Hic-5 and actin. Plekhh2 and Hic-5 coprecipitated and colocalized at the soles of podocyte foot processes in situ and Hic-5 partially relocated from focal adhesions to lamellipodia in Plekhh2-expressing podocytes. In addition, Plekhh2 stabilizes the cortical actin cytoskeleton by attenuating actin depolymerization. Our findings suggest a structural and functional role for Plekhh2 in the podocyte foot processes. PMID:22832517

  8. Signaling during platelet adhesion and activation

    PubMed Central

    Li, Zhenyu; Delaney, M. Keegan; O’Brien, Kelly A.; Du, Xiaoping

    2011-01-01

    Upon vascular injury, platelets are activated by adhesion to adhesive proteins like von Willebrand factor and collagen, or by soluble platelet agonists like ADP, thrombin, and thromboxane A2. These adhesive proteins and soluble agonists induce signal transduction via their respective receptors. The various receptor-specific platelet activation signaling pathways converge into common signaling events, which stimulate platelet shape change, granule secretion, and ultimately induce the “inside-out” signaling process leading to activation of the ligand binding function of integrin αIIbβ3. Ligand binding to integrin αIIbβ3 mediates platelet adhesion and aggregation and triggers “outside-in” signaling, resulting in platelet spreading, additional granule secretion, stabilization of platelet adhesion and aggregation, and clot retraction. It has become increasingly evident that agonist-induced platelet activation signals also crosstalk with integrin “outside-in” signals to regulate platelet responses. Platelet activation involves a series of rapid positive feedback loops that greatly amplify initial activation signals, and enable robust platelet recruitment and thrombus stabilization. Recent studies have provided novel insight into the molecular mechanisms of these processes. PMID:21071698

  9. Organization and post-transcriptional processing of focal adhesion kinase gene

    PubMed Central

    Corsi, Jean-Marc; Rouer, Evelyne; Girault, Jean-Antoine; Enslen, Hervé

    2006-01-01

    Background Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase critical for processes ranging from embryo development to cancer progression. Although isoforms with specific molecular and functional properties have been characterized in rodents and chicken, the organization of FAK gene throughout phylogeny and its potential to generate multiple isoforms are not well understood. Here, we study the phylogeny of FAK, the organization of its gene, and its post-transcriptional processing in rodents and human. Results A single orthologue of FAK and the related PYK2 was found in non-vertebrate species. Gene duplication probably occurred in deuterostomes after the echinoderma embranchment, leading to the evolution of PYK2 with distinct properties. The amino acid sequence of FAK and PYK2 is conserved in their functional domains but not in their linker regions, with the absence of autophosphorylation site in C. elegans. Comparison of mouse and human FAK genes revealed the existence of multiple combinations of conserved and non-conserved 5'-untranslated exons in FAK transcripts suggesting a complex regulation of their expression. Four alternatively spliced coding exons (13, 14, 16, and 31), previously described in rodents, are highly conserved in vertebrates. Cis-regulatory elements known to regulate alternative splicing were found in conserved alternative exons of FAK or in the flanking introns. In contrast, other reported human variant exons were restricted to Homo sapiens, and, in some cases, other primates. Several of these non-conserved exons may correspond to transposable elements. The inclusion of conserved alternative exons was examined by RT-PCR in mouse and human brain during development. Inclusion of exons 14 and 16 peaked at the end of embryonic life, whereas inclusion of exon 13 increased steadily until adulthood. Study of various tissues showed that inclusion of these exons also occurred, independently from each other, in a tissue-specific fashion

  10. Akt1 binds focal adhesion kinase via the Akt1 kinase domain independently of the pleckstrin homology domain.

    PubMed

    Basson, M D; Zeng, B; Wang, S

    2015-10-01

    Akt1 and focal adhesion kinase (FAK) are protein kinases that play key roles in normal cell signaling. Individually, aberrant expression of these kinases has been linked to a variety of cancers. Together, Akt1/FAK interactions facilitate cancer metastasis by increasing cell adhesion under conditions of increased extracellular pressure. Pathological and iatrogenic sources of pressure arise from tumor growth against constraining stroma or direct perioperative manipulation. We previously reported that 15 mmHg increased extracellular pressure causes Akt1 to both directly interact with FAK and to phosphorylate and activate it. We investigated the nature of the Akt1/FAK binding by creating truncations of recombinant FAK, conjugated to glutathione S-transferase (GST), to pull down full-length Akt1. Western blots probing for Akt1 showed that FAK/Akt1 binding persisted in FAK truncations consisting of only amino acids 1-126, FAK(NT1), which contains the F1 subdomain of its band 4.1, ezrin, radixin, and moesin (FERM) domain. Using FAK(NT1) as bait, we then pulled down truncated versions of recombinant Akt1 conjugated to HA (human influenza hemagglutinin). Probes for GST-FAK(NT1) showed Akt1-FAK binding to occur in the absence of the both the Akt1 (N)-terminal pleckstrin homology (PH) domain and its adjacent hinge region. The Akt1 (C)-terminal regulatory domain was equally unnecessary for Akt1/FAK co-immunoprecipitation. Truncations involving the Akt1 catalytic domain showed that the domain by itself was enough to pull down FAK. Additionally, a fragment spanning from the PH domain to half way through the catalytic domain demonstrated increased FAK binding compared to full length Akt1. These results begin to delineate the Akt1/FAK interaction and can be used to manipulate their force-activated signal interactions. Furthermore, the finding that the N-terminal half of the Akt1 catalytic domain binds so strongly to FAK when cleaved from the rest of the protein may suggest a means

  11. Pelargonidin attenuates PDGF-BB-induced aortic smooth muscle cell proliferation and migration by direct inhibition of focal adhesion kinase.

    PubMed

    Son, Joe Eun; Jeong, Hyein; Kim, Heejoo; Kim, Yeong A; Lee, Eunjung; Lee, Hyong Joo; Lee, Ki Won

    2014-05-15

    Pelargonidin is a natural red pigment found in fruits and vegetables, and has been reported to exhibit various effects potentially beneficial for human health. However, the possible preventive effects of pelargonidin toward atherosclerosis and mechanisms involved have not been investigated to date. Here, we compared the effects of pelargonidin and its glucoside-conjugated form, pelargonidin-3-glucoside (P3G), on proliferation and migration induced by platelet-derived growth factor (PDGF)-BB in human aortic smooth muscle cells (HASMCs). Pelargonidin, but not P3G, exhibited strong inhibitory effects against PDGF-BB-induced HASMC proliferation and migration, while suppressing PDGF-BB-induced ex vivo rat aortic ring sprouting. Immunoblot analysis revealed that pelargonidin inhibited PDGF-BB-induced phosphorylation of focal adhesion kinase (FAK) as well as F-actin reduction, whereas Src, mitogen-activated protein kinases (MAPKs) and Akt phosphorylation status were not altered. We also observed that the anti-proliferative and migratory effects of both pelargonidin and P3G corresponded with the extent of FAK inhibition. Both in vitro and ex vivo pull-down assays revealed that pelargonidin binds directly with FAK in an adenosine triphosphate-competitive manner, suggesting that FAK could be a molecular target of pelargonidin. Interestingly, pelargonidin did not exhibit inhibitory effects on the proliferation, migration or FAK phosphorylation of human umbilical vein endothelial cells (HUVECs). Taken together, our results suggest that pelargonidin exhibits potential preventive effects toward atherosclerosis through the attenuation of HASMC proliferation and migration, as well as aortic sprouting via the direct inhibition of FAK activity. PMID:24582770

  12. Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis.

    PubMed

    Zhang, J; Gao, Q; Zhou, Y; Dier, U; Hempel, N; Hochwald, S N

    2016-04-14

    Cancer cells often gains a growth advantage by taking up glucose at a high rate and undergoing aerobic glycolysis through intrinsic cellular factors that reprogram glucose metabolism. Focal adhesion kinase (FAK), a key transmitter of growth factor and anchorage stimulation, is aberrantly overexpressed or activated in most solid tumors, including pancreatic ductal adenocarcinomas (PDACs). We determined whether FAK can act as an intrinsic driver to promote aerobic glycolysis and tumorigenesis. FAK inhibition decreases and overexpression increases intracellular glucose levels during unfavorable conditions, including growth factor deficiency and cell detachment. Amplex glucose assay, fluorescence and carbon-13 tracing studies demonstrate that FAK promotes glucose consumption and glucose-to-lactate conversion. Extracellular flux analysis indicates that FAK enhances glycolysis and decreases mitochondrial respiration. FAK increases key glycolytic proteins, including enolase, pyruvate kinase M2 (PKM2), lactate dehydrogenase and monocarboxylate transporter. Furthermore, active/tyrosine-phosphorylated FAK directly binds to PKM2 and promotes PKM2-mediated glycolysis. On the other hand, FAK-decreased levels of mitochondrial complex I can result in reduced oxidative phosphorylation (OXPHOS). Attenuation of FAK-enhanced glycolysis re-sensitizes cancer cells to growth factor withdrawal, decreases cell viability and reduces growth of tumor xenografts. These observations, for the first time, establish a vital role of FAK in cancer glucose metabolism through alterations in the OXPHOS-to-glycolysis balance. Broadly targeting the common phenotype of aerobic glycolysis and more specifically FAK-reprogrammed glucose metabolism will disrupt the bioenergetic and biosynthetic supply for uncontrolled growth of tumors, particularly glycolytic PDAC. PMID:26119934

  13. Focal adhesion kinase: predictor of tumour response and risk factor for recurrence after neoadjuvant chemoradiation in rectal cancer.

    PubMed

    Gómez Del Pulgar, Teresa; Cebrián, Arancha; Fernández-Aceñero, Maria Jesús; Borrero-Palacios, Aurea; Del Puerto-Nevado, Laura; Martínez-Useros, Javier; Marín-Arango, Juan Pablo; Caramés, Cristina; Vega-Bravo, Ricardo; Rodríguez-Remírez, María; Cruz-Ramos, Marlid; Manzarbeitia, Félix; García-Foncillas, Jesús

    2016-09-01

    Rectal cancer represents about 30% of colorectal cancers, being around 50% locally advanced at presentation. Chemoradiation (CRT) followed by total mesorectal excision is the standard of care for these locally advanced stages. However, it is not free of adverse effects and toxicity and the complete pathologic response rate is between 10% and 30%. This makes it extremely important to define factors that can predict response to this therapy. Focal adhesion kinase (FAK) expression has been correlated with worse prognosis in several tumours and its possible involvement in cancer radio- and chemosensitivity has been suggested; however, its role in rectal cancer has not been analysed yet. To analyse the association of FAK expression with tumour response to CRT in locally advanced rectal cancer. This study includes 73 patients with locally advanced rectal cancer receiving standard neoadjuvant CRT followed by total mesorectal excision. Focal adhesion kinase protein levels were immunohistochemically analysed in the pre-treatment biopsies of these patients and correlated with tumour response to CRT and patients survival. Low FAK expression was significantly correlated with local and distant recurrence (P = 0.013). Low FAK expression was found to be a predictive marker of tumour response to neoadjuvant therapy (P = 0.007) and patients whose tumours did not express FAK showed a strong association with lower disease-free survival (P = 0.01). Focal adhesion kinase expression predicts neoadjuvant CRT response in rectal cancer patients and it is a clinically relevant risk factor for local and distant recurrence. PMID:27171907

  14. Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays

    PubMed Central

    Torres, Alexis J.; Vasudevan, Lavanya; Holowka, David; Baird, Barbara A.

    2008-01-01

    Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-FcεRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-FcεRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-FcεRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-FcεRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (α5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-FcεRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-FcεRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the FcεRI β subunit but enhances stimulated Ca2+ release from intracellular stores. The results suggest that paxillin associated with clustered IgE-FcεRI has a net positive effect on FcεRI signaling. PMID:19004813

  15. Micro-adhesion rings surrounding TCR microclusters are essential for T cell activation.

    PubMed

    Hashimoto-Tane, Akiko; Sakuma, Machie; Ike, Hiroshi; Yokosuka, Tadashi; Kimura, Yayoi; Ohara, Osamu; Saito, Takashi

    2016-07-25

    The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro-adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro-adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro-adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro-adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals. PMID:27354546

  16. A vascular injury model using focal heat-induced activation of endothelial cells

    PubMed Central

    Sylman, J.L.; Artzer, D.T.; Rana, K.; Neeves, K.B.

    2015-01-01

    Endothelial cells (EC) both inhibit and promote platelet function depending on their activation state. Quiescent EC inhibit platelet activation by constitutive secretion of platelet inhibitors. Activated EC promote platelet adhesion by secretion of von Willebrand factor (vWF). EC also secrete an extracellular matrix that support platelet adhesion when exposed following vascular injury. Previous studies of EC-platelet interactions under flow activate entire monolayers of cells by chemical activation. In this study, EC cultured in microfluidic channels were focally activated by heat from an underlying microelectrode. Based on finite element modeling, microelectrodes induced peak temperature increases of 10–40 °C above 37 °C after applying 5–9 V for 30 s resulting in three zones: (1) A quiescent zone corresponded to peak temperatures of less than 15 °C characterized by no EC activation or platelet accumulation. (2) An activation zone corresponding to an increase of 16–22 °C yielded EC that were viable, secreted elevated levels of vWF, and were P-selectin positive. Platelets accumulated in the retracted spaces between EC in the activation zone at a wall shear rate of 150 s−1. Experiments with blocking antibodies show that platelets adhere via GPIbα-vWF and α6β1-laminin interactions. (3) A kill zone corresponded to peak temperatures of greater than 23 °C where EC were not viable and did not support platelet adhesion. These data define heating conditions for the activation of EC, causing the secretion of vWF and the exposure of a subendothelial matrix that support platelet adhesion and aggregation. This model provides for spatially defined zones of EC activation that could be a useful tool for measuring the relative roles of anti- and prothrombotic roles of EC at the site of vascular injury. PMID:26087748

  17. Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation

    PubMed Central

    Liu, Ching-Yi; Lin, Hsi-Hui; Tang, Ming-Jer; Wang, Yang-Kao

    2015-01-01

    Modulations of cytoskeletal organization and focal adhesion turnover correlate to tumorigenesis and epithelial-mesenchymal transition (EMT), the latter process accompanied by the loss of epithelial markers and the gain of mesenchymal markers (e.g., vimentin). Clinical microarray results demonstrated that increased levels of vimentin mRNA after chemotherapy correlated to a poor prognosis of breast cancer patients. We hypothesized that vimentin mediated the reorganization of cytoskeletons to maintain the mechanical integrity in EMT cancer cells. By using knockdown strategy, the results showed reduced cell proliferation, impaired wound healing, loss of directional migration, and increased large membrane extension in MDA-MB 231 cells. Vimentin depletion also induced reorganization of cytoskeletons and reduced focal adhesions, which resulted in impaired mechanical strength because of reduced cell stiffness and contractile force. In addition, overexpressing vimentin in MCF7 cells increased cell stiffness, elevated cell motility and directional migration, reoriented microtubule polarity, and increased EMT phenotypes due to the increased β1-integrin and the loss of junction protein E-cadherin. The EMT-related transcription factor slug was also mediated by vimentin. The current study demonstrated that vimentin serves as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the balance of cell force generation in EMT cancer cells. PMID:25965826

  18. Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors.

    PubMed

    Riehl, Brandon D; Lee, Jeong Soon; Ha, Ligyeom; Lim, Jung Yul

    2015-03-01

    The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear. PMID:25589570

  19. Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors

    PubMed Central

    Riehl, Brandon D.; Lee, Jeong Soon; Ha, Ligyeom; Lim, Jung Yul

    2015-01-01

    The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear. PMID:25589570

  20. Role of stress fibers and focal adhesions as a mediator for mechano-signal transduction in endothelial cells in situ

    PubMed Central

    Katoh, Kazuo; Kano, Yumiko; Ookawara, Shigeo

    2008-01-01

    Fluid shear stress is the mechanical force generated by the blood flow which is applied over the apical surface of endothelial cells in situ. The findings of a recent study suggest that stress fibers and its associated focal adhesions play roles in mechano-signal transduction mechanism. Stress fibers are present along the apical and the basal portion of the endothelial cells. Endothelial cells respond to fluid shear stress and change their morphological characteristics in both their cell shape and cytoskeletal organization. Atherosclerosis is a common disease of the arteries and it occurs in areas around the branching site of blood vessels where the cells are exposed to low fluid shear stress. The organization of stress fibers and focal adhesions are strongly influenced by shear stress, and therefore the generation of atherosclerotic lesions seem to be associated with the cytoskeletal components of endothelial cells. This review describes the possible role of the cytoskeleton as a mechano-transducer in endothelial cells in situ. PMID:19337541

  1. Autophagy Promotes Focal Adhesion Disassembly and Cell Motility of Metastatic Tumor Cells through the Direct Interaction of Paxillin with LC3.

    PubMed

    Sharifi, Marina N; Mowers, Erin E; Drake, Lauren E; Collier, Chris; Chen, Hong; Zamora, Marta; Mui, Stephanie; Macleod, Kay F

    2016-05-24

    Autophagy is a conserved catabolic process that plays a housekeeping role in eliminating protein aggregates and organelles and is activated during nutrient deprivation to generate metabolites and energy. Autophagy plays a significant role in tumorigenesis, although opposing context-dependent functions of autophagy in cancer have complicated efforts to target autophagy for therapeutic purposes. We demonstrate that autophagy inhibition reduces tumor cell migration and invasion in vitro and attenuates metastasis in vivo. Numerous abnormally large focal adhesions (FAs) accumulate in autophagy-deficient tumor cells, reflecting a role for autophagy in FA disassembly through targeted degradation of paxillin. We demonstrate that paxillin interacts with processed LC3 through a conserved LIR motif in the amino-terminal end of paxillin and that this interaction is regulated by oncogenic SRC activity. Together, these data establish a function for autophagy in FA turnover, tumor cell motility, and metastasis. PMID:27184837

  2. Phg2, a Kinase Involved in Adhesion and Focal Site Modeling in Dictyostelium

    PubMed Central

    Gebbie, Leigh; Benghezal, Mohammed; Cornillon, Sophie; Froquet, Romain; Cherix, Nathalie; Malbouyres, Marilyne; Lefkir, Yaya; Grangeasse, Christophe; Fache, Sébastien; Dalous, Jérémie; Brückert, Franz; Letourneur, François; Cosson, Pierre

    2004-01-01

    The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility. PMID:15194808

  3. Therapeutic effects of tyroservatide on metastasis of lung cancer and its mechanism affecting integrin–focal adhesion kinase signal transduction

    PubMed Central

    Huang, Yu-ting; Zhao, Lan; Fu, Zheng; Zhao, Meng; Song, Xiao-meng; Jia, Jing; Wang, Song; Li, Jin-ping; Zhu, Zhi-feng; Lin, Gang; Lu, Rong; Yao, Zhi

    2016-01-01

    Tyroservatide (YSV) can inhibit the growth and metastasis of mouse lung cancer significantly. This study investigated the therapeutic effects of tripeptide YSV on metastasis of human lung cancer cells and explored its possible mechanism that affects integrin–focal adhesion kinase (FAK) signal transduction in tumor cells. YSV significantly inhibited the adhesion and the invasion of highly metastatic human lung cancer cell lines 95D, A549, and NCI-H1299. In addition, YSV significantly inhibited phosphorylation of FAK Tyr397 and FAK Tyr576/577 in the 95D, A549, and NCI-H1299 human lung cancer cells in vitro. And the mRNA level and protein expression of FAK in these human lung cancer cells decreased at the same time. YSV also significantly inhibited mRNA and protein levels of integrin β1 and integrin β3 in the 95D, A549, and NCI-H1299 human lung cancer cells. Our research showed that YSV inhibited adhesion and invasion of human lung cancer cells and exhibited therapeutic effects on metastasis of lung cancer. PMID:27041993

  4. PI3K{gamma} activation by CXCL12 regulates tumor cell adhesion and invasion

    SciTech Connect

    Monterrubio, Maria; Mellado, Mario; Carrera, Ana C.

    2009-10-16

    Tumor dissemination is a complex process, in which certain steps resemble those in leukocyte homing. Specific chemokine/chemokine receptor pairs have important roles in both processes. CXCL12/CXCR4 is the most commonly expressed chemokine/chemokine receptor pair in human cancers, in which it regulates cell adhesion, extravasation, metastatic colonization, angiogenesis, and proliferation. All of these processes require activation of signaling pathways that include G proteins, phosphatidylinositol-3 kinase (PI3K), JAK kinases, Rho GTPases, and focal adhesion-associated proteins. We analyzed these pathways in a human melanoma cell line in response to CXCL12 stimulation, and found that PI3K{gamma} regulates tumor cell adhesion through mechanisms different from those involved in cell invasion. Our data indicate that, following CXCR4 activation after CXCL12 binding, the invasion and adhesion processes are regulated differently by distinct downstream events in these signaling cascades.

  5. An occludin-focal adhesion kinase protein complex at the blood-testis barrier: a study using the cadmium model.

    PubMed

    Siu, Erica R; Wong, Elissa W P; Mruk, Dolores D; Sze, K L; Porto, Catarina S; Cheng, C Yan

    2009-07-01

    Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII-IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl(2) to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics. PMID:19213829

  6. An Occludin-Focal Adhesion Kinase Protein Complex at the Blood-Testis Barrier: A Study Using the Cadmium Model

    PubMed Central

    Siu, Erica R.; Wong, Elissa W. P.; Mruk, Dolores D.; Sze, K. L.; Porto, Catarina S.; Cheng, C. Yan

    2009-01-01

    Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII–IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl2 to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics. PMID:19213829

  7. Pulsed ultrasound promotes melanoblast migration through upregulation of macrophage colony-stimulating factor/focal adhesion kinase autocrine signaling and paracrine mechanisms.

    PubMed

    Liao, Yi-Hua; Huang, Yu-Ting; Deng, Jhu-Yun; Chen, Wen-Shiang; Jee, Shiou-Hwa

    2013-09-01

    Repigmentation of vitiliginous lesions relies on the proliferation and migration of melanoblasts from hair follicles to the epidermis. Pulsed ultrasound has been demonstrated to have stimulatory effects on cell proliferation and migration and has been applied clinically to enhance tissue repair. To clarify the biologic effects and signaling mechanisms of pulsed ultrasound on melanoblast proliferation and migration, two melanoblast cell lines, the undifferentiated NCCmelb4 cells and the differentiated NCCmelan5 cells, were examined. We demonstrated that pulsed ultrasound increased cell migration in a dose-dependent manner without altering cell proliferation. Pulsed ultrasound enhanced autocrine secretion of macrophage colony-stimulating factor (M-CSF), which subsequently activated the focal adhesion kinase (FAK) pathway to promote melanoblast migration. Furthermore, conditioned medium from mouse embryonic fibroblasts NIH 3T3 and primary human keratinocytes treated with pulsed ultrasound could stimulate melanoblast migration through a paracrine effect. Our results provide a novel mechanism to promote migration of melanoblasts by pulsed ultrasound stimulation. PMID:23725022

  8. Inhibition of focal adhesion kinase suppresses the adverse phenotype of endocrine-resistant breast cancer cells and improves endocrine response in endocrine-sensitive cells.

    PubMed

    Hiscox, Stephen; Barnfather, Peter; Hayes, Edd; Bramble, Pamela; Christensen, James; Nicholson, Robert I; Barrett-Lee, Peter

    2011-02-01

    Acquired resistance to endocrine therapy in breast cancer is a major clinical problem. Previous reports have demonstrated that cell models of acquired endocrine resistance have altered cell-matrix adhesion and a highly migratory phenotype, features which may impact on tumour spread in vivo. Focal adhesion kinase (FAK) is an intracellular kinase that regulates signalling pathways central to cell adhesion, migration and survival and its expression is frequently deregulated in breast cancer. In this study, we have used the novel FAK inhibitor PF573228 to address the role of FAK in the development of endocrine resistance. Whilst total-FAK expression was similar between endocrine-sensitive and endocrine-resistant MCF7 cells, FAK phosphorylation status (Y397 or Y861) was altered in resistance. PF573228 promoted a dose-dependent inhibition of FAK phosphorylation at Y397 but did not affect other FAK activation sites (pY407, pY576 and pY861). Endocrine-resistant cells were more sensitive to these inhibitory effects versus MCF7 (mean IC(50) for FAK pY397 inhibition: 0.43 μM, 0.05 μM and 0.13 μM for MCF7, TamR and FasR cells, respectively). Inhibition of FAK pY397 was associated with a reduction in TamR and FasR adhesion to, and migration over, matrix components. PF573228 as a single agent (0-1 μM) did not affect the growth of MCF7 cells or their endocrine-resistant counterparts. However, treatment of endocrine-sensitive cells with PF573228 and tamoxifen combined resulted in greater suppression of proliferation versus single agent treatment. Together these data suggest the importance of FAK in the process of endocrine resistance, particularly in the development of an aggressive, migratory cell phenotype and demonstrate the potential to improve endocrine response through combination treatment. PMID:20354780

  9. Expression of focal adhesion kinase in the eutopic endometrium of women with adenomyosis varies with dysmenorrhea and pelvic pain

    PubMed Central

    MU, LIN; CHEN, WEIMIN; MA, YANYAN; ZHENG, WEI

    2015-01-01

    The aim of the present study was to examine whether the expression of focal adhesion kinase (FAK) is altered in the eutopic endometrium of female patients with adenomyosis, as compared with that of females without adenomyosis. The expression of FAK was assessed by immunohistochemical, western blot and reverse transcription-quantitative polymerase chain reaction analyses. An elevated expression of FAK mRNA and protein was identified in the eutopic endometrium of patients with adenomyosis compared with patients without adenomyosis (P<0.05). In addition, a positive correlation was detected between FAK protein expression and dysmenorrhea and pelvic pain in females with adenomyosis (P<0.05). The significant increase of FAK expression identified in the eutopic endometrium of females with adenomyosis, as well as the association of FAK protein expression with dysmenorrhea and pelvic pain, suggested that FAK may play a role in the pathogenesis of adenomyosis. PMID:26640570

  10. Secreted Frizzled-related protein 1 (sFRP1) regulates spermatid adhesion in the testis via dephosphorylation of focal adhesion kinase and the nectin-3 adhesion protein complex

    PubMed Central

    Wong, Elissa W. P.; Lee, Will M.; Cheng, C. Yan

    2013-01-01

    Development of spermatozoa in adult mammalian testis during spermatogenesis involves extensive cell migration and differentiation. Spermatogonia that reside at the basal compartment of the seminiferous epithelium differentiate into more advanced germ cell types that migrate toward the apical compartment until elongated spermatids are released into the tubule lumen during spermiation. Apical ectoplasmic specialization (ES; a testis-specific anchoring junction) is the only cell junction that anchors and maintains the polarity of elongating/elongated spermatids (step 8–19 spermatids) in the epithelium. Little is known regarding the signaling pathways that trigger the disassembly of the apical ES at spermiation. Here, we show that secreted Frizzled-related protein 1 (sFRP1), a putative tumor suppressor gene that is frequently down-regulated in multiple carcinomas, is a crucial regulatory protein for spermiation. The expression of sFRP1 is tightly regulated in adult rat testis to control spermatid adhesion and sperm release at spermiation. Down-regulation of sFRP1 during testicular development was found to coincide with the onset of the first wave of spermiation at approximately age 45 d postpartum, implying that sFRP1 might be correlated with elongated spermatid adhesion conferred by the apical ES before spermiation. Indeed, administration of sFRP1 recombinant protein to the testis in vivo delayed spermiation, which was accompanied by down-regulation of phosphorylated (p)-focal adhesion kinase (FAK)-Tyr397 and retention of nectin-3 adhesion protein at the apical ES. To further investigate the functional relationship between p-FAK-Tyr397 and localization of nectin-3, we overexpressed sFRP1 using lentiviral vectors in the Sertoli-germ cell coculture system. Consistent with the in vivo findings, overexpression of sFRP1 induced down-regulation of p-FAK-Tyr397, leading to a decline in phosphorylation of nectin-3. In summary, this report highlights the critical role of s

  11. The mysterious nature of bacterial surface (gliding) motility: A focal adhesion-based mechanism in Myxococcus xanthus.

    PubMed

    Islam, Salim T; Mignot, Tâm

    2015-10-01

    Motility of bacterial cells promotes a range of important physiological phenomena such as nutrient detection, harm avoidance, biofilm formation, and pathogenesis. While much research has been devoted to the mechanism of bacterial swimming in liquid via rotation of flagellar filaments, the mechanisms of bacterial translocation across solid surfaces are poorly understood, particularly when cells lack external appendages such as rotary flagella and/or retractile type IV pili. Under such limitations, diverse bacteria at the single-cell level are still able to "glide" across solid surfaces, exhibiting smooth translocation of the cell along its long axis. Though multiple gliding mechanisms have evolved in different bacterial classes, most remain poorly characterized. One exception is the gliding motility mechanism used by the Gram-negative social predatory bacterium Myxococcus xanthus. The available body of research suggests that M. xanthus gliding motility is mediated by trafficked multi-protein (Glt) cell envelope complexes, powered by proton-driven flagellar stator homologues (Agl). Through coupling to the substratum via polysaccharide slime, Agl-Glt assemblies can become fixed relative to the substratum, forming a focal adhesion site. Continued directional transport of slime-associated substratum-fixed Agl-Glt complexes would result in smooth forward movement of the cell. In this review, we have provided a comprehensive synthesis of the latest mechanistic and structural data for focal adhesion-mediated gliding motility in M. xanthus, with emphasis on the role of each Agl and Glt protein. Finally, we have also highlighted the possible connection between the motility complex and a new type of spore coat assembly system, suggesting that gliding and cell envelope synthetic complexes are evolutionarily linked. PMID:26520023

  12. Quantitative changes in focal adhesion kinase and its inhibitor, FRNK, drive load-dependent expression of costamere components.

    PubMed

    Klossner, Stephan; Li, Ruowei; Ruoss, Severin; Durieux, Anne-Cécile; Flück, Martin

    2013-09-15

    Costameres are mechanosensory sites of focal adhesion in the sarcolemma that reinforce the muscle-fiber composite and provide an anchor for myofibrillogenesis. We hypothesized that elevated content of the integrin-associated regulator of costamere turnover in culture, focal adhesion kinase (FAK), drives changes in costamere component content in antigravity muscle in a load-dependent way in correspondence with altered muscle weight. The content of FAK in soleus muscle being phosphorylated at autoregulatory tyrosine 397 (FAK-pY397) was increased after 20 s of stretch. FAK-pY397 content remained elevated after 24 h of stretch-overload due to upregulated FAK content. Overexpression of FAK in soleus muscle fibers by means of gene electrotransfer increased the β1-integrin (+56%) and meta-vinculin (+88%) content. α7-Integrin (P = 0.46) and γ-vinculin (P = 0.18) content was not altered after FAK overexpression. Co-overexpression of the FAK inhibitor FAK-related nonkinase (FRNK) reduced FAK-pY397 content by 33% and increased the percentage of fast-type fibers that arose in connection with hybrid fibers with gene transfer. Transplantation experiments confirmed the association of FRNK expression with slow-to-fast fiber transformation. Seven days of unloading blunted the elevation of FAK-pY397, β1-integrin, and meta-vinculin content with FAK overexpression, and this was reversed by 1 day of reloading. The results highlight that the expression of components for costameric attachment sites of myofibrils is under load- and fiber type-related control via FAK and its inhibitor FRNK. PMID:23904105

  13. Structural Basis of Focal Adhesion Localization of LIM-only Adaptor PINCH by Integrin-linked Kinase*S⃞

    PubMed Central

    Yang, Yanwu; Wang, Xiaoxia; Hawkins, Cheryl A.; Chen, Kan; Vaynberg, Julia; Mao, Xian; Tu, Yizeng; Zuo, Xiaobing; Wang, Jinbu; Wang, Yun-xing; Wu, Chuanyue; Tjandra, Nico; Qin, Jun

    2009-01-01

    The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK·PINCH complex (28 kDa, KD ∼ 68 nm) involving the N-terminal ankyrin repeat domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication. PMID:19117955

  14. Structural Basis of Focal Adhesion Localization of LIM-only Adaptor PINCH by Integrin-linked Kinase

    SciTech Connect

    Yang, Yanwu; Wang, Xiaoxia; Hawkins, Cheryl A.; Chen, Kan; Vaynberg, Julia; Mao, Xian; Tu, Yizeng; Zuo, Xiaobing; Wang, Jinbu; Wang, Yun-xing; Wu, Chuanyue; Tjandra, Nico; Qin, Jun

    2010-11-22

    The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK {center_dot} PINCH complex (28 kDa, K{sub D} {approx} 68 nm) involving the N-terminal ankyrin repeat domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication.

  15. Ambient and focal visual processing of naturalistic activity.

    PubMed

    Eisenberg, Michelle L; Zacks, Jeffrey M

    2016-01-01

    When people inspect a picture, they progress through two distinct phases of visual processing: an ambient, or exploratory, phase that emphasizes input from peripheral vision and rapid acquisition of low-frequency information, followed by a focal phase that emphasizes central vision, salient objects, and high-frequency information. Does this qualitative shift occur during dynamic scene viewing? If so, when? One possibility is that shifts to exploratory processing are triggered at subjective event boundaries. This shift would be adaptive, because event boundaries typically occur when activity features change and when activity becomes unpredictable. Here, we used a perceptual event segmentation task, in which people identified boundaries between meaningful units of activity, to test this hypothesis. In two studies, an eye tracker recorded eye movements and pupil size while participants first watched movies of actors engaged in everyday activities and then segmented them into meaningful events. Saccade amplitudes and fixation durations during the initial viewings suggest that event boundaries function much like the onset of a new picture during static picture presentation: Viewers initiate an ambient processing phase and then progress to focal viewing as the event progresses. These studies suggest that this shift in processing mode could play a role in the formation of mental representations of the current environment. PMID:27002550

  16. The Interaction between Cancer Stem Cell Marker CD133 and Src Protein Promotes Focal Adhesion Kinase (FAK) Phosphorylation and Cell Migration.

    PubMed

    Liu, Chanjuan; Li, Yinan; Xing, Yang; Cao, Benjin; Yang, Fan; Yang, Tianxiao; Ai, Zhilong; Wei, Yuanyan; Jiang, Jianhai

    2016-07-22

    CD133, a widely known cancer stem cell marker, has been proved to promote tumor metastasis. However, the mechanism by which CD133 regulates metastasis remains largely unknown. Here, we report that CD133 knockdown inhibits cancer cell migration, and CD133 overexpression promotes cell migration. CD133 expression is beneficial to activate the Src-focal adhesion kinase (FAK) signaling pathway. Further studies show that CD133 could interact with Src, and the region between amino acids 845 and 857 in the CD133 C-terminal domain is indispensable for its interaction with Src. The interaction activates Src to phosphorylate its substrate FAK and to promote cell migration. Likewise, a Src binding-deficient CD133 mutant loses the abilities to increase Src and FAK phosphorylation and to promote cell migration. Inhibition of Src activity by PP2, a known Src activity inhibitor, could block the activation of FAK phosphorylation and cell migration induced by CD133. In summary, our data suggest that activation of FAK by the interaction between CD133 and Src promotes cell migration, providing clues to understand the migratory mechanism of CD133(+) tumor cells. PMID:27226554

  17. Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation

    PubMed Central

    van Miltenburg, M H A M; van Nimwegen, M J; Tijdens, I; Lalai, R; Kuiper, R; Klarenbeek, S; Schouten, P C; de Vries, A; Jonkers, J; van de Water, B

    2014-01-01

    Background: Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development. Methods: To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53lox/lox/FAK+/+/WapCre, p53lox/lox/FAKflox/+/WapCre and p53lox/lox/FAKflox/−/WapCre mice, and mice with WapCre-mediated conditional expression of p53R270H, the mouse equivalent of human p53R273H hot spot mutation, together with conditional deletion of FAK, P53R270H/+/FAKlox/+/WapCre and p53R270H/+/FAKflox/−/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours. Results: Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53R270H-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures. Conclusions: Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion

  18. PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

    PubMed Central

    Madaro, Luca; Marrocco, Valeria; Fiore, Piera; Aulino, Paola; Smeriglio, Piera; Adamo, Sergio; Molinaro, Mario; Bouché, Marina

    2011-01-01

    Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKCθ, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKCθ is strongly up-regulated following freeze injury–induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKCθ knockout and muscle-specific PKCθ dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKCθ mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKCθ mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKCθ in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKCθ-null myoblasts. We thus propose that PKC

  19. Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

    PubMed

    Shynlova, Oksana; Chow, Michelle; Lye, Stephen J

    2009-10-01

    The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions. PMID:19602722

  20. Physical Activity Performance of Focal Middle School Students

    ERIC Educational Resources Information Center

    Erfle, Stephen E.; Gelbaugh, Corey M.

