Science.gov

Sample records for focal adhesion complex

  1. Bacillus cereus Certhrax ADP-ribosylates vinculin to disrupt focal adhesion complexes and cell adhesion.

    PubMed

    Simon, Nathan C; Barbieri, Joseph T

    2014-04-11

    Bacillus cereus is often associated with mild to moderate gastroenteritis; however, some recent isolates cause inhalational anthrax-like diseases and death. These potential emerging human pathogens express multiple virulence factors. B. cereus strain G9241 expresses anthrax toxin, several polysaccharide capsules, and the novel ADP-ribosyltransferase, Certhrax. In this study, we show that Certhrax ADP-ribosylates Arg-433 of vinculin, a protein that coordinates actin cytoskeleton and extracellular matrix interactions. ADP-ribosylation of vinculin disrupted focal adhesion complexes and redistributed vinculin to the cytoplasm. Exogenous vinculin rescued these phenotypes. This provides a mechanism for strain G9241 to breach host barrier defenses and promote bacterial growth and spread. Certhrax is the first bacterial toxin to add a post-translational modification to vinculin to disrupt the actin cytoskeleton.

  2. 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

  3. 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

  4. The role of focal adhesion complexes in fibroblast mechanotransduction during scar formation.

    PubMed

    Rustad, Kristine C; Wong, Victor W; Gurtner, Geoffrey C

    2013-10-01

    Historically, great efforts have been made to elucidate the biochemical pathways that direct the complex process of wound healing; however only recently has there been recognition of the importance that mechanical signals play in the process of tissue repair and scar formation. The body's physiologic response to injury involves a dynamic interplay between mechanical forces and biochemical cues which directs a cascade of signals leading ultimately to the formation of fibrotic scar. Fibroblasts are a highly mechanosensitive cell type and are also largely responsible for the generation of the fibrotic matrix during scar formation and are thus a critical player in the process of mechanotransduction during tissue repair. Mechanotransduction is initiated at the interface between the cell membrane and the extracellular matrix where mechanical signals are first translated into a biochemical response. Focal adhesions are dynamic multi-protein complexes through which the extracellular matrix links to the intracellular cytoskeleton. These focal adhesion complexes play an integral role in the propagation of this initial mechanical cue into an extensive network of biochemical signals leading to widespread downstream effects including the influx of inflammatory cells, stimulation of angiogenesis, keratinocyte migration, fibroblast proliferation and collagen synthesis. Increasing evidence has demonstrated the importance of the biomechanical milieu in healing wounds and suggests that an integrated approach to the discovery of targets to decrease scar formation may prove more clinically efficacious than previous purely biochemical strategies.

  5. 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

  6. Focal adhesion kinase

    PubMed Central

    Stone, Rebecca L; Baggerly, Keith A; Armaiz-Pena, Guillermo N; Kang, Yu; Sanguino, Angela M; Thanapprapasr, Duangmani; Dalton, Heather J; Bottsford-Miller, Justin; Zand, Behrouz; Akbani, Rehan; Diao, Lixia; Nick, Alpa M; DeGeest, Koen; Lopez-Berestein, Gabriel; Coleman, Robert L; Lutgendorf, Susan; Sood, Anil K

    2014-01-01

    This investigation describes the clinical significance of phosphorylated focal adhesion kinase (FAK) at the major activating tyrosine site (Y397) in epithelial ovarian cancer (EOC) cells and tumor-associated endothelial cells. FAK gene amplification as a mechanism for FAK overexpression and the effects of FAK tyrosine kinase inhibitor VS-6062 on tumor growth, metastasis, and angiogenesis were examined. FAK and phospho-FAKY397 were quantified in tumor (FAK-T; pFAK-T) and tumor-associated endothelial (FAK-endo; pFAK-endo) cell compartments of EOCs using immunostaining and qRT-PCR. Associations between expression levels and clinical variables were evaluated. Data from The Cancer Genome Atlas were used to correlate FAK gene copy number and expression levels in EOC specimens. The in vitro and in vivo effects of VS-6062 were assayed in preclinical models. FAK-T and pFAK-T overexpression was significantly associated with advanced stage disease and increased microvessel density (MVD). High MVD was observed in tumors with elevated endothelial cell FAK (59%) and pFAK (44%). Survival was adversely affected by FAK-T overexpression (3.03 vs 2.06 y, P = 0.004), pFAK-T (2.83 vs 1.78 y, P < 0.001), and pFAK-endo (2.33 vs 2.17 y, P = 0.005). FAK gene copy number was increased in 34% of tumors and correlated with expression levels (P < 0.001). VS-6062 significantly blocked EOC and endothelial cell migration as well as endothelial cell tube formation in vitro. VS-6062 reduced mean tumor weight by 56% (P = 0.005), tumor MVD by 40% (P = 0.0001), and extraovarian metastasis (P < 0.01) in orthotopic EOC mouse models. FAK may be a unique therapeutic target in EOC given the dual anti-angiogenic and anti-metastatic potential of FAK inhibitors. PMID:24755674

  7. Regulation of Cell Adhesion Strength by Peripheral Focal Adhesion Distribution

    PubMed Central

    Elineni, Kranthi Kumar; Gallant, Nathan D.

    2011-01-01

    Cell adhesion to extracellular matrices is a tightly regulated process that involves the complex interplay between biochemical and mechanical events at the cell-adhesive interface. Previous work established the spatiotemporal contributions of adhesive components to adhesion strength and identified a nonlinear dependence on cell spreading. This study was designed to investigate the regulation of cell-adhesion strength by the size and position of focal adhesions (FA). The cell-adhesive interface was engineered to direct FA assembly to the periphery of the cell-spreading area to delineate the cell-adhesive area from the cell-spreading area. It was observed that redistributing the same adhesive area over a larger cell-spreading area significantly enhanced cell-adhesion strength, but only up to a threshold area. Moreover, the size of the peripheral FAs, which was interpreted as an adhesive patch, did not directly govern the adhesion strength. Interestingly, this is in contrast to the previously reported functional role of FAs in regulating cellular traction where sizes of the peripheral FAs play a critical role. These findings demonstrate, to our knowledge for the first time, that two spatial regimes in cell-spreading area exist that uniquely govern the structure-function role of FAs in regulating cell-adhesion strength. PMID:22208188

  8. JUNCTIONAL COMPLEX AND FOCAL ADHESION REARRANGEMENT MEDIATES PULMONARY ENDOTHELIAL BARRIER ENHANCEMENT BY FTY720 S-PHOSPHONATE

    PubMed Central

    Wang, Lichun; Bittman, Robert; Garcia, Joe G.N.; Dudek, Steven M.

    2015-01-01

    Rationale Modulation of pulmonary vascular barrier function is an important clinical goal given the devastating effects of vascular leak in acute lung injury (ALI). We previously demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine 1-phosphate (S1P) and FTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in mouse models of ALI. Tys preserves expression of the barrier-promoting S1P1 receptor (S1PR1), whereas S1P and FTY720 induce its ubiquitination and degradation. Here we further characterize the novel barrier promoting effects of Tys in cultured human pulmonary endothelial cells (EC). Methods/Results : In human lung EC, Tys significantly increased peripheral redistribution of adherens junction proteins VE-cadherin and β-catenin and tight junction protein ZO-1. Inhibition of VE-cadherin with blocking antibody significantly attenuated Tys-induced transendothelial resistance (TER) elevation, while ZO-1 siRNA partially inhibited this elevation. Tys significantly increased focal adhesion formation and phosphorylation of focal adhesion kinase (FAK). Pharmacologic inhibition of FAK significantly attenuated Tys-induced TER elevation. Tys significantly increased phosphorylation and peripheral redistribution of the actin-binding protein, cortactin, while cortactin siRNA partially attenuated Tys-induced TER elevation. Although Tys significantly increased phosphorylation of Akt and GSK3β, neither PI3 kinase nor GSK3β inhibition altered Tys-induced TER elevation. Tys significantly increased Rac1 activity, while inhibition of Rac1 activity significantly attenuated Tys-induced VE-cadherin redistribution and TER elevation. Conclusion Junctional complex, focal adhesion rearrangement and Rac1 activation play critical roles in Tys-mediated barrier protection in pulmonary EC. These results provide mechanistic insights into the effects of this potential ALI therapy. PMID:25862132

  9. 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.

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

    PubMed Central

    2013-01-01

    Background Focal adhesions are integrin-based cell-matrix contacts that transduce and integrate mechanical and biochemical cues from the environment. They develop from smaller and more numerous focal complexes under the influence of mechanical force and are key elements for many physiological and disease-related processes, including wound healing and metastasis. More than 150 different proteins localize to focal adhesions and have been systematically classified in the adhesome project (http://www.adhesome.org). First RNAi-screens have been performed for focal adhesions and the effect of knockdown of many of these components on the number, size, shape and location of focal adhesions has been reported. Results We have developed a kinetic model for RNA interference of focal adhesions which represents some of its main elements: a spatially layered structure, signaling through the small GTPases Rac and Rho, and maturation from focal complexes to focal adhesions under force. The response to force is described by two complementary scenarios corresponding to slip and catch bond behavior, respectively. Using estimated and literature values for the model parameters, three time scales of the dynamics of RNAi-influenced focal adhesions are identified: a sub-minute time scale for the assembly of focal complexes, a sub-hour time scale for the maturation to focal adhesions, and a time scale of days that controls the siRNA-mediated knockdown. Our model shows bistability between states dominated by focal complexes and focal adhesions, respectively. Catch bonding strongly extends the range of stability of the state dominated by focal adhesions. A sensitivity analysis predicts that knockdown of focal adhesion components is more efficient for focal adhesions with slip bonds or if the system is in a state dominated by focal complexes. Knockdown of Rho leads to an increase of focal complexes. Conclusions The suggested model provides a kinetic description of the effect of RNA

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

    PubMed

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

    2013-05-01

    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-dimethyl-amino)-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.

  12. 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

  13. Intermediate filaments and the regulation of focal adhesion.

    PubMed

    Leube, Rudolf E; Moch, Marcin; Windoffer, Reinhard

    2015-02-01

    Focal adhesions are localized actin filament-anchoring signalling centres at the cell-extracellular matrix interface. The currently emerging view is that they fulfil an all-embracing coordinating function for the entire cytoskeleton. This review highlights the tight relationship between focal adhesions and the intermediate filament cytoskeleton. We summarize the accumulating evidence for direct binding of intermediate filaments to focal adhesion components and their mutual cross-talk through signalling molecules. Examples are presented to emphasize the high degree of complexity of these interactions equipping cells with a precisely controlled machinery for context-dependent adjustment of their biomechanical properties.

  14. 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

    FRP1 in regulating spermiation via its effects on the FAK signaling and retention of nectin-3 adhesion complex at the apical ES.—Wong, E. W. P., Lee, W. M., Cheng, C. Y. 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. PMID:23073828

  15. A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures.

    PubMed Central

    Harden, N; Lee, J; Loh, H Y; Ong, Y M; Tan, I; Leung, T; Manser, E; Lim, L

    1996-01-01

    Changes in cell morphology are essential in the development of a multicellular organism. The regulation of the cytoskeleton by the Rho subfamily of small GTP-binding proteins is an important determinant of cell shape. The Rho subfamily has been shown to participate in a variety of morphogenetic processes during Drosophila melanogaster development. We describe here a Drosophila homolog, DPAK, of the serine/threonine kinase PAK, a protein which is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK bound to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK was elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of the embryo. We have previously shown that the accumulation of cytoskeletal elements initiating cell shape changes in these cells could be inhibited by expression of a dominant-negative DRacA transgene. We show that leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. We propose that DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure. PMID:8628256

  16. 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

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

    PubMed

    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

  18. 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

  19. C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion

    PubMed Central

    2013-01-01

    Background Previous studies by our group and others have shown that C3G interacts with Bcr-Abl through its SH3-b domain. Results In this work we show that C3G and Bcr-Abl form complexes with the focal adhesion (FA) proteins CrkL, p130Cas, Cbl and Abi1 through SH3/SH3-b interactions. The association between C3G and Bcr-Abl decreased upon Abi1 or p130Cas knock-down in K562 cells, which suggests that Abi1 and p130Cas are essential partners in this interaction. On the other hand, C3G, Abi1 or Cbl knock-down impaired adhesion to fibronectin, while p130Cas silencing enhanced it. C3G, Cbl and p130Cas-SH3-b domains interact directly with common proteins involved in the regulation of cell adhesion and migration. Immunoprecipitation and immunofluorescence studies revealed that C3G form complexes with the FA proteins paxillin and FAK and their phosphorylated forms. Additionally, C3G, Abi1, Cbl and p130Cas regulate the expression and phosphorylation of paxillin and FAK. p38α MAPK also participates in the regulation of adhesion in chronic myeloid leukemia cells. It interacts with C3G, CrkL, FAK and paxillin and regulates the expression of paxillin, CrkL and α5 integrin, as well as paxillin phosphorylation. Moreover, double knock-down of C3G/p38α decreased adhesion to fibronectin, similarly to the single silencing of one of these genes, either C3G or p38α. These suggest that C3G and p38α MAPK are acting through a common pathway to regulate cell adhesion in K562 cells, as previously described for the regulation of apoptosis. Conclusions Our results indicate that C3G-p38αMAPK pathway regulates K562 cell adhesion through the interaction with FA proteins and Bcr-Abl, modulating the formation of different protein complexes at FA. PMID:23343344

  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. ADAMTS-10 and -6 differentially regulate cell-cell junctions and focal adhesions

    PubMed Central

    Cain, Stuart A.; Mularczyk, Ewa J.; Singh, Mukti; Massam-Wu, Teresa; Kielty, Cay M.

    2016-01-01

    ADAMTS10 and ADAMTS6 are homologous metalloproteinases with ill-defined roles. ADAMTS10 mutations cause Weill-Marchesani syndrome (WMS), implicating it in fibrillin microfibril biology since some fibrillin-1 mutations also cause WMS. However little is known about ADAMTS6 function. ADAMTS10 is resistant to furin cleavage, however we show that ADAMTS6 is effectively processed and active. Using siRNA, over-expression and mutagenesis, it was found ADAMTS6 inhibits and ADAMTS10 is required for focal adhesions, epithelial cell-cell junction formation, and microfibril deposition. Either knockdown of ADAMTS6, or disruption of its furin processing or catalytic sites restores focal adhesions, implicating its enzyme activity acts on targets in the focal adhesion complex. In ADAMTS10-depleted cultures, expression of syndecan-4 rescues focal adhesions and cell-cell junctions. Recombinant C-termini of ADAMTS10 and ADAMTS6, both of which induce focal adhesions, bind heparin and syndecan-4. However, cells overexpressing full-length ADAMTS6 lack heparan sulphate and focal adhesions, whilst depletion of ADAMTS6 induces a prominent glycocalyx. Thus ADAMTS10 and ADAMTS6 oppositely affect heparan sulphate-rich interfaces including focal adhesions. We previously showed that microfibril deposition requires fibronectin-induced focal adhesions, and cell-cell junctions in epithelial cultures. Here we reveal that ADAMTS6 causes a reduction in heparan sulphate-rich interfaces, and its expression is regulated by ADAMTS10. PMID:27779234

  2. Integrin Molecular Tension within Motile Focal Adhesions.

    PubMed

    Wang, Xuefeng; Sun, Jie; Xu, Qian; Chowdhury, Farhan; Roein-Peikar, Mehdi; Wang, Yingxiao; Ha, Taekjip

    2015-12-01

    Forces transmitted by integrins regulate many important cellular functions. Previously, we developed tension gauge tether (TGT) as a molecular force sensor and determined the threshold tension across a single integrin-ligand bond, termed integrin tension, required for initial cell adhesion. Here, we used fluorescently labeled TGTs to study the magnitude and spatial distribution of integrin tension on the cell-substratum interface. We observed two distinct levels of integrin tension. A >54 pN molecular tension is transmitted by clustered integrins in motile focal adhesions (FAs) and such force is generated by actomyosin, whereas the previously reported ∼40 pN integrin tension is transmitted by integrins before FA formation and is independent of actomyosin. We then studied FA motility using a TGT-coated surface as a fluorescent canvas, which records the history of integrin force activity. Our data suggest that the region of the strongest integrin force overlaps with the center of a motile FA within 0.2 μm resolution. We also found that FAs move in pairs and that the asymmetry in the motility of an FA pair is dependent on the initial FA locations on the cell-substratum interface.

  3. 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

  4. Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation

    PubMed Central

    Roa-Espitia, Ana L.; Hernández-Rendón, Eva R.; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J.; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto

    2016-01-01

    ABSTRACT Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca2+ dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton. PMID:27402964

  5. Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation.

    PubMed

    Roa-Espitia, Ana L; Hernández-Rendón, Eva R; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto; Hernández-González, Enrique O

    2016-01-01

    Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca(2+) dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton. PMID:27402964

  6. Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation.

    PubMed

    Roa-Espitia, Ana L; Hernández-Rendón, Eva R; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto; Hernández-González, Enrique O

    2016-09-15

    Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca(2+) dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton.

  7. 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

  8. 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

  9. Focal Adhesion Induction at the Tip of a Functionalized Nanoelectrode

    PubMed Central

    Fuentes, Daniela E.; Bae, Chilman; Butler, Peter J.

    2012-01-01

    Cells dynamically interact with their physical micro-environment through the assembly of nascent focal contacts and focal adhesions. The dynamics and mechanics of these contact points are controlled by transmembrane integrins and an array of intracellular adaptor proteins. In order to study the mechanics and dynamics of focal adhesion assembly, we have developed a technique for the timed induction of a nascent focal adhesion. Bovine aortic endothelial cells were approached at the apical surface by a nanoelectrode whose position was controlled with a resolution of 10s of nanometers using changes in electrode current to monitor distance from the cell surface. Since this probe was functionalized with fibronectin, a focal contact formed at the contact location. Nascent focal adhesion assembly was confirmed using time-lapse confocal fluorescent images of red fluorescent protein (RFP) – tagged talin, an adapter protein that binds to activated integrins. Binding to the cell was verified by noting a lack of change of electrode current upon retraction of the electrode. This study demonstrates that functionalized nanoelectrodes can enable precisely-timed induction and 3-D mechanical manipulation of focal adhesions and the assay of the detailed molecular kinetics of their assembly. PMID:22247742

  10. 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

  11. 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

  12. ISOLATION OF INTEGRIN-BASED ADHESION COMPLEXES

    PubMed Central

    Jones, Matthew C.; Humphries, Jonathan D.; Byron, Adam; Millon-Frémillon, Angelique; Robertson, Joseph; Paul, Nikki R.; Ng, Daniel H. J.; Askari, Janet A.; Humphries, Martin J.

    2015-01-01

    The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signalling across the plasma membrane and as such help to coordinate and / or modulate the multitude of physical or chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes together with their local plasma membrane / cytosolic environments from cells in culture. In the first protocol integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components by subsequent downstream analysis by Western blotting or mass spectrometry. PMID:25727331

  13. 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.

  14. 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

  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. Talin1 Promotes Tumor Invasion and Metastasis via Focal Adhesion Signaling and Anoikis Resistance

    PubMed Central

    Sakamoto, Shinichi; McCann, Richard O.; Dhir, Rajiv; Kyprianou, Natasha

    2010-01-01

    Talin1 is a focal adhesion complex protein that regulates integrin interactions with the extracellular matrix (ECM). This study investigated the significance of talin1 in prostate cancer progression to metastasis in vitro and in vivo. Talin1 overexpression enhanced prostate cancer cell adhesion, migration and invasion by activating survival signals and conferring resistance to anoikis. ShRNA-mediated talin1 loss led to a significant suppression of prostate cancer cell migration and transendothelial invasion in vitro and a significant inhibition of prostate cancer metastasis in vivo. Talin1 regulated cell survival signals via phosphorylation of focal adhesion complex proteins such as focal adhesion kinase (FAK) and Src, and downstream activation of AKT. Targeting AKT activation led to a significant reduction of talin1-mediated prostate cancer cell invasion. Furthermore, talin1 immunoreactivity directly correlated with prostate tumor progression to metastasis in the TRAMP mouse model. Talin1 profiling in human prostate specimens revealed a significantly higher expression of cytoplasmic talin1 in metastatic tissue compared to primary prostate tumors (P<0.0001). These findings suggest: (a) a therapeutic significance of disrupting talin1 signaling/focal adhesion interactions in targeting metastatic prostate cancer and (b) a potential value for talin1 as a marker of tumor progression to metastasis. PMID:20160039

  17. Mechanosensing through focal adhesion-anchored intermediate filaments.

    PubMed

    Gregor, Martin; Osmanagic-Myers, Selma; Burgstaller, Gerald; Wolfram, Michael; Fischer, Irmgard; Walko, Gernot; Resch, Guenter P; Jörgl, Almut; Herrmann, Harald; Wiche, Gerhard

    2014-02-01

    Integrin-based mechanotransduction involves a complex focal adhesion (FA)-associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up-regulation of the compensatory feedback loop acting on RhoA and myosin light chain. In line with these findings, we show strongly reduced FA turnover rates in P0 fibroblasts combined with impaired directional migration, formation of protrusions, and up-regulation of "stretched" high-affinity integrin complexes. By exploiting tension-independent conditions, we were able to mechanistically link these defects to diminished cytoskeletal tension in both P0 and V0 cells. Our data provide important new insights into molecular mechanisms underlying cytoskeleton-regulated mechanosensing, a feature that is fundamental for controlled cell movement and tumor progression.

  18. Mulberry water extracts inhibit atherosclerosis through suppression of the integrin-β₃/focal adhesion kinase complex and downregulation of nuclear factor κB signaling in vivo and in vitro.

    PubMed

    Chan, Kuei-Chuan; Ho, Hsieh-Hsun; Lin, Ming-Cheng; Yen, Chi-Hua; Huang, Chien-Ning; Huang, Hui-Pei; Wang, Chau-Jong

    2014-10-01

    Previous studies have shown that mulberry water extracts (MWEs), which contain polyphenolic compounds, have an antiatherosclerotic effect in vivo and in vitro through stimulating apoptosis of vascular smooth muscle cells (VSMCs). Histological analysis was performed on atherosclerotic lesions from high-cholesterol diet (HCD)-fed rabbits after treatment with 0.5-1% MWEs for 10 weeks. Immunohistochemistry showed that the expressions of SMA, Ras, and matrix metalloproteinase-2 in the VSMCs were dose-dependently inhibited after MWE treatment. The antimigratory effects of MWEs on A7r5 VSMCs were assessed by western blot analysis of migration-related proteins, visualization of F-actin cytoskeleton, and reverse transcription polymerase chain reaction. The results showed that MWEs inhibited VSMC migration through reducing interactions of the integrin-β3/focal adhesion kinase complex, alterations of the cytoskeleton, and downregulation of glycogen synthase kinase 3β/nuclear factor κB signaling. Taken together, MWEs inhibited HCD-induced rabbit atherogenesis through blocking VSMC migration via reducing interactions of integrin-β3 and focal adhesion kinase and downregulating migration-related proteins.

  19. 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.

  20. 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

  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. Induction of focal adhesions and motility in Drosophila S2 cells.

    PubMed

    Ribeiro, Susana A; D'Ambrosio, Michael V; Vale, Ronald D

    2014-12-01

    Focal adhesions are dynamic structures that interact with the extracellular matrix on the cell exterior and actin filaments on the cell interior, enabling cells to adhere and crawl along surfaces. We describe a system for inducing the formation of focal adhesions in normally non-ECM-adherent, nonmotile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a nonpolarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. A subset of nonmotile, nonpolarized cells also exhibited focal adhesions that rapidly assembled and disassembled around the cell perimeter. Such cooperative and dynamic fluctuations of focal adhesions were decreased by RNA interference (RNAi) depletion of myosin II and focal adhesion kinase, suggesting that this behavior requires force and focal adhesion maturation. These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility.

  4. Essential role for focal adhesion kinase in regulating stress hematopoiesis

    PubMed Central

    Ramdas, Baskar; Hanneman, Philip; Martin, Joseph; Beggs, Hilary E.

    2010-01-01

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been extensively studied in fibroblasts; however its function in hematopoiesis remains an enigma. FAK is thought to be expressed in myeloid and erythroid progenitors, and its expression is enhanced in response to cytokines such as granu-locyte macrophage colony-stimulating factor. Furthermore, bone marrow cells cultured in granulocyte macrophage colony-stimulating factor show active migration and chemoattractant-induced polarization, which correlates with FAK induction. While loss of FAK in mice results in embryonic lethality, we have deleted FAK in the adult bone marrow. We show an essential role for FAK in regulating hemolytic, myelotoxic, as well as acute inflammatory stress responses in vivo. In vitro, loss of FAK in erythroid and myeloid progenitor's results in impaired cytokine induced growth and survival, as well as defects in the activation and expression of antiapoptotic proteins caspase 3 and Bcl-xL. Additionally, reduced migration and adhesion of myeloid cells on extracellular matrix proteins, as well as impaired activation of Rac GTPase is also observed in the absence of FAK. Our studies reveal an essential role for FAK in integrating growth/survival and adhesion based functions in myeloid and erythroid cells predominantly under conditions of stress. PMID:20664055

  5. 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

  6. 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

  7. Crosstalk between focal adhesions and material mechanical properties governs cell mechanics and functions.

    PubMed

    Fusco, Sabato; Panzetta, Valeria; Embrione, Valerio; Netti, Paolo A

    2015-09-01

    Mechanical properties of materials strongly influence cell fate and functions. Focal adhesions are involved in the extremely important processes of mechanosensing and mechanotransduction. To address the relationship between the mechanical properties of cell substrates, focal adhesion/cytoskeleton assembly and cell functions, we investigated the behavior of NIH/3T3 cells over a wide range of stiffness (3-1000kPa) using two of the most common synthetic polymers for cell cultures: polyacrylamide and polydimethylsiloxane. An overlapping stiffness region was created between them to compare focal adhesion characteristics and cell functions, taking into account their different time-dependent behavior. Indeed, from a rheological point of view, polyacrylamide behaves like a strong gel (elastically), whereas polydimethylsiloxane like a viscoelastic solid. First, focal adhesion characteristics and dynamics were addressed in terms of material stiffness, then cell spreading area, migration rate and cell mechanical properties were correlated with focal adhesion size and assembly. Focal adhesion size was found to increase in the whole range of stiffness and to be in agreement in the overlapping rigidity region for the investigated materials. Cell mechanics directly correlated with focal adhesion lengths, whereas migration rate followed an inverse correlation. Cell spreading correlated with the substrate stiffness on polyacrylamide hydrogel, while no specific trend was found on polydimethylsiloxane. Substrate mechanics can be considered as a key physical cue that regulates focal adhesion assembly, which in turn governs important cellular properties and functions. PMID:26004223

  8. 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

  9. Why do receptor-ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

    NASA Astrophysics Data System (ADS)

    Gao, Zhiwen; Gao, Yanfei

    2016-10-01

    Cell adhesion often exhibits the clustering of the receptor-ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation for the clustering/assembling of the receptor-ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor-ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.

  10. Complex source description of focal regions.

    PubMed

    Monzon, Cesar; Forester, Donald W; Moore, Peter

    2006-04-01

    Closed-form solutions of the two-dimensional homogeneous wave equation are presented that provide focal-region descriptions corresponding to a converging bundle of rays. The solutions do have evanescent wave content and can be described as a source-sink pair or particle-antiparticle pair, collocated in complex space, with the complex location being critical in the determination of beam shape and focal region size. The wave solutions are not plagued by singularities, have a finite energy, and have a limitation on how small the focal size can get, with a penalty for limiting small spot sizes in the form of impractically high associated reactive energy. The electric-field-defined spot-size limiting value is 0.35lambda x 0.35lambda, which is about 38% of the Poynting-vector-defined minimum spot size (0.8lambda x 0.4lambda) and corresponds to a condition related to the maximum possible beam angle. A multiple set of solutions is introduced, and the elementary solutions are used to produce new solutions via superposition, resulting in fields with chiral character or with increased depth of focus. We do not claim generality, as the size of focal regions exhibited by the closed-form solutions has a lower bound and hence is not able to account for Pendry's "ideal lens" scenario. PMID:16604758

  11. Complex source description of focal regions.

    PubMed

    Monzon, Cesar; Forester, Donald W; Moore, Peter

    2006-04-01

    Closed-form solutions of the two-dimensional homogeneous wave equation are presented that provide focal-region descriptions corresponding to a converging bundle of rays. The solutions do have evanescent wave content and can be described as a source-sink pair or particle-antiparticle pair, collocated in complex space, with the complex location being critical in the determination of beam shape and focal region size. The wave solutions are not plagued by singularities, have a finite energy, and have a limitation on how small the focal size can get, with a penalty for limiting small spot sizes in the form of impractically high associated reactive energy. The electric-field-defined spot-size limiting value is 0.35lambda x 0.35lambda, which is about 38% of the Poynting-vector-defined minimum spot size (0.8lambda x 0.4lambda) and corresponds to a condition related to the maximum possible beam angle. A multiple set of solutions is introduced, and the elementary solutions are used to produce new solutions via superposition, resulting in fields with chiral character or with increased depth of focus. We do not claim generality, as the size of focal regions exhibited by the closed-form solutions has a lower bound and hence is not able to account for Pendry's "ideal lens" scenario.

  12. 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

  13. 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

  14. 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

  15. Interactions of the Protein-tyrosine Phosphatase-α with the Focal Adhesion Targeting Domain of Focal Adhesion Kinase Are Involved in Interleukin-1 Signaling in Fibroblasts*

    PubMed Central

    Wang, Qin; Wang, Yongqiang; Fritz, Dominik; Rajshankar, Dhaarmini; Downey, Gregory P.; McCulloch, Christopher A.

    2014-01-01

    Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca2+ release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα. PMID:24821720

  16. Interactions of the protein-tyrosine phosphatase-α with the focal adhesion targeting domain of focal adhesion kinase are involved in interleukin-1 signaling in fibroblasts.

    PubMed

    Wang, Qin; Wang, Yongqiang; Fritz, Dominik; Rajshankar, Dhaarmini; Downey, Gregory P; McCulloch, Christopher A

    2014-06-27

    Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca(2+) release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα.

  17. 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

  18. 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

  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. 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

  1. Fibronectin is not Present in the Focal Adhesions Formed between Normal Cultured Fibroblasts and Their Substrata

    NASA Astrophysics Data System (ADS)

    Chen, Wen-Tien; Singer, S. J.

    1980-12-01

    Fibronectin is an extracellular matrix protein that has been implicated in the spreading and adhesion of cultured fibroblasts to their substrata. In this paper, double immunoelectron microscopic labeling experiments for fibronectin and for concanavalin A-binding proteins on the cell surface were carried out on ultrathin frozen sections of cultures of embryonic chicken heart fibroblasts. On cross sections through the focal adhesions of the cell to the substratum there was substantial labeling for concanavalin A-binding proteins but no detectable labeling for fibronectin, whereas both the binding proteins and fibronectin were extensively labeled elsewhere on the cell surface and substratum. These results demonstrate that fibronectin is not present within the sites of focal adhesions. Therefore, the functions of fibronectin in cell spreading and adhesion are not directly mediated through its binding at focal adhesion sites. An alternative model is presented which can account for such fibronectin functions.

  2. 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

  3. 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.

  4. FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion

    PubMed Central

    Deramaudt, Therese B.; Dujardin, Denis; Hamadi, Abdelkader; Noulet, Fanny; Kolli, Kaouther; De Mey, Jan; Takeda, Kenneth; Rondé, Philippe

    2011-01-01

     Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAK−/− mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130CAS/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion. PMID:21289086

  5. 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

  6. Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins

    NASA Astrophysics Data System (ADS)

    Kirchenbüchler, David; Born, Simone; Kirchgeßner, Norbert; Houben, Sebastian; Hoffmann, Bernd; Merkel, Rudolf

    2010-05-01

    Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile

  7. A plastic relationship between vinculin-mediated tension and adhesion complex area defines adhesion size and lifetime

    NASA Astrophysics Data System (ADS)

    Hernández-Varas, Pablo; Berge, Ulrich; Lock, John G.; Strömblad, Staffan

    2015-06-01

    Cell-matrix adhesions are central mediators of mechanotransduction, yet the interplay between force and adhesion regulation remains unclear. Here we use live cell imaging to map time-dependent cross-correlations between vinculin-mediated tension and adhesion complex area, revealing a plastic, context-dependent relationship. Interestingly, while an expected positive cross-correlation dominated in mid-sized adhesions, small and large adhesions display negative cross-correlation. Furthermore, although large changes in adhesion complex area follow vinculin-mediated tension alterations, small increases in area precede vinculin-mediated tension dynamics. Modelling based on this mapping of the vinculin-mediated tension-adhesion complex area relationship confirms its biological validity, and indicates that this relationship explains adhesion size and lifetime limits, keeping adhesions focal and transient. We also identify a subpopulation of steady-state adhesions whose size and vinculin-mediated tension become stabilized, and whose disassembly may be selectively microtubule-mediated. In conclusion, we define a plastic relationship between vinculin-mediated tension and adhesion complex area that controls fundamental cell-matrix adhesion properties.

  8. 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.

  9. 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.

  10. Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions.

    PubMed

    Legant, Wesley R; Choi, Colin K; Miller, Jordan S; Shao, Lin; Gao, Liang; Betzig, Eric; Chen, Christopher S

    2013-01-15

    Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a high-spatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces. PMID:23277584

  11. 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.

  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. Inhibitory effects of Yangzheng Xiaoji on angiogenesis and the role of the focal adhesion kinase pathway.

    PubMed

    Jiang, Wen G; Ye, Lin; Ji, Ke; Frewer, Natasha; Ji, Jiafu; Mason, Malcolm D

    2012-11-01

    Angiogenesis is an essential event during the excessive growth and metastatic spread of solid tumours. Anti-angiogenic agents have become a new choice of therapy for patients with cancer. In the present study, we investigated the potential effect of Yangzheng Xiaoji, a traditional Chinese medicinal formula presently used in the treatment of several solid tumours including liver cancer and gastric cancer, on angiogenesis, in vitro. The human vascular endothelial cell line HECV was used. A Matrigel-based sandwich tubule formation assay was employed to assess in vitro angiogenesis, a colorimetric method for assessing in vitro cell growth. Electric cell-substrate impedance sensing (ECIS) was used to evaluate the adhesion and migration of endothelial cells. The effects on activation of focal adhesion kinase (FAK) were evaluated using western blotting and immunofluorescence methods. Yangzhen Xiaoji extract DME25 significantly inhibited tube formation (p=0.046 vs control). This was seen together with a concentration-dependent inhibition on cell-matrix adhesion and cellular migration. It was demonstrated that the focal adhesion kinase (FAK) inhibitor PF557328 had a significant synergistic effect on DME25-induced inhibition of cell adhesion, migration and tube formation. The study showed that DME25 inhibited the phosphorylation of FAK in endothelial cells. In conclusion, Yangzhen Xiaoji has a marked effect on angiogenesis, in vitro and that this effect is at least partly mediated by the focal adhesion kinase (FAK) pathway. PMID:22971748

  14. 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.

  15. 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.

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

    PubMed

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

    2014-01-01

    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.

  17. Myxococcus xanthus Gliding Motors Are Elastically Coupled to the Substrate as Predicted by the Focal Adhesion Model of Gliding Motility

    PubMed Central

    Balagam, Rajesh; Litwin, Douglas B.; Czerwinski, Fabian; Sun, Mingzhai; Kaplan, Heidi B.; Shaevitz, Joshua W.; Igoshin, Oleg A.

    2014-01-01

    Myxococcus xanthus is a model organism for studying bacterial social behaviors due to its ability to form complex multi-cellular structures. Knowledge of M. xanthus surface gliding motility and the mechanisms that coordinated it are critically important to our understanding of collective cell behaviors. Although the mechanism of gliding motility is still under investigation, recent experiments suggest that there are two possible mechanisms underlying force production for cell motility: the focal adhesion mechanism and the helical rotor mechanism, which differ in the biophysics of the cell–substrate interactions. Whereas the focal adhesion model predicts an elastic coupling, the helical rotor model predicts a viscous coupling. Using a combination of computational modeling, imaging, and force microscopy, we find evidence for elastic coupling in support of the focal adhesion model. Using a biophysical model of the M. xanthus cell, we investigated how the mechanical interactions between cells are affected by interactions with the substrate. Comparison of modeling results with experimental data for cell-cell collision events pointed to a strong, elastic attachment between the cell and substrate. These results are robust to variations in the mechanical and geometrical parameters of the model. We then directly measured the motor-substrate coupling by monitoring the motion of optically trapped beads and find that motor velocity decreases exponentially with opposing load. At high loads, motor velocity approaches zero velocity asymptotically and motors remain bound to beads indicating a strong, elastic attachment. PMID:24810164

  18. Focal adhesion sites and the removal of substratum-bound fibronectin

    PubMed Central

    1986-01-01

    Fibronectin was not removed from the substratum beneath focal adhesion sites when fibroblasts spread in serum-free medium on adsorbed fibronectin substrata, or when fibroblasts spread in serum-containing medium on covalently cross-linked fibronectin substrata. Under these conditions, there was colocalization between 140-kD fibronectin receptors and focal adhesion sites. It was concluded that removal of adsorbed fibronectin from beneath focal adhesion sites was a mechanical process that required serum. The effect of serum was nonspecific since serum could be replaced by equivalent concentrations of serum albumin, ovalbumin, or gamma globulins. Quantitative measurements indicated that the presence of proteins in the incubation medium weakens the interaction of fibronectin with the substratum, thereby allowing the adsorbed protein to be removed from the substratum at sites of high stress. After removing fibronectin from the substratum, cells reorganized this material into patches and fibrils beneath cells, and the reorganized fibronectin colocalized with fibronectin receptors. Some of the patches of fibronectin were phagocytosed. The fibronectin fibrils were observed to be in register with actin filament bundles and sometimes translocated to the upper cell surfaces. It is proposed that removal of fibronectin from beneath focal adhesion sites is an example of how cells can modify their extracellular matrices through contractile activity. PMID:2947902

  19. Occurrence and resolution of focal epithelial hyperplasia in two siblings with leukocyte adhesion deficiency.

    PubMed

    Mealey, B L; Hallmon, W W; Waldrop, T C

    1993-02-01

    Focal epithelial hyperplasia is an uncommon disorder characterized by formation of multiple asymptomatic oral mucosal papular and/or nodular lesions. This article relates the occurrence and clinical course of FEH in 2 adolescent siblings with leukocyte adhesion deficiency. Histological findings are described and insights into potential causes are discussed.

  20. Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement.

    PubMed

    Yoshigi, Masaaki; Hoffman, Laura M; Jensen, Christopher C; Yost, H Joseph; Beckerle, Mary C

    2005-10-24

    Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues. PMID:16247023

  1. Quantitative measurement of changes in adhesion force involving focal adhesion kinase during cell attachment, spread, and migration.

    PubMed

    Wu, Chia-Ching; Su, Hsiao-Wen; Lee, Chen-Chen; Tang, Ming-Jer; Su, Fong-Chin

    2005-04-01

    Focal adhesion kinase (FAK) is a critical protein for the regulation of integrin-mediated cellular functions and it can enhance cell motility in Madin-Darby canine kidney (MDCK) cells by hepatocyte growth factor (HGF) induction. We utilized optical trapping and cytodetachment techniques to measure the adhesion force between pico-Newton and nano-Newton (nN) for quantitatively investigating the effects of FAK on adhesion force during initial binding (5 s), beginning of spreading (30 min), spreadout (12 h), and migration (induced by HGF) in MDCK cells with overexpressed FAK (FAK-WT), FAK-related non-kinase (FRNK), as well as normal control cells. Optical tweezers was used to measure the initial binding force between a trapped cell and glass coverslide or between a trapped bead and a seeded cell. In cytodetachment, the commercial atomic force microscope probe with an appropriate spring constant was used as a cyto-detacher to evaluate the change of adhesion force between different FAK expression levels of cells in spreading, spreadout, and migrating status. The results demonstrated that FAK-WT significantly increased the adhesion forces as compared to FRNK cells throughout all the different stages of cell adhesion. For cells in HGF-induced migration, the adhesion force decreased to almost the same level (approximately 600 nN) regardless of FAK levels indicating that FAK facilitates cells to undergo migration by reducing the adhesion force. Our results suggest FAK plays a role of enhancing cell adhesive ability in the binding and spreading, but an appropriate level of adhesion force is required for HGF-induced cell migration.

  2. Activity and Distribution of Paxillin, Focal Adhesion Kinase, and Cadherin Indicate Cooperative Roles during Zebrafish Morphogenesis

    PubMed Central

    Crawford, Bryan D.; Henry, Clarissa A.; Clason, Todd A.; Becker, Amanda L.; Hille, Merrill B.