    2013-01-01

    Histograms of push-ups and curl-ups from a sample of more than 9,000 students show periodic spikes at five and 10 unit intervals. This article argues that these spikes are related to focal points, a game theoretic concept popularized by Nobel Laureate Thomas Schelling. Being focal on one test makes one more likely to be focal on the other. Focal…

  1. Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion

    NASA Astrophysics Data System (ADS)

    Heuslein, Joshua L.; Murrell, Kelsey P.; Leiphart, Ryan J.; Llewellyn, Ryan A.; Meisner, Joshua K.; Price, Richard J.

    2016-05-01

    Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase’s (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6Chi and Ly6Clo blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK.

  2. Cell nanomechanics and focal adhesions are regulated by retinol and conjugated linoleic acid in a dose-dependent manner

    NASA Astrophysics Data System (ADS)

    Silberberg, Yaron R.; Yakubov, Gleb E.; Horton, Michael A.; Pelling, Andrew E.

    2009-07-01

    Retinol and conjugated linoleic acid (CLA) have previously been shown to have an important role in gene expression and various cellular processes, including differentiation, proliferation and cell death. In this study we have investigated the effect of retinol and CLA, both individually and in combination, on the intracellular cytoskeleton, focal adhesions (FAs) and the nanomechanical properties of 3T3 fibroblasts. We observed a dose-dependent decrease in the formation of FAs following treatment with either compound, which was directly correlated to an increase in cell height (>30%) and a decrease in the measured Young's modulus (~28%). Furthermore, treatments with both compounds demonstrated an increased effect and led to a reduction of>70% in the average number of FAs per cell and a decrease of >50% in average cell stiffness. These data reveal that retinol and CLA disrupt FA formation, leading to an increase in cell height and a significant decrease in stiffness. These results may broaden our understanding of the interplay between cell nanomechanics and cellular contact with the external microenvironment, and help to shed light on the important role of retinoids and CLA in health and disease.

  3. Down-regulation of integrin β1 and focal adhesion kinase in renal glomeruli under various hemodynamic conditions.

    PubMed

    Yuan, Xiaoli; Wang, Wei; Wang, Juan; Yin, Xiaohui; Zhai, Xiaoyue; Wang, Lining; Li, Kai; Li, Zilong

    2014-01-01

    Given that integrin β1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK) is the most essential intracellular integrator in the integrin β1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possible interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT) and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin β1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin β1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin β1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin β1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases. PMID:24705394

  4. Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion

    PubMed Central

    Heuslein, Joshua L.; Murrell, Kelsey P.; Leiphart, Ryan J.; Llewellyn, Ryan A.; Meisner, Joshua K.; Price, Richard J.

    2016-01-01

    Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase’s (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6Chi and Ly6Clo blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK. PMID:27244251

  5. Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion.

    PubMed

    Heuslein, Joshua L; Murrell, Kelsey P; Leiphart, Ryan J; Llewellyn, Ryan A; Meisner, Joshua K; Price, Richard J

    2016-01-01

    Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase's (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6C(hi) and Ly6C(lo) blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK. PMID:27244251

  6. Stonin1 mediates endocytosis of the proteoglycan NG2 and regulates focal adhesion dynamics and cell motility

    PubMed Central

    Feutlinske, Fabian; Browarski, Marietta; Ku, Min-Chi; Trnka, Philipp; Waiczies, Sonia; Niendorf, Thoralf; Stallcup, William B.; Glass, Rainer; Krause, Eberhard; Maritzen, Tanja

    2015-01-01

    Cellular functions, ranging from focal adhesion (FA) dynamics and cell motility to tumour growth, are orchestrated by signals cells receive from outside via cell surface receptors. Signalling is fine-tuned by the exo–endocytic cycling of these receptors to control cellular responses such as FA dynamics, which determine cell motility. How precisely endocytosis regulates turnover of the various cell surface receptors remains unclear. Here we identify Stonin1, an endocytic adaptor of unknown function, as a regulator of FA dynamics and cell motility, and demonstrate that it facilitates the internalization of the oncogenic proteoglycan NG2, a co-receptor of integrins and platelet-derived growth factor receptor. Embryonic fibroblasts obtained from Stonin1-deficient mice display a marked surface accumulation of NG2, increased cellular signalling and defective FA disassembly as well as altered cellular motility. These data establish Stonin1 as a specific adaptor for the endocytosis of NG2 and as an important factor for FA dynamics and cell migration. PMID:26437238

  7. Rac1 recruitment to the archipelago structure of the focal adhesion through the fluid membrane as revealed by single-molecule analysis.

    PubMed

    Shibata, Akihiro C E; Chen, Limin H; Nagai, Rie; Ishidate, Fumiyoshi; Chadda, Rahul; Miwa, Yoshihiro; Naruse, Keiji; Shirai, Yuki M; Fujiwara, Takahiro K; Kusumi, Akihiro

    2013-03-01

    The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress-fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter-island channels in the FA zone rather freely, and the integrins in the FA-protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G-protein regulating FA formation, and its activators αPIX and βPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively-active Rac1 mutant exhibited temporary and all-time immobilizations in the FA zone, suggesting that upon PIX-induced Rac1 activation at the FA-protein islands, Rac1 tends to be immobilized at the FA-protein islands. PMID:23341328

  8. Ling Zhi-8 reduces lung cancer mobility and metastasis through disruption of focal adhesion and induction of MDM2-mediated Slug degradation.

    PubMed

    Lin, Tung-Yi; Hsu, Hsien-Yeh

    2016-06-01

    We recently reported that recombinant Ling Zhi-8 (rLZ-8), a medicinal mushroom Ganoderma lucidum recombinant protein, effectively prevents lung cancer cells proliferation in vivo mice model. In our current study, we demonstrated that rLZ-8 suppressed tumor metastasis and increased the survival rate in Lewis lung carcinoma cell-bearing mice. The epithelial to mesenchymal transition (EMT) process is regarded as the critical event in tumor metastasis. Herein, we showed that rLZ-8 effectively induced changes in EMT by interfering with cell adhesion and focal adhesion kinase (FAK) functions in lung cancer cells. Slug, a transcription factor, represses E-cadherin transcription and is regarded as a critical event in EMT and tumor metastasis. Functional studies revealed that downregulation of Slug as a result of rLZ-8-induced FAK inactivation enhanced E-cadherin expression and repressed cancer cell mobility. Moreover, we found that rLZ-8 enhanced the ubiquitination proteasome pathway (UPP)-mediated degradation of Slug in CL1-5 cells. Mechanistically, we demonstrated that rLZ-8 promoted the interaction between MDM2 and Slug, resulting in Slug degradation; however, MDM2-shRNA abolished rLZ-8-enhanced Slug degradation. This study is the first to determine anti-metastatic activity of rLZ-8 and its potential mechanism, with how the regulation of EMT and cell mobility is via the negative modulation of FAK, and thereby leading to the ubiquitination and degradation of Slug. Our findings suggest that the targets of FAK play a key role in metastasis. Moreover, rLZ-8 may be useful as a chemotherapeutic agent for treating lung cancer. PMID:26992741

  9. Adhesion

    MedlinePlus

    ... adhesions Ovarian cyst References Munireddy S, Kavalukas SL, Barbul A. Intra-abdominal healing: gastrointestinal tract and adhesions. Surg Clin N Am Kulaylat MN, Dayton, MT. Surgical complications. In: Townsend CM Jr, Beauchamp RD, Evers BM, Mattox KL, ...

  10. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

    PubMed

    González-Fernández, Lauro; Macías-García, Beatriz; Loux, Shavahn C; Varner, Dickson D; Hinrichs, Katrin

    2013-06-01

    Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways. PMID:23595906

  11. Focal adhesion kinase is a load-dependent governor of the slow contractile and oxidative muscle phenotype

    PubMed Central

    Durieux, Anne-Cécile; D’Antona, Giuseppe; Desplanches, Dominique; Freyssenet, Damien; Klossner, Stephan; Bottinelli, Roberto; Flück, Martin

    2009-01-01

    Striated muscle exhibits a pronounced structural–functional plasticity in response to chronic alterations in loading. We assessed the implication of focal adhesion kinase (FAK) signalling in mechano-regulated differentiation of slow-oxidative muscle. Load-dependent consequences of FAK signal modulation were identified using a multi-level approach after electrotransfer of rat soleus muscle with FAK-expression plasmid vs. empty plasmid-transfected contralateral controls. Muscle fibre-targeted over-expression of FAK in anti-gravitational muscle for 9 days up-regulated transcript levels of gene ontologies underpinning mitochondrial metabolism and contraction in the transfected belly portion. Concomitantly, mRNA expression of the major fast-type myosin heavy chain (MHC) isoform, MHC2A, was reduced. The promotion of the slow-oxidative expression programme by FAK was abolished after co-expression of the FAK inhibitor FAK-related non-kinase (FRNK). Elevated protein content of MHC1 (+9%) and proteins of mitochondrial respiration (+165–610%) with FAK overexpression demonstrated the translation of transcript differentiation in targeted muscle fibres towards a slow-oxidative muscle phenotype. Coincidentally MHC2A protein was reduced by 50% due to protection of muscle from de-differentiation with electrotransfer. Fibre cross section in FAK-transfected muscle was elevated by 6%. The FAK-modulated muscle transcriptome was load-dependent and regulated in correspondence to tyrosine 397 phosphorylation of FAK. In the context of overload, the FAK-induced gene expression became manifest at the level of contraction by a slow transformation and the re-establishment of normal muscle force from the lowered levels with transfection. These results highlight the analytic power of a systematic somatic transgene approach by mapping a role of FAK in the dominant mechano-regulation of muscular motor performance via control of gene expression. PMID:19470782

  12. Preferential phosphorylation of focal adhesion kinase tyrosine 861 is critical for mediating an anti-apoptotic response to hyperosmotic stress.

    PubMed

    Lunn, J Adrian; Jacamo, Rodrigo; Rozengurt, Enrique

    2007-04-01

    The results presented here demonstrate that focal adhesion kinase (FAK) Tyr-861 is the predominant tyrosine phosphorylation site stimulated by hyperosmotic stress in a variety of cell types, including epithelial cell lines (ileum-derived IEC-18, colon-derived Caco2, and stomach-derived NCI-N87), FAK null fibroblasts re-expressing FAK, and Src family kinase triple null fibroblasts (SYF cells) in which c-Src has been restored (YF cells). We show that hyperosmotic stress-stimulated FAK phosphorylation in epithelial cells is inhibited by Src family kinase inhibitors PP2 and SU6656 and that it does not occur in SYF cells. Unexpectedly, hyperosmotic stress-induced phosphorylation of FAK at Tyr-397, Tyr-576, and most dramatically at Tyr-861 was completely insensitive to the F-actin-disrupting agents, latrunculin A and cytochalasin D. Finally, we show that in FAK null cells exposed to hyperosmotic stress or growth factor withdrawal, re-expression of wild type FAK restored cell survival, whereas re-expression of FAK mutated from tyrosine to phenylalanine at position 861 (FAKY861F) did not. Our results indicate that FAK Tyr-861 phosphorylation is required for mammalian cell survival of hyperosmotic stress. Furthermore, the results suggest that FAK is an upstream regulator (rather than downstream effector) of F-actin reorganization in response to hyperosmotic stress. We propose that FAK/c-Src bipartite enzyme is a sensor of cytoplasmic shrinkage, and that the phosphorylation on FAK Tyr-861 by Src and subsequent reorganization of F-actin can initiate an anti-apoptotic signaling pathway that protects cells from hyperosmotic stress. PMID:17289681

  13. Synaptopodin-2 induces assembly of peripheral actin bundles and immature focal adhesions to promote lamellipodia formation and prostate cancer cell migration.

    PubMed

    Kai, FuiBoon; Fawcett, James P; Duncan, Roy

    2015-05-10

    Synaptopodin-2 (Synpo2), an actin-binding protein and invasive cancer biomarker, induces formation of complex stress fiber networks in the cell body and promotes PC3 prostate cancer cell migration in response to serum stimulation. The role of these actin networks in enhanced cancer cell migration is unknown. Using time-course analysis and live cell imaging of mock- and Synpo2-transduced PC3 cells, we now show that Synpo2 induces assembly of actin fibers near the cell periphery and Arp2/3-dependent lamellipodia formation. Lamellipodia formed in a non-directional manner or repeatedly changed direction, explaining the enhanced chemokinetic activity of PC3 cells in response to serum stimulation. Myosin contraction promotes retrograde flow of the Synpo2-associated actin filaments at the leading edge and their merger with actin networks in the cell body. Enhanced PC3 cell migration correlates with Synpo2-induced formation of lamellipodia and immature focal adhesions (FAs), but is not dependent on myosin contraction or FA maturation. The previously reported correlation between Synpo2-induced stress fiber assembly and enhanced PC3 cell migration therefore reflects the role of Synpo2 as a newly identified regulator of actin bundle formation and nascent FA assembly near the leading cell edge. PMID:25883213

  14. Non-Smad transforming growth factor-β signaling regulated by focal adhesion kinase binding the p85 subunit of phosphatidylinositol 3-kinase.

    PubMed

    Hong, Min; Wilkes, Mark C; Penheiter, Sumedha G; Gupta, Shiv K; Edens, Maryanne; Leof, Edward B

    2011-05-20

    TGF-β modulates numerous diverse cellular phenotypes including growth arrest in epithelial cells and proliferation in fibroblasts. Although the Smad pathway is fundamental for the majority of these responses, recent evidence indicates that non-Smad pathways may also have a critical role. Here we report a novel mechanism whereby the nonreceptor tyrosine focal adhesion kinase (FAK) functions as an adaptor necessary for cell type-specific responses to TGF-β. We show that in contrast to Smad actions, non-Smad pathways, including c-Abl, PAK2, and Akt, display an obligate requirement for FAK. Interestingly, this occurs in Src null SYF cells and is independent of FAK tyrosine phosphorylation, kinase activity, and/or proline-rich sequences in the C-terminal FAT domain. FAK binds the phosphatidylinositol 3-kinase (PI3K) p85 regulatory subunit following TGF-β treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-β-stimulated anchorage-independent growth and migration. Together, these results uncover a new scaffolding role for FAK as the most upstream component regulating the profibrogenic action of TGF-β and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases. PMID:21454615

  15. Focal Adhesion Kinase Controls pH-Dependent Epidermal Barrier Homeostasis by Regulating Actin-Directed Na+/H+ Exchanger 1 Plasma Membrane Localization

    PubMed Central

    Ilic, Dusko; Mao-Qiang, Man; Crumrine, Debra; Dolganov, Gregory; Larocque, Nicholas; Xu, Pu; Demerjian, Marianne; Brown, Barbara E.; Lim, Ssang-Taek; Ossovskaya, Valeria; Schlaepfer, David D.; Fisher, Susan J.; Feingold, Kenneth R.; Elias, Peter M.; Mauro, Theodora M.

    2007-01-01

    Ubiquitously expressed focal adhesion kinase (FAK), linked to multiple intracellular signaling pathways, has previously been shown to control cell motility, invasion, proliferation, and survival. Using mice with a keratinocyte-restricted deletion of fak (FAKK5 KO), we report here a novel role for FAK: maintenance of adult epidermal permeability barrier homeostasis. Abundant lacunae of unprocessed lipids in stratum corneum (SC) of FAKK5 KO mice and delayed barrier recovery pointed to malfunction of pH-dependent enzymes active in extracellular space of SC. Measuring the SC pH gradient showed significantly more neutral pH values in FAKK5 KO mice, suggesting the importance of FAK for acidification. Moreover, normal functions were restored when FAKK5 KO mice were exposed to a surface pH typical of mouse SC (pH = 5.5). Baseline levels and response to barrier disruption of secretory phospholipase A2 isoforms, enzymes that mediate generation of free fatty acids in epidermis, appeared similar in both FAKK5 KO and control littermates. We found that the critical SC acidification regulator Na+/H+ exchanger 1 failed to localize to the plasma membrane in FAK-deficient keratinocytes both in vivo and in vitro. Thus, for plasma membrane localization in terminally differentiated keratinocytes, Na+/H+ exchanger 1 requires an intact actin cytoskeleton, which is impaired in FAK-deficient cells. PMID:17525272

  16. CFTR Cl– channel functional regulation by phosphorylation of focal adhesion kinase at tyrosine 407 in osmosensitive ion transporting mitochondria rich cells of euryhaline killifish

    PubMed Central

    Marshall, William S.; Watters, Kaitlyn D.; Hovdestad, Leah R.; Cozzi, Regina R. F.; Katoh, Fumi

    2009-01-01

    Summary Cystic fibrosis transmembrane conductance regulator (CFTR) anion channels are the regulated exit pathway in Cl– secretion by teleost mitochondria rich salt secreting (MR) cells of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry, immunogold transmission electron microscopy (TEM), and co-immunoprecipitation, using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase focal adhesion kinase (FAK) phosphorylated at Y407 (FAK pY407) are colocalized in the apical membrane and in subjacent membrane vesicles of MR cells. We showed previously that basolateral FAK pY407, unlike other FAK phosphorylation sites, is osmosensitive and dephosphorylates during hypotonic shock of epithelial cells (Marshall et al., 2008). In the present study, we found that hypotonic shock and the α2-adrenergic agonist clonidine (neither of which affects cAMP levels) rapidly and reversibly inhibit Cl– secretion by isolated opercular membranes, simultaneous with dephosphorylation of FAK pY407, located in the apical membrane. FAK pY407 is rephosphorylated and Cl– secretion rapidly restored by hypertonic shock as well as by forskolin and isoproterenol, which operate via cAMP and protein kinase A. We conclude that hormone mediated, cAMP dependent and osmotically mediated, cAMP independent pathways converge on a mechanism to activate CFTR and Cl– secretion, possibly through tyrosine phosphorylation of CFTR by FAK. PMID:19617429

  17. Kank2 activates talin, reduces force transduction across integrins and induces central adhesion formation.

    PubMed

    Sun, Zhiqi; Tseng, Hui-Yuan; Tan, Steven; Senger, Fabrice; Kurzawa, Laetitia; Dedden, Dirk; Mizuno, Naoko; Wasik, Anita A; Thery, Manuel; Dunn, Alexander R; Fässler, Reinhard

    2016-09-01

    Integrin-based adhesions play critical roles in cell migration. Talin activates integrins and flexibly connects integrins to the actomyosin cytoskeleton, thereby serving as a 'molecular clutch' that transmits forces to the extracellular matrix to drive cell migration. Here we identify the evolutionarily conserved Kank protein family as novel components of focal adhesions (FAs). Kank proteins accumulate at the lateral border of FAs, which we term the FA belt, and in central sliding adhesions, where they directly bind the talin rod domain through the Kank amino-terminal (KN) motif and induce talin and integrin activation. In addition, Kank proteins diminish the talin-actomyosin linkage, which curbs force transmission across integrins, leading to reduced integrin-ligand bond strength, slippage between integrin and ligand, central adhesion formation and sliding, and reduced cell migration speed. Our data identify Kank proteins as talin activators that decrease the grip between the integrin-talin complex and actomyosin to regulate cell migration velocity. PMID:27548916

  18. Focal adhesion kinase is required for IGF-I-mediated growth of skeletal muscle cells via a TSC2/mTOR/S6K1-associated pathway

    PubMed Central

    Crossland, Hannah; Kazi, Abid A.; Lang, Charles H.; Timmons, James A.; Pierre, Philippe; Wilkinson, Daniel J.; Smith, Kenneth; Szewczyk, Nathaniel J.

    2013-01-01

    Focal adhesion kinase (FAK) is an attachment complex protein associated with the regulation of muscle mass through as-of-yet unclear mechanisms. We tested whether FAK is functionally important for muscle hypertrophy, with the hypothesis that FAK knockdown (FAK-KD) would impede cell growth associated with a trophic stimulus. C2C12 skeletal muscle cells harboring FAK-targeted (FAK-KD) or scrambled (SCR) shRNA were created using lentiviral transfection techniques. Both FAK-KD and SCR myotubes were incubated for 24 h with IGF-I (10 ng/ml), and additional SCR cells (±IGF-1) were incubated with a FAK kinase inhibitor before assay of cell growth. Muscle protein synthesis (MPS) and putative FAK signaling mechanisms (immunoblotting and coimmunoprecipitation) were assessed. IGF-I-induced increases in myotube width (+41 ± 7% vs. non-IGF-I-treated) and total protein (+44 ± 6%) were, after 24 h, attenuated in FAK-KD cells, whereas MPS was suppressed in FAK-KD vs. SCR after 4 h. These blunted responses were associated with attenuated IGF-I-induced FAK Tyr397 phosphorylation and markedly suppressed phosphorylation of tuberous sclerosis complex 2 (TSC2) and critical downstream mTOR signaling (ribosomal S6 kinase, eIF4F assembly) in FAK shRNA cells (all P < 0.05 vs. IGF-I-treated SCR cells). However, binding of FAK to TSC2 or its phosphatase Shp-2 was not affected by IGF-I or cell phenotype. Finally, FAK-KD-mediated suppression of cell growth was recapitulated by direct inhibition of FAK kinase activity in SCR cells. We conclude that FAK is required for IGF-I-induced muscle hypertrophy, signaling through a TSC2/mTOR/S6K1-dependent pathway via means requiring the kinase activity of FAK but not altered FAK-TSC2 or FAK-Shp-2 binding. PMID:23695213

  19. Doxycycline reduces the migration of tuberous sclerosis complex-2 null cells - effects on RhoA-GTPase and focal adhesion kinase

    PubMed Central

    Ng, Ho Yin; Oliver, Brian Gregory George; Burgess, Janette Kay; Krymskaya, Vera P; Black, Judith Lee; Moir, Lyn M

    2015-01-01

    Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2-positive and TSC2-negative mouse embryonic fibroblasts (MEF), 323-TSC2-positive and 323-TSC2-null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2-negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2-null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2–59 μM. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC2 dysfunction. PMID:26282580

  20. Doxycycline reduces the migration of tuberous sclerosis complex-2 null cells - effects on RhoA-GTPase and focal adhesion kinase.

    PubMed

    Ng, Ho Yin; Oliver, Brian Gregory George; Burgess, Janette Kay; Krymskaya, Vera P; Black, Judith Lee; Moir, Lyn M

    2015-11-01

    Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2-positive and TSC2-negative mouse embryonic fibroblasts (MEF), 323-TSC2-positive and 323-TSC2-null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2-negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2-null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2-59 μM. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC2 dysfunction. PMID:26282580

  1. Glycogen synthase kinase 3β sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway.

    PubMed

    Chikano, Yuri; Domoto, Takahiro; Furuta, Takuya; Sabit, Hemragul; Kitano-Tamura, Ayako; Pyko, Ilya V; Takino, Takahisa; Sai, Yoshimichi; Hayashi, Yutaka; Sato, Hiroshi; Miyamoto, Ken-ichi; Nakada, Mitsutoshi; Minamoto, Toshinari

    2015-02-01

    The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3β (GSK3β), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3β compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3β also contributes to the highly invasive nature of glioblastoma. The effects of GSK3β inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3β attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3β as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance. PMID:25504636

  2. Disruption of focal adhesion kinase and p53 interaction with small molecule compound R2 reactivated p53 and blocked tumor growth

    PubMed Central

    2013-01-01

    Background Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor kinase that plays a major role in cancer cell survival and metastasis. Methods We performed computer modeling of the p53 peptide containing the site of interaction with FAK, predicted the peptide structure and docked it into the three-dimensional structure of the N-terminal domain of FAK involved in the complex with p53. We screened small molecule compounds that targeted the site of the FAK-p53 interaction and identified compounds (called Roslins, or R compounds) docked in silico to this site. Results By different assays in isogenic HCT116p53+/+ and HCT116 p53-/- cells we identified a small molecule compound called Roslin 2 (R2) that bound FAK, disrupted the binding of FAK and p53 and decreased cancer cell viability and clonogenicity in a p53-dependent manner. In addition, dual-luciferase assays demonstrated that the R2 compound increased p53 transcriptional activity that was inhibited by FAK using p21, Mdm-2, and Bax-promoter targets. R2 also caused increased expression of p53 targets: p21, Mdm-2 and Bax proteins. Furthermore, R2 significantly decreased tumor growth, disrupted the complex of FAK and p53, and up-regulated p21 in HCT116 p53+/+ but not in HCT116 p53-/- xenografts in vivo. In addition, R2 sensitized HCT116p53+/+ cells to doxorubicin and 5-fluorouracil. Conclusions Thus, disruption of the FAK and p53 interaction with a novel small molecule reactivated p53 in cancer cells in vitro and in vivo and can be effectively used for development of FAK-p53 targeted cancer therapy approaches. PMID:23841915

  3. Quantitative relationship among integrin-ligand binding, adhesion, and signaling via focal adhesion kinase and extracellular signal-regulated kinase 2.

    PubMed

    Asthagiri, A R; Nelson, C M; Horwitz, A F; Lauffenburger, D A

    1999-09-17

    ERK2. These measures of FAK and ERK2 activity were found to correlate with short term cell-substratum adhesivity, indicating that signaling via FAK and ERK2 is proportional to the number of integrin-fibronectin bonds. PMID:10480927

  4. Focal Adhesion Kinase Directly Interacts with TSC2 Through Its FAT Domain and Regulates Cell Proliferation in Cashmere Goat Fetal Fibroblasts.

    PubMed

    Zheng, Xu; Bao, Wenlei; Yang, Jiaofu; Zhang, Tao; Sun, Dongsheng; Liang, Yan; Li, Shuyu; Wang, Yanfeng; Feng, Xue; Hao, Huifang; Wang, Zhigang

    2016-09-01

    Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor tyrosine kinase that senses a variety of extracellular signals, such as growth factors and integrins, to control the process of cell proliferation and metabolism. We cloned three goat FAK transcript variants (KM655805, KM658268, and KM658269) that encode 1052, 1006, and 962 amino-acid residue proteins. Bioinformatics analysis indicated that the putative FAK protein contains an FERM domain, a PTK domain, two Proline-rich regions, and a focal adhesion-targeting (FAT) domain. All the three transcript variants of FAK were detected in seven different goat tissues, and variant 1 had the most accumulation whereas variant 2 and variant 3 had lower accumulation. Treatment of goat fetal fibroblasts (GFbs) with a specific FAK inhibitor, TAE226, inhibited cell proliferation (p < 0.05) and induced damage to the cell morphology in a dose- and time-dependent manner. Further research demonstrated that FAK directly interacted with TSC2 (Tuberous sclerosis 2) tuberin domain through its C-terminus, which contains the complete FAT domain. In conclusion, our results indicated that FAK may be widely expressed in Cashmere goat tissues and its products participate in the mammalian target of rapamycin signaling pathway and cell proliferation through a direct interaction with TSC2 in GFBs. PMID:27380318

  5. Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour

    PubMed Central

    Fokkelman, Michiel; Balcıoğlu, Hayri E.; Klip, Janna E.; Yan, Kuan; Verbeek, Fons J.; Danen, Erik H. J.; van de Water, Bob

    2016-01-01

    Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour. PMID:27531518

  6. JAK2/STAT3 targeted therapy suppresses tumor invasion via disruption of the EGFRvIII/JAK2/STAT3 axis and associated focal adhesion in EGFRvIII-expressing glioblastoma

    PubMed Central

    Zheng, Qifan; Han, Lei; Dong, Yucui; Tian, Jing; Huang, Wei; Liu, Zhaoyu; Jia, Xiuzhi; Jiang, Tao; Zhang, Jianning; Li, Xia; Kang, Chunsheng; Ren, Huan

    2014-01-01

    Background As a commonly mutated form of the epidermal growth factor receptor, EGFRvIII strongly promotes glioblastoma (GBM) tumor invasion and progression, but the mechanisms underlying this promotion are not fully understood. Methods Through gene manipulation, we established EGFRvIII-, wild-type EGFR-, and vector-expressing GBM cells. We used cDNA microarrays, bioinformatics analysis, target-blocking migration and invasion assays, Western blotting, and an orthotopic U87MG GBM model to examine the phenotypic shifts and treatment effects of EGFRvIII expression in vitro and in vivo. Confocal imaging, co-immunoprecipitation, and siRNA assays detected the focal adhesion-associated complex and their relationships to the EGFRvIII/JAK2/STAT3 axis in GBM cells. Results The activation of JAK2/STAT3 signaling is vital for promoting migration and invasion in EGFRvIII-GBM cells. AG490 or WP1066, the JAK2/STAT3 inhibitors, specifically destroyed EGFRvIII/JAK2/STAT3-related focal adhesions and depleted the activation of EGFR/Akt/FAK and JAK2/STAT3 signaling, thereby abolishing the ability of EGFRvIII-expressing GBM cells to migrate and invade. Furthermore, the RNAi silencing of JAK2 in EGFRvIII-expressing GBM cells significantly attenuated their ability to migrate and invade; however, as a result of a potential EGFRvIII-JAK2-STAT3 activation loop, neither EGFR nor STAT3 knockdown yielded the same effects. Moreover, AG490 or JAK2 gene knockdown greatly suppressed tumor invasion and progression in the U87MG-EGFRvIII orthotopic models. Conclusion Taken together, our data demonstrate that JAK2/STAT3 signaling is essential for EGFRvIII-driven migration and invasion by promoting focal adhesion and stabilizing the EGFRvIII/JAK2/STAT3 axis. Targeting JAK2/STAT3 therapy, such as AG490, may have potential clinical implications for the tailored treatment of GBM patients bearing EGFRvIII-positive tumors. PMID:24861878

  7. Epstein–Barr virus LMP1 induces focal adhesions and epithelial cell migration through effects on integrin-α5 and N-cadherin

    PubMed Central

    Wasil, L R; Shair, K H Y

    2015-01-01

    Epstein–Barr virus (EBV) is a γ-herpesvirus associated with human epithelial and B-cell malignancies. The EBV latent membrane protein (LMP) 1 is expressed in nasopharyngeal carcinoma (NPC) and promotes oncogenic intracellular signaling mechanisms. LMP1 also promotes a pro-migratory phenotype through potential effects on cell surface proteins, as expression of LMP1 induces an epithelial–mesenchymal transition (EMT) in epithelial cell lines. In this study, LMP1 was examined for potential effects on cadherin and integrin surface interactions, and assessed for biological effects on adhesion and motility to fibronectin. Expression of LMP1 in the non-tumorigenic epithelial cell line MCF10a induced an EMT-associated cadherin switch. The induced N-cadherin was ligated and localized to the cell surface as determined by triton-solubility and immunofluorescence assays. In addition, LMP1 induced the assembly of focal adhesions (FAs) with increased production of fibronectin in MCF10a and NP460hTERT-immortalized nasopharyngeal cells. Biochemical enrichment of fibronectin-associated proteins indicated that LMP1 selectively promoted the recruitment of integrin-α5 and Src family kinase proteins to FA complexes. Neutralizing antibodies to N-cadherin and integrin-α5, but not integrin-αV, blocked the adhesion and transwell motility of MCF10a cells to fibronectin induced by LMP1. LMP1-induced transwell motility was also decreased by Src inhibition with the PP2 kinase inhibitor and short hairpin RNAs. These studies reveal that LMP1 has multiple mechanisms to promote the adhesive and migratory properties of epithelial cells through induction of fibronectin and modulation of cell surface interactions involving integrin-α5 and N-cadherin, which may contribute to the metastatic potential of NPC. PMID:26479443

  8. MiR-138 and MiR-135 directly target focal adhesion kinase, inhibit cell invasion, and increase sensitivity to chemotherapy in cancer cells.

    PubMed

    Golubovskaya, Vita M; Sumbler, Brittany; Ho, Baotran; Yemma, Michael; Cance, William G

    2014-01-01

    Focal Adhesion Kinase is a 125 kDa non-receptor kinase and overexpressed in many types of tumors. Recently, short noncoding RNAs, called microRNAs have been discovered as regulators of gene expression mainly through binding to the untranslated region (UTR) of mRNA. In this report we show that MiR-138 and MiR-135 down-regulated FAK expression in cancer cells. MiR-138 and MiR-135 inhibited FAK protein expression in different cancer cell lines. The computer analysis of 3'FAK-untranslated region (FAKUTR) identified one conserved MiR-138 binding site (CACCAGCA) at positions 3514-3521 and one conserved MiR-135 (AAGCCAU) binding site at positions 4278-4284 in the FAK-UTR. By a dual-luciferase assay we demonstrate that MiR-138 and MiR-135 directly bound the FAK untranslated region using FAK-UTR-Target (FAK-UTR) luciferase plasmid and inhibited its luciferase activity. The sitedirected mutagenesis of the MiR-138 and MiR-135 binding sites in the FAK-UTR abrogated MiR-138 and MiR-135-directed inhibition of FAK-UTR. Real-time PCR demonstrated that cells transfected with MiR-138 and MiR-135 expressed decreased FAK mRNA levels. Moreover, stable expression of MiR-138 and MiR-135 in 293 and HeLa cells decreased cell invasion and increased sensitivity to 5- fluorouracil (5-FU), FAK inhibitor, Y15, and doxorubicin. In addition, MiR-138 significantly decreased 293 xenograft tumor growth in vivo. This is the first report on regulation of FAK expression by MiR-135 and MiR138 that affected invasion, drug sensitivity, and tumor growth in cancer cells, which is important to the development of FAK-targeted therapeutics and understanding their novel regulations and functions. PMID:23438844

  9. Deregulation of focal adhesion pathway mediated by miR-659-3p is implicated in bone marrow infiltration of stage M neuroblastoma patients

    PubMed Central

    Lagazio, Corrado; Persico, Luca; Carlini, Barbara; Varesio, Luigi; Morandi, Fabio; Morini, Martina; Gigliotti, Anna Rita; Esposito, Maria Rosaria; Viscardi, Elisabetta; Cecinati, Valerio; Conte, Massimo; Corrias, Maria Valeria

    2015-01-01

    To get insights on the metastatic process of human neuroblastoma (NB), the miRNA expression profile of bone marrow (BM)-infiltrating cells has been determined and compared to that of primary tumors. Twenty-two BM-infiltrating cells, 22 primary tumors, and 4 paired samples from patients with metastatic NB aged > 12 months were analyzed for the expression of 670 miRNAs by stem-loop RT-qPCR. The miRNAs whose expression was significantly different were subjected to selection criteria, and 20 selected miRNAs were tested in 10 additional BM-infiltrating cells and primary tumors. Among the miRNAs confirmed to be differentially expressed, miR-659-3p was further analyzed. Transfection of miR-659-3p mimic and inhibitor demonstrated the specific suppression and over-expression, respectively, of the miR-659-3p target gene CNOT1, a regulator of transcription of genes containing AU-rich element (ARE) sequence. Among the ARE-containing genes, miR-659-3p mimic and inhibitor specifically modified the expression of AKT3, BCL2, CYR61 and THSB2, belonging to the focal adhesion pathway. Most importantly, in BM-infiltrating cells CNOT1 expression was significantly higher, and that of AKT3, BCL2, THSB2 and CYR61 was significantly lower than in primary tumors. Thus, our study suggests a role of the focal adhesion pathway, regulated by miR-659-3p through CNOT1, in the human NB metastatic process. PMID:25980492

  10. ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

    PubMed Central

    Henríquez, Mauricio; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Alvarez, Alvaro; Kong, Milene; Muñoz, Nicolás; Eisner, Verónica; Jaimovich, Enrique; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

    2011-01-01

    Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion. PMID:21502139

  11. Epac Activation Regulates Human Mesenchymal Stem Cells Migration and Adhesion.

    PubMed

    Yu, Jiao-Le; Deng, Ruixia; Chung, Sookja K; Chan, Godfrey Chi-Fung

    2016-04-01

    How to enhance the homing of human mesenchymal stem cells (hMSCs) to the target tissues remains a clinical challenge nowadays. To overcome this barrier, the mechanism responsible for the hMSCs migration and engraftment has to be defined. Currently, the exact mechanism involved in migration and adhesion of hMSCs remains unknown. Exchange protein directly activated by cAMP (Epac), a novel protein discovered in cAMP signaling pathway, may have a potential role in regulating cells adhesion and migration by triggering the downstream Rap family signaling cascades. However, the exact role of Epac in cells homing is elusive. Our study evaluated the role of Epac in the homing of hMSCs. We confirmed that hMSCs expressed functional Epac and its activation enhanced the migration and adhesion of hMSCs significantly. The Epac activation was further found to be contributed directly to the chemotactic responses induced by stromal cell derived factor-1 (SDF-1) which is a known chemokine in regulating hMSCs homing. These findings suggested Epac is connected to the SDF-1 signaling cascades. In conclusion, our study revealed that Epac plays a role in hMSCs homing by promoting adhesion and migration. Appropriate manipulation of Epac may enhance the homing of hMSCs and facilitate their future clinical applications. Stem Cells 2016;34:948-959. PMID:26727165

  12. [Site and propagation of focal epileptic activity: multichannel MEG/EEG analysis].

    PubMed

    Stefan, H; Abraham-Fuchs, K; Schüler, P; Schneider, S; Neubauer, P U; Huk, H J; Neundörfer, B

    1991-12-01

    Electrophysiological examinations provide the basis for a deeper pathophysiological understanding of focal epileptic activity. In addition to electroencephalography, magnetoencephalography from field measurements is now available for biomagnetic diagnosis. As magnetoencephalography (MEG) is basically better suited for the localization of focal epileptic activity than EEG, an increase in MEG measurements has taken place over the last years. In this study we discuss magnetic source localization which was combined with anatomical 3-D-MR-images and compared with the results of EEG-registration carried out simultaneously and with other investigative procedures of presurgical diagnosis. The results of investigation show that simultaneous magnetic field measurements over one hemisphere of the skull allow localization of sources both in the temporal lobe and in deeper areas of the brain. Furthermore, propagation of epileptic activity can be registered not only in neighbouring areas of the epileptogenic source but also in regions localized deeper in the temporal lobe. This opens new possibilities for presurgical evaluation as well as an understanding of partial and generalized epilepsies. The results of investigation show primary focal epileptic activity neocortex laterally or surrounding a mesio-temporal lesion in all investigated patients with partial (temporal, frontal) and secondary generalized epilepsies. Furthermore, a pattern of propagation of focal epileptic activity which is directed from neocortical-lateral to mediobasal-limbic brain structures is found in most of these patients. PMID:1795752

  13. A ternary complex comprising FAK, PTPα and IP3 receptor 1 functionally engages focal adhesions and the endoplasmic reticulum to mediate IL-1-induced Ca2+ signalling in fibroblasts.

    PubMed

    Wang, Qin; Wang, Yongqiang; Downey, Gregory P; Plotnikov, Sergey; McCulloch, Christopher A

    2016-02-15

    Ca(2+) release is tightly sequestered in eukaryotic cells to enable fine spatio-temporal control of signalling but how Ca(2+) release from the endoplasmic reticulum (ER) is linked to cell adhesions is not defined. We examined the spatial restriction of Ca(2+) release through the inositol 1,4,5-triphosphate receptor 1 (IP3R1) in response to interleukin-1 (IL-1) and the functions of the adhesion-associated proteins, focal adhesion kinase (FAK) and protein tyrosine phosphatase-α (PTPα). In cultured fibroblasts IL-1 treatment promoted co-localization of PTPα and FAK with the ER and increased association of IP3R1 with PTPα and FAK at focal adhesions (FAs). GST pull-down assays of purified proteins demonstrated that PTPα and FAK directly interacted with IP3R1. These interactions depended on the focal adhesion-targeting (FAT) and band4.1-ezrin-radixin-moesin (FERM) domains of FAK. PTPα was required for the association of IP3R1 with Src, which mediated IP3R1 phosphorylation and consequently ER Ca(2+) release. Collectively, these data indicate that PTPα and FAK, which are enriched in FAs, interact with IP3R1 at adjacent ER sites to spatially sequester IL-1-induced Ca(2+) signalling. PMID:26611753

  14. Complement Activation in Patients with Focal Segmental Glomerulosclerosis

    PubMed Central

    Thurman, Joshua M.; Wong, Maria; Renner, Brandon; Frazer-Abel, Ashley; Giclas, Patricia C.; Joy, Melanie S.; Jalal, Diana; Radeva, Milena K.; Gassman, Jennifer; Gipson, Debbie S.; Kaskel, Frederick; Friedman, Aaron; Trachtman, Howard

    2015-01-01

    Background Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process. Study Design Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed. Setting and Participants We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis. Outcomes Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR). Measurements Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine. Results Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study. Limitations Limited number of patients with samples from all time-points. Conclusions The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of

  15. Monocyte Trafficking to Hepatic Sites of Bacterial Infection Is Chemokine Independent and Directed by Focal Intercellular Adhesion Molecule-1 Expression

    PubMed Central

    Shi, Chao; Velázquez, Peter; Hohl, Tobias M.; Leiner, Ingrid; Dustin, Michael L.; Pamer, Eric G.

    2010-01-01

    Recruitment of CCR2+Ly6Chigh monocytes to sites of infection is essential for efficient clearance of microbial pathogens. Although CCR2-mediated signals promote monocyte emigration from bone marrow, the contribution of CCR2 to later stages of monocyte recruitment remains unresolved. In this article, we show that CCR2 deficiency markedly worsens hepatic Listeria monocytogenes infection because Ly6Chigh monocytes are retained in the bone marrow. Intravenously transferred, CCR2-deficient Ly6Chigh monocytes traffic normally to hepatic foci of infection and contribute to bacterial clearance. Pertussis toxin treatment of adoptively transferred monocytes does not impair their intrahepatic trafficking, suggesting that chemokine signaling, once CCR2+ Ly6Chigh monocytes emigrate from the bone marrow, is not required for monocyte localization to sites of bacterial infection in the liver. Expression of ICAM-1 is induced in close proximity to foci of bacterial infection in the liver, including on CD31+ endothelial cells, and blockade of CD11b and CD44 diminishes monocyte localization to these hepatic foci. Our studies demonstrated that Ly6Chigh monocyte recruitment from the bloodstream to the L. monocytogenes-infected liver does not require chemokine receptor-mediated signals but instead is principally dependent on integrin- and extracellular matrix-mediated monocyte adhesion. PMID:20435926

  16. Nascent Integrin Adhesions Form on All Matrix Rigidities after Integrin Activation.

    PubMed

    Changede, Rishita; Xu, Xiaochun; Margadant, Felix; Sheetz, Michael P

    2015-12-01

    Integrin adhesions assemble and mature in response to ligand binding and mechanical factors, but the molecular-level organization is not known. We report that ∼100-nm clusters of ∼50 β3-activated integrins form very early adhesions under a wide variety of conditions on RGD surfaces. These adhesions form similarly on fluid and rigid substrates, but most adhesions are transient on rigid substrates. Without talin or actin polymerization, few early adhesions form, but expression of either the talin head or rod domain in talin-depleted cells restores early adhesion formation. Mutation of the integrin binding site in the talin rod decreases cluster size. We suggest that the integrin clusters constitute universal early adhesions and that they are the modular units of cell matrix adhesions. They require the association of activated integrins with cytoplasmic proteins, in particular talin and actin, and cytoskeletal contraction on them causes adhesion maturation for cell motility and growth. PMID:26625956

  17. Dynamic Regulation of Activated Leukocyte Cell Adhesion Molecule–mediated Homotypic Cell Adhesion through the Actin CytoskeletonV⃞

    PubMed Central

    Nelissen, Judith M. D. T.; Peters, Inge M.; de Grooth, Bart G.; van Kooyk, Yvette; Figdor, Carl G.

    2000-01-01

    Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM–ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM–ALCAM interactions. This induction of cell adhesion is likely due to clustering of ALCAM at the cell surface, which is observed after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after CytD treatment. In contrast, both surface distribution and adhesion of a glycosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to CytD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatment. This demonstrates that clustering of ALCAM is essential for cell adhesion, whereas enhanced diffusion of ALCAM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the freely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduced over time, suggesting strengthening of the cytoskeleton linkage. From these findings we conclude that activation of ALCAM-mediated adhesion is dynamically regulated through actin cytoskeleton-dependent clustering. PMID:10848629

  18. Active Focal Zone Sharpening for High-Precision Treatment Using Histotripsy

    PubMed Central

    Wang, Tzu-Yin; Xu, Zhen; Hall, Timothy L.; Fowlkes, J. Brian; Roberts, William W.; Cain, Charles A.

    2011-01-01

    The goal of this study is to develop a focal zone sharpening strategy that produces more precise lesions for pulsed cavitational ultrasound therapy, or histotripsy. Precise and well-confined lesions were produced by locally suppressing cavitation in the periphery of the treatment focus without affecting cavitation in the center. The local suppression of cavitation was achieved using cavitation nuclei preconditioning pulses to actively control cavitation in the periphery of the focus. A 1-MHz 513-element therapeutic array was used to generate both the therapy and the nuclei preconditioning pulses. For therapy, 10-cycle bursts at 100-Hz pulse repetition frequency with P−/P+ pressure of 21/76 MPa were delivered to the geometric focus of the therapeutic array. For nuclei preconditioning, a different pulse was delivered to an annular region immediately surrounding the focus before each therapy pulse. A parametric study on the effective pressure, pulse duration, and delivery time of the preconditioning pulse was conducted in red blood cell-gel phantoms, where cavitational damage was indicated by the color change resulting from local cell lysis. Results showed that a short-duration (20 µs) preconditioning pulse at a medium pressure (P−/P+ pressure of 7.2/13.6 MPa) delivered shortly before (30 µs) the therapy pulse substantially suppressed the peripheral damage by 77 ± 13% while complete fractionation in the focal center was maintained. High-speed imaging of the bubble cloud showed a substantial decrease in the maximum width of the bubble cloud by 48 ± 24% using focal zone sharpening. Experiments in ex vivo livers confirmed that highly confined lesions were produced in real tissues as well as in the phantoms. This study demonstrated the feasibility of active focal zone sharpening using cavitation nuclei preconditioning, allowing for increased treatment precision compared with the natural focal width of the therapy transducer. PMID:21342816

  19. The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

    PubMed Central

    Schiefermeier, Natalia; Scheffler, Julia M.; de Araujo, Mariana E.G.; Stasyk, Taras; Yordanov, Teodor; Ebner, Hannes L.; Offterdinger, Martin; Munck, Sebastian; Hess, Michael W.; Wickström, Sara A.; Lange, Anika; Wunderlich, Winfried; Fässler, Reinhard; Teis, David

    2014-01-01

    Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14–MP1 (LAMTOR2/3) complex in FA dynamics. p14–MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end–directed traffic of p14–MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14–MP1 scaffold complex to the vicinity of FAs. PMID:24841562

  20. Inhibition of the focal adhesion kinase and vascular endothelial growth factor receptor-3 interaction leads to decreased survival in human neuroblastoma cell lines.

    PubMed

    Beierle, Elizabeth A; Ma, Xiaojie; Stewart, Jerry E; Megison, Michael; Cance, William G; Kurenova, Elena V

    2014-03-01

    Neuroblastoma continues to be a devastating childhood solid tumor and is responsible for over 15% of all childhood cancer-related deaths. Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in a number of human cancers, including neuroblastoma. These two kinases can directly interact and provide survival signals to cancer cells. In this study, we utilized siRNA to VEGFR-3 to demonstrate the biologic importance of this kinase in neuroblastoma cell survival. We also used confocal microscopy and immunoprecipitation to show that FAK and VEGFR-3 bind in neuroblastoma. Finally, employing a 12-amino-acid peptide (AV3) specific to VEGFR-3, we showed that the colocalization between FAK and VEGFR-3 could be disrupted, and that disruption resulted in decreased neuroblastoma cell survival. These studies provide insight to the FAK-VEGFR-3 interaction in neuroblastoma and demonstrate its importance in this tumor type. Focusing upon the FAK-VEGFR-3 interaction may provide a novel therapeutic target for the development of new strategies for treatment of neuroblastoma. PMID:23065847

  1. Adhesive disbond detection using piezoelectric wafer active sensors

    NASA Astrophysics Data System (ADS)

    Roth, William; Giurgiutiu, Victor

    2015-04-01

    The aerospace industry continues to increase the use of adhesives for structural bonding due to the increased joint efficiency (reduced weight), even distribution of the load path and decreases in stress concentrations. However, the limited techniques for verifying the strength of adhesive bonds has reduced its use on primary structures and requires an intensive inspection schedule. This paper discusses a potential structural health monitoring (SHM) technique for the detection of disbonds through the in situ inspection of adhesive joints. This is achieved through the use of piezoelectric wafer active sensors (PWAS), thin unobtrusive sensors which are permanently bonded to the aircraft structure. The detection method discussed in this study is electromechanical impedance spectroscopy (EMIS), a local vibration method. This method detects disbonds from the change in the mechanical impedance of the structure surrounding the disbond. This paper will discuss how predictive modeling can provide valuable insight into the inspection method, and provide better results than empirical methods alone. The inspection scheme was evaluated using the finite element method, and the results were verified experimentally using a large aluminum test article, and included both pristine and disbond coupons.

  2. Immunocytochemical analysis of proliferative activity of endometrial and myometrial cell populations in focal and stromal adenomyosis.

    PubMed

    Nepomnyashchikh, L M; Lushnikova, E L; Molodykh, O P; Pichigina, A K

    2013-08-01

    Immunocytochemical study has shown that Ki-67 antigen is detected in adenomyosis in both endometrial and myometrial cell populations (in the eutopic and ectopic endometrial glandular epithelium, stromal cells, smooth muscle cells, and vascular endotheliocytes of the endometrium and myometrium), the label index differing significantly in different cell populations. The highest labeled cell index is found in the endometrial gland epitheliocytes in focal adenomyosis (23.2 ± 2.9%); in the stromal variant this index is by 2.8 times lower despite the fact that this variant is associated with endometrial glandular hyperplasia in the majority of cases. Proliferative activity of secretory epitheliocytes is significantly lower in both adenomyosis variants than in the normal eutopic endometrium. Stromal adenomyosis is characterized by 2-fold higher proliferative activity of the cytogenic stroma than that in focal adenomyosis. PMID:24143380

  3. Src and FAK mediate cell-matrix adhesion-dependent activation of Met during transformation of breast epithelial cells.

    PubMed

    Hui, Angela Y; Meens, Jalna A; Schick, Colleen; Organ, Shawna L; Qiao, Hui; Tremblay, Eric A; Schaeffer, Erik; Uniyal, Shashi; Chan, Bosco M C; Elliott, Bruce E

    2009-08-15

    Cell-matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand-independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non-neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto-activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with beta1 integrins and is co-localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src-dependent phosphorylation of Met requires cell-matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src-induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF-dependent and Src/integrin-dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells. PMID:19533669

  4. Focal Adhesion Kinase Signaling Mediated the Enhancement of Osteogenesis of Human Mesenchymal Stem Cells Induced by Extracorporeal Shockwave

    PubMed Central

    Hu, Jun; Liao, Haojie; Ma, Zebin; Chen, Hongjiang; Huang, Zhonglian; Zhang, Yuantao; Yu, Menglei; Chen, Youbin; Xu, Jiankun

    2016-01-01

    Extracorporeal shockwave (ESW) has been shown of great potential in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but it is unknown whether this osteogenic promotion effect can also be achieved in other MSCs (i.e., tendon-derived stem cells (TDSCs) and adipose-derived stem cells (ADSCs)). In the current study, we aimed not only to compare the osteogenic effects of BMSCs induced by ESW to those of TDSCs and ADSCs; but also to investigate the underlying mechanisms. We show here that ESW (0.16 mj/mm2) significantly promoted the osteogenic differentiation in all the tested types of MSCs, accompanied with the downregulation of miR-138, but the activation of FAK, ERK1/2, and RUNX2. The enhancement of osteogenesis in these MSCs was consistently abolished when the cells were pretreated with one of the following conditions: overexpression of miR-138, FAK knockdown using specific siRNA, and U0126, implying that all of these elements are indispensable for mediating the effect of ESW. Moreover, our study provides converging genetic and molecular evidence that the miR-138-FAK-ERK1/2-RUNX2 machinery can be generally activated in ESW-preconditioned MSCs, suggesting that ESW may be a promising therapeutic strategy for the enhancement of osteogenesis of MSCs, regardless of their origins. PMID:26863924

  5. Focal Adhesion Kinase Signaling Mediated the Enhancement of Osteogenesis of Human Mesenchymal Stem Cells Induced by Extracorporeal Shockwave

    NASA Astrophysics Data System (ADS)

    Hu, Jun; Liao, Haojie; Ma, Zebin; Chen, Hongjiang; Huang, Zhonglian; Zhang, Yuantao; Yu, Menglei; Chen, Youbin; Xu, Jiankun

    2016-02-01

    Extracorporeal shockwave (ESW) has been shown of great potential in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but it is unknown whether this osteogenic promotion effect can also be achieved in other MSCs (i.e., tendon-derived stem cells (TDSCs) and adipose-derived stem cells (ADSCs)). In the current study, we aimed not only to compare the osteogenic effects of BMSCs induced by ESW to those of TDSCs and ADSCs; but also to investigate the underlying mechanisms. We show here that ESW (0.16 mj/mm2) significantly promoted the osteogenic differentiation in all the tested types of MSCs, accompanied with the downregulation of miR-138, but the activation of FAK, ERK1/2, and RUNX2. The enhancement of osteogenesis in these MSCs was consistently abolished when the cells were pretreated with one of the following conditions: overexpression of miR-138, FAK knockdown using specific siRNA, and U0126, implying that all of these elements are indispensable for mediating the effect of ESW. Moreover, our study provides converging genetic and molecular evidence that the miR-138-FAK-ERK1/2-RUNX2 machinery can be generally activated in ESW-preconditioned MSCs, suggesting that ESW may be a promising therapeutic strategy for the enhancement of osteogenesis of MSCs, regardless of their origins.

  6. Changes in focal adhesion kinase expression in rats with collagen-induced arthritis and efficacy of intervention with disease modifying anti-rheumatic drugs alone or in combination

    PubMed Central

    Gao, Hui-Ying; Luo, Jing; Li, Xiao-Feng; Lv, Qian; Wen, Hong-Yan; Song, Qing-Zhen; Zhao, Wen-Peng; Zhao, Xiang-Cong; Zhang, Ting-Ting; Zhang, Si-Yu; Zhi, Jian-Ming

    2015-01-01

    Focal adhesion kinase (FAK) is known to promote the proliferation, migration and survival of synovial cells and plays an important role in the occurrence, development and pathological process of rheumatoid arthritis (RA). The aim of the present study was to observe FAK changes in synovial cells of rats with collagen-induced arthritis (CIA) and after intervention with disease modifying anti-rheumatic drugs (DMARDs) alone or in combination in a CIA female SD rat model induced by collagen type II. The rats were randomized to 8 groups: normal control group, CIA model control group, methotrexate (MTX, 0.9 mg/kg/w) group, cyclophosphamide (CTX, 24 mg/kg/3 w) group, leflunomide (LEF, 1.2 mg/kg/d) group, MTX + CTX group, LEF + CTX group, and MTX + LEF group. They were intervened with DMARDs alone or in combination for six weeks. The experiment lasted a total of 9 weeks in vivo. Articular inflammation was measured during the process of drug intervention in terms of the degree of swelling degree in the right hind foot using a venire caliper. All animals were sacrificed by breaking the neck after 9 weeks. Then, the ankle was fixed, decalcified, embedded, and HE stained, and prepared into slices to observe pathological changes in the synovial tissue. FAK expression in synovial cells was assayed by immunohistochemistry and the mean optical density (OD) value was measured using the HPIAS-2000 image analysis system. It was found that FAK expression was negative in normal control group, positive in CIA model control group, and decreased in the three DMARD combination treatment groups significantly as compared with that in the three single-drug groups (P < 0.05). FAK expression in LEF + CTX group or MTX + CTX group decreased more significantly than that in MTX + LEF group (P < 0.05), and there was no statistically significant difference between LEF + CTX and MTX + CTX groups. The arthritis index and pathological change in the synovial tissue in LEF + CTX group or MTX + CTX group

  7. Alterations in Daytime and Nighttime Activity in Piglets after Focal and Diffuse Brain Injury.

    PubMed

    Olson, Emily; Badder, Carlie; Sullivan, Sarah; Smith, Colin; Propert, Kathleen; Margulies, Susan S

    2016-04-15

    We have developed and implemented a noninvasive, objective neurofunctional assessment for evaluating the sustained effects of traumatic brain injury (TBI) in piglets with both diffuse and focal injury types. Derived from commercial actigraphy methods in humans, this assessment continuously monitors the day/night activity of piglets using close-fitting jackets equipped with tri-axial accelerometers to monitor movements of the thorax. Acceleration metrics were correlated (N = 7 naïve piglets) with video images to define values associated with a range of activities, from recumbancy (rest) to running. Both focal (N = 8) and diffuse brain injury (N = 9) produced alterations in activity that were significant 4 days post-TBI. Compared to shams (N = 6) who acclimated to the animal facility 4 days after an anesthesia experience by blurring the distinction between day and night activity, post-TBI time-matched animals had larger fractions of inactive periods during the daytime than nighttime, and larger fractions of active time in the night were spent in high activity (e.g., constant walking, intermittent running) than during the day. These persistent disturbances in rest and activity are similar to those observed in human adults and children post-TBI, establishing actigraphy as a translational metric, used in both humans and large animals, for assessment of injury severity, progressions, and intervention. PMID:26414329

  8. Divergent modulation of Rho‐kinase and Ca2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle

    PubMed Central

    Shaifta, Yasin; Irechukwu, Nneka; Prieto‐Lloret, Jesus; MacKay, Charles E; Marchon, Keisha A; Ward, Jeremy P T

    2015-01-01

    Background and Purpose The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src‐family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR‐mediated ASM contraction and associated signalling events. Experimental Approach Contraction was recorded in intact or α‐toxin permeabilized rat bronchioles. Phosphorylation of SrcFK, FAK, myosin light‐chain‐20 (MLC20) and myosin phosphatase targeting subunit‐1 (MYPT‐1) was evaluated in cultured human ASM cells (hASMC). [Ca2+]i was evaluated in Fura‐2 loaded hASMC. Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined. Key Results Contractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho‐kinase, while after α‐toxin permeabilization, they were sensitive to inhibition of SrcFK and Rho‐kinase, but not FAK. CCh and BK increased phosphorylation of MYPT‐1 and MLC20 and auto‐phosphorylation of SrcFK and FAK. MYPT‐1 phosphorylation was sensitive to inhibition of Rho‐kinase and SrcFK, but not FAK. Contraction induced by SR Ca2+ depletion and equivalent [Ca2+]i responses in hASMC were sensitive to inhibition of both SrcFK and FAK, while depolarization‐induced contraction was sensitive to FAK inhibition only. SrcFK auto‐phosphorylation was partially FAK‐dependent, while FAK auto‐phosphorylation was SrcFK‐independent. Conclusions and Implications SrcFK mediates Ca2+‐sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca2+ influx pathways. Tyrosine phosphorylation is therefore a key upstream signalling event in ASM contraction and may be a viable target for modulating ASM tone in respiratory disease. PMID:26294392

  9. Janus kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction.

    PubMed Central

    Girault, J. A.; Labesse, G.; Mornon, J. P.; Callebaut, I.

    1998-01-01

    The band 4.1 domain was first identified in the red blood cell protein band 4.1, and subsequently in ezrin, radixin, and moesin (ERM proteins) and other proteins, including tumor suppressor merlin/schwannomin, talin, unconventional myosins VIIa and X, and protein tyrosine phosphatases. Recently, the presence of a structurally related domain has been demonstrated in the N-terminal region of two groups of tyrosine kinases: the focal adhesion kinases (FAK) and the Janus kinases (JAK). Additional proteins containing the 4.1/JEF (JAK, ERM, FAK) domain include plant kinesin-like calmodulin-binding proteins (KCBP) and a number of uncharacterized open reading frames identified by systematic DNA sequencing. Phylogenetic analysis of amino acid sequences suggests that band 4.1/JEF domains can be grouped in several families that have probably diverged early during evolution. Hydrophobic cluster analysis indicates that the band 4.1/JEF domains might consist of a duplicated module of approximately 140 residues and a central hinge region. A conserved property of the domain is its capacity to bind to the membrane-proximal region of the C-terminal cytoplasmic tail of proteins with a single transmembrane segment. Many proteins with band 4.1/JEF domains undergo regulated intra- or intermolecular homotypic interactions. Additional properties common to band 4.1/JEF domains of several proteins are binding of phosphoinositides and regulation by GTPases of the Rho family. Many proteins with band 4. 1/JEF domains are associated with the actin-based cytoskeleton and are enriched at points of contact with other cells or the extracellular matrix, from which they can exert control over cell growth. Thus, proteins with band 4.1/JEF domain are at the crossroads between cytoskeletal organization and signal transduction in multicellular organisms. Their importance is underlined by the variety of diseases that can result from their mutations. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:9990861

  10. Airway Hyperresponsiveness in Asthma Model Occurs Independently of Secretion of β1 Integrins in Airway Wall and Focal Adhesions Proteins Down Regulation.

    PubMed

    Álvarez-Santos, Mayra; Carbajal, Verónica; Tellez-Jiménez, Olivia; Martínez-Cordero, Erasmo; Ruiz, Victor; Hernández-Pando, Rogelio; Lascurain, Ricardo; Santibañez-Salgado, Alfredo; Bazan-Perkins, Blanca

    2016-10-01

    The extracellular domains of some membrane proteins can be shed from the cell. A similar phenomenon occurs with β1 integrins (α1β1 and α2β1) in guinea pig. The putative role of β1 integrin subunit alterations due to shedding in airway smooth muscle (ASM) in an allergic asthma model was evaluated. Guinea pigs were sensitized and challenged with antigen. Antigenic challenges induced bronchoobstruction and hyperresponsiveness at the third antigenic challenge. Immunohistochemistry and immunoelectronmicroscopy studies showed that the cytosolic and extracellular domains of the β1 integrin subunit shared the same distribution in airway structures in both groups. Various polypeptides with similar molecular weights were detected with both the cytosolic and extracellular β1 integrin subunit antibodies in isolated airway myocytes and the connective tissue that surrounds the ASM bundle. Flow cytometry and Western blot studies showed that the expression of cytosolic and extracellular β1 integrin subunit domains in ASM was similar between groups. An increment of ITGB1 mRNA in ASM was observed in the asthma model group. RACE-PCR of ITGB1 in ASM did not show splicing variants. The expression levels of integrin-linked kinase (ILK) and paxillin diminished in the asthma model, but not talin. The levels of phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr(696) increased in asthma model. Our work suggests that β1 integrin is secreted in guinea pig airway wall. This secretion is not altered in asthma model; nevertheless, β1 integrin cytodomain assembly proteins in focal cell adhesions in which ILK and paxillin are involved are altered in asthma model. J. Cell. Biochem. 117: 2385-2396, 2016. © 2016 Wiley Periodicals, Inc. PMID:26969873

  11. P130Cas Src-Binding and Substrate Domains Have Distinct Roles in Sustaining Focal Adhesion Disassembly and Promoting Cell Migration

    PubMed Central

    Meenderink, Leslie M.; Ryzhova, Larisa M.; Donato, Dominique M.; Gochberg, Daniel F.; Kaverina, Irina; Hanks, Steven K.

    2010-01-01

    The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas −/− mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation. PMID:20976150

  12. Role of Suspended Fiber Structural Stiffness and Curvature on Single-Cell Migration, Nucleus Shape, and Focal-Adhesion-Cluster Length

    PubMed Central

    Meehan, Sean; Nain, Amrinder S.

    2014-01-01

    It has been shown that cellular migration, persistence, and associated cytoskeletal arrangement are highly dependent on substrate stiffness (modulus: N/m2 and independent of geometry), but little is known on how cells respond to subtle changes in local geometry and structural stiffness (N/m). Here, using fibers of varying diameter (400, 700, and 1200 nm) and length (1 and 2 mm) deposited over hollow substrates, we demonstrate that single mouse C2C12 cells attached to single suspended fibers form spindle morphologies that are sensitive to fiber mechanical properties. Over a wide range of increasing structural stiffness (2 to 100+ mN/m), cells exhibited decreases in migration speed and average nucleus shape index of ∼57% (from 58 to 25 μm/h) and ∼26% (from 0.78 to 0.58), respectively, whereas the average paxillin focal-adhesion-cluster (FAC, formed at poles) length increased by ∼38% (from 8 to 11 μm). Furthermore, the increase in structural stiffness directly correlates with cellular persistence, with 60% of cells moving in the direction of increasing structural stiffness. At similar average structural stiffness (25 ± 5 mN/m), cells put out longer FAC lengths on smaller diameters, suggesting a conservation of FAC area, and also exhibited higher nucleus shape index and migration speeds on larger-diameter fibers. Interestingly, cells were observed to deform fibers locally or globally through forces applied through the FAC sites and cells undergoing mitosis were found to be attached to the FAC sites by single filamentous tethers. These varied reactions have implications in developmental and disease biology models as they describe a strong dependence of cellular behavior on the cell’s immediate mechanistic environment arising from alignment and geometry of fibers. PMID:25468339

  13. Activation of the canonical Wnt/{beta}-catenin pathway enhances monocyte adhesion to endothelial cells

    SciTech Connect

    Lee, Dong Kun . E-mail: leedk@memorialhealthsource.com; Nathan Grantham, R.; Trachte, Aaron L.; Mannion, John D.; Wilson, Colleen L.

    2006-08-18

    Monocyte adhesion to vascular endothelium has been reported to be one of the early processes in the development of atherosclerosis. In an attempt to develop strategies to prevent or delay atherosclerosis progression, we analyzed effects of the Wnt/{beta}-catenin signaling pathway on monocyte adhesion to various human endothelial cells. Adhesion of fluorescein-labeled monocytes to various human endothelial cells was analyzed under a fluorescent microscope. Unlike sodium chloride, lithium chloride enhanced monocyte adhesion to endothelial cells in a dose-dependent manner. We further demonstrated that inhibitors for glycogen synthase kinase (GSK)-3{beta} or proteosome enhanced monocyte-endothelial cell adhesion. Results of semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that activation of Wnt/{beta}-catenin pathway did not change expression levels of mRNA for adhesion molecules. In conclusion, the canonical Wnt/{beta}-catenin pathway enhanced monocyte-endothelial cell adhesion without changing expression levels of adhesion molecules.

  14. Absence status associated with focal activity and polydipsia-induced hyponatremia

    PubMed Central

    Azuma, Hideki; Akechi, Tatsuo; Furukawa, Toshi A

    2008-01-01

    We report a case of de novo absence status associated with focal discharge and polydipsia-induced hyponatremia. Nonconvulsive status epilepticus (NCSE) is classified as absence status or complex partial status. Absence status is characterized by bilateral synchronized spike and wave complex bursts and a variety of conscious disturbances. Possible precipitating factors for NCSE include benzodiazepine withdrawal, excessive use of psychotropic drugs, and electrolyte imbalances. Hyponatremia is a rare precipitating factor. In this case, the patient was 59 years old and had suffered from primary insomnia but had no history of epilepsy. NCSE improved by means of saline infusion. However after recovery from NCSE EEG revealed some spikes in the left frontal area. Absence seizures can also show generalized spike and slow waves, and cases of focal lesion-associated absence seizures have been reported. Although absence seizures and absence status are two distinct conditions, they should not be considered together. We assumed that hyponatremia induced by polydipsia precipitated epileptogenicity in the left frontal area, and then focal activity secondarily generalized and resulted in absence status. PMID:18728738

  15. LXW7 ameliorates focal cerebral ischemia injury and attenuates inflammatory responses in activated microglia in rats

    PubMed Central

    Fang, T.; Zhou, D.; Lu, L.; Tong, X.; Wu, J.; Yi, L.

    2016-01-01

    Inflammation plays a pivotal role in ischemic stroke, when activated microglia release excessive pro-inflammatory mediators. The inhibition of integrin αvβ3 improves outcomes in rat focal cerebral ischemia models. However, the mechanisms by which microglia are neuroprotective remain unclear. This study evaluated whether post-ischemic treatment with another integrin αvβ3 inhibitor, the cyclic arginine-glycine-aspartic acid (RGD) peptide-cGRGDdvc (LXW7), alleviates cerebral ischemic injury. The anti-inflammatory effect of LXW7 in activated microglia within rat focal cerebral ischemia models was examined. A total of 108 Sprague-Dawley rats (250–280 g) were subjected to middle cerebral artery occlusion (MCAO). After 2 h, the rats were given an intravenous injection of LXW7 (100 μg/kg) or phosphate-buffered saline (PBS). Neurological scores, infarct volumes, brain water content (BWC) and histology alterations were determined. The expressions of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)], and Iba1-positive activated microglia, within peri-ischemic brain tissue, were assessed with ELISA, western blot and immunofluorescence staining. Infarct volumes and BWC were significantly lower in LXW7-treated rats compared to those in the MCAO + PBS (control) group. The LXW7 treatment lowered the expression of pro-inflammatory cytokines. There was a reduction of Iba1-positive activated microglia, and the TNF-α and IL-1β expressions were attenuated. However, there was no difference in the Zea Longa scores between the ischemia and LXW7 groups. The results suggest that LXW7 protected against focal cerebral ischemia and attenuated inflammation in activated microglia. LXW7 may be neuroprotective during acute MCAO-induced brain damage and microglia-related neurodegenerative diseases. PMID:27533766

  16. Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials

    NASA Astrophysics Data System (ADS)

    Lee, Ted T.; García, José R.; Paez, Julieta I.; Singh, Ankur; Phelps, Edward A.; Weis, Simone; Shafiq, Zahid; Shekaran, Asha; Del Campo, Aránzazu; García, Andrés J.

    2015-03-01

    Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

  17. Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials.

    PubMed

    Lee, Ted T; García, José R; Paez, Julieta I; Singh, Ankur; Phelps, Edward A; Weis, Simone; Shafiq, Zahid; Shekaran, Asha; Del Campo, Aránzazu; García, Andrés J

    2015-03-01

    Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials. PMID:25502097

  18. Light-triggered in vivo Activation of Adhesive Peptides Regulates Cell Adhesion, Inflammation and Vascularization of Biomaterials

    PubMed Central

    Lee, Ted T.; García, José R.; Paez, Julieta; Singh, Ankur; Phelps, Edward A.; Weis, Simone; Shafiq, Zahid; Shekaran, Asha; del Campo, Aránzazu; García, Andrés J.

    2014-01-01

    Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have been recently realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials. PMID:25502097

  19. Noninvasive transcranial focal stimulation via tripolar concentric ring electrodes lessens behavioral seizure activity of recurrent pentylenetetrazole administrations in rats.