    2003-01-01

    We investigated the focal adhesion proteins paxillin and Fak, and the cell-cell adhesion protein cadherin in developing zebrafish (Danio rerio) embryos. Cadherins are expressed in presomitic mesoderm where they delineate cells. The initiation of somite formation coincides with an increase in the phosphorylation of Fak, and the accumulation of Fak, phosphorylated Fak, paxillin, and fibronectin at nascent somite boundaries. In the notochord, cadherins are expressed on cells during intercalation, and phosphorylated Fak accumulates in circumferential rings where the notochord cells contact laminin in the perichordal sheath. Subsequently, changes in the orientations of collagen fibers in the sheath suggest that Fak-mediated adhesion allows longitudinal expansion of the notochord, but not lateral expansion, resulting in notochord elongation. Novel observations showed that focal adhesion kinase and paxillin concentrate at sites of cell-cell adhesion in the epithelial enveloping layer and may associate with actin cytoskeleton at epithelial junctions containing cadherins. Fak is phosphorylated at these epithelial junctions but is not phosphorylated on Tyr397, implicating a noncanonical mechanism of regulation. These data suggest that Fak and paxillin may function in the integration of cadherin-based and integrin-based cell adhesion during the morphogenesis of the early zebrafish embryo. PMID:12925747

  3. 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

  4. 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

  5. Adhesive ligand tether length affects the size and length of focal adhesions and influences cell spreading and attachment

    PubMed Central

    Attwood, Simon J.; Cortes, Ernesto; Haining, Alexander William M.; Robinson, Benjamin; Li, Danyang; Gautrot, Julien; del Río Hernández, Armando

    2016-01-01

    Cells are known to respond to physical cues from their microenvironment such as matrix rigidity. Discrete adhesive ligands within flexible strands of fibronectin connect cell surface integrins to the broader extracellular matrix and are thought to mediate mechanosensing through the cytoskeleton-integrin-ECM linkage. We set out to determine if adhesive ligand tether length is another physical cue that cells can sense. Substrates were covalently modified with adhesive arginylglycylaspartic acid (RGD) ligands coupled with short (9.5 nm), medium (38.2 nm) and long (318 nm) length inert polyethylene glycol tethers. The size and length of focal adhesions of human foreskin fibroblasts gradually decreased from short to long tethers. Furthermore, we found cell adhesion varies in a linker length dependent manner with a remarkable 75% reduction in the density of cells on the surface and a 50% reduction in cell area between the shortest and longest linkers. We also report the interplay between RGD ligand concentration and tether length in determining cellular spread area. Our findings show that without varying substrate rigidity or ligand density, tether length alone can modulate cellular behaviour. PMID:27686622

  6. Adhesive ligand tether length affects the size and length of focal adhesions and influences cell spreading and attachment

    NASA Astrophysics Data System (ADS)

    Attwood, Simon J.; Cortes, Ernesto; Haining, Alexander William M.; Robinson, Benjamin; Li, Danyang; Gautrot, Julien; Del Río Hernández, Armando

    2016-09-01

    Cells are known to respond to physical cues from their microenvironment such as matrix rigidity. Discrete adhesive ligands within flexible strands of fibronectin connect cell surface integrins to the broader extracellular matrix and are thought to mediate mechanosensing through the cytoskeleton-integrin-ECM linkage. We set out to determine if adhesive ligand tether length is another physical cue that cells can sense. Substrates were covalently modified with adhesive arginylglycylaspartic acid (RGD) ligands coupled with short (9.5 nm), medium (38.2 nm) and long (318 nm) length inert polyethylene glycol tethers. The size and length of focal adhesions of human foreskin fibroblasts gradually decreased from short to long tethers. Furthermore, we found cell adhesion varies in a linker length dependent manner with a remarkable 75% reduction in the density of cells on the surface and a 50% reduction in cell area between the shortest and longest linkers. We also report the interplay between RGD ligand concentration and tether length in determining cellular spread area. Our findings show that without varying substrate rigidity or ligand density, tether length alone can modulate cellular behaviour.

  7. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions.

    PubMed

    Walkiewicz, Katarzyna W; Girault, Jean-Antoine; Arold, Stefan T

    2015-10-01

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein 'nanomachines' to become activated in a site-specific manner.

  8. Structural Insight into the Role of Thrombospondin-1 Binding to Calreticulin in Calreticulin-Induced Focal Adhesion Disassembly†

    PubMed Central

    Yan, Qi; Murphy-Ullrich, Joanne E.; Song, Yuhua

    2010-01-01

    Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface stimulates association of CRT with LDL receptor-related protein (LRP1) to signal focal adhesion disassembly and engagement of cellular activities. The structural basis for this phenomenon is unknown. We studied the binding thermodynamics of the TSP1–CRT complex and the conformational changes in CRT induced by binding to TSP1 with combined binding free energy analysis, molecular dynamics simulation, and anisotropic network model restrained molecular dynamics simulation. Results showed that mutations of Lys 24 and Lys 32 in TSP1 to Ala and of amino acids 24–26 and 32–34 in CRT to Ala significantly weakened the binding of TSP1 and CRT, which is consistent with experimental results. Upon validation of the calculated binding affinity changes of the TSP1–CRT complex by mutations in key residues in TSP1 and CRT with the experimental results, we performed conformational analyses to understand the role of TSP1 binding to CRT in the induction of conformational changes in CRT. Conformational analyses showed that TSP1 binding to CRT resulted in a more “open” conformation and a significant rotational change for the CRT N-domain with respect to the CRT P-domain, which could expose the potential binding site(s) in CRT for binding to LRP1 to signal focal adhesion disassembly. Results offer structural insight into the role of TSP1 binding to CRT in CRT-induced focal adhesion disassembly. PMID:20337411

  9. 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.

  10. 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

  11. Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity

    PubMed Central

    1994-01-01

    The molecular mechanisms whereby hyaluronan (HA) stimulates cell motility was investigated in a C-H-ras transformed 10T 1/2 fibroblast cell line (C3). A significant (p < 0.001) stimulation of C3 cell motility with HA (10 ng/ml) was accompanied by an increase in protein tyrosine phosphorylation as detected by anti-phosphotyrosine antibodies using immunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and transient with phosphorylation occurring within 1 min of HA addition and dissipating below control levels 10-15 min later. These responses were also elicited by an antibody generated against a peptide sequence within the HA receptor RHAMM. Treatment of cells with tyrosine kinase inhibitors (genistein, 10 micrograms/ml or herbimycin A, 0.5 micrograms/ml) or microinjection of anti-phosphotyrosine antibodies inhibited the transient protein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimulated tyrosine phosphorylation and the resulting cell locomotion, cytoskeletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapid assembly and disassembly of focal adhesions as revealed by immunofluorescent localization of vinculin. The time course with which HA and antibody induced focal adhesion turnover exactly paralleled the induction of transient protein tyrosine phosphorylation. In addition, phosphotyrosine staining colocalized with vinculin within structures in the lamellapodia of these cells. Notably, the focal adhesion kinase, pp125FAK, was rapidly phosphorylated and dephosphorylated after HA stimulation. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAMM. They also provide a possible molecular basis for focal adhesion turnover, a process that is

  12. Insights into the Utility of the Focal Adhesion Scaffolding Proteins in the Anaerobic Fungus Orpinomyces sp. C1A

    PubMed Central

    Calkins, Shelby; Youssef, Noha H.

    2016-01-01

    Focal adhesions (FAs) are large eukaryotic multiprotein complexes that are present in all metazoan cells and function as stable sites of tight adhesion between the extracellular matrix (ECM) and the cell’s cytoskeleton. FAs consist of anchor membrane protein (integrins), scaffolding proteins (e.g. α-actinin, talin, paxillin, and vinculin), signaling proteins of the IPP complex (e.g. integrin-linked kinase, α-parvin, and PINCH), and signaling kinases (e.g. focal adhesion kinase (FAK) and Src kinase). While genes encoding complete focal adhesion machineries are present in genomes of all multicellular Metazoa; incomplete machineries were identified in the genomes of multiple non-metazoan unicellular Holozoa, basal fungal lineages, and amoebozoan representatives. Since a complete FA machinery is required for functioning, the putative role, if any, of these incomplete FA machineries is currently unclear. We sought to examine the expression patterns of FA-associated genes in the anaerobic basal fungal isolate Orpinomyces sp. strain C1A under different growth conditions and at different developmental stages. Strain C1A lacks clear homologues of integrin, and the two signaling kinases FAK and Src, but encodes for all scaffolding proteins, and the IPP complex proteins. We developed a protocol for synchronizing growth of C1A cultures, allowing for the collection and mRNA extraction from flagellated spores, encysted germinating spores, active zoosporangia, and late inactive sporangia of strain C1A. We demonstrate that the genes encoding the FA scaffolding proteins α-actinin, talin, paxillin, and vinculin are indeed transcribed under all growth conditions, and at all developmental stages of growth. Further, analysis of the observed transcriptional patterns suggests the putative involvement of these components in alternative non-adhesion-specific functions, such as hyphal tip growth during germination and flagellar assembly during zoosporogenesis. Based on these results

  13. Plakophilin 2 Affects Cell Migration by Modulating Focal Adhesion Dynamics and Integrin Protein Expression

    PubMed Central

    Koetsier, Jennifer L.; Amargo, Evangeline V.; Todorović, Viktor; Green, Kathleen J.; Godsel, Lisa M.

    2014-01-01

    Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cell–cell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ~30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ~2× larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, β4 and β1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing β1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of β1 integrin and RhoA in integrating cell–cell and cell–substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis. PMID:23884246

  14. Redistribution of microfilament-associated proteins during the formation of focal contacts and adhesions in chick fibroblasts.

    PubMed

    Couchman, J R; Badley, R A; Rees, D A

    1983-12-01

    The roles of the microfilament-associated proteins vinculin, alpha-actinin, myosin and filamin have been studied by immunofluorescence and double fluorescence in conjunction with interference reflection microscopy (IRM), during the development of focal contacts and focal adhesions in a chick fibroblast system which initially has no such adhesion specializations but then develops them sequentially over a 48 h period. Without exception, all focal contacts and focal adhesions contain both vinculin and alpha-actinin at every stage that we can detect by IRM or by double staining to reveal the associated microfilament bundles. Indeed the appearance of small bodies containing alpha-actinin and vinculin is shown to precede focal contact formation in our model system and such structures (not visible by IRM) are proposed to be the precursors of focal contacts and adhesions. Myosin and filamin are distributed generally with some reticular patterning in the early motile cells which lack the focal specializations, but as focal contacts and adhesions form these proteins become progressively recruited into the associated microfilament bundles. Only then do we see the marked depletion that has been reported earlier of diffusely distributed myosin and filamin in the leading lamella. Although this is not initially associated with any change in the motile status of the cells, the recruitment of these microfilament-associated proteins into stress fibres is proposed to occur in preparation for anchorage and bracing of cells to the substratum when they later become stationary. PMID:6421873

  15. 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

  16. 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

  17. 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

  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. 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

  20. Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer.

    PubMed

    Seong, Jihye; Ouyang, Mingxing; Kim, Taejin; Sun, Jie; Wen, Po-Chao; Lu, Shaoying; Zhuo, Yue; Llewellyn, Nicholas M; Schlaepfer, David D; Guan, Jun-Lin; Chien, Shu; Wang, Yingxiao

    2011-07-26

    Proper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane. Here we show that, on cell adhesion to extracellular matrix proteins or stimulation by platelet-derived growth factor (PDGF), the FRET responses of DRM-targeting FAK biosensor are stronger than that at non-DRM regions, suggesting that FAK activation can occur at DRM microdomains. Further experiments reveal that the PDGF-induced FAK activation is mediated and maintained by Src activity, whereas FAK activation on cell adhesion is independent of, and in fact essential for the Src activation. Therefore, FAK is activated at membrane microdomains with distinct activation mechanisms in response to different physiological stimuli.

  1. 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

  2. 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

  3. Liprin-α1 and ERC1 control cell edge dynamics by promoting focal adhesion turnover

    PubMed Central

    Astro, Veronica; Tonoli, Diletta; Chiaretti, Sara; Badanai, Sabrina; Sala, Kristyna; Zerial, Marino; de Curtis, Ivan

    2016-01-01

    Liprin-α1 and ERC1 are interacting scaffold proteins regulating the motility of normal and tumor cells. They act as part of plasma membrane-associated platforms at the edge of motile cells to promote protrusion by largely unknown mechanisms. Here we identify an amino-terminal region of the liprin-α1 protein (liprin-N) that is sufficient and necessary for the interaction with other liprin-α1 molecules. Similar to liprin-α1 or ERC1 silencing, expression of the liprin-N negatively affects tumor cell motility and extracellular matrix invasion, acting as a dominant negative by interacting with endogenous liprin-α1 and causing the displacement of the endogenous ERC1 protein from the cell edge. Interfering with the localization of ERC1 at the cell edge inhibits the disassembly of focal adhesions, impairing protrusion. Liprin-α1 and ERC1 proteins colocalize with active integrin β1 clusters distinct from those colocalizing with cytoplasmic focal adhesion proteins, and influence the localization of peripheral Rab7-positive endosomes. We propose that liprin-α1 and ERC1 promote protrusion by displacing cytoplasmic adhesion components to favour active integrin internalization into Rab7-positive endosomes. PMID:27659488

  4. 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

  5. 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

  6. 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

  7. Antitumour effects of Yangzheng Xiaoji in human osteosarcoma: the pivotal role of focal adhesion kinase signalling.

    PubMed

    Jiang, Wen G; Ye, Lin; Ji, Ke; Ruge, Fiona; Wu, Yiling; Gao, Yong; Ji, Jiafu; Mason, Malcolm D

    2013-09-01

    The present study examined, in vitro and in vivo, the potential antitumour effects of Yangzheng Xiaoji (YZXJ), a traditional Chinese medical formula used in cancer treatment, on osteosarcoma, a tumour type recently found to be sensitive to YZXJ. The human osteosarcoma cell line MG63 was used in cell-matrix adhesion and cell growth assays. The same cell line was used in an in vivo tumour model by establishing subcutaneous osteosarcoma xenografts. Oral and intraperitoneal routes were used to deliver the YZXJ extract. The effect of YZXJ on the activation of focal adhesion kinase (FAK) and paxillin was evaluated by immunofluorescence methods. It was found that YZXJ exhibited a significant inhibitory effect on cell-matrix adhesion as demonstrated by a cell-based assay and electric cell-substrate impedance sensing (ECIS) analysis. The effect was observed together with a reduction in phospho-FAK and phospho-paxillin in the cells when treated with YZXJ. In the in vivo tumour model, YZXJ was found to significantly inhibit the growth of osteosarcoma with a sustained effect observed when YZXJ was delivered intraperitoneally. YZXJ sensitized cells to the effect of FAK inhibitor in vitro and in vivo. It is concluded that Yangzheng Xiaoji plays a significant role in cell-matrix adhesion and tumour growth, likely by inhibiting the activation of the FAK pathway. The therapeutic role of Yangzheng Xiaoji in osteosarcoma warrants further investigation. PMID:23828123

  8. Focal Cortical Dysplasia (FCD) lesion analysis with complex diffusion approach.

    PubMed

    Rajan, Jeny; Kannan, K; Kesavadas, C; Thomas, Bejoy

    2009-10-01

    Identification of Focal Cortical Dysplasia (FCD) can be difficult due to the subtle MRI changes. Though sequences like FLAIR (fluid attenuated inversion recovery) can detect a large majority of these lesions, there are smaller lesions without signal changes that can easily go unnoticed by the naked eye. The aim of this study is to improve the visibility of focal cortical dysplasia lesions in the T1 weighted brain MRI images. In the proposed method, we used a complex diffusion based approach for calculating the FCD affected areas. Based on the diffused image and thickness map, a complex map is created. From this complex map; FCD areas can be easily identified. MRI brains of 48 subjects selected by neuroradiologists were given to computer scientists who developed the complex map for identifying the cortical dysplasia. The scientists were blinded to the MRI interpretation result of the neuroradiologist. The FCD could be identified in all the patients in whom surgery was done, however three patients had false positive lesions. More lesions were identified in patients in whom surgery was not performed and lesions were seen in few of the controls. These were considered as false positive. This computer aided detection technique using complex diffusion approach can help detect focal cortical dysplasia in patients with epilepsy. PMID:19560319

  9. PEGylated human plasma fibronectin is proteolytically stable, supports cell adhesion, cell migration, focal adhesion assembly, and fibronectin fibrillogenesis.

    PubMed

    Zhang, Chen; Hekmatfar, Sogol; Ramanathan, Anand; Karuri, Nancy W

    2013-01-01

    Delayed wound healing in many chronic wounds has been linked to the degradation of fibronectin (FN) by abnormally high protease levels. We sought to develop a proteolytically stable and functionally active form of FN. For this purpose, we conjugated 3.35 kDa polyethylene glycol diacrylate (PEGDA) to human plasma fibronectin (HPFN). Conjugation of PEGDA to HPFN or HPFN PEGylation was characterized by an increase of approximately 16 kDa in the average molecular weight of PEGylated HPFN compared to native HPFN in SDS-PAGE gels. PEGylated HPFN was more resistant to α chymotrypsin or neutrophil elastase digestion than native HPFN: after 30 min incubation with α chymotrypsin, 56 and 90% of native and PEGylated HPFN respectively remained intact. PEGylated HPFN and native HPFN supported NIH 3T3 mouse fibroblast adhesion and spreading, migration and focal adhesion formation in a similar manner. Fluorescence microscopy showed that both native and PEGylated HPFN in the culture media were assembled into extracellular matrix (ECM) fibrils. Interestingly, when coated on surfaces, native but not PEGylated HPFN was assembled into the ECM of fibroblasts. The proteolytically stable PEGylated HPFN developed herein could be used to replenish FN levels in the chronic wound bed and promote tissue repair.

  10. Septins promote stress fiber–mediated maturation of focal adhesions and renal epithelial motility

    PubMed Central

    Dolat, Lee; Hunyara, John L.; Bowen, Jonathan R.; Karasmanis, Eva Pauline; Elgawly, Maha; Galkin, Vitold E.

    2014-01-01

    Organogenesis and tumor metastasis involve the transformation of epithelia to highly motile mesenchymal-like cells. Septins are filamentous G proteins, which are overexpressed in metastatic carcinomas, but their functions in epithelial motility are unknown. Here, we show that a novel network of septin filaments underlies the organization of the transverse arc and radial (dorsal) stress fibers at the leading lamella of migrating renal epithelia. Surprisingly, septin depletion resulted in smaller and more transient and peripheral focal adhesions. This phenotype was accompanied by a highly disorganized lamellar actin network and rescued by the actin bundling protein α-actinin-1. We show that preassembled actin filaments are cross-linked directly by Septin 9 (SEPT9), whose expression is increased after induction of renal epithelial motility with the hepatocyte growth factor. Significantly, SEPT9 overexpression enhanced renal cell migration in 2D and 3D matrices, whereas SEPT9 knockdown decreased migration. These results suggest that septins promote epithelial motility by reinforcing the cross-linking of lamellar stress fibers and the stability of nascent focal adhesions. PMID:25349260

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

    PubMed Central

    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

  12. Serine palmitoyltransferase subunit 1 is present in the endoplasmic reticulum, nucleus and focal adhesions, and functions in cell morphology.

    PubMed

    Wei, Jia; Yerokun, Tokunbo; Leipelt, Martina; Haynes, Chris A; Radhakrishna, Harish; Momin, Amin; Kelly, Samuel; Park, Hyejung; Wang, Elaine; Carton, Jill M; Uhlinger, David J; Merrill, Alfred H

    2009-08-01

    Serine palmitoyltransferase (SPT) has been localized to the endoplasmic reticulum (ER) by subcellular fractionation and enzymatic assays, and fluorescence microscopy of epitope-tagged SPT; however, our studies have suggested that SPT subunit 1 might be present also in focal adhesions and the nucleus. These additional locations have been confirmed by confocal microscopy using HEK293 and HeLa cells, and for focal adhesions by the demonstration that SPT1 co-immunoprecipitates with vinculin, a focal adhesion marker protein. The focal adhesion localization of SPT1 is associated with cell morphology, and possibly cell migration, because it is seen in most cells before they reach confluence but disappears when they become confluent, and is restored by a standard scratch-wound healing assay. Conversely, elimination of SPT1 using SPTLC1 siRNA causes cell rounding. Thus, in addition to its "traditional" localization in the ER for de novo sphingolipid biosynthesis, SPT1 is present in other cellular compartments, including focal adhesions where it is associated with cell morphology. PMID:19362163

  13. Allosteric Regulation of Focal Adhesion Kinase by PIP2 and ATP

    PubMed Central

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

    2015-01-01

    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. Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins.

    PubMed

    Seong, Jihye; Tajik, Arash; Sun, Jie; Guan, Jun-Lin; Humphries, Martin J; Craig, Susan E; Shekaran, Asha; García, Andrés J; Lu, Shaoying; Lin, Michael Z; Wang, Ning; Wang, Yingxiao

    2013-11-26

    Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

  15. Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoAD⃞

    PubMed Central

    Nelson, Celeste M.; Pirone, Dana M.; Tan, John L.; Chen, Christopher S.

    2004-01-01

    Changes in vascular endothelial (VE)-cadherin–mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin–mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells. PMID:15075376

  16. Different cytokeratin and neuronal cell adhesion molecule staining patterns in focal nodular hyperplasia and hepatic adenoma and their significance

    PubMed Central

    Iyer, Anita; Robert, Marie E.; Bifulco, Carlo B.; Salem, Ronald R.; Jain, Dhanpat

    2013-01-01

    Summary Differentiating focal nodular hyperplasia from hepatic adenoma can be challenging. Cytokeratin 7, neuronal cell adhesion molecule, and cytokeratin 19 are differentially expressed in hepatocytes, biliary epithelium, and possibly hepatic progenitor/stem cells. CD34 is known to have altered expression patterns in the hepatic endothelium in conditions associated with abnormal perfusion and in hepatocellular carcinoma. The purpose of this study was to examine the expression pattern of these markers in focal nodular hyperplasia and hepatic adenoma and assess their diagnostic use. Ten resection specimens each of hepatic adenoma and focal nodular hyperplasia (including a case of telangiectatic focal nodular hyperplasia) were selected for the study. Immunohistochemical analysis was performed using antibodies against cytokeratin 7, cytokeratin 19, neuronal cell adhesion molecule, and CD34 on formalin-fixed, paraffin-embedded sections from each case. The staining patterns and intensity for each marker were analyzed. In hepatic adenoma, the cytokeratin 7 stain revealed strong positivity in hepatocytes in patches, with a gradual decrease in the staining intensity as the cells differentiated towards mature hepatocytes. Although bile ducts were typically absent in hepatic adenoma, occasional ductules could be identified with cytokeratin 7 stain. In focal nodular hyperplasia, cytokeratin 7 showed strong staining of the biliary epithelium within the fibrous septa and staining of the peripheral hepatocytes of most lobules that was focal and weaker than hepatic adenoma. Cytokeratin 19 and neuronal cell adhesion molecule showed patchy and moderate staining in the biliary epithelium of the ductules in focal nodular hyperplasia. While in the hepatic adenoma, cytokeratin 19 showed only rare positivity in occasional cells within ductules, and neuronal cell adhesion molecule marked occasional isolated cells in the lesion. CD34 showed staining of sinusoids in the inflow areas

  17. 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

  18. Rab'ing tumor cell migration and invasion: focal adhesion disassembly driven by Rab5.

    PubMed

    Torres, Vicente A

    2014-01-01

    The small GTPase Rab5 has been extensively studied in the context of endocytic trafficking because it is critical in the regulation of early endosome dynamics. In addition to this canonical role, evidence obtained in recent years implicates Rab5 in the regulation of cell migration. This novel role of Rab5 is based not only on an indirect relationship between cell migration and endosomal trafficking as separate processes, but also on the direct regulation of signaling proteins implicated in cell migration. However, the precise mechanisms underlying this connection have remained elusive. Recent studies have shown that the activation of Rab5 is a critical event for maintaining the dynamics of focal adhesions, which is fundamental in regulating not only cell migration but also tumor cell invasion.

  19. 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

    2015-06-24

    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.

  20. 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

  1. Identification of a new actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties

    PubMed Central

    Thompson, Peter M.; Tolbert, Caitlin E.; Shen, Kai; Kota, Pradeep; Palmer, Sean M.; Plevock, Karen M.; Orlova, Albina; Galkin, Vitold E.; Burridge, Keith; Egelman, Edward H.; Dokholyan, Nikolay V.; Superfine, Richard; Campbell, Sharon L.

    2014-01-01

    SUMMARY Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin, and developed an alternative model to better define this novel actin-binding surface, using negative-stain EM, discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this new actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes which promote actin bundling. PMID:24685146

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Performance characterization of a PIAA complex focal plane mask

    NASA Astrophysics Data System (ADS)

    Newman, Kevin; Belikov, Ruslan; Guyon, Olivier; Pluzhnik, Eugene

    2015-01-01

    The Phase Induced Amplitude Apodization Complex Mask Coronagraph (PIAACMC) is an architecture for directly observing extrasolar planets, and can achieve performance near the theoretical limits for any direct-detection instrument. PIAACMC can be designed for centrally-obscured and segmented apertures, which is particularly useful for next-generation telescopes. The PIAACMC architecture includes aspheric PIAA optics, and a complex phase-shifting focal plane mask that provides a pi phase shift to a portion of the on-axis starlight. The phase-shifted starlight is forced to interfere destructively with the un-shifted starlight, causing the starlight to be eliminated, and allowing a region for high-contrast imaging near the star.The main challenge in designing the complex focal plane mask is to achieve deep contrast over a wide spectral band. Another challenge for the mask design is to avoid sharp features, which can be difficult to manufacture. We present a solution to the design challenge by dividing the mask into sections and optimizing the phase shift produced by each section. We also demonstrate a method to design the mask with a smooth profile. One remaining challenge is to measure the performance of the mask. We present a method to compute the phase profile of the mask based on measurements of the diffraction pattern. The computed phase profile is used to simulate the expected coronagraph performance.

  8. Nuclear Signaling from Cadherin Adhesion Complexes

    PubMed Central

    McCrea, Pierre D.; Maher, Meghan T.; Gottardi, Cara J.

    2015-01-01

    The arrival of multicellularity in evolution facilitated cell–cell signaling in conjunction with adhesion. As the ectodomains of cadherins interact with each other directly in trans (as well as in cis), spanning the plasma membrane and associating with multiple other entities, cadherins enable the transduction of “outside-in” or “inside-out” signals. We focus this review on signals that originate from the larger family of cadherins that are inwardly directed to the nucleus, and thus have roles in gene control or nuclear structure–function. The nature of cadherin complexes varies considerably depending on the type of cadherin and its context, and we will address some of these variables for classical cadherins versus other family members. Substantial but still fragmentary progress has been made in understanding the signaling mediators used by varied cadherin complexes to coordinate the state of cell–cell adhesion with gene expression. Evidence that cadherin intracellular binding partners also localize to the nucleus is a major point of interest. In some models, catenins show reduced binding to cadherin cytoplasmic tails favoring their engagement in gene control. When bound, cadherins may serve as stoichiometric competitors of nuclear signals. Cadherins also directly or indirectly affect numerous signaling pathways (e.g., Wnt, receptor tyrosine kinase, Hippo, NFκB, and JAK/STAT), enabling cell–cell contacts to touch upon multiple biological outcomes in embryonic development and tissue homeostasis. PMID:25733140

  9. Tissue factor pathway inhibitor (TFPI) interferes with endothelial cell migration by inhibition of both the Erk pathway and focal adhesion proteins.

    PubMed

    Provençal, Mathieu; Michaud, Marisol; Beaulieu, Edith; Ratel, David; Rivard, Georges-Etienne; Gingras, Denis; Béliveau, Richard

    2008-03-01

    Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that is mainly known for its inhibition of tissue factor-mediated coagulation. In addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. In this work we demonstrate that at concentrations within the physiological range, TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro. These effects were specific to endothelial cells since no inhibitory effect was observed on the migration of tumor (glioblastoma) cells. Inhibition of endothelial cell migration was correlated with a concomitant loss in cell adhesion, suggesting an alteration of focal adhesion complex integrity. Accordingly, we observed that TFPI inhibited the phosphorylation of focal adhesion kinase and paxillin, two key proteins involved in the scaffolding of these complexes, and that this effect was specific to endothelial cells. These results suggest that TFPI influences the angiogenic process via a non-haemostatic pathway, by downregulating the migratory mechanisms of endothelial cells. PMID:18327407

  10. Cross-Correlated Fluctuation Analysis Reveals Phosphorylation-Regulated Paxillin-FAK Complexes in Nascent Adhesions

    PubMed Central

    Choi, Colin K.; Zareno, Jessica; Digman, Michelle A.; Gratton, Enrico; Horwitz, Alan Rick

    2011-01-01

    We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions. PMID:21281572

  11. Diagnostic use of cytokeratins, CD34, and neuronal cell adhesion molecule staining in focal nodular hyperplasia and hepatic adenoma.

    PubMed

    Ahmad, Imran; Iyer, Anita; Marginean, Celia E; Yeh, Matthew M; Ferrell, Linda; Qin, Lihui; Bifulco, Carlo B; Jain, Dhanpat

    2009-05-01

    Cytokeratins 7 and 19 and neuronal cell adhesion molecule (CD56) are differentially expressed in the hepatocytes and biliary epithelium. CD34 is an endothelial marker that is expressed in hepatic sinusoids in conditions associated with altered vascular flow and neoplasms. Distinct staining patterns using these markers have been shown in resected specimens of focal nodular hyperplasia, telangiectatic focal nodular hyperplasia, and hepatic adenoma. The purpose of this study was to examine the diagnostic use of these markers in needle biopsies. Needle biopsies from focal nodular hyperplasia (n = 21), telangiectatic focal nodular hyperplasia (n = 2), and hepatic adenoma (n = 14) were included in the study. These cases represent typical examples of each entity that have been diagnosed on the basis of clinical, imaging, and histologic features. Corresponding resection specimens available in 9 cases were also included in the study for comparison. Immunohistochemical analysis was performed on 4-mum-thick formalin-fixed and paraffin-embedded sections using antibodies against cytokeratin 7, cytokeratin 19, neuronal cell adhesion molecule, and CD34. The staining patterns and intensity for each marker were analyzed in a blinded fashion, and the patterns were recorded as focal nodular hyperplasia-like, hepatic adenoma-like, or indeterminate for each case. Presence of normal tissue was also recorded in each case. The hepatic adenoma-like pattern is characterized by strong cytokeratin 7 positivity in hepatocytes in patches with a gradual decrease in the staining intensity as the cells differentiate toward mature hepatocytes. Hepatic adenomas lack bile ducts and ductules as highlighted by cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule stains. The focal nodular hyperplasia-like pattern is characterized by milder and focal cytokeratin 7 staining of hepatocytes. Cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule show a strong staining of bile

  12. The Regulation of RhoA at Focal Adhesions by StarD13 is Important for Astrocytoma Cell Motility

    PubMed Central

    Khalil, Bassem D.; Hanna, Samer; Saykali, Bechara A.; El-Sitt, Sally; Nasrallah, Anita; Marston, Daniel; El-Sabban, Marwan; Hahn, Klaus M.; Symons, Marc; El-Sibai, Mirvat

    2015-01-01

    Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Rho GTPases are small monomeric G proteins that play important roles in cytoskeleton rearrangement, cell motility, and tumor invasion. In the present study, we show that the knock down of StarD13, a GTPase activating protein (GAP) for RhoA and Cdc42, inhibits astrocytoma cell migration through modulating focal adhesion dynamics and cell adhesion. This effect is mediated by the resulting constitutive activation of RhoA and the subsequent indirect inhibition of Rac. Using Total Internal Reflection Fluorescence (TIRF)-based Förster Resonance Energy Transfer (FRET), we show that RhoA activity localizes with focal adhesions at the basal surface of astrocytoma cells. Moreover, the knock down of StarD13 inhibits the cycling of RhoA activation at the rear edge of cells, which makes them defective in retracting their tail. This study highlights the importance of the regulation of RhoA activity in focal adhesions of astrocytoma cells and establishes StarD13 as a GAP playing a major role in this process. PMID:24333506

  13. 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

  14. 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

  15. Photonic force microscopy of local tension at cell surface focal adhesions

    NASA Astrophysics Data System (ADS)

    Bordeleau, François; Bessard, Judicaël; Marceau, Normand; Sheng, Yunlong

    2006-09-01

    The ability of cells to sustain mechanical stress is essential. It is however not very well understood how tension is expressed from the inside of the cell to the exterior. Here we show that these forces can be measured by photonic force microscopy (PFM), which is able to apply a force to cells and to probe their response to the physical stress. Our setup consists of an inverted microscope coupled with an optical trap from a 5W ND:YVO 4 fiber laser. Forces are applied to the cell by the trap through a 5μm polystyrene bead coated with fibronectin, pre-incubated with cells to allow bead attachment and creation of focal adhesions. The reaction of the cell is monitored by sensing the position of the bead relative to the trap center by a quadrant photodiode, when the bead is in an equilibrium state between the photonics forces and the membrane elasticity and cell stiffness. The detection system is calibrated using a piezo nano-positioner and thermal noise analysis. We observed increased deformation of H4 cells treated with cytocholasin D, which disrupts the actin microfilaments. This observation is correlated to an overall decrease in the force by the photonics force microscope. Our results suggest that cell stiffness can be assessed by the PFM, which allows quantification of a tension within cells with sufficient precision.

  16. 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

  17. 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

  18. Phosphorylation of Focal Adhesion Kinase at Tyr397 in Gastric Carcinomas and its Clinical Significance

    PubMed Central

    Lai, I-Rue; Chu, Pei-Yu; Lin, Hsiao-Sheng; Liou, Jun-Yang; Jan, Yee-Jee; Lee, Jen-Chieh; Shen, Tang-Long

    2010-01-01

    Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various cancers; however, it remains unclear how FAK participates in tumor malignancy in vivo. This study seeks to understand the role of FAK activation in gastric cancer progression. Using immunohistochemical staining and Western blotting, we found that pY397 FAK, an autophosphorylation site on FAK activation, was abundant in the cancerous tissues of 21 of 59 patients with gastric carcinomas. We attempted to correlate clinicopathological parameters, including histological types, TNM staging, and cancer recurrence, with the expression of FAK and pY397 FAK in cancerous tissues. Intriguingly, patients with higher levels of pY397 FAK displayed higher incidences of gastric cancer recurrence after surgery and poor 5-year recurrence-free survival. Furthermore, multivariate analyses showed that pY397 FAK was an independent predictor of gastric cancer recurrence. As a result, expression of pY397 FAK is a significant prognostic factor for the recurrence of gastric cancer. Additionally, in vitro studies showed that overexpression of Y397F, a dominant-negative mutant of FAK, in AGS human gastric carcinoma cells impaired cell migration, invasion, and proliferation compared with cells overexpressing wild-type FAK. Thus, activation of FAK through autophosphorylation at Tyr397 leads to the progression of gastric carcinomas by promoting cell migration, invasion, and proliferation. Collectively, our results have provided valuable insights for the development of novel diagnoses and therapeutic targets for gastric cancer treatments. PMID:20724588

  19. 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

  20. 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

  1. Heat stress activates AKT via focal adhesion kinase-mediated pathway in neonatal rat ventricular myocytes.

    PubMed

    Wei, Hongguang; Vander Heide, Richard S

    2008-08-01

    Heat stress (HS)-induced cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). The purpose of this study was 1) to examine the subcellular signaling pathways activated by HS; 2) to determine whether myocardial stress organizes and activates an integrated survival pathway; and 3) to investigate potential downstream cytoprotective proteins activated by HS. After HS, NRVM were subjected to chemical inhibitors (CI) designed to simulate ischemia by inhibiting both glycolysis and mitochondrial respiration. Protein kinase B (AKT) expression (wild type) was increased selectively with an adenoviral vector. Cell signaling was analyzed with Western blot analysis, while oncosis/apoptosis was assayed by measuring Trypan blue exclusion and/or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. HS increased phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 but did not adversely affect the viability of NRVM before CI. HS increased association between FAK and phosphatidylinositol 3-kinase as well as causing a significant increase in AKT activity. Increased expression of wild-type AKT protected myocytes from both oncotic and apoptotic cell death. Increased expression of a FAK inhibitor, FRNK, reduced AKT phosphorylation in response to HS both at time 0 and after 10 min of CI compared with myocytes expressing empty virus. We conclude that myocardial stress activates cytoskeleton-based signaling pathways that are associated with protection from lethal cell injury.

  2. Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes.

    PubMed Central

    Manser, E; Huang, H Y; Loo, T H; Chen, X Q; Dong, J M; Leung, T; Lim, L

    1997-01-01

    The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mol. Cell. Biol. 16:1896-1908, 1996), where it colocalizes with structures resembling focal complexes. We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes. PMID:9032240

  3. 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

  4. Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

    PubMed Central

    Xing, Z; Chen, H C; Nowlen, J K; Taylor, S J; Shalloway, D; Guan, J L

    1994-01-01

    The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules. Images PMID:8054685

  5. Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration

    PubMed Central

    Zhang, Yijun; Luo, Youguang; Lyu, Rui; Chen, Jie; Liu, Ruming; Li, Dengwen; Liu, Min; Zhou, Jun

    2016-01-01

    Cell migration, a complex process critical for tumor progression and metastasis, requires a dynamic crosstalk between microtubules (MTs) and focal adhesions (FAs). However, the molecular mechanisms underlying this event remain elusive. Herein we identify the proto-oncogenic protein Src as an important player in the regulation of the MT-FA crosstalk. Src interacts with and phosphorylates end-binding protein 1 (EB1), a member of MT plus end-tracking proteins (+TIPs), both in cells and in vitro. Systematic mutagenesis reveals that tyrosine-247 (Y247) is the primary residue of EB1 phosphorylated by Src. Interestingly, both constitutively activated Src and Y247-phosphorylated EB1 localize to the centrosome and FAs. Src-mediated EB1 phosphorylation diminishes its interactions with other +TIPs, including adenomatous polyposis coli (APC) and mitotic centromere associated kinesin (MCAK). In addition, EB1 phosphorylation at Y247 enhances the rate of MT catastrophe and significantly stimulates cell migration. These findings thus demonstrate that the Src-EB1 axis plays a crucial role in regulating the crosstalk between MTs and FAs to promote cell migration.

  6. Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration

    PubMed Central

    Zhang, Yijun; Luo, Youguang; Lyu, Rui; Chen, Jie; Liu, Ruming; Li, Dengwen; Liu, Min; Zhou, Jun

    2016-01-01

    Cell migration, a complex process critical for tumor progression and metastasis, requires a dynamic crosstalk between microtubules (MTs) and focal adhesions (FAs). However, the molecular mechanisms underlying this event remain elusive. Herein we identify the proto-oncogenic protein Src as an important player in the regulation of the MT-FA crosstalk. Src interacts with and phosphorylates end-binding protein 1 (EB1), a member of MT plus end-tracking proteins (+TIPs), both in cells and in vitro. Systematic mutagenesis reveals that tyrosine-247 (Y247) is the primary residue of EB1 phosphorylated by Src. Interestingly, both constitutively activated Src and Y247-phosphorylated EB1 localize to the centrosome and FAs. Src-mediated EB1 phosphorylation diminishes its interactions with other +TIPs, including adenomatous polyposis coli (APC) and mitotic centromere associated kinesin (MCAK). In addition, EB1 phosphorylation at Y247 enhances the rate of MT catastrophe and significantly stimulates cell migration. These findings thus demonstrate that the Src-EB1 axis plays a crucial role in regulating the crosstalk between MTs and FAs to promote cell migration. PMID:27698945

  7. 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.

  8. High-performance mussel-inspired adhesives of reduced complexity

    PubMed Central

    Ahn, B. Kollbe; Das, Saurabh; Linstadt, Roscoe; Kaufman, Yair; Martinez-Rodriguez, Nadine R.; Mirshafian, Razieh; Kesselman, Ellina; Talmon, Yeshayahu; Lipshutz, Bruce H.; Israelachvili, Jacob N.; Waite, J. Herbert

    2015-01-01

    Despite the recent progress in and demand for wet adhesives, practical underwater adhesion remains limited or non-existent for diverse applications. Translation of mussel-inspired wet adhesion typically entails catechol functionalization of polymers and/or polyelectrolytes, and solution processing of many complex components and steps that require optimization and stabilization. Here we reduced the complexity of a wet adhesive primer to synthetic low-molecular-weight catecholic zwitterionic surfactants that show very strong adhesion (∼50 mJ m−2) and retain the ability to coacervate. This catecholic zwitterion adheres to diverse surfaces and self-assembles into a molecularly smooth, thin (<4 nm) and strong glue layer. The catecholic zwitterion holds particular promise as an adhesive for nanofabrication. This study significantly simplifies bio-inspired themes for wet adhesion by combining catechol with hydrophobic and electrostatic functional groups in a small molecule. PMID:26478273

  9. High-performance mussel-inspired adhesives of reduced complexity

    NASA Astrophysics Data System (ADS)

    Ahn, B. Kollbe; Das, Saurabh; Linstadt, Roscoe; Kaufman, Yair; Martinez-Rodriguez, Nadine R.; Mirshafian, Razieh; Kesselman, Ellina; Talmon, Yeshayahu; Lipshutz, Bruce H.; Israelachvili, Jacob N.; Waite, J. Herbert

    2015-10-01

    Despite the recent progress in and demand for wet adhesives, practical underwater adhesion remains limited or non-existent for diverse applications. Translation of mussel-inspired wet adhesion typically entails catechol functionalization of polymers and/or polyelectrolytes, and solution processing of many complex components and steps that require optimization and stabilization. Here we reduced the complexity of a wet adhesive primer to synthetic low-molecular-weight catecholic zwitterionic surfactants that show very strong adhesion (~50 mJ m-2) and retain the ability to coacervate. This catecholic zwitterion adheres to diverse surfaces and self-assembles into a molecularly smooth, thin (<4 nm) and strong glue layer. The catecholic zwitterion holds particular promise as an adhesive for nanofabrication. This study significantly simplifies bio-inspired themes for wet adhesion by combining catechol with hydrophobic and electrostatic functional groups in a small molecule.

  10. High-performance mussel-inspired adhesives of reduced complexity.

    PubMed

    Ahn, B Kollbe; Das, Saurabh; Linstadt, Roscoe; Kaufman, Yair; Martinez-Rodriguez, Nadine R; Mirshafian, Razieh; Kesselman, Ellina; Talmon, Yeshayahu; Lipshutz, Bruce H; Israelachvili, Jacob N; Waite, J Herbert

    2015-10-19

    Despite the recent progress in and demand for wet adhesives, practical underwater adhesion remains limited or non-existent for diverse applications. Translation of mussel-inspired wet adhesion typically entails catechol functionalization of polymers and/or polyelectrolytes, and solution processing of many complex components and steps that require optimization and stabilization. Here we reduced the complexity of a wet adhesive primer to synthetic low-molecular-weight catecholic zwitterionic surfactants that show very strong adhesion (∼50 mJ m(-2)) and retain the ability to coacervate. This catecholic zwitterion adheres to diverse surfaces and self-assembles into a molecularly smooth, thin (<4 nm) and strong glue layer. The catecholic zwitterion holds particular promise as an adhesive for nanofabrication. This study significantly simplifies bio-inspired themes for wet adhesion by combining catechol with hydrophobic and electrostatic functional groups in a small molecule.

  11. 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.

  12. Focal adhesion kinase antagonizes doxorubicin cardiotoxicity via p21(Cip1.).

    PubMed

    Cheng, Zhaokang; DiMichele, Laura A; Rojas, Mauricio; Vaziri, Cyrus; Mack, Christopher P; Taylor, Joan M

    2014-02-01

    Clinical application of potent anthracycline anticancer drugs, especially doxorubicin (DOX), is limited by a toxic cardiac side effect that is not fully understood and preventive strategies are yet to be established. Studies in genetically modified mice have demonstrated that focal adhesion kinase (FAK) plays a key role in regulating adaptive responses of the adult myocardium to pathological stimuli through activation of intracellular signaling cascades that facilitate cardiomyocyte growth and survival. The objective of this study was to determine if targeted myocardial FAK activation could protect the heart from DOX-induced de-compensation and to characterize the underlying mechanisms. To this end, mice with myocyte-restricted FAK knock-out (MFKO) or myocyte-specific expression of an active FAK variant (termed SuperFAK) were subjected to DOX treatment. FAK depletion enhanced susceptibility to DOX-induced myocyte apoptosis and cardiac dysfunction, while elevated FAK activity provided remarkable cardioprotection. Our mec6hanistic studies reveal a heretofore unappreciated role for the protective cyclin-dependent kinase inhibitor p21 in the repression of the pro-apoptotic BH3-only protein Bim and the maintenance of mitochondrial integrity and myocyte survival. DOX treatment induced proteasomal degradation of p21, which exacerbated mitochondrial dysfunction and cardiomyocyte apoptosis. FAK was both necessary and sufficient for maintaining p21 levels following DOX treatment and depletion of p21 compromised FAK-dependent protection from DOX. These findings identify p21 as a key determinant of DOX resistance downstream of FAK in cardiomyocytes and indicate that cardiac-restricted enhancement of the FAK/p21 signaling axis might be an effective strategy to preserve myocardial function in patients receiving anthracycline chemotherapy. PMID:24342076

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

    PubMed

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

    2015-12-01

    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.

  15. 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

  16. 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

  17. 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.

  18. Prognostic Value of Focal Adhesion Kinase (FAK) in Human Solid Carcinomas: A Meta-Analysis

    PubMed Central

    Ma, Li-Li; Tseng, Yu-Jen; Zhao, Nai-Qing; Chen, Shi-Yao

    2016-01-01

    Background Recently, the number of reports on focal adhesion kinase (FAK) as a vital therapeutic target in solid carcinomas has increased; however, the prognostic role of FAK status remains poorly understood. This study aims to evaluate the prognostic effect of FAK by means of a meta-analysis. Methods We performed a systematic literature search in order to examine the correlation between expression of FAK and overall survival(OS). The hazard ratio (HR) of OS was used to measure survival. A random-effects model was used to pool study statistics. Sensitivity and publication bias analyses were also conducted. Results Thirty eligible studies involving 4702 patients were included. The median expression rate of FAK was 54%. Meta-analysis of the HRs demonstrated that high FAK expression was associated with worse OS (average HR = 2.073, 95%confidence interval[CI]:1.712–2.510, p = 0.000). Regarding cancer type, FAK was associated with worse OS in gastric cancer (HR = 2.646,95% CI:1.743–4.017, p = 0.000), hepatocellular carcinoma (HR = 1.788,95% CI:1.228–2.602, p = 0.002), ovarian cancer (HR = 1.815, 95% CI: 1.193–2.762, p = 0.005), endometrial cancer (HR = 4.149, 95% CI:2.832–6.079, p = 0.000), gliomas (HR = 2.650, 95% CI: 1.205–5.829, p = 0.015), and squamous cell carcinoma (HR = 1,696, 95% CI: 1.030–2.793, p = 0.038). No association was found between HR and disease staging according to our meta-regression analysis. Conclusions Our study shows that high expression of FAK is associated with a worse OS in patients with carcinomas, but the association between FAK and prognosis varies according to cancer type. The value of FAK status in clinical prognosis in cancer needs further research. PMID:27637100

  19. Conditional Knockout of Myocyte Focal Adhesion Kinase Abrogates Ischemic Preconditioning in Adult Murine Hearts

    PubMed Central

    Perricone, Adam J.; Bivona, Benjamin J.; Jackson, Fannie R.; Vander Heide, Richard S.

    2013-01-01

    Background Our laboratory has previously demonstrated the importance of a cytoskeletal‐based survival signaling pathway using in vitro models of ischemia/reperfusion (IR). However, the importance of this pathway in mediating stress‐elicited survival signaling in vivo is unknown. Methods and Results The essential cytoskeletal signaling pathway member focal adhesion kinase (FAK) was selectively deleted in adult cardiac myocytes using a tamoxifen‐inducible Cre‐Lox system (α‐MHC‐MerCreMer). Polymerase chain reaction (PCR) and Western blot were performed to confirm FAK knockout (KO). All mice were subjected to a 40‐minute coronary occlusion followed by 24 hours of reperfusion. Ischemic preconditioning (IP) was performed using a standard protocol. Control groups included wild‐type (WT) and tamoxifen‐treated α‐MHC‐MerCreMer+/−/FAKWT/WT (experimental control) mice. Infarct size was expressed as a percentage of the risk region. In WT mice IP significantly enhanced the expression of activated/phosphorylated FAK by 36.3% compared to WT mice subjected to a sham experimental protocol (P≤0.05; n=6 hearts [sham], n=4 hearts [IP]). IP significantly reduced infarct size in both WT and experimental control mice (43.7% versus 19.8%; P≤0.001; 44.7% versus 17.5%; P≤0.001, respectively). No difference in infarct size was observed between preconditioned FAK KO and nonpreconditioned controls (37.1% versus 43.7% versus 44.7%; FAK KO versus WT versus experimental control; P=NS). IP elicited a 67.2%/88.8% increase in activated phosphatidylinositol‐3‐kinase (PI3K) p85/activated Akt expression in WT mice, but failed to enhance the expression of either in preconditioned FAK KO mice. Conclusions Our results indicate that FAK is an essential mediator of IP‐elicited cardioprotection and provide further support for the hypothesis that cytoskeletal‐based signaling is an important component of stress‐elicited survival signaling. PMID:24080910

  20. Focal adhesion kinase protein regulates Wnt3a gene expression to control cell fate specification in the developing neural plate

    PubMed Central

    Fonar, Yuri; Gutkovich, Yoni E.; Root, Heather; Malyarova, Anastasia; Aamar, Emil; Golubovskaya, Vita M.; Elias, Sarah; Elkouby, Yaniv M.; Frank, Dale

    2011-01-01

    Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase protein localized to regions called focal adhesions, which are contact points between cells and the extracellular matrix. FAK protein acts as a scaffold to transfer adhesion-dependent and growth factor signals into the cell. Increased FAK expression is linked to aggressive metastatic and invasive tumors. However, little is known about its normal embryonic function. FAK protein knockdown during early Xenopus laevis development anteriorizes the embryo. Morphant embryos express increased levels of anterior neural markers, with reciprocally reduced posterior neural marker expression. Posterior neural plate folding and convergence-extension is also inhibited. This anteriorized phenotype resembles that of embryos knocked down zygotically for canonical Wnt signaling. FAK and Wnt3a genes are both expressed in the neural plate, and Wnt3a expression is FAK dependent. Ectopic Wnt expression rescues this FAK morphant anteriorized phenotype. Wnt3a thus acts downstream of FAK to balance anterior–posterior cell fate specification in the developing neural plate. Wnt3a gene expression is also FAK dependent in human breast cancer cells, suggesting that this FAK–Wnt linkage is highly conserved. This unique observation connects the FAK- and Wnt-signaling pathways, both of which act to promote cancer when aberrantly activated in mammalian cells. PMID:21551070

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

    PubMed Central

    Zhang, Jianliang; Gao, Qile; Zhou, Ying; Dier, Usawadee; Hempel, Nadine; Hochwald, Steven N.

    2015-01-01

    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

  2. Direct observation of dynamic force propagation between focal adhesions of cells on microposts by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Okada, Akinori; Mizutani, Yusuke; Subagyo, Agus; Hosoi, Hirotaka; Nakamura, Motonori; Sueoka, Kazuhisa; Kawahara, Koichi; Okajima, Takaharu

    2011-12-01

    We investigated dynamic force propagation between focal adhesions of fibroblast cells cultured on polydimethylsiloxane micropost substrates, by atomic force microscopy. Live cells were mechanically modulated by the atomic force microscopy probe bound to cell apical surfaces at 0.01-0.5 Hz, while microposts served as a force sensor at basal surfaces. We observed that cells exhibited rheological behavior at the apical surface but had no apparent out-of-phase response at the basal surface, indicating that the dynamic force propagating through cytoskeletal filaments behaves in an elastic manner. Moreover, the direction of the propagated force was observed to be intimately associated with the prestress.

  3. The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

    PubMed Central

    Ellis, Stephanie J.; Lostchuck, Emily; Goult, Benjamin T.; Bouaouina, Mohamed; Fairchild, Michael J.; López-Ceballos, Pablo; Calderwood, David A.; Tanentzapf, Guy

    2014-01-01

    Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development. PMID:25393120

  4. Complex coacervates as a foundation for synthetic underwater adhesives

    PubMed Central

    Stewart, Russell J.; Wang, Ching Shuen; Shao, Hui

    2011-01-01

    Complex coacervation was proposed to play a role in the formation of the underwater bioadhesive of the Sandcastle worm (Phragmatopoma californica) based on the polyacidic and polybasic nature of the glue proteins and the balance of opposite charges at physiological pH. Morphological studies of the secretory system suggested the natural process does not involve complex coacervation as commonly defined. The distinction may not be important because electrostatic interactions likely play an important role in formation of the sandcastle glue. Complex coacervation has also been invoked in the formation of adhesive underwater silk fibers of caddisfly larvae and the adhesive plaques of mussels. A process similar to complex coacervation, that is, condensation and dehydration of biopolyelectrolytes through electrostatic associations, seems plausible for the caddisfly silk. This much is clear, the sandcastle glue complex coacervation model provided a valuable blueprint for the synthesis of a biomimetic, waterborne, underwater adhesive with demonstrated potential for repair of wet tissue. PMID:21081223

  5. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ

    PubMed Central

    Durand, Nisha; Bastea, Ligia I.; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-01-01

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs’ lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process. PMID:27775029

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Glycogen synthase kinase 3β dictates podocyte motility and focal adhesion turnover by modulating paxillin activity: implications for the protective effect of low-dose lithium in podocytopathy.

    PubMed

    Xu, Weiwei; Ge, Yan; Liu, Zhihong; Gong, Rujun

    2014-10-01

    Aberrant focal adhesion turnover is centrally involved in podocyte actin cytoskeleton disorganization and foot process effacement. The structural and dynamic integrity of focal adhesions is orchestrated by multiple cell signaling molecules, including glycogen synthase kinase 3β (GSK3β), a multitasking kinase lately identified as a mediator of kidney injury. However, the role of GSK3β in podocytopathy remains obscure. In doxorubicin (Adriamycin)-injured podocytes, lithium, a GSK3β inhibitor and neuroprotective mood stabilizer, obliterated the accelerated focal adhesion turnover, rectified podocyte hypermotility, and restored actin cytoskeleton integrity. Mechanistically, lithium counteracted the doxorubicin-elicited GSK3β overactivity and the hyperphosphorylation and overactivation of paxillin, a focal adhesion-associated adaptor protein. Moreover, forced expression of a dominant negative kinase dead mutant of GSK3β highly mimicked, whereas ectopic expression of a constitutively active GSK3β mutant abolished, the effect of lithium in doxorubicin-injured podocytes, suggesting that the effect of lithium is mediated, at least in part, through inhibition of GSK3β. Furthermore, paxillin interacted with GSK3β and served as its substrate. In mice with doxorubicin nephropathy, a single low dose of lithium ameliorated proteinuria and glomerulosclerosis. Consistently, lithium therapy abrogated GSK3β overactivity, blunted paxillin hyperphosphorylation, and reinstated actin cytoskeleton integrity in glomeruli associated with an early attenuation of podocyte foot process effacement. Thus, GSK3β-modulated focal adhesion dynamics might serve as a novel therapeutic target for podocytopathy.

  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. 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

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

    PubMed

    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.

  15. 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

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

    PubMed

    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

  17. 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.

  18. Protein-tyrosine phosphatase-alpha and Src functionally link focal adhesions to the endoplasmic reticulum to mediate interleukin-1-induced Ca2+ signaling.

    PubMed

    Wang, Qin; Rajshankar, Dhaarmini; Branch, Donald R; Siminovitch, Katherine A; Herrera Abreu, Maria Teresa; Downey, Gregory P; McCulloch, Christopher A

    2009-07-31

    Calcium (Ca2+) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) alpha in regulating IL-1-induced Ca2+ signaling in fibroblasts. IL-1 promoted recruitment of PTPalpha to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca2+ release channel IP3R. In response to IL-1, catalytically active PTPalpha was required for Ca2+ release from the ER, Src-dependent phosphorylation of IP3R1 and accumulation of IP3R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPalpha was required for the association of PTPalpha with IP3R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPalpha acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca2+ signaling.

  19. Protein-tyrosine Phosphatase-α and Src Functionally Link Focal Adhesions to the Endoplasmic Reticulum to Mediate Interleukin-1-induced Ca2+ Signaling*

    PubMed Central

    Wang, Qin; Rajshankar, Dhaarmini; Branch, Donald R.; Siminovitch, Katherine A.; Abreu, Maria Teresa Herrera; Downey, Gregory P.; McCulloch, Christopher A.

    2009-01-01

    Calcium (Ca2+) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) α in regulating IL-1-induced Ca2+ signaling in fibroblasts. IL-1 promoted recruitment of PTPα to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca2+ release channel IP3R. In response to IL-1, catalytically active PTPα was required for Ca2+ release from the ER, Src-dependent phosphorylation of IP3R1 and accumulation of IP3R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPα was required for the association of PTPα with IP3R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPα acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca2+ signaling. PMID:19497848

  20. Renewable Interfaces: Surface Topography Actuation for Complex Biological Adhesion Control

    NASA Astrophysics Data System (ADS)

    Pocivavsek, Luka; Ye, Sangho; Cao, Kathleen; Lee, Ka Yee C.; Velankar, Sachin; Wagner, William

    2015-03-01

    Controlling adhesion at biological interfaces is a complex problem with great biomedical importance. We use dynamic wrinkling, generated with PDMS/UVO chemistry under different macroscopic strains (ɛij ~ 0 . 3), to create a mechanical interfacial term that frustrates particle adhesion. This device actuates surface topography between flat (zero surface confinement χij) and wrinkled surfaces (χij ~(A / λ) 2 , where A and λ are wrinkle amplitude and wavelength, respectively), with a maximum rate of 0.6 Hz. Un-actuated PDMS placed in contact with whole sheep blood shows near total surface coverage with adhered platelets over 90 min. Actuation showed a nearly 100-fold decrease in platelet adhesion. Interestingly, topographic actuation is four times as effective compared to flat surface actuation in controlling platelet adhesion. Our model explores the competition between surface tension terms (Uγ = γɛij) and interfacial elastic terms (Uχ =Eij (t .ɛij2 +t3 . (χij /λ2)) generated because of actuation and wrinkling, where Eij is platelet modulus and t is characteristic platelet length scale. The condition for de-adhesion is Uχ >Uγ .

  1. Super-complexes of adhesion GPCRs and neural guidance receptors

    PubMed Central

    Jackson, Verity A.; Mehmood, Shahid; Chavent, Matthieu; Roversi, Pietro; Carrasquero, Maria; del Toro, Daniel; Seyit-Bremer, Goenuel; Ranaivoson, Fanomezana M.; Comoletti, Davide; Sansom, Mark S. P.; Robinson, Carol V.; Klein, Rüdiger; Seiradake, Elena

    2016-01-01

    Latrophilin adhesion-GPCRs (Lphn1–3 or ADGRL1–3) and Unc5 cell guidance receptors (Unc5A–D) interact with FLRT proteins (FLRT1–3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger ‘super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes. PMID:27091502

  2. Super-complexes of adhesion GPCRs and neural guidance receptors.

    PubMed

    Jackson, Verity A; Mehmood, Shahid; Chavent, Matthieu; Roversi, Pietro; Carrasquero, Maria; Del Toro, Daniel; Seyit-Bremer, Goenuel; Ranaivoson, Fanomezana M; Comoletti, Davide; Sansom, Mark S P; Robinson, Carol V; Klein, Rüdiger; Seiradake, Elena

    2016-01-01

    Latrophilin adhesion-GPCRs (Lphn1-3 or ADGRL1-3) and Unc5 cell guidance receptors (Unc5A-D) interact with FLRT proteins (FLRT1-3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger 'super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes. PMID:27091502

  3. 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.

  4. Celecoxib exhibits an anti-gastric cancer effect by targeting focal adhesion and leukocyte transendothelial migration-associated genes

    PubMed Central

    Jin, Guo-Hua; Xu, Wei; Shi, Yang; Wang, Li-Bo

    2016-01-01

    Gastric cancer (GC) is a prevalent cancer, which remains incurable, and therefore requires an alternative treatment method. Celecoxib is a nonsteroidal anti-inflammatory drug that targets cyclooxygenase-2, and exhibits anticancer effects. The present study aimed to investigate the anti-GC mechanism of celecoxib using bioinformatics methods. Gene expression datasets GSE56807 (GC tissues and normal gastric tissues) and GSE54657 (celecoxib-treated and non-treated human GC epithelial AGS cells) were downloaded from the Gene Expression Omnibus database. Two groups of differentially expressed genes (DEGs) were identified using limma package in R language. The criterion for GSE56807 was a false discovery rate of <0.05, while that for GSE54657 was P<0.01. Overlapping DEGs from the two datasets were screened out. Subsequently, pathway enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery software (P<0.1; gene count ≥2). In addition, the protein-protein interactions (PPIs) among the overlapped DEGs were obtained based on IntAct, Database of Interacting Proteins, Biomolecular Interaction Network Database and Human Protein Reference Database. Finally, a PPI network was visualized using Cytoscape software. A total of 137 overlapped DEGs were obtained, and DEGs with opposite regulation directions in the two datasets were significantly enriched in focal adhesion and leukocyte transendothelial migration. Subsequently, a PPI network of overlapped DEGs was constructed. Comprehensively, a total of 8 key DEGs [cysteine and glycine rich protein 1 (CSRP1), thrombospondin 1 (THBS1), myosin light chain 9 (MYL9), filamin A (FLNA), actinin alpha 1 (ACTN1), vinculin (VCL), laminin subunit gamma 2 (LAMC2) and claudin 1 (CLDN1)] were upregulated in GC tissues and downregulated in celecoxib-treated cells. In conclusion, celecoxib may exhibit anti-GC effects by suppressing the expression of CSRP1, THBS1, MYL9, FLNA, ACTN1, VCL, LAMC2 and CLDN1

  5. Celecoxib exhibits an anti-gastric cancer effect by targeting focal adhesion and leukocyte transendothelial migration-associated genes

    PubMed Central

    Jin, Guo-Hua; Xu, Wei; Shi, Yang; Wang, Li-Bo

    2016-01-01

    Gastric cancer (GC) is a prevalent cancer, which remains incurable, and therefore requires an alternative treatment method. Celecoxib is a nonsteroidal anti-inflammatory drug that targets cyclooxygenase-2, and exhibits anticancer effects. The present study aimed to investigate the anti-GC mechanism of celecoxib using bioinformatics methods. Gene expression datasets GSE56807 (GC tissues and normal gastric tissues) and GSE54657 (celecoxib-treated and non-treated human GC epithelial AGS cells) were downloaded from the Gene Expression Omnibus database. Two groups of differentially expressed genes (DEGs) were identified using limma package in R language. The criterion for GSE56807 was a false discovery rate of <0.05, while that for GSE54657 was P<0.01. Overlapping DEGs from the two datasets were screened out. Subsequently, pathway enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery software (P<0.1; gene count ≥2). In addition, the protein-protein interactions (PPIs) among the overlapped DEGs were obtained based on IntAct, Database of Interacting Proteins, Biomolecular Interaction Network Database and Human Protein Reference Database. Finally, a PPI network was visualized using Cytoscape software. A total of 137 overlapped DEGs were obtained, and DEGs with opposite regulation directions in the two datasets were significantly enriched in focal adhesion and leukocyte transendothelial migration. Subsequently, a PPI network of overlapped DEGs was constructed. Comprehensively, a total of 8 key DEGs [cysteine and glycine rich protein 1 (CSRP1), thrombospondin 1 (THBS1), myosin light chain 9 (MYL9), filamin A (FLNA), actinin alpha 1 (ACTN1), vinculin (VCL), laminin subunit gamma 2 (LAMC2) and claudin 1 (CLDN1)] were upregulated in GC tissues and downregulated in celecoxib-treated cells. In conclusion, celecoxib may exhibit anti-GC effects by suppressing the expression of CSRP1, THBS1, MYL9, FLNA, ACTN1, VCL, LAMC2 and CLDN1

  6. Effect of bioactive extruded PLA/HA composite films on focal adhesion formation of preosteoblastic cells.

    PubMed

    Persson, Maria; Lorite, Gabriela S; Kokkonen, Hanna E; Cho, Sung-Woo; Lehenkari, Petri P; Skrifvars, Mikael; Tuukkanen, Juha

    2014-09-01

    The quality of the initial cell attachment to a biomaterial will influence any further cell function, including spreading, proliferation, differentiation and viability. Cell attachment is influenced by the material's ability to adsorb proteins, which is related to the surface chemistry and topography of the material. In this study, we incorporated hydroxyapatite (HA) particles into a poly(lactic acid) (PLA) composite and evaluated the surface structure and the effects of HA density on the initial cell attachment in vitro of murine calvarial preosteoblasts (MC3T3-EI). Scanning electron microscopy (SEM), atomic force microscopy (AFM) and infrared spectroscopy (FTIR) showed that the HA particles were successfully incorporated into the PLA matrix and located at the surface which is of importance in order to maintain the bioactive effect of the HA particles. SEM and AFM investigation revealed that the HA density (particles/area) as well as surface roughness increased with HA loading concentration (i.e. 5, 10, 15 and 20wt%), which promoted protein adsorption. Furthermore, the presence of HA on the surface enhanced cell spreading, increased the formation of actin stress fibers and significantly improved the expression of vinculin in MC3T3-E1 cells which is a key player in the regulation of cell adhesion. These results suggest the potential utility of PLA/HA composites as biomaterials for use as a bone substitute material and in tissue engineering applications. PMID:24986753

  7. 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.

  8. 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

  9. Inhibition of osteopontin reduce the cardiac myofibrosis in dilated cardiomyopathy via focal adhesion kinase mediated signaling pathway

    PubMed Central

    Zhao, Hui; Wang, Wei; Zhang, Jie; Liang, Tuo; Fan, Guang-Pu; Wang, Zhi-Wei; Zhang, Pei-De; Wang, Xu; Zhang, Jing

    2016-01-01

    Background: Osteopontin (OPN) is a pleiotropic cytokine, which has been shown to a close relationship with cardiac fibrosis. Overexpression of OPN in cardiomyocytes induces dilated cardiomyopathy (DCM). This research is to study whether inhibition of OPN could reduce myocardial remodelling in DCM, and if this process is focal adhesion kinase (FAK) dependent, which is recently found an important signal molecule in fibrosis. Method: Eight-week-old cTnTR141W transgenic mouse of DCM were injected with OPN-shRNA in left ventricular free wall, which could inhibit the OPN expression. Six weeks later, echocardiographic examinations were performed to test left ventricle function and heart tissues were harvested to test the quality of FAK by western blot and severity of fibrosis by masson staining. Human cardiac fibroblast was administrated with OPN, and FAK inhibition by PP2 was treated 2 h before OPN was given. Expression of α-SMA and collagen-I were tested by western blot and real-time PCR assay. Results: OPN-shRNA group has a relatively high ejection fraction (EF), fractional shortening (FS), LV free wall thickness and a less sever cardiac fibrosis. In vitro, OPN could increase collagen-I and α-SMA expression, and this process can be inhibited by FAK inhibitor. Conclusion: Inhibition of OPN could reduce the LV remodeling and dysfunction in DCM mice, which may attribute to the suppression of collagen-I secretion in fibroblast through a FAK/Akt dependent pathway. PMID:27725847

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

    PubMed

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

    2009-07-15

    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 (approximately 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. PMID:19546491

  11. Exploring the interaction between human focal adhesion kinase and inhibitors: a molecular dynamic simulation and free energy calculations.

    PubMed

    Zhan, Jiu-Yu; Zhang, Ji-Long; Wang, Yan; Li, Ye; Zhang, Hong-Xing; Zheng, Qing-Chuan

    2016-11-01

    Focal adhesion kinase is an important target for the treatment of many kinds of cancers. Inhibitors of FAK are proposed to be the anticancer agents for multiple tumors. The interaction characteristic between FAK and its inhibitors is crucial to develop new inhibitors. In the present article, we used Molecular Dynamic (MD) simulation method to explore the characteristic of interaction between FAK and three inhibitors (PHM16, TAE226, and ligand3). The MD simulation results together with MM-GB/SA calculations show that the combinations are enthalpy-driven process. Cys502 and Asp564 are both essential residues due to the hydrogen bond interactions with inhibitors, which was in good agreement with experimental data. Glu500 can form a non-classical hydrogen bond with each inhibitor. Arg426 can form electrostatic interactions with PHM16 and ligand3, while weaker with TAE226. The electronic static potential was employed, and we found that the ortho-position methoxy of TAE226 has a weaker negative charge than the meta-position one in PHM16 or ligand3. Ile428, Val436, Ala452, Val484, Leu501, Glu505, Glu506, Leu553, Gly563 Leu567, Ser568 are all crucial residues in hydrophobic interactions. The key residues in this work will be available for further inhibitor design of FAK and also give assistance to further research of cancer.

  12. Roles for focal adhesion kinase (FAK) in blastomere abscission and vesicle trafficking during cleavage in the sea urchin embryo

    PubMed Central

    Schumpert, Brenda; García, María Guadalupe; Wessel, Gary M.; Wordeman, Linda; Hille, Merrill B.

    2014-01-01

    Is focal adhesion kinase (FAK) needed for embryonic cleavage? FAK is expressed during early cleavage divisions of sea urchin embryos as determined by polyclonal antibodies to the Lytechinus variegatus protein. FAK is absent in eggs and zygotes and then cycles in abundance during the first cleavages after fertilization, and is maximal at anaphase. Such cycling is consistent with the occurrence of a destruction box in the N-terminal sequence of L. variegatus FAK and the behavior of cyclins in sea urchin eggs. To investigate whether FAK is needed during early cleavage, we interfered with its function by microinjecting eggs with FAK antisense morpholino oligonucleotides or with anti-FAK antibodies. Both treatments led to regression of the cleavage furrow. FAK knockdown with morpholino oligonucleotides or antibodies also resulted in an over-accumulation of endocytic vesicles. Thus, FAK could be restricting endocytosis or increasing exocytosis in localized areas important for abscission. FAK appears to be necessary for successful cleavage. These results are the first to document a functional role for FAK during embryonic cleavage. PMID:23313141

  13. 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.

  14. Conditional deletion of the focal adhesion kinase FAK alters remodeling of the blood-brain barrier in glioma

    PubMed Central

    Lee, Jisook; Borboa, Alexandra; Chun, Hyun Bae; Baird, Andrew; Eliceiri, Brian

    2010-01-01

    Gliomas generally infiltrate the surrounding normal brain parenchyma, a process associated with increased vascular permeability (VP) and dysregulation of the blood-brain barrier (BBB). However, the molecular mechanisms underlying glioma-induced VP in the brain remain poorly understood. Utilizing a conditional, endothelial-specific deletion of the focal adhesion kinase FAK in the mouse (FAK CKO), we show that FAK is critical for destabilization of the tumor endothelium in tumor-bearing mice, with mutant mice exhibiting a relatively stabilized vasculature to wild-type mice (FAK WT). Tumor vessels in the FAK CKO mice displayed reduced VP compared to FAK WT mice, resulting in reduced tumor growth. Additionally, FAK CKO mice displayed partial restoration of cell-cell junction proteins in the tumor vessels and astrocyte-endothelial interactions in tumors, revealing an additional role of astrocytes in mediating tumor-induced VP. Together, these results provide genetic evidence that FAK is a mediator of tumor-induced VP in the brain. Our findings may help understand how therapeutics might be used to regulate cell type-specific interactions to restore BBB structure/function in cancer and perhaps other pathological conditions. PMID:21159635

  15. Defining central themes in breast cancer biology by differential proteomics: conserved regulation of cell spreading and focal adhesion kinase.

    PubMed

    Bateman, Nicholas W; Sun, Mai; Hood, Brian L; Flint, Melanie S; Conrads, Thomas P

    2010-10-01

    Breast cancer is a highly heterogeneous disease, an observation that underscores the importance of elucidating conserved molecular characteristics, such as gene and protein expression, across breast cancer cell types toward providing a greater understanding of context-specific features central to this disease. Motivated by the goal of defining central biological themes across breast cancer cell subtypes, we conducted a global proteomic analysis of three breast cancer cell lines, MCF7, SK-BR-3, and MDA-MB-231, and compared these to a model of nontransformed mammary cells (MCF10A). Our results demonstrate modulation of proteins localized to the extracellular matrix, plasma membrane, and nucleus, along with coordinate decreases in proteins that regulate "cell spreading," a cellular event previously shown to be dysregulated in transformed cells. Protein interaction network analysis revealed the clustering of focal adhesion kinase (FAK), a fundamental regulator of cell spreading, with several proteins identified as mutually, differentially abundant across breast cancer cell lines that impact expression and activity of FAK, such as neprilysin and keratin 19. These analyses provide insights into conservation of protein expression across breast cancer cell subtypes, a subset of which warrants further investigation for their roles in the regulation of cell spreading and FAK in breast cancer.

  16. 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.

  17. 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

  18. 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.

  19. Arp2/3 Inhibition Induces Amoeboid-Like Protrusions in MCF10A Epithelial Cells by Reduced Cytoskeletal-Membrane Coupling and Focal Adhesion Assembly

    PubMed Central

    Stricker, Jonathan; Sayegh, Kareem; Campillo, Clement; Gardel, Margaret L.

    2014-01-01

    Here we demonstrate that Arp2/3 regulates a transition between mesenchymal and amoeboid protrusions in MCF10A epithelial cells. Using genetic and pharmacological means, we first show Arp2/3 inhibition impairs directed cell migration. Arp2/3 inhibition results in a dramatically impaired cell adhesion, causing deficient cell attachment and spreading to ECM as well as an 8-fold decrease in nascent adhesion assembly at the leading edge. While Arp2/3 does not play a significant role in myosin-dependent adhesion growth, mature focal adhesions undergo large scale movements against the ECM suggesting reduced coupling to the ECM. Cell edge protrusions occur at similar rates when Arp2/3 is inhibited but their morphology is dramatically altered. Persistent lamellipodia are abrogated and we observe a markedly increased incidence of blebbing and unstable pseuodopods. Micropipette-aspiration assays indicate that Arp2/3-inhibited cells have a weak coupling between the cell cortex and the plasma membrane, and suggest a potential mechanism for increased pseudopod and bleb formation. Pseudopods are not sensitive to reduced in formin or myosin II activity. Collectively, these results indicate that Arp2/3 is not necessary for rapid protrusion of the cell edge but plays a crucial role in assembling focal adhesions required for its stabilization. PMID:24967897

  20. Stimulation of human monocytes with macrophage colony-stimulating factor induces a Grb2-mediated association of the focal adhesion kinase pp125FAK and dynamin.

    PubMed Central

    Kharbanda, S; Saleem, A; Yuan, Z; Emoto, Y; Prasad, K V; Kufe, D

    1995-01-01

    Macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. In the present studies using human monocytes, we show that M-CSF induces interaction of the Grb2 adaptor protein with the focal adhesion kinase pp125FAK. The results demonstrate that tyrosine-phosphorylated pp125FAK directly interacts with the SH2 domain of Grb2. The findings indicate that a pYENV site at Tyr-925 in pp125FAK is responsible for this interaction. We also demonstrate that the Grb2-FAK complex associates with the GTPase dynamin. Dynamin interacts with the SH3 domains of Grb2 and exhibits M-CSF-dependent tyrosine phosphorylation in association with pp125FAK. These findings suggest that M-CSF-induced signaling involves independent Grb2-mediated pathways, one leading to Ras activation and another involving pp125FAK and a GTPase implicated in receptor internalization. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7597091

  1. CCM1 and the second life of proteins in adhesion complexes

    PubMed Central

    van den Berg, Maaike CW; Burgering, Boudewijn MT

    2014-01-01

    It is well recognized that a number of proteins present within adhesion complexes perform discrete signaling functions outside these adhesion complexes, including transcriptional control. In this respect, β-catenin is a well-known example of an adhesion protein present both in cadherin complexes and in the nucleus where it regulates the TCF transcription factor. Here we discuss nuclear functions of adhesion complex proteins with a special focus on the CCM-1/KRIT-1 protein, which may turn out to be yet another adhesion complex protein with a second life. PMID:24714220

  2. Accumulation of talin in nodes at the edge of the lamellipodium and separate incorporation into adhesion plaques at focal contacts in fibroblasts

    PubMed Central

    1991-01-01

    The focal contact forms beneath F-actin-rich ribs, or cytoplasmic precursors, present in the lamellipodia of fibroblasts. The basal part of the precursor is retained at the contact as the initial adhesion plaque. We have examined the distribution of talin in the lamellipodia and adhesion plaques of chicken embryo fibroblasts relative to the process of focal contact formation. Motility of single cells was recorded with differential interference contrast or interference reflection microscopy before fixation and fluorescent staining for talin, F-actin, and vinculin. Talin is present along the extreme edge of the lamellipodium, where it is further concentrated into a series of nodes. The nodes of talin are present at the tips of both larger and finer F-actin-rich ribs and at small structural nodes at the edge of the lamellipodium. We suggest that the talin in the nodes functions, via a cross-linking activity, in the convergence of actin filaments at the membrane during development of the ribs. Talin accumulates de novo in the adhesion plaque, independent of that at the tip of the precursor, in response to contact with the substrate. This second accumulation of talin at the focal contact starts before vinculin, consistent with a sequential binding of talin at the membrane and of vinculin to talin. The results imply that talin functions independently at two steps during formation of the focal contact: the development of the F-actin-rich precursor of the contact; and development of the contact-associated adhesion plaque, both involving organization of F- actin at the membrane. PMID:1904445

  3. 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

  4. Friction and adhesion mediated by supramolecular host-guest complexes.

    PubMed

    Guerra, Roberto; Benassi, Andrea; Vanossi, Andrea; Ma, Ming; Urbakh, Michael

    2016-04-01

    The adhesive and frictional response of an AFM tip connected to a substrate through supramolecular host-guest complexes is investigated by dynamic Monte Carlo simulations. Here, the variation of the pull-off force with the unloading rate recently observed in experiments is unraveled by evidencing simultaneous (progressive) breaking of the bonds at fast (slow) rates. The model reveals the origin of the observed plateaus in the retraction force as a function of the tip-surface distance, showing that they result from the tip geometrical features. In lateral sliding, the model exhibits a wide range of dynamic behaviors ranging from smooth sliding to stick-slip at different velocities, with the average friction force determined by the characteristic formation/rupture rates of the complexes. In particular, it is shown that for some molecular complexes friction can become almost constant over a wide range of velocities. Also, we show the possibility of exploiting the ageing effect through slide-hold-slide experiments, in order to infer the characteristic formation rate. Finally, our model predicts a novel "anti-ageing" effect which is characterized by a decrease of the static friction force with the hold time. Such an effect is explained in terms of enhancement of adhesion during sliding, especially observed at high driving velocities. PMID:26975343

  5. Fenretinide Perturbs Focal Adhesion Kinase in Premalignant and Malignant Human Oral Keratinocytes. Fenretinide’s chemopreventive mechanisms include ECM interactions

    PubMed Central

    Han, Byungdo B.; Li, Suyang; Tong, Meng; Holpuch, Andrew S.; Spinney, Richard; Wang, Daren; Border, Michael B.; Liu, Zhongfa; Sarode, Sachin; Pei, Ping; Schwendeman, Steven; Mallery, Susan R.

    2015-01-01

    The membrane-associated protein, focal adhesion kinase (FAK), modulates cell-extracellular matrix interactions and also conveys pro-survival and proliferative signals. Notably, increased intraepithelial FAK levels accompany transformation of premalignant oral intraepithelial neoplasia (OIN) to oral squamous cell carcinoma (OSCC). OIN chemoprevention is a patient-centric, optimal strategy to prevent OSCC’s co-morbidities and mortality. The cancer chemopreventive and synthetic vitamin A derivative, fenretinide, has demonstrated protein-binding capacities e.g. mTOR and retinol binding protein interactions. These studies employed a continuum of human oral keratinocytes (normal-HPV E6/E7-transduced-OSCC) to assess potential fenretinide-FAK drug protein interactions and functional consequences on cellular growth regulation and motility. Molecular modeling studies demonstrated fenretinide has ~200-fold greater binding affinity relative to the natural ligand (ATP) at FAK’s kinase domain. Fenretinide also shows intermediate binding at FAK’s FERM domain and interacts at the ATP-binding site of the closest FAK analogue, Pyk2. Fenretinide significantly suppressed proliferation via induction of apoptosis and G2/M cell cycle blockade. Fenretinide-treated cells also demonstrated F-actin disruption, significant inhibition of both directed migration and invasion of a synthetic basement membrane, and decreased phosphorylation of growth-promoting kinases. A commercially available FAK inhibitor did not suppress cell invasion. Notably, while FAK’s FERM domain directs cell invasion, FAK inhibitors target the kinase domain. In addition, FAK-specific siRNA treated cells showed an intermediate cell migration capacity; data which suggest co-contribution of the established migrating-enhancing Pyk2. Our data imply that fenretinide is uniquely capable of disrupting FAK’s and Pyk2’s pro-survival and mobility-enhancing effects and further extend fenretinide’s chemopreventive

  6. 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

  7. Progesterone promotes focal adhesion formation and migration in breast cancer cells through induction of protease-activated receptor-1.

    PubMed

    Diaz, Jorge; Aranda, Evelyn; Henriquez, Soledad; Quezada, Marisol; Espinoza, Estefanía; Bravo, Maria Loreto; Oliva, Bárbara; Lange, Soledad; Villalon, Manuel; Jones, Marius; Brosens, Jan J; Kato, Sumie; Cuello, Mauricio A; Knutson, Todd P; Lange, Carol A; Leyton, Lisette; Owen, Gareth I

    2012-08-01

    Progesterone and progestins have been demonstrated to enhance breast cancer cell migration, although the mechanisms are still not fully understood. The protease-activated receptors (PARs) are a family of membrane receptors that are activated by serine proteases in the blood coagulation cascade. PAR1 (F2R) has been reported to be involved in cancer cell migration and overexpressed in breast cancer. We herein demonstrate that PAR1 mRNA and protein are upregulated by progesterone treatment of the breast cancer cell lines ZR-75 and T47D. This regulation is dependent on the progesterone receptor (PR) but does not require PR phosphorylation at serine 294 or the PR proline-rich region mPRO. The increase in PAR1 mRNA was transient, being present at 3  h and returning to basal levels at 18  h. The addition of a PAR1-activating peptide (aPAR1) to cells treated with progesterone resulted in an increase in focal adhesion (FA) formation as measured by the cellular levels of phosphorylated FA kinase. The combined but not individual treatment of progesterone and aPAR1 also markedly increased stress fiber formation and the migratory capacity of breast cancer cells. In agreement with in vitro findings, data mining from the Oncomine platform revealed that PAR1 expression was significantly upregulated in PR-positive breast tumors. Our observation that PAR1 expression and signal transduction are modulated by progesterone provides new insight into how the progestin component in hormone therapies increases the risk of breast cancer in postmenopausal women.

  8. 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

  9. Adhesion

    MedlinePlus

    ... as the shoulder Eyes Inside the abdomen or pelvis Adhesions can become larger or tighter over time. ... Other causes of adhesions in the abdomen or pelvis include: Appendicitis , most often when the appendix breaks ...

  10. Selective binding and lateral clustering of α5β1 and αvβ3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

    PubMed Central

    Schaufler, Viktoria; Czichos-Medda, Helmi; Hirschfeld-Warnecken, Vera; Neubauer, Stefanie; Rechenmacher, Florian; Medda, Rebecca; Kessler, Horst; Geiger, Benjamin; Spatz, Joachim P.; Cavalcanti-Adam, E. Ada

    2016-01-01

    ABSTRACT Coordination of the specific functions of α5β1 and αvβ3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvβ3 and α5β1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5β1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvβ3 ligand at 30 and 60 nm spacings. Analysis of αvβ3 and α5β1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5β1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvβ3 ligand. αvβ3 integrin clusters are more pronounced on αvβ3 ligand, though they can also be detected in cells adhering to α5β1 ligand. Furthermore, α5β1 integrin clusters are present in cells adhering to α5β1 ligand, and often colocalize with αvβ3 clusters. Taken together, these findings indicate that the activation of αvβ3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions. PMID:27003228

  11. alpha2-Adrenoceptor stimulation promotes actin polymerization and focal adhesion in 3T3F442A and BFC-1beta preadipocytes.

    PubMed

    Bétuing, S; Daviaud, D; Valet, P; Bouloumié, A; Lafontan, M; Saulnier-Blache, J S

    1996-12-01

    We previously demonstrated that in white fat cell precursors alpha2-adrenoceptor stimulation lead to the phosphorylation of p44 and p42 mitogen-activated protein kinases and an increase in cell number. Regulation of cell adhesion and cell cytoskeleton plays a crucial role in the control of cell growth by various growth factors. Here, we report that in mouse 3T3F442A preadipocytes expressing 2500 fmol/mg protein of the human alpha2C10-adrenoceptor (alpha2AF2 cells), alpha2-adrenergic stimulation rapidly restored the spreading of cells previously retracted by serum withdrawal. This effect was pertussis toxin sensitive and was blocked by pretreatment of the cells with dihydrocytochalasin B (a blocker of actin polymerization), genistein (a tyrosine kinase inhibitor), or agents that increase cell cAMP content. Spreading was accompanied by cell membrane ruffling, formation of lamelipodia and filipodia, appearance of focal adhesion plaques, and induction of actin stress fibers. alpha2-Adrenergic stimulation also lead to a rapid Gi- and actin-dependent tyrosine phosphorylation of the pp125 focal adhesion kinase (FAK) as well as of the p42 and p44 mitogen-activated protein kinases. alpha2-Adrenergic-dependent spreading and FAK and mitogen-activated protein kinase phosphorylation were also observed in 3T3F442A preadipocytes permanently expressing 20 fmol/mg protein of the human alpha2C10-adrenoceptor (alpha2AF3 cells) as well as in BFC-1beta preadipocytes, which constitutively express 25 fmol/mg protein of mouse alpha2A-adrenoceptors. In BFC-1beta preadipocytes, alpha2-adrenergic-dependent spreading and pp125FAK phosphorylation were counteracted by beta-adrenergic stimulation. Our results suggest that alpha2-adrenergic control of actin polymerization and focal adhesion assembly could play a crucial role in the regulation of preadipocyte growth by the sympathetic nervous system.