    PubMed

    Makeyev, Oleksandr; Luna-Munguía, Hiram; Rogel-Salazar, Gabriela; Liu, Xiang; Besio, Walter G

    2013-05-01

    Epilepsy affects approximately 1% of the world population. Antiepileptic drugs are ineffective in approximately 30% of patients and have side effects. We have been developing a noninvasive transcranial focal electrical stimulation with our novel tripolar concentric ring electrodes as an alternative/complementary therapy for seizure control. In this study we demonstrate the effect of focal stimulation on behavioral seizure activity induced by two successive pentylenetetrazole administrations in rats. Seizure onset latency, time of the first behavioral change, duration of seizure, and maximal seizure severity score were studied and compared for focal stimulation treated (n = 9) and control groups (n = 10). First, we demonstrate that no significant difference was found in behavioral activity for focal stimulation treated and control groups after the first pentylenetetrazole administration. Next, comparing first and second pentylenetetrazole administrations, we demonstrate there was a significant change in behavioral activity (time of the first behavioral change) in both groups that was not related to focal stimulation. Finally, we demonstrate focal stimulation provoking a significant change in seizure onset latency, duration of seizure, and maximal seizure severity score. We believe that these results, combined with our previous reports, suggest that transcranial focal stimulation may have an anticonvulsant effect. PMID:22692938

  20. Temperatures Achieved in Human and Canine Neocortex During Intraoperative Passive or Active Focal Cooling

    PubMed Central

    Han, Rowland H.; Yarbrough, Chester K.; Patterson, Edward E.; Yang, Xiao-Feng; Miller, John W.; Rothman, Steven M.; D'Ambrosio, Raimondo

    2015-01-01

    Focal cortical cooling inhibits seizures and prevents acquired epileptogenesis in rodents. To investigate the potential clinical utility of this treatment modality, we examined the thermal characteristics of canine and human brain undergoing active and passive surface cooling in intraoperative settings. Four patients with intractable epilepsy were treated in a standard manner. Before the resection of a neocortical epileptogenic focus, multiple intraoperative studies of active (custom-made cooled irrigation-perfused grid) and passive (stainless steel probe) cooling were performed. We also actively cooled the neocortices of two dogs with perfused grids implanted for 2 hours. Focal surface cooling of the human brain causes predictable depth-dependent cooling of the underlying brain tissue. Cooling of 0.6–2°C was achieved both actively and passively to a depth of 10–15 mm from the cortical surface. The perfused grid permitted comparable and persistent cooling of canine neocortex when the craniotomy was closed. Thus, the human cortex can easily be cooled with the use of simple devices such as a cooling grid or a small passive probe. These techniques provide pilot data for the design of a permanently implantable device to control intractable epilepsy. PMID:25902001

  1. Temperatures achieved in human and canine neocortex during intraoperative passive or active focal cooling.

    PubMed

    Smyth, Matthew D; Han, Rowland H; Yarbrough, Chester K; Patterson, Edward E; Yang, Xiao-Feng; Miller, John W; Rothman, Steven M; D'Ambrosio, Raimondo

    2015-06-01

    Focal cortical cooling inhibits seizures and prevents acquired epileptogenesis in rodents. To investigate the potential clinical utility of this treatment modality, we examined the thermal characteristics of canine and human brain undergoing active and passive surface cooling in intraoperative settings. Four patients with intractable epilepsy were treated in a standard manner. Before the resection of a neocortical epileptogenic focus, multiple intraoperative studies of active (custom-made cooled irrigation-perfused grid) and passive (stainless steel probe) cooling were performed. We also actively cooled the neocortices of two dogs with perfused grids implanted for 2 hours. Focal surface cooling of the human brain causes predictable depth-dependent cooling of the underlying brain tissue. Cooling of 0.6-2°C was achieved both actively and passively to a depth of 10-15 mm from the cortical surface. The perfused grid permitted comparable and persistent cooling of canine neocortex when the craniotomy was closed. Thus, the human cortex can easily be cooled with the use of simple devices such as a cooling grid or a small passive probe. These techniques provide pilot data for the design of a permanently implantable device to control intractable epilepsy. PMID:25902001

  2. Effects of small doses of cytochalasins on fibroblasts: preferential changes of active edges and focal contacts.

    PubMed Central

    Domnina, L V; Gelfand, V I; Ivanova, O Y; Leonova, E V; Pletjushkina, O Y; Vasiliev, J M; Gelfand, I M

    1982-01-01

    The effects of low doses of cytochalasin B (2 micrograms/ml) and cytochalasin D (0.2 microgram/ml) on the spreading of normal mouse fibroblasts in culture were investigated to find out which components of cell-substrate interactions are most sensitive to alterations of the state of actin cytoskeleton. Cytochalasin B disorganized the cortical layer of actin microfilaments and caused partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Diffuse close contacts were apparently insensitive to cytochalasin B. Low doses of cytochalasin B did not inhibit the outgrowth and maintenance of lamellas at the cell periphery. However, in contrast to controls, these lamellas had no distal zones with convex outer edges and ruffles at the upper surfaces. The disappearance of these ruffling active edges was accompanied by loss of the ability to clear the surface of the lamellas from the concanavalin A receptors crosslinked by the corresponding ligand. The effects of cytochalasin D were similar to those of cytochalasin B. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of crosslinked surface receptors. Images PMID:6961447

  3. Focal Activation of Cells by Plasmon Resonance Assisted Optical Injection of Signaling Molecules

    PubMed Central

    2015-01-01

    Experimental methods for single cell intracellular delivery are essential for probing cell signaling dynamics within complex cellular networks, such as those making up the tumor microenvironment. Here, we show a quantitative and general method of interrogation of signaling pathways. We applied highly focused near-infrared laser light to optically inject gold-coated liposomes encapsulating bioactive molecules into single cells for focal activation of cell signaling. For this demonstration, we encapsulated either inositol trisphosphate (IP3), an endogenous cell signaling second messenger, or adenophostin A (AdA), a potent analogue of IP, within 100 nm gold-coated liposomes, and injected these gold-coated liposomes and their contents into the cytosol of single ovarian carcinoma cells to initiate calcium (Ca2+) release from intracellular stores. Upon optical injection of IP3 or AdA at doses above the activation threshold, we observed increases in cytosolic Ca2+ concentration within the injected cell initiating the propagation of a Ca2+ wave throughout nearby cells. As confirmed by octanol-induced inhibition, the intercellular Ca2+ wave traveled via gap junctions. Optical injection of gold-coated liposomes represents a quantitative method of focal activation of signaling cascades of broad interest in biomedical research. PMID:24877558

  4. The intermediate filament protein vimentin binds specifically to a recombinant integrin {alpha}2/{beta}1 cytoplasmic tail complex and co-localizes with native {alpha}2/{beta}1 in endothelial cell focal adhesions

    SciTech Connect

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly . E-mail: kieffer@cu.lu

    2005-04-15

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short {alpha} and {beta} cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin {alpha}2{beta}1 is a major collagen receptor but to date, only few proteins have been shown to interact with the {alpha}2 cytoplasmic tail or with the {alpha}2{beta}1 complex. In order to identify novel binding partners of a {alpha}2{beta}1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-{alpha}2 and GST-Jun {alpha}2 bound His-tagged calreticulin while GST-{beta}1 and GST-Fos {beta}1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun {alpha}2/GST-Fos {beta}1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with {alpha}v{beta}3-positive focal contacts. Here, we provide evidence that this interaction also occurs with {alpha}2{beta}1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.

  5. Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

    PubMed Central

    Strazza, Marianne; Azoulay-Alfaguter, Inbar; Pedoeem, Ariel; Mor, Adam

    2014-01-01

    T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits αL and β2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g. VLA-4 - fibronectin). PMID:24961998

  6. Noninvasive Imaging of the High Frequency Brain Activity in Focal Epilepsy Patients

    PubMed Central

    Lu, Yunfeng; Worrell, Gregory A.; Zhang, Huishi Clara; Yang, Lin; Brinkmann, Benjamin; Nelson, Cindy

    2014-01-01

    High frequency (HF) activity represents a potential biomarker of the epileptogenic zone in epilepsy patients, the removal of which is considered to be crucial for seizure-free surgical outcome. We proposed a high frequency source imaging (HFSI) approach to noninvasively image the brain sources of scalp recorded high frequency EEG activity. Both computer simulation and clinical patient data analysis were performed to investigate the feasibility of using the HFSI approach to image the sources of HF activity from noninvasive scalp EEG recordings. The HF activity was identified from high-density scalp recordings after high-pass filtering the EEG data and the EEG segments with HF activity were concatenated together to form repetitive HF activity. Independent component analysis was utilized to extract the components corresponding to the HF activity. Noninvasive EEG source imaging using realistic geometric boundary element head modeling was then applied to image the sources of the pathological HF brain activity. Five medically intractable focal epilepsy patients were studied and the estimated sources were found to be concordant with the surgical resection or intracranial recordings of the patients. The present study demonstrates, for the first time, that source imaging from the scalp HF activity could help to localize the seizure onset zone (SOZ) and provide a novel noninvasive way of studying the epileptic brain in humans. This study also indicates the potential application of studying HF activity in the pre-surgical planning of medically intractable epilepsy patients. PMID:24845275

  7. Experimental Study to Produce Multiple Focal Points of Acoustic Field for Active Path Selection of Microbubbles through Multi-bifurcation

    NASA Astrophysics Data System (ADS)

    Koda, Ren; Koido, Jun; Ito, Takumi; Mochizuki, Takashi; Masuda, Kohji; Ikeda, Seiichi; Arai, Fumihito; Miyamoto, Yoshitaka; Chiba, Toshio

    2013-07-01

    We previously reported our attempt to propel microbubbles in a flow by a primary Bjerknes force, which is a physical phenomenon where an acoustic wave pushes an obstacle along its direction of propagation. However, when ultrasound was emitted from the surface of the body, controlling bubbles in an against-flow was necessary. It is unpractical to use multiple transducers to produce the same number of focal points because single-element transducers cannot produce more than two focal points. In this study, we introduced a complex artificial blood vessel according to a capillary model and a two-dimensional (2D) array transducer to produce multiple focal points for the active control of microbubbles in an against-flow. From the results, about 15% more microbubbles were led to the desired path with multiple focal points of ultrasound relative to the no-emission case.

  8. Inhibition of tumor vasculogenic mimicry and prolongation of host survival in highly aggressive gallbladder cancers by norcantharidin via blocking the ephrin type a receptor 2/focal adhesion kinase/paxillin signaling pathway.

    PubMed

    Wang, Hui; Sun, Wei; Zhang, Wen-Zhong; Ge, Chun-Yan; Zhang, Jing-Tao; Liu, Zhong-Yan; Fan, Yue-Zu

    2014-01-01

    Vasculogenic mimicry (VM) is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2)/focal adhesion kinase (FAK)/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD) as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D) matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway. PMID:24811250

  9. MAP-kinase activity necessary for TGFbeta1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397.

    PubMed

    Hayashida, Tomoko; Wu, Ming-Hua; Pierce, Amy; Poncelet, Anne-Christine; Varga, John; Schnaper, H William

    2007-12-01

    The signals mediating transforming growth factor beta (TGFbeta)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFbeta-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFbeta1-dependent. By immunocytochemistry, TGFbeta1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFbeta-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFbeta-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFbeta-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFbeta-induced responses to regulate collagen production. PMID:18032789

  10. Imaging Active Surface Processes in Barnacle Adhesive Interfaces.

    PubMed

    Golden, Joel P; Burden, Daniel K; Fears, Kenan P; Barlow, Daniel E; So, Christopher R; Burns, Justin; Miltenberg, Benjamin; Orihuela, Beatriz; Rittshof, Daniel; Spillmann, Christopher M; Wahl, Kathryn J; Tender, Leonard M

    2016-01-19

    Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor Amphibalanus (=Balanus) amphitrite barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct

  11. Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

    SciTech Connect

    Whelan, Jarrett T.; Wang, Lei; Chen, Jianming; Metts, Meagan E.; Nasser, Taj A.; McGoldrick, Liam J.; Bridges, Lance C.

    2014-11-28

    Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH){sub 2}D{sub 3}, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.

  12. Effect of active packaging incorporated with triclosan on bacteria adhesion.

    PubMed

    Camilloto, Geany P; Pires, Ana Clarissa S; Soares, Nilda de Fátima F; Araújo, Emiliane A; Andrade, Nélio J; Ferreira, Sukarno O

    2010-10-01

    Antimicrobial polyethylene and cellulose based films incorporated with triclosan were studied. The antimicrobial efficacy, the hydrophobicity, microscopic and the mechanical characteristics of the films, as well free energy of adhesion between bacteria and antimicrobial films were evaluated. It was observed that both polyethylene and cellulose based films incorporated with the antimicrobial were homogeneous. Furthermore, the addition of triclosan did not affect mechanical characteristics of the films (P > 0.05). However, triclosan incorporated into polyethylene films reduced its hydrophobicity while antimicrobial cellulose based films became more hydrophobic. The adhesion was thermodynamically favorable between tested bacteria and polyethylene films. On the other hand, the adhesion to triclosan cellulose based film was thermodynamically unfavorable to Staphylococcus aureus and Escherichia coli and favorable to Listeria innocua and Pseudomonas aeruginosa. Polyethylene and cellulose based films showed inhibitory effect against S. aureus and E. coli, being the inhibition halo higher for polyethylene films. This study improves the knowledge about antimicrobial films. PMID:21535496

  13. Interleukin-1 activation of vascular endothelium. Effects on procoagulant activity and leukocyte adhesion.

    PubMed Central

    Bevilacqua, M. P.; Pober, J. S.; Wheeler, M. E.; Cotran, R. S.; Gimbrone, M. A.

    1985-01-01

    Interleukin-1 (IL-1), an inflammatory/immune mediator, acts directly and selectively on cultured human vascular endothelial cells to alter two important functional properties. First, IL-1 induces endothelial cell biosynthesis and surface expression of a tissue factor-like procoagulant activity. Second, IL-1 dramatically increases the adhesiveness of the endothelial cell surface for human peripheral blood polymorphonuclear leukocytes (6-42-fold increase) and monocytes (2-5-fold increase), as well as the related leukocyte cell lines HL-60 and U937. These IL-1 effects are concentration-dependent (maximum, 5-10 U/ml), time-dependent (peak 4-6 hours), and reversible. Cycloheximide and actinomycin D block these IL-1 actions on endothelium, which suggests the requirement for de novo protein synthesis. Human-monocyte-derived IL-1, cell-line--derived IL-1, and recombinant IL-1 exhibited comparable biologic activities in our assays, whereas two other mediators, IL-2 and immune interferon, were without effect. IL-1 stimulated procoagulant activity and leukocyte adhesion in human endothelial cells cultured from both umbilical veins and adult saphenous veins but not in other cultured cell types, including SV-40-transformed human endothelial cells and human dermal fibroblasts. Similar actions of IL-1 on vascular endothelium in vivo may contribute to the development of intravascular coagulation and enhanced leukocyte--vessel wall adhesion at sites of inflammation. Images Figure 2 PMID:3878084

  14. Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation.

    PubMed

    Fiorilli, Paul; Partridge, Darren; Staniszewska, Izabela; Wang, Jin Y; Grabacka, Maja; So, Kelvin; Marcinkiewicz, Cezary; Reiss, Krzysztof; Khalili, Kamel; Croul, Sidney E

    2008-11-01

    Medulloblastoma spreads by leptomeningeal dissemination rather than by infiltration that characterizes other CNS tumors, eg, gliomas. This study represents an initial attempt to identify both the molecules that mediate medulloblastoma adhesion to leptomeninges and the pathways that are key to survival and proliferation of tumor following adhesion. As a first step in molecule identification, we produced adhesion of D283 medulloblastoma cells to the extracellular matrix (ECM) of H4 glioma cells in vitro. Within this context, D283 cells preferentially expressed the alpha9 and beta1 integrin subunits; antibody and disintegrin blockade of alpha9 and beta1 binding eliminated the adhesion. The H4 ECM was enriched in tenascin, a binding partner for the alpha9beta1 integrin heterodimer. Purified tenascin-C supported D283 cell adhesion. The adhesion was blocked by antibodies to alpha9 and beta1 integrin. In vivo data were similar; immunohistochemistry of primary human medulloblastomas with leptomeningeal extension demonstrated increased expression of alpha9 and beta1 integrins as well as tenascin at the interface of brain and leptomeningeal tumor. These data suggest that tumor-cell expressions of alpha9 and beta1 integrins in combination with extracellular tenascin are necessary for medulloblastoma adhesion to the leptomeninges. As a first step in the identification of pathways that mediate survival and proliferation of tumor following adhesion, we demonstrated that adhesion to H4 ECM was associated with survival and proliferation of D283 cells as well as activation of the MAPK pathway in a growth factor deficient environment. Antibody blockade of alpha9 and beta1 integrin binding that eliminated adhesion also eliminated the in vitro survival benefit. These data suggest that adhesion of medulloblastoma to the meninges is necessary for the survival and proliferation of these tumor cells at the secondary site. PMID:18794852

  15. Focal hyperemia followed by spreading oligemia and impaired activation of rCBF in classic migraine

    SciTech Connect

    Olesen, J.; Larsen, B.; Lauritzen, M.

    1981-04-01

    Regional cerebral blood flow (rCBF) was measured in 254 areas of a hemisphere with the xenon 133 intraarterial injection method. Six cases of classic migraine were followed from the normal state into the prodromal phase, and in 3 cases further into the headache phase. One patient with common migraine was similarly followed during his only classic attack. The attacks were initiated by focal hyperemia in 3 patients. During prodromes all patients displayed occipitoparietal rCBF reduction (oligemia), but in only 1 case did the reduction approach critical values. Oligemia gradually spread anteriorly in the course of 15 to 45 minutes. In 4 patients a global oligemia was observed. In 4 patients severe headache was present concomitantly with oligemia and with no sign of hyperemia or nonhomogeneous brain perfusion. The normal rCBF increase during cortical activity (hand movement, speech, and similar activities) was impaired in 6 patients. The results indicate that the vasospastic model of the migraine attack is too simplistic.

  16. Mechanisms of endothelial cell-dependent leukocyte adhesion stimulated by platelet-activating factor.

    PubMed

    Ding, Z; Li, S; Wu, Z

    1992-04-01

    Platelet-activating factor (PAF) stimulates leukocyte-endothelial cell (EC) adhesion through its effects either on leukocytes or on ECs. ECs may be injured, synthesize, or express new adhesive proteins to increase leukocyte adhesion. Intermediary mediators produced by activated ECs are also likely involved in promoting leukocyte adhesion. Our experiments demonstrated that PAF induced no obvious damage to bovine pulmonary artery ECs evaluated by lactic dehydrogenase release rate, angiotensin-converting enzyme activity, and cellular malondialdehyde content. Treatment of EC monolayers with 10(-9) M PAF increased polymorphonuclear leukocyte (PMN) adhesion. Increasing PAF concentration did not induce more PMN adherence. PAF elicited both a rapid and prolonged increment of PMN adherence to EC monolayers. The rapid adherence was greatly attenuated by pretreatment of ECs with PAF receptor antagonist SRI 63-441 but was not affected by pretreatment of PMNs with SRI 63-441, suggesting that PAF increases PMN adherence rapidly through its effects on specific receptors on ECs. Increased PMN adherence lasted if PAF treatment of ECs was sustained for 3 or 6 h. Pretreatment of ECs with actinomycin D, a protein synthesis inhibitor, significantly decreased PAF-induced sustained PMN adherence, but the inhibition is incomplete, suggesting that other mechanisms than protein synthesis also participated in the prolonged PMN adherence. We concluded from the results that PAF may induce both rapid and prolonged PMN adhesion to ECs. The effects are receptor mediated. The prolonged PMN adhesion is partly the result of protein synthesis. PMID:1592489

  17. Inhibition of cyclooxygenase-2 prevents intra-abdominal adhesions by decreasing activity of peritoneal fibroblasts

    PubMed Central

    Wei, Guangbing; Chen, Xin; Wang, Guanghui; Jia, Pengbo; Xu, Qinhong; Ping, Gaofeng; Wang, Kang; Li, Xuqi

    2015-01-01

    Background Postoperative intra-abdominal adhesions are common complications after abdominal surgery. The exact molecular mechanisms that are responsible for these complications remain unclear, and there are no effective methods for preventing adhesion formation or reformation. The aim of the study reported here was to investigate the preventive effects and underlying potential molecular mechanisms of selective cyclooxygenase-2 (COX-2) inhibitors in a rodent model of postoperative intra-abdominal adhesions. Materials and methods The expression of COX-2 in postoperative intra-abdominal adhe-sions and normal peritoneal tissue was examined by immunohistochemistry and Western blot analysis. Assays were performed to elucidate the effect of COX-2 inhibition on hypoxia-induced fibroblast activity in vitro and on intra-abdominal adhesion formation in vivo. Results Hypoxia-induced COX-2 expression in peritoneal fibroblasts was increased in postoperative intra-abdominal adhesions. Inhibition of COX-2 attenuated the activating effect of hypoxia on normal peritoneal fibroblasts in vitro. Data indicate that selective COX-2 inhibitor prevents in vivo intra-abdominal adhesion by inhibition of basic fibroblast growth factor and transforming growth factor-beta expression, but not through an antiangiogenic mechanism. Furthermore, using selective COX-2 inhibitors to prevent intra-abdominal adhesions did not adversely affect the weight, bowel motility, or healing of intestinal anastomoses in a rat model. Conclusion These results show that hypoxia-induced COX-2 expression in peritoneal fibroblasts is involved in the formation of intra-abdominal adhesions. Inhibition of COX-2 prevents postoperative intra-abdominal adhesions through suppression of inflammatory cytokines. PMID:26109851

  18. Detection of Bidirectional Signaling During Integrin Activation and Neutrophil Adhesion

    PubMed Central

    Altman, Stuart M.; Dixit, Neha; Simon, Scott I.

    2014-01-01

    Neutrophil arrest and migration on inflamed endothelium is dependent upon a conformational shift in CD11a/CD18 (LFA-1) from a low to high affinity and clustered state which determines the strength and lifetime of bond formation with intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal adaptor proteins kindlin-3 and talin-1 anchor clustered LFA-1 to the cytoskeleton and support the transition from neutrophil rolling to arrest. We employ microfluidic flow channels and total internal reflection fluorescence microscopy to evaluate the spatiotemporal regulation of LFA-1 affinity and bond formation that facilitate the transition from neutrophil rolling to arrest. Methodology is presented to correlate the relationship between integrin conformation, bond formation with ICAM-1, and cytoskeletal engagement and adhesion strengthening necessary to achieve a migratory phenotype. PMID:24504956

  19. Does inhibition of proteolytic activity improve adhesive luting?

    PubMed

    Lührs, Anne-Katrin; De Munck, Jan; Geurtsen, Werner; Van Meerbeek, Bart

    2013-04-01

    Endogenous enzymes may be involved in the biodegradation of adhesive restoration-tooth interfaces. Inhibitors of matrix metalloproteinases (MMPs) have been suggested to retard the bond-degradation process. Limited data are available on whether composite cements may also benefit from MMP inhibitors. Therefore, the aim of this study was to determine the effect of two MMP inhibitors--chlorhexidine digluconate (CHX) and galardin--on the microtensile bond strength (μTBS) of two self-adhesive composite cements to dentin. Ceramic specimens were cemented to bur-cut dentin surfaces using the self-adhesive composite cements RelyX Unicem 2 (3M ESPE) or Clearfil SA (Kuraray), or the etch-and-rinse composite cement Nexus 3 (Kerr) that served as the control. The surfaces were left untreated or were pretreated with MMP inhibitors (2% CHX or 0.2 mM galardin). The μTBS was determined 'immediately' and upon ageing (water storage for 6 months). Statistical analysis revealed a significant effect of the factors 'composite cement' and 'storage', as well as all interactions, but no effect of the MMP inhibitors. After 6 months of ageing, the μTBS decreased for all cements, except for the multistep etch-and-rinse luting composite when it was applied without MMP inhibitors. The MMP inhibitors could not prevent the decrease in μTBS upon ageing and therefore do not improve the luting durability of the composite cements tested. PMID:23489902

  20. [Effect of cultivation conditions on the adhesive activity of rhodococci towards n-hexadecane].

    PubMed

    Rubtsova, E V; Kuiukina, M S; Ivshina, I B

    2012-01-01

    The effect of cultivation conditions (the composition, acidity, and salinity of the cultivation medium; temperature; and the hydrodynamic conditions of cultivation) on the adhesion of actinobacteria of the genus Rhodococcus to n-hexadecane has been investigated. A study performed showed that the adhesive activity of rhodococci depends on the composition of the cultivation medium and on the cultivation temperature. The possible mechanisms underlying the effect of growth conditions on the adhesion ofrhodococci to liquid hydrocarbons and involving changes in the cell lipid content or the zeta potential of cells are addressed. Rhodococcal strains displaying high adhesive activity (80-90%) at a low temperature (18 degrees C), high salinity (5.0% NaCl), and acidity (pH 6.0) of the cultivation medium have been selected as a result of the present work; these strains have a considerable potential for use in bioremediation of soil and water contaminated by hydrocarbons. PMID:23101387

  1. Shedding of APP limits its synaptogenic activity and cell adhesion properties

    PubMed Central

    Stahl, Ronny; Schilling, Sandra; Soba, Peter; Rupp, Carsten; Hartmann, Tobias; Wagner, Katja; Merdes, Gunter; Eggert, Simone; Kins, Stefan

    2014-01-01

    The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD) and has essential synapse promoting functions. Synaptogenic activity as well as cell adhesion properties of APP presumably depend on trans-cellular dimerization via its extracellular domain. Since neuronal APP is extensively processed by secretases, it raises the question if APP shedding affects its cell adhesion and synaptogenic properties. We show that inhibition of APP shedding using cleavage deficient forms of APP or a dominant negative α-secretase strongly enhanced its cell adhesion and synaptogenic activity suggesting that synapse promoting function of APP is tightly regulated by α-secretase mediated processing, similar to other trans-cellular synaptic adhesion molecules. PMID:25520622

  2. Traffic Sign Recognition with Invariance to Lighting in Dual-Focal Active Camera System

    NASA Astrophysics Data System (ADS)

    Gu, Yanlei; Panahpour Tehrani, Mehrdad; Yendo, Tomohiro; Fujii, Toshiaki; Tanimoto, Masayuki

    In this paper, we present an automatic vision-based traffic sign recognition system, which can detect and classify traffic signs at long distance under different lighting conditions. To realize this purpose, the traffic sign recognition is developed in an originally proposed dual-focal active camera system. In this system, a telephoto camera is equipped as an assistant of a wide angle camera. The telephoto camera can capture a high accuracy image for an object of interest in the view field of the wide angle camera. The image from the telephoto camera provides enough information for recognition when the accuracy of traffic sign is low from the wide angle camera. In the proposed system, the traffic sign detection and classification are processed separately for different images from the wide angle camera and telephoto camera. Besides, in order to detect traffic sign from complex background in different lighting conditions, we propose a type of color transformation which is invariant to light changing. This color transformation is conducted to highlight the pattern of traffic signs by reducing the complexity of background. Based on the color transformation, a multi-resolution detector with cascade mode is trained and used to locate traffic signs at low resolution in the image from the wide angle camera. After detection, the system actively captures a high accuracy image of each detected traffic sign by controlling the direction and exposure time of the telephoto camera based on the information from the wide angle camera. Moreover, in classification, a hierarchical classifier is constructed and used to recognize the detected traffic signs in the high accuracy image from the telephoto camera. Finally, based on the proposed system, a set of experiments in the domain of traffic sign recognition is presented. The experimental results demonstrate that the proposed system can effectively recognize traffic signs at low resolution in different lighting conditions.

  3. Orthogonal electrode catheter array for mapping of endocardial focal site of ventricular activation

    SciTech Connect

    Desai, J.M.; Nyo, H.; Vera, Z.; Seibert, J.A.; Vogelsang, P.J. )

    1991-04-01

    Precise location of the endocardial site of origin of ventricular tachycardia may facilitate surgical and catheter ablation of this arrhythmia. The endocardial catheter mapping technique can locate the site of ventricular tachycardia within 4-8 cm2 of the earliest site recorded by the catheter. This report describes an orthogonal electrode catheter array (OECA) for mapping and radiofrequency ablation (RFA) of endocardial focal site of origin of a plunge electrode paced model of ventricular activation in dogs. The OECA is an 8 F five pole catheter with four peripheral electrodes and one central electrode (total surface area 0.8 cm{sup 2}). In eight mongrel dogs, mapping was performed by arbitrarily dividing the left ventricle (LV) into four segments. Each segment was mapped with OECA to find the earliest segment. Bipolar and unipolar electrograms were obtained. The plunge electrode (not visible on fluoroscopy) site was identified by the earliest wave front arrival times of -30 msec or earlier at two or more electrodes (unipolar electrograms) with reference to the earliest recorded surface ECG (I, AVF, and V1). Validation of the proximity of the five electrodes of the OECA to the plunge electrode was performed by digital radiography and RFA. Pathological examination was performed to document the proximity of the OECA to the plunge electrode and also for the width, depth, and microscopic changes of the ablation. To find the segment with the earliest LV activation a total of 10 {plus minus} 3 (mean {plus minus} SD) positions were mapped. Mean arrival times at the two earlier electrodes were -39 {plus minus} 4 msec and -35 {plus minus} 3 msec. Digital radiography showed the plunge electrode to be within the area covered by all five electrodes in all eight dogs. The plunge electrode was within 1 cm2 area of the region of RFA in all eight dogs.

  4. Inhibition on Apoptosis Induced by Elevated Hydrostatic Pressure in Retinal Ganglion Cell-5 via Laminin Upregulating β1-integrin/Focal Adhesion Kinase/Protein Kinase B Signaling Pathway

    PubMed Central

    Li, Yi; Chen, Yan-Ming; Sun, Ming-Ming; Guo, Xiao-Dan; Wang, Ya-Chen; Zhang, Zhong-Zhi

    2016-01-01

    Background: Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs). High intraocular pressure (HIOP), the main risk factor, causes the optic nerve damage. However, the precise mechanism of HIOP-induced RGC death is not yet completely understood. This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures, explore whether laminin is associated with apoptosis under pressure, whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival. Methods: RGC-5 cells were exposed to 0, 20, 40, and 60 mmHg in a pressurized incubator for 6, 12, and 24 h, respectively. The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Western blotting of cleaved caspase-3 protein. Location and expression of laminin were detected by immunofluorescence. The expression of β1-integrin, phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB, or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis. Results: Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells. Pressure with 40 mmHg for 24 h induced a maximum apoptosis. Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h. After pretreating with laminin, RGC-5 cells survived from elevated pressure. Furthermore, β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group. Conclusions: The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure. Laminin can protect RGC-5 cells against high pressure via β1-integrin/FAK/AKT signaling pathway. These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure

  5. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism.

    PubMed

    Riveline, D; Zamir, E; Balaban, N Q; Schwarz, U S; Ishizaki, T; Narumiya, S; Kam, Z; Geiger, B; Bershadsky, A D

    2001-06-11

    The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force. PMID:11402062

  6. Endocardial focal activation originating from Purkinje fibers plays a role in the maintenance of long duration ventricular fibrillation

    PubMed Central

    Lin, Changjian; Jin, Qi; Zhang, Ning; Zhou, Jian; Pang, Yang; Xin, Yangxun; Liu, Shaohua; Wu, Qiong; Wu, Liqun

    2014-01-01

    Aim To determine the role of repetitive endocardial focal activations and Purkinje fibers in the maintenance of long duration ventricular fibrillation (LDVF, VF>1 minute) in canine hearts in vivo. Methods The study was conducted in electrophysiological laboratory of Shanghai Ruijin hospital from July 2010 to August 2012. A 64-electrode basket was introduced through a carotid artery into the left ventricle (LV) of 11 beagle dogs for global endocardial electrical mapping. In the Lugol’s solution group (n = 5), the subendocardium was ablated by washing with Lugol’s solution. In the control group, (n = 6) saline was used for ablation. Before and after saline or Lugol ablation, we determined QRS duration and QT/QTc interval in sinus rhythm (SR). We also measured the activation rates in the first 2 seconds of each minute during 7 minutes of VF for each group. If VF terminated spontaneously in less than 7 minutes, the VF segments used in activation rate analysis were reduced accordingly. Results At the beginning of VF there was no difference between the groups in the activation rate. However, after 1 minute of LDVF the Lugol’s solution group had significantly slower activation rate than the control group. In the control group, all episodes of LDVF (6/6) were successfully sustained for 7 minutes, while in the Lugol’s solution group 4/5 episodes of LDVF spontaneously terminated before 7 minutes (4.8 ± 1.4 minutes) (P = 0.015). In the control group, at 5.1 ± 1.3 minutes of LDVF, a successive, highly organized focal LV endocardial activation pattern was observed. During this period, activations partly arose in PF and spread to the working ventricular myocardium. Mapping analysis showed that these events were consistent with repetitive endocardial focal activations. No evidence of similar focal activations was observed in the Lugol’s solution group. Conclusions Repetitive endocardial focal activations in the LV endocardium may be associated with

  7. Crosstalk in the measurement of focal brain activation by near-infrared topography

    NASA Astrophysics Data System (ADS)

    Okui, N.; Okada, E.

    2005-08-01

    Crosstalk between oxy- and deoxy-haemoglobin observed in near-infrared topography is investigated. The light propagation in an adult head model is predicted by Monte Carlo simulation to obtain the change in intensity detected with source-detector pairs on the scalp caused by a focal absorption change in the brain. The topographic images of changes in oxy- and deoxy-haemoglobin are obtained from the changes in intensity detected with source-detector pairs on the scalp. The crosstalk depends on the relative position of the focal absorption change to source-detector pairs. The crosstalk is minimised when the focal absorption change is located below a measurement point that is the midpoint between a source and a detector. Appropriate selection of wavelength pair is effective to reduce the crosstalk in the topographic image.

  8. Therapeutic polymers for dental adhesives: Loading resins with bio-active components

    PubMed Central

    Imazato, Satoshi; Ma, Sai; Chen, Ji-hua; Xu, Hockin H.K.

    2014-01-01

    Objectives Many recent adhesives on the market exhibit reasonable clinical performance. Future innovations in adhesive materials should therefore seek out novel properties rather than simply modifying existing technologies. It is proposed that adhesive materials that are “bio-active” could contribute to better prognosis of restorative treatments. Methods This review examines the recent approaches used to achieve therapeutic polymers for dental adhesives by incorporating bio-active components. A strategy to maintain adhesive restorations is the focus of this paper. Results Major trials on therapeutic dental adhesives have looked at adding antibacterial activities or remineralization effects. Applications of antibacterial resin monomers based on quaternary ammonium compounds have received much research attention, and the loading of nano-sized bioactive particles or multiple ion-releasing glass fillers have been perceived as advantageous since they are not expected to influence the mechanical properties of the carrier polymer. Significance The therapeutic polymer approaches described here have the potential to provide clinical benefits. However, not many technological applications in this category have been successfully commercialized. Clinical evidence as well as further advancement of these technologies can be a driving force to make these new types of materials clinically available. PMID:23899387

  9. Trimeric Autotransporters Require Trimerization of the Passenger Domain for Stability and Adhesive Activity

    PubMed Central

    Cotter, Shane E.; Surana, Neeraj K.; Grass, Susan; St. Geme, Joseph W.

    2006-01-01

    In recent years, structural studies have identified a number of bacterial, viral, and eukaryotic adhesive proteins that have a trimeric architecture. The prototype examples in bacteria are the Haemophilus influenzae Hia adhesin and the Yersinia enterocolitica YadA adhesin. Both Hia and YadA are members of the trimeric-autotransporter subfamily and are characterized by an internal passenger domain that harbors adhesive activity and a short C-terminal translocator domain that inserts into the outer membrane and facilitates delivery of the passenger domain to the bacterial surface. In this study, we examined the relationship between trimerization of the Hia and YadA passenger domains and the capacity for adhesive activity. We found that subunit-subunit interactions and stable trimerization are essential for native folding and stability and ultimately for full-level adhesive activity. These results raise the possibility that disruption of the trimeric architecture of trimeric autotransporters, and possibly other trimeric adhesins, may be an effective strategy to eliminate adhesive activity. PMID:16855229

  10. [AMP-activated protein kinase activation regulates adhesion of monocytes to vascular endothelial cells and the underlying mechanism].

    PubMed

    Bai, Hong-Bo; Wang, Yun; Zhang, Yu-Hua; Zhang, Yuan

    2016-02-25

    The present study was aimed to explore the effect of AMP-activated protein kinase (AMPK) on monocyte adhesion to vascular endothelial cells and underlying molecular mechanism. Tumor necrosis factor α (TNFα)-activated human aortic endothelial cells (HAECs) were treated with different concentrations of AMPK agonist 5-Aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) or AMPK inhibitor compound C. And other HAECs were overexpressed with constitutive active or dominant negative AMPK protein and then treated with TNFα. The rates of monocytes adhering to endothelial cells were detected by fluorescent staining. Intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels and protein secretions were detected by quantitative PCR and ELISA, respectively. Acetylation of NF-κB p65 at lysine 221 site was assessed by Western blot. NF-κB p65 DNA binding activity was analyzed by an ELISA-based method. By using small interfering RNA based strategy, p300 expression in HAECs was down-regulated and then cells were incubated with TNFα. NF-κB p65 DNA binding activity, ICAM-1 and VCAM-1 expressions and adhesion rates were detected, respectively. The activity of p300 was also detected by ELISA. The results showed that AICAR treatment significantly reduced monocyte-endothelial adhesion rate, as well as ICAM-1 and VCAM-1 mRNA levels and protein secretions, in TNFα-activated HAECs. Moreover, transfection of constitutive active AMPKα but not dominant negative AMPKα strongly diminished TNFα-induced upregulation of ICAM-1 and VCAM-1 mRNA expressions and secretions, as well as monocyte-endothelial adhesion. Furthermore, AMPK activation decreased TNFα-mediated acetylation of NF-κB p65 at Lys221 site and reduced NF-κB p65 DNA binding activity. Silencing p300 by siRNA significantly abolished the effect of TNFα- induced adhesion molecules expression and monocyte-endothelial adhesion. Blocking AMPK activation by compound C almost

  11. Small molecule inhibitors of the Pyk2 and FAK kinases modulate chemoattractant-induced migration, adhesion and Akt activation in follicular and marginal zone B cells.

    PubMed

    Tse, Kathy W K; Lin, Kevin B L; Dang-Lawson, May; Guzman-Perez, Angel; Aspnes, Gary E; Buckbinder, Leonard; Gold, Michael R

    2012-01-01

    B-lymphocytes produce protective antibodies but also contribute to autoimmunity. In particular, marginal zone (MZ) B cells recognize both microbial components and self-antigens. B cell trafficking is critical for B cell activation and is controlled by chemoattactants such as CXCL13 and sphingosine 1-phosphate (S1P). The related tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) regulate cell migration and adhesion but their roles in B cells are not fully understood. Using a novel Pyk2-selective inhibitor described herein (PF-719), as well as a FAK-selective inhibitor, we show that both Pyk2 and FAK are important for CXCL13- and S1P-induced migration of B-2 cells and MZ B cells. In contrast, LFA-1-mediated adhesion required only Pyk2 whereas activation of the Akt pro-survival kinase required FAK but not Pyk2. Thus Pyk2 and FAK mediate critical processes in B cells and these inhibitors can be used to further elucidate their functions in B cells. PMID:22507871

  12. A characteristic time sequence of epileptic activity in EEG during dynamic penicillin-induced focal epilepsy--a preliminary study.