  12. Correlation between matrix metalloproteinase expression and activation of the focal adhesion kinase signaling pathway in herpes stromal keratitis

    PubMed Central

    CAO, TING; XING, YIQIAO; YANG, YANNING; MEI, HAIFENG

    2014-01-01

    The present study aimed to investigate the correlation between matrix metalloproteinase-2 (MMP-2) expression and activation of the focal adhesion kinase (FAK) signaling pathway in herpes stromal keratitis (HSK). The cornea of 24 BALB/c mice was infected with herpes simplex virus type 1 (HSV-1) to construct a model of HSK. Six additional mice served as negative controls. Immunohistochemical staining was used to detect FAK expression levels. Human corneal epithelial (HCE) cells cultured in vitro were infected with HSV-1 and the expression levels of MMP-2, FAK and phosphorylated-FAK (p-FAK) in HCE cells were detected using reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis and immunohistochemistry at 2, 20 and 40 h following infection. In the HSK rat model, the corneal epithelial cells appeared deranged and the number of neutrophils and FAK-positive cells was significantly increased compared with that of the negative control group (P<0.05). Repeated measures analysis of variance of RT-PCR showed no significant differences in MMP-2 and FAK mRNA expression levels in the infected cells at various time points, and no significant differences between infected cells and the negative control group were observed. There was no interaction between groups and time points. Pairwise comparisons showed that MMP-2 and FAK mRNA expression levels were significantly increased in virus-infected cells compared with those of the control group. Over time, MMP-2 and FAK mRNA expression levels did not differ significantly in virus-infected cells or in control cells. Western blot analysis indicated no significant differences in p-FAK, FAK and MMP-2 expression levels between the infected and control cells at 2 h (P>0.05). Infected cells showed a significant increase in MMP-2 and p-FAK expression levels than that of the control cells at 20 and 40 h (P<0.05). p-FAK, FAK and MMP-2 expression levels in virus-infected cells at 2 h differed significantly from those at 20

  13. Boronate Complex Formation with Dopa Containing Mussel Adhesive Protein Retards pH-Induced Oxidation and Enables Adhesion to Mica

    PubMed Central

    Israelachvili, Jacob N.; Chen, Yunfei; Waite, J. Herbert

    2014-01-01

    The biochemistry of mussel adhesion has inspired the design of surface primers, adhesives, coatings and gels for technological applications. These mussel-inspired systems often focus on incorporating the amino acid 3,4-dihydroxyphenyl-L-alanine (Dopa) or a catecholic analog into a polymer. Unfortunately, effective use of Dopa is compromised by its susceptibility to auto-oxidation at neutral pH. Oxidation can lead to loss of adhesive function and undesired covalent cross-linking. Mussel foot protein 5 (Mfp-5), which contains ∼30 mole % Dopa, is a superb adhesive under reducing conditions but becomes nonadhesive after pH-induced oxidation. Here we report that the bidentate complexation of borate by Dopa to form a catecholato-boronate can be exploited to retard oxidation. Although exposure of Mfp-5 to neutral pH typically oxidizes Dopa, resulting in a>95% decrease in adhesion, inclusion of borate retards oxidation at the same pH. Remarkably, this Dopa-boronate complex dissociates upon contact with mica to allow for a reversible Dopa-mediated adhesion. The borate protection strategy allows for Dopa redox stability and maintained adhesive function in an otherwise oxidizing environment. PMID:25303409

  14. An extracellular adhesion molecule complex patterns dendritic branching and morphogenesis.

    PubMed

    Dong, Xintong; Liu, Oliver W; Howell, Audrey S; Shen, Kang

    2013-10-10

    Robust dendrite morphogenesis is a critical step in the development of reproducible neural circuits. However, little is known about the extracellular cues that pattern complex dendrite morphologies. In the model nematode Caenorhabditis elegans, the sensory neuron PVD establishes stereotypical, highly branched dendrite morphology. Here, we report the identification of a tripartite ligand-receptor complex of membrane adhesion molecules that is both necessary and sufficient to instruct spatially restricted growth and branching of PVD dendrites. The ligand complex SAX-7/L1CAM and MNR-1 function at defined locations in the surrounding hypodermal tissue, whereas DMA-1 acts as the cognate receptor on PVD. Mutations in this complex lead to dramatic defects in the formation, stabilization, and organization of the dendritic arbor. Ectopic expression of SAX-7 and MNR-1 generates a predictable, unnaturally patterned dendritic tree in a DMA-1-dependent manner. Both in vivo and in vitro experiments indicate that all three molecules are needed for interaction. PMID:24120131

  15. Separation of distinct adhesion complexes and associated cytoskeleton by a micro-stencil-printing method.

    PubMed

    Caballero, David; Osmani, Naël; Georges-Labouesse, Elisabeth; Labouesse, Michel; Riveline, Daniel

    2012-01-01

    Adhesion between cells and the extracellular matrix is mediated by different types of transmembraneous proteins. Their associations to specific partners lead to the assembly of contacts such as focal adhesions and hemidesmosomes. The spatial overlap between both contacts within cells has however limited the study of each type of contact. Here we show that with "stampcils" focal contacts and hemidesmosomes can be spatially separated: cells are plated within the cavities of a stencil and the grids of the stencil serve as stamps for grafting an extracellular matrix protein-fibronectin. Cells engage new contacts on stamped zones leading to the segregation of adhesions and their associated cytoskeletons, i.e., actin and intermediate filaments of keratins. This new method should provide new insights into cell contacts compositions and dynamics. PMID:23076134

  16. Rgnef (p190RhoGEF) Knockout Inhibits RhoA Activity, Focal Adhesion Establishment, and Cell Motility Downstream of Integrins

    PubMed Central

    Miller, Nichol L. G.; Lawson, Christine; Chen, Xiao Lei; Lim, Ssang-Taek; Schlaepfer, David D.

    2012-01-01

    Background Cell migration is a highly regulated process that involves the formation and turnover of cell-matrix contact sites termed focal adhesions. Rho-family GTPases are molecular switches that regulate actin and focal adhesion dynamics in cells. Guanine nucleotide exchange factors (GEFs) activate Rho-family GTPases. Rgnef (p190RhoGEF) is a ubiquitous 190 kDa GEF implicated in the control of colon carcinoma and fibroblast cell motility. Principal Findings Rgnef exon 24 floxed mice (Rgnefflox) were created and crossed with cytomegalovirus (CMV)-driven Cre recombinase transgenic mice to inactivate Rgnef expression in all tissues during early development. Heterozygous RgnefWT/flox (Cre+) crosses yielded normal Mendelian ratios at embryonic day 13.5, but Rgnefflox/flox (Cre+) mice numbers at 3 weeks of age were significantly less than expected. Rgnefflox/flox (Cre+) (Rgnef−/−) embryos and primary mouse embryo fibroblasts (MEFs) were isolated and verified to lack Rgnef protein expression. When compared to wildtype (WT) littermate MEFs, loss of Rgnef significantly inhibited haptotaxis migration, wound closure motility, focal adhesion number, and RhoA GTPase activation after fibronectin-integrin stimulation. In WT MEFs, Rgnef activation occurs within 60 minutes upon fibronectin plating of cells associated with RhoA activation. Rgnef−/− MEF phenotypes were rescued by epitope-tagged Rgnef re-expression. Conclusions Rgnef−/− MEF phenotypes were due to Rgnef loss and support an essential role for Rgnef in RhoA regulation downstream of integrins in control of cell migration. PMID:22649559

  17. Integrating focal adhesion dynamics, cytoskeleton remodeling, and actin motor activity for predicting cell migration on 3D curved surfaces of the extracellular matrix.

    PubMed

    Kim, Min-Cheol; Kim, Choong; Wood, Levi; Neal, Devin; Kamm, Roger D; Asada, H Harry

    2012-11-01

    An integrative cell migration model incorporating focal adhesion (FA) dynamics, cytoskeleton and nucleus remodeling and actin motor activity is developed for predicting cell migration behaviors on 3-dimensional curved surfaces, such as cylindrical lumens in the 3-D extracellular matrix (ECM). The work is motivated by 3-D microfluidic migration experiments suggesting that the migration speed and direction may vary depending on the cross sectional shape of the lumen along which the cell migrates. In this paper, the mechanical structure of the cell is modeled as double elastic membranes of cell and nucleus. The two elastic membranes are connected by stress fibers, which are extended from focal adhesions on the cell surface to the nuclear membrane. The cell deforms and gains traction as transmembrane integrins distributed over the outer cell membrane bind to ligands on the ECM, form focal adhesions, and activate stress fibers. Probabilities at which integrin ligand-receptor bonds are formed as well as ruptures are affected by the surface geometry, resulting in diverse migration behaviors that depend on the curvature of the surface. Monte Carlo simulations of the integrative model reveal that (a) the cell migration speed is dependent on the cross sectional area of the lumen with a maximum speed at a particular diameter or width, (b) as the lumen diameter increases, the cell tends to spread and migrate around the circumference of the lumen, while it moves in the longitudinal direction as the lumen diameter narrows, (c) once the cell moves in one direction, it tends to stay migrating in the same direction despite the stochastic nature of migration. The relationship between the cell migration speed and the lumen width agrees with microfluidic experimental data for cancer cell migration.

  18. Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion.

    PubMed

    Cerna, Joel; Cerecedo, Doris; Ortega, Arturo; García-Sierra, Francisco; Centeno, Federico; Garrido, Efrain; Mornet, Dominique; Cisneros, Bulmaro

    2006-10-01

    Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of beta1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisense-Dp71 clones to analyze in detail the potential involvement of Dp71f isoform with the beta1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell beta1-integrin adhesion complex is composed of beta1-integrin, talin, paxillin, alpha-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the beta1-integrin complex components (beta1-integrin, FAK, alpha-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the beta1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and beta1-integrin. Our data indicate that Dp71f is a structural component of the beta1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance.

  19. Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion

    PubMed Central

    Cerna, Joel; Cerecedo, Doris; Ortega, Arturo; García-Sierra, Francisco; Centeno, Federico; Garrido, Efrain; Mornet, Dominique; Cisneros, Bulmaro

    2006-01-01

    Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of β1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisenseDp71 clones to analyze in detail the potential involvement of Dp71f isoform with the β1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell β1-integrin adhesion complex is composed of β1-integrin, talin, paxillin, α-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the β1-integrin complex components (β1-integrin, FAK, α-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the β1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and β1-integrin. Our data indicate that Dp71f is a structural component of the β1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance. PMID:16935300

  20. 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

  1. Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).

    PubMed

    Reinhard, M; Jouvenal, K; Tripier, D; Walter, U

    1995-08-15

    VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.

  2. Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).

    PubMed Central

    Reinhard, M; Jouvenal, K; Tripier, D; Walter, U

    1995-01-01

    VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 PMID:7644520

  3. Adhesion and formation of microbial biofilms in complex microfluidic devices

    SciTech Connect

    Kumar, Aloke; Karig, David K; Neethirajan, Suresh; Suresh, Anil K; Srijanto, Bernadeta R; Mukherjee, Partha P; Retterer, Scott T; Doktycz, Mitchel John

    2012-01-01

    Shewanella oneidensis is a metal reducing bacterium, which is of interest for bioremediation and clean energy applications. S. oneidensis biofilms play a critical role in several situations such as in microbial energy harvesting devices. Here, we use a microfluidic device to quantify the effects of hydrodynamics on the biofilm morphology of S. oneidensis. For different rates of fluid flow through a complex microfluidic device, we studied the spatiotemporal dynamics of biofilms, and we quantified several morphological features such as spatial distribution, cluster formation and surface coverage. We found that hydrodynamics resulted in significant differences in biofilm dynamics. The baffles in the device created regions of low and high flow in the same device. At higher flow rates, a nonuniform biofilm develops, due to unequal advection in different regions of the microchannel. However, at lower flow rates, a more uniform biofilm evolved. This depicts competition between adhesion events, growth and fluid advection. Atomic force microscopy (AFM) revealed that higher production of extra-cellular polymeric substances (EPS) occurred at higher flow velocities.

  4. E-Cadherin-Dependent Stimulation of Traction Force at Focal Adhesions via the Src and PI3K Signaling Pathways

    PubMed Central

    Jasaitis, Audrius; Estevez, Maruxa; Heysch, Julie; Ladoux, Benoit; Dufour, Sylvie

    2012-01-01

    The interplay between cadherin- and integrin-dependent signals controls cell behavior, but the precise mechanisms that regulate the strength of adhesion to the extracellular matrix remains poorly understood. We deposited cells expressing a defined repertoire of cadherins and integrins on fibronectin (FN)-coated polyacrylamide gels (FN-PAG) and on FN-coated pillars used as a micro-force sensor array (μFSA), and analyzed the functional relationship between these adhesion receptors to determine how it regulates cell traction force. We found that cadherin-mediated adhesion stimulated cell spreading on FN-PAG, and this was modulated by the substrate stiffness. We compared S180 cells with cells stably expressing different cadherins on μFSA and found that traction forces were stronger in cells expressing cadherins than in parental cells. E-cadherin-mediated contact and mechanical coupling between cells are required for this increase in cell-FN traction force, which was not observed in isolated cells, and required Src and PI3K activities. Traction forces were stronger in cells expressing type I cadherins than in cells expressing type II cadherins, which correlates with our previous observation of a higher intercellular adhesion strength developed by type I compared with type II cadherins. Our results reveal one of the mechanisms whereby molecular cross talk between cadherins and integrins upregulates traction forces at cell-FN adhesion sites, and thus provide additional insight into the molecular control of cell behavior. PMID:22853894

  5. The algorithmic complexity of neural spike trains increases during focal seizures.

    PubMed

    Rapp, P E; Zimmerman, I D; Vining, E P; Cohen, N; Albano, A M; Jiménez-Montaño, M A

    1994-08-01

    The interspike interval spike trains of spontaneously active cortical neurons can display nonrandom internal structure. The degree of nonrandom structure can be quantified and was found to decrease during focal epileptic seizures. Greater statistical discrimination between the two physiological conditions (normal vs seizure) was obtained with measurements of context-free grammar complexity than by measures of the distribution of the interspike intervals such as the mean interval, its standard deviation, skewness, or kurtosis. An examination of fixed epoch data sets showed that two factors contribute to the complexity: the firing rate and the internal structure of the spike train. However, calculations with randomly shuffled surrogates of the original data sets showed that the complexity is not completely determined by the firing rate. The sequence-sensitive structure of the spike train is a significant contributor. By combining complexity measurements with statistically related surrogate data sets, it is possible to classify neurons according to the dynamical structure of their spike trains. This classification could not have been made on the basis of conventional distribution-determined measures. Computations with more sophisticated kinds of surrogate data show that the structure observed using complexity measures cannot be attributed to linearly correlated noise or to linearly correlated noise transformed by a static monotonic nonlinearity. The patterns in spike trains appear to reflect genuine nonlinear structure. The limitations of these results are also discussed. The results presented in this article do not, of themselves, establish the presence of a fine-structure encoding of neural information.

  6. Adhesions

    MedlinePlus

    ... surfaces so they can shift easily as the body moves. Adhesions cause tissues and organs to stick together. They might connect the loops of the intestines to each other, to nearby ... can occur anywhere in the body. But they often form after surgery on the ...

  7. 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.

  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. Molecular Architecture of a Complex between an Adhesion Protein from the Malaria Parasite and Intracellular Adhesion Molecule 1*

    PubMed Central

    Brown, Alan; Turner, Louise; Christoffersen, Stig; Andrews, Katrina A.; Szestak, Tadge; Zhao, Yuguang; Larsen, Sine; Craig, Alister G.; Higgins, Matthew K.

    2013-01-01

    The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria. The PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from CIDR (cysteine-rich interdomain regions) and DBL (Duffy-binding-like) domains and show extensive variation in sequence, size, and domain organization. Here we use biophysical methods to characterize the entire ∼300-kDa ectodomain from IT4VAR13, a protein that interacts with the host receptor, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLβ domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1 ectodomain in complex with its ligand. They show that it combines a modular domain arrangement consisting of individual ligand binding domains, with a defined higher order architecture that exposes the ICAM-1 binding surface to allow adhesion. PMID:23297413

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

    PubMed

    Stigliani, Sara; Scaruffi, Paola; 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

    2015-05-30

    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

  11. Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

    PubMed

    Dormond, Olivier; Ponsonnet, Lionel; Hasmim, Meriem; Foletti, Alessandro; Rüegg, Curzio

    2004-07-01

    Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

  12. Letting Go of JuNK by Disassembly of Adhesive Complexes.

    PubMed

    Farley, Jonathan E; Freeman, Marc R

    2015-12-01

    Immature neural circuits form excessive synaptic connections that are later refined through pruning of exuberant branches. In this issue, Bornstein et al. identify a role for JNK signaling in selective axon elimination through disassembly of cell adhesion complexes. PMID:26637791

  13. Rac1 Controls the Subcellular Localization of the Rho Guanine Nucleotide Exchange Factor Net1A To Regulate Focal Adhesion Formation and Cell Spreading

    PubMed Central

    Carr, Heather S.; Morris, Christopher A.; Menon, Sarita; Song, Eun Hyeon

    2013-01-01

    RhoA is overexpressed in human cancer and contributes to aberrant cell motility and metastatic progression; however, regulatory mechanisms controlling RhoA activity in cancer are poorly understood. Neuroepithelial transforming gene 1 (Net1) is a RhoA guanine nucleotide exchange factor that is overexpressed in human cancer. It encodes two isoforms, Net1 and Net1A, which cycle between the nucleus and plasma membrane. Net1 proteins must leave the nucleus to activate RhoA, but mechanisms controlling the extranuclear localization of Net1 isoforms have not been described. Here, we show that Rac1 activation causes relocalization of Net1 isoforms outside the nucleus and stimulates Net1A catalytic activity. These effects do not require Net1A catalytic activity, its pleckstrin homology domain, or its regulatory C terminus. We also show that Rac1 activation protects Net1A from proteasome-mediated degradation. Replating cells on collagen stimulates endogenous Rac1 to relocalize Net1A, and inhibition of proteasome activity extends the duration and magnitude of Net1A relocalization. Importantly, we demonstrate that Net1A, but not Net1, is required for cell spreading on collagen, myosin light chain phosphorylation, and focal adhesion maturation. These data identify the first physiological mechanism controlling the extranuclear localization of Net1 isoforms. They also demonstrate a previously unrecognized role for Net1A in regulating cell adhesion. PMID:23184663

  14. The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion*

    PubMed Central

    Thwaites, Tristan; Nogueira, Ana T.; Campeotto, Ivan; Silva, Ana P.; Grieshaber, Scott S.; Carabeo, Rey A.

    2014-01-01

    Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34Arc, and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector. PMID:25193659

  15. 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

  16. 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

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

    PubMed

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

    2015-11-26

    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.

  18. 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

  19. A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

    PubMed

    Chakraborty, T; Ebel, F; Domann, E; Niebuhr, K; Gerstel, B; Pistor, S; Temm-Grove, C J; Jockusch, B M; Reinhard, M; Walter, U

    1995-04-01

    The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.

  20. Combination of heat shock protein 90 and focal adhesion kinase inhibitors synergistically inhibits the growth of non-small cell lung cancer cells.

    PubMed

    Webber, Philip J; Park, Chanhee; Qui, Min; Ramalingam, Suresh S; Khuri, Fadlo R; Fu, Haian; Du, Yuhong

    2015-01-01

    Discovery of effective drug combinations is a promising strategy to improve patient survival. This study explores the impact of heat shock protein 90 (Hsp90) inhibition in combination with focal adhesion kinase (FAK) inhibitor on the growth of non-small cell lung cancer cells (NSCLC cells). Our data show that 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG), a well-studied Hsp90 inhibitor, synergized with FAK inhibitor, PF-573228, on the growth inhibition of NSCLC cells. This combination effect was confirmed using additional chemically distinct Hsp90 inhibitor, STA-9090, which is currently undergoing phase 3 clinical evaluation. Co-treatment of NSCLC cells with Hsp90 and FAK inhibitors significantly enhanced the inhibition on long-term colony formation compared to that with single agent. Inhibition of FAK exacerbated the G2 cell cycle arrest and annexin-V apoptotic staining induced by 17-AAG. Further mechanistic studies revealed that the combination of Hsp90 and FAK inhibitors reduced the activity of canonical proliferative and survival Akt-mTOR signaling, and increased pro-apoptotic caspase activation. Interestingly, FAK inhibition alone induced feedback activation of pro-survival Erk signaling, which was abrogated by co-treatment with Hsp90 inhibitors. Both Hsp90 and FAK inhibitors are undergoing clinical evaluation. Our studies suggest the tandem of Hsp90 and FAK inhibitors may provide an effective treatment option for NSCLC patients.

  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. PKCδ Influences p190 Phosphorylation and Activity: Events Independent of PKCδ-Mediated Regulation of Endothelial Cell Stress Fiber and Focal Adhesion Formation and Barrier Function

    PubMed Central

    Fordjour, Akua K.; Harrington, Elizabeth O.

    2009-01-01

    Background We have shown that protein kinase Cδ (PKCδ) inhibition results in increased endothelial cell (EC) permeability and decreased RhoA activity; which correlated with diminished stress fibers (SF) and focal adhesions (FA). We have also shown co-precipitation of p190RhoGAP (p190) with PKCδ. Here, we investigated if PKCδ regulates p190 and whether PKCδ-mediated changes in SF and FA or permeability were dependent upon p190. Methods Protein-protein interaction and activity analyses were performed using co-precipitation assays. Analysis of p190 phosphorylation was performed using in vitro kinase assays. SF and FA were analyzed by immunofluorescence analyses. EC monolayer permeability was measured using electrical cell impedance sensor (ECIS) technique. Results Inhibition of PKCδ increased p190 activity, while PKCδ overexpression diminished p190 activity. PKCδ bound to and phosphorylated both p190FF and p190GTPase domains. p190 protein overexpression diminished SF and FA formation and RhoA activity. Disruption of SF and FA or increased permeability induced upon PKCδ inhibition, were not attenuated in EC in which the p190 isoforms were suppressed individually or concurrently. Conclusion and General Significance Our findings suggest that while PKCδ can regulate p190 activity, possibly at the FF and/ or GTPase domains, the effect of PKCδ inhibition on SF and FA and barrier dysfunction occurs through a pathway independent of p190. PMID:19632305

  3. [Functional connectivity and complex networks in focal epilepsy. Pathophysiology and therapeutic implications].

    PubMed

    Pastor, Jesús; Sola, Rafael G; Vega-Zelaya, Lorena; Garnes, Óscar; Ortega, Guillermo J

    2014-05-01

    Introduccion. El modelo tradicional de cirugia de la epilepsia focal consiste en la localizacion y reseccion de la zona epileptogena. Sin embargo, una minoria significativa de pacientes epilepticos sigue teniendo crisis tras la reseccion, lo que muestra el nivel de conocimiento incompleto que actualmente se tiene sobre esta materia. Objetivo. Introducir algunos conceptos de conectividad funcional y redes complejas y su aplicacion al estudio de registros neurofisiologicos de pacientes con epilepsia focal farmacorresistente, principalmente del lobulo temporal. Desarrollo. Con el objeto de entender con plenitud los nuevos desarrollos en el area de redes complejas y sus aplicaciones al estudio de la epilepsia, se revisaran los conceptos fundamentales de la metodologia de redes complejas, sincronizacion y conectividad funcional. Asimismo, se revisaran algunos de los trabajos mas recientes en los que se analizan registros neurofisiologicos y resonancias funcionales de pacientes con epilepsia focal bajo esta nueva perspectiva. Conclusiones. Una vision mas amplia en la perspectiva para el entendimiento de la epilepsia focal, como la tratada en este trabajo, permitira tambien plantear nuevas alternativas al tratamiento quirurgico de la epilepsia focal, tanto con el desarrollo de nuevos metodos no invasivos como con la mejora y optimizacion de algunos de los metodos invasivos ya existentes.

  4. Integrin-mediated adhesion complex: Cooption of signaling systems at the dawn of Metazoa.

    PubMed

    Sebé-Pedrós, Arnau; Ruiz-Trillo, Iñaki

    2010-09-01

    The integrin-mediated adhesion machinery is the primary cell-matrix adhesion mechanism in Metazoa. The integrin adhesion complex, which modulates important aspects of the cell physiology, is composed of integrins (alpha and beta subunits) and several scaffolding and signaling proteins. Integrins appeared to be absent in all non-metazoan eukaryotes so-far analyzed, including fungi, plants and choanoflagellates, the sister-group to Metazoa. Thus, integrins and, therefore, the integrin-mediated adhesion and signaling mechanism was considered a metazoan innovation. Recently, a broad comparative genomic analysis including new genome data from several unicellular organisms closely related to fungi and metazoans shattered previous views. The integrin adhesion and signaling complex is not specific to Metazoa, but rather it is present in apusozoans and holozoan protists. Thus, this important signaling and adhesion system predated the origin of Fungi and Metazoa, and was subsequently lost in fungi and choanoflagellates. This finding suggests that cooption played a more important role in the origin of Metazoa than previously believed. Here, we hypothesize that the integrin adhesome was ancestrally involved in signaling.

  5. Underwater contact adhesion and microarchitecture in polyelectrolyte complexes actuated by solvent exchange

    NASA Astrophysics Data System (ADS)

    Zhao, Qiang; Lee, Dong Woog; Ahn, B. Kollbe; Seo, Sungbaek; Kaufman, Yair; Israelachvili, Jacob N.; Waite, J. Herbert

    2016-04-01

    Polyelectrolyte complexation is critical to the formation and properties of many biological and polymeric materials, and is typically initiated by aqueous mixing followed by fluid-fluid phase separation, such as coacervation. Yet little to nothing is known about how coacervates evolve into intricate solid microarchitectures. Inspired by the chemical features of the cement proteins of the sandcastle worm, here we report a versatile and strong wet-contact microporous adhesive resulting from polyelectrolyte complexation triggered by solvent exchange. After premixing a catechol-functionalized weak polyanion with a polycation in dimethyl sulphoxide (DMSO), the solution was applied underwater to various substrates whereupon electrostatic complexation, phase inversion, and rapid setting were simultaneously actuated by water-DMSO solvent exchange. Spatial and temporal coordination of complexation, inversion and setting fostered rapid (~25 s) and robust underwater contact adhesion (Wad >= 2 J m-2) of complexed catecholic polyelectrolytes to all tested surfaces including plastics, glasses, metals and biological materials.

  6. Yes-mediated phosphorylation of focal adhesion kinase at tyrosine 861 increases metastatic potential of prostate cancer cells.

    PubMed

    Chatterji, Tanushree; Varkaris, Andreas S; Parikh, Nila U; Song, Jian H; Cheng, Chien-Jui; Schweppe, Rebecca E; Alexander, Stephanie; Davis, John W; Troncoso, Patricia; Friedl, Peter; Kuang, Jian; Lin, Sue-Hwa; Gallick, Gary E

    2015-04-30

    To study the role of FAK signaling complexes in promoting metastatic properties of prostate cancer (PCa) cells, we selected stable, highly migratory variants, termed PC3 Mig-3 and DU145 Mig-3, from two well-characterized PCa cell lines, PC3 and DU145. These variants were not only increased migration and invasion in vitro, but were also more metastatic to lymph nodes following intraprostatic injection into nude mice. Both PC3 Mig-3 and DU145 Mig-3 were specifically increased in phosphorylation of FAK Y861. We therefore examined potential alterations in Src family kinases responsible for FAK phosphorylation and determined only Yes expression was increased. Overexpression of Yes in PC3 parental cells and src-/-fyn-/-yes-/- fibroblasts selectively increased FAK Y861 phosphorylation, and increased migration. Knockdown of Yes in PC3 Mig-3 cells decreased migration and decreased lymph node metastasis following orthotopic implantation of into nude mice. In human specimens, Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis. These results demonstrate a unique role for Yes in phosphorylation of FAK and in promoting PCa metastasis. Therefore, phosphorylated FAK Y861 and increased Yes expression may be predictive markers for PCa metastasis.

  7. Ultrastable cellulosome-adhesion complex tightens under load

    PubMed Central

    Schoeler, Constantin; Malinowska, Klara H.; Bernardi, Rafael C.; Milles, Lukas F.; Jobst, Markus A.; Durner, Ellis; Ott, Wolfgang; Fried, Daniel B.; Bayer, Edward A.; Schulten, Klaus; Gaub, Hermann E.; Nash, Michael A.

    2014-01-01

    Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand–receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand–receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600–750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass. PMID:25482395

  8. Adhesive complex coacervate inspired by the sandcastle worm as a sealant for fetoscopic defects

    NASA Astrophysics Data System (ADS)

    Kaur, Sarbjit

    Inspired by the Sandcastle Worm, biomimetic of the water-borne adhesive was developed by complex coacervation of the synthetic copolyelectrolytes, mimicking the chemistries of the worm glue. The developed underwater adhesive was designed for sealing fetal membranes after fetoscopic surgery in twin-to-twin transfusion syndrome (TTTS) and sealing neural tissue of a fetus in aminiotic sac for spina bifida condition. Complex coacervate with increased bond strength was created by entrapping polyethylene glycol diacrylate (PEG-dA) monomer within the cross-linked coacervate network. Maximum shear bond strength of ~ 1.2 MPa on aluminum substrates was reached. The monomer-filled coacervate had complex flow behavior, thickening at low shear rates and then thinning suddenly with a 16-fold drop in viscosity at shear rates near 6 s-1. The microscale structure of the complex coacervates resembled a three-dimensional porous network of interconnected tubules. This complex coacervate adhesive was used in vitro studies to mimic the uterine wall-fetal membrane interface using a water column with one end and sealed with human fetal membranes and poultry breast, and a defect was created with an 11 French trocar. The coacervate adhesive in conjunction with the multiphase adhesive was used to seal the defect. The sealant withstood an additional traction of 12 g for 30-60 minutes and turbulence of the water column without leakage of fluid or slippage. The adhesive is nontoxic when in direct contact with human fetal membranes in an organ culture setting. A stable complex coacervate adhesive for long-term use in TTTS and spina bifida application was developed by methacrylating the copolyelectrolytes. The methacrylated coacervate was crosslinked chemically for TTTS and by photopolymerization for spina bifida. Tunable mechanical properties of the adhesive were achieved by varying the methacrylation of the polymers. Varying the amine to phosphate (A/P) ratio in the coacervate formation

  9. 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

    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

  10. A novel orally available inhibitor of focal adhesion signaling increases survival in a xenograft model of diffuse large B-cell lymphoma with central nervous system involvement.

    PubMed

    Bosch, Rosa; Moreno, María José; Dieguez-Gonzalez, Rebeca; Céspedes, María Virtudes; Gallardo, Alberto; Trias, Manuel; Grañena, Albert; Sierra, Jorge; Casanova, Isolda; Mangues, Ramon

    2013-08-01

    Central nervous system dissemination is a relatively uncommon but almost always fatal complication in diffuse large B-cell lymphoma patients. Optimal therapy for central nervous involvement in this malignancy has not been established. In this paper, we aimed to evaluate the therapeutic effect of E7123, a celecoxib derivative that inhibits focal adhesion signaling, in a novel xenograft model of diffuse large B-cell lymphoma with central nervous system involvement. Cells obtained after disaggregation of HT subcutaneous tumors (HT-SC cells) were intravenously injected in NOD/SCID mice. These mice received oral vehicle or 75 mg/kg of E7123 daily until they were euthanized for weight loss or signs of sickness. The antitumor effect of E7123 was validated in an independent experiment using a bioluminescent mouse model. Intravenously injected HT-SC cells showed higher take rate and higher central nervous system tropism (associated with increased expression of β1-integrin and p130Cas proteins) than HT cells. The oral administration of E7123 significantly increased survival time in 2 independent experiments using mice injected with unmodified or bioluminescent HT-SC cells. We have developed a new xenograft model of diffuse large B-cell lymphoma with central nervous system involvement that can be used in the pre-clinical evaluation of new drugs for this malignancy. E7123 is a new, well-tolerated and orally available therapeutic agent that merits further investigation since it may improve current management of diffuse large B-cell lymphoma patients with central nervous system involvement.

  11. 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

  12. Osteoprotegerin (OPG) activates integrin, focal adhesion kinase (FAK), and Akt signaling in ovarian cancer cells to attenuate TRAIL-induced apoptosis

    PubMed Central

    2013-01-01

    Background Resistance to apoptosis is a major problem in ovarian cancer (OC) and correlates with poor prognosis. Osteoprotegerin (OPG) is a soluble secreted factor that acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). OPG has been reported to attenuate TRAIL-induced apoptosis in a variety of cancer cells, including OC cells. OPG-mediated protection against TRAIL has been attributed to its decoy receptor function. However, OPG activates integrin/focal adhesion kinase (FAK) signaling in endothelial cells. In OC cells, activation of integrin/FAK signaling inhibits TRAIL-induced apoptosis. Based on these observations, we hypothesized that OPG could attenuate TRAIL-induced apoptosis in OC cells through integrin/FAK signaling. Methods In vitro experiments including immunoblots, colony formation assays, and apoptosis measurements were used to assess the effect of OPG on TRAIL-induced apoptosis. Results Exogenous OPG protected from TRAIL-induced apoptosis in a TRAIL binding-independent manner and OPG protection was αvβ3 and αvβ5 integrin/FAK signaling-dependent. Moreover, OPG-mediated activation of integrin/FAK signaling resulted in the activation of Akt. Inhibition of both integrin/FAK and Akt signaling significantly inhibited OPG-mediated attenuation of TRAIL-induced apoptosis. Although OPG also stimulated ERK1/2 phosphorylation, inhibition of ERK1/2 signaling did not significantly altered OPG protection. Conclusions Our studies provide evidence, for the first time, that OPG can attenuate TRAIL-induced apoptosis in a TRAIL binding-independent manner through the activation of integrin/FAK/Akt signaling in OC cells. PMID:24267510

  13. 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

  14. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    PubMed

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

  15. Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers.

    PubMed

    Basuroy, Shyamali; Dunagan, Mitzi; Sheth, Parimal; Seth, Ankur; Rao, R K

    2010-07-01

    Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl]pyrazolo[3-4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-Src(Y527F) enhanced cell migration, whereas the expression of dominant negative c-Src(K296R/Y528F) attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130(CAS) in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism. PMID:20378826

  16. 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.

  17. The potential of sarcospan in adhesion complex replacement therapeutics for the treatment of muscular dystrophy.

    PubMed

    Marshall, Jamie L; Kwok, Yukwah; McMorran, Brian J; Baum, Linda G; Crosbie-Watson, Rachelle H

    2013-09-01

    Three adhesion complexes span the sarcolemma and facilitate critical connections between the extracellular matrix and the actin cytoskeleton: the dystrophin- and utrophin-glycoprotein complexes and α7β1 integrin. Loss of individual protein components results in a loss of the entire protein complex and muscular dystrophy. Muscular dystrophy is a progressive, lethal wasting disease characterized by repetitive cycles of myofiber degeneration and regeneration. Protein-replacement therapy offers a promising approach for the treatment of muscular dystrophy. Recently, we demonstrated that sarcospan facilitates protein-protein interactions amongst the adhesion complexes and is an important potential therapeutic target. Here, we review current protein-replacement strategies, discuss the potential benefits of sarcospan expression, and identify important experiments that must be addressed for sarcospan to move to the clinic. PMID:23601082

  18. Role of dystrophins and utrophins in platelet adhesion process.

    PubMed

    Cerecedo, Doris; Mondragón, Ricardo; Cisneros, Bulmaro; Martínez-Pérez, Francisco; Martínez-Rojas, Dalila; Rendón, Alvaro

    2006-07-01

    Platelets are crucial at the site of vascular injury, adhering to the sub-endothelial matrix through receptors on their surface, leading to cell activation and aggregation to form a haemostatic plug. Platelets display focal adhesions as well as stress fibres to contract and facilitate expulsion of growth and pro-coagulant factors contained in the granules and to constrict the clot. The interaction of F-actin with different actin-binding proteins determines the properties and composition of the focal adhesions. Recently, we demonstrated the presence of dystrophin-associated protein complex corresponding to short dystrophin isoforms (Dp71d and Dp71) and the uthophin gene family (Up400 and Up71), which promote shape change, adhesion, aggregation, and granule centralisation. To elucidate participation of both complexes during the platelet adhesion process, their potential association with integrin beta-1 fraction and the focal adhesion system (alpha-actinin, vinculin and talin) was evaluated by immunofluorescence and immunoprecipitation assays. It was shown that the short dystrophin-associated protein complex participated in stress fibre assembly and in centralisation of cytoplasmic granules, while the utrophin-associated protein complex assembled and regulated focal adhesions. The simultaneous presence of dystrophin and utrophin complexes indicates complementary structural and signalling mechanisms to the actin network, improving the platelet haemostatic role.

  19. Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly.

    PubMed

    Horton, Edward R; Byron, Adam; Askari, Janet A; Ng, Daniel H J; Millon-Frémillon, Angélique; Robertson, Joseph; Koper, Ewa J; Paul, Nikki R; Warwood, Stacey; Knight, David; Humphries, Jonathan D; Humphries, Martin J

    2015-12-01

    Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome is likely to represent a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community. PMID:26479319

  20. Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly

    PubMed Central

    Askari, Janet A.; Ng, Daniel H. J.; Millon-Frémillon, Angélique; Robertson, Joseph; Koper, Ewa J.; Paul, Nikki R.; Warwood, Stacey; Knight, David; Humphries, Jonathan D.; Humphries, Martin J.

    2015-01-01

    Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is currently lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this dataset reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome likely represents a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community. PMID:26479319

  1. Mediation of the migration of endothelial cells and fibroblasts on polyurethane nanocomposites by the activation of integrin-focal adhesion kinase signaling.

    PubMed

    Hung, Huey-Shan; Chu, Mei-Yun; Lin, Chien-Hsun; Wu, Chia-Ching; Hsu, Shan-hui

    2012-01-01

    Model surfaces of polyurethane-gold nanocomposites (PU-Au) were used to examine cell behavior on nanophase-segregated materials. Previously we showed that endothelial cell (EC) migration on these materials was modulated by the PI3K/Akt/eNOS pathway. The present study, investigated the expressions of alpha5/beta3 (α5β3) integrin, focal adhesion kinase (FAK), and other downstream signal molecules such as the Rho family and matrix metalloproteinases 2 (MMP-2) induced by the materials in two different cells, that is bovine arterial endothelial cells (BAEC) and human skin fibroblasts (HSF). Both cells proliferated better on the more phase-separated PU-Au 43.5 ppm than on the less phase-separated controls (PU and PU-Au 174 ppm). On PU-Au 43.5 ppm, BAEC compared to HSF had denser actin fibers and were more extended. BAEC became rounded with Y-27632 treatment and shrunk with LY294002 treatment. Treatment by inhibitors only caused slight changes in HSF. The migration distance of BAEC on PU-Au 43.5 ppm was greater than that of HSF, and was significantly reduced by LY294002 or Y-27632 but not SU-1498. The expressions of p-FAK, p-RhoA, p-Rac/Cdc42, MMP2, and α5β3 integrin induced by PU-Au 43.5 ppm were more pronounced in BAEC versus HSF. Further enhancement in MMP2 and α5β3 integrin expressions by FAK-GFP transfection was more remarkable for cells on PU-Au 43.5 ppm. Our findings suggested that the integrin α5β3/FAK pathway may be induced by nanophase-separated materials in both ECs and fibroblasts to promote their proliferation/migration, while the crosstalk between the PI3K/Akt/eNOS pathway and FAK/Rho-GTPase activation may account for the greater effect in ECs than in fibroblasts.

  2. 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

  3. Underwater contact adhesion and microarchitecture in polyelectrolyte complexes actuated by solvent exchange

    PubMed Central

    Seo, Sungbaek; Kaufman, Yair; Israelachvili, Jacob N.; Waite, J. Herbert

    2016-01-01

    Polyelectrolyte complexation is critical to the formation and properties of many biological and polymeric materials, and is typically initiated by aqueous mixing1 followed by fluid–fluid phase separation, such as coacervation2–5. Yet little to nothing is known about how coacervates evolve into intricate solid microarchitectures. Inspired by the chemical features of the cement proteins of the sandcastle worm, here we report a versatile and strong wet-contact microporous adhesive resulting from polyelectrolyte complexation triggered by solvent exchange. After premixing a catechol-functionalized weak polyanion with a polycation in dimethyl sulphoxide (DMSO), the solution was applied underwater to various substrates whereupon electrostatic complexation, phase inversion, and rapid setting were simultaneously actuated by water–DMSO solvent exchange. Spatial and temporal coordination of complexation, inversion and setting fostered rapid (~25 s) and robust underwater contact adhesion (Wad ≥ 2 J m−2) of complexed catecholic polyelectrolytes to all tested surfaces including plastics, glasses, metals and biological materials. PMID:26779881

  4. Underwater contact adhesion and microarchitecture in polyelectrolyte complexes actuated by solvent exchange.

    PubMed

    Zhao, Qiang; Lee, Dong Woog; Ahn, B Kollbe; Seo, Sungbaek; Kaufman, Yair; Israelachvili, Jacob N; Waite, J Herbert

    2016-04-01

    Polyelectrolyte complexation is critical to the formation and properties of many biological and polymeric materials, and is typically initiated by aqueous mixing followed by fluid-fluid phase separation, such as coacervation. Yet little to nothing is known about how coacervates evolve into intricate solid microarchitectures. Inspired by the chemical features of the cement proteins of the sandcastle worm, here we report a versatile and strong wet-contact microporous adhesive resulting from polyelectrolyte complexation triggered by solvent exchange. After premixing a catechol-functionalized weak polyanion with a polycation in dimethyl sulphoxide (DMSO), the solution was applied underwater to various substrates whereupon electrostatic complexation, phase inversion, and rapid setting were simultaneously actuated by water-DMSO solvent exchange. Spatial and temporal coordination of complexation, inversion and setting fostered rapid (∼25 s) and robust underwater contact adhesion (Wad ≥ 2 J m(-2)) of complexed catecholic polyelectrolytes to all tested surfaces including plastics, glasses, metals and biological materials.

  5. Underwater contact adhesion and microarchitecture in polyelectrolyte complexes actuated by solvent exchange.

    PubMed

    Zhao, Qiang; Lee, Dong Woog; Ahn, B Kollbe; Seo, Sungbaek; Kaufman, Yair; Israelachvili, Jacob N; Waite, J Herbert

    2016-04-01

    Polyelectrolyte complexation is critical to the formation and properties of many biological and polymeric materials, and is typically initiated by aqueous mixing followed by fluid-fluid phase separation, such as coacervation. Yet little to nothing is known about how coacervates evolve into intricate solid microarchitectures. Inspired by the chemical features of the cement proteins of the sandcastle worm, here we report a versatile and strong wet-contact microporous adhesive resulting from polyelectrolyte complexation triggered by solvent exchange. After premixing a catechol-functionalized weak polyanion with a polycation in dimethyl sulphoxide (DMSO), the solution was applied underwater to various substrates whereupon electrostatic complexation, phase inversion, and rapid setting were simultaneously actuated by water-DMSO solvent exchange. Spatial and temporal coordination of complexation, inversion and setting fostered rapid (∼25 s) and robust underwater contact adhesion (Wad ≥ 2 J m(-2)) of complexed catecholic polyelectrolytes to all tested surfaces including plastics, glasses, metals and biological materials. PMID:26779881

  6. Focal cerebral vasculitis associated with circulating immune complexes and brain irradiation

    SciTech Connect

    Groothuis, D.R.; Mikhael, M.A.

    1986-06-01

    In this report we describe a patient with a benign glioma treated with surgery and radiation. After a period of stability he developed subacute bacterial endocarditis, and deteriorated neurologically. Computed tomographic scans did not show recurrent tumor. An angiogram showed vasculitis restricted to the previously irradiated area. Secondary to subacute bacterial endocarditis was the presence of high levels of circulating immune complexes. His neurological status was unchanged after antibiotics, but improved after treatment with dexamethasone. We interpret the clinical course as an immune-complex-mediated vasculitis superimposed on a subclinical radiation vasculitis. This case supports the hypothesis that immune mechanisms may be involved in delayed radiation injury to the nervous system.

  7. Retention of a cell adhesion complex at the paranodal junction requires the cytoplasmic region of Caspr.

    PubMed

    Gollan, Leora; Sabanay, Helena; Poliak, Sebastian; Berglund, Erik O; Ranscht, Barbara; Peles, Elior

    2002-06-24

    An axonal complex of cell adhesion molecules consisting of Caspr and contactin has been found to be essential for the generation of the paranodal axo-glial junctions flanking the nodes of Ranvier. Here we report that although the extracellular region of Caspr was sufficient for directing it to the paranodes in transgenic mice, retention of the Caspr-contactin complex at the junction depended on the presence of an intact cytoplasmic domain of Caspr. Using immunoelectron microscopy, we found that a Caspr mutant lacking its intracellular domain was often found within the axon instead of the junctional axolemma. We further show that a short sequence in the cytoplasmic domain of Caspr mediated its binding to the cytoskeleton-associated protein 4.1B. Clustering of contactin on the cell surface induced coclustering of Caspr and immobilized protein 4.1B at the plasma membrane. Furthermore, deletion of the protein 4.1B binding site accelerated the internalization of a Caspr-contactin chimera from the cell surface. These results suggest that Caspr serves as a "transmembrane scaffold" that stabilizes the Caspr/contactin adhesion complex at the paranodal junction by connecting it to cytoskeletal components within the axon. PMID:12082082

  8. Vanadium(IV) complexes inhibit adhesion, migration and colony formation of UMR106 osteosarcoma cells.

    PubMed

    Molinuevo, María S; Cortizo, Ana M; Etcheverry, Susana B

    2008-04-01

    Vanadium is a trace element widely distributed in the environment. In vertebrates it is mainly stored in bone tissue. The unique cellular environment in the bone and the variety of interactions that mediate cancer metastasis determine that certain types of cancer, such as breast and prostate cancer, preferentially metastize in the skeleton. Since this effect usually signifies serious morbidity and grave prognosis there is an increasing interest in the development of new treatments for this pathology. The present work shows that vanadium complexes can inhibit some parameters related to cancer metastasis such as cell adhesion, migration and clonogenicity. We have also investigated the role of protein kinase A in these processes.

  9. 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

  10. Effect of different adhesion strategies on bond strength of resin composite to composite-dentin complex.

    PubMed

    Özcan, M; Pekkan, G

    2013-01-01

    Service life of discolored and abraded resin composite restorations could be prolonged by repair or relayering actions. Composite-composite adhesion can be achieved successfully using some surface conditioning methods, but the most effective adhesion protocol for relayering is not known when the composite restorations are surrounded with dentin. This study evaluated the effect of three adhesion strategies on the bond strength of resin composite to the composite-dentin complex. Intact maxillary central incisors (N=72, n=8 per subgroup) were collected and the coronal parts of the teeth were embedded in autopolymerized poly(methyl tfr54methacrylate) surrounded by a polyvinyl chloride cylinder. Cylindrical cavities (diameter: 2.6 mm; depth: 2 mm) were opened in the middle of the labial surfaces of the teeth using a standard diamond bur, and the specimens were randomly divided into three groups. Two types of resin composite, namely microhybrid (Quadrant Anterior Shine; AS) and nanohybrid (Grandio; G), were photo-polymerized incrementally in the cavities according to each manufacturer's recommendations. The composite-enamel surfaces were ground finished to 1200-grit silicone carbide paper until the dentin was exposed. The surfaces of the substrate composites and the surrounding dentin were conditioned according to one of the following adhesion protocols: protocol 1: acid-etching (dentin) + silica coating (composite) + silanization (composite) + primer (dentin) + bonding agent (dentin + composite); protocol 2: silica coating (composite) + acid-etching (dentin) + silanization (composite) + primer (dentin) + bonding agent (dentin + composite); and protocol 3: acid-etching (dentin) + primer (dentin) + silanization (composite) + bonding agent (dentin + composite). Applied primer and bonding agents were the corresponding materials of the composite manufacturer. Silica coating (CoJet sand, 30 μm) was achieved using a chairside air-abrasion device (distance: 10 mm; duration

  11. Effect of different adhesion strategies on bond strength of resin composite to composite-dentin complex.

    PubMed

    Özcan, M; Pekkan, G

    2013-01-01

    Service life of discolored and abraded resin composite restorations could be prolonged by repair or relayering actions. Composite-composite adhesion can be achieved successfully using some surface conditioning methods, but the most effective adhesion protocol for relayering is not known when the composite restorations are surrounded with dentin. This study evaluated the effect of three adhesion strategies on the bond strength of resin composite to the composite-dentin complex. Intact maxillary central incisors (N=72, n=8 per subgroup) were collected and the coronal parts of the teeth were embedded in autopolymerized poly(methyl tfr54methacrylate) surrounded by a polyvinyl chloride cylinder. Cylindrical cavities (diameter: 2.6 mm; depth: 2 mm) were opened in the middle of the labial surfaces of the teeth using a standard diamond bur, and the specimens were randomly divided into three groups. Two types of resin composite, namely microhybrid (Quadrant Anterior Shine; AS) and nanohybrid (Grandio; G), were photo-polymerized incrementally in the cavities according to each manufacturer's recommendations. The composite-enamel surfaces were ground finished to 1200-grit silicone carbide paper until the dentin was exposed. The surfaces of the substrate composites and the surrounding dentin were conditioned according to one of the following adhesion protocols: protocol 1: acid-etching (dentin) + silica coating (composite) + silanization (composite) + primer (dentin) + bonding agent (dentin + composite); protocol 2: silica coating (composite) + acid-etching (dentin) + silanization (composite) + primer (dentin) + bonding agent (dentin + composite); and protocol 3: acid-etching (dentin) + primer (dentin) + silanization (composite) + bonding agent (dentin + composite). Applied primer and bonding agents were the corresponding materials of the composite manufacturer. Silica coating (CoJet sand, 30 μm) was achieved using a chairside air-abrasion device (distance: 10 mm; duration

  12. Interfacial tension of complex coacervated mussel adhesive protein according to the Hofmeister series.

    PubMed

    Lim, Seonghye; Moon, Dustin; Kim, Hyo Jeong; Seo, Jeong Hyun; Kang, In Seok; Cha, Hyung Joon

    2014-02-01

    Complex coacervation is a liquid-liquid phase separation in a colloidal system of two oppositely charged polyelectrolytes or colloids. The interfacial tension of the coacervate phase is the key parameter for micelle formation and interactions with the encapsulating material. However, the relationship between interfacial tensions and various salt solutions is poorly understood in complex coacervation. In the present work, the complex coacervate dynamics of recombinant mussel adhesive protein (MAP) with hyaluronic acid (HA) were determined in the presence of Hofmeister series salt ions. Using measurements of absorbance, hydrodynamic diameter, capillary force, and receding contact angle in the bulk phase, the interfacial tensions of complex coacervated MAP/HA were determined to be 0.236, 0.256, and 0.287 mN/m in 250 mM NaHCOO, NaCl, and NaNO3 solutions, respectively. The sequences of interfacial tensions and contact angles of the complex coacervates in the presence of three sodium salts with different anions were found to follow the Hofmeister ordering. The tendency of interfacial tension between the coacervate and dilute phases in the presence of different types of Hofmeister salt ions could provide a better understanding of Hofmeister effects on complex coacervated materials based on the protein-polysaccharide system. This information can also be utilized for microencapsulation and adsorption by controlling intramolecular interactions. In addition, the injection molding dynamics of mussel byssus formation was potentially explained based on the measured interfacial tension of coacervated MAP. PMID:24490867

  13. Simvastatin disrupts cytoskeleton and decreases cardiac fibroblast adhesion, migration and viability.

    PubMed

    Copaja, Miguel; Venegas, Daniel; Aranguiz, Pablo; Canales, Jimena; Vivar, Raul; Avalos, Yennifer; Garcia, Lorena; Chiong, Mario; Olmedo, Ivonne; Catalán, Mabel; Leyton, Lisette; Lavandero, Sergio; Díaz-Araya, Guillermo

    2012-03-29

    Statins reduce the isoprenoids farnesyl and geranylgeranyl pyrophosphate, essential intermediates, which control a diversity of cellular events such as cytoskeleton integrity, adhesion, migration and viability. Cardiac fibroblasts are the major non-myocyte cell constituent in the normal heart, and play a key role in the maintenance of extracellular matrix. The effects of simvastatin on cardiac fibroblast processes previously mentioned remain unknown. Our aims were to investigate the effects of simvastatin on cytoskeleton structure and focal adhesion complex assembly and their relationships with cell adhesion, migration and viability in cultured cardiac fibroblasts. To this end, cells were treated with simvastatin for 24 h and changes in actin cytoskeleton, levels of vimentin and paxillin as well as their subcellular localization were analyzed by Western blot and immunocytochemistry, respectively. Cell adhesion to plastic or collagen coated dishes, migration in Transwell chambers, and cell viability were analyzed after simvastatin treatment. Our results show that simvastatin disrupts actin cytoskeleton and focal adhesion complex evaluated by phalloidin stain and immunocytochemistry for paxillin and vinculin. All these effects occurred by a cholesterol synthesis-independent mechanism. Simvastatin decreased cell adhesion, migration and viability in a concentration-dependent manner. Finally, simvastatin decreased angiotensin II-induced phospho-paxillin levels and cell adhesion. We concluded that simvastatin disrupts cytoskeleton integrity and focal adhesion complex assembly in cultured cardiac fibroblasts by a cholesterol-independent mechanism and consequently decreases cell migration, adhesion and viability. PMID:22306966

  14. 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

  15. Structure of Reovirus σ1 in Complex with Its Receptor Junctional Adhesion Molecule-A

    PubMed Central

    Kirchner, Eva; Guglielmi, Kristen M.; Strauss, Holger M.; Dermody, Terence S.; Stehle, Thilo

    2008-01-01

    Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. Mammalian reoviruses are highly useful experimental models for studies of viral pathogenesis and show promise as vectors for oncolytics and vaccines. Reoviruses engage cells by binding to carbohydrates and the immunoglobulin superfamily member, junctional adhesion molecule-A (JAM-A). JAM-A exists at the cell surface as a homodimer formed by extensive contacts between its N-terminal immunoglobulin-like domains. We report the crystal structure of reovirus attachment protein σ1 in complex with a soluble form of JAM-A. The σ1 protein disrupts the JAM-A dimer, engaging a single JAM-A molecule via virtually the same interface that is used for JAM-A homodimerization. Thus, reovirus takes advantage of the adhesive nature of an immunoglobulin-superfamily receptor by usurping the ligand-binding site of this molecule to attach to the cell surface. The dissociation constant (KD) of the interaction between σ1 and JAM-A is 1,000-fold lower than that of the homophilic interaction between JAM-A molecules, indicating that JAM-A strongly prefers σ1 as a ligand. Analysis of reovirus mutants engineered by plasmid-based reverse genetics revealed residues in σ1 required for binding to JAM-A and infectivity of cultured cells. These studies define biophysical mechanisms of reovirus cell attachment and provide a platform for manipulating reovirus tropism to enhance vector targeting. PMID:19079583

  16. The PSGL-1–L-selectin signaling complex regulates neutrophil adhesion under flow

    PubMed Central

    Stadtmann, Anika; Germena, Giulia; Block, Helena; Boras, Mark; Rossaint, Jan; Sundd, Prithu; Lefort, Craig; Fisher, Charles I.; Buscher, Konrad; Gelschefarth, Bernadette; Urzainqui, Ana; Gerke, Volker; Ley, Klaus

    2013-01-01

    Neutrophils are recruited from the blood to sites of inflammation, where they contribute to immune defense but may also cause tissue damage. During inflammation, neutrophils roll along the microvascular endothelium before arresting and transmigrating. Arrest requires conformational activation of the integrin lymphocyte function–associated antigen 1 (LFA-1), which can be induced by selectin engagement. Here, we demonstrate that a subset of P-selectin glycoprotein ligand-1 (PSGL-1) molecules is constitutively associated with L-selectin. Although this association does not require the known lectin-like interaction between L-selectin and PSGL-1, the signaling output is dependent on this interaction and the cytoplasmic tail of L-selectin. The PSGL-1–L-selectin complex signals through Src family kinases, ITAM domain–containing adaptor proteins, and other kinases to ultimately result in LFA-1 activation. The PSGL-1–L-selectin complex–induced signaling effects on neutrophil slow rolling and recruitment in vivo demonstrate the functional importance of this pathway. We conclude that this is a signaling complex specialized for sensing adhesion under flow. PMID:24127491

  17. High affinity neurexin binding to cell adhesion G-protein-coupled receptor CIRL1/latrophilin-1 produces an intercellular adhesion complex.

    PubMed

    Boucard, Antony A; Ko, Jaewon; Südhof, Thomas C

    2012-03-16

    The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for α-latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice site 4 (SS4). Using saturation binding assays, we showed that neurexins lacking an insert at SS4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/threonine-rich, hormone-binding, and G-protein-coupled receptor autoproteolysis-inducing (GAIN) domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction can be trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well characterized interaction with intracellular SH3 and multiple ankyrin repeats adaptor proteins (SHANK) and could form a trans-synaptic complex with presynaptic neurexins.

  18. Plasticity of hydrogen bond networks regulates mechanochemistry of cell adhesion complexes

    PubMed Central

    Chakrabarti, Shaon; Hinczewski, Michael; Thirumalai, D.

    2014-01-01

    Mechanical forces acting on cell adhesion receptor proteins regulate a range of cellular functions by formation and rupture of noncovalent interactions with ligands. Typically, force decreases the lifetimes of intact complexes (“slip bonds”), making the discovery that these lifetimes can also be prolonged (“catch bonds”) a surprise. We created a microscopic analytic theory by incorporating the structures of selectin and integrin receptors into a conceptual framework based on the theory of stochastic equations, which quantitatively explains a wide range of experimental data (including catch bonds at low forces and slip bonds at high forces). Catch bonds arise due to force-induced remodeling of hydrogen bond networks, a finding that also accounts for unbinding in structurally unrelated integrin–fibronectin and actomyosin complexes. For the selectin family, remodeling of hydrogen bond networks drives an allosteric transition resulting in the formation of the maximum number of hydrogen bonds determined only by the structure of the receptor and independent of the ligand. A similar transition allows us to predict the increase in the number of hydrogen bonds in a particular allosteric state of α5β1 integrin–fibronectin complex, a conformation which is yet to be crystallized. We also make a testable prediction that a single point mutation (Tyr51Phe) in the ligand associated with selectin should dramatically alter the nature of the catch bond compared with the wild type. Our work suggests that nature uses a ductile network of hydrogen bonds to engineer function over a broad range of forces. PMID:24927549

  19. Implications for complex cognition from the hafting of tools with compound adhesives in the Middle Stone Age, South Africa

    PubMed Central

    Wadley, Lyn; Hodgskiss, Tamaryn; Grant, Michael

    2009-01-01

    Compound adhesives made from red ochre mixed with plant gum were used in the Middle Stone Age (MSA), South Africa. Replications reported here suggest that early artisans did not merely color their glues red; they deliberately effected physical transformations involving chemical changes from acidic to less acidic pH, dehydration of the adhesive near wood fires, and changes to mechanical workability and electrostatic forces. Some of the steps required for making compound adhesive seem impossible without multitasking and abstract thought. This ability suggests overlap between the cognitive abilities of modern people and people in the MSA. Our multidisciplinary analysis provides a new way to recognize complex cognition in the MSA without necessarily invoking the concept of symbolism. PMID:19433786

  20. Activation of CD8-dependent cytotoxic T lymphocyte adhesion and degranulation by peptide class I antigen complexes.

    PubMed

    Kane, K P; Mescher, M F

    1993-06-01

    Activation of CTL requires engagement of both the TCR and the CD8 coreceptor. Immobilized class I proteins and in vitro-formed peptide class I Ag complexes have been used to examine the relative contributions of TCR and CD8 to the adhesion and response of cloned, class I-restricted CTL. The extent of degranulation was found to be directly proportional to the concentration of peptide used to pulse class I, suggesting that activation is a direct function of TCR occupancy level. In contrast, activation of degranulation as a function of the amount of class I on the surface displayed a marked threshold density dependence. Essentially the same density dependence was found for the response of CTL to fluid phase anti-TCR mAb and non-Ag class I, indicating that CD8-class I interaction must exceed a threshold before effective cosignaling can occur. Adhesion and degranulation of CTL was minimal in response to in vitro peptide-class I complexes prepared at a class I density below the threshold. However, the same density of peptide class I initiated both adhesion and response if additional non-Ag class I was coimmobilized on the same surface at levels above threshold. Thus, when surface levels of peptide class I complex are low, as is likely to be the case under physiologic conditions, the level of TCR occupancy achieved is, by itself, insufficient to mediate cell adhesion or activate degranulation. The results demonstrate, however, that low TCR occupancy is sufficient to provide the signal to prime CD8. Provided that the surface density of class I is sufficiently high, CD8 then mediates strong adhesion and provides the costimulatory signal(s) to activate response.

  1. Endothelial activation by hydrogen peroxide. Selective increases of intercellular adhesion molecule-1 and major histocompatibility complex class I.

    PubMed Central

    Bradley, J. R.; Johnson, D. R.; Pober, J. S.

    1993-01-01

    Products of activated leukocytes may alter vascular endothelial cell (EC) function. For example, ECs respond to leukocyte-derived cytokines, such as tumor necrosis factor (TNF) or interleukin-1, by reversibly altering levels of expression of specific gene products that promote inflammation. In contrast, hydrogen peroxide, a product of TNF-activated neutrophils, can produce irreversible EC injury and death. In this study, we have investigated the effects of subinjurious concentrations of hydrogen peroxide on EC inflammatory functions. Treatment with 50 to 100 mumol/L hydrogen peroxide selectively increases surface expression of intercellular adhesion molecule-1 and major histocompatibility complex class I, but not endothelial leukocyte adhesion molecule-1 (also known as E-selectin), vascular cell adhesion molecule-1, or gp96, a constitutively expressed EC surface protein. Increased major histocompatibility complex class I and intercellular adhesion molecule-1 surface expression is associated with specifically increased messenger RNA levels, suggesting selective endothelial gene activation. Hydrogen peroxide does not activate the transcription factor Nuclear Factor kappa B, an important mediator of TNF-induced gene expression. Co-treatment with hydrogen peroxide inhibits TNF-induced gene expression at 4 hours, an effect which can be attributed to reversible inhibition of TNF binding to EC surface receptors. Hydrogen peroxide also antagonizes the actions of interleukin-1. At 24 hours, TNF and hydrogen peroxide produce, at most, additive increases in intercellular adhesion molecule-1 and major histocompatibility complex class I. These results suggest that subinjurious concentrations of hydrogen peroxide can activate endothelium and that the effects of hydrogen peroxide on ECs differ from those of inflammatory cytokines. Images Figure 3 Figure 4 Figure 5 PMID:8098585

  2. The direct effect of Focal Adhesion Kinase (FAK), dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

    PubMed Central

    2009-01-01

    Background Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. Methods To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible) system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD), and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Results Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p < 0.05) by FAKsiRNA. Conclusion Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal

  3. Surface modulation of complex stiffness via layer-by-layer assembly as a facile strategy for selective cell adhesion.

    PubMed

    Chang, Hao; Zhang, He; Hu, Mi; Chen, Xia-chao; Ren, Ke-feng; Wang, Jin-lei; Ji, Jian

    2015-02-01

    In-stent restenosis and thrombosis are the main severe problems that occur after the percutaneous vascular intervention. The competition between endothelial cells (ECs) and smooth muscle cells (SMCs) plays a key role during these pathological changes. The regulation of this competition offers new opportunities to design biomaterials in the cardiovascular fields. Bioactive molecules have been typically employed to increase EC adhesion and thereafter to enhance EC competitiveness; however, this method is associated with limitations from the point of view of practical and industrial applications. Herein, we present an approach to enhance EC competitiveness over that of SMC through the selective EC adhesion, which is achieved by modulating a complex surface stiffness based on the technique of layer-by-layer (LbL) assembly. This complex stiffness can be achieved by regulating the thickness of multilayer films coordinating with a rigid underlying substrate. The selective cell adhesion is attributed to changes in the complex surface stiffness and a different intrinsic property between ECs and SMCs. This study provides a facile and broadly applicable approach for the purpose of the enhancement of EC competitiveness over that of SMC, which has great potential for the development of cell-based functional biomaterials in the cardiovascular field. PMID:26218126

  4. 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

  5. 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

  6. A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration

    PubMed Central

    Villari, Giulia; Jayo, Asier; Zanet, Jennifer; Fitch, Briana; Serrels, Bryan; Frame, Margaret; Stramer, Brian M.; Goult, Benjamin T.; Parsons, Maddy

    2015-01-01

    ABSTRACT Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here, we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK, also known as PTK2) and Src, and that this signalling pathway lies downstream of fascin–microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and might have implications for the design of therapies to target fascin in metastatic disease. PMID:26542021

  7. Microbial Adhesion and Biofilm Formation on Microfiltration Membranes: A Detailed Characterization Using Model Organisms with Increasing Complexity

    PubMed Central

    Vanysacker, L.; Denis, C.; Declerck, P.; Piasecka, A.; Vankelecom, I. F. J.

    2013-01-01

    Since many years, membrane biofouling has been described as the Achilles heel of membrane fouling. In the present study, an ecological assay was performed using model systems with increasing complexity: a monospecies assay using Pseudomonas aeruginosa or Escherichia coli separately, a duospecies assay using both microorganisms, and a multispecies assay using activated sludge with or without spiked P. aeruginosa. The microbial adhesion and biofilm formation were evaluated in terms of bacterial cell densities, species richness, and bacterial community composition on polyvinyldifluoride, polyethylene, and polysulfone membranes. The data show that biofouling formation was strongly influenced by the kind of microorganism, the interactions between the organisms, and the changes in environmental conditions whereas the membrane effect was less important. The findings obtained in this study suggest that more knowledge in species composition and microbial interactions is needed in order to understand the complex biofouling process. This is the first report describing the microbial interactions with a membrane during the biofouling development. PMID:23986906

  8. Adhesives in larynx repair.

    PubMed

    Lyons, M B; Lyons, G D; Webster, D; Wheeler, V R

    1989-04-01

    Guinea pig laryngeal fractures were used as a model to compare the ease of application and effectiveness of the fibrinogen-adhesive system with the ease of application and effectiveness of cyanoacrylate glue and control fractures stinted with contralateral gelatin film. Seven fibrin adhesive-treated and two cyanoacrylate glue-treated guinea pigs were perfused after 60 and 35 days, respectively. The larynges were serial sectioned, and the wound sites were compared. The fibrinogen adhesive system was easier to dispense than cyanoacrylate glue, did not require a completely dry surface, and stabilized within 3 minutes. Cartilage segment alignment with focal, complete fracture healing and symmetrical chondrocyte proliferation were seen in fibrogen adhesive-stinted larynges. In the cyanoacrylate glue-treated larynges, there was no alignment and minimal, asymmetrical chondrocyte proliferation. Gelatin film-stinted controls exhibited similar features. Thus, fibrogen adhesive was easier to apply and more effectively bound laryngeal fractures than cyanoacrylate glue or gelatin film.

  9. CD4 binding to major histocompatibility complex class II antigens induces LFA-1-dependent and -independent homotypic adhesion of B lymphocytes.

    PubMed

    Kansas, G S; Cambier, J C; Tedder, T F

    1992-01-01

    T helper cells recognize processed antigen (Ag) in the context of major histocompatibility complex (MHC) class II antigens present on the surface of B cells and other Ag-presenting cells. This interaction is mediated through the T cell receptor complex with associate recognition of class II molecules by the CD4 molecule. In this study, the binding of a soluble recombinant CD4/Ig heavy chain fusion protein (CD4-gamma 3) or monoclonal antibody (mAb) to class II antigens on human B cells was shown to induce rapid and specific homotypic adhesion of B cells and most B lymphoblastoid cell lines. mAb reactive with CD4 inhibited CD4-gamma 3-induced adhesion and a mutant B lymphoblastoid cell line deficient in class II antigens failed to respond. Induction of homotypic adhesion was dependent on energy metabolism and a functional cytoskeleton, and class II+ pre-B cells did not exhibit adhesion in response to these stimuli, suggesting that cross-linking of class II molecules generated a transmembrane signal and did not simply aggregate cells. In addition, MHC class II-induced adhesion was Fc receptor independent, as 15 mAb of different Ig isotypes reactive with HLA-D or HLA-DQ gene products induced adhesion. Anti-class II mAb and CD4-gamma 3 were able to induce adhesion at concentrations as low as 10 ng/ml and 100 ng/ml, respectively. Suboptimal stimulation of B cell lines through HLA-D antigens induced homotypic adhesion that was dependent on the activation of LFA-1 (CD11a/CD18), and which could be blocked by specific mAb. However, at greater signal strengths, adhesion was not blocked by mAb against the known adhesion receptors, suggesting the induction of a novel adhesion pathway. Consistent with this, homotypic adhesion induced by engagement of MHC class II antigens was observed with LFA-1-deficient B cell lines, and was independent of CD49d or CD18 expression. Thus, the direct engagement of B cell class II antigens by CD4 is likely to generate transmembrane signals which

  10. Magnaporthe oryzae Glycine-Rich Secretion Protein, Rbf1 Critically Participates in Pathogenicity through the Focal Formation of the Biotrophic Interfacial Complex

    PubMed Central

    Ishii-Minami, Naoko; Kawahara, Yoshihiro; Yoshida, Yuri; Okada, Kazunori; Ando, Sugihiro; Matsumura, Hideo; Terauchi, Ryohei; Minami, Eiichi; Nishizawa, Yoko

    2016-01-01

    Magnaporthe oryzae, the fungus causing rice blast disease, should contend with host innate immunity to develop invasive hyphae (IH) within living host cells. However, molecular strategies to establish the biotrophic interactions are largely unknown. Here, we report the biological function of a M. oryzae-specific gene, Required-for-Focal-BIC-Formation 1 (RBF1). RBF1 expression was induced in appressoria and IH only when the fungus was inoculated to living plant tissues. Long-term successive imaging of live cell fluorescence revealed that the expression of RBF1 was upregulated each time the fungus crossed a host cell wall. Like other symplastic effector proteins of the rice blast fungus, Rbf1 accumulated in the biotrophic interfacial complex (BIC) and was translocated into the rice cytoplasm. RBF1-knockout mutants (Δrbf1) were severely deficient in their virulence to rice leaves, but were capable of proliferating in abscisic acid-treated or salicylic acid-deficient rice plants. In rice leaves, Δrbf1 inoculation caused necrosis and induced defense-related gene expression, which led to a higher level of diterpenoid phytoalexin accumulation than the wild-type fungus did. Δrbf1 showed unusual differentiation of IH and dispersal of the normally BIC-focused effectors around the short primary hypha and the first bulbous cell. In the Δrbf1-invaded cells, symplastic effectors were still translocated into rice cells but with a lower efficiency. These data indicate that RBF1 is a virulence gene essential for the focal BIC formation, which is critical for the rice blast fungus to suppress host immune responses. PMID:27711180

  11. Structural analysis of the synaptic protein neuroligin and its beta-neurexin complex: determinants for folding and cell adhesion.

    PubMed

    Fabrichny, Igor P; Leone, Philippe; Sulzenbacher, Gerlind; Comoletti, Davide; Miller, Meghan T; Taylor, Palmer; Bourne, Yves; Marchot, Pascale

    2007-12-20

    The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a beta-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca2+-dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an alpha/beta-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism.

  12. Structural Analysis of the Synaptic Protein Neuroligin and Its β-Neurexin Complex: Determinants for Folding and Cell Adhesion

    PubMed Central

    Fabrichny, Igor P.; Leone, Philippe; Sulzenbacher, Gerlind; Comoletti, Davide; Miller, Meghan T.; Taylor, Palmer; Bourne, Yves; Marchot, Pascale

    2009-01-01

    SUMMARY The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a β-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca2+-dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an α/β-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism. PMID:18093521

  13. Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin

    PubMed Central

    1995-01-01

    Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E- , P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins termed catenins, including alpha-catenin, beta- catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that alpha- actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Moreover, we show that alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin- independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin. PMID:7790378

  14. Estrogen-Dependent Uterine Secretion of Osteopontin Activates Blastocyst Adhesion Competence

    PubMed Central

    Egashira, Mahiro; Bai, Rulan; Nomura, Nana; Nomura, Shintaro; Hirota, Yasushi; Sakurai, Toshihiro; Imakawa, Kazuhiko

    2012-01-01

    Embryo implantation is a highly orchestrated process that involves blastocyst-uterine interactions. This process is confined to a defined interval during gestation referred to as the “window of embryo implantation receptivity”. In mice this receptive period is controlled by ovarian estrogen and involves a coordination of blastocyst adhesion competence and uterine receptivity. Mechanisms coordinating the acquisition of blastocyst adhesion competence and uterine receptivity are largely unknown. Here, we show that ovarian estrogen indirectly regulates blastocyst adhesion competence. Acquisition of blastocyst adhesion competence was attributed to integrin activation (e.g. formation of adhesion complexes) rather than de novo integrin synthesis. Osteopontin (OPN) was identified as an estrogen-dependent uterine endometrial gland secretory factor responsible for activating blastocyst adhesion competence. Increased adhesion complex assembly in OPN-treated blastocysts was mediated through focal adhesion kinase (FAK)- and phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. These findings define for the first time specific regulatory components of an estrogen-dependent pathway coordinating blastocyst adhesion competence and uterine receptivity. PMID:23152823

  15. Engagement of major histocompatibility complex class I and class II molecules up-regulates intercellular adhesion of human B cells via a CD11/CD18-independent mechanism.

    PubMed

    Alcover, A; Juillard, V; Acuto, O

    1992-02-01

    We have studied the role of major histocompatibility complex (MHC) molecules in the regulation of intercellular adhesion of human B cells. We found that molecules able to bind to MHC class II molecules, such as monoclonal antibodies or staphylococcal enterotoxins, induced rapid and sustained homotypic adhesion of Epstein-Barr virus (EBV)-transformed B cell lines as well as peripheral blood B lymphocytes. Moreover, anti-MHC class I monoclonal antibodies also stimulated intercellular adherence. Adhesion induced upon MHC engagement was faster and stronger than that triggered by phorbol esters. It needed active metabolism, but divalent cations were not required. Monoclonal antibodies directed against LFA-1 (CD11a/CD18) or its ligand ICAM-1 (CD54) did not inhibit MHC class II-induced homotypic adhesion of various EBV-transformed B cell lines, nor of a variant of the B cell line Raji expressing very low LFA-1 surface levels. Moreover, EBV-transformed B cells from a severe lymphocyte adhesion deficiency patient, lacking surface CD11/CD18, also aggregated in response to anti-MHC class I or class II monoclonal antibodies. Together these data indicate that engagement of MHC molecules may transduce signals to B cells resulting in up-regulation of intercellular adhesion, via an LFA-1-independent mechanism. This may play a role in the stabilization of T cell/antigen-presenting cell conjugates at the moment of antigen recognition.

  16. [The focal renal lesions].

    PubMed

    Tuma, Jan

    2013-06-01

    The focal renal lesions are altogether common. Most frequently are found Columna Bertini hypertrophies (so called pseudotumors) and simple renal cysts. The role of sonography in the practice is to distinguish pseudotumors from real renal tumors, and simple renal cysts from complex cysts. The differentiation of complex renal cysts is possible with the help of the CEUS (= contrast enhanced ultrasound) and other imaging modalities such as CT or MRI. In these cases, the CEUS imaging agent has clear advantages over CT and MRI, because it is composed of gas bubbles, which are only slightly smaller than red blood cells and remains exclusively intravascularly while the CT and MRI contrast agents diffuse into the interstitial space without any real perfusion. The real tumors can be differentiated from certain focal non-tumorous changes based on the ultrasound and clinic. The further differentiation of individual kidney tumors and metastases using ultrasound, MRI, CT and CEUS is only partly possible. In all uncertain or unclear cases, therefore, an open or ultrasound-guided biopsy is useful.

  17. Alpha actinin-1 regulates cell-matrix adhesion organization in keratinocytes: consequences for skin cell motility.

    PubMed

    Hamill, Kevin J; Hiroyasu, Sho; Colburn, Zachary T; Ventrella, Rosa V; Hopkinson, Susan B; Skalli, Omar; Jones, Jonathan C R

    2015-04-01

    The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton, and matrix adhesions. In this study, we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via a small hairpin RNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity, and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower compared with their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells, and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion, and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics, and directed migration. PMID:25431851

  18. Drug delivery and cell interaction of adhesive poly(ethyleneimine)/sulfated polysaccharide complex particle films.

    PubMed

    Müller, Martin; Torger, Bernhard; Wehrum, Diana; Vehlow, David; Urban, Birgit; Woltmann, Beatrice; Hempel, Ute

    2015-01-01

    Herein, the authors report and review polyelectrolyte complex (PEC) nanoparticles (NPs) loaded with zoledronate (ZOL) and simvastatin and their effects on bone cells. PEC NPs are intended for modification of bone substitute materials. For characterization, they can be solution casted on germanium (Ge) substrates serving as analytically accessible model substrate. PEC NPs were generated by mixing poly(ethyleneimine) (PEI) either with linear cellulose sulfate (CS) or with branched dextransulfate (DS). Four important requirements for drug loaded PEC NPs and their films are addressed herein, which are the colloidal stability of PEC dispersions (1), interfacial stability (2), cytocompatibility (3), and retarded drug release (4). Dynamic light scattering measurements (DLS) showed that both PEI/CS and PEI/DS PEC NP were obtained with hydrodynamic radii in the range of 35-170 nm and were colloidally stable up to several months. Transmission FTIR spectroscopy evidenced that films of both systems were stable in contact to the release medium up to several days. ZOL-loaded PEI/CS nanoparticles, which were immobilized on an osteoblast-derived extracellular matrix, reduced significantly the resorption and the metabolic activity of human monocyte-derived osteoclasts. FTIR spectroscopy at cast PEC/drug films at Ge substrates revealed retarded drug releases in comparison to the pure drug films.

  19. Glycosylation Genes Expressed in Seam Cells Determine Complex Surface Properties and Bacterial Adhesion to the Cuticle of Caenorhabditis elegans

    PubMed Central

    Gravato-Nobre, Maria J.; Stroud, Dave; O'Rourke, Delia; Darby, Creg; Hodgkin, Jonathan

    2011-01-01

    The surface of the nematode Caenorhabditis elegans is poorly understood but critical for its interactions with the environment and with pathogens. We show here that six genes (bus-2, bus-4, and bus-12, together with the previously cloned srf-3, bus-8, and bus-17) encode proteins predicted to act in surface glycosylation, thereby affecting disease susceptibility, locomotory competence, and sexual recognition. Mutations in all six genes cause resistance to the bacterial pathogen Microbacterium nematophilum, and most of these mutations also affect bacterial adhesion and biofilm formation by Yersinia species, demonstrating that both infection and biofilm formation depend on interaction with complex surface carbohydrates. A new bacterial interaction, involving locomotory inhibition by a strain of Bacillus pumilus, reveals diversity in the surface properties of these mutants. Another biological property—contact recognition of hermaphrodites by males during mating—was also found to be impaired in mutants of all six genes. An important common feature is that all are expressed most strongly in seam cells, rather than in the main hypodermal syncytium, indicating that seam cells play the major role in secreting surface coat and consequently in determining environmental interactions. To test for possible redundancies in gene action, the 15 double mutants for this set of genes were constructed and examined, but no synthetic phenotypes were observed. Comparison of the six genes shows that each has distinctive properties, suggesting that they do not act in a linear pathway. PMID:20980242

  20. Molecular mechanisms involved in TFF3 peptide-mediated modulation of the E-cadherin/catenin cell adhesion complex.

    PubMed

    Meyer zum Büschenfelde, Dirk; Hoschützky, Heinz; Tauber, Rudolf; Huber, Otmar

    2004-05-01

    TFF3 is a member of the TFF-domain peptide family which is constitutively expressed in mucous epithelial tissues where it acts as a motogenic factor and plays an important role during epithelial restitution after wounding and during inflammation. In contrast to these beneficial functions, TFFs were also reported to be involved in cell scattering and tumor invasion. These changes in epithelial cell morphology and motility are associated with a modulation of cell contacts. In this respect, we here investigated the E-cadherin/catenin cell adhesion complex in FLAG-hTFF3-transfected HT29/B6 and MDCK cells. In hTFF3-transfected cells the amount of E-cadherin is reduced with a concomitant reduction of alpha- and beta-catenin levels. On one hand, E-cadherin expression is lowered at the transcriptional level as shown by multiplex RT-PCR analysis. This decrease does not depend on differences in the promoter methylation status as shown by methylation-specific PCR. On the other hand, pulse-chase experiments showed a reduction in the E-cadherin half-life in hTFF3-transfected cells reflecting increased E-cadherin degradation. In summary, hTFF3 induces transcriptional and posttranslational processes resulting in a modulation of E-cadherin-mediated cell-cell contacts that may play an important role in the paradoxical benefical and pathogenic function of TFF peptides.

  1. Gene expression analysis of tuberous sclerosis complex cortical tubers reveals increased expression of adhesion and inflammatory factors

    PubMed Central

    Boer, Karin; Crino, Peter B.; Gorter, Jan A.; Nellist, Mark; Jansen, Floor E.; Spliet, Wim G.M.; van Rijen, Peter C.; Wittink, Floyd R.A.; Breit, Timo M.; Troost, Dirk; Wadman, Wytse J.; Aronica, Eleonora

    2009-01-01

    Cortical tubers in patients with tuberous sclerosis complex are associated with disabling neurological manifestations, including intractable epilepsy. While these malformations are believed to result from the effects of TSC1 or TSC2 gene mutations, the molecular mechanisms leading to tuber formation, as well as the onset of seizures remain largely unknown. We used the Affymetrix Gene Chip platform to provide the first genome wide investigation of gene expression in surgically resected tubers, compared with histological normal perituberal tissue from the same patients or autopsy control tissue. We identified 2501 differentially expressed genes in cortical tubers compared with autopsy controls. Expression of genes associated with cell adhesion e.g., VCAM1, integrins and CD44, or with the inflammatory response, including complement factors, serpinA3, CCL2 and several cytokines, was increased in cortical tubers, whereas genes related to synaptic transmission e.g., the glial glutamate transporter GLT-1, and voltage-gated channel activity, exhibited lower expression. Gene expression in perituberal cortex was distinct from autopsy control cortex suggesting that even in the absence of tissue pathology the transcriptome is altered in TSC. Changes in gene expression yield insights into new candidate genes that may contribute to tuber formation or seizure onset, representing new targets for potential therapeutic development. PMID:19912235

  2. The Development of Complex Sentence Interpretation in Typically Developing Children Compared with Children with Specific Language Impairments or Early Unilateral Focal Lesions

    ERIC Educational Resources Information Center

    Dick, Frederic; Wulfeck, Beverly; Krupa-Kwiatkowski, Magda; Bates, Elizabeth

    2004-01-01

    This study compared sentence comprehension skills in typically developing children 5-17 years of age, children with language impairment (LI) and children with focal brain injuries (FL) acquired in the pre/perinatal period. Participants were asked to process sentences "on-line", choosing the agent in sentences that varied in syntactic complexity…

  3. 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.

  4. Characterization of the Cadherin-Catenin Complex of the Sea Anemone Nematostella vectensis and Implications for the Evolution of Metazoan Cell-Cell Adhesion.

    PubMed

    Clarke, Donald Nathaniel; Miller, Phillip W; Lowe, Christopher J; Weis, William I; Nelson, William James

    2016-08-01

    The cadherin-catenin complex (CCC) mediates cell-cell adhesion in bilaterian animals by linking extracellular cadherin-based adhesions to the actin cytoskeleton. However, it is unknown whether the basic organization of the complex is conserved across all metazoans. We tested whether protein interactions and actin-binding properties of the CCC are conserved in a nonbilaterian animal, the sea anemone Nematostella vectensis We demonstrated that N. vectensis has a complete repertoire of cadherin-catenin proteins, including two classical cadherins, one α-catenin, and one β-catenin. Using size-exclusion chromatography and multi-angle light scattering, we showed that α-catenin and β-catenin formed a heterodimer that bound N. vectensis Cadherin-1 and -2. Nematostella vectensis α-catenin bound F-actin with equivalent affinity as either a monomer or an α/β-catenin heterodimer, and its affinity for F-actin was, in part, regulated by a novel insert between the N- and C-terminal domains. Nematostella vectensis α-catenin inhibited Arp2/3 complex-mediated nucleation of actin filaments, a regulatory property previously thought to be unique to mammalian αE-catenin. Thus, despite significant differences in sequence, the key interactions of the CCC are conserved between bilaterians and cnidarians, indicating that the core function of the CCC as a link between cell adhesions and the actin cytoskeleton is ancestral in the eumetazoans. PMID:27189570

  5. Upregulation of adhesion complex proteins and fibronectin by human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin).