    PubMed

    Silfverhuth, Minna J; Kortelainen, Jukka; Ruohonen, Jyrki; Suominen, Kalervo; Niinimäki, Jaakko; Sonkajärvi, Eila; Kiviniemi, Vesa; Alahuhta, Seppo; Jäntti, Ville; Tervonen, Osmo; Seppänen, Tapio

    2011-09-01

    Penicillin-induced focal epilepsy is a well-known model in experimental epilepsy. However, the dynamic evolution of waveforms, DC-level changes, spectral content and coherence are rarely reported. Stimulated by earlier fMRI findings, we also seek for the early signs preceding spiking activity from frequency domain of EEG signal. In this study, EEG data is taken from previous EEG/fMRI series (six pigs, 20-24kg) of an experimental focal epilepsy model, which includes dynamic induction of epileptic activity with penicillin (6000IU) injection into the somatosensory cortex during deep isoflurane anaesthesia. No ictal discharges were recorded with this dose. Spike waveforms, DC-level, time-frequency content and coherence of EEG were analysed. Development of penicillin induced focal epileptic activity was not preceded with specific spectral changes. The beginning of interictal spiking was related to power increase in the frequencies below 6Hz or 20Hz, and continued to a widespread spectral increase. DC-level and coherence changes were clear in one animal. Morphological evolution of epileptic activity was a collection of the low-amplitude monophasic, bipolar, triple or double spike-wave forms, with an increase in amplitude, up to large monophasic spiking. In conclusion, in the time sequence of induced epileptic activity, immediate shifts in DC-level EEG are plausible, followed by the spike activity-related widespread increase in spectral content. Morphological evolution does not appear to follow a clear continuum; rather, intermingled and variable spike or multispike waveforms generally lead to stabilised activity of high-amplitude monophasic spikes. PMID:21511498

  13. Integrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model

    PubMed Central

    Granato, Daniela C.; Zanetti, Mariana R.; Kawahara, Rebeca; Yokoo, Sami; Domingues, Romênia R.; Aragão, Annelize Z.; Agostini, Michelle; Carazzolle, Marcelo F.; Vidal, Ramon O.; Flores, Isadora L.; Korvala, Johanna; Cervigne, Nilva K.; Silva, Alan R. S.; Coletta, Ricardo D.; Graner, Edgard; Sherman, Nicholas E.; Leme, Adriana F. Paes

    2014-01-01

    Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules. PMID:24858105

  14. Rapidly light-activated surgical protein glue inspired by mussel adhesion and insect structural crosslinking.

    PubMed

    Jeon, Eun Young; Hwang, Byeong Hee; Yang, Yun Jung; Kim, Bum Jin; Choi, Bong-Hyuk; Jung, Gyu Yong; Cha, Hyung Joon

    2015-10-01

    Currently approved surgical tissue glues do not satisfy the requirements for ideal bioadhesives due to limited adhesion in wet conditions and severe cytotoxicity. Herein, we report a new light-activated, mussel protein-based bioadhesive (LAMBA) inspired by mussel adhesion and insect dityrosine crosslinking chemistry. LAMBA exhibited substantially stronger bulk wet tissue adhesion than commercially available fibrin glue and good biocompatibility in both in vitro and in vivo studies. Besides, the easily tunable, light-activated crosslinking enabled an effective on-demand wound closure and facilitated wound healing. Based on these outstanding properties, LAMBA holds great potential as an ideal surgical tissue glue for diverse medical applications, including sutureless wound closures of skin and internal organs. PMID:26197411

  15. Quantal concept of T-cell activation: adhesion domains as immunological synapses

    NASA Astrophysics Data System (ADS)

    Sackmann, Erich

    2011-06-01

    Adhesion micro-domains (ADs) formed during encounters of lymphocytes with antigen-presenting cells (APC) mediate the genetic expression of quanta of cytokines interleukin-2 (IL-2). The IL-2-induced activation of IL-2 receptors promotes the stepwise progression of the T-cells through the cell cycle, hence their name, immunological synapses. The ADs form short-lived reaction centres controlling the recruitment of activators of the biochemical pathway (the kinases Lck and ZAP) while preventing the access of inhibitors (phosphatase CD45) through steric repulsion forces. CD45 acts as the generator of adhesion domains and, through its role as a spacer protein, also as the promoter of the reaction. In a second phase of T-cell-APC encounters, long-lived global reaction spaces (called supramolecular activation complexes (SMAC)) form by talin-mediated binding of the T-cell integrin (LFA-1) to the counter-receptor ICAM-1, resulting in the formation of ring-like tight adhesion zones (peripheral SMAC). The ADs move to the centre of the intercellular adhesion zone forming the central SMAC, which serve in the recycling of the AD. We propose that cell stimulation is triggered by integrating the effect evoked by the short-lived adhesion domains. Similar global reaction platforms are formed by killer cells to destruct APC. We present a testable mechanical model showing that global reaction spaces (SMAC or dome-like contacts between cytotoxic cells and APC) form by self-organization through delayed activation of the integrin-binding affinity and stabilization of the adhesion zones by F-actin recruitment. The mechanical stability and the polarization of the adhering T-cells are mediated by microtubule-actin cross-talk.

  16. Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.

    PubMed

    Honda, S; Campbell, J J; Andrew, D P; Engelhardt, B; Butcher, B A; Warnock, R A; Ye, R D; Butcher, E C

    1994-04-15

    Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands. PMID:7511663

  17. Focal vibration in neurorehabilitation.

    PubMed

    Murillo, N; Valls-Sole, J; Vidal, J; Opisso, E; Medina, J; Kumru, H

    2014-04-01

    During the last decade, many studies have been carried out to understand the effects of focal vibratory stimuli at various levels of the central nervous system and to study pathophysiological mechanisms of neurological disorders as well as the therapeutic effects of focal vibration in neurorehabilitation. This review aimed to describe the effects of focal vibratory stimuli in neurorehabilitation including the neurological diseases or disorders like stroke, spinal cord injury, multiple sclerosis, Parkinson's' disease and dystonia. In conclusion, focal vibration stimulation is well tolerated, effective and easy to use, and it could be used to reduce spasticity, to promote motor activity and motor learning within a functional activity, even in gait training, independent from etiology of neurological pathology. Further studies are needed in the future well-designed trials with bigger sample size to determine the most effective frequency, amplitude and duration of vibration application in the neurorehabilitation. PMID:24842220

  18. Dynamic transcription factor activity networks in response to independently altered mechanical and adhesive microenvironmental cues.

    PubMed

    Peñalver Bernabé, Beatriz; Shin, Seungjin; Rios, Peter D; Broadbelt, Linda J; Shea, Lonnie D; Seidlits, Stephanie K

    2016-08-01

    Multiple aspects of the local extracellular environment profoundly affect cell phenotype and function. Physical and chemical cues in the environment trigger intracellular signaling cascades that ultimately activate transcription factors (TFs) - powerful regulators of the cell phenotype. TRACER (TRanscriptional Activity CEll aRrays) was employed for large-scale, dynamic quantification of TF activity in human fibroblasts cultured on hydrogels with a controlled elastic modulus and integrin ligand density. We identified three groups of TFs: responders to alterations in ligand density alone, substrate stiffness or both. Dynamic networks of regulatory TFs were constructed computationally and revealed distinct TF activity levels, directionality (i.e., activation or inhibition), and dynamics for adhesive and mechanical cues. Moreover, TRACER networks predicted conserved hubs of TF activity across multiple cell types, which are significantly altered in clinical fibrotic tissues. Our approach captures the distinct and overlapping effects of adhesive and mechanical stimuli, identifying conserved signaling mechanisms in normal and disease states. PMID:27470442

  19. Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion

    PubMed Central

    Oberoi, Raghav; Schuett, Jutta; Schuett, Harald; Koch, Ann-Kathrin; Luchtefeld, Maren

    2016-01-01

    Objective It is well known that atherosclerotic inflammatory vascular disease is critically driven by oxidized lipids and cytokines. In this regard, tumor necrosis factor (TNF)-α is known as a crucial mediator of early pro-atherosclerotic events. Epidemiologic data suggest that blockade of TNF-α has beneficial effects on vascular outcomes in patients with rheumatoid arthritis, however, detailed mechanistic studies are still lacking. This study aims to elucidate effects of TNF-α blockade by adalimumab–which is approved for several inflammatory disorders–on endothelial activation and monocyte adhesion under pro-atherosclerotic conditions. Methods and Results Phorbol myristate acetate (PMA) differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein and subsequent analysis of this conditioned media (oxLDL CM) revealed a strong release of TNF-α. The TNF-α rich supernatant led to activation of human umbilical vein endothelial cells (HUVEC) as shown by enhanced expression of major adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin which was suppressed by the TNF-α inhibitor adalimumab. Accordingly, adalimumab effectively prevented THP-1 monocyte adhesion to endothelial cells under static as well as under flow conditions. Furthermore, adalimumab suppressed endothelial leakage as shown by Evan's blue diffusion across a confluent endothelial monolayer. Of note, after intraperitoneal injection we detected abundant deposition of fluorophore-labelled adalimumab in atherosclerotic plaques of hypercholesterolemic mice. Conclusion Our results show that adalimumab prevents major inflammatory effects of TNF-α on endothelial activation, endothelial monocyte adhesion, endothelial leakage and therefore extends the therapeutic options of adalimumab to limit vascular inflammation. PMID:27467817

  20. Platelet adhesion and activation on polyethylene glycol modified polyurethane surfaces. Measurement of cytoplasmic calcium.

    PubMed

    Park, K D; Suzuki, K; Lee, W K; Lee, J E; Kim, Y H; Sakurai, Y; Okano, T

    1996-01-01

    Polyurethane (PU) surfaces were modified by coupling polyethylene glycol (PEG; molecular weight, 1,000) chains carrying different terminal groups (PU-PEG1K-OH, PU-PEG1K-NH2, PU-PEG1K-SO3) and longer PEG chains (MW, 3,350; PU-PEG3.4K-OH). The modified PU surfaces have the same PEG (1K) chain density. Surface induced platelet activation was evaluated by measuring cytoplasmic free calcium concentration in platelets contacting modified surfaces, and platelet adhesion onto modified surfaces was investigated in vitro. Cytoplasmic free calcium levels in platelets contacting PU-PEG-SO3 remained relatively constant, in contrast to the significant increase observed for PU-PEG-NH2, PU-PEG-OH, and control PU surfaces. The degree of platelet adhesion clearly demonstrates that all PEG graft surfaces prevented platelet adhesion. Among PEG1K surfaces, PU-PEG-SO3 shows the lowest platelet adhesion. In the case of relatively longer PEG grafted surfaces (PU-PEG3.4K-OH and PU-PEG3.4K-Hep), both surfaces were found to prevent the increase in both cytoplasmic free calcium and platelet adhesion. These results suggest that longer PEG chain grafting is more effective than shorter grafting in preventing platelet activation and adhesion because of the highly dynamic movement of hydrated PEG chains at the interface. In addition, in vitro platelet interaction is dependent upon terminal groups of PEG chains on PEG1K series surfaces. PMID:8945010

  1. Thin active region, type II superlattice photodiode arrays: Single-pixel and focal plane array characterization

    NASA Astrophysics Data System (ADS)

    Little, J. W.; Svensson, S. P.; Beck, W. A.; Goldberg, A. C.; Kennerly, S. W.; Hongsmatip, T.; Winn, M.; Uppal, P.

    2007-02-01

    We have measured the radiometric properties of two midwave infrared photodiode arrays (320×256pixel2 format) fabricated from the same wafer comprising a thin (0.24μm), not intentionally doped InAs /GaSb superlattice between a p-doped GaSb layer and a n-doped InAs layer. One of the arrays was indium bump bonded to a silicon fanout chip to allow for the measurement of properties of individual pixels, and one was bonded to a readout integrated circuit to enable array-scale measurements and infrared imaging. The superlattice layer is thin enough that it is fully depleted at zero bias, and the collection efficiency of photogenerated carriers in the intrinsic region is close to unity. This simplifies the interpretation of photocurrent data as compared with previous measurements made on thick superlattices with complex doping profiles. Superlattice absorption coefficient curves, obtained from measurements of the external quantum efficiency using two different assumptions for optical coupling into the chip, bracket values calculated using an eight-band k •p model. Measurements of the quantum efficiency map of the focal plane array were in good agreement with the single-pixel measurements. Imagery obtained with this focal plane array demonstrates the high uniformity and crystal quality of the type II superlattice material.

  2. Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

    PubMed

    De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

    2016-07-01

    Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. PMID:27189837

  3. In vivo imaging of activated microglia in a mouse model of focal cerebral ischemia by two-photon microscopy.

    PubMed

    Bok, Seoyeon; Wang, Taejun; Lee, Chan-Ju; Jeon, Seong-Uk; Kim, Young-Eun; Kim, Jeongwoo; Hong, Beom-Ju; Yoon, Calvin Jinse; Kim, Sungjee; Lee, Seung-Hoon; Kim, Hak Jae; Kim, Il Han; Kim, Ki Hean; Ahn, G-One

    2015-09-01

    Microglia are brain resident macrophages rapidly responding to various stimuli to exert appropriate inflammatory responses. Although they have recently been exploited as an attractive candidate for imaging neuroinflammation, it is still difficult to visualize them at the cellular and molecular levels. Here we imaged activated microglia by establishing intracranial window chamber (ICW) in a mouse model of focal cerebral ischemia by using two-photon microscopy (TPM), in vivo. Intravenous injection of fluorescent antibodies allowed us to detect significantly elevated levels of Iba-1 and CD68 positive activated microglia in the ipsilateral compared to the contralateral side of the infarct. We further observed that indomethacin, a non-steroidal anti-inflammatory drug significantly attenuated CD68-positive microglial activation in ICW, which was further confirmed by qRT-PCR biochemical analyses. In conclusion, we believe that in vivo TPM imaging of ICW would be a useful tool to screen for therapeutic interventions lowering microglial activation hence neuroinflammation. PMID:26417502

  4. In vivo imaging of activated microglia in a mouse model of focal cerebral ischemia by two-photon microscopy

    PubMed Central

    Bok, Seoyeon; Wang, Taejun; Lee, Chan-Ju; Jeon, Seong-Uk; Kim, Young-Eun; Kim, Jeongwoo; Hong, Beom-Ju; Yoon, Calvin Jinse; Kim, Sungjee; Lee, Seung-Hoon; Kim, Hak Jae; Kim, Il Han; Kim, Ki Hean; Ahn, G-One

    2015-01-01

    Microglia are brain resident macrophages rapidly responding to various stimuli to exert appropriate inflammatory responses. Although they have recently been exploited as an attractive candidate for imaging neuroinflammation, it is still difficult to visualize them at the cellular and molecular levels. Here we imaged activated microglia by establishing intracranial window chamber (ICW) in a mouse model of focal cerebral ischemia by using two-photon microscopy (TPM), in vivo. Intravenous injection of fluorescent antibodies allowed us to detect significantly elevated levels of Iba-1 and CD68 positive activated microglia in the ipsilateral compared to the contralateral side of the infarct. We further observed that indomethacin, a non-steroidal anti-inflammatory drug significantly attenuated CD68-positive microglial activation in ICW, which was further confirmed by qRT-PCR biochemical analyses. In conclusion, we believe that in vivo TPM imaging of ICW would be a useful tool to screen for therapeutic interventions lowering microglial activation hence neuroinflammation. PMID:26417502

  5. Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen*

    PubMed Central

    Marjoram, Robin J.; Voss, Bryan; Pan, Yumei; Dickeson, S. Kent; Zutter, Mary M.; Hamm, Heidi E.; Santoro, Samuel A.

    2009-01-01

    Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the α2β1 integrin (α2β1) and the glycoprotein VI (GPVI)/FcRγ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (α2-CRP) containing the α2β1-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to α2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was α2β1-dependent and GPVI/FcRγ-independent as revealed in experiments with α2β1- or FcRγ-deficient mouse platelets. We further show that suboptimal activation of other platelet Gq-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to α2-CRP. The enhanced α2β1-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of αIIbβ3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet Gq-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α2β1 binding to collagens containing GFOGER sites. PMID:19815553

  6. Activation of GPR4 by Acidosis Increases Endothelial Cell Adhesion through the cAMP/Epac Pathway

    PubMed Central

    Leffler, Nancy R.; Asch, Adam S.; Witte, Owen N.; Yang, Li V.

    2011-01-01

    Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2′,5′-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a Gi signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the Gs/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the Gs/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells. PMID:22110680

  7. Screening of immunomodulatory and adhesive Lactobacillus with antagonistic activities against Salmonella from fermented vegetables.

    PubMed

    Feng, Junchang; Liu, Pilong; Yang, Xin; Zhao, Xin

    2015-12-01

    The purpose of this study was to select strains of lactic acid bacteria (LAB) by their in vitro adhesive and immunomodulatory properties for potential use as probiotics. In this study, 16 randomly selected LAB strains from fermented vegetables (sauerkraut, bean and cabbage) were first screened for their tolerance to acid, bile salts, pepsin and pancreatin, bacterial inhibitory activities and abilities to adherence to Caco-2 cells. Then, 4 strains with the highest adhesion abilities were selected for further studies of their immunomodulatory properties and inhibitory effects against Salmonella adhesion and invasion to Caco-2 cells in vitro. The results showed that these 16 LAB strains effectively survived in simulated gastrointestinal condition and inhibited growth of six tested pathogens. Lactobacillus rhamnosus P1, Lactobacillus plantarum P2, Lactobacillus rhamnosus P3 and Lactobacillus casei P4 had the highest abilities to adhere to Caco-2 cells. Furthermore, L. plantarum P2 strain showed higher abilities to induce expression of tumor necrosis factor-α and interleukin-12 by splenic monocytes and strongly inhibited the adhesion and invasion of S. enteritidis ATCC13076 to Caco-2 cells. These results suggest that Lactobacillus strains P2 could be used as a probiotic candidate in food against Salmonella infection. PMID:26340935

  8. Kupffer cells and activation of endothelial TLR4 coordinate neutrophil adhesion within liver sinusoids during endotoxemia.

    PubMed

    McDonald, Braedon; Jenne, Craig N; Zhuo, Lisheng; Kimata, Koji; Kubes, Paul

    2013-12-01

    A key pathological feature of the systemic inflammatory response of sepsis/endotoxemia is the accumulation of neutrophils within the microvasculature of organs such as the liver, where they cause tissue damage and vascular dysfunction. There is emerging evidence that the vascular endothelium is critical to the orchestration of inflammatory responses to blood-borne microbes and microbial products in sepsis/endotoxemia. In this study, we aimed to understand the role of endothelium, and specifically endothelial TLR4 activation, in the regulation of neutrophil recruitment to the liver during endotoxemia. Intravital microscopy of bone marrow chimeric mice revealed that TLR4 expression by non-bone marrow-derived cells was required for neutrophil recruitment to the liver during endotoxemia. Furthermore, LPS-induced neutrophil adhesion in liver sinusoids was equivalent between wild-type mice and transgenic mice that express TLR4 only on endothelium (tlr4(-/-)Tie2(tlr4)), revealing that activation of endothelial TLR4 alone was sufficient to initiate neutrophil adhesion. Neutrophil arrest within sinusoids of endotoxemic mice requires adhesive interactions between neutrophil CD44 and endothelial hyaluronan. Intravital immunofluorescence imaging demonstrated that stimulation of endothelial TLR4 alone was sufficient to induce the deposition of serum-derived hyaluronan-associated protein (SHAP) within sinusoids, which was required for CD44/hyaluronan-dependent neutrophil adhesion. In addition to endothelial TLR4 activation, Kupffer cells contribute to neutrophil recruitment via a distinct CD44/HA/SHAP-independent mechanism. This study sheds new light on the control of innate immune activation within the liver vasculature during endotoxemia, revealing a key role for endothelial cells as sentinels in the detection of intravascular infections and coordination of neutrophil recruitment to the liver. PMID:24113769

  9. Antibiotic-decorated titanium with enhanced antibacterial activity through adhesive polydopamine for dental/bone implant

    PubMed Central

    He, Shu; Zhou, Ping; Wang, Linxin; Xiong, Xiaoling; Zhang, Yifei; Deng, Yi; Wei, Shicheng

    2014-01-01

    Implant-associated infections, which are normally induced by microbial adhesion and subsequent biofilm formation, are a major cause of morbidity and mortality. Therefore, practical approaches to prevent implant-associated infections are in great demand. Inspired by adhesive proteins in mussels, here we have developed a novel antibiotic-decorated titanium (Ti) material with enhanced antibacterial activity. In this study, Ti substrate was coated by one-step pH-induced polymerization of dopamine followed by immobilization of the antibiotic cefotaxime sodium (CS) onto the polydopamine-coated Ti through catechol chemistry. Contact angle measurement and X-ray photoelectron spectroscopy confirmed the presence of CS grafted on the Ti surface. Our results demonstrated that the antibiotic-grafted Ti substrate showed good biocompatibility and well-behaved haemocompatibility. In addition, the antibiotic-grafted Ti could effectively prevent adhesion and proliferation of Escherichia coli (Gram-negative) and Streptococcus mutans (Gram-positive). Moreover, the inhibition of biofilm formation on the antibiotic-decorated Ti indicated that the grafted CS could maintain its long-term antibacterial activity. This modified Ti substrate with enhanced antibacterial activity holds great potential as implant material for applications in dental and bone graft substitutes. PMID:24647910

  10. Antibiotic-decorated titanium with enhanced antibacterial activity through adhesive polydopamine for dental/bone implant.

    PubMed

    He, Shu; Zhou, Ping; Wang, Linxin; Xiong, Xiaoling; Zhang, Yifei; Deng, Yi; Wei, Shicheng

    2014-06-01

    Implant-associated infections, which are normally induced by microbial adhesion and subsequent biofilm formation, are a major cause of morbidity and mortality. Therefore, practical approaches to prevent implant-associated infections are in great demand. Inspired by adhesive proteins in mussels, here we have developed a novel antibiotic-decorated titanium (Ti) material with enhanced antibacterial activity. In this study, Ti substrate was coated by one-step pH-induced polymerization of dopamine followed by immobilization of the antibiotic cefotaxime sodium (CS) onto the polydopamine-coated Ti through catechol chemistry. Contact angle measurement and X-ray photoelectron spectroscopy confirmed the presence of CS grafted on the Ti surface. Our results demonstrated that the antibiotic-grafted Ti substrate showed good biocompatibility and well-behaved haemocompatibility. In addition, the antibiotic-grafted Ti could effectively prevent adhesion and proliferation of Escherichia coli (Gram-negative) and Streptococcus mutans (Gram-positive). Moreover, the inhibition of biofilm formation on the antibiotic-decorated Ti indicated that the grafted CS could maintain its long-term antibacterial activity. This modified Ti substrate with enhanced antibacterial activity holds great potential as implant material for applications in dental and bone graft substitutes. PMID:24647910

  11. Transmeningeal muscimol can prevent focal EEG seizures in the rat neocortex without stopping multineuronal activity in the treated area.

    PubMed

    Ludvig, Nandor; Tang, Hai M; Artan, N Sertac; Mirowski, Piotr; Medveczky, Geza; Baptiste, Shirn L; Darisi, Sindhu; Kuzniecky, Ruben I; Devinsky, Orrin; French, Jacqueline A

    2011-04-18

    Muscimol has potent antiepileptic efficacy after transmeningeal administration in animals. However, it is unknown whether this compound stops local neuronal firing at concentrations that prevent seizures. The purpose of this study was to test the hypothesis that epidurally administered muscimol can prevent acetylcholine (Ach)-induced focal seizures in the rat neocortex without causing cessation of multineuronal activity. Rats were chronically implanted with a modified epidural cup over the right frontal cortex, with microelectrodes positioned underneath the cup. In each postsurgical experimental day, either saline or 0.005-, 0.05-, 0.5- or 5.0-mM muscimol was delivered through the cup, followed by a 20-min monitoring of the multineuronal activity and the subsequent delivery of Ach in the same way. Saline and muscimol pretreatment in the concentration range of 0.005-0.05 mM did not prevent EEG seizures. In contrast, 0.5-mM muscimol reduced the average EEG Seizure Duration Ratio value from 0.30±0.04 to 0. At this muscimol concentration, the average baseline multineuronal firing rate of 10.9±4.4 spikes/s did not change significantly throughout the 20-min pretreatment. Muscimol at 5.0mM also prevented seizures, but decreased significantly the baseline multineuronal firing rate of 7.0±1.8 to 3.7±0.9 spikes/s in the last 10 min of pretreatment. These data indicate that transmeningeal muscimol in a submillimolar concentration range can prevent focal neocortical seizures without stopping multineuronal activity in the treated area, and thus this treatment is unlikely to interrupt local physiological functions. PMID:21338591

  12. Activation of extracellular regulated kinase and mechanistic target of rapamycin pathway in focal cortical dysplasia.

    PubMed

    Patil, Vinit V; Guzman, Miguel; Carter, Angela N; Rathore, Geetanjali; Yoshor, Daniel; Curry, Daniel; Wilfong, Angus; Agadi, Satish; Swann, John W; Adesina, Adekunle M; Bhattacharjee, Meenakshi B; Anderson, Anne E

    2016-04-01

    Neuropathology of resected brain tissue has revealed an association of focal cortical dysplasia (FCD) with drug-resistant epilepsy (DRE). Recent studies have shown that the mechanistic target of rapamycin (mTOR) pathway is hyperactivated in FCD as evidenced by increased phosphorylation of the ribosomal protein S6 (S6) at serine 240/244 (S(240/244) ), a downstream target of mTOR. Moreover, extracellular regulated kinase (ERK) has been shown to phosphorylate S6 at serine 235/236 (S(235/236) ) and tuberous sclerosis complex 2 (TSC2) at serine 664 (S(664) ) leading to hyperactive mTOR signaling. We evaluated ERK phosphorylation of S6 and TSC2 in two types of FCD (FCD I and FCD II) as a candidate mechanism contributing to mTOR pathway dysregulation. Tissue samples from patients with tuberous sclerosis (TS) served as a positive control. Immunostaining for phospho-S6 (pS6(240/244) and pS6(235/236) ), phospho-ERK (pERK), and phospho-TSC2 (pTSC2) was performed on resected brain tissue with FCD and TS. We found increased pS6(240/244) and pS6(235/236) staining in FCD I, FCD II and TS compared to normal-appearing tissue, while pERK and pTSC2 staining was increased only in FCD IIb and TS tissue. Our results suggest that both the ERK and mTOR pathways are dysregulated in FCD and TS; however, the signaling alterations are different for FCD I as compared to FCD II and TS. PMID:26381727

  13. Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif.

    PubMed

    Jacob, Reeba S; George, Edna; Singh, Pradeep K; Salot, Shimul; Anoop, Arunagiri; Jha, Narendra Nath; Sen, Shamik; Maji, Samir K

    2016-03-01

    Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids. PMID:26742841

  14. Functional imaging of focal brain activation in conscious rats: impact of [(14)C]glucose metabolite spreading and release.

    PubMed

    Cruz, Nancy F; Ball, Kelly K; Dienel, Gerald A

    2007-11-15

    Labeled glucose and its analogs are widely used in imaging and metabolic studies of brain function, astrocyte-neuron interactions, and neurotransmission. Metabolite shuttling among astrocytes and neurons is essential for cell-cell transfer of neurotransmitter precursors and supply and elimination of energy metabolites, but dispersion and release of labeled compounds from activated tissue would reduce signal registration in metabolic labeling studies, causing underestimation of focal functional activation. Processes and pathways involved in metabolite trafficking and release were therefore assessed in the auditory pathway of conscious rats. Unilateral monotonic stimulation increased glucose utilization (CMR(glc)) in tonotopic bands in the activated inferior colliculus by 35-85% compared with contralateral tissue when assayed with [(14)C]deoxyglucose (DG), whereas only 20-30% increases were registered with [1- or 6-(14)C]glucose. Tonotopic bands were not evident with [1-(14)C]glucose unless assayed during halothane anesthesia or pretreatment with probenecid but were detectable with [6-(14)C]glucose. Extracellular lactate levels transiently doubled during acoustic stimulation, so metabolite spreading was assessed by microinfusion of [(14)C]tracers into the inferior colliculus. The volume of tissue labeled by [1-(14)C]glucose exceeded that by [(14)C]DG by 3.2- and 1.4-fold during rest and acoustic activation, respectively. During activation, the tissue volume labeled by U-(14)C-labeled glutamine and lactate rose, whereas that by glucose fell 50% and that by DG was unchanged. Dispersion of [1-(14)C]glucose and its metabolites during rest was also reduced 50% by preinfusion of gap junction blockers. To summarize, during brain activation focal CMR(glc) is underestimated with labeled glucose because of decarboxylation reactions, spreading within tissue and via the astrocyte syncytium, and release from activated tissue. These findings help explain the fall in CMR(O2)/CMR

  15. Three-dimensional neuronal brain activity estimation using shrinking smooth weighted-minimum-norm focal underdetermined-system solver methods

    NASA Astrophysics Data System (ADS)

    Zouch, Wassim; Slima, Mohamed Ben; Feki, Imed; Derambure, Philippe; Taleb-Ahmed, Abdelmalik; Hamida, Ahmed Ben

    2010-12-01

    A new nonparametric method, based on the smooth weighted-minimum-norm (WMN) focal underdetermined-system solver (FOCUSS), for electrical cerebral activity localization using electroencephalography measurements is proposed. This method iteratively adjusts the spatial sources by reducing the size of the lead-field and the weighting matrix. Thus, an enhancement of source localization is obtained, as well as a reduction of the computational complexity. The performance of the proposed method, in terms of localization errors, robustness, and computation time, is compared with the WMN-FOCUSS and nonshrinking smooth WMN-FOCUSS methods as well as with standard generalized inverse methods (unweighted minimum norm, WMN, and FOCUSS). Simulation results for single-source localization confirm the effectiveness and robustness of the proposed method with respect to the reconstruction accuracy of a simulated single dipole.

  16. Indomethacin treatment reduces microglia activation and increases numbers of neuroblasts in the subventricular zone and ischaemic striatum after focal ischaemia.

    PubMed

    Lopes, Rosana S; Cardoso, Marcelo M; Sampaio, Arthur O; Barbosa, Mario Santos; Souza, Celice C; DA Silva, Michelle C; Ferreira, Elane Magno N; Freire, Marco Aurelio M; Lima, Rafael Rodrigues; Gomes-Leal, Walace

    2016-09-01

    Neuroblasts from the subventricular zone (SVZ) migrate to striatum following stroke, but most of them die in the ischaemic milieu and this can be related to exacerbated microglial activation. Here, we explored the effects of the non-steroidal anti-inflammatory indomethacin on microglial activation, neuronal preservation and neuroblast migration following experimental striatal stroke in adult rats. Animals were submitted to endothelin-1 (ET-1)-induced focal striatal ischaemia and were treated with indomethacin or sterile saline (i.p.) for 7 days, being perfused after 8 or 14 days. Immunohistochemistry was performed to assess neuronal loss (anti-NeuN), microglial activation (anti-Iba1, ED1) and migrating neuroblasts (anti-DCX) by counting NeuN, ED1 and DCX-positive cells in the ischaemic striatum or SVZ. Indomethacin treatment reduced microglia activation and the number of ED1+ cells in both 8 and 14 days post injury as compared with controls. There was an increase in the number of DCX+ cells in both SVZ and striatum at the same survival times. Moreover, there was a decrease in the number of NeuN+ cells in indomethacin-treated animals as compared with the control group at 8 days but not after 14 days post injury. Our results suggest that indomethacin treatment modulates microglia activation, contributing to increased neuroblast proliferation in the SVZ and migration to the ischaemic striatum following stroke. PMID:27581930

  17. In-vivo tissue repair using light-activated surgical adhesive in a porcine model

    NASA Astrophysics Data System (ADS)

    McNally-Heintzelman, Karen M.; Riley, Jill N.; Dickson, Tonya J.; Hou, Dong Ming; Rogers, Pamela; March, Keith L.

    2001-05-01

    An in vivo study was conducted to investigate the feasibility, mechanical function, and chronic biocompatibility of a new light-activated surgical adhesive for achieving rapid hemostasis of the puncture site following diagnostic catheterization and interventional cardiac procedures. Porcine carotid arteries (nequals6) and femoral arteries (nequals6) were exposed, and an incision was made in the arterial walls using a 16G needle. The surgical adhesive, composed of a poly(L-lactic-co-glycolic acid) scaffold doped with the traditional protein solder mix of serum albumin and indocyanine green dye, was used to close the incisions in conjunction with an 805-nm diode laser. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The strength and hemostatic abilities of the new surgical adhesive were evaluated in the context of arterial pressure, persistence of hemostatis and presence of any inflammatory reaction after 3 days. After this evaluation period, the surgical procedure was repeated on the carotid arteries (nequals6) and femoral arteries (nequals6) of three additional animals that had been heparinized prior to surgery to closer approximate the conditions seen in a typical vascular surgical setting.

  18. Vascular activation of adhesion molecule mRNA and cell surface expression by ionizing radiation.

    PubMed

    Heckmann, M; Douwes, K; Peter, R; Degitz, K

    1998-01-10

    During cutaneous inflammatory reactions the recruitment of circulating leukocytes into the tissue critically depends on the regulated expression of endothelial cell adhesion molecules (CAMs). Various proinflammatory stimuli upregulate endothelial CAMs, including cytokines and UV irradiation. We have investigated the effects of ionizing radiation (IR) on endothelial CAM expression. Organ cultures of normal human skin as well as cultured human dermal microvascular endothelial cells (HDMEC) were exposed to IR. Expression of three major endothelial CAMs was studied in skin organ cultures by immunohistochemistry and in cell culture by Northern blot analysis and flow cytometry. In skin organ cultures vascular immunoreactivity for ICAM-1, E-selectin, and VCAM-1 was strongly induced 24 h after exposure to 5 or 10 Gy of IR, while immunoreactivity for CD31/PECAM-1, a constitutively expressed endothelial cell adhesion molecule, remained unchanged. In cultured HDMEC IR upregulated ICAM-1, VCAM-1, and E-selectin mRNAs and cell surface expression in a time- and dose-dependent fashion. Cellular morphology and viability remained unaltered by IR up to 24 h postirradiation. This study characterizes microvascular activation of adhesion molecule expression in response to ionizing radiation in a clinically relevant IR dose range. The findings also underscore the ability of endothelial cells to integrate environmental electromagnetic stimuli. PMID:9457067

  19. hTERT promotes cell adhesion and migration independent of telomerase activity

    PubMed Central

    Liu, Haiying; Liu, Qianqian; Ge, Yuanlong; Zhao, Qi; Zheng, Xiaohui; Zhao, Yong

    2016-01-01

    hTERT, a catalytic component of human telomerase, is undetectable in normal somatic cells but up-regulated in cancer and stem cells where telomere length is maintained by telomerase. Accumulated evidence indicates that hTERT may have noncanonical functions beyond telomerase by regulating the expression of particular genes. However, comprehensive identification of the genes regulated by hTERT is unavailable. In this report, we expressed WT hTERT and hTERTmut which displays dysfunctional catalytic activity, in human U2OS cancer cells and VA-13 immortalized fibroblast cells, both of which lack endogenous hTERT and hTR expression. Changes in gene expression induced by hTERT and hTERT-mut expression were determined by genome-wide RNA-seq and verified by qPCR. Our results showed that hTERT affects different genes in two cell lines, implying that the regulation of gene expression by hTERT is indirect and cell type dependent. Moreover, functional analysis identifies cell adhesion-related genes that have been changed by hTERT in both cell lines. Adhesion experiments revealed that hTERT expression significantly increases cell adhesion. Monolayer wound healing and transwell assays demonstrated increased cell migration upon hTERT expression. These results provide new evidence to support a noncanonical function for hTERT in promoting tumorigenesis. PMID:26971878

  20. hTERT promotes cell adhesion and migration independent of telomerase activity.

    PubMed

    Liu, Haiying; Liu, Qianqian; Ge, Yuanlong; Zhao, Qi; Zheng, Xiaohui; Zhao, Yong

    2016-01-01

    hTERT, a catalytic component of human telomerase, is undetectable in normal somatic cells but up-regulated in cancer and stem cells where telomere length is maintained by telomerase. Accumulated evidence indicates that hTERT may have noncanonical functions beyond telomerase by regulating the expression of particular genes. However, comprehensive identification of the genes regulated by hTERT is unavailable. In this report, we expressed WT hTERT and hTERTmut which displays dysfunctional catalytic activity, in human U2OS cancer cells and VA-13 immortalized fibroblast cells, both of which lack endogenous hTERT and hTR expression. Changes in gene expression induced by hTERT and hTERT-mut expression were determined by genome-wide RNA-seq and verified by qPCR. Our results showed that hTERT affects different genes in two cell lines, implying that the regulation of gene expression by hTERT is indirect and cell type dependent. Moreover, functional analysis identifies cell adhesion-related genes that have been changed by hTERT in both cell lines. Adhesion experiments revealed that hTERT expression significantly increases cell adhesion. Monolayer wound healing and transwell assays demonstrated increased cell migration upon hTERT expression. These results provide new evidence to support a noncanonical function for hTERT in promoting tumorigenesis. PMID:26971878

  1. Shuttle active thermal control system development testing. Volume 7: Improved radiator coating adhesive tests

    NASA Technical Reports Server (NTRS)

    Reed, M. W.

    1973-01-01

    Silver/Teflon thermal control coatings have been tested on a modular radiator system projected for use on the space shuttle. Seven candidate adhesives have been evaluated in a thermal vacuum test on radiator panels similar to the anticipated flight hardware configuration. Several classes of adhesives based on polyester, silicone, and urethane resin systems were tested. These included contact adhesives, heat cured adhesives, heat and pressure cured adhesives, pressure sensitive adhesives, and two part paint on or spray on adhesives. The coatings attached with four of the adhesives, two silicones and two urethanes, had no changes develop during the thermal vacuum test. The two silicone adhesives, both of which were applied to the silver/Teflon as transfer laminates to form a tape, offered the most promise based on application process and thermal performance. Each of the successful silicone adhesives required a heat and pressure cure to adhere during the cryogenic temperature excursion of the thermal-vacuum test.

  2. Effect of viscous macromolecules on peritoneal plasminogen activator activity: a potential mechanism for their ability to reduce postoperative adhesion formation.

    PubMed

    Mayer, M; Yedgar, S; Hurwitz, A; Palti, Z; Finzi, Z; Milwidsky, A

    1988-10-01

    Activity of peritoneal plasminogen activator and its regulation by dextran and other macromolecules that clinically suppress postoperative adhesions was studied. Plasminogen activator activity was assayed by a two-stage globinolytic assay that monitors formation of plasmin, as well as by cleavage of a chromogenic peptide substrate (S-2444) in the presence of aprotinin (Trasylol). Plasminogen activator activity was located on the outer surface of human peritoneum. Incubation of peritoneal tissue with buffer in vitro (conditioning) prompted release of plasminogen activator into the conditioning medium. The released plasminogen activator formed a single band on sodium dodecyl sulfate-gel electrophoresis at an apparent molecular weight of 174,000 and was markedly suppressed by antiserum raised against human melanoma tissue-type plasminogen activator. Nonspecific proteolytic activity did not accumulate in the medium during conditioning. The presence of dextran 80 during conditioning of peritoneum reversibly suppressed tissue-bound plasminogen activator activity and reduced plasminogen activator activity in the spent medium. A similar inhibition of peritoneal plasminogen activator was induced by dextran 500, methyl cellulose, and polyvinylpyrrolidone. Dextran, when added to the medium after conditioning, had no direct inhibitory effect on plasminogen activator activity. Dextran did not induce peritoneal production of inhibitor(s) of trypsin, chymotrypsin, or urokinase. On the basis of these findings, two possible mechanisms for the effect of viscous polymers in the reduction of adhesion formation are proposed. These mechanisms consider the importance of peritoneal tissue-type plasminogen activator for removal of fibrin clots and suggest that polymer coating either prevents the shedding of plasminogen activator into the abdominal cavity or reduces the access of fibrin clots to the serosal surfaces. PMID:2459968

  3. Erythromycin exerts in vivo anti-inflammatory activity downregulating cell adhesion molecule expression

    PubMed Central

    Sanz, María-Jesús; Nabah, Yafa Naim Abu; Cerdá-Nicolás, Miguel; O'Connor, José-Enrique; Issekutz, Andrew C; Cortijo, Julio; Morcillo, Esteban J

    2004-01-01

    Macrolides have long been used as anti-bacterial agents; however, there is some evidence that may exert anti-inflammatory activity. Therefore, erythromycin was used to characterize the mechanisms involved in their in vivo anti-inflammatory activity. Erythromycin pretreatment (30 mg kg−1 day−1 for 1 week) reduced the lipopolysaccharide (LPS; intratracheal, 0.4 mg kg−1)-induced increase in neutrophil count and elastase activity in the bronchoalveolar lavage fluid (BALF) and lung tissue myeloperoxidase activity, but failed to decrease tumor necrosis factor-α and macrophage-inflammatory protein-2 augmented levels in BALF. Erythromycin pretreatment also prevented lung P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA upregulation in response to airway challenge with LPS. Mesentery superfusion with LPS (1 μg ml−1) induced a significant increase in leukocyte–endothelial cell interactions at 60 min. Erythromycin pretreatment abolished the increases in these parameters. LPS exposure of the mesentery for 4 h caused a significant increase in leukocyte rolling flux, adhesion and emigration, which were inhibited by erythromycin by 100, 93 and 95%, respectively. Immunohistochemical analysis showed that LPS exposure of the mesentery for 4 h caused a significant enhancement in P-selectin, E-selectin, ICAM-1 and VCAM-1 expression that was downregulated by erythromycin pretreatment. Flow cytometry analysis indicated that erythromycin pretreatment inhibited LPS-induced CD11b augmented expression in rat neutrophils. In conclusion, erythromycin inhibits leukocyte recruitment in the lung and this effect appears mediated through downregulation of CAM expression. Therefore, macrolides may be useful in the control of neutrophilic pulmonary diseases. PMID:15665859

  4. Protease activity at invadopodial focal digestive areas is dependent on NHE1-driven acidic pHe.

    PubMed

    Greco, Maria Raffaella; Antelmi, Ester; Busco, Giovanni; Guerra, Lorenzo; Rubino, Rosa; Casavola, Valeria; Reshkin, Stephan Joel; Cardone, Rosa Angela

    2014-02-01

    Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease-dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the invadopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na+/H+ exchanger 1 (NHE1)-driven extracellular acidification, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs preferentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1‑dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidification and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia. PMID:24337203

  5. The localization of focal heart activity via body surface potential measurements: tests in a heterogeneous torso phantom

    NASA Astrophysics Data System (ADS)

    Wetterling, F.; Liehr, M.; Schimpf, P.; Liu, H.; Haueisen, J.

    2009-09-01

    The non-invasive localization of focal heart activity via body surface potential measurements (BSPM) could greatly benefit the understanding and treatment of arrhythmic heart diseases. However, the in vivo validation of source localization algorithms is rather difficult with currently available measurement techniques. In this study, we used a physical torso phantom composed of different conductive compartments and seven dipoles, which were placed in the anatomical position of the human heart in order to assess the performance of the Recursively Applied and Projected Multiple Signal Classification (RAP-MUSIC) algorithm. Electric potentials were measured on the torso surface for single dipoles with and without further uncorrelated or correlated dipole activity. The localization error averaged 11 ± 5 mm over 22 dipoles, which shows the ability of RAP-MUSIC to distinguish an uncorrelated dipole from surrounding sources activity. For the first time, real computational modelling errors could be included within the validation procedure due to the physically modelled heterogeneities. In conclusion, the introduced heterogeneous torso phantom can be used to validate state-of-the-art algorithms under nearly realistic measurement conditions.

  6. The localization of focal heart activity via body surface potential measurements: tests in a heterogeneous torso phantom.

    PubMed

    Wetterling, F; Liehr, M; Schimpf, P; Liu, H; Haueisen, J

    2009-09-21

    The non-invasive localization of focal heart activity via body surface potential measurements (BSPM) could greatly benefit the understanding and treatment of arrhythmic heart diseases. However, the in vivo validation of source localization algorithms is rather difficult with currently available measurement techniques. In this study, we used a physical torso phantom composed of different conductive compartments and seven dipoles, which were placed in the anatomical position of the human heart in order to assess the performance of the Recursively Applied and Projected Multiple Signal Classification (RAP-MUSIC) algorithm. Electric potentials were measured on the torso surface for single dipoles with and without further uncorrelated or correlated dipole activity. The localization error averaged 11 +/- 5 mm over 22 dipoles, which shows the ability of RAP-MUSIC to distinguish an uncorrelated dipole from surrounding sources activity. For the first time, real computational modelling errors could be included within the validation procedure due to the physically modelled heterogeneities. In conclusion, the introduced heterogeneous torso phantom can be used to validate state-of-the-art algorithms under nearly realistic measurement conditions. PMID:19700819

  7. Down-regulated Na+/K+-ATPase activity in ischemic penumbra after focal cerebral ischemia/reperfusion in rats

    PubMed Central

    Huang, Hao; Chen, Yang-Mei; Zhu, Fei; Tang, Shi-Ting; Xiao, Ji-Dong; Li, Lv-Li; Lin, Xin-Jing

    2015-01-01

    This study was aimed to examine whether the Na+/K+ adenosine triphosphatase (Na+/K+-ATPase) activity in ischemic penumbra is associated with the pathogenesis of ischemia/reperfusion-induced brain injury. An experimental model of cerebral ischemia/reperfusion was made by transient middle cerebral artery occlusion (tMCAO) in rats and the changes of Na+/K+-ATPase activity in the ischemic penumbra was examined by Enzyme Assay Kit. Extensive infarction was observed in the frontal and parietal cortical and subcortical areas at 6 h, 24 h, 48 h, 3 d and 7 d after tMCAO. Enzyme Assay analyses revealed the activity of Na+/K+-ATPase was decreased in the ischemic penumbra of model rats after focal cerebral ischemia/reperfusion compared with sham-operated rats, and reduced to its minimum at 48 h, while the infarct volume was enlarged gradually. In addition, accompanied by increased brain water content, apoptosis-related bcl-2 and Bax proteins, apoptotic index and neurologic deficits Longa scores, but fluctuated the ratio of bcl-2/Bax. Correlation analysis showed that the infarct volume, apoptotic index, neurologic deficits Longa scores and brain water content were negatively related with Na+/K+-ATPase activity, while the ratio of bcl-2/Bax was positively related with Na+/K+-ATPase activity. Our results suggest that down-regulated Na+/K+-ATPase activity in ischemic penumbra might be involved in the pathogenesis of cerebral ischemia/reperfusion injury presumably through the imbalance ratio of bcl-2/Bax and neuronal apoptosis, and identify novel target for neuroprotective therapeutic intervention in cerebral ischemic disease. PMID:26722460

  8. Integrin adhesion drives the emergent polarization of active cytoskeletal stresses to pattern cell delamination

    PubMed Central

    Meghana, C.; Ramdas, Nisha; Hameed, Feroz Meeran; Rao, Madan; Shivashankar, G. V.; Narasimha, Maithreyi

    2011-01-01

    Tissue patterning relies on cellular reorganization through the interplay between signaling pathways and mechanical stresses. Their integration and spatiotemporal coordination remain poorly understood. Here we investigate the mechanisms driving the dynamics of cell delamination, diversely deployed to extrude dead cells or specify distinct cell fates. We show that a local mechanical stimulus (subcellular laser perturbation) releases cellular prestress and triggers cell delamination in the amnioserosa during Drosophila dorsal closure, which, like spontaneous delamination, results in the rearrangement of nearest neighbors around the delaminating cell into a rosette. We demonstrate that a sequence of “emergent cytoskeletal polarities” in the nearest neighbors (directed myosin flows, lamellipodial growth, polarized actomyosin collars, microtubule asters), triggered by the mechanical stimulus and dependent on integrin adhesion, generate active stresses that drive delamination. We interpret these patterns in the language of active gels as asters formed by active force dipoles involving surface and body stresses generated by each cell and liken delamination to mechanical yielding that ensues when these stresses exceed a threshold. We suggest that differential contributions of adhesion, cytoskeletal, and external stresses must underlie differences in spatial pattern. PMID:21571643

  9. Chitosan hydrogels enriched with polyphenols: Antibacterial activity, cell adhesion and growth and mineralization.

    PubMed

    Lišková, Jana; Douglas, Timothy E L; Beranová, Jana; Skwarczyńska, Agata; Božič, Mojca; Samal, Sangram Keshari; Modrzejewska, Zofia; Gorgieva, Selestina; Kokol, Vanja; Bačáková, Lucie

    2015-09-20

    Injectable hydrogels for bone regeneration consisting of chitosan, sodium beta-glycerophosphate (Na-β-GP) and alkaline phosphatase (ALP) were enriched with the polyphenols phloroglucinol (PG) and gallic acid (GA) and characterized physicochemically and biologically with respect to properties relevant for applications in bone regeneration, namely gelation kinetics, mineralizability, antioxidant properties, antibacterial activity, cytocompatibility and ability to support adhesion and growth of human osteoblast-like MG63 cells. Enrichment with PG and GA had no negative effect on gelation kinetics and mineralizability. PG and GA both enhanced antioxidant activity of unmineralized hydrogels. Mineralization reduced antioxidant activity of hydrogels containing GA. Hydrogels containing GA, PG and without polyphenols reduced colony forming ability of Escherichia coli after 1h, 3h and 6h incubation and slowed E. coli growth in liquid culture for 150min. Hydrogels containing GA were cytotoxic and supported cell growth more poorly than polyphenol-free hydrogels. PG had no negative effect on cell adhesion and growth. PMID:26050898

  10. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation

    PubMed Central

    Cuff, Carolyn A.; Kothapalli, Devashish; Azonobi, Ijeoma; Chun, Sam; Zhang, Yuanming; Belkin, Richard; Yeh, Christine; Secreto, Anthony; Assoian, Richard K.; Rader, Daniel J.; Puré, Ellen

    2001-01-01

    Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD44 to atherosclerosis, we bred CD44-null mice to atherosclerosis-prone apoE-deficient mice. We found a 50–70% reduction in aortic lesions in CD44-null mice compared with CD44 heterozygote and wild-type littermates. We demonstrate that CD44 promotes the recruitment of macrophages to atherosclerotic lesions. Furthermore, we show that CD44 is required for phenotypic dedifferentiation of medial smooth muscle cells to the “synthetic” state as measured by expression of VCAM-1. Finally, we demonstrate that hyaluronan, the principal ligand for CD44, is upregulated in atherosclerotic lesions of apoE-deficient mice and that the low-molecular-weight proinflammatory forms of hyaluronan stimulate VCAM-1 expression and proliferation of cultured primary aortic smooth muscle cells, whereas high-molecular-weight forms of hyaluronan inhibit smooth muscle cell proliferation. We conclude that CD44 plays a critical role in the progression of atherosclerosis through multiple mechanisms. PMID:11581304

  11. Adhesive properties of the purified plasminogen activator Pla of Yersinia pestis.

    PubMed

    Lobo, Leandro Araujo

    2006-09-01

    The beta-barrel outer membrane protease Pla from Yersinia pestis is an important virulence factor in plague and enables initiation of the bubonic plague. Pla is a multifunctional protease whose expression also enhances bacterial adherence to extracellular matrix. It has remained uncertain whether the increase in cellular adhesiveness results from modification of the bacterial surface by Pla, or whether the Pla molecule is an adhesin. Pla was purified as a His6-fusion protein from Escherichia coli and reconstituted with lipopolysaccharide to an enzymatically active form. Purified His6-Pla was coated onto fluorescent micro-particles (FMPs) that expressed plasminogen activity. Pla-coated FMPs also bound to laminin and to reconstituted basement membrane (Matrigel) immobilized on permanox slides, whereas only poor activity was seen with lipopolysaccharide-coated FMPs or bovine serum albumin-coated FMPs. The results show that the Pla molecule has intrinsic adhesive properties and that purified transmembrane proteins coated onto FMPs can be used for functional assays. PMID:16923070

  12. Kinetics of LFA-1 mediated adhesion of human neutrophils to ICAM-1-role of E-selectin signaling post-activation.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    LFA-1 and Mac-1 are the two integrins involved in the arrest and firm adhesion of neutrophils. LFA-1 plays a role in the early stage of cell arrest while Mac-1 stabilizes firm adhesion. Here, we further elucidated the kinetics of LFA-1 activation and its role in mediating neutrophil adhesion to ICAM...

  13. Matrine attenuates focal cerebral ischemic injury by improving antioxidant activity and inhibiting apoptosis in mice

    PubMed Central

    ZHAO, PENG; ZHOU, RU; ZHU, XIAO-YUN; HAO, YIN-JU; LI, NAN; WANG, JIE; NIU, YANG; SUN, TAO; LI, YU-XIANG; YU, JIAN-QIANG

    2015-01-01

    Matrine, an active constituent of the Chinese herb, Sophora flavescens Ait., and it is known for its antioxidant, anti-inflammatory and antitumor activities. It has been demonstrated that matrine exerts protective effects against heart failure by decreasing the expression of caspase-3 and Bax, and increasing Bcl-2 levels. In this study, we aimed to determine whether these protective effects of matrine can be applied to cerebral ischemia. Following 7 successive days of treatment with matrine (7.5, 15 and 30 mg/kg) and nimodipine (1 mg/kg) by intraperitoneal injection, male Institute of Cancer Research (ICR) mice were subjected to middle cerebral artery occlusion (MCAO). Following reperfusion, the neurobehavioral score and brain infarct volume were estimated, and morphological changes were analyzed by hematoxylin and eosin (H&E) staining and electron microscopy. The percentage of apoptotic neurons was determined by flow cytometry. The levels of oxidative stress were assessed by measuring the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the total antioxidant capacity (T-AOC). Western blot analysis and immunofluorescence staining were used to examine the expression of the apoptosis-related proteins, caspase-3, Bax and Bcl-2. Our results revealed that pre-treatment with matrine significantly decreased the infarct volume and improved the neurological scores. Matrine also reduced the percentage of apoptotic neurons and relieved neuronal morphological damage. Furthermore, matrine markedly decreased the MDA levels, and increased SOD, GSH-Px and CAT activity, and T-AOC. Western blot analysis and immunofluorescence staining revealed a marked decrease in caspase-3 expression and an increase in the Bcl-2/Bax ratio in the group pre-treated with matrine (30 mg/kg) as compared with the vehicle-treated group. The findings of the present study demonstrate that matrine exerts neuroprotective effects against

  14. Surface morphology of platelet adhesion influenced by activators, inhibitors and shear stress

    NASA Astrophysics Data System (ADS)

    Watson, Melanie Groan

    Platelet activation involves multiple events, one of which is the generation and release of nitric oxide (NO), a platelet aggregation inhibitor. Platelets simultaneously send and receive various agents that promote a positive and negative feedback control system during hemostasis. Although the purpose of platelet-derived NO is not fully understood, NO is known to inhibit platelet recruitment. NO's relatively large diffusion coefficient allows it to diffuse more rapidly than platelet agonists. It may thus be able to inhibit recruitment of platelets near the periphery of a growing thrombus before agonists have substantially accumulated in those regions. Results from two studies in our laboratory differed in the extent to which platelet-derived NO decreased platelet adhesion. Frilot studied the effect of L-arginine (L-A) and NG-Methyl-L-arginine acetate salt (L-NMMA) on platelet adhesion to collagen under static conditions in a Petri dish. Eshaq examined the percent coverage on collagen-coated and fibrinogen-coated microchannels under shear conditions with different levels of L-A and Adenosine Diphosphate (ADP). Frilot's results showed no effect of either L-A or L-NMMA on surface coverage, thrombus size or serotonin release, while Eshaq's results showed a decrease in surface coverage with increased levels of L-A. A possible explanation for these contrasting results is that platelet-derived NO may be more important under flow conditions than under static conditions. For this project, the effects of L-A. ADP and L-NMMA on platelet adhesion were studied at varying shear stresses on protein-coated glass slides. The surface exposed to platelet-rich-plasma in combination with each chemical solution was observed under AFM, FE-SEM and fluorescence microscopy. Quantitative and qualitative comparisons of images obtained with these techniques confirmed the presence of platelets on the protein coatings. AFM images of fibrinogen and collagen-coated slides presented characteristic

  15. Doxycycline inhibits MMPs via modulation of plasminogen activators in focal cerebral ischemia.

    PubMed

    Burggraf, Dorothe; Trinkl, Andreas; Dichgans, Martin; Hamann, Gerhard F

    2007-03-01

    Tetracyclines inhibit matrix metalloproteinases (MMPs) and reduce infarction volume following cerebral ischemia. In this thesis an involvement of urokinase could be proven. Cerebral ischemia in rats was induced for 3 h followed by 24 h reperfusion (suture model). Each 6 animals received orally either doxycycline or water. Doxycycline treatment began 10 days before ischemia. MMP-2 and MMP-9 were substantially decreased. The possibility of involvement of the endogenous MMP inhibitors in the MMP inhibiting mechanisms was excluded. The plasminogen activator uPA was significantly decreased by doxycycline indicating an MMP inhibiting mechanism including the plasminogen/plasmin system. In the doxycycline group, this resulted in a decreased damage to the cerebral microvessels and less loss of the basal lamina antigen collagen type IV. Hemoglobin extravasation was also significantly reduced. Our results suggest that doxycycline may have a potential use as an anti-ischemic compound since it provides microvascular protection by inhibiting the plasminogen system. PMID:17166729

  16. Heat transfer model to characterize the focal cooling necessary to suppress spontaneous epileptiform activity (Invited Paper)

    NASA Astrophysics Data System (ADS)

    Guerra, Reynaldo G.; Davalos, Rafael V.; Garcia, Paul A.; Rubinsky, Boris; Berger, Mitchel

    2005-04-01

    Epilepsy is characterized by paroxysmal transient disturbances of the electrical activity of the brain. Symptoms are manifested as impairment of motor, sensory, or psychic function with or without loss of consciousness or convulsive seizures. This paper presents an initial post-operative heat transfer analysis of surgery performed on a 41 year-old man with medically intractable Epilepsy. The surgery involved tumor removal and the resection of adjacent epileptogenic tissue. Electrocorticography was performed before resection. Cold saline was applied to the resulting interictal spike foci resulting in transient, complete cessation of spiking. A transient one dimensional semi-infinite finite element model of the surface of the brain was developed to simulate the surgery. An approximate temperature distribution of the perfused brain was developed by applying the bioheat equation. The model quantifies the surface heat flux reached in achieving seizure cessation to within an order of magnitude. Rat models have previously shown that the brain surface temperature range to rapidly terminate epileptogenic activity is 20-24°C. The developed model predicts that a constant heat flux of approximately -13,000W/m2, applied at the surface of the human brain, would achieve a surface temperature in this range in approximately 3 seconds. A parametric study was subsequently performed to characterize the effects of brain metabolism and brain blood perfusion as a function of the determined heat flux. The results of these findings can be used as a first approximation in defining the specifications of a cooling device to suppress seizures in human models.

  17. Research of surface activating influence on formation of adhesion between gas-thermal coating and steel substrate

    NASA Astrophysics Data System (ADS)

    Kovalevskaya, Z.; Klimenov, V.; Zaitsev, K.

    2015-09-01

    Estimation of influence of physical and thermal activating on adhesion between steel substrates and thermal coatings has been performed. The substrates with surfaces obtained by and ultrasonic surface plastic deformation were used. To evaluate physical activating, preheating of the substrates to 600°C was performed. To evaluate the effect of thermal activating, the substrate surfaces after interfacial detachment were examined. Bonded areas on the substrate surfaces were measured by means of optical profilometry. The experiments have shown that surface physical activating is the main factor in formation of the adhesive bond between the coating and the substrate processed with the proposed methods.

  18. Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains isolated from Tuber borchii ascocarps.

    PubMed

    Sbrana, C; Bagnoli, G; Bedini, S; Filippi, C; Giovannetti, M; Nuti, M P

    2000-03-01

    Pseudomonas spp. isolates from Tuber borchii ascocarps, known to be able to produce phytoregulatory and biocontrol substances in pure culture, were used to perform studies on their possible physiological role in nature. Antimycotic activity was confirmed against fungal contaminants isolated from the ascocarps, suggesting that populations associated with Tuber borchii fruit bodies may play a role in the maintenance of ascocarp health. Fifty-five percent of strains tested were also able to release metabolites which affected T. borchii mycelial growth and morphogenesis in culture. On the contrary, growth of the arbuscular mycorrhizal fungus Glomus mosseae and the ectomycorrhizal fungus Laccaria bicolor, putative competitors of Tuber for mycorrhizal infection sites on roots, was not influenced by the presence of any bacterial strain. The possibility that these bacteria, which show antifungal activity and fungal growth modulation activities, might be incorporated in the developing ascocarp by means of their preferential adhesion to Tuber mycelium is discussed. PMID:10749539

  19. Porcine E. coli: Virulence-Associated Genes, Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

    PubMed Central

    Schierack, Peter; Rödiger, Stefan; Kuhl, Christoph; Hiemann, Rico; Roggenbuck, Dirk; Li, Ganwu; Weinreich, Jörg; Berger, Enrico; Nolan, Lisa K.; Nicholson, Bryon; Römer, Antje; Frömmel, Ulrike; Wieler, Lothar H.; Schröder, Christian

    2013-01-01

    We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars. PMID:23658605

  20. Active Compounds of Rhubarb Root and Rhizome in Animal Model Experiments of Focal Cerebral Ischemia

    PubMed Central

    Liu, Ai-ju; Song, Liang; Li, Yan; Zhang, Xiao-guang; Chen, Zi-xian; Huang, Li-bo; Zhang, Hong-feng; Zheng, Guo-qing

    2015-01-01

    Rhubarb root and rhizome (RRR) has been clinically used for stroke at least 2000 years and is still used in modern times in both China and elsewhere worldwide. The objective of present study was to evaluate the efficacy of active compounds of RRR (ACRRR) for experimental ischemic stroke. Studies of ACRRR in animal models of ischemic stroke were identified from 5 databases until April 2014. Study quality for each included article was evaluated according to the CAMARADES 10-item checklist. Outcome measures were neurological deficit score and infarct size. All the data were analyzed using RevMan 5.1 software. As a result, 20 studies were identified describing procedures involving 577 animals. The quality score of studies ranges from 2 to 6, and the median was 3.4. Six studies showed significant effects of ACRRR for improving infarct size compared with model group (P < 0.01). Six studies indicated significant effects of ACRRR for improving the neurological deficit scores according to Zea longa criterion or eight-point criterion (P < 0.01). In conclusion, these findings demonstrated a possible efficacy of ACRRR that have potential neuroprotective effect for experimental ischemic stroke. However, these apparently positive findings should be interpreted with caution because of the methodological flaws. PMID:26495006

  1. Cysteine Protease Activity of Feline Tritrichomonas foetus Promotes Adhesion-Dependent Cytotoxicity to Intestinal Epithelial Cells

    PubMed Central

    Tolbert, M. K.; Stauffer, S. H.; Brand, M. D.

    2014-01-01

    Trichomonads are obligate protozoan parasites most renowned as venereal pathogens of the reproductive tract of humans and cattle. Recently, a trichomonad highly similar to bovine venereal Tritrichomonas foetus but having a unique tropism for the intestinal tract was recognized as a significant cause of colitis in domestic cats. Despite a high prevalence, worldwide distribution, and lack of consistently effective drugs for treatment of the infection, the cellular mechanisms of T. foetus pathogenicity in the intestinal tract have not been examined. The aims of this study were to determine the pathogenic effect of feline T. foetus on porcine intestinal epithelial cells, the dependence of T. foetus pathogenicity on adhesion of T. foetus to the intestinal epithelium, and the identity of mediators responsible for these effects. Using an in vitro coculture approach to model feline T. foetus infection of the intestinal epithelium, these studies demonstrate that T. foetus promotes a direct contact-dependent activation of intestinal epithelial cell apoptosis signaling and progressive monolayer destruction. Moreover, these pathological effects were demonstrated to be largely dependent on T. foetus cell-associated cysteine protease activity. Finally, T. foetus cysteine proteases were identified as enabling cytopathic effects by promoting adhesion of T. foetus to the intestinal epithelium. The present studies are the first to examine the cellular mechanisms of pathogenicity of T. foetus toward the intestinal epithelium and support further investigation of the cysteine proteases as virulence factors in vivo and as potential therapeutic targets for ameliorating the pathological effects of intestinal trichomonosis. PMID:24752513

  2. Cysteine protease activity of feline Tritrichomonas foetus promotes adhesion-dependent cytotoxicity to intestinal epithelial cells.

    PubMed

    Tolbert, M K; Stauffer, S H; Brand, M D; Gookin, J L

    2014-07-01

    Trichomonads are obligate protozoan parasites most renowned as venereal pathogens of the reproductive tract of humans and cattle. Recently, a trichomonad highly similar to bovine venereal Tritrichomonas foetus but having a unique tropism for the intestinal tract was recognized as a significant cause of colitis in domestic cats. Despite a high prevalence, worldwide distribution, and lack of consistently effective drugs for treatment of the infection, the cellular mechanisms of T. foetus pathogenicity in the intestinal tract have not been examined. The aims of this study were to determine the pathogenic effect of feline T. foetus on porcine intestinal epithelial cells, the dependence of T. foetus pathogenicity on adhesion of T. foetus to the intestinal epithelium, and the identity of mediators responsible for these effects. Using an in vitro coculture approach to model feline T. foetus infection of the intestinal epithelium, these studies demonstrate that T. foetus promotes a direct contact-dependent activation of intestinal epithelial cell apoptosis signaling and progressive monolayer destruction. Moreover, these pathological effects were demonstrated to be largely dependent on T. foetus cell-associated cysteine protease activity. Finally, T. foetus cysteine proteases were identified as enabling cytopathic effects by promoting adhesion of T. foetus to the intestinal epithelium. The present studies are the first to examine the cellular mechanisms of pathogenicity of T. foetus toward the intestinal epithelium and support further investigation of the cysteine proteases as virulence factors in vivo and as potential therapeutic targets for ameliorating the pathological effects of intestinal trichomonosis. PMID:24752513

  3. Evaluation of disease activity in rheumatic patients by leucocyte adhesiveness/aggregation.

    PubMed Central

    Berliner, S; Fried, M; Caspi, D; Weinberger, A; Yaron, M; Pinkhas, J; Aronson, M

    1988-01-01

    Previous work has shown that leucocyte adhesiveness/aggregation (LAA), as measured by the leukergy test, correlates well with disease severity in rheumatic patients. As LAA is probably a manifestation of the acute phase reaction various components of the acute phase reaction were measured in order to identify the best marker of disease activity. In addition to LAA, the following variables were measured in 79 patients with various rheumatic diseases and in 10 controls: white blood cell and platelet counts, erythrocyte sedimentation rate, haptoglobin, fibrinogen, C reactive protein, albumin, globulin, caeruloplasmin, alpha 1, alpha 2, beta, and gamma globulin, and haemoglobin concentrations. Patients were graded according to the state of their disease as mild, moderate, or severe. The extent of leucocyte adhesiveness/aggregation in peripheral blood proved to be the best laboratory variable for the grading of disease activity. Correct grading was obtained in 63% of the patients by means of the LAA, compared with 48% with C reactive protein, 41% with caeruloplasmin, 40% with haptoglobin, and 32% with haemoglobin. It is suggested that LAA of the peripheral blood during inflammation may be used as a reliable marker of disease severity. PMID:3260093

  4. Early detection of human focal seizures based on cortical multiunit activity.

    PubMed

    Park, Yun S; Hochberg, Leigh R; Eskandar, Emad N; Cash, Sydney S; Truccolo, Wilson

    2014-01-01

    Approximately 50 million people in the world suffer from epileptic seizures. Reliable early seizure detection could bring significantly beneficial therapeutic alternatives. In recent decades, most approaches have relied on scalp EEG and intracranial EEG signals, but practical early detection for closed-loop seizure control remains challenging. In this study, we present preliminary analyses of an early detection approach based on intracortical neuronal multiunit activity (MUA) recorded from a 96-microelectrode array (MEA). The approach consists of (1) MUA detection from broadband field potentials recorded at 30 kHz by the MEA; (2) MUA feature extraction; (3) cost-sensitive support vector machine classification of ictal and interictal samples; and (4) Kalman-filtering postprocessing. MUA was here defined as the number of threshold crossing (spike counts) applied to the 300 Hz-6 kHz bandpass filtered local field potentials in 0.1 sec time windows. MUA features explored in this study included the mean, variance, and Fano-factor, computed across the MEA channels. In addition, we used the leading eigenvalues of MUA spatial and temporal correlation matrices computed in 1-sec moving time windows. We assessed the seizure detection approach on out-of-sample data from one-participant recordings with six seizure events and 4.73-hour interictal data. The proposed MUA-based detection approach yielded a 100% sensitivity (6/6) and no false positives, and a latency of 4.17 ± 2.27 sec (mean ± SD) with respect to ECoG-identified seizure onsets. These preliminary results indicate intracortical MUA may be a useful signal for early detection of human epileptic seizures. PMID:25571313

  5. Characterisation of a novel light activated adhesive scaffold: Potential for device attachment.

    PubMed

    Ark, Morris; Boughton, Philip; Lauto, Antonio; Tran, Giang T; Chen, Yongjuan; Cosman, Peter H; Dunstan, Colin R

    2016-09-01

    The most common methods for attaching a device to the internal tissues of the human body are via sutures, clips or staples. These attachment techniques require penetration and manipulation of the tissue. Tears and leaks can often be a complication post-attachment, and scarring usually occurs around the attachment sites. To resolve these issues, it is proposed to develop a soft tissue scaffold impregnated with Rose Bengal/Chitosan solution (RBC-scaffold, 0.01% w/v Rose Bengal, 1.7% w/v Medium Molecular Weight Chitosan). This scaffold will initially attach to the tissue via a light activation method. The light activates the dye in the scaffold which causes cross-links to form between the scaffold and tissue, thus adhering them together. This is done without mechanically manipulating the surrounding tissue, thus avoiding the issues associated with current techniques. Eventually, the scaffold will be resorbed and tissue will integrate for long-term attachment. A variety of tests were performed to characterise the RBC-scaffold. Porosity, interconnectivity, and mechanical strength were measured. Light activation was performed with a broad spectrum (380-780nm) 10W LED lamp exposed to various time lengths (2-15min, Fluence range 0.4-3J/cm(2) ). Adhesive strength of the light-activated bond was measured with lap-shear tests performed on porcine stomach tissue. Cell culture viability was also assessed to confirm tissue integration potential. These properties were compared to Variotis™, an aliphatic polyester soft tissue scaffold which has proven to be viable for soft tissue regeneration. The RBC-scaffolds were found to have high porosity (86.46±2.95%) and connectivity, showing rapid fluid movement. The elastic modulus of the RBC-scaffolds (3.55±1.28MPa) was found to be significantly higher than the controls (0.15±0.058MPa, p<0.01) and approached reported values for human gastrointestinal tissue (2.3MPa). The maximum adhesion strength achieved of the RBC-scaffolds was 8

  6. Transcriptional activation of endothelial cells by TGFβ coincides with acute microvascular plasticity following focal spinal cord ischaemia/reperfusion injury.

    PubMed

    Benton, Richard L; Maddie, Melissa A; Dincman, Toros A; Hagg, Theo; Whittemore, Scott R

    2009-01-01

    Microvascular dysfunction, loss of vascular support, ischaemia and sub-acute vascular instability in surviving blood vessels contribute to secondary injury following SCI (spinal cord injury). Neither the precise temporal profile of the cellular dynamics of spinal microvasculature nor the potential molecular effectors regulating this plasticity are well understood. TGFβ (transforming growth factor β) isoforms have been shown to be rapidly increased in response to SCI and CNS (central nervous system) ischaemia, but no data exist regarding their contribution to microvascular dysfunction following SCI. To examine these issues, in the present study we used a model of focal spinal cord ischaemia/reperfusion SCI to examine the cellular response(s) of affected microvessels from 30 min to 14 days post-ischaemia. Spinal endothelial cells were isolated from affected tissue and subjected to focused microarray analysis of TGFβ-responsive/related mRNAs 6 and 24 h post-SCI. Immunohistochemical analyses of histopathology show neuronal disruption/loss and astroglial regression from spinal microvessels by 3 h post-ischaemia, with complete dissolution of functional endfeet (loss of aquaporin-4) by 12 h post-ischaemia. Coincident with this microvascular plasticity, results from microarray analyses show 9 out of 22 TGFβ-responsive mRNAs significantly up-regulated by 6 h post-ischaemia. Of these, serpine 1/PAI-1 (plasminogen-activator inhibitor 1) demonstrated the greatest increase (>40-fold). Furthermore, uPA (urokinase-type plasminogen activator), another member of the PAS (plasminogen activator system), was also significantly increased (>7.5-fold). These results, along with other select up-regulated mRNAs, were confirmed biochemically or immunohistochemically. Taken together, these results implicate TGFβ as a potential molecular effector of the anatomical and functional plasticity of microvessels following SCI. PMID:19663807

  7. Transcriptional activation of endothelial cells by TGFβ coincides with acute microvascular plasticity following focal spinal cord ischaemia/reperfusion injury

    PubMed Central

    Benton, Richard L; Maddie, Melissa A; Dincman, Toros A; Hagg, Theo; Whittemore, Scott R

    2009-01-01

    Microvascular dysfunction, loss of vascular support, ischaemia and sub-acute vascular instability in surviving blood vessels contribute to secondary injury following SCI (spinal cord injury). Neither the precise temporal profile of the cellular dynamics of spinal microvasculature nor the potential molecular effectors regulating this plasticity are well understood. TGFβ (transforming growth factor β) isoforms have been shown to be rapidly increased in response to SCI and CNS (central nervous system) ischaemia, but no data exist regarding their contribution to microvascular dysfunction following SCI. To examine these issues, in the present study we used a model of focal spinal cord ischaemia/reperfusion SCI to examine the cellular response(s) of affected microvessels from 30 min to 14 days post-ischaemia. Spinal endothelial cells were isolated from affected tissue and subjected to focused microarray analysis of TGFβ-responsive/related mRNAs 6 and 24 h post-SCI. Immunohistochemical analyses of histopathology show neuronal disruption/loss and astroglial regression from spinal microvessels by 3 h post-ischaemia, with complete dissolution of functional endfeet (loss of aquaporin-4) by 12 h post-ischaemia. Coincident with this microvascular plasticity, results from microarray analyses show 9 out of 22 TGFβ-responsive mRNAs significantly up-regulated by 6 h post-ischaemia. Of these, serpine 1/PAI-1 (plasminogen-activator inhibitor 1) demonstrated the greatest increase (>40-fold). Furthermore, uPA (urokinase-type plasminogen activator), another member of the PAS (plasminogen activator system), was also significantly increased (>7.5-fold). These results, along with other select up-regulated mRNAs, were confirmed biochemically or immunohistochemically. Taken together, these results implicate TGFβ as a potential molecular effector of the anatomical and functional plasticity of microvessels following SCI. PMID:19663807

  8. Activity and social factors affect cohesion among individuals in female Japanese macaques: A simultaneous focal-follow study.