    PubMed

    Phan, T T; Allen, J; Hughes, M A; Cherry, G; Wojnarowska, F

    2000-01-01

    The fresh leaves and extract of the plant Chromolaena odorata are a traditional herbal treatment in developing countries for burns, soft tissue wounds and skin infections. We have previously shown that the extract had an effect on the growth and proliferation of keratinocytes and fibroblasts in culture. This study has demonstrated that Eupolin extract increased expression of several components of the adhesion complex and fibronectin by human keratinocytes. Using indirect immunofluorescence we found increased expression (dose-dependent) of laminin 5, laminin 1, collagen IV, and fibronectin. The expression of the b1 and b4 integrins was upregulated by the extract at low concentrations (0.1 and 1 microg/ml), but the expression was decreased at higher doses of Eupolin (10 microg-150 microg/ml). A number of clinical studies carried out by Vietnamese and international medical investigators have demonstrated the efficacy of this extract on the wound healing process. In this study we have shown that Eupolin stimulated the expression of many proteins of the adhesion complex and fibronectin by human keratinocytes. The adhesion complex proteins are essential to stabilise epithelium and this effect could contribute to the clinical efficacy of Eupolin in healing.

  6. Characterization of the Cadherin–Catenin Complex of the Sea Anemone Nematostella vectensis and Implications for the Evolution of Metazoan Cell–Cell Adhesion

    PubMed Central

    Clarke, Donald Nathaniel; Miller, Phillip W.; Lowe, Christopher J.; Weis, William I.; Nelson, William James

    2016-01-01

    The cadherin–catenin complex (CCC) mediates cell–cell adhesion in bilaterian animals by linking extracellular cadherin-based adhesions to the actin cytoskeleton. However, it is unknown whether the basic organization of the complex is conserved across all metazoans. We tested whether protein interactions and actin-binding properties of the CCC are conserved in a nonbilaterian animal, the sea anemone Nematostella vectensis. We demonstrated that N. vectensis has a complete repertoire of cadherin–catenin proteins, including two classical cadherins, one α-catenin, and one β-catenin. Using size-exclusion chromatography and multi-angle light scattering, we showed that α-catenin and β-catenin formed a heterodimer that bound N. vectensis Cadherin-1 and -2. Nematostella vectensis α-catenin bound F-actin with equivalent affinity as either a monomer or an α/β-catenin heterodimer, and its affinity for F-actin was, in part, regulated by a novel insert between the N- and C-terminal domains. Nematostella vectensis α-catenin inhibited Arp2/3 complex-mediated nucleation of actin filaments, a regulatory property previously thought to be unique to mammalian αE-catenin. Thus, despite significant differences in sequence, the key interactions of the CCC are conserved between bilaterians and cnidarians, indicating that the core function of the CCC as a link between cell adhesions and the actin cytoskeleton is ancestral in the eumetazoans. PMID:27189570

  7. Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

    NASA Astrophysics Data System (ADS)

    Kal Van Tam, Janice; Uto, Koichiro; Ebara, Mitsuhiro; Pagliari, Stefania; Forte, Giancarlo; Aoyagi, Takao

    2012-12-01

    The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell-matrix interaction, using poly-ɛ-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.

  8. Mini-review: barnacle adhesives and adhesion.

    PubMed

    Kamino, Kei

    2013-01-01

    Barnacles are intriguing, not only with respect to their importance as fouling organisms, but also in terms of the mechanism of underwater adhesion, which provides a platform for biomimetic and bioinspired research. These aspects have prompted questions regarding how adult barnacles attach to surfaces under water. The multidisciplinary and interdisciplinary nature of the studies makes an overview covering all aspects challenging. This mini-review, therefore, attempts to bring together aspects of the adhesion of adult barnacles by looking at the achievements of research focused on both fouling and adhesion. Biological and biochemical studies, which have been motivated mainly by understanding the nature of the adhesion, indicate that the molecular characteristics of barnacle adhesive are unique. However, it is apparent from recent advances in molecular techniques that much remains undiscovered regarding the complex event of underwater attachment. Barnacles attached to silicone-based elastomeric coatings have been studied widely, particularly with respect to fouling-release technology. The fact that barnacles fail to attach tenaciously to silicone coatings, combined with the fact that the mode of attachment to these substrata is different to that for most other materials, indicates that knowledge about the natural mechanism of barnacle attachment is still incomplete. Further research on barnacles will enable a more comprehensive understanding of both the process of attachment and the adhesives used. Results from such studies will have a strong impact on technology aimed at fouling prevention as well as adhesion science and engineering.

  9. A talin-dependent LFA-1 focal zone is formed by rapidly migrating T lymphocytes.

    PubMed

    Smith, Andrew; Carrasco, Yolanda R; Stanley, Paula; Kieffer, Nelly; Batista, Facundo D; Hogg, Nancy

    2005-07-01

    Cells such as fibroblasts and endothelial cells migrate through the coordinated responses of discrete integrin-containing focal adhesions and complexes. In contrast, little is known about the organization of integrins on the highly motile T lymphocyte. We have investigated the distribution, activity, and cytoskeletal linkage of the integrin lymphocyte function associated antigen-1 (LFA-1) on human T lymphocytes migrating on endothelial cells and on ligand intercellular adhesion molecule-1 (ICAM-1). The pattern of total LFA-1 varies from low expression in the lamellipodia to high expression in the uropod. However, high affinity, clustered LFA-1 is restricted to a mid-cell zone that remains stable over time and over a range of ICAM-1 densities. Talin is essential for the stability and formation of the LFA-1 zone. Disruption of the talin-integrin link leads to loss of zone integrity and a substantial decrease in speed of migration on ICAM-1. This adhesive structure, which differs from the previously described integrin-containing attachments displayed by many other cell types, we have termed the "focal zone."

  10. Control cell adhesion with dynamic bilayer films

    NASA Astrophysics Data System (ADS)

    Kourouklis, Andreas; Lerum, Ronald; Bermudez, Harry

    2012-02-01

    Interfacially-directed assembly of amphiphilic block copolymers was employed to create ultrathin films having the potential to correlate the dynamics of ECM cues with cell adhesion and cytoskeletally-generated forces. The mobility of the polymeric bilayer films were tuned by the incorporation of hydrophobic homopolymer chains, which are thought to reduce interlayer friction. Labeling of the block copolymer chains with an adhesive peptide ligand (RGD) provided a specific means to study integrin-mediated cellular processes and the corresponding mechanotransduction. By seeding anchorage-dependent cells on ``dynamic'' (laterally mobile) and ``static'' films that display the same amount of RGD, we have found that cells recognize the difference in RGD diffusivity and develop distinct responses over time. We intend to examine changes in cell response by controlling the extent of cytoskeletally-generated forces and the assembly dynamics of focal adhesion complexes. Such films provide a unique platform to unveil the biomechanical signals related with ECM dynamics, and may ultimately facilitate a deeper understanding of cellular processes.

  11. Switchable bio-inspired adhesives

    NASA Astrophysics Data System (ADS)

    Kroner, Elmar

    2015-03-01

    Geckos have astonishing climbing abilities. They can adhere to almost any surface and can run on walls and even stick to ceilings. The extraordinary adhesion performance is caused by a combination of a complex surface pattern on their toes and the biomechanics of its movement. These biological dry adhesives have been intensely investigated during recent years because of the unique combination of adhesive properties. They provide high adhesion, allow for easy detachment, can be removed residue-free, and have self-cleaning properties. Many aspects have been successfully mimicked, leading to artificial, bio-inspired, patterned dry adhesives, and were addressed and in some aspects they even outperform the adhesion capabilities of geckos. However, designing artificial patterned adhesion systems with switchable adhesion remains a big challenge; the gecko's adhesion system is based on a complex hierarchical surface structure and on advanced biomechanics, which are both difficult to mimic. In this paper, two approaches are presented to achieve switchable adhesion. The first approach is based on a patterned polydimethylsiloxane (PDMS) polymer, where adhesion can be switched on and off by applying a low and a high compressive preload. The switch in adhesion is caused by a reversible mechanical instability of the adhesive silicone structures. The second approach is based on a composite material consisting of a Nickel- Titanium (NiTi) shape memory alloy and a patterned adhesive PDMS layer. The NiTi alloy is trained to change its surface topography as a function of temperature, which results in a change of the contact area and of alignment of the adhesive pattern towards a substrate, leading to switchable adhesion. These examples show that the unique properties of bio-inspired adhesives can be greatly improved by new concepts such as mechanical instability or by the use of active materials which react to external stimuli.

  12. Statistical earthquake focal mechanism forecasts

    NASA Astrophysics Data System (ADS)

    Kagan, Yan Y.; Jackson, David D.

    2014-04-01

    Forecasts of the focal mechanisms of future shallow (depth 0-70 km) earthquakes are important for seismic hazard estimates and Coulomb stress, and other models of earthquake occurrence. Here we report on a high-resolution global forecast of earthquake rate density as a function of location, magnitude and focal mechanism. In previous publications we reported forecasts of 0.5° spatial resolution, covering the latitude range from -75° to +75°, based on the Global Central Moment Tensor earthquake catalogue. In the new forecasts we have improved the spatial resolution to 0.1° and the latitude range from pole to pole. Our focal mechanism estimates require distance-weighted combinations of observed focal mechanisms within 1000 km of each gridpoint. Simultaneously, we calculate an average rotation angle between the forecasted mechanism and all the surrounding mechanisms, using the method of Kagan & Jackson proposed in 1994. This average angle reveals the level of tectonic complexity of a region and indicates the accuracy of the prediction. The procedure becomes problematical where longitude lines are not approximately parallel, and where shallow earthquakes are so sparse that an adequate sample spans very large distances. North or south of 75°, the azimuths of points 1000 km away may vary by about 35°. We solved this problem by calculating focal mechanisms on a plane tangent to the Earth's surface at each forecast point, correcting for the rotation of the longitude lines at the locations of earthquakes included in the averaging. The corrections are negligible between -30° and +30° latitude, but outside that band uncorrected rotations can be significantly off. Improved forecasts at 0.5° and 0.1° resolution are posted at http://eq.ess.ucla.edu/kagan/glob_gcmt_index.html.

  13. Focal dystonia in musicians.

    PubMed

    Lie-Nemeth, Theresa J

    2006-11-01

    In conclusion, musicians' focal dystonia is a significant and potentially career-ending neurological condition of which physiatrists and other performing arts medicine clinicians should be aware. Pathology has been identified in the somatosensory cortex, and in the motor cortex and basal ganglia. Although advances have been made in the elucidating some of the pathologic changes in focal dystonia, better understanding is needed. Current treatments such as retraining, splinting, oral medications, and botulinum toxin injections are limited. Therefore, the ultimate goal for focal dystonia is to prevent this disabling disorder of instrumental musicians.

  14. Partial (focal) seizure

    MedlinePlus

    ... Jacksonian seizure; Seizure - partial (focal); Temporal lobe seizure; Epilepsy - partial seizures ... Abou-Khalil BW, Gallagher MJ, Macdonald RL. Epilepsies. In: Daroff ... Practice . 7th ed. Philadelphia, PA: Elsevier; 2016:chap 101. ...

  15. The Adhesion Molecule KAL-1/anosmin-1 Regulates Neurite Branching through a SAX-7/L1CAM-EGL-15/FGFR Receptor Complex.

    PubMed

    Díaz-Balzac, Carlos A; Lázaro-Peña, María I; Ramos-Ortiz, Gibram A; Bülow, Hannes E

    2015-06-01

    Neurite branching is essential for correct assembly of neural circuits, yet it remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann syndrome, regulates neurite branching through mechanisms largely unknown. Here, we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-factor with EGL-17/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the fibroblast growth factor receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig) domains of SAX-7/L1CAM and the FN(III) domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system. PMID:26004184

  16. Mechanics in Mechanosensitivity of Cell Adhesion and its Roles in Cell Migration

    NASA Astrophysics Data System (ADS)

    Zhong, Yuan; He, Shijie; Ji, Baohua

    2012-12-01

    Cells sense and respond to external stimuli and properties of their environment through focal adhesion complexes (FACs) to regulate a broad range of physiological and pathological processes, including cell migration. Currently, the basic principles in mechanics of the mechanosensitivity of cell adhesion and migration have not been fully understood. In this paper, an FEM-based mechano-chemical coupling model is proposed for studying the cell migration behaviors in which the dynamics of stability of FACs and the effect of cell shape on cell traction force distribution are considered. We find that the driving force of cell migration is produced by the competition of stability of cell adhesion between the cell front and cell rear, which consequently controls the speed of cell migration. We show that the rigidity gradient of matrix can bias this competition which allows cell to exhibit a durotaxis behavior, i.e. the larger the gradient, the higher the cell speed.

  17. SNAP focal plane

    SciTech Connect

    Lampton, Michael L.; Kim, A.; Akerlof, C.W.; Aldering, G.; Amanullah, R.; Astier, P.; Barrelet, E.; Bebek, C.; Bergstrom, L.; Berkovitz, J.; Bernstein, G.; Bester, M.; Bonissent, A.; Bower, C.; Carithers Jr., W.C.; Commins, E.D.; Day, C.; Deustua, S.E.; DiGennaro,R.; Ealet, A.; Ellis, R.S.; Eriksson, M.; Fruchter, A.; Genat, J.-F.; Goldhaber, G.; Goobar, A.; Groom, D.; Harris, S.E.; Harvey, P.R.; Heetderks, H.D.; Holland, S.E.; Huterer, D.; Karcher, A.; Kolbe, W.; Krieger, B.; Lafever, R.; Lamoureux, J.; Levi, M.E.; Levin, D.S.; Linder,E.V.; Loken, S.C.; Malina, R.; Massey, R.; McKay, T.; McKee, S.P.; Miquel, R.; Mortsell, E.; Mostek, N.; Mufson, S.; Musser, J.; Nugent, P.; Oluseyi, H.; Pain, R.; Palaio, N.; Pankow, D.; Perlmutter, S.; Pratt, R.; Prieto, E.; Refregier, A.; Rhodes, J.; Robinson, K.; Roe, N.; Sholl, M.; Schubnell, M.; Smadja, G.; Smoot, G.; Spadafora, A.; Tarle, G.; Tomasch,A.; von der Lippe, H.; Vincent, R.; Walder, J.-P.; Wang, G.

    2002-07-29

    The proposed SuperNova/Acceleration Probe (SNAP) mission will have a two-meter class telescope delivering diffraction-limited images to an instrumented 0.7 square-degree field sensitive in the visible and near-infrared wavelength regime. We describe the requirements for the instrument suite and the evolution of the focal plane design to the present concept in which all the instrumentation--visible and near-infrared imagers, spectrograph, and star guiders--share one common focal plane.

  18. Innate Non-Specific Cell Substratum Adhesion

    PubMed Central

    Loomis, William F.; Fuller, Danny; Gutierrez, Edgar; Groisman, Alex; Rappel, Wouter-Jan

    2012-01-01

    Adhesion of motile cells to solid surfaces is necessary to transmit forces required for propulsion. Unlike mammalian cells, Dictyostelium cells do not make integrin mediated focal adhesions. Nevertheless, they can move rapidly on both hydrophobic and hydrophilic surfaces. We have found that adhesion to such surfaces can be inhibited by addition of sugars or amino acids to the buffer. Treating whole cells with αlpha-mannosidase to cleave surface oligosaccharides also reduces adhesion. The results indicate that adhesion of these cells is mediated by van der Waals attraction of their surface glycoproteins to the underlying substratum. Since glycoproteins are prevalent components of the surface of most cells, innate adhesion may be a common cellular property that has been overlooked. PMID:22952588

  19. A theoretical analysis for the effect of focal contact formation on cell-substrate attachment strength.

    PubMed Central

    Ward, M D; Hammer, D A

    1993-01-01

    For many cell types, growth, differentiation, and motility are dependent on receptor-mediated adhesion to ligand-coated surfaces. Focal contacts are strong, specialized, adhesive connections between cell and substrate in which receptors aggregate and connect extracellular ligand to intracellular cytoskeletal molecules. In this paper, we present a mathematical model to examine how focal contact formation affects cellular adhesive strength. To calculate adhesive strength with and without focal contacts, we use a one-dimensional tape peeling analysis to determine the critical tension necessary to peel the membrane. Receptor-ligand bonds are modeled as adhesive springs. In the absence of focal contacts, we derive analytic expressions for the critical tension at low and high ligand densities and show how membrane morphology affects adhesion. Then, focal contacts are modeled as cytoplasmic nucleation centers which bind adhesion receptors. The extent of adhesive strengthening upon focal contact formation depends on the elastic rigidity of the cytoskeletal connections, which determines the structural integrity of the focal contact itself. We consider two limits to this elasticity, very weak and rigid. Rigid cytoskeletal connections give much greater attachment strengths. The dependence of attachment strength on measurable model parameters is quite different in these two limits, which suggests focal contact structure might be deduced from properly performed adhesion experiments. Finally, we compare our model to the adhesive strengthening response reported for glioma cell adhesion to fibronectin (Lotz et al., 1989. J. Cell Biol. 109:1795-1805). Our model successfully predicts the observed detachment forces at 4 degrees C and yields values for the number of fibronectin receptors per glioma cell and the density of cytoskeletal connection molecules (talin) involved in receptor clusters which are consistent with measurements for other cell types. Comparison of the model with

  20. Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics

    PubMed Central

    Cerecedo, Doris; Martínez-Rojas, Dalila; Chávez, Oscar; Martínez-Pérez, Francisco; García-Sierra, Francisco; Rendon, Alvaro; Mornet, Dominique; Mondragón, Ricardo

    2005-01-01

    Summary Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71d, as well as the Up71 isoform and the dystrophin-associated proteins, α and β-dystrobrevins. Distribution of Dp71d/Dp71Δ110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71Δ110m~DAPC and Up400/Up71~DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71Δ110m~DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71Δ110m~DAPC and Up400/Up71~DAPC in the biological roles of the platelets is discussed. PMID:16411395

  1. Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics.

    PubMed

    Cerecedo, Doris; Martínez-Rojas, Dalila; Chávez, Oscar; Martínez-Pérez, Francisco; García-Sierra, Francisco; Rendon, Alvaro; Mornet, Dominique; Mondragón, Ricardo

    2005-12-01

    Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71 d, as well as the Up71 isoform and the dystrophin-associated proteins, alpha and beta -dystrobrevins. Distribution of Dp71d/Dp71delta110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71delta100m approximately DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC in the biological roles of the platelets is discussed.

  2. Adhesion rings surround invadopodia and promote maturation

    PubMed Central

    Branch, Kevin M.; Hoshino, Daisuke; Weaver, Alissa M.

    2012-01-01

    Summary Invasion and metastasis are aggressive cancer phenotypes that are highly related to the ability of cancer cells to degrade extracellular matrix (ECM). At the cellular level, specialized actin-rich structures called invadopodia mediate focal matrix degradation by serving as exocytic sites for ECM-degrading proteinases. Adhesion signaling is likely to be a critical regulatory input to invadopodia, but the mechanism and location of such adhesion signaling events are poorly understood. Here, we report that adhesion rings surround invadopodia shortly after formation and correlate strongly with invadopodium activity on a cell-by-cell basis. By contrast, there was little correlation of focal adhesion number or size with cellular invadopodium activity. Prevention of adhesion ring formation by inhibition of RGD-binding integrins or knockdown (KD) of integrin-linked kinase (ILK) reduced the number of ECM-degrading invadopodia and reduced recruitment of IQGAP to invadopodium actin puncta. Furthermore, live cell imaging revealed that the rate of extracellular MT1-MMP accumulation at invadopodia was greatly reduced in both integrin-inhibited and ILK-KD cells. Conversely, KD of MT1-MMP reduced invadopodium activity and dynamics but not the number of adhesion-ringed invadopodia. These results suggest a model in which adhesion rings are recruited to invadopodia shortly after formation and promote invadopodium maturation by enhancing proteinase secretion. Since adhesion rings are a defining characteristic of podosomes, similar structures formed by normal cells, our data also suggest further similarities between invadopodia and podosomes. PMID:23213464

  3. Low density lipoprotein receptor-related protein 1 mediated endocytosis of β1-integrin influences cell adhesion and cell migration.

    PubMed

    Rabiej, Verena K; Pflanzner, Thorsten; Wagner, Timo; Goetze, Kristina; Storck, Steffen E; Eble, Johannes A; Weggen, Sascha; Mueller-Klieser, Wolfgang; Pietrzik, Claus U

    2016-01-01

    The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with β1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and β1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of β1-integrin and decreased β1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model. Cells formed more focal adhesion complexes, whereby in vitro cell migration rates were decreased. Similar results could be observed in a corresponding mouse model, the C57Bl6 LRP1 NPxYxxL knock in mice, therefore, the biochemistry of cellular adhesion was altered in primary cortical neurons. In vivo cell migration experiments demonstrated a disturbance of neuroblast cell migration along the rostral migratory stream. In summary, our results indicate that LRP1 interacts with β1-integrin mediating integrin internalization and thus correlates with downstream signaling of β1-integrin such as focal adhesion dynamics. Consequently, the disturbance of this interaction resulted in a dysfunction in in vivo and in vitro cell adhesion and cell migration.

  4. Oral focal epithelial hyperplasia.

    PubMed

    Bassioukas, K; Danielides, V; Georgiou, I; Photos, E; Zagorianakou, P; Skevas, A

    2000-01-01

    Focal epithelial hyperplasia (FEH) or Heck disease, is a rare viral infection of the oral mucosa caused by HPV 13 or HPV 32. In Caucasians there have been only a few cases reported. We present the first case in Greece in a young Caucasian girl in which HPV 13 was detected with PCR analysis. The patient was successfully treated with CO2 laser.

  5. [Focal infections in otorhinolaryngology].

    PubMed

    Pal'chun, V T

    2016-01-01

    This publication is focused on the mechanisms underlying the clinical course of acute focal infections concomitant with ENT pathology, factors responsible for their chronization and the development of complications. Also discussed are the methods for the early adequate conservative and surgical treatment of these conditions. Special emphasis is placed on the principles of management of chronic tonsillitis. PMID:26977559

  6. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

    PubMed Central

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  7. Incidence of bovine leukocyte adhesion deficiency, complex vertebral malformation, and deficiency of uridine-5-monophosphate synthase carriers in Brazilian Girolando cattle.

    PubMed

    Paiva, D S; Fonseca, I; Pinto, I S B; Ianella, P; Campos, T A; Caetano, A R; Paiva, S R; Silva, M V G B; Martins, M F

    2013-01-01

    Among the various hereditary diseases that have been widely studied in dairy cattle, bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine-5-monophosphate synthase (DUMPS), and complex vertebral malformation (CVM) are noteworthy because of their high impact on overall herd productivity as a consequence of increased calf mortality. The aim of this study was to verify the frequency of carriers of BLAD, CVM, and DUMPS mutant alleles in cows and bulls from the National Girolando Progeny Test carried out in Brazil by using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR assays. A total of 777 animals were genotyped for BLAD, 783 for CVM, and 122 for DUMPS. The frequencies of carriers for BLAD and CVM were 0.77 and 1.53%, respectively, whereas no carriers of DUMPS were observed. PMID:24065661

  8. Multi-focal multiphoton lithography.

    PubMed

    Ritschdorff, Eric T; Nielson, Rex; Shear, Jason B

    2012-03-01

    Multiphoton lithography (MPL) provides unparalleled capabilities for creating high-resolution, three-dimensional (3D) materials from a broad spectrum of building blocks and with few limitations on geometry, qualities that have been key to the design of chemically, mechanically, and biologically functional microforms. Unfortunately, the reliance of MPL on laser scanning limits the speed at which fabrication can be performed, making it impractical in many instances to produce large-scale, high-resolution objects such as complex micromachines, 3D microfluidics, etc. Previously, others have demonstrated the possibility of using multiple laser foci to simultaneously perform MPL at numerous sites in parallel, but use of a stage-scanning system to specify fabrication coordinates resulted in the production of identical features at each focal position. As a more general solution to the bottleneck problem, we demonstrate here the feasibility for performing multi-focal MPL using a dynamic mask to differentially modulate foci, an approach that enables each fabrication site to create independent (uncorrelated) features within a larger, integrated microform. In this proof-of-concept study, two simultaneously scanned foci produced the expected two-fold decrease in fabrication time, and this approach could be readily extended to many scanning foci by using a more powerful laser. Finally, we show that use of multiple foci in MPL can be exploited to assign heterogeneous properties (such as differential swelling) to micromaterials at distinct positions within a fabrication zone.

  9. Multi-focal multiphoton lithography.

    PubMed

    Ritschdorff, Eric T; Nielson, Rex; Shear, Jason B

    2012-03-01

    Multiphoton lithography (MPL) provides unparalleled capabilities for creating high-resolution, three-dimensional (3D) materials from a broad spectrum of building blocks and with few limitations on geometry, qualities that have been key to the design of chemically, mechanically, and biologically functional microforms. Unfortunately, the reliance of MPL on laser scanning limits the speed at which fabrication can be performed, making it impractical in many instances to produce large-scale, high-resolution objects such as complex micromachines, 3D microfluidics, etc. Previously, others have demonstrated the possibility of using multiple laser foci to simultaneously perform MPL at numerous sites in parallel, but use of a stage-scanning system to specify fabrication coordinates resulted in the production of identical features at each focal position. As a more general solution to the bottleneck problem, we demonstrate here the feasibility for performing multi-focal MPL using a dynamic mask to differentially modulate foci, an approach that enables each fabrication site to create independent (uncorrelated) features within a larger, integrated microform. In this proof-of-concept study, two simultaneously scanned foci produced the expected two-fold decrease in fabrication time, and this approach could be readily extended to many scanning foci by using a more powerful laser. Finally, we show that use of multiple foci in MPL can be exploited to assign heterogeneous properties (such as differential swelling) to micromaterials at distinct positions within a fabrication zone. PMID:22282105

  10. Focal and generalized alopecia.

    PubMed

    O'Dair, H A; Foster, A P

    1995-07-01

    Focal or generalized alopecia is defined as hair loss affecting the ventral, lateral, perineal, and dorsal aspects of the trunk of the cat, usually in a symmetric pattern. This may be attributable to failure of hair coat production, excess loss of hair due to self trauma, or excess shedding of whole hairs. Self trauma is the most common cause of hair loss and is associated particularly with flea allergy dermatitis. Other causes of hair loss are reviewed.

  11. Oral focal epithelial hyperplasia.

    PubMed

    López-Jornet, Pía; Camacho-Alonso, Fabio; Berdugo, Lucero

    2010-01-01

    Focal epithelial hyperplasia (FEH) is a benign, asymptomatic disease. It appears as papules, principally on the lower lip, although it can also be found on the retro-commissural mucosa and tongue and, less frequently, on the upper lip, gingiva and palate. FEH is caused by human papillomavirus subtype 13 or 32. The condition occurs in many populations and ethnic groups. We present the clinical case of a 31-year-old male with lesions that clinically and histologically corresponded to FEH.

  12. The possibility of generating focal regions of complex configurations in application to the problems of stimulation of human receptor structures by focused ultrasound

    NASA Astrophysics Data System (ADS)

    Gavrilov, L. R.

    2008-03-01

    Studies of the stimulating effect of ultrasound on human receptor structures have recently become more intensive in connection with the development of promising robotic techniques and systems, sensors, and automated control systems, as well as with the use of taction in the design of a human-machine interface. One of the promising fields of research is the development of tactile displays for transmission of sensory data to a human by an acoustic method based on the effect of radiation pressure. In this case, it is necessary to generate rapidly changing patterns on a display (symbols, letters, digits, etc.), which may often have a complex shape. It is demonstrated that such patterns can be created by the generation of multiple-focus ultrasonic fields with the help of two-dimensional phased arrays whose elements are randomly positioned on the surface. The parameters for such an array are presented. It is shown that the arrays make it possible to form the regions of action by focused ultrasound with various necessary shapes and the sidelobe (or other secondary peak) intensity level acceptable for practical purposes. Using these arrays, it is possible to move the set of foci off the array axis to a distance of at least ±5 mm, which corresponds to the display dimensions. It is possible, on the screen of a tactile display, to generate the regions of action with a very complex shape, for example, Latin letters. This opportunity may be of interest, for example, for the development of systems that enable a blind person to perceive the displayed text information by using the sense of touch.

  13. Natural Underwater Adhesives

    PubMed Central

    Stewart, Russell J.; Ransom, Todd C.; Hlady, Vladimir

    2011-01-01

    The general topic of this review is protein-based underwater adhesives produced by aquatic organisms. The focus is on mechanisms of interfacial adhesion to native surfaces and controlled underwater solidification of natural water-borne adhesives. Four genera that exemplify the broad range of function, general mechanistic features, and unique adaptations are discussed in detail: blue mussels, acorn barnacles, sandcastle worms, and freshwater caddisfly larva. Aquatic surfaces in nature are charged and in equilibrium with their environment, populated by an electrical double layer of ions as well as adsorbed natural polyelectrolytes and microbial biofilms. Surface adsorption of underwater bioadhesives likely occurs by exchange of surface bound ligands by amino acid sidechains, driven primarily by relative affinities and effective concentrations of polymeric functional groups. Most aquatic organisms exploit modified amino acid sidechains, in particular phosphorylated serines and hydroxylated tyrosines (dopa), with high-surface affinity that form coordinative surface complexes. After delivery to the surfaces as a fluid, permanent natural adhesives solidify to bear sustained loads. Mussel plaques are assembled in a manner superficially reminiscent of in vitro layer-by-layer strategies, with sequentially delivered layers associated through Fe(dopa)3 coordination bonds. The adhesives of sandcastle worms, caddisfly larva, and barnacles may be delivered in a form somewhat similar to in vitro complex coacervation. Marine adhesives are secreted, or excreted, into seawater that has a significantly higher pH and ionic strength than the internal environment. Empirical evidence suggests these environment triggers could provide minimalistic, fail-safe timing mechanisms to prevent premature solidification (insolubilization) of the glue within the secretory system, yet allow rapid solidification after secretion. Underwater bioadhesives are further strengthened by secondary covalent

  14. Rsu1 contributes to regulation of cell adhesion and spreading by PINCH1-dependent and - independent mechanisms.

    PubMed

    Gonzalez-Nieves, Reyda; Desantis, Akiko Iwahari; Cutler, Mary L

    2013-12-01

    Cell adhesion and migration are complex processes that require integrin activation, the formation and dissolution of focal adhesion (FAs), and linkage of actin cytoskeleton to the FAs. The IPP (ILK, PINCH, Parvin) complex regulates FA formation via binding of the adaptor protein ILK to β1 integrin, PINCH and parvin. The signaling protein Rsu1 is linked to the complex via binding PINCH1. The role of Rsu1 and PINCH1 in adhesion and migration was examined in non-transformed mammary epithelial cells. Confocal microscopy revealed that the depletion of either Rsu1 or PINCH1 by siRNA in MCF10A cells decreased the number of focal adhesions and altered the distribution and localization of β1 integrin, vinculin, talin and paxillin without affecting the levels of FA protein expression. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. In addition, constitutive phosphorylation of actin regulatory proteins occurred in the absence of PINCH1. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function by regulating levels of PINCH1. However, while both Rsu1- or PINCH1-depleted cells retained the ability to activate adhesion signaling in response to EGF stimulation, only Rsu1 was required for EGF-induced p38 Map Kinase phosphorylation and ATF2 activation, suggesting an Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with an Rsu1 mutant that does not bind to PINCH1 failed to restore FAs or migration but did promote spreading and constitutive p38 activation. These data show that Rsu1-PINCH1 association with ILK and the IPP complex is required for regulation of adhesion and migration but that Rsu1 has a critical role in linking integrin-induced adhesion to activation of p38 Map kinase signaling and cell spreading. Moreover, it suggests that Rsu1 may regulate p38 signaling from the IPP complex affecting other functions including

  15. [Focal epithelial hyperplasia].

    PubMed

    Delgado, Yolanda; Torrelo, Antonio; Colmenero, Isabel; Zambrano, Antonio

    2005-12-01

    Focal epithelial hyperplasia (FEH) is a benign proliferation of the oral mucosa with well defined clinical and histological characteristics. It has been associated with infection of the oral mucosa by types 13 and 32 of the human papillomavirus (HPV), and to a lesser extent, with other types. Its clinical course is variable, although it usually persists for months or years; cases with spontaneous resolution have been described, as have others with prolonged persistence. We present the case of an Ecuadorian boy whose visit was motivated by lesions in the oral mucosa consistent with a diagnosis of FEH, which were confirmed in the histological study, and in which HPV type 13 DNA was identified.

  16. [Focal epithelial hyperplasia].

    PubMed

    Carlino, P; Di Felice, R; Fiore-Donno, G; Samson, J

    1991-05-01

    Five cases of "focal epithelial hyperplasia" (FEH) of the oral mucosa observed in Switzerland are reported. The patients were of Turkish and North African extraction. The lesions of FEH were multiple, painless, located at various sites of the oral mucosa including the tongue in the form of either soft papules or hard nodules. Evidence of a human papilloma virus origin was ascertained. Among the 1067 cases reported in the literature and reviewed for this study, this condition has been described to occur among American Indians, Eskimos and North African, also in Israeli and European cases the disorder was often reported in individuals of Turkish or North African extraction.

  17. Partial recovery of the damaged rat blood-brain barrier is mediated by adherens junction complexes, extracellular matrix remodeling and macrophage infiltration following focal astrocyte loss

    PubMed Central

    Willis, Colin L.; Camire, Ryan B.; Brule, Stephanie A.; Ray, David E.

    2013-01-01

    Blood-brain barrier (BBB) dysfunction is a feature of many neurodegenerative disorders. The mechanisms and interactions between astrocytes, extracellular matrix and vascular endothelial cells in regulating the mature BBB are poorly understood. We have previously shown that transitory GFAP-astrocyte loss, induced by systemic administration of 3-chloropropanediol, leads to reversible disruption of tight junction complexes and BBB integrity to a range of markers. However, early restoration of BBB integrity to dextran (10-70 kDa) and fibrinogen was seen in the absence of paracellular tight junction proteins claudin-5 and occludin. In the present study we show that in the GFAP-astrocyte lesioned rat inferior colliculus, paracellular expression of adherens junction proteins (VE-cadherin and β-catenin) was maintained in vascular endothelial cells that lacked paracellular claudin-5 expression and which showed reversible post-translational occludin modification. Claudin-1 expression paralleled the loss and recovery of claudin-5, while claudin -3 or -12 immunoreactivity was not detected. In addition, the extracellular matrix, as visualized by laminin and fibronectin, underwent extensive reversible remodeling and perivascular CD169 macrophages become abundant throughout the lesioned inferior colliculus. At a time that GFAP-astrocytes repopulated the lesion area and tight junction proteins were returned to paracellular domains, the extracellular matrix and leukocyte profiles normalized and resembled profiles seen in control tissue. This study supports the hypothesis that a combination of paracellular adherens junctional proteins, remodeled basement membrane and the presence perivascular leukocytes provide a temporary barrier to limit extravasation of macromolecules and potentially neurotoxic substances into the brain parenchyma until tight junction proteins are restored to paracellular domains. PMID:23845748

  18. Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin

    PubMed Central

    Marshall, Jamie L.; Oh, Jennifer; Chou, Eric; Lee, Joy A.; Holmberg, Johan; Burkin, Dean J.; Crosbie-Watson, Rachelle H.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin–glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7β1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan ‘rescue’ of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7β1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7β1 integrin. PMID:25504048

  19. Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1

    PubMed Central

    1991-01-01

    Many ligands of adhesion molecules mediate costimulation of T cell activation. The generality of this emerging concept is best determined by using model systems which exploit physiologically relevant ligands. We developed such an "antigen-specific" model system for stimulation of resting CD4+ human T cells using the following purified ligands: (a) major histocompatibility complex class II plus the superantigen Staphylococcus enterotoxin A, to engage the T cell receptor (TCR); (b) adhesion proteins vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1), to provide potential cell surface costimulatory signals; and (c) recombinant interleukin 1 beta (rIL-1 beta)/rIL-6 as costimulatory cytokines. In this biochemically defined system, we find that resting CD4+ T cells require costimulation in order to respond to TCR engagement. This costimulation can be provided by VCAM-1 or ICAM-1; however adhesion alone is not sufficient since ELAM-1 mediates adhesion but not costimulation. The cytokines IL-1 beta and IL-6 by themselves cannot mediate costimulation, but augment the adhesion ligand-mediated costimulation. Direct comparison with the model of TCR/CD3 engagement by CD3 monoclonal antibody demonstrated comparable costimulatory requirements in both systems, thereby authenticating the commonly used CD3 model. The costimulation mediated by the activation-dependent interaction of the VLA-4 and LFA-1 integrins with their respective ligands VCAM-1 and ICAM-1 leads to increased IL-2R alpha (CD25) expression and proliferation in both CD45RA+ CD4+ and CD45RO+ CD4+ T cells. The integrins also regulate the secretion of IL-2, IL-4, and granulocyte/macrophage colony-stimulating factor. In contrast the activation-independent adhesion of CD4+ T cell to ELAM-1 molecules does not lead to T cell stimulation as measured by proliferation, IL-2R alpha expression, or cytokine release. These findings imply

  20. Geology and mineral resources of the Sheldon-Hart Mountain National Wildlife Refuge Complex (Oregon and Nevada), the Southeastern Oregon and North-Central Nevada, and the Southern Idaho and Northern Nevada (and Utah) Sagebrush Focal Areas: Chapter B in Mineral resources of the Sagebrush Focal Areas of Idaho, Montana, Nevada, Oregon, Utah, and Wyoming

    USGS Publications Warehouse

    Vikre, Peter G.; Benson, Mary Ellen; Bleiwas, Donald I.; Colgan, Joseph P.; Cossette, Pamela M.; DeAngelo, Jacob; Dicken, Connie L.; Drake, Ronald M.; du Bray, Edward A.; Fernette, Gregory L.; Glen, Jonathan M.G.; Haacke, Jon E.; Hall, Susan M.; Hofstra, Albert H.; John, David A.; Ludington, Stephen; Mihalasky, Mark J.; Rytuba, James J.; Shaffer, Brian N.; Stillings, Lisa L.; Wallis, John C.; Williams, Colin F.; Yager, Douglas B.; Zürcher, Lukas

    2016-10-04

    This report is temporarily unavailableSummaryThe U.S. Department of the Interior has proposed to withdraw approximately 10 million acres of Federal lands from mineral entry (subject to valid existing rights) from 12 million acres of lands defined as Sagebrush Focal Areas (SFAs) in Idaho, Montana, Nevada, Oregon, Utah, and Wyoming (for further discussion on the lands involved see Scientific Investigations Report 2016–5089–A). The purpose of the proposed action is to protect the greater sage-grouse (Centrocercus urophasianus) and its habitat from potential adverse effects of locatable mineral exploration and mining. The U.S. Geological Survey Sagebrush Mineral-Resource Assessment (SaMiRA) project was initiated in November 2015 and supported by the Bureau of Land Management to (1) assess locatable mineral-resource potential and (2) to describe leasable and salable mineral resources for the seven SFAs and Nevada additions.This chapter summarizes the current status of locatable, leasable, and salable mineral commodities and assesses the potential of selected locatable minerals in lands proposed for withdrawal that span the Nevada, Oregon, Idaho, and Utah borders. In this report, the four study areas evaluated were (1) the Sheldon-Hart Mountain National Wildlife Refuge Complex SFA in Washoe County, Nevada, and Harney and Lake Counties, Oregon; (2) the Southeastern Oregon and North-Central Nevada SFA in Humboldt County, Nevada, and Harney and Malheur Counties, Oregon; (3) the Southern Idaho and Northern Nevada SFA in Cassia, Owyhee, and Twin Falls Counties, Idaho, Elko County, Nevada, and Box Elder County, Utah; and (4) the Nevada additions in Humboldt and Elko Counties, Nevada.

  1. Geology and mineral resources of the Sheldon-Hart Mountain National Wildlife Refuge Complex (Oregon and Nevada), the Southeastern Oregon and North-Central Nevada, and the Southern Idaho and Northern Nevada (and Utah) Sagebrush Focal Areas: Chapter B in Mineral resources of the Sagebrush Focal Areas of Idaho, Montana, Nevada, Oregon, Utah, and Wyoming

    USGS Publications Warehouse

    Vikre, Peter G.; Benson, Mary Ellen; Bleiwas, Donald I.; Colgan, Joseph P.; Cossette, Pamela M.; DeAngelo, Jacob; Dicken, Connie L.; Drake, Ronald M.; du Bray, Edward A.; Fernette, Gregory L.; Glen, Jonathan M.G.; Haacke, Jon E.; Hall, Susan M.; Hofstra, Albert H.; John, David A.; Ludington, Stephen; Mihalasky, Mark J.; Rytuba, James J.; Shaffer, Brian N.; Stillings, Lisa L.; Wallis, John C.; Williams, Colin F.; Yager, Douglas B.; Zürcher, Lukas

    2016-10-04

    SummaryThe U.S. Department of the Interior has proposed to withdraw approximately 10 million acres of Federal lands from mineral entry (subject to valid existing rights) from 12 million acres of lands defined as Sagebrush Focal Areas (SFAs) in Idaho, Montana, Nevada, Oregon, Utah, and Wyoming (for further discussion on the lands involved see Scientific Investigations Report 2016–5089–A). The purpose of the proposed action is to protect the greater sage-grouse (Centrocercus urophasianus) and its habitat from potential adverse effects of locatable mineral exploration and mining. The U.S. Geological Survey Sagebrush Mineral-Resource Assessment (SaMiRA) project was initiated in November 2015 and supported by the Bureau of Land Management to (1) assess locatable mineral-resource potential and (2) to describe leasable and salable mineral resources for the seven SFAs and Nevada additions.This chapter summarizes the current status of locatable, leasable, and salable mineral commodities and assesses the potential of selected locatable minerals in lands proposed for withdrawal that span the Nevada, Oregon, Idaho, and Utah borders. In this report, the four study areas evaluated were (1) the Sheldon-Hart Mountain National Wildlife Refuge Complex SFA in Washoe County, Nevada, and Harney and Lake Counties, Oregon; (2) the Southeastern Oregon and North-Central Nevada SFA in Humboldt County, Nevada, and Harney and Malheur Counties, Oregon; (3) the Southern Idaho and Northern Nevada SFA in Cassia, Owyhee, and Twin Falls Counties, Idaho, Elko County, Nevada, and Box Elder County, Utah; and (4) the Nevada additions in Humboldt and Elko Counties, Nevada.

  2. Asterixis in focal brain lesions.

    PubMed

    Degos, J D; Verroust, J; Bouchareine, A; Serdaru, M; Barbizet, J

    1979-11-01

    Asterixis was observed in 20 cases of focal brain lesions. Metabolic or toxic factors were excluded. An electromyogram study of asterixis was carried out in nine cases to establish the diagnosis. The site of the focal lesion was either parietal or mesencephalic and was always contralateral to the asterixis. "Focal asterixis" could result from a dysfunction of the sensorimotor integration in the parietal lobe and the midbrain.

  3. Enhanced adhesion by gecko-inspired hierarchical fibrillar adhesives.

    PubMed

    Murphy, Michael P; Kim, Seok; Sitti, Metin

    2009-04-01

    The complex structures that allow geckos to repeatably adhere to surfaces consist of multilevel branching fibers with specialized tips. We present a novel technique for fabricating similar multilevel structures from polymer materials and demonstrate the fabrication of arrays of two- and three-level structures, wherein each level terminates in flat mushroom-type tips. Adhesion experiments are conducted on two-level fiber arrays on a 12-mm-diameter glass hemisphere, which exhibit both increased adhesion and interface toughness over one-level fiber samples and unstructured control samples. These adhesion enhancements are the result of increased surface conformation as well as increased extension during detachment.

  4. Adhesive plasters

    DOEpatents

    Holcombe, Jr., Cressie E.; Swain, Ronald L.; Banker, John G.; Edwards, Charlene C.

    1978-01-01

    Adhesive plaster compositions are provided by treating particles of Y.sub.2 O.sub.3, Eu.sub.2 O.sub.3, Gd.sub.2 O.sub.3 or Nd.sub.2 O.sub.3 with dilute acid solutions. The resulting compositions have been found to spontaneously harden into rigid reticulated masses resembling plaster of Paris. Upon heating, the hardened material is decomposed into the oxide, yet retains the reticulated rigid structure.

  5. IGF-1 Receptor and Adhesion Signaling: An Important Axis in Determining Cancer Cell Phenotype and Therapy Resistance

    PubMed Central

    Cox, Orla T.; O’Shea, Sandra; Tresse, Emilie; Bustamante-Garrido, Milan; Kiran-Deevi, Ravi; O’Connor, Rosemary

    2015-01-01

    IGF-1R expression and activation levels generally cannot be correlated in cancer cells, suggesting that cellular proteins may modulate IGF-1R activity. Strong candidates for such modulation are found in cell-matrix and cell–cell adhesion signaling complexes. Activated IGF-1R is present at focal adhesions, where it can stabilize β1 integrin and participate in signaling complexes that promote invasiveness associated with epithelial mesenchymal transition (EMT) and resistance to therapy. Whether IGF-1R contributes to EMT or to non-invasive tumor growth may be strongly influenced by the degree of extracellular matrix engagement and the presence or absence of key proteins in IGF-1R-cell adhesion complexes. One such protein is PDLIM2, which promotes both cell polarization and EMT by regulating the stability of transcription factors including NFκB, STATs, and beta catenin. PDLIM2 exhibits tumor suppressor activity, but is also highly expressed in certain invasive cancers. It is likely that distinct adhesion complex proteins modulate IGF-1R signaling during cancer progression or adaptive responses to therapy. Thus, identifying the key modulators will be important for developing effective therapeutic strategies and predictive biomarkers. PMID:26191041

  6. Amplicon DNA Melting Analysis for the Simultaneous Detection of Brucella spp and Mycobacterium tuberculosis Complex. Potential Use in Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis

    PubMed Central

    Sanjuan-Jimenez, Rocio; Colmenero, Juan D.; Bermúdez, Pilar; Alonso, Antonio; Morata, Pilar

    2013-01-01

    Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a

  7. Endothelial cell adhesion in real time. Measurements in vitro by tandem scanning confocal image analysis.

    PubMed Central

    Davies, P F; Robotewskyj, A; Griem, M L

    1993-01-01

    Real time measurements of cell-substratum adhesion in endothelial cells were obtained by tandem scanning confocal microscopy of sites of focal contact (focal adhesions) at the abluminal cell surface. Focal contact sites were sharply defined (low radiance levels) in the living cell such that the images could be enhanced, digitized, and isolated from other cellular detail. Sites of focal contact are the principal determinant of cell-substratum adhesion. Measurements of (a) the focal contact area and (b) the closeness of contact (inverse radiance) were used to nominally define the adhesion of a single cell or field of cells, and to record spontaneous and induced changes of cell adhesion in real time. The topography of focal contacts was estimated by calculating separation distances from radiance values using a calibration technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and substratum at or near the center of each focal contact, separation distances throughout the adhesion regions were always < 50 nm. Subtraction of consecutive images revealed continuous spontaneous remodeling of individual focal adhesions in unperturbed cells during periods of < 1 min. Despite extensive remodeling of focal contact sites, however, cell adhesion calculated for an entire cell over extended periods varied by < 10%. When cytoskeletal stability was impaired by exposure to cytochalasin or when cells were exposed to proteolytic enzyme, endothelial adhesion declined rapidly. Such changes were recorded at the level of single cells, groups of cells, and at single focal adhesions. In both unperturbed and manipulated cells, the dynamics of remodeling and cell adhesion characteristics varied greatly between individual sites within the same cell; disappearance of existing sites and appearance of new ones often occurred within minutes while adjacent sites underwent minimal remodelling. Tandem scanning confocal microscopy image analysis of

  8. Screening for bovine leukocyte adhesion deficiency, deficiency of uridine monophosphate synthase, complex vertebral malformation, bovine citrullinaemia, and factor XI deficiency in Holstein cows reared in Turkey

    PubMed Central

    2010-01-01

    Background Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins. Methods Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not. Results Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID). Conclusion This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population. PMID:20929557

  9. Implication of complex vertebral malformation and bovine leukocyte adhesion deficiency DNA-based testing on disease frequency in the Holstein population.

    PubMed

    Schütz, E; Scharfenstein, M; Brenig, B

    2008-12-01

    Two inherited lethal disorders, bovine leukocyte adhesion deficiency (BLAD) and complex vertebral malformation (CVM), play a major role in breeding of Holstein cattle. Both inherited diseases are based on single nucleotide polymorphisms that have been known for 12 and 7 yr, respectively. A total of 25,753 cattle were genotyped for BLAD (18,200 tests) and CVM (14,493 tests) in our laboratory since the beginning of the genotyping programs for these diseases. Based on founder effects, the CVM mutation is thought to be linked to milk production. The BLAD was genotyped using RFLP until 2001; then a fluorescence resonance energy transfer assay on a LightCycler was used, as for CVM genotyping. By using single nucleotide polymorphism-aided breeding, the allelic frequency of the BLAD and CVM mutations in the active sire population was reduced from 9.4% in 1997 to 0.3% in 2007 (BLAD) and from 8.3% in 2002 to 2.3% in 2007 (CVM), with calculated half-life of the mutant allele of 2.1 yr for BLAD and 3.6 yr for CVM. An observed increase of BLAD frequency in 1999 could be attributed to the massive use of a BLAD-positive sire tested falsely negative in another laboratory. These data show that marker-assisted selection is capable of substantially reducing the frequency of a mutation within a period of not more than 5 yr. The different selection strategies against the lethal recessive allele in CVM and BLAD are reflected in the different reduction rates of the specific allele frequencies. PMID:19038961

  10. Inhibition of Rac and ROCK signalling influence osteoblast adhesion, differentiation and mineralization on titanium topographies.

    PubMed

    Prowse, Paul D H; Elliott, Christopher G; Hutter, Jeff; Hamilton, Douglas W

    2013-01-01

    Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK)). Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated) and SLA (sandblasted large grit, acid etched) topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications.

  11. Inhibition of Rac and ROCK Signalling Influence Osteoblast Adhesion, Differentiation and Mineralization on Titanium Topographies

    PubMed Central

    Prowse, Paul D. H.; Elliott, Christopher G.; Hutter, Jeff; Hamilton, Douglas W.

    2013-01-01

    Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK)). Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated) and SLA (sandblasted large grit, acid etched) topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications. PMID:23505566

  12. 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

  13. The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state?

    PubMed

    Murphy-Ullrich, J E

    2001-04-01

    The process of cellular de-adhesion is potentially important for the ability of a cell to participate in morphogenesis and to respond to injurious stimuli. Cellular de-adhesion is induced by the highly regulated matricellular proteins TSP1 and 2, tenascin-C, and SPARC. These proteins induce a rapid transition to an intermediate state of adhesiveness characterized by loss of actin-containing stress fibers and restructuring of the focal adhesion plaque that includes loss of vinculin and alpha-actinin, but not of talin or integrin. This process involves intracellular signaling mediators, which are engaged in response to matrix protein-receptor interactions. Each of these proteins employs different receptors and signaling pathways to achieve this common morphologic endpoint. What is the function of this intermediate adhesive state and what is the physiologic significance of this action of the matricellular proteins? Given that matricellular proteins are expressed in response to injury and during development, one can speculate that the intermediate adhesive state is an adaptive condition that facilitates expression of specific genes that are involved in repair and adaptation. Since cell shape is maintained in weakly adherent cells, this state might induce survival signals to prevent apoptosis due to loss of strong cell adhesion, but yet allow for cell locomotion. The three matricellular proteins considered here might each preferentially facilitate one or more aspects of this adaptive response rather than all of these equally. Currently, we have only preliminary data to support the specific ideas proposed in this article. It will be interesting in the next several years to continue to elucidate the biological roles of the intermediate adhesive state induced by these matricellular proteins. and focal adhesions in a cell that nevertheless maintains a spread, extended morphology and integrin clustering. TSP1, tenascin-C, and SPARC induce the intermediate adhesive state, as

  14. Cell Adhesion and Growth on the Anodized Aluminum Oxide Membrane.

    PubMed

    Park, Jeong Su; Moon, Dalnim; Kim, Jin-Seok; Lee, Jin Seok

    2016-03-01

    Nanotopological cues are popular tools for in vivo investigation of the extracellular matrix (ECM) and cellular microenvironments. The ECM is composed of multiple components and generates a complex microenvironment. The development of accurate in vivo methods for the investigation of ECM are important for disease diagnosis and therapy, as well as for studies on cell behavior. Here, we fabricated anodized aluminum oxide (AAO) membranes using sulfuric and oxalic acid under controlled voltage and temperature. The membranes were designed to possess three different pore and interpore sizes, AAO-1, AAO-2, and AAO-3 membranes, respectively. These membranes were used as tools to investigate nanotopology-signal induced cell behavior. Cancerous cells, specifically, the OVCAR-8 cell-line, were cultured on porous AAO membranes and the effects of these membranes on cell shape, proliferation, and viability were studied. AAO-1 membranes bearing small sized pores were found to maintain the spreading shape of the cultured cells. Cells cultured on AAO-2 and AAO-3 membranes, bearing large pore-sized AAO membranes, changed shape from spreading to rounding. Furthermore, cellular area decreased when cells were cultured on all three AAO membranes that confirmed decreased levels of focal adhesion kinase (FAK). Additionally, OVCAR-8 cells exhibited increased proliferation on AAO membranes possessing various pore sizes, indicating the importance of the nanosurface structure in regulating cell behaviors, such as cell proliferation. Our results suggest that porous-AAO membranes induced nanosurface regulated cell behavior as focal adhesion altered the intracellular organization of the cytoskeleton. Our results may find potential applications as tools in in vivo cancer research studies. PMID:27280255

  15. Cell Adhesion and Growth on the Anodized Aluminum Oxide Membrane.

    PubMed

    Park, Jeong Su; Moon, Dalnim; Kim, Jin-Seok; Lee, Jin Seok

    2016-03-01

    Nanotopological cues are popular tools for in vivo investigation of the extracellular matrix (ECM) and cellular microenvironments. The ECM is composed of multiple components and generates a complex microenvironment. The development of accurate in vivo methods for the investigation of ECM are important for disease diagnosis and therapy, as well as for studies on cell behavior. Here, we fabricated anodized aluminum oxide (AAO) membranes using sulfuric and oxalic acid under controlled voltage and temperature. The membranes were designed to possess three different pore and interpore sizes, AAO-1, AAO-2, and AAO-3 membranes, respectively. These membranes were used as tools to investigate nanotopology-signal induced cell behavior. Cancerous cells, specifically, the OVCAR-8 cell-line, were cultured on porous AAO membranes and the effects of these membranes on cell shape, proliferation, and viability were studied. AAO-1 membranes bearing small sized pores were found to maintain the spreading shape of the cultured cells. Cells cultured on AAO-2 and AAO-3 membranes, bearing large pore-sized AAO membranes, changed shape from spreading to rounding. Furthermore, cellular area decreased when cells were cultured on all three AAO membranes that confirmed decreased levels of focal adhesion kinase (FAK). Additionally, OVCAR-8 cells exhibited increased proliferation on AAO membranes possessing various pore sizes, indicating the importance of the nanosurface structure in regulating cell behaviors, such as cell proliferation. Our results suggest that porous-AAO membranes induced nanosurface regulated cell behavior as focal adhesion altered the intracellular organization of the cytoskeleton. Our results may find potential applications as tools in in vivo cancer research studies.

  16. A contactin-receptor-like protein tyrosine phosphatase beta complex mediates adhesive communication between astroglial cells and gonadotrophin-releasing hormone neurones.

    PubMed

    Parent, A-S; Mungenast, A E; Lomniczi, A; Sandau, U S; Peles, E; Bosch, M A; Rønnekleiv, O K; Ojeda, S R

    2007-11-01

    Although it is well established that gonadotrophin-releasing hormone (GnRH) neurones and astrocytes maintain an intimate contact throughout development and adult life, the cell-surface molecules that may contribute to this adhesiveness remain largely unknown. In the peripheral nervous system, the glycosylphosphatidyl inositol (GPI)-anchored protein contactin is a cell-surface neuronal protein required for axonal-glial adhesiveness. A glial transmembrane protein recognised by neuronal contactin is receptor-like protein tyrosine phosphatase beta (RPTP beta), a phosphatase with structural similarities to cell adhesion molecules. In the present study, we show that contactin, and its preferred in cis partner Caspr1, are expressed in GnRH neurones. We also show that the RPTP beta mRNA predominantly expressed in hypothalamic astrocytes encodes an RPTP beta isoform (short RPTP beta) that uses its carbonic anhydrase (CAH) extracellular subdomain to interact with neuronal contactin. Immunoreactive contactin is most abundant in GnRH nerve terminals projecting to both the organum vasculosum of the lamina terminalis and median eminence, implying GnRH axons as an important site of contactin-dependent cell adhesiveness. GT1-7 immortalised GnRH neurones adhere to the CAH domain of RPTPbeta, and this adhesiveness is blocked when contactin GPI anchoring is disrupted or contactin binding capacity is immunoneutralised, suggesting that astrocytic RPTP beta interacts with neuronal contactin to mediate glial-GnRH neurone adhesiveness. Because the abundance of short RPTP beta mRNA increases in the female mouse hypothalamus (but not in the cerebral cortex) before puberty, it appears that an increased interaction between GnRH axons and astrocytes mediated by RPTP beta-contactin is a dynamic mechanism of neurone-glia communication during female sexual development. PMID:17927663

  17. Characterization of the oligodeoxynucleotide-mediated inhibition of interferon-gamma-induced major histocompatibility complex class I and intercellular adhesion molecule-1.

    PubMed

    Ramanathan, M; Lantz, M; MacGregor, R D; Garovoy, M R; Hunt, C A

    1994-10-01

    The major histocompatibility complex (MHC) Class I and II genes and intercellular adhesion molecule-1 (ICAM-1) are regulated by interferon-gamma in a variety of cell types. We have previously shown that the oligodeoxynucleotide 5'-GGG GTT GGT TGT GTT GGG TGT TGT GT-RNH2 (oligo I) inhibits the interferon-gamma-mediated enhancement of MHC Class I and ICAM-1 proteins in the K562 cell line. We have now investigated the mechanism of action of oligo I and report that it acts by inhibiting the binding of interferon-gamma to cells. We also show that the dose-response curves, the selectivity profile, and the kinetics of oligo I are consistent with this novel mechanism of action. The dose-response curves for oligo I, obtained using antibodies against the MHC Class I heavy chain, beta 2-microglobulin, or ICAM-1, are almost superimposable at each observation time. MHC Class I induction by 6400 units/ml interferon-alpha or interferon-beta or ICAM-1 enhancement by 800 units/ml tumor necrosis factor-alpha is not inhibited by oligo I. However, the synergistic induction of MHC Class I by mixtures of tumor necrosis factor-alpha and interferon-gamma is inhibited. Oligo I belongs to a class of active oligodeoxynucleotides that inhibits interferon-gamma-induced MHC Class I and ICAM-1 in K562 cells. The activity and potency is sequence-dependent, but remarkably different sequences can have comparable effects. The activity of oligo I in the HeLa S3 cell line inhibits the interferon-gamma-mediated enhancement of both ICAM-1 and MHC Class II DR and the interferon-gamma-mediated reduction in transferrin receptor expression. Thus, oligo I appears to specifically inhibit interferon-gamma-induced changes in protein expression, which is consistent with oligo I acting at an early step(s) in the induction process. Taken together, our results show that oligo I exerts its effects by inhibiting the association of interferon-gamma with the cell surface, which is a novel mechanism of action for

  18. Characterization of Palladin, a Novel Protein Localized to Stress Fibers and Cell Adhesions

    PubMed Central

    Parast, Mana M.; Otey, Carol A.

    2000-01-01

    Here, we describe the identification of a novel phosphoprotein named palladin, which colocalizes with α-actinin in the stress fibers, focal adhesions, cell–cell junctions, and embryonic Z-lines. Palladin is expressed as a 90–92-kD doublet in fibroblasts and coimmunoprecipitates in a complex with α-actinin in fibroblast lysates. A cDNA encoding palladin was isolated by screening a mouse embryo library with mAbs. Palladin has a proline-rich region in the NH2-terminal half of the molecule and three tandem Ig C2 domains in the COOH-terminal half. In Northern and Western blots of chick and mouse tissues, multiple isoforms of palladin were detected. Palladin expression is ubiquitous in embryonic tissues, and is downregulated in certain adult tissues in the mouse. To probe the function of palladin in cultured cells, the Rcho-1 trophoblast model was used. Palladin expression was observed to increase in Rcho-1 cells when they began to assemble stress fibers. Antisense constructs were used to attenuate expression of palladin in Rcho-1 cells and fibroblasts, and disruption of the cytoskeleton was observed in both cell types. At longer times after antisense treatment, fibroblasts became fully rounded. These results suggest that palladin is required for the normal organization of the actin cytoskeleton and focal adhesions. PMID:10931874

  19. Continuously variable focal length lens

    DOEpatents

    Adams, Bernhard W; Chollet, Matthieu C

    2013-12-17

    A material preferably in crystal form having a low atomic number such as beryllium (Z=4) provides for the focusing of x-rays in a continuously variable manner. The material is provided with plural spaced curvilinear, optically matched slots and/or recesses through which an x-ray beam is directed. The focal length of the material may be decreased or increased by increasing or decreasing, respectively, the number of slots (or recesses) through which the x-ray beam is directed, while fine tuning of the focal length is accomplished by rotation of the material so as to change the path length of the x-ray beam through the aligned cylindrical slows. X-ray analysis of a fixed point in a solid material may be performed by scanning the energy of the x-ray beam while rotating the material to maintain the beam's focal point at a fixed point in the specimen undergoing analysis.

  20. Complex Laparoscopic Myomectomy with Severe Adhesions Performed with Proper Preventive Measures and Power Morcellation Provides a Safe Choice in Certain Infertility Cases.

    PubMed

    Alfaro-Alfaro, Jaime; Flores-Manzur, María de Los Ángeles; Nevarez-Bernal, Roberto; Ayala-Yáñez, Rodrigo

    2016-01-01

    Laparoscopic myomectomy offers a real benefit to infertile patients with uterine fibroids and peritoneal adhesions. The procedure requires a skilled surgeon and laparoscopy technique to minimize adhesion formation and other proven benefits. Restrictions arise since this procedure requires power morcellation for fibroid tissue extraction. Two years ago, the Food and Drug Administration in the United States of America (FDA) issued the alert on power morcellation for uterine leiomyomas, addressing the risk of malignant cell spreading within the abdominal cavity (actual risk assessment from 1 in 360 to 1 in 7400 cases). We review a 30-year-old female, without previous gestations, hypermenorrhea, intermenstrual bleeding, and chronic pelvic pain. Transvaginal ultrasound reports multiple fibroids in the right portion of a bicornuate uterus. Relevant history includes open myomectomy 6 years before and a complicated appendectomy, developing peritonitis within a year. Laparoscopy revealed multiple adhesions blocking uterine access, a bicornuate uterus, and myomas in the expected site. Myomectomy was performed utilizing power morcellation with good results. FDA recommendations have diminished this procedure's selection, converting many to open variants. This particular case was technically challenging, requiring morcellation, and safety device deployment was impossible, yet the infertility issue was properly addressed. Patient evaluation, safety measures, and laparoscopy benefits may outweigh the risks in particular cases as this one. PMID:27668110

  1. Complex Laparoscopic Myomectomy with Severe Adhesions Performed with Proper Preventive Measures and Power Morcellation Provides a Safe Choice in Certain Infertility Cases

    PubMed Central

    Alfaro-Alfaro, Jaime; Flores-Manzur, María de los Ángeles; Nevarez-Bernal, Roberto

    2016-01-01

    Laparoscopic myomectomy offers a real benefit to infertile patients with uterine fibroids and peritoneal adhesions. The procedure requires a skilled surgeon and laparoscopy technique to minimize adhesion formation and other proven benefits. Restrictions arise since this procedure requires power morcellation for fibroid tissue extraction. Two years ago, the Food and Drug Administration in the United States of America (FDA) issued the alert on power morcellation for uterine leiomyomas, addressing the risk of malignant cell spreading within the abdominal cavity (actual risk assessment from 1 in 360 to 1 in 7400 cases). We review a 30-year-old female, without previous gestations, hypermenorrhea, intermenstrual bleeding, and chronic pelvic pain. Transvaginal ultrasound reports multiple fibroids in the right portion of a bicornuate uterus. Relevant history includes open myomectomy 6 years before and a complicated appendectomy, developing peritonitis within a year. Laparoscopy revealed multiple adhesions blocking uterine access, a bicornuate uterus, and myomas in the expected site. Myomectomy was performed utilizing power morcellation with good results. FDA recommendations have diminished this procedure's selection, converting many to open variants. This particular case was technically challenging, requiring morcellation, and safety device deployment was impossible, yet the infertility issue was properly addressed. Patient evaluation, safety measures, and laparoscopy benefits may outweigh the risks in particular cases as this one. PMID:27668110

  2. Complex Laparoscopic Myomectomy with Severe Adhesions Performed with Proper Preventive Measures and Power Morcellation Provides a Safe Choice in Certain Infertility Cases

    PubMed Central

    Alfaro-Alfaro, Jaime; Flores-Manzur, María de los Ángeles; Nevarez-Bernal, Roberto

    2016-01-01

    Laparoscopic myomectomy offers a real benefit to infertile patients with uterine fibroids and peritoneal adhesions. The procedure requires a skilled surgeon and laparoscopy technique to minimize adhesion formation and other proven benefits. Restrictions arise since this procedure requires power morcellation for fibroid tissue extraction. Two years ago, the Food and Drug Administration in the United States of America (FDA) issued the alert on power morcellation for uterine leiomyomas, addressing the risk of malignant cell spreading within the abdominal cavity (actual risk assessment from 1 in 360 to 1 in 7400 cases). We review a 30-year-old female, without previous gestations, hypermenorrhea, intermenstrual bleeding, and chronic pelvic pain. Transvaginal ultrasound reports multiple fibroids in the right portion of a bicornuate uterus. Relevant history includes open myomectomy 6 years before and a complicated appendectomy, developing peritonitis within a year. Laparoscopy revealed multiple adhesions blocking uterine access, a bicornuate uterus, and myomas in the expected site. Myomectomy was performed utilizing power morcellation with good results. FDA recommendations have diminished this procedure's selection, converting many to open variants. This particular case was technically challenging, requiring morcellation, and safety device deployment was impossible, yet the infertility issue was properly addressed. Patient evaluation, safety measures, and laparoscopy benefits may outweigh the risks in particular cases as this one.

  3. Thermomechanical architecture of the VIS focal plane for Euclid

    NASA Astrophysics Data System (ADS)

    Martignac, Jérôme; Carty, Michaël.; Tourette, Thierry; Bachet, Damien; Berthé, Michel; Augueres, Jean-Louis; Amiaux, Jérôme; Fontignie, Jean; Horeau, Benoît; Renaud, Diana; Pottinger, Sabrina; Denniston, James; Winter, Berend; Guttridge, Phillip; Cole, Richard; Cropper, Mark; Niemi, Sami; Coker, John; Hunt, Thomas

    2014-08-01

    One of the main challenges for current and near future space experiments is the increase of focal plane complexity in terms of amount of pixels. In the frame work of the ESA Euclid mission to be launched in 2020, the Euclid Consortium is developing an extremely large and stable focal plane for the VIS instrument. CEA has developed the thermomechanical architecture of that Focal Plane taking into account all the very stringent performance and mission related requirements. The VIS Focal Plane Assembly integrates 36 CCDs (operated at 150K) connected to their front end electronics (operated at 280K) as to obtain one of the largest focal plane (˜0.6 billion pixels) ever built for space application after the GAIA one. The CCDs are CCD273 type specially designed and provided by the e2v company under ESA contract, front end electronics is studied and provided by MSSL. In this paper we first recall the specific requirements that have driven the overall architecture of the VIS-FPA and especially the solutions proposed to cope with the scientific needs of an extremely stable focal plane, both mechanically and thermally. The mechanical structure based on SiC material used for the cold sub assembly supporting the CCDs is detailed. We describe also the modular architecture concept that we have selected taking into account AIT-AIV and programmatic constraints.

  4. mTOR signaling pathway genes in focal epilepsies.

    PubMed

    Baulac, S

    2016-01-01

    Focal epilepsies, where seizures initiate in spatially limited networks, are the most frequent epilepsy type, accounting for two-thirds of patients. Focal epilepsies have long been thought to be acquired disorders; several focal epilepsy syndromes are now proven to be (genetically heterogeneous) monogenic disorders. While earlier genetic studies have demonstrated a strong contribution of ion channel and neurotransmitter receptor genes, or synaptic secreted protein genes, later work has revealed a new class of genes encoding components of the mechanistic target of rapamycin (mTOR) signal transduction pathway. The mTOR pathway controls a myriad of biological processes among which cell growth and protein synthesis in response to several extracellular and intracellular. Recently, germline mutations have been found in genes encoding the components of the GATOR1 complex (DEPDC5, NPRL2, NPRL3), a repressor of mTORC1. These mutations are increasingly recognized as causing a wide and yet evolving spectrum of focal epilepsy syndromes, with and without cortical structural abnormalities (usually focal cortical dysplasia). Brain somatic mutations in the gene encoding mTOR (MTOR) have recently been linked to focal cortical dysplasia and other associated brain pathologies including hemimegalencephaly. This chapter reviews the genetics and neurobiology of DEPDC5, NPRL2, and NPRL3, and summarizes the clinical and molecular spectrum of GATOR1-related epilepsies. PMID:27323939

  5. Thermal Characterization of Adhesive

    NASA Technical Reports Server (NTRS)

    Spomer, Ken A.

    1999-01-01

    The current Space Shuttle Reusable Solid Rocket Motor (RSRM) nozzle adhesive bond system is being replaced due to obsolescence. Down-selection and performance testing of the structural adhesives resulted in the selection of two candidate replacement adhesives, Resin Technology Group's Tiga 321 and 3M's EC2615XLW. This paper describes rocket motor testing of these two adhesives. Four forty-pound charge motors were fabricated in configurations that would allow side by side comparison testing of the candidate replacement adhesives and the current RSRM adhesives. The motors provided an environment where the thermal performance of adhesives in flame surface bondlines was compared. Results of the FPC testing show that: 1) The phenolic char depths on radial bond lines is approximately the same and vary depending on the position in the blast tube regardless of which adhesive was used; 2) The adhesive char depth of the candidate replacement adhesives is less than the char depth of the current adhesives; 3) The heat-affected depth of the candidate replacement adhesives is less than the heat-affected depth of the current adhesives; and 4) The ablation rates for both replacement adhesives are slower than that of the current adhesives.

  6. Integrin adhesions suppress syncytium formation in the Drosophila larval epidermis

    PubMed Central

    Wang, Yan; Antunes, Marco; Anderson, Aimee E.; Kadrmas, Julie L.; Jacinto, Antonio; Galko, Michael J.

    2015-01-01

    Summary Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how Integrins and associated proteins maintain the regular morphology of epithelia is not well understood. We found that targeted knockdown of the integrin focal adhesion (FA) complex components βIntegrin, PINCH, and Integrin-linked kinase (ILK), caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the Integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation including that induced by wounding, genetic loss-of FA-proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss of function phenotype of Integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. Our results reveal a novel and unexpected role for proper Integrin-based adhesion in suppressing larval epidermal cell-cell fusion– a role that may be conserved in other epithelia. PMID:26255846

  7. Integrin Adhesions Suppress Syncytium Formation in the Drosophila Larval Epidermis.

    PubMed

    Wang, Yan; Antunes, Marco; Anderson, Aimee E; Kadrmas, Julie L; Jacinto, Antonio; Galko, Michael J

    2015-08-31

    Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how integrins and associated proteins maintain the regular morphology of epithelia is not well understood. We found that targeted knockdown of the integrin focal adhesion (FA) complex components β-integrin, PINCH, and integrin-linked kinase (ILK) caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation, including that induced by wounding, genetic loss of FA proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss-of-function phenotype of integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. Our results reveal a novel and unexpected role for proper integrin-based adhesion in suppressing larval epidermal cell-cell fusion--a role that may be conserved in other epithelia.

  8. Design and fabrication of polymer based dry adhesives inspired by the gecko adhesive system

    NASA Astrophysics Data System (ADS)

    Jin, Kejia

    There has been significant interest in developing dry adhesives mimicking the gecko adhesive system, which offers several advantages compared to conventional pressure sensitive adhesives. Specifically, gecko adhesive pads have anisotropic adhesion properties: the adhesive pads (spatulae) stick strongly when sheared in one direction but are non-adherent when sheared in the opposite direction. This anisotropy property is attributed to the complex topography of the array of fine tilted and curved columnar structures (setae) that bear the spatulae. In this thesis, easy, scalable methods, relying on conventional and unconventional techniques are presented to incorporate tilt in the fabrication of synthetic polymer-based dry adhesives mimicking the gecko adhesive system, which provide anisotropic adhesion properties. In the first part of the study, the anisotropic adhesion and friction properties of samples with various tilt angles to test the validity of a nanoscale tape-peeling model of spatular function are measured. Consistent with the Peel Zone model, samples with lower tilt angles yielded larger adhesion forces. Contact mechanics of the synthetic array were highly anisotropic, consistent with the frictional adhesion model and gecko-like. Based on the original design, a new design of gecko-like dry adhesives was developed which showed superior tribological properties and furthermore showed anisotropic adhesive properties without the need for tilt in the structures. These adhesives can be used to reversibly suspend weights from vertical surfaces (e.g., walls) and, for the first time to our knowledge, horizontal surfaces (e.g., ceilings) by simultaneously and judiciously activating anisotropic friction and adhesion forces. Furthermore, adhesion properties between artificial gecko-inspired dry adhesives and rough substrates with varying roughness are studied. The results suggest that both adhesion and friction forces on a rough substrate depends significantly on the

  9. 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

  10. Understanding Marine Mussel Adhesion

    PubMed Central

    Roberto, Francisco F.

    2007-01-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion. PMID:17990038

  11. Understanding Marine Mussel Adhesion

    SciTech Connect

    H. G. Silverman; F. F. Roberto

    2007-12-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are waterimpervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.

  12. Anfibatide, a novel GPIb complex antagonist, inhibits platelet adhesion and thrombus formation in vitro and in vivo in murine models of thrombosis.

    PubMed

    Lei, Xi; Reheman, Adili; Hou, Yan; Zhou, Hui; Wang, Yiming; Marshall, Alexandra H; Liang, Chaofan; Dai, Xiangrong; Li, Benjamin Xiaoyi; Vanhoorelbeke, Karen; Ni, Heyu

    2014-02-01

    Platelet adhesion and aggregation at the sites of vascular injury are key events for thrombosis and haemostasis. It has been well demonstrated that interaction between glycoprotein (GP) Ibα and von Willebrand factor (VWF) initiates platelet adhesion and contributes to platelet aggregation, particularly at high shear. GPIb has long been suggested as a desirable antithrombotic target, but anti-GPIb therapy has never been successfully developed. Here, we evaluated the antithrombotic potential of Anfibatide, a novel snake venom-derived GPIb antagonist.We found Anfibatide inhibited washed murine platelet aggregation induced by ristocetin and recombinant murine VWF. It also blocked botrocetin-induced binding of murine plasma VWF to recombinant human GPIbα. Interestingly, Anfibatide did not inhibit botrocetin-induced aggregation of platelet-rich plasma, indicating that its binding site may differ from other snake venom-derived GPIb antagonists. Anfibatide strongly inhibited platelet adhesion, aggregation, and thrombus formation in perfusion chambers at high shear conditions and efficiently dissolved preformed thrombi. Anfibatide also inhibited thrombus growth at low shear conditions, though less than at high shear. Using intravital microscopy, we found that Anfibatide markedly inhibited thrombosis in laser-injured cremaster vessels and prevented vessel occlusion in FeCl3-injured mesenteric vessels. Importantly, Anfibatide further inhibited residual thrombosis in VWF-deficient mice, suggesting that Anfibatide has additional antithrombotic effect beyond its inhibitory role in GPIb-VWF interaction. Anfibatide did not significantly cause platelet activation, prolong tail bleeding time, or cause bleeding diathesis in mice. Thus, consistent with the data from an ongoing clinical trial, the data from this study suggests that Anfibatide is a potent and safe antithrombotic agent. PMID:24172860

  13. Design and fabrication of gecko-inspired adhesives.

    PubMed

    Jin, Kejia; Tian, Yu; Erickson, Jeffrey S; Puthoff, Jonathan; Autumn, Kellar; Pesika, Noshir S

    2012-04-01

    Recently, there has been significant interest in developing dry adhesives mimicking the gecko adhesive system, which offers several advantages compared to conventional pressure-sensitive adhesives. Specifically, gecko adhesive pads have anisotropic adhesion properties; the adhesive pads (spatulae) stick strongly when sheared in one direction but are non-adherent when sheared in the opposite direction. This anisotropy property is attributed to the complex topography of the array of fine tilted and curved columnar structures (setae) that bear the spatulae. In this study, we present an easy, scalable method, relying on conventional and unconventional techniques, to incorporate tilt in the fabrication of synthetic polymer-based dry adhesives mimicking the gecko adhesive system, which provides anisotropic adhesion properties. We measured the anisotropic adhesion and friction properties of samples with various tilt angles to test the validity of a nanoscale tape-peeling model of spatular function. Consistent with the peel zone model, samples with lower tilt angles yielded larger adhesion forces. The tribological properties of the synthetic arrays were highly anisotropic, reminiscent of the frictional adhesion behavior of gecko setal arrays. When a 60° tilt sample was actuated in the gripping direction, a static adhesion strength of ~1.4 N/cm(2) and a static friction strength of ~5.4 N/cm(2) were obtained. In contrast, when the dry adhesive was actuated in the releasing direction, we measured an initial repulsive normal force and negligible friction.

  14. Ultrastructural localization of plasma membrane-associated urokinase- type plasminogen activator at focal contacts

    PubMed Central

    1988-01-01

    We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PA-containing focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5% FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion. PMID:3123496

  15. Focal hyperhidrosis: diagnosis and management.

    PubMed

    Haider, Aamir; Solish, Nowell

    2005-01-01

    Hyperhidrosis, a condition characterized by excessive sweating, can be generalized or focal. Generalized hyperhidrosis involves the entire body and is usually part of an underlying condition, most often an infectious, endocrine or neurologic disorder. Focal hyperhidrosis is idiopathic, occurring in otherwise healthy people. It affects 1 or more body areas, most often the palms, armpits, soles or face. Almost 3% of the general population, largely people aged between 25 and 64 years, experience hyperhidrosis. The condition carries a substantial psychological and social burden, since it interferes with daily activities. However, patients rarely seek a physician's help because many are unaware that they have a treatable medical disorder. Early detection and management of hyperhidrosis can significantly improve a patient's quality of life. There are various topical, systemic, surgical and nonsurgical treatments available with efficacy rates greater than 90%-95%.

  16. Focal hyperhidrosis: diagnosis and management

    PubMed Central

    Haider, Aamir; Solish, Nowell

    2005-01-01

    HYPERHIDROSIS, A CONDITION CHARACTERIZED by excessive sweating, can be generalized or focal. Generalized hyperhidrosis involves the entire body and is usually part of an underlying condition, most often an infectious, endocrine or neurologic disorder. Focal hyperhidrosis is idiopathic, occurring in otherwise healthy people. It affects 1 or more body areas, most often the palms, armpits, soles or face. Almost 3% of the general population, largely people aged between 25 and 64 years, experience hyperhidrosis. The condition carries a substantial psychological and social burden, since it interferes with daily activities. However, patients rarely seek a physician's help because many are unaware that they have a treatable medical disorder. Early detection and management of hyperhidrosis can significantly improve a patient's quality of life. There are various topical, systemic, surgical and nonsurgical treatments available with efficacy rates greater than 90%–95%. PMID:15632408

  17. Variable focal length deformable mirror

    DOEpatents

    Headley, Daniel; Ramsey, Marc; Schwarz, Jens

    2007-06-12

    A variable focal length deformable mirror has an inner ring and an outer ring that simply support and push axially on opposite sides of a mirror plate. The resulting variable clamping force deforms the mirror plate to provide a parabolic mirror shape. The rings are parallel planar sections of a single paraboloid and can provide an on-axis focus, if the rings are circular, or an off-axis focus, if the rings are elliptical. The focal length of the deformable mirror can be varied by changing the variable clamping force. The deformable mirror can generally be used in any application requiring the focusing or defocusing of light, including with both coherent and incoherent light sources.

  18. Integrin-Mediated Adhesion and Proliferation of Human MCs Elicited by A Hydroxyproline-Lacking, Collagen-like Peptide

    PubMed Central

    Krishna, Ohm D.; Jha, Amit K.; Jia, Xinqiao; Kiick, Kristi L.

    2011-01-01

    In this study, we evaluated the competence of a rationally designed collagen-like peptide (CLP-Cys) sequence - containing the minimal essential Glycine-Glutamic acid-Arginine (GER) triplet but lacking the hydroxyproline residue - for supporting human mesenchymal stem cell (hMSC) adhesion, spreading and proliferation. Cellular responses to the CLP-Cys sequence were analyzed by conjugating the peptide to two different substrates – a hard, planar glass surface and a soft hyaluronic acid (HA) particle-based hydrogel. Integrin-mediated cell spreading and adhesion were observed for hMSCs cultivated on the CLP-Cys functionalized surfaces, whereas on control surfaces lacking the peptide motif, cells either did not adhere or maintained a round morphology. On the glass surface, CLP-Cys-mediated spreading led to the formation of extended and well developed stress fibers composed of F-actin bundles and focal adhesion complexes while on the soft gel surface, less cytoskeletal reorganization was observed. The hMSCs proliferated significantly on the surfaces presenting CLP-Cys, compared to the control surfaces lacking CLP-Cys. Competitive binding assay employing soluble CLP-Cys revealed a dose-dependent inhibition of hMSC adhesion to the CLP-Cys-presenting surfaces. Blocking the α2β1 receptor on hMSC also resulted in a reduction of cell adhesion on both types of CLP-Cys surfaces, confirming the affinity of CLP-Cys to α2β1 receptors. These results established the competence of the hydroxyproline-free CLP-Cys for eliciting integrin-mediated cellular responses including adhesion, spreading and proliferation. Thus, CLP-Cys-modified HA hydrogels are attractive candidates as bioactive scaffolds for tissue engineering applications. PMID:21658756

  19. [Asterixis in focal brain lesions].

    PubMed

    Velasco, F; Gomez, J C; Zarranz, J J; Lambarri, I; Ugalde, J

    2004-05-01

    Asterixis is a motor control disorder characterized by the presence of abnormal movements of the lower limbs in the vertical plane during posture maintenance. Asterixis is usually bilateral and associated with toxic-metabolic metabolic encephalopathies. Unilateral asterixis is less frequent and it normally indicates focal brain damage. We report the cases of four patients (two males/two females), aged 57 to 83 years, suffering from uni or bilateral asterixis associated with focal brain damage. All patients underwent CT brain scan and a neurophysiological study (parietal EMG and/or PES). In addition, any toxic-metabolic cause that could be produced by this clinical phenomenon was ruled out with the appropriate testing. Unilateral asterixis is a clinical symptom that may indicate the presence of focal brain damage. Often, it is ignored or overlooked during routine neurological examinations. On the other hand, the presence of a bilateral asterixis is not always indicative of a toxic-metabolic encephalopathy.Rarely, such as in one of the cases herein presented, bilateral asterixis can also appear associated with structural brain lesions. Although asterixis diagnosis is fundamentally clinical, the neurophysiological study contributes to verify the diagnosis.

  20. MIPAS focal-plane optics

    NASA Astrophysics Data System (ADS)

    Bokhove, Henk; Smorenburg, C.; Visser, H.