    PubMed

    Nishikawa, Mari; Suzuki, Mariko; Sprague, David S

    2014-07-01

    Understanding cohesion among individuals within a group is necessary to reveal the social system of group-living primates. Japanese macaques (Macaca fuscata) are female-philopatric primates that reside in social groups. We investigated whether individual activity and social factors can affect spatio-temporal cohesion in wild female Japanese macaques. We conducted behavioral observation on a group, which contained 38 individuals and ranged over ca. 60 ha during the study period. Two observers carried out simultaneous focal-animal sampling of adult female pairs during full-day follows using global positioning system which enabled us to quantify interindividual distances (IIDs), group members within visual range (i.e., visual unit), and separation duration beyond visual range as indicators of cohesion among individuals. We found considerable variation in spatio-temporal group cohesion. The overall mean IID was 99.9 m (range = 0-618.2 m). The percentage of IIDs within visual range was 23.1%, within auditory range was 59.8%, and beyond auditory range was 17.1%. IIDs varied with activity; they were shorter during grooming and resting, and longer during foraging and traveling. Low-ranking females showed less cohesion than high-ranking ones. Kin females stayed nearly always within audible range. The macaques were weakly cohesive with small mean visual unit size (3.15 counting only adults, 5.99 counting all individuals). Both-sex units were the most frequently observed visual unit type when they were grooming/resting. Conversely, female units were the most frequently observed visual unit type when they were foraging. The overall mean visual separation duration was 25.7 min (range = 3-513 min). Separation duration was associated with dominance rank. These results suggest that Japanese macaques regulate cohesion among individuals depending on their activity and on social relationships; they were separated to adapt food distribution and aggregated to maintain social

  9. Polyurethane blended with polylactides for improved cell adhesion and reduced platelet activation.

    PubMed

    Hsu, S H; Tseng, H J; Fang, Z K

    1999-10-01

    Poly(L-lactide) (PLLA) or 50:50 poly(lactide-co-glycolide) (PLGA) was blended with a commercial polyurethane (PU), Pellethane 2103-80A, using a mutual solvent technique. After preparation by salt casting, the mechanical properties of the porous matrices were measured, and the surface element was characterized by electron spectroscopy for chemical analysis. The attachment and growth of fibroblasts as well as human umbilical vein endothelial cells on the substrates were investigated by the cell culture test. Platelet activation on different substrates was also studied. It was found that blending with these biodegradable polymers, especially PLGA. enhanced the cellular attachment and growth. This enhancement may be correlated with the higher oxygen/carbon (0/C) atomic ratio on the surface of the blends. Blending with either PLLA or PLGA was also found to decrease platelet adhesion and activation in vitro. These characteristics make PU/polylactide blends potential substrates for cardiovascular applications. PMID:10610681

  10. Syntenin-1 and Ezrin Proteins Link Activated Leukocyte Cell Adhesion Molecule to the Actin Cytoskeleton*

    PubMed Central

    Tudor, Cicerone; te Riet, Joost; Eich, Christina; Harkes, Rolf; Smisdom, Nick; Bouhuijzen Wenger, Jessica; Ameloot, Marcel; Holt, Matthew; Kanger, Johannes S.; Figdor, Carl G.; Cambi, Alessandra; Subramaniam, Vinod

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side. PMID:24662291

  11. EphB2 activity plays a pivotal role in pediatric medulloblastoma cell adhesion and invasion

    PubMed Central

    Sikkema, Arend H.; den Dunnen, Wilfred F.A.; Hulleman, Esther; van Vuurden, Dannis G.; Garcia-Manero, Guillermo; Yang, Hui; Scherpen, Frank J.G.; Kampen, Kim R.; Hoving, Eelco W.; Kamps, Willem A.; Diks, Sander H.; Peppelenbosch, Maikel P.; de Bont, Eveline S.J.M.

    2012-01-01

    Eph/ephrin signaling has been implicated in various types of key cancer-enhancing processes, like migration, proliferation, and angiogenesis. In medulloblastoma, invading tumor cells characteristically lead to early recurrence and a decreased prognosis. Based on kinase-activity profiling data published recently, we hypothesized a key role for the Eph/ephrin signaling system in medulloblastoma invasion. In primary medulloblastoma samples, a significantly higher expression of EphB2 and the ligand ephrin-B1 was observed compared with normal cerebellum. Furthermore, medulloblastoma cell lines showed high expression of EphA2, EphB2, and EphB4. Stimulation of medulloblastoma cells with ephrin-B1 resulted in a marked decrease in in vitro cell adhesion and an increase in the invasion capacity of cells expressing high levels of EphB2. The cell lines that showed an ephrin-B1–induced phenotype possessed increased levels of phosphorylated EphB2 and, to a lesser extent, EphB4 after stimulation. Knockdown of EphB2 expression by short hairpin RNA completely abolished ephrin ligand–induced effects on adhesion and migration. Analysis of signal transduction identified p38, Erk, and mTOR as downstream signaling mediators potentially inducing the ephrin-B1 phenotype. In conclusion, the observed deregulation of Eph/ephrin expression in medulloblastoma enhances the invasive phenotype, suggesting a potential role in local tumor cell invasion and the formation of metastases. PMID:22723427

  12. Impact of simulated microgravity on the secretory and adhesive activity of cultured human vascular endothelial cells.

    NASA Astrophysics Data System (ADS)

    Rudimov, Evgeny; Buravkova, Ludmila; Pogodina, Margarita; Andrianova, Irina

    The layer of vascular endothelial cells (ECs) is a dynamic,disseminated organ that perform the function of an interface between the blood and vascular wall. The endothelial monolayer is able to quickly respond to changes in the microenvironment due to its synthesis of vasoactive substances, chemokines, adhesion molecules expression, etc. ECs are highly sensitive to gravitational changes and capable of short-term and long-term responses (Sangha et al., 2001; Buravkova et al., 2005; Infanger et al., 2006, 2007. However, the question remains how to reflect the impact of microgravity on endothelium under the inflammatory process. Therefore, the aim of this study was to investigate secretory and adhesive activity of human umbilical vein endothelial cells (HUVECs) during simulated microgravity and TNF-a activation. HUVECs were isolated according to Gimbrone et al. (1978) in modification A. Antonov (1981) and used for experiments at 2-4 passages. HUVECs were activated by low level of TNF-a (2 ng/ml). Microgravity was generated by Random Positioning Machine (RPM, Dutch Space, Leiden) placed into the thermostat at 37°C. After 24 hours of clinorotation we measured adhesion molecules expression on the cell surface (ICAM-1, VCAM-1, PECAM-1, E-selectin, CD144, endoglin (CD105)) and cell viability using a flow cytometry. To evaluate the level of target gene expression was used the real time RT-PCR. IL-6 and IL-8 concentration was measured in the conditioned medium of HUVECs by using the ELISA test. We found that simulated microgravity within 24 hours caused a decrease of ICAM-1, CD144, and E-selectin expression, at the same time not affect the cell viability, endoglin and PECAM-1 expression on the surface HUVEC. Furthermore, there were no changes of the level of IL-6 and IL-8 gene expression and their products in the culture medium. TNF-activated HUVECs showed an increase in gene expression of interleukins and molecules involved in the adhesion process, which also was confirmed

  13. Role of Extracellular Matrix Renal Tubulo-interstitial Nephritis Antigen (TINag) in Cell Survival Utilizing Integrin αvβ3/Focal Adhesion Kinase (FAK)/Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B-Serine/Threonine Kinase (AKT) Signaling Pathway*

    PubMed Central

    Xie, Ping; Kondeti, Vinay K.; Lin, Sun; Haruna, Yoshisuke; Raparia, Kirtee; Kanwar, Yashpal S.

    2011-01-01

    Tubulo-interstitial nephritis antigen (TINag) is an extracellular matrix protein expressed in tubular basement membranes. Combined mutations in TINag and nephrocystin-1 genes lead to nephronophthisis with reduced cell survival. Because certain extracellular matrix proteins are known to modulate cell survival, studies were initiated in Lewis rats lacking TINag to assess if they are more susceptible to cisplatin-induced injury. Cisplatin induced a higher degree of tubular cell damage and apoptosis in regions where TINag is expressed in a parental Wistar strain. This was accompanied by an accentuated increase in serum creatinine and Kim-1 RNA and renal expression of Bax, p53, and its nuclear accumulation, mtDNA fragmentation, and a decrease of Bcl-2. Cisplatin induced fulminant apoptosis of HK-2 cells with increased caspase3/7 activity, mtDNA fragmentation, and a reduced cell survival. These effects were partially reversed in cells maintained on TINag substratum. Far Western/solid phase assays established TINag binding with integrin αvβ3 comparable with vitronectin. Transfection of cells with αv-siRNA accentuated cisplatin-induced apoptosis, aberrant translocation of cytochrome c and Bax, and reduced cell survival. The αv-siRNA decreased expression of integrin-recruited focal adhesion kinase (FAK) and p-FAK, while increasing the expression of p53 and p-p53. Similarly, p-AKT was reduced although ILK was unaffected. Inhibition of PI3K had similar adverse cellular effects. These effects were ameliorated in cells on TINag substratum. In vivo, a higher degree of decrease in the expression of p-FAK and pAKT was observed in Lewis rats following cisplatin treatment. These in vivo and in vitro studies demonstrate an essential role of TINag in cellular survival to maintain proper tubular homeostasis utilizing integrin αvβ3 and downstream effectors. PMID:21795690

  14. Integrin-Matrix Clusters Form Podosome-like Adhesions in the Absence of Traction Forces

    PubMed Central

    Yu, Cheng-han; Rafiq, Nisha Bte Mohd; Krishnasamy, Anitha; Hartman, Kevin L.; Jones, Gareth E.; Bershadsky, Alexander D.; Sheetz, Michael P.

    2013-01-01

    Summary Matrix-activated integrins can form different adhesion structures. We report that nontransformed fibroblasts develop podosome-like adhesions when spread on fluid Arg-Gly-Asp peptide (RGD)-lipid surfaces, whereas they habitually form focal adhesions on rigid RGD glass surfaces. Similar to classic macrophage podosomes, the podosome-like adhesions are protrusive and characterized by doughnut-shaped RGD rings that surround characteristic core components including F-actin, N-WASP, and Arp2/Arp3. Furthermore, there are 18 podosome markers in these adhesions, though they lack matrix metalloproteinases that characterize invadopodia and podosomes of Src-transformed cells. When nontransformed cells develop force on integrin-RGD clusters by pulling RGD lipids to prefabricated rigid barriers (metal lines spaced by 1–2 μm), these podosomes fail to form and instead form focal adhesions. The formation of podosomes on fluid surfaces is mediated by local activation of phosphoinositide 3-kinase (PI3K) and the production of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) in a FAK/PYK2-dependent manner. Enrichment of PIP3 precedes N-WASP activation and the recruitment of RhoA-GAP ARAP3. We propose that adhesion structures can be modulated by traction force development and that production of PIP3 stimulates podosome formation and subsequent RhoA downregulation in the absence of traction force. PMID:24290759

  15. A brain slice experimental model to study the generation and the propagation of focally-induced epileptiform activity

    PubMed Central

    Losi, Gabriele; Marcon, Iacopo; Mariotti, Letizia; Sessolo, Michele; Chiavegato, Angela; Carmignoto, Giorgio

    2016-01-01

    The early cellular events that in a brain network lead to seizure generation and govern seizure propagation are probably based on different cellular mechanisms. Experimental models in which these events can be separately studied would contribute to improve our understanding of epilepsy. We recently described an in vitro model in entorhinal cortex slices from young rats in which focal seizure-like discharges (SLDs) can be induced in spatially defined regions and at predictable times by local NMDA applications performed in the presence of 4-amimopyridine (4-AP) and low extracellular Mg2+. Through the use of single-dual cell patch-clamp and field potential recordings, and Ca2+ imaging from large ensembles of neurons, interneurons and astrocytes, we here extend this model to entorhinal and temporal cortex slices of rat and mouse brain, providing evidence that multiple SLDs exhibiting the typical tonic–clonic discharge pattern can be also evoked in these cortical regions by successive NMDA applications. Importantly, the temporal cortex is more accessible to viral vector injections than the entorhinal cortex: this makes it feasible in the former region the selective expression in inhibitory interneurons or principal neurons of genetically encoded Ca2+ indicators (GECI) or light-gated opsins. In this model, an optogenetic approach allows to activate specific neuronal types at spatially defined locations, i.e., the focus or the propagating region, and at precise time, i.e., before or during SLD. The NMDA/4-AP model can, therefore, represent a valuable tool to gain insights into the role of specific cell populations in seizure generation, propagation and cessation. PMID:25863141

  16. Ebselen attenuates oxidative DNA damage and enhances its repair activity in the thalamus after focal cortical infarction in hypertensive rats.

    PubMed

    He, Meixia; Xing, Shihui; Yang, Bo; Zhao, Liqun; Hua, Haiying; Liang, Zhijian; Zhou, Wenliang; Zeng, Jinsheng; Pei, Zhong

    2007-11-21

    Oxidative DNA damage has been proposed to be a major contributor to focal cerebral ischemic injury. However, little is known about the role of oxidative DNA damage in remote damage secondary to the primary infarction. In the present study, we investigated oxidative damage within the ventroposterior nucleus (VPN) after distal middle cerebral artery occlusion (MCAO) in hypertensive rats. We also examined the possible protective effect of ebselen, one glutathione peroxidase mimic, on delayed degeneration in the VPN after distal MCAO. Neuronal damage in the ipsilateral VPN was examined by Nissl staining. Oxidative DNA damage and base repair enzyme activity were assessed by analyzing immunoreactivity of 8-hydroxy-2'-deoxyguanosine (8-ohdG) and 8-oxoguanine DNA glycosylase (OGG1), respectively. The number of intact neurons in the ipsilateral VPN decreased by 52% compared to the contralateral side in ischemia group 2 weeks after distal cerebral cortical infarction. The immunoreactivity of 8-ohdG significantly increased while OGG1 immunoreactivity significantly decreased in the ipsilateral VPN 2 weeks after distal cortical infarction (all p<0.01). Compared with vehicle treatment, ebselen significantly attenuated the neuron loss, ameliorated ischemia-induced increase in 8-ohdG level as well as decrease in OGG1 level within the ipsilateral VPN (all p<0.01). OGG1 was further demonstrated to mainly express in neurons. These findings strongly suggest that oxidative DNA damage may be involved in the delayed neuronal death in the VPN region following distal MCAO. Furthermore, ebselen protects against the delayed damage in the VPN when given at 24 h following distal MCAO. PMID:17920569

  17. Ambroxol inhibits neutrophil respiratory burst activated by alpha chain integrin adhesion.

    PubMed

    Peroni, D G; Moser, S; Gallo, G; Pigozzi, R; Tenero, L; Zanoni, L; Boner, A L; Piacentini, G L

    2013-01-01

    The purpose of the present study was to investigate the possible anti-oxidant effect(s) of Ambroxol on neutrophils activated by ligand-binding of the drug with membrane-associated adhesion integrin CD11a and to estimate dose-response changes in oxygen free radical production. The amount of free radical production by anti-CD11a- and anti-CD4-coated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP) and challenged with increasing concentration of Ambroxol, was evaluated within a time frame of 90 minutes. A significant dose-dependent effect response of Ambroxol on O2‾ production by cells coated with anti-CD11a antibody was observed. This preliminary study opens a new perspective on the therapeutic role of Ambroxol as an antioxidant drug and for its potential use in controlling oxidative stress, particularly in leukocyte-dependent inflammation. PMID:24355223

  18. Active Metal Brazing and Adhesive Bonding of Titanium to C/C Composites for Heat Rejection System

    NASA Technical Reports Server (NTRS)

    Singh, M.; Shpargel, Tarah; Cerny, Jennifer

    2006-01-01

    Robust assembly and integration technologies are critically needed for the manufacturing of heat rejection system (HRS) components for current and future space exploration missions. Active metal brazing and adhesive bonding technologies are being assessed for the bonding of titanium to high conductivity Carbon-Carbon composite sub components in various shapes and sizes. Currently a number of different silver and copper based active metal brazes and adhesive compositions are being evaluated. The joint microstructures were examined using optical microscopy, and scanning electron microscopy (SEM) coupled with energy dispersive spectrometry (EDS). Several mechanical tests have been employed to ascertain the effectiveness of different brazing and adhesive approaches in tension and in shear that are both simple and representative of the actual system and relatively straightforward in analysis. The results of these mechanical tests along with the fractographic analysis will be discussed. In addition, advantages, technical issues and concerns in using different bonding approaches will also be presented.

  19. Osthole, a natural coumarin, improves neurobehavioral functions and reduces infarct volume and matrix metalloproteinase-9 activity after transient focal cerebral ischemia in rats.

    PubMed

    Mao, Xuexuan; Yin, Wei; Liu, Mengfei; Ye, Minzhong; Liu, Peiqing; Liu, Jianxin; Lian, Qishen; Xu, Suowen; Pi, Rongbiao

    2011-04-18

    Previously we demonstrated that Osthole, a natural coumarin, protects against focal cerebral ischemia/reperfusion-induced injury in rats. In the present study, the effects of Osthole on neurobehavioral functions, infarct volume and matrix metalloproteinase-9 (MMP-9) in a rat 2h focal cerebral ischemia model were investigated. Osthole (100mg/kg per dose) was administrated intraperitoneally 30min before ischemic insult and immediately after reperfusion. Osthole treatment significantly reduced neurological deficit score and infarct volume by 38.5% and 33.8%, respectively, as compared with the untreated animals. Osthole reversed ischemia-reperfusion-induced increase in MMP-9 protein level/activity as evidenced by Western blotting and gelatin zymography. Taken together, these results for the first time demonstrate that Osthole reduces infarct volume, restores neurobehavioral functions and downregulates MMP-9 protein level/activity in ischemia/reperfused brain. PMID:21316348

  20. Development of Buccal Adhesive Tablet with Prolonged Antifungal activity: Optimization and ex vivo Deposition Studies

    PubMed Central

    Madgulkar, A.; Kadam, S.; Pokharkar, V.

    2009-01-01

    The purpose of the present work was to prepare buccal adhesive tablets of miconazole nitrate. The simplex centroid experimental design was used to arrive at optimum ratio of carbopol 934P, hydroxypropylmethylcellulose K4M and polyvinylpyrollidone, which will provide desired drug release and mucoadhesion. Swelling index, mucoadhesive strength and in vitro drug release of the prepared tablet was determined. The drug release and bioadhesion was dependent on type and relative amounts of the polymers. The optimized combination was subjected to in vitro antifungal activity, transmucosal permeation, drug deposition in mucosa, residence time and bioadhesion studies. IR spectroscopy was used to investigate any interaction between drug and excipients. Dissolution of miconazole from tablets was sustained for 6 h. based on the results obtained, it can be concluded that the prepared slow release buccoadhesive tablets of miconazole would markedly prolong the duration of antifungal activity. Comparison of in vitro antifungal activity of tablet with marketed gel showed that drug concentrations above the minimum inhibitory concentration were achieved immediately from both formulations but release from tablet was sustained up to 6 h, while the gel showed initially fast drug release, which did not sustain later. Drug permeation across buccal mucosa was minimum from the tablet as well as marketed gel; the deposition of drug in mucosa was higher in case of tablet. In vitro residence time and bioadhesive strength of tablet was higher than gel. Thus the buccoadhesive tablet of miconazole nitrate may offer better control of antifungal activity as compared to the gel formulation. PMID:20490296

  1. Development of Buccal Adhesive Tablet with Prolonged Antifungal activity: Optimization and ex vivo Deposition Studies.

    PubMed

    Madgulkar, A; Kadam, S; Pokharkar, V

    2009-05-01

    The purpose of the present work was to prepare buccal adhesive tablets of miconazole nitrate. The simplex centroid experimental design was used to arrive at optimum ratio of carbopol 934P, hydroxypropylmethylcellulose K4M and polyvinylpyrollidone, which will provide desired drug release and mucoadhesion. Swelling index, mucoadhesive strength and in vitro drug release of the prepared tablet was determined. The drug release and bioadhesion was dependent on type and relative amounts of the polymers. The optimized combination was subjected to in vitro antifungal activity, transmucosal permeation, drug deposition in mucosa, residence time and bioadhesion studies. IR spectroscopy was used to investigate any interaction between drug and excipients. Dissolution of miconazole from tablets was sustained for 6 h. based on the results obtained, it can be concluded that the prepared slow release buccoadhesive tablets of miconazole would markedly prolong the duration of antifungal activity. Comparison of in vitro antifungal activity of tablet with marketed gel showed that drug concentrations above the minimum inhibitory concentration were achieved immediately from both formulations but release from tablet was sustained up to 6 h, while the gel showed initially fast drug release, which did not sustain later. Drug permeation across buccal mucosa was minimum from the tablet as well as marketed gel; the deposition of drug in mucosa was higher in case of tablet. In vitro residence time and bioadhesive strength of tablet was higher than gel. Thus the buccoadhesive tablet of miconazole nitrate may offer better control of antifungal activity as compared to the gel formulation. PMID:20490296

  2. Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

    SciTech Connect

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu . E-mail: dxliu001@yahoo.com

    2007-07-13

    Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.

  3. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

    PubMed Central

    Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R.

    2016-01-01

    Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages. PMID:26934296

  4. CD44 sensitivity of platelet activation, membrane scrambling and adhesion under high arterial shear rates.

    PubMed

    Liu, Guilai; Liu, Guoxing; Alzoubi, Kousi; Chatterjee, Madhumita; Walker, Britta; Münzer, Patrick; Luo, Dong; Umbach, Anja T; Elvira, Bernat; Chen, Hong; Voelkl, Jakob; Föller, Michael; Mak, Tak W; Borst, Oliver; Gawaz, Meinrad; Lang, Florian

    2016-01-01

    CD44 is required for signalling of macrophage migration inhibitory factor (MIF), an anti-apoptotic pro-inflammatory cytokine. MIF is expressed and released from blood platelets, key players in the orchestration of occlusive vascular disease. Nothing is known about a role of CD44 in the regulation of platelet function. The present study thus explored whether CD44 modifies degranulation (P-selectin exposure), integrin activation, caspase activity, phosphatidylserine exposure on the platelet surface, platelet volume, Orai1 protein abundance and cytosolic Ca(2+)-activity ([Ca2+]i). Platelets from mice lacking CD44 (cd44(-/-)) were compared to platelets from corresponding wild-type mice (cd44(+/+)). In resting platelets, P-selectin abundance, α(IIb)β3 integrin activation, caspase-3 activity and phosphatidylserine exposure were negligible in both genotypes and Orai1 protein abundance, [Ca2+]i, and volume were similar in cd44(-/-) and cd44(+/+) platelets. Platelet degranulation and α(IIb)β3 integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca(2+)-store depletion with thapsigargin (1 µM), effects more pronounced in cd44(-/-) than in cd44(+/+) platelets. Thrombin (0.02 U/ml) increased platelet [Ca2+]i, caspase-3 activity, phosphatidylserine exposure and Orai1 surface abundance, effects again significantly stronger in cd44(-/-) than in cd44(+/+) platelets. Thrombin further decreased forward scatter in cd44(-/-) and cd44(+/+) platelets, an effect which tended to be again more pronounced in cd44(-/-) than in cd44(+/+) platelets. Platelet adhesion and in vitro thrombus formation under high arterial shear rates (1,700 s(-1)) were significantly augmented in cd44(-/-) mice. In conclusion, genetic deficiency of CD44 augments activation, apoptosis and pro-thrombotic potential of platelets. PMID:26355696

  5. PYK2 is an adhesion kinase in macrophages, localized in podosomes and activated by beta(2)-integrin ligation.

    PubMed

    Duong, L T; Rodan, G A

    2000-11-01

    Pyk2 is a member of the focal adhesion kinase (FAK) family, highly expressed in the central nervous system and haemopoietic cells. Although Pyk2 is homologous to FAK, its role in signaling pathways was shown to be distinct from that of FAK. We show here that Pyk2 is highly expressed in peritoneal IC-21 macrophage and is tyrosine phosphorylated in response to cell attachment to fibronectin and fibrinogen. Upon IC-21 cell adhesion, Pyk2 tyrosine phosphorylation is inhibited by blocking antibodies to the integrin subunits alpha(M) and beta(2). Furthermore, Pyk2 is rapidly tyrosine phosphorylated in response to ligation of beta(2) integrins by antibodies. In migrating macrophages, Pyk2 localizes to perinuclear regions and to podosomes, where it is clustered with tyrosine phosphorylated proteins. Furthermore, in the podosomal ring structure, which surrounds the central actin core, Pyk2 co-localizes with vinculin, talin, and paxillin. In the podosomes, Pyk2 also co-localizes with the integrin alpha(M)beta(2). Lastly, reduction of Pyk2 expression in macrophages leads to inhibition of cell migration. We propose that Pyk2 is functionally linked to the formation of podosomes where it mediates the integrin-cytoskeleton interface and regulates cell spreading and migration. PMID:11056520

  6. Antimicrobial activity of a novel adhesive containing chlorhexidine gluconate (CHG) against the resident microflora in human volunteers

    PubMed Central

    Carty, Neal; Wibaux, Anne; Ward, Colleen; Paulson, Daryl S.; Johnson, Peter

    2014-01-01

    Objectives To evaluate the antimicrobial activity of a new, transparent composite film dressing, whose adhesive contains chlorhexidine gluconate (CHG), against the native microflora present on human skin. Methods CHG-containing adhesive film dressings and non-antimicrobial control film dressings were applied to the skin on the backs of healthy human volunteers without antiseptic preparation. Dressings were removed 1, 4 or 7 days after application. The bacterial populations underneath were measured by quantitative cultures (cylinder-scrub technique) and compared with one another as a function of time. Results The mean baseline microflora recovery was 3.24 log10 cfu/cm2. The mean log reductions from baseline measured from underneath the CHG-containing dressings were 0.87, 0.78 and 1.30 log10 cfu/cm2 on days 1, 4 and 7, respectively, compared with log reductions of 0.67, −0.87 and −1.29 log10 cfu/cm2 from underneath the control film dressings. There was no significant difference between the log reductions of the two treatments on day 1, but on days 4 and 7 the log reduction associated with the CHG adhesive was significantly higher than that associated with the control adhesive. Conclusions The adhesive containing CHG was associated with a sustained antimicrobial effect that was not present in the control. Incorporating the antimicrobial into the adhesive layer confers upon it bactericidal properties in marked contrast to the non-antimicrobial adhesive, which contributed to bacterial proliferation when the wear time was ≥4 days. PMID:24722839

  7. Inhibitory activity of cranberry extract on the bacterial adhesiveness in the urine of women: an ex-vivo study.

    PubMed

    Tempera, G; Corsello, S; Genovese, C; Caruso, F E; Nicolosi, D

    2010-01-01

    Strains of uropathogenic E. coli are responsible for approximately 90% of community-acquired, uncomplicated cystitis, and fimbriae represent the adhesive factors enabling E. coli to be anchored to uroepithelial cells in the first step of the infectious process. Recently, a few studies have shown that a correlation between the consumption of cranberry (Vaccinium macrocarpon) and prevention of UTI is related to the ability of proanthocyanidins to reduce the bacterial adhesion to uroepithelial cells. In this study we evaluate the inhibitory activity of urine of healthy women treated with tablets containing cranberry extract on the adhesiveness of E. coli to uroepithelial human cells. Two groups of 12 female volunteers each, aged between 18 and 65 years, were enrolled, one group with negative history and one group with positive history of recurrent cystitis. Subjects were treated with the active product or placebo in a random, cross-over, double-blinded sequence for one week in each of the two treatment sequences. Urine samples were collected at the beginning and the end of each study period. Tests of bacterial adhesiveness were performed with two strains of E. coli (ATCC 25922 and ATCC 35218) on HT1376 human bladder carcinoma cells. Significant reductions of bacterial adhesiveness were observed in women who received cranberry extract (-50.9%; p less than 0.0001), regardless of their medical history and the treatment period in the cross-over sequence. No changes were observed with placebo (-0.29%; n.s.). This ex-vivo study showed that the assumption of cranberry extract in suitable amounts can have an anti-adhesive activity on uropathogenic E. coli. PMID:20646356

  8. A comparative phenotypical analysis of rheumatoid nodules and rheumatoid synovium with special reference to adhesion molecules and activation markers

    PubMed Central

    Elewaut, D.; De Keyser, F.; De Wever, N.; Baeten, D.; Van Damme, N.; Verbruggen, G.; Cuvelier, C.; Veys, E.

    1998-01-01

    OBJECTIVES—(1)To analyse the in situ expression of adhesion molecules in rheumatoid nodules. (2) To compare the endothelial expression of adhesion molecules in synovial tissue and subcutaneous nodules obtained from the same patients. (3) To compare the expression of adhesion molecules and activation markers on T cell lines from nodules and synovium.
METHODS—(1) Immunohistochemical analysis by APAAP technique of E selectin, CD44, ICAM-1, PECAM-1, and VCAM-1 was performed on 10 rheumatoid nodules from seven patients with rheumatoid arthritis (RA); nodules and synovium were simultaneously analysed from three patients. (2) T cell lines were generated from RA nodules (n=7) and synovium (n=7) by interleukin 2 expansion, and subsequently characterised by flow cytometry for surface expression of αEβ7, α4β7, CD44, L selectin, LFA-1a, PECAM-1, and CD30.
RESULTS—(1) In rheumatoid nodules, the palisading layer strongly stains for ICAM-1 and PECAM-1, but less pronounced for CD44. VCAM-1 staining was usually negative. ICAM-1 is upregulated in the vessels surrounding the central zone of fibrinoid necrosis. The immunohistological picture in different nodules derived from the same patient was similar. (2) The endothelial expression of adhesion molecules is comparable in RA nodules and synovium on an individual level, except for E selectin, which is overexpressed in nodule endothelium. (3) T cell lines from nodules and synovium display similar adhesion molecule profiles. However, the expression of CD30, a T cell activation marker linked with Th2 subsets, is higher in nodules compared with synovium.
CONCLUSION—These data support a recirculation hypothesis of T cells between articular and extra-articular manifestations in RA, although the activation state of the T cells in each of these localisations may differ.

 Keywords: T cells; adhesion molecules; rheumatoid nodules; rheumatoid synovium PMID:9797554

  9. Modulation of Cell-adhesive Activity of Fibronectin by the Alternatively Spliced EDA Segment

    PubMed Central

    Manabe, Ri-ichiroh; Oh-e, Naoko; Maeda, Toshinaga; Fukuda, Tomohiko; Sekiguchi, Kiyotoshi

    1997-01-01

    Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA− FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin α5 and β1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin α5β1. In support of this conclusion, purified integrin α5β1 bound more avidly to EDA+ FN than to EDA− FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA− FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin α5β1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180° at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local

  10. Activated Leukocyte Cell Adhesion Molecule (ALCAM or CD166) Modulates Bone Phenotype and Hematopoiesis

    PubMed Central

    Hooker, R. Adam; Chitteti, Brahmananda R.; Egan, Patrick H.; Cheng, Ying-Hua; Himes, Evan R.; Meijome, Tomas; Srour, Edward F.; Fuchs, Robyn K.; Kacena, Melissa A.

    2015-01-01

    Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166), is expressed on osteoblasts (OB) and hematopoietic stem cells (HSC) residing in the hematopoietic niche, and may have important regulatory roles in bone formation. Because HSC numbers are reduced 77% in CD166−/− mice, we hypothesized that changes in bone phenotype and consequently the endosteal niche may partially be responsible for this alteration. Therefore, we investigated bone phenotype and OB function in CD166−/− mice. Although osteoclastic measures were not affected by loss of CD166, CD166−/− mice exhibited a modest increase in trabecular bone fraction (42%), and increases in osteoid deposition (72%), OB number (60%), and bone formation rate (152%). Cortical bone geometry was altered in CD166−/− mice resulting in up to 81% and 49% increases in stiffness and ultimate force, respectively. CD166−/− OB displayed elevated alkaline phosphatase (ALP) activity and mineralization, and increased mRNA expression of Fra 1, ALP, and osteocalcin. Overall, CD166−/− mice displayed modestly elevated trabecular bone volume fraction with increased OB numbers and deposition of osteoid, and increased OB differentiation in vitro, possibly suggesting more mature OB are secreting more osteoid. This may explain the decline in HSC number in vivo because immature OB are mainly responsible for hematopoiesis enhancing activity. PMID:25730656

  11. Micro-structuring of polycarbonate-urethane surfaces in order to reduce platelet activation and adhesion.

    PubMed

    Clauser, Johanna; Gester, Kathrin; Roggenkamp, Jan; Mager, Ilona; Maas, Judith; Jansen, Sebastian V; Steinseifer, Ulrich

    2014-01-01

    In the development of new hemocompatible biomaterials, surface modification appears to be a suitable method in order to reduce the thrombogenetic potential of such materials. In this study, polycarbonate-urethane (PCU) tubes with different surface microstructures to be used for aortic heart valve models were investigated with regard to the thrombogenicity. The surface structures were produced by using a centrifugal casting process for manufacturing PCU tubes with defined casting mold surfaces which are conferred to the PCU surface during the process. Tubes with different structures defined by altering groove widths were cut into films and investigated under dynamic flow conditions in contact with porcine blood. The analysis was carried out by laser scanning microscopy which allowed for counting various morphological types of platelets with regard to the grade of activation. The comparison between plain and shaped PCU samples showed that the surface topography led to a decline of the activation of the coagulation cascade and thus to the reduction of the fibrin synthesis. Comparing different types of structures revealed that smooth structures with a small groove width (d ~ 3 μm) showed less platelet activation as well as less adhesion in contrast to a distinct wave structure (d ~ 90 μm). These results prove surface modification of polymer biomaterials to be a suitable method for reducing thrombogenicity and hence give reason for further alterations and improvements. PMID:24484511

  12. Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces.

    PubMed

    Finley, Matthew J; Rauova, Lubica; Alferiev, Ivan S; Weisel, John W; Levy, Robert J; Stachelek, Stanley J

    2012-08-01

    CD47 is a ubiquitously expressed transmembrane protein that, through signaling mechanisms mediated by signal regulatory protein alpha (SIRPα1), functions as a biological marker of 'self-recognition'. We showed previously that inflammatory cell attachment to polymeric surfaces is inhibited by the attachment of biotinylated recombinant CD47 (CD47B). We test herein the hypothesis that CD47 modified blood conduits can reduce platelet and neutrophil activation under clinically relevant conditions. We appended a poly-lysine tag to the C-terminus of recombinant CD47 (CD47L) allowing for covalent linkage to the polymer. SIRPα1 expression was confirmed in isolated platelets. We then compared biocompatibility between CD47B and CD47L functionalized polyvinyl chloride (PVC) surfaces and unmodified control PVC surfaces. Quantitative and Qualitative analysis of blood cell attachment to CD47B and CD47L surfaces, via scanning electron microscopy, showed strikingly fewer platelets attached to CD47 modified surfaces compared to control. Flow cytometry analysis showed that activation markers for neutrophils (CD62L) and platelets (CD62P) exposed to CD47 modified PVC were equivalent to freshly acquired control blood, while significantly elevated in the unmodified PVC tubing. In addition, ethylene oxide gas sterilization did not inhibit the efficacy of the CD47 modification. In conclusion, CD47 modified PVC inhibits both the adhesion and activation of platelets and neutrophils. PMID:22613135

  13. Anthocyanins and their gut metabolites reduce the adhesion of monocyte to TNFα-activated endothelial cells at physiologically relevant concentrations.

    PubMed

    Krga, Irena; Monfoulet, Laurent-Emmanuel; Konic-Ristic, Aleksandra; Mercier, Sylvie; Glibetic, Maria; Morand, Christine; Milenkovic, Dragan

    2016-06-01

    An increasing number of evidence suggests a protective role of dietary anthocyanins against cardiovascular diseases. Anthocyanins' extensive metabolism indicates that their metabolites could be responsible for the protective effects associated with consumption of anthocyanin-rich foods. The aim of this work was to investigate the effect of plasma anthocyanins and their metabolites on the adhesion of monocytes to TNFα-activated endothelial cells and on the expression of genes encoding cell adhesion molecules. Human umbilical vein endothelial cells (HUVECs) were exposed to circulating anthocyanins: cyanidin-3-arabinoside, cyanidin-3-galactoside, cyanidin-3-glucoside, delphinidin-3-glucoside, peonidin-3-glucoside, anthocyanin degradation product: 4-hydroxybenzaldehyde, or to their gut metabolites: protocatechuic, vanillic, ferulic and hippuric acid, at physiologically-relevant concentrations (0.1-2 μM) and time of exposure. Both anthocyanins and gut metabolites decreased the adhesion of monocytes to HUVECs, with a magnitude ranging from 18.1% to 47%. The mixture of anthocyanins and that of gut metabolites also reduced monocyte adhesion. However, no significant effect on the expression of genes encoding E-selectin, ICAM1 and VCAM1 was observed, suggesting that other molecular targets are involved in the observed effect. In conclusion, this study showed the potency of anthocyanins and their gut metabolites to modulate the adhesion of monocytes to endothelial cells, the initial step in atherosclerosis development, under physiologically-relevant conditions. PMID:26873533

  14. Decreased cell adhesion promotes angiogenesis in a Pyk2-dependent manner

    SciTech Connect

    Shen, Colette J.; Raghavan, Srivatsan; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205 ; Xu, Zhe; Baranski, Jan D.; Yu, Xiang; Wozniak, Michele A.; Miller, Jordan S.; Gupta, Mudit; Buckbinder, Leonard; Chen, Christopher S.

    2011-08-01

    Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.

  15. Degree of conversion of simplified contemporary adhesive systems as influenced by extended air-activated or passive solvent volatilization modes.

    PubMed

    Borges, Boniek C D; Souza-Junior, Eduardo Jose; Brandt, William C; Loguercio, Alessandro D; Montes, Marcos A J R; Puppin-Rontani, Regina M; Sinhoreti, Mario Alexandre Coelho

    2012-01-01

    This study evaluated the effect of five methods of solvent volatilization on the degree of conversion (DC) of nine one-bottle adhesive systems using Fourier transform infrared/attenuated total reflectance (FTIR/ATR) analysis. Nine adhesives were tested: Adper Single Bond 2 (SB), Adper Easy One (EO), One Up Bond F Plus (OUP), One Coat Bond SL (OC), XP Bond (XP), Ambar (AM), Natural Bond (NB), GO, and Stae. The adhesive systems were applied to a zinc-selenide pellet and 1) cured without solvent volatilization, 2) left undisturbed for 10 seconds before curing, 3) left undisturbed for 60 seconds before curing, 4) air-dried with an air stream for 10 seconds before curing, and 5) air-dried with an air stream for 60 seconds before curing. FTIR/ATR spectra were obtained, and the DC was calculated by comparing the aliphatic bonds/reference peaks before and after light activation for 10 seconds (FlashLite 1401). The DC means of each material were analyzed by one-way analysis of variance and post hoc Tukey test (p<0.05). The DC of GO and Stae adhesive systems was not affected by the five evaporation conditions. Air-drying for 60 seconds before curing yielded the highest DC for SB, EO, and OC. Extended solvent volatilization time (60 seconds) either with or without air-drying before curing provided the highest DC for AM, NB, XP, and OUP. Thus, the monomer conversion of adhesive systems was material dependent. In general, the 60-second passive or active air-drying modes to volatilize solvents before curing enhanced the degree of conversion for the one-bottle simplified adhesive systems. PMID:22313268

  16. Adhesive Properties and Acid-Forming Activity of Lactobacilli and Streptococci Under Inhibitory Substances, Such as Nitrates.

    PubMed

    Hakobyan, L; Harutyunyan, K; Harutyunyan, N; Melik-Andreasyan, G; Trchounian, A

    2016-06-01

    One of the main requirements for probiotics is their ability to survive during passage through gastrointestinal tract and to maintain their activity at different adverse conditions. The aim of the study was to look for the strains of lactobacilli and streptococci with high adhesive properties even affected by inhibitory substances, such as nitrates (NO3 (-)). To study the adhesion properties hemagglutination reaction of bacterial cells with red blood cells of different animals and humans was used. The acid formation ability of bacteria was determined by the method of titration after 7 days of incubation in the sterile milk. These properties were investigated at different concentrations of NO3 (-). The high concentration (mostly ≥2.0 %) NO3 (-) inhibited the growth of both lactobacilli and streptococci, but compared with streptococcal cultures lactobacilli, especially Lactobacillus acidophilus Ep 317/402, have shown more stability and higher adhesive properties. In addition, the concentrations of NO3 (-) of 0.5-2.0 % decreased the acid-forming activity of the strains, but even under these conditions they coagulated milk and, in comparison to control, formed low acidity in milk. Thus, the L. acidophilus Ep 317/402 with high adhesive properties has demonstrated a higher activity of NO3 (-) transformation. PMID:26942420

  17. Soluble adhesion molecules correlate with surface expression in an in vitro model of endothelial activation.

    PubMed

    Kjaergaard, Anders G; Dige, Anders; Krog, Jan; Tønnesen, Else; Wogensen, Lise

    2013-10-01

    Endothelial activation is a pivotal event in the development and progression of inflammation. Central to endothelial activation is the up-regulation of cellular adhesion molecules (CAMs) including E-selectin (CD62E), ICAM-1 (CD54), VCAM-1 (CD106) and PECAM-1 (CD31). These CAMs are also found in soluble forms (sCAMs). In this in vitro study of endothelial activation, we examined whether the levels of sCAMs correlate with the endothelial surface expression of CAMs in a dose-dependent and time-dependent manner. Such a correlation would support the use of sCAMs as surrogate markers for endothelial activation in inflammatory conditions. Human umbilical vein endothelial cells (HUVEC) were cultured with various concentrations of TNF-α for 8 hr and at a fixed concentration of TNF-α for various durations. The levels of soluble and surface-bound E-selectin, ICAM-1, VCAM-1 and PECAM-1 were quantified by flow cytometry. TNF-α stimulation increased CAM and sCAM expression in a dose-dependent and time-dependent manner. There was a significant positive correlation between the levels of ICAM-1 and sICAM-1 and between the levels of VCAM and sVCAM-1 in both the dose-response and time-response experiments. A positive correlation between the levels of E-selectin and sE-selectin was observed in the time-response experiment. This study supports the use of sCAMs as potential biomarkers of endothelial activation. In particular, the use of sICAM-1, sVCAM-1 and sE-selectin seems promising. PMID:23724832

  18. α-Tocopheryl Succinate as a Scaffold to Develop Potent Inhibitors of Breast Cancer Cell Adhesion

    PubMed Central

    Wang, Dasheng; Chuang, Hsiao-Ching; Weng, Shu-Chuan; Huang, Po-Hsien; Hsieh, Hao-Yu; Kulp, Samuel K.; Chen, Ching-Shih

    2009-01-01

    This study is aimed at the pharmacological exploitation of α-tocopheryl succinate (1) to develop potent anti-adhesion agents. Considering the structural cooperativity between the phytyl chain and the carboxylic terminus in determining the anti-adhesion activity, our structural optimization led to compound 5 ([2-(4,8-dimethyl-non-1-enyl)-2,5,7,8-tetramethyl-chroman-6-yloxy]-acetic acid), which exhibited an-order-of-magnitude higher potency than 1 in blocking the adhesion of 4T1 metastatic breast cancer cells to extracellular matrix proteins (IC50, 0.6 μM versus 10 μM). Evidence indicates that the ability of compound 5 to block cell adhesion and migration was attributable to its effect on disrupting focal adhesion and actin cytoskeletal integrity by facilitating the degradation of focal adhesion kinase. Interactions between tumor cells and the ECM in the tumor microenvironment have been increasingly recognized as critical modulators of the metastatic potential of tumor cells. Consequently, the ability of compound 5 to block such interactions provides a unique pharmacological tool to shed light onto mechanisms that govern cell adhesion and tumor metastasis. PMID:19708661

  19. TRPM7 Regulates Cell Adhesion by Controlling the Calcium-dependent Protease Calpain*S

    PubMed Central

    Su, Li-Ting; Agapito, Maria A.; Li, Mingjiang; Simonson, William T. N.; Huttenlocher, Anna; Habas, Raymond; Yue, Lixia; Runnels, Loren W.

    2011-01-01

    m-Calpain is a protease implicated in the control of cell adhesion through focal adhesion disassembly. The mechanism by which the enzyme is spatially and temporally controlled is not well understood, particularly because the dependence of calpain on calcium exceeds the submicromolar concentrations normally observed in cells. Here we show that the channel kinase TRPM7 localizes to peripheral adhesion complexes with m-calpain, where it regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the channel of the protein but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells. Together, our results suggest that the ion channel TRPM7 regulates cell adhesion through m-calpain by mediating the local influx of calcium into peripheral adhesion complexes. PMID:16436382

  20. Activation of matrix metalloproteinase-26 by HOXA10 promotes embryo adhesion in vitro.

    PubMed

    Jiang, Yue; Yan, Guijun; Zhang, Hui; Shan, Huizhi; Kong, Chengcai; Yan, Qiang; Xue, Bai; Diao, Zhenyu; Hu, Yali; Sun, Haixiang

    2014-03-14

    Successful embryonic implantation requires an effective maternal-embryonic molecular dialogue. However, the detailed mechanisms of epithelial-embryo adhesion remain poorly understood. Here, we report that matrix metalloproteinase-26 (MMP-26) is a novel downstream target gene of homeobox a 10 (HOXA10) in human endometrial cells. HOXA10 binds directly to a conserved TTAT unit (-442 to -439) located within the 5' regulatory region of the MMP-26 gene and regulates the expression and secretion of MMP-26 in a concentration-dependent manner. Moreover, the adenovirus-mediated overexpression of MMP-26 in Ishikawa cells markedly increased BeWo spheroid adhesion. An antibody blocking assay further demonstrated that the promotion of BeWo spheroid adhesion by HOXA10 and MMP-26 was significantly inhibited by pre-treatment with a specific antibody against MMP-26. These results demonstrate that the HOXA10-mediated expression of MMP-26 promotes embryo adhesion during the process of embryonic implantation. PMID:24565841

  1. Lamellipodial actin mechanically links myosin activity with adhesion-site formation.

    PubMed

    Giannone, Grégory; Dubin-Thaler, Benjamin J; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P

    2007-02-01

    Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  2. Breast cancer metastasis suppressor 1 (BRMS1) suppresses attachment and spreading of breast cancer cells on 2D and 3D extracellular matrix components by altering focal adhesion-associated signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Metastatic dissemination of cancer cells from primary tumor to secondary sites is a multi-step process that depends heavily on the ability of cancer cells to respond to the microenvironmental cues, such as changes in composition of surrounding extracellular matrix (ECM), by adapting their adhesion a...

  3. Focal epileptiform activity in the prefrontal cortex is associated with long-term attention and sociability deficits

    PubMed Central

    Hernan, Amanda E.; Alexander, Abigail; Jenks, Kyle R.; Barry, Jeremy; Lenck-Santini, Pierre-Pascal; Isaeva, Elena; Holmes, Gregory L.; Scott, Rod C.

    2015-01-01

    There is a well-described association between childhood epilepsy and pervasive cognitive and behavioral deficits. Often these children not only have ictal EEG events, but also frequent interictal abnormalities. The precise role of these interictal discharges in cognition remains unclear. In order to understand the relationship between frequent epileptiform discharges during neurodevelopment and cognition later in life, we developed a model of frequent focal interictal spikes (IIS). Postnatal day (p) 21 rats received injections of bicuculline methiodine into the prefrontal cortex (PFC). Injections were repeated in order to achieve 5 consecutive days of transient inhibitory/excitatory imbalance resulting in IIS. Short-term plasticity (STP) and behavioral outcomes were studied in adulthood. IIS is associated with a significant increase in STP bilaterally in the PFC. IIS rats did not show working memory deficits, but rather showed marked inattentiveness without significant alterations in motivation, anxiety or hyperactivity. Rats also demonstrated significant deficits in social behavior. We conclude that GABAergic blockade during early-life and resultant focal IIS in the PFC disrupt neural networks and are associated with long-term consequences for behavior at a time when IIS are no longer present, and thus may have important implications for ADHD and autism spectrum disorder associated with childhood epilepsy. PMID:24269731

  4. Interface Immobilization Chemistry of cRGD-based Peptides Regulates Integrin Mediated Cell Adhesion

    PubMed Central

    Pallarola, Diego; Bochen, Alexander; Boehm, Heike; Rechenmacher, Florian; Sobahi, Tariq R; Spatz, Joachim P; Kessler, Horst

    2014-01-01

    The interaction of specific surface receptors of the integrin family with different extracellular matrix-based ligands is of utmost importance for the cellular adhesion process. A ligand consists of an integrin-binding group, here cyclic RGDfX, a spacer molecule that lifts the integrin-binding group from the surface and a surface anchoring group. c(-RGDfX-) peptides are bound to gold nanoparticle structured surfaces via polyproline, polyethylene glycol or aminohexanoic acid containing spacers of different lengths. Although keeping the integrin-binding c(-RGDfX-) peptides constant for all compounds, changes of the ligand's spacer chemistry and length reveal significant differences in cell adhesion activation and focal adhesion formation. Polyproline-based peptides demonstrate improved cell adhesion kinetics and focal adhesion formation compared with common aminohexanoic acid or polyethylene glycol spacers. Binding activity can additionally be improved by applying ligands with two head groups, inducing a multimeric effect. This study gives insights into spacer-based differences in integrin-driven cell adhesion processes and remarkably highlights the polyproline-based spacers as suitable ligand-presenting templates for surface functionalization. PMID:25810710

  5. Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

    SciTech Connect

    Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier; Noppe, Gauthier; Horman, Sandrine; Morel, Nicole

    2013-11-22

    Highlights: •The three ERM proteins are expressed in vascular smooth muscle cell. •ERM depletion inhibited PDGF-evoked migration redundantly. •ERM depletion increased cell adhesion redundantly. •ERM depletion did not affect PDGF-evoked Ca signal, Rac1 activation, proliferation. •ERM proteins control PDGF-induced migration by regulating adhesion. -- Abstract: Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca{sup 2+} signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

  6. Thyroid hormone regulates adhesion, migration and matrix metalloproteinase 9 activity via αvβ3 integrin in myeloma cells.

    PubMed

    Cohen, Keren; Flint, Nir; Shalev, Shachar; Erez, Daniel; Baharal, Tal; Davis, Paul J; Hercbergs, Aleck; Ellis, Martin; Ashur-Fabian, Osnat

    2014-08-15

    Thyroid hormone (3,5,3'-triiodothyronine, T3; L-thyroxine, T4) enhances cancer cell proliferation, invasion and angiogenesis via a discrete receptor located near the RGD recognition site on αvβ3 integrin. Tetraiodothyroacetic acid (tetrac) and its nanoparticulate formulation interfere with binding of T3/T4 to the integrin. This integrin is overexpressed in multiple myeloma (MM) and other cancers. MM cells interact with αvβ3 integrin to support growth and invasion. Matrix metalloproteinases (MMPs) are a family of enzymes active in tissue remodeling and cancer. The association between integrins and MMPs secretion and action is well established. In the current study, we examined the effects of thyroid hormone on myeloma cell adhesion, migration and MMP activity. We show that T3 and T4 increased myeloma adhesion to fibronectin and induced αvβ3 clustering. In addition, the hormones induced MMP-9 expression and activation via αvβ3 and MAPK induction. Bortezomib, a standard myeloma treatment, caused a decrease in activity/quantity of MMPs and thyroid hormone opposed this effect. RGD peptide and tetrac impaired the production of MMP-9 in cell lines and in primary BM cells from myeloma patients. In conclusion, thyroid hormone-dependent regulation via αvβ3 of myeloma cell adhesion and MMP-9 production may play a role in myeloma migration and progression. PMID:25071016

  7. Thyroid hormone regulates adhesion, migration and matrix metalloproteinase 9 activity via αvβ3 integrin in myeloma cells

    PubMed Central

    Cohen, Keren; Flint, Nir; Shalev, Shachar; Erez, Daniel; Baharal, Tal; Davis, Paul J.; Hercbergs, Aleck; Ellis, Martin; Ashur-Fabian, Osnat

    2014-01-01

    Thyroid hormone (3,5,3′-triiodothyronine, T3; L-thyroxine, T4) enhances cancer cell proliferation, invasion and angiogenesis via a discrete receptor located near the RGD recognition site on αvβ3 integrin. Tetraiodothyroacetic acid (tetrac) and its nanoparticulate formulation interfere with binding of T3/T4 to the integrin. This integrin is overexpressed in multiple myeloma (MM) and other cancers. MM cells interact with αvβ3 integrin to support growth and invasion. Matrix metalloproteinases (MMPs) are a family of enzymes active in tissue remodeling and cancer. The association between integrins and MMPs secretion and action is well established. In the current study, we examined the effects of thyroid hormone on myeloma cell adhesion, migration and MMP activity. We show that T3 and T4 increased myeloma adhesion to fibronectin and induced αvβ3 clustering. In addition, the hormones induced MMP-9 expression and activation via αvβ3 and MAPK induction. Bortezomib, a standard myeloma treatment, caused a decrease in activity/quantity of MMPs and thyroid hormone opposed this effect. RGD peptide and tetrac impaired the production of MMP-9 in cell lines and in primary BM cells from myeloma patients. In conclusion, thyroid hormone-dependent regulation via αvβ3 of myeloma cell adhesion and MMP-9 production may play a role in myeloma migration and progression. PMID:25071016

  8. The role of adhesion strength in human mesenchymal stem cell osteoblastic differentiation on biodegradable polymers

    NASA Astrophysics Data System (ADS)

    Krizan, Sylva Jana

    Human mesenchymal stem cells (hMSC) are promising candidates for promoting bone growth on biodegradable polymer scaffolds however little is known about early hMSC-polymer interactions. Adhesion is highly dynamic and during adhesive reinforcement, numerous proteins form adhesion plaques linking the cell's cytoskeleton with the extracellular environment. These proteins are known to affect cellular function but their role in hMSC differentiation is less clear. Adhesion plaques are associated with adhesive force, still a detachment force of hMSC on polycaprolactone (PCL), poly-lactide-co-glycolide (PLGA) or alginate has never been described or shown to affect downstream function. We demonstrate that hMSC attached to PCL, PLGA and alginate exhibit different adhesion strengths (tau50) as determined by both fluid shear and spinning disk systems, with PLGA demonstrating the greatest tau 50. Elastic modulus and hydrophobicity were characterized for these surfaces and correlated positively with tau50 to an optimum. Attachment studies of hMSC showed that adhesion plateau timespans were independent of cell line and surface but both morphology and focal adhesion expression varied by polymer type. Differentiation studies of hMSC on PLGA and PCL showed a strong association between markers of differentiation (alkaline phosphatase activity and mineral content) and tau50 within polymer groups, but a poor relationship was found between tau50 and differentiation across polymer groups, suggesting that other polymer properties may be important for differentiation. Subsequently, we examined the role of focal adhesion kinase (FAK) and Rho-GTPase (RhoA) on hMSC adhesion and differentiation when plated onto PLGA. hMSC were retrovirally transduced with mutant constructs of FAK and RhoA cDNA. Alternatively, hMSC were treated with Rho-kinase inhibitor, Y27632. Both cells transduced with mutant RhoA or FAK constructs, or those treated with Y27632 displayed aberrant cell morphology and changes

  9. GABAergic networks jump-start focal seizures.

    PubMed

    de Curtis, Marco; Avoli, Massimo

    2016-05-01

    Abnormally enhanced glutamatergic excitation is commonly believed to mark the onset of a focal seizure. This notion, however, is not supported by firm evidence, and it will be challenged here. A general reduction of unit firing has been indeed observed in association with low-voltage fast activity at the onset of seizures recorded during presurgical intracranial monitoring in patients with focal, drug-resistant epilepsies. Moreover, focal seizures in animal models start with increased γ-aminobutyric acid (GABA)ergic interneuronal activity that silences principal cells. In vitro studies have shown that synchronous activation of GABAA receptors occurs at seizure onset and causes sizeable elevations in extracellular potassium, thus facilitating neuronal recruitment and seizure progression. A paradoxical involvement of GABAergic networks is required for the initiation of focal seizures characterized by low-voltage fast activity, which represents the most common seizure-onset pattern in focal epilepsies. PMID:27061793

  10. Cytokines induce urokinase-dependent adhesion of human myeloid cells. A regulatory role for plasminogen activator inhibitors.

    PubMed Central

    Waltz, D A; Sailor, L Z; Chapman, H A

    1993-01-01

    Differentiation of monocytic precursors often results in adhesive properties thought to be important in migration. In this study, the influence of cytokines, known to induce macrophage differentiation, on the adhesiveness of the monocytic cell line U937 was examined in vitro. Despite development of a macrophage morphology, < 5% of cytokine-stimulated U937 cells were adherent at 24 h. Addition of 1-10 nM urokinase-type plasminogen activator (uPA) induced adherence in the presence of transforming growth factor type beta-1, 1,25-(OH)2 vitamin D3, granulocyte macrophage colony-stimulating factor, or tumor necrosis factor alpha. uPA-dependent adhesiveness was reversible after 24 h of stimulation with cytokines and uPA as adherence was prevented by the subsequent addition of anti-uPA antibodies. Adherence induced by diisopropylfluorophosphate-inactivated uPA was severalfold greater than that seen with active uPA. This difference was largely due to cell-surface turnover of active uPA complexed with plasminogen activator inhibitor (PAI). These data indicate that cytokines prime monocyte progenitors for uPA receptor-mediated signals leading to adherence, continued uPA receptor occupancy is required for adherence, and PAI decreases adherence by promoting clearance of uPA/PAI complexes. Thus the interaction of uPA and PAI at the cell surface, known to affect extracellular matrix proteolysis and hence myeloid cell migration, also regulates adhesion. The coordinated regulation of these two uPA functions by PAI may enhance the migratory potential of monocytic cells. Images PMID:8386190

  11. Peel resistance characterization of localized polymer film bonding via thin film adhesive thermally activated by scanned CO2 laser

    NASA Astrophysics Data System (ADS)

    Dowding, Colin; Dowding, Robert; Griffiths, Jonathan; Lawrence, Jonathan

    2013-06-01

    Thermal laser polymer bonding is a non-contact process for the joining of polymer laminates using thermally activated adhesives. Conventional, contact based bonding techniques suffer from mechanical wear, geometric inflexibility and poor energy efficiency. The application of lasers offers the potential for highly localized delivery of energy and increased process flexibility whilst achieving controlled and repeatable bonding of polymer laminates in a contact free process. Unlike previously reported techniques, here it is reported that laser based non-contact bonding is both viable and highly desirable due to the increased levels of control it affords the user. In this work, laser polymer bonding of 75 μm thick linear low density polyethylene (LLDPE) film backed with a thermally activated adhesive to a 640 μm thick polypropylene (PP) substrate was conducted using continuous wave 10.6 μm laser radiation and scanning galvanometric optics. The effect of laser power and scanning traverse speed on the peel resistance properties of the bonded polymer laminates is presented, with a threshold specific energy density for successful adhesive activation determined.

  12. Cardiomyocytes In Vitro Adhesion Is Actively Influenced by Biomimetic Synthetic Peptides for Cardiac Tissue Engineering

    PubMed Central

    Huerta-Cantillo, Rocio; Comisso, Marina; Danesin, Roberta; Ghezzo, Francesca; Naso, Filippo; Gastaldello, Alessandra; Schittullo, Eleonora; Buratto, Edward; Spina, Michele; Gerosa, Gino; Dettin, Monica

    2012-01-01

    Scaffolds for tissue engineering must be designed to direct desired events such as cell attachment, growth, and differentiation. The incorporation of extracellular matrix-derived peptides into biomaterials has been proposed to mimic biochemical signals. In this study, three synthetic fragments of fibronectin, vitronectin, and stromal-derived factor-1 were investigated for the first time as potential adhesive sequences for cardiomyocytes (CMs) compared to smooth muscle cells. CMs are responsive to all peptides to differing degrees, demonstrating the existence of diverse adhesion mechanisms. The pretreatment of nontissue culture well surfaces with the (Arginine-Glycine-Aspartic Acid) RGD sequence anticipated the appearance of CMs' contractility compared to the control (fibronectin-coated well) and doubled the length of cell viability. Future prospects are the inclusion of these sequences into biomaterial formulation with the improvement in cell adhesion that could play an important role in cell retention during dynamic cell seeding. PMID:22011064

  13. Studies of adhesion-dependent platelet activation: distinct roles for different participating receptors can be dissociated by proteolysis of collagen.

    PubMed

    Siljander, P; Lassila, R

    1999-12-01

    The molecular differences between native-type collagen type I fibrils (NC) and their pepsinated monomers (PC) were used to uncover receptors involved in platelet-collagen interaction along the adhesion-activation axis. The platelet-depositing capacity of NC and PC under blood flow and their adhesive properties and respective morphologies, aggregation, procoagulant capacity, and tyrosine phosphorylation were compared under different cationic milieus, including or excluding the glycoprotein (GP) Ia/IIa. NC was consistently a more preferable and activating substrate than PC during flow (5 minutes) and in platelet aggregation. In PPACK-treated blood, both NC (3.3-fold) and PC (2.7-fold) increased platelet attachment on elevation of the shear rate from 500 to 1640 s(-1), whereas in citrated blood, adhesion and thrombus growth on PC were negligible under the high shear rate, unlike on NC (1.9-fold increase). The complete lack of platelet deposition on PC in citrated blood could be overcome by restoring physiological Mg(2+) concentration, and in contrast to NC, platelets interacting with PC were highly dependent on Mg(2+) during adhesion, aggregation, and procoagulant response. Monoclonal antibody (mAb 131.7) against GP IV inhibited platelet deposition to NC in citrated blood (2 minutes) by 49%, which was not further increased by coincubation with mAb against GP Ia (6F1). These results stress the importance of GP Ia/IIa in shear-resistant platelet deposition on collagen monomers. In native fibers, however, the preserved quaternary structure with telopeptides activates additional platelet receptors capable of substituting GP Ia/IIa and GP IV. PMID:10591685

  14. Pneumatic displacement with perfluoropropane gas and intravitreal tissue plasminogen activator for subretinal subfoveal hemorrhage after focal laser photocoagulation in central serous chorioretinopathy.

    PubMed

    Al Rubaie, Khalid; Espinoza, Juan V; Lasave, Andres F; Savino-Zari, Dario; Arevalo, Fernando A; Arevalo, J Fernando

    2014-01-01

    Objective. To report the visual and anatomic outcomes of pneumatic displacement with perfluoropropane (C3F8) gas and intravitreal tissue plasminogen activator (IVTPA) for subretinal subfoveal hemorrhage after focal laser photocoagulation in central serous chorioretinopathy (CSCR). Method. Interventional, retrospective case report of one eye (one patient). Outcome measures included visual acuity (VA), central macular thickness (CMT), and size of the lesion at two weeks of followup. Fluorescein angiography (FA) and optical coherent tomography (OCT) were used to measure anatomic outcomes. Results. A 35-year-old man with history of chronic CSCR received focal laser photocoagulation in the right eye two days before presentation. At initial examination, VA was 20/200 (ETDRS chart), CMT was 398 μ, and a subretinal subfoveal hemorrhage was seen. Tissue plasminogen activator (tPA) at a dose of 25 µg/0.1 mL was injected intravitreally before intravitreal C3F8 injection, and prone positioning was indicated postoperatively. At 24 hours, the hemorrhage had been displaced inferiorly and VA improved to 20/100. Two weeks later, VA improved to 20/80, CMT decreased to 225 μ, and the hemorrhage decreased without foveal involvement. Conclusions. The technique seems safe and effective in treating visually significant subretinal subfoveal hemorrhage. PMID:25485161

  15. Pneumatic Displacement with Perfluoropropane Gas and Intravitreal Tissue Plasminogen Activator for Subretinal Subfoveal Hemorrhage after Focal Laser Photocoagulation in Central Serous Chorioretinopathy

    PubMed Central

    Espinoza, Juan V.; Lasave, Andres F.; Savino-Zari, Dario; Arevalo, Fernando A.

    2014-01-01

    Objective. To report the visual and anatomic outcomes of pneumatic displacement with perfluoropropane (C3F8) gas and intravitreal tissue plasminogen activator (IVTPA) for subretinal subfoveal hemorrhage after focal laser photocoagulation in central serous chorioretinopathy (CSCR). Method. Interventional, retrospective case report of one eye (one patient). Outcome measures included visual acuity (VA), central macular thickness (CMT), and size of the lesion at two weeks of followup. Fluorescein angiography (FA) and optical coherent tomography (OCT) were used to measure anatomic outcomes. Results. A 35-year-old man with history of chronic CSCR received focal laser photocoagulation in the right eye two days before presentation. At initial examination, VA was 20/200 (ETDRS chart), CMT was 398 μ, and a subretinal subfoveal hemorrhage was seen. Tissue plasminogen activator (tPA) at a dose of 25 µg/0.1 mL was injected intravitreally before intravitreal C3F8 injection, and prone positioning was indicated postoperatively. At 24 hours, the hemorrhage had been displaced inferiorly and VA improved to 20/100. Two weeks later, VA improved to 20/80, CMT decreased to 225 μ, and the hemorrhage decreased without foveal involvement. Conclusions. The technique seems safe and effective in treating visually significant subretinal subfoveal hemorrhage. PMID:25485161

  16. Amygdalin influences bladder cancer cell adhesion and invasion in vitro.

    PubMed

    Makarević, Jasmina; Rutz, Jochen; Juengel, Eva; Kaulfuss, Silke; Tsaur, Igor; Nelson, Karen; Pfitzenmaier, Jesco; Haferkamp, Axel; Blaheta, Roman A

    2014-01-01

    The cyanogenic diglucoside amygdalin, derived from Rosaceae kernels, is employed by many patients as an alternative anti-cancer treatment. However, whether amygdalin indeed acts as an anti-tumor agent is not clear. Metastasis blocking properties of amygdalin on bladder cancer cell lines was, therefore, investigated. Amygdalin (10 mg/ml) was applied to UMUC-3, TCCSUP or RT112 bladder cancer cells for 24 h or for 2 weeks. Tumor cell adhesion to vascular endothelium or to immobilized collagen as well as tumor cell migration was examined. Effects of drug treatment on integrin α and β subtypes, on integrin-linked kinase (ILK) and total and activated focal adhesion kinase (FAK) were also determined. Integrin knock-down was carried out to evaluate integrin influence on migration and adhesion. A 24 h or 2 week amygdalin application distinctly reduced tumor cell adhesion and migration of UMUC-3 and RT112 cells. TCCSUP adhesion was also reduced, but migration was elevated under amygdalin. Integrin subtype expression was significantly and specifically altered by amygdalin depending on the cell line. ILK was moderately, and activated FAK strongly, lost in all tumor cell lines in the presence of amygdalin. Knock down of β1 integrin caused a significant decrease in both adhesion and migration of UMUC-3 cells, but a significant increase in TCCSUP adhesion. Knock down of β4 integrin caused a significant decrease in migration of RT112 cells. Since the different actions of amygdalin on the different cell lines was mirrored by β1 or β4 knock down, it is postulated that amygdalin influences adhesion and migratory properties of bladder cancer cells by modulating β1 or β4 integrin expression. The amygdalin induced increase in TCCSUP migratory behavior indicates that any anti-tumor benefits from amygdalin (seen with the other two cell lines) may depend upon the cancer cell type. PMID:25333694

  17. Amygdalin Influences Bladder Cancer Cell Adhesion and Invasion In Vitro

    PubMed Central

    Makarević, Jasmina; Rutz, Jochen; Juengel, Eva; Kaulfuss, Silke; Tsaur, Igor; Nelson, Karen; Pfitzenmaier, Jesco

    2014-01-01

    The cyanogenic diglucoside amygdalin, derived from Rosaceae kernels, is employed by many patients as an alternative anti-cancer treatment. However, whether amygdalin indeed acts as an anti-tumor agent is not clear. Metastasis blocking properties of amygdalin on bladder cancer cell lines was, therefore, investigated. Amygdalin (10 mg/ml) was applied to UMUC-3, TCCSUP or RT112 bladder cancer cells for 24 h or for 2 weeks. Tumor cell adhesion to vascular endothelium or to immobilized collagen as well as tumor cell migration was examined. Effects of drug treatment on integrin α and β subtypes, on integrin-linked kinase (ILK) and total and activated focal adhesion kinase (FAK) were also determined. Integrin knock-down was carried out to evaluate integrin influence on migration and adhesion. A 24 h or 2 week amygdalin application distinctly reduced tumor cell adhesion and migration of UMUC-3 and RT112 cells. TCCSUP adhesion was also reduced, but migration was elevated under amygdalin. Integrin subtype expression was significantly and specifically altered by amygdalin depending on the cell line. ILK was moderately, and activated FAK strongly, lost in all tumor cell lines in the presence of amygdalin. Knock down of β1 integrin caused a significant decrease in both adhesion and migration of UMUC-3 cells, but a significant increase in TCCSUP adhesion. Knock down of β4 integrin caused a significant decrease in migration of RT112 cells. Since the different actions of amygdalin on the different cell lines was mirrored by β1 or β4 knock down, it is postulated that amygdalin influences adhesion and migratory properties of bladder cancer cells by modulating β1 or β4 integrin expression. The amygdalin induced increase in TCCSUP migratory behavior indicates that any anti-tumor benefits from amygdalin (seen with the other two cell lines) may depend upon the cancer cell type. PMID:25333694

  18. ICAM-1-activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration.

    PubMed

    Liu, Guoquan; Place, Aaron T; Chen, Zhenlong; Brovkovych, Viktor M; Vogel, Stephen M; Muller, William A; Skidgel, Randal A; Malik, Asrar B; Minshall, Richard D

    2012-08-30

    Polymorphonuclear neutrophil (PMN) extravasation requires selectin-mediated tethering, intercellular adhesion molecule-1 (ICAM-1)-dependent firm adhesion, and platelet/endothelial cell adhesion molecule 1 (PECAM-1)-mediated transendothelial migration. An important unanswered question is whether ICAM-1-activated signaling contributes to PMN transmigration mediated by PECAM-1. We tested this concept and the roles of endothelial nitric oxide synthase (eNOS) and Src activated by PMN ligation of ICAM-1 in mediating PECAM-1-dependent PMN transmigration. We observed that lung PMN infiltration in vivo induced in carrageenan-injected WT mice was significantly reduced in ICAM-1(-/-) and eNOS(-/-) mice. Crosslinking WT mouse ICAM-1 expressed in human endothelial cells (ECs), but not the phospho-defective Tyr(518)Phe ICAM-1 mutant, induced SHP-2-dependent Src Tyr530 dephosphorylation that resulted in Src activation. ICAM-1 activation also stimulated phosphorylation of Akt (p-Ser473) and eNOS (p-Ser1177), thereby increasing NO production. PMN migration across EC monolayers was abolished in cells expressing the Tyr(518)Phe ICAM-1 mutant or by pretreatment with either the Src inhibitor PP2 or eNOS inhibitor L-NAME. Importantly, phospho-ICAM-1 induction of Src signaling induced PECAM-1 Tyr686 phosphorylation and increased EC surface anti-PECAM-1 mAb-binding activity. These results collectively show that ICAM-1-activated Src and eNOS signaling sequentially induce PECAM-1-mediated PMN transendothelial migration. Both Src and eNOS inhibition may be important therapeutic targets to prevent or limit vascular inflammation. PMID:22806890

  19. Human neutrophil adhesion to bovine aortic endothelium. Evidence for endothelial lipoxygenase activity.

    PubMed

    Damtew, B; Goldsmith, G; Tserng, K Y; Spagnuolo, P J

    1993-06-01

    We examined the effect of phorbol myristate acetate on cultured bovine aortic endothelial cells to determine the role of endothelial cells in neutrophil-endothelial cell adhesive interactions. Confluent endothelial cells were preincubated with phorbol myristate acetate and other inflammatory signals including N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe), the ionophore A23187, and thrombin; washed extensively; and incubated with 51Cr-labeled neutrophils. Preincubation of endothelium with A23187, phorbol ester, or thrombin increased adherence of neutrophils by 3.1-, 5.7-, and 3.7-fold over baseline. In contrast, f-Met-Leu-Phe preincubation failed to increase adhesion over baseline. Supernatants from endothelium preincubated with phorbol failed to augment adherence of untreated endothelial cells. Preincubation of endothelium with lipoxygenase inhibitors nordihydroguaiaretic acid (50 microM), 5,8,11,14-eicosatetraenoic acid (50 microM), and BW755C (50 microM) inhibited the effect of phorbol preincubation of endothelium significantly by 55, 27, and 22%, respectively. In contrast, inhibitors of cyclooxygenase and thromboxane synthase or thromboxane receptor antagonists had no effect on phorbol-induced adhesion. Specific desensitization of neutrophil adhesion to phorbol-treated endothelium could be demonstrated by prior exposure of neutrophils to low concentrations of leukotriene B4 (3.8 x 10(-10) M). Endothelium preincubated with phorbol but not f-Met-Leu-Phe or thrombin produced several fatty acid peaks at 280 nm, one of which comigrated with authentic leukotriene B4 (LTB4). This peak, isolated and purified, increased endothelial cell adherence in a temporal fashion in the same way as LTB4 and was demonstrated to be LTB4 by ultraviolet spectroscopy, high-performance liquid chrom