    1993-11-01

    The Michelson Interferometer for Passive Atmospheric Sounding (MIPAS) has been selected by ESA for the ENVISAT-Mission, scheduled for launch in 1998. The instrument will measure the concentration of a number of atmospheric trace gases in the earth atmosphere in a spectral region from 4.15 - 14.6 micrometers . Within this region measurements are performed with high spectral resolution. The MIPAS optical system consists of scan mirrors, a telescope, a Michelson interferometer, an afocal reducer and a focal plane assembly. TNO Institute of Applied Physics is involved in the design and development of the afocal reducer and the focal plane assembly. The beam reducing optics of the afocal reducer consist of 2 concave and one convex mirror. Both the housing and the mirrors are made of aluminum to ensure temperature invariance. The optics of the focal plane assembly consist of aluminum mirrors, dichroic beamsplitters and Ge lenses in front of the detectors. The optical/mechanical design is developed to the level that phase C2/D activities can start.

  1. PH dependent adhesive peptides

    DOEpatents

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  2. 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…

  3. Evidence for an actin-containing cytoplasmic precursor of the focal contact and the timing of incorporation of vinculin at the focal contact

    PubMed Central

    1987-01-01

    The distribution of F-actin and vinculin in chicken embryo fibroblasts has been examined by nitrobenzoxadiazol (NBD)-phallacidin and indirect immunofluorescent staining, respectively, and related to the process of focal contact formation by recording the motility of the cell with differential interference contrast (DIC) or interference reflection microscopy (IRM) before fixation for staining. Linear cytoplasmic precursors of the focal contact, present within unattached lamellipodia, stained intensely with NBD-phallacidin. Without exception new focal contacts, 8 s and older at fixation, were associated with either a longer F-actin rib in the lamellipodium or, in older contacts, an F-actin structure of similar dimensions to the contact. This change in distribution of F-actin over the new contacts was accounted for by the segregation of the structural precursor into an attached part over the focal contact and a separate motile part. These results show that F- actin accumulates in the precursor adjacent to areas of the membrane competent to form the focal contact, and are consistent with the interpretation that this F-actin contributes to the initial adhesion plaque associated with the new contact. Vinculin was essentially absent from motile lamellipodia, showed no preferential association with F- actin rich precursors or very young focal contacts, but accumulated over new contacts during a 90-s period. Therefore, the association of F- actin with the membrane that precedes and persists in the initial focal contact is independent of vinculin, and the role of vinculin in development of the focal contact remains unclear. PMID:3121637

  4. Idiopathic focal segmental glomerulosclerosis and HLA antigens.

    PubMed

    Gerbase-DeLima, M; Pereira-Santos, A; Sesso, R; Temin, J; Aragão, E S; Ajzen, H

    1998-03-01

    The objective of the present study was to investigate a possible association between HLA class II antigens and idiopathic focal segmental glomerulosclerosis (FSGS). HLA-A, -B, -DR and -DQ antigens were determined in 19 Brazilian patients (16 white subjects and three subjects of Japanese origin) with biopsy-proven FSGS. Comparison of the HLA antigen frequencies between white patients and white local controls showed a significant increase in HLA-DR4 frequency among FSGS patients (37.7 vs 17.2%, P < 0.05). In addition, the three patients of Japanese extraction, not included in the statistical analysis, also presented HLA-DR4. In conclusion, our data confirm the association of FSGS with HLA-DR4 previously reported by others, thus providing further evidence for a role of genes of the HLA complex in the susceptibility to this disease. PMID:9698788

  5. Compliance of bio-adhesive substrates controls the kinetics of membrane-substrate association.

    PubMed

    Sarvestani, Alireza S

    2010-10-21

    Mechanical stiffness of bio-adhesive substrates is one of the major regulators of the cell adhesion and migration. In this study, we propose a theoretical model for the spontaneous growth of focal adhesion (FA) sites, on compliant elastic substrates, at the early stages of cellular adhesion. Using a purely thermodynamic approach, we demonstrate that the rate of membrane-substrate association decreases with increasing the compliance of the substrate. This can be considered as a reason for smaller spread area of the FA points after the stabilization of adhesion on compliant substrates, as reported by experiments. We also show that the extent to which the compliance of the substrate modulates the growth rate of adhesion site depends on the areal density of cell-adhesive ligands on the substrate. PMID:20655930

  6. PAK4: a pluripotent kinase that regulates prostate cancer cell adhesion

    PubMed Central

    Wells, Claire M.; Whale, Andrew D.; Parsons, Maddy; Masters, John R. W.; Jones, Gareth E.

    2010-01-01

    Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Migration and invasion require coordinated reorganisation of the actin cytoskeleton and regulation of cell-adhesion dynamics. Rho-family GTPases orchestrate both of these cellular processes. p21-activated kinase 4 (PAK4), a specific effector of the Rho GTPase Cdc42, is activated by HGF, and we have previously shown that activated PAK4 induces a loss of both actin stress fibres and focal adhesions. We now report that DU145 human prostate cancer cells with reduced levels of PAK4 expression are unable to successfully migrate in response to HGF, have prominent actin stress fibres, and an increase in the size and number of focal adhesions. Moreover, these cells have a concomitant reduction in cell-adhesion turnover rates. We find that PAK4 is localised at focal adhesions, is immunoprecipitated with paxillin and phosphorylates paxillin on serine 272. Furthermore, we demonstrate that PAK4 can regulate RhoA activity via GEF-H1. Our results suggest that PAK4 is a pluripotent kinase that can regulate both actin cytoskeletal rearrangement and focal-adhesion dynamics. PMID:20406887

  7. Molecular Architecture and Function of Matrix Adhesions

    PubMed Central

    Geiger, Benjamin; Yamada, Kenneth M.

    2011-01-01

    Cell adhesions mediate important bidirectional interactions between cells and the extracellular matrix. They provide an interactive interface between the extracellular chemical and physical environment and the cellular scaffolding and signaling machinery. This dynamic, reciprocal regulation of intracellular processes and the matrix is mediated by membrane receptors such as the integrins, as well as many other components that comprise the adhesome. Adhesome constituents assemble themselves into different types of cell adhesion structures that vary in molecular complexity and change over time. These cell adhesions play crucial roles in cell migration, proliferation, and determination of cell fate. PMID:21441590

  8. VASP, zyxin and TES are tension-dependent members of Focal Adherens Junctions independent of the α-catenin-vinculin module

    PubMed Central

    Oldenburg, Joppe; van der Krogt, Gerard; Twiss, Floor; Bongaarts, Annika; Habani, Yasmin; Slotman, Johan A.; Houtsmuller, Adriaan; Huveneers, Stephan; de Rooij, Johan

    2015-01-01

    Mechanical forces are integrated at cadherin-based adhesion complexes to regulate morphology and strength of cell-cell junctions and organization of associated F-actin. A central mechanosensor at the cadherin complex is α-catenin, whose stretching recruits vinculin to regulate adhesion strength. The identity of the F-actin regulating signals that are also activated by mechanical forces at cadherin-based junctions has remained elusive. Here we identify the actin-regulators VASP, zyxin and TES as members of punctate, tensile cadherin-based junctions called Focal Adherens Junctions (FAJ) and show that they display mechanosensitive recruitment similar to that of vinculin. However, this recruitment is not altered by destroying or over-activating the α-catenin/vinculin module. Structured Illumination Microscopy (SIM) indicates that these tension sensitive proteins concentrate at locations within FAJs that are distinct from the core cadherin complex proteins. Furthermore, localization studies using mutated versions of VASP and zyxin indicate that these two proteins require binding to each other in order to localize to the FAJs. We conclude that there are multiple force sensitive modules present at the FAJ that are activated at distinct locations along the cadherin-F-actin axis and regulate specific aspects of junction dynamics. PMID:26611125

  9. [Cutaneous myxoma (focal dermal mucinosis)].

    PubMed

    Senff, H; Kuhlwein, A; Jänner, M; Schäfer, R

    1988-09-01

    Two cases of cutaneous myxoma are presented. In case 1 the cutaneous myxoma was localized on the left thumb and clinically resembled a pyogenic granuloma. In case 2 it was found at the left nipple. The benign cutaneous tumor may herald a cardiac myxoma and other conditions. Thus, a cutaneous myxoma should be accepted as an indication for thorough investigation of the whole body at regular intervals. As there are neither clinically nor histologically adequate criteria for differentiation, cutaneous myxoma and focal dermal mucinosis can be considered as variants of a single entity.

  10. Focal epithelial hyperplasia: Heck disease.

    PubMed

    Cohen, P R; Hebert, A A; Adler-Storthz, K

    1993-09-01

    Two sisters of Mexican ancestry had focal epithelial hyperplasia (FEH). The lesions on the oral mucosa of the older child were initially misinterpreted as representing sexual abuse. Microscopic evaluation of a hematoxylin and eosin-stained section from a lower lip papule demonstrated the histologic features of FEH. Although human papillomavirus (HPV) type 13 and HPV32 have been most consistently present in FEH lesions, types 6, 11, 13, and 32 were not detected in the paraffin-embedded tissue specimen of our patient using an in situ hybridization technique. The lesions persisted or recurred during management using destructive modalities; subsequently, they completely resolved spontaneously.

  11. Focal epithelial hyperplasia in Sweden.

    PubMed

    Axéll, T; Hammarström, L; Larsson, A

    1981-01-01

    A prevalence of 0.11% of focal epithelial hyperplasia (FEH) was found among 20,333 adult Swedes. There was no sex difference, the lesion was most prevalent in age groups above 45 years and the lesion was most frequent on the tongue. The frequency of FEH in 15,132 consecutive routine biopsies was 0.26%. Four FEH-cells were ultrastructurally examined. They exhibited a clear cytoplasm with scattered ribosomes, a peripheral condensation of tonofilaments, a central aggregation of chromatin clumps with loss of nuclear membrane and an accumulation of desmosome fragments. No viral particles could be identified in these FEH-cells.

  12. SNAP Satellite Focal Plane Development

    SciTech Connect

    Bebek, C.; Akerlof, C.; Aldering, G.; Amanullah, R.; Astier, P.; Baltay, C.; Barrelet, E.; Basa, S.; Bercovitz, J.; Bergstrom, L.; Berstein, G.P.; Bester, M.; Bohlin, R.; Bonissent, A.; Bower, C.; Campbell, M.; Carithers, W.; Commins, E.; Day, C.; Deustua, S.; DiGennaro, R.; Ealet, A.; Ellis, R.; Emmett, W.; Eriksson, M.; Fouchez,D.; Fruchter, A.; Genat, J-F.; Goldhaber, G.; Goobar, A.; Groom, D.; Heetderks, H.; Holland, S.; Huterer, D.; Johnson, W.; Kadel, R.; Karcher,A.; Kim, A.; Kolbe, W.; Lafever, R.; Lamoureaux, J.; Lampton, M.; Lefevre, O.; Levi, M.; Levin, D.; Linder, E.; Loken, S.; Malina, R.; Mazure, A.; McKay, T.; McKee, S.; Miquel, R.; Morgan, N.; Mortsell, E.; Mostek, N.; Mufson, S.; Musser, J.; Roe, N.; Nugent, P.; Oluseyi, H.; Pain, R.; Palaio, N.; Pankow, D.; Perlmutter, S.; Prieto, E.; Rabinowitz,D.; Refregier, A.; Rhodes, J.; Schubnell, M.; Sholl, M.; Smadja, G.; Smith, R.; Smoot, G.; Snyder, J.; Spadafora, A.; Szymkowiak, A.; Tarle,G.; Taylor, K.; Tilquin, A.; Tomasch, A.; Vincent, D.; von der Lippe, H.; Walder, J-P.; Wang, G.

    2003-07-07

    The proposed SuperNova/Acceleration Probe (SNAP) mission will have a two-meter class telescope delivering diffraction-limited images to an instrumented 0.7 square degree field in the visible and near-infrared wavelength regime. The requirements for the instrument suite and the present configuration of the focal plane concept are presented. A two year R&D phase, largely supported by the Department of Energy, is just beginning. We describe the development activities that are taking place to advance our preparedness for mission proposal in the areas of detectors and electronics.

  13. Focal Plane Instrumentation of VERITAS

    NASA Astrophysics Data System (ADS)

    Nagai, T.; McKay, R.; Sleege, G.; Petry, D.

    VERITAS is a new atmospheric Cherenkov imaging telescope array to detect very high energy gamma rays above 100 GeV. The array is located in southern Arizona, USA, at an altitude of 1270m above see level. The array currently consists of four 12 m telescopes, structurally resembling the Davis-Cotton design of the Whipple 10 m telescope. The VERITAS focal plane instruments are equipped with high-resolution (499 pixels) fast photo-multiplier-tube (PMT) cameras covering a 3.5 degree field of view with 0.148 degree pixel separation. Light concentrators reduce the dead-space between PMTs to 25% and shield the PMTs from ambient light. The PMTs are connected to high-speed pre-amplifiers improving the signal to noise ratio and allow single photoelectron measurements in situ at operating voltage. Current monitor circuits in the focus box provide real-time monitoring of the anode currents for each pixel and ambient conditions of the focus box. A charge injection system installed in the focus box allows daytime testing of the trigger and data acquisition system by injecting pulses of variable amplitude and length into pre-amplifier stage. A detailed description of the VERITAS focal plane instruments will be given in this presentation.

  14. Adhesion at metal interfaces

    NASA Technical Reports Server (NTRS)

    Banerjea, Amitava; Ferrante, John; Smith, John R.

    1991-01-01

    A basic adhesion process is defined, the theory of the properties influencing metallic adhesion is outlined, and theoretical approaches to the interface problem are presented, with emphasis on first-principle calculations as well as jellium-model calculations. The computation of the energies of adhesion as a function of the interfacial separation is performed; fully three-dimensional calculations are presented, and universality in the shapes of the binding energy curves is considered. An embedded-atom method and equivalent-crystal theory are covered in the framework of issues involved in practical adhesion.

  15. Gecko adhesion: evolutionary nanotechnology.

    PubMed

    Autumn, Kellar; Gravish, Nick

    2008-05-13

    If geckos had not evolved, it is possible that humans would never have invented adhesive nanostructures. Geckos use millions of adhesive setae on their toes to climb vertical surfaces at speeds of over 1ms-1. Climbing presents a significant challenge for an adhesive in requiring both strong attachment and easy rapid removal. Conventional pressure-sensitive adhesives (PSAs) are either strong and difficult to remove (e.g. duct tape) or weak and easy to remove (e.g. sticky notes). The gecko adhesive differs dramatically from conventional adhesives. Conventional PSAs are soft viscoelastic polymers that degrade, foul, self-adhere and attach accidentally to inappropriate surfaces. In contrast, gecko toes bear angled arrays of branched, hair-like setae formed from stiff, hydrophobic keratin that act as a bed of angled springs with similar effective elastic modulus to that of PSAs. Setae are self-cleaning and maintain function for months during repeated use in dirty conditions. Setae are an anisotropic 'frictional adhesive' in that adhesion requires maintenance of a proximally directed shear load, enabling either a tough bond or spontaneous detachment. Gecko-like synthetic adhesives may become the glue of the future-and perhaps the screw of the future as well.

  16. Electro-dry-adhesion.

    PubMed

    Krahn, Jeffrey; Menon, Carlo

    2012-03-27

    This work presents novel conductive bioinspired dry adhesives with mushroom caps that enable the use of a synergistic combination of electrostatic and van der Waals forces (electro-dry-adhesion). An increase in shear adhesion bond strength of up to 2046% on a wide range of materials is measured when a maximum electrical field of 36.4 V μm(-1) is applied. A suction effect, due to the shape of the dry adhesive fibers, on overall adhesion was not noted for electro-dry-adhesives when testing was performed at both atmospheric and reduced pressure. Utilization of electrostatics to apply a preloading force to dry adhesive fiber arrays allows increased adhesion even after electrostatic force generation has been halted by ensuring the close contact necessary for van der Waals forces to be effective. A comparison is made between self-preloading of the electro-dry-adhesives and the direct application of a normal preloading pressure resulting in nearly the same shear bond strength with an applied voltage of 3.33 kV on the same sample.

  17. Electro-dry-adhesion.

    PubMed

    Krahn, Jeffrey; Menon, Carlo

    2012-03-27

    This work presents novel conductive bioinspired dry adhesives with mushroom caps that enable the use of a synergistic combination of electrostatic and van der Waals forces (electro-dry-adhesion). An increase in shear adhesion bond strength of up to 2046% on a wide range of materials is measured when a maximum electrical field of 36.4 V μm(-1) is applied. A suction effect, due to the shape of the dry adhesive fibers, on overall adhesion was not noted for electro-dry-adhesives when testing was performed at both atmospheric and reduced pressure. Utilization of electrostatics to apply a preloading force to dry adhesive fiber arrays allows increased adhesion even after electrostatic force generation has been halted by ensuring the close contact necessary for van der Waals forces to be effective. A comparison is made between self-preloading of the electro-dry-adhesives and the direct application of a normal preloading pressure resulting in nearly the same shear bond strength with an applied voltage of 3.33 kV on the same sample. PMID:22397643

  18. Reversible Thermoset Adhesives

    NASA Technical Reports Server (NTRS)

    Mac Murray, Benjamin C. (Inventor); Tong, Tat H. (Inventor); Hreha, Richard D. (Inventor)

    2016-01-01

    Embodiments of a reversible thermoset adhesive formed by incorporating thermally-reversible cross-linking units and a method for making the reversible thermoset adhesive are provided. One approach to formulating reversible thermoset adhesives includes incorporating dienes, such as furans, and dienophiles, such as maleimides, into a polymer network as reversible covalent cross-links using Diels Alder cross-link formation between the diene and dienophile. The chemical components may be selected based on their compatibility with adhesive chemistry as well as their ability to undergo controlled, reversible cross-linking chemistry.

  19. Tissue Mechanics and Adhesion during Embryo Development

    PubMed Central

    Shawky, Joseph H.; Davidson, Lance A.

    2014-01-01

    During development cells interact mechanically with their microenvironment through cell-cell and cell-matrix adhesions. Many proteins involved in these adhesions serve both mechanical and signaling roles. In this review we will focus on the mechanical roles of these proteins and their complexes in transmitting force or stress from cell to cell or from cell to the extracellular matrix. As forces operate against tissues they establish tissue architecture, extracellular matrix assembly, and pattern cell shapes. As tissues become more established, adhesions play a major role integrating cells with the mechanics of their local environment. Adhesions may serve as both a molecular-specific glue, holding defined populations of cells together, and as a lubricant, allowing tissues to slide past one another. We review the biophysical principles and experimental tools used to study adhesion so that we may aid efforts to understand how adhesions guide these movements and integrate their signaling functions with mechanical function. As we conclude we review efforts to develop predictive models of adhesion that can be used to interpret experiments and guide future efforts to control and direct the process of tissue self-assembly during development. PMID:25512299

  20. Adhesion in ceramics and magnetic media

    NASA Technical Reports Server (NTRS)

    Miyoshi, Kazuhisa

    1989-01-01

    When a ceramic is brought into contact with a metal or a polymeric material such as a magnetic medium, strong bonds form between the materials. For ceramic-to-metal contacts, adhesion and friction are strongly dependent on the ductility of the metals. Hardness of metals plays a much more important role in adhesion and friction than does the surface energy of metals. Adhesion, friction, surface energy, and hardness of a metal are all related to its Young's modulus and shear modulus, which have a marked dependence on the electron configuration of the metal. An increase in shear modulus results in a decrease in area of contact that is greater than the corresponding increase in surface energy (the fond energy) with shear modulus. Consequently, the adhesion and friction decrease with increasing shear modulus. For ceramics in contact with polymeric magnetic tapes, environment is extremely important. For example, a nitrogen environment reduces adhesion and friction when ferrite contacts polymeric tape, whereas a vacuum environment strengthens the ferrite-to-tape adhesion and increases friction. Adhesion and friction are strongly dependent on the particle loading of the tape. An increase in magnetic particle concentration increases the complex modulus of the tape, and a lower real area of contact and lower friction result.

  1. Isolation and biochemical characterization of underwater adhesives from diatoms.

    PubMed

    Poulsen, Nicole; Kröger, Nils; Harrington, Matthew J; Brunner, Eike; Paasch, Silvia; Buhmann, Matthias T

    2014-01-01

    Many aquatic organisms are able to colonize surfaces through the secretion of underwater adhesives. Diatoms are unicellular algae that have the capability to colonize any natural and man-made submerged surfaces. There is great technological interest in both mimicking and preventing diatom adhesion, yet the biomolecules responsible have so far remained unidentified. A new method for the isolation of diatom adhesive material is described and its amino acid and carbohydrate composition determined. The adhesive materials from two model diatoms show differences in their amino acid and carbohydrate compositions, but also share characteristic features including a high content of uronic acids, the predominance of hydrophilic amino acid residues, and the presence of 3,4-dihydroxyproline, an extremely rare amino acid. Proteins containing dihydroxyphenylalanine, which mediate underwater adhesion of mussels, are absent. The data on the composition of diatom adhesives are consistent with an adhesion mechanism based on complex coacervation of polyelectrolyte-like biomolecules.

  2. Supramolecular adhesives to hard surfaces: adhesion between host hydrogels and guest glass substrates through molecular recognition.

    PubMed

    Takashima, Yoshinori; Sahara, Taiga; Sekine, Tomoko; Kakuta, Takahiro; Nakahata, Masaki; Otsubo, Miyuki; Kobayashi, Yuichiro; Harada, Akira

    2014-10-01

    Supramolecular materials based on host-guest interactions should exhibit high selectivity and external stimuli-responsiveness. Among various stimuli, redox and photo stimuli are useful for its wide application. An external stimuli-responsive adhesive system between CD host-gels (CD gels) and guest molecules modified glass substrates (guest Sub) is focused. Here, the selective adhesion between host gels and guest substrates where adhesion depends on molecular complementarity is reported. Initially, it is thought that adhesion of a gel material onto a hard material might be difficult unless many guest molecules modified linear polymers immobilize on the surface of hard materials. However, reversible adhesion of the CD gels is observed by dissociating and re-forming inclusion complex in response to redox and photo stimuli.

  3. Focal Plane Phase Masks for PIAA: Design and Manufacturing

    NASA Astrophysics Data System (ADS)

    Newman, K.; Conway, J.; Belikov, R.; Guyon, O.

    2016-05-01

    The Phase Induced Amplitude Apodization Complex Mask Coronagraph (PIAACMC) is a coronagraph architecture for the direct detection of extrasolar planets, which can achieve close to the theoretical performance limit of any direct detection system. The primary components of a PIAACMC system are the Phase Induced Amplitude Apodization (PIAA) optics and the complex phase-shifting focal plane mask. PIAA optics have been produced and demonstrated with high coronagraph performance. In this paper, we describe the design process for the phase-shifting focal plane mask, and strategies for smoothing the mask profile. We describe the mask manufacturing process and show manufacturing results. Errors in the fabricated mask profile degrade the system performance, but we can recover performance by refining the manufacturing process and implementing wavefront control.

  4. Primary focal hyperhidrosis: diagnosis and management. .

    PubMed

    Wang, Rena; Solish, Nowell; Murray, Christian A

    2008-12-01

    Primary focal hyperhidrosis is a common and serious medical condition that causes considerable psychosocial morbidity. Diagnostic and effective management strategies can improve patients' quality of living dramatically.

  5. Optimal focal-plane restoration

    NASA Technical Reports Server (NTRS)

    Reichenbach, Stephen E.; Park, Stephen K.

    1989-01-01

    Image restoration can be implemented efficiently by calculating the convolution of the digital image and a small kernel during image acquisition. Processing the image in the focal-plane in this way requires less computation than traditional Fourier-transform-based techniques such as the Wiener filter and constrained least-squares filter. Here, the values of the convolution kernel that yield the restoration with minimum expected mean-square error are determined using a frequency analysis of the end-to-end imaging system. This development accounts for constraints on the size and shape of the spatial kernel and all the components of the imaging system. Simulation results indicate the technique is effective and efficient.

  6. Focal epithelial hyperplasia - an update.

    PubMed

    Said, Ahmed K; Leao, Jair C; Fedele, Stefano; Porter, Stephen R

    2013-07-01

    Focal epithelial hyperplasia (FEH) is an asymptomatic benign mucosal disease, which is mostly observed in specific groups in certain geographical regions. FEH is usually a disease of childhood and adolescence and is generally associated with people who live in poverty and of low socioeconomic status. Clinically, FEH is typically characterized by multiple, painless, soft, sessile papules, plaques or nodules, which may coalesce to give rise to larger lesions. Human papillomavirus (HPV), especially genotypes 13 and 32, have been associated and detected in the majority of FEH lesions. The clinical examination and social history often allow diagnosis, but histopathological examination of lesional tissue is usually required to confirm the exact diagnosis. FEH sometimes resolves spontaneously however, treatment is often indicated as a consequence of aesthetic effects or any interference with occlusion. There remains no specific therapy for FEH, although surgical removal, laser excision or possibly topical antiviral agents may be of benefit. There remains no evidence that FEH is potentially malignant.

  7. Ins and Outs of Microbial Adhesion

    NASA Astrophysics Data System (ADS)

    Virji, Mumtaz

    Microbial adhesion is generally a complex process, involving multiple adhesins on a single microbe and their respective target receptors on host cells. In some situations, various adhesins of a microbe may co-operate in an apparently hierarchical and sequential manner whereby the first adhesive event triggers the target cell to express receptors for additional microbial adhesins. In other instances, adhesins may act in concert leading to high avidity interactions, often a prelude to cellular invasion and tissue penetration. Mechanisms used to target the host include both lectin-like interactions and protein-protein interactions; the latter are often highly specific for the host or a tissue within the host. This reflective chapter aims to offer a point of view on microbial adhesion by presenting some experiences and thoughts especially related to respiratory pathogens and explore if there can be any future hope of controlling bacterial infections via preventing adhesion or invasion stages of microbial pathogenesis.

  8. Ice adhesions in relation to freeze stress.

    PubMed

    Olien, C R; Smith, M N

    1977-10-01

    In freezing, competitive interaction between ice and hydrophilic plant substances causes an energy of adhesion to develop through the interstitial liquid. The thermodynamic basis for the adhesion energy is discussed, with estimates of the energies involved. In this research, effects of adhesion energy were observed microscopically in conjunction with energies of crystallization and frost desiccation. The complex character of ice in intact crown tissue of winter barley (Hordeum vulgare L.) and the problems of sectioning frozen tissue without producing artifacts led to an alternative study of single barley cells in a mesh of ice and cell wall polymers. Adhesions between ice, cell wall polymers, and the plasmalemma form a complexly interacting system in which the pattern of crystallization is a major factor in determination of stress and injury. PMID:16660124

  9. Surface Modification of PMMA to Improve Adhesion to Corneal Substitutes in a Synthetic Core-Skirt Keratoprosthesis.

    PubMed

    Riau, Andri K; Mondal, Debasish; Yam, Gary H F; Setiawan, Melina; Liedberg, Bo; Venkatraman, Subbu S; Mehta, Jodhbir S

    2015-10-01

    Patients with advanced corneal disease do poorly with conventional corneal transplantation and require a keratoprosthesis (KPro) for visual rehabilitation. The most widely used KPro is constructed using poly(methyl methacrylate) (PMMA) in the central optical core and a donor cornea as skirt material. In many cases, poor adherence between the PMMA and the soft corneal tissue is responsible for device "extrusion" and bacterial infiltration. The interfacial adhesion between the tissue and the PMMA was therefore critical to successful implantation and device longevity. In our approach, we modified the PMMA surface using oxygen plasma (plasma group); plasma followed by calcium phosphate (CaP) coating (p-CaP); dopamine followed by CaP coating (d-CaP); or plasma followed by coating with (3-aminopropyl)triethoxysilane (3-APTES). To create a synthetic KPro model, we constructed and attached 500 μm thick collagen type I hydrogel on the modified PMMA surfaces. Surface modifications produced significantly improved interfacial adhesion strength compared to untreated PMMA (p < 0.001). The p-CaP group yielded the best interfacial adhesion with the hydrogel (177 ± 27 mN/cm(2)) followed by d-CaP (168 ± 31 mN/cm(2)), 3-APTES (145 ± 12 mN/cm(2)), and plasma (119 ± 10 mN/cm(2)). Longer-term stability of the adhesion was achieved by d-CaP, which, after 14 and 28 days of incubation in phosphate buffered saline, yielded 164 ± 25 mN/cm(2) (p = 0.906 compared to adhesion at day 1) and 131 ± 20 mN/cm(2) (p = 0.053), respectively. In contrast, significant reduction of adhesion strength was observed in p-CaP group over time (p < 0.001). All surface coatings were biocompatible to human corneal stromal fibroblasts, except for the 3-APTES group, which showed no live cells at 72 h of culture. In contrast, cells on d-CaP surface showed good anchorage, evidenced by the expression of focal adhesion complex (paxillin and vinculin), and prominent filopodia protrusions. In conclusion, d-CaP can

  10. Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell

    PubMed Central

    1993-01-01

    A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell- substrate adhesion. We have previously shown that neither uvomorulin (E- cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F- actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins. PMID:8491782

  11. Neuron adhesion and strengthening

    NASA Astrophysics Data System (ADS)

    Rocha, Aracely; Jian, Kuihuan; Ko, Gladys; Liang, Hong

    2010-07-01

    Understanding the neuron/material adhesion is important for neuron stimulation and growth. The current challenges remain in the lack of precision of measuring techniques and understanding the behavior of neuron. Here, we report a fluid shear method to investigate adhesion at the neuron/poly-D-lysine interface. In this study, the adhesion of 12-day-old chick embryo-retina neurons cultured on poly-D-lysine coated glass coverslips was measured via parallel disk rotational flow. The shear stress experienced by the cells increases with the disk radius. There is a critical point along the radius (Rc) where the stress experienced by the neurons equals their adhesion. The measured Rc can be used to calculate the neuron adhesion. Our results demonstrate that neurons adhered to the poly-D-lysine had a strain hardening effect. The adhesive shear stress of the neuron-material increased with applied shear (τa). When the τa reached or exceeded the value of 40 dyn/cm2, the adhesion remained constant at approximately 30 dyn/cm2. The present work allowed us not only to quantify the adhesive strength and force but also to evaluate the value of strain hardening at the neuron/poly-D-lysine interface.

  12. Postoperative Peritoneal Adhesions

    PubMed Central

    Ryan, Graeme B.; Grobéty, Jocelyne; Majno, Guido

    1971-01-01

    This paper describes an experimental model of peritoneal adhesions, in the rat, based on two relatively minor accidents that may occur during abdominal surgery in man: drying of the serosa, and bleeding. Drying alone had little effect; drying plus bleeding consistently produced adhesions to the dried area. Fresh blood alone produced adhesions between the three membranous structures [omentum and pelvic fat bodies (PFBs)]. The formation of persistent adhesions required whole blood. Preformed clots above a critical size induced adhesions even without previous serosal injury; they were usually captured by the omentum and PFBs. If all three membranous structures were excised, the clots caused visceral adhesions. The protective role of the omentum, its structure, and the mechanism of omental adhesions, are discussed. These findings are relevant to the pathogenesis of post-operative adhesions in man. ImagesFig 3Fig 4Fig 5Fig 6Fig 7Fig 12Fig 13Fig 1Fig 2Fig 14Fig 15Fig 8Fig 9Fig 10Fig 11 PMID:5315369

  13. Instant acting adhesive system

    NASA Technical Reports Server (NTRS)

    Davis, T. R.; Haines, R. C.

    1971-01-01

    Adhesive developes 80 percent of minimum bond strength of 250 psi less than 30 sec after activation is required. Adhesive is stable, handles easily, is a low toxic hazard, and is useful in industrial and domestic prototype bonding and clamping operations.

  14. An unusual presentation of Carney complex with diffuse primary pigmented nodular adrenocortical disease on one adrenal gland and a nonpigmented adrenocortical adenoma and focal primary pigmented nodular adrenocortical disease on the other.

    PubMed

    Tung, Shih-Chen; Hwang, Daw-Yang; Yang, Joseph W; Chen, Wei-Jen; Lee, Chien-Te

    2012-01-01

    A 24-year-old female patient with cushingoid appearance was admitted in May 2000. The endocrine studies showed ACTH-independent Cushing's syndrome. A 2-day high-dose dexamethasone suppression test (HDDST) revealed paradoxical increase of 24 h urinary free cortisol (UFC). Abdominal computed tomography demonstrated a left adrenal nodule (3 x 2 cm in diameter). An adrenal scintigram with ¹³¹I-6β-iodomethyl-19-norcholesterol showed uptake of the isotope in the left adrenal gland and non-visualization in the right adrenal gland throughout the examination course. A retroperitoneoscopic left total adrenalectomy was performed in July 2000. The cut surface of the left adrenal was yellow-tan grossly. Microscopically, the left adrenal nodule contained a nonpigmented adrenocortical adenoma (NP) and another focal primary pigmented nodular adrenocortical disease (PPNAD, FP) mixed lesion. The immunohistochemical studies of CYP17 demonstrate positive in NP and FP of the left adrenal gland. Very low baseline morning plasma cortisol (0.97 μg/dL) and subnormal ACTH (8.16 pg/mL) levels were measured 1.5 months after left adrenalectomy. Right adrenal gland recovered its function 6 months after left adrenalectomy. Plasma cortisol could be suppressed to 3.47 μg/dL by overnight low-dose dexamethasone suppression test 65 months after left adrenalectomy. Cushingoid features still did not appear 122 months after left adrenalectomy. In May 2011, this patient was readmitted due to cushingoid characteristics. Paradoxical rise of 24-h UFC to 2-day HDDST was demonstrated. Ultrasonography of thyroid showed bilateral thyroid cysts. Subtotal right adrenalectomy about 80% of right adrenal was performed. Diffuse PPNAD of the right adrenal was proved pathologically. Immunohischemical stain for CYP17 is positive in the right adrenal gland but weaker positive than that in the left adrenal gland. The genetic study of the peripheral blood, left adrenocortical nodule, and right PPNAD all showed p.R16X

  15. PDMS bonding to a bio-friendly photoresist via self-polymerized poly(dopamine) adhesive for complex protein micropatterning inside microfluidic channels.

    PubMed

    Kim, Miju; Song, Kwang Hoon; Doh, Junsang

    2013-12-01

    Protein micropatterned surfaces integrated with microfluidics are useful in numerous bioanalytical and biological applications. In this study, we demonstrated the fabrication of complex protein micropatterned surfaces within poly(dimethylsiloxane) (PDMS) microfluidic channels by attaching the PDMS channels to bio-friendly photoresist films and subsequently performing microscope projection photolithography (MPP). A muscle-inspired poly(dopamine) (PDA) coating was employed to mediate the bonding between the PDMS and the bio-friendly photoresist poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP). By adjusting the dip-coating time for the PDA coating, we could successfully introduce sufficient amounts of functional groups on the PDMP surfaces to mediate strong bonding between the PDMS channels and the PDA-coated PDMP thin films with minimal alteration of the surface properties of the PDMP thin films that are critical for protein micropatterning. Using this novel bonding strategy, we successfully fabricated multiple protein micropatterns and gradient micropatterns of proteins within microfluidic channels. The technique developed in this study will be useful for the fabrication of complex biochips for multiplex bioassays and fundamental cell biological studies.

  16. Treatment of focal dystonias with botulinum neurotoxin

    PubMed Central

    Benecke, Reiner; Blitzer, Andrew; Comella, Cynthia L.

    2016-01-01

    This is a review on the use of injections of botulinum toxin for the treatment of focal dystonias. Disorders covered include cranial dystonia, cervical dystonia, spasmodic dysphonia, and focal hand dystonia. Considered are clinical aspects, alternative treatment strategies and principles of use of botulinum toxin injections. PMID:19103214

  17. Cytotoxicity of denture adhesives.

    PubMed

    de Gomes, Pedro Sousa; Figueiral, Maria Helena; Fernandes, Maria Helena R; Scully, Crispian

    2011-12-01

    Ten commercially available denture adhesives, nine soluble formulations (six creams, three powders) and one insoluble product (pad), were analyzed regarding the cytotoxicity profile in direct and indirect assays using L929 fibroblast cells. In the direct assay, fibroblasts were seeded over the surface of a thick adhesive gel (5%, creams; 2.5%, powders and pad). In the indirect assay, cells were cultured in the presence of adhesive extracts prepared in static and dynamic conditions (0.5-2%, creams; 0.25-1%, powders and pad). Cell toxicity was assessed for cell viability/proliferation (MTT assay) and cell morphology (observation of the F-actin cytoskeleton organization by confocal laser scanning microscopy). Direct contact of the L929 fibroblasts with the thick adhesive gels caused no, or only a slight, decrease in cell viability/proliferation. The adhesive extracts (especially those prepared in dynamic conditions) caused significantly higher growth inhibition of fibroblasts and, in addition, caused dose- and time-dependent effects, throughout the 6-72 h exposure time. Also, dose-dependent effects on cell morphology, with evident disruption of the F-actin cytoskeleton organization, were seen in the presence of most adhesives. In conclusion, the adhesives possessed different degrees of cytotoxicity, but similar dose- and time-dependent biological profiles.

  18. Polyelectrolytes Multilayers to Modulate Cell Adhesion: A Study of the Influence of Film Composition and Polyelectrolyte Interdigitation on the Adhesion of the A549 Cell Line.

    PubMed

    Muzzio, Nicolás E; Pasquale, Miguel A; Gregurec, Danijela; Diamanti, Eleftheria; Kosutic, Marija; Azzaroni, Omar; Moya, Sergio E

    2016-04-01

    Polyelectrolyte multilayers (PEMs) with different polycation/polyanion pairs are fabricated by the layer-by-layer technique employing synthetic, natural, and both types of polyelectrolytes. The impact of the chemical composition of PEMs on cell adhesion is assessed by studying cell shape, spreading area, focal contacts, and cell proliferation for the A549 cell line. Cells exhibit good adhesion on PEMs containing natural polycations and poly(sodium 4-styrenesulfonate) (PSS) as polyanion, but limited adhesion is observed on PEMs fabricated from both natural polyelectrolytes. PEMs are then assembled, depositing a block of natural polyelectrolytes on top of a stiffer block with PSS as polyanion. Cell adhesion is enhanced on top of the diblock PEMs compared to purely natural PEMs. This fact could be explained by the interdigitation between polyelectrolytes from the two blocks. Diblock PEM assembly provides a simple means to tune cell adhesion on biocompatible PEMs.

  19. Focal brain inflammation and autism.

    PubMed

    Theoharides, Theoharis C; Asadi, Shahrzad; Patel, Arti B

    2013-04-09

    Increasing evidence indicates that brain inflammation is involved in the pathogenesis of neuropsychiatric diseases. Autism spectrum disorders (ASD) are characterized by social and learning disabilities that affect as many as 1/80 children in the USA. There is still no definitive pathogenesis or reliable biomarkers for ASD, thus significantly curtailing the development of effective therapies. Many children with ASD regress at about age 3 years, often after a specific event such as reaction to vaccination, infection, stress or trauma implying some epigenetic triggers, and may constitute a distinct phenotype. ASD children respond disproportionally to stress and are also affected by food and skin allergies. Corticotropin-releasing hormone (CRH) is secreted under stress and together with neurotensin (NT) stimulates mast cells and microglia resulting in focal brain inflammation and neurotoxicity. NT is significantly increased in serum of ASD children along with mitochondrial DNA (mtDNA). NT stimulates mast cell secretion of mtDNA that is misconstrued as an innate pathogen triggering an auto-inflammatory response. The phosphatase and tensin homolog (PTEN) gene mutation, associated with the higher risk of ASD, which leads to hyper-active mammalian target of rapamycin (mTOR) signalling that is crucial for cellular homeostasis. CRH, NT and environmental triggers could hyperstimulate the already activated mTOR, as well as stimulate mast cell and microglia activation and proliferation. The natural flavonoid luteolin inhibits mTOR, mast cells and microglia and could have a significant benefit in ASD.

  20. Early vision and focal attention

    NASA Astrophysics Data System (ADS)

    Julesz, Bela

    1991-07-01

    At the thirty-year anniversary of the introduction of the technique of computer-generated random-dot stereograms and random-dot cinematograms into psychology, the impact of the technique on brain research and on the study of artificial intelligence is reviewed. The main finding-that stereoscopic depth perception (stereopsis), motion perception, and preattentive texture discrimination are basically bottom-up processes, which occur without the help of the top-down processes of cognition and semantic memory-greatly simplifies the study of these processes of early vision and permits the linking of human perception with monkey neurophysiology. Particularly interesting are the unexpected findings that stereopsis (assumed to be local) is a global process, while texture discrimination (assumed to be a global process, governed by statistics) is local, based on some conspicuous local features (textons). It is shown that the top-down process of "shape (depth) from shading" does not affect stereopsis, and some of the models of machine vision are evaluated. The asymmetry effect of human texture discrimination is discussed, together with recent nonlinear spatial filter models and a novel extension of the texton theory that can cope with the asymmetry problem. This didactic review attempts to introduce the physicist to the field of psychobiology and its problems-including metascientific problems of brain research, problems of scientific creativity, the state of artificial intelligence research (including connectionist neural networks) aimed at modeling brain activity, and the fundamental role of focal attention in mental events.

  1. Focal liver lesions found incidentally.

    PubMed

    Algarni, Abdullah A; Alshuhri, Abdullah H; Alonazi, Majed M; Mourad, Moustafa Mabrouk; Bramhall, Simon R

    2016-03-28

    Incidentally found focal liver lesions are a common finding and a reason for referral to hepatobiliary service. They are often discovered in patients with history of liver cirrhosis, colorectal cancer, incidentally during work up for abdominal pain or in a trauma setting. Specific points should considered during history taking such as risk factors of liver cirrhosis; hepatitis, alcohol consumption, substance exposure or use of oral contraceptive pills and metabolic syndromes. Full blood count, liver function test and tumor markers can act as a guide to minimize the differential diagnosis and to categorize the degree of liver disease. Imaging should start with B-mode ultrasound. If available, contrast enhanced ultrasound is a feasible, safe, cost effective option and increases the ability to reach a diagnosis. Contrast enhanced computed tomography should be considered next. It is more accurate in diagnosis and better to study anatomy for possible operation. Contrast enhanced magnetic resonance is the gold standard with the highest sensitivity. If doubt still remains, the options are biopsy or surgical excision. PMID:27028805

  2. [Endothelial cell adhesion molecules].

    PubMed

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  3. Cell adhesion force microscopy

    PubMed Central

    Sagvolden, G.; Giaever, I.; Pettersen, E. O.; Feder, J.

    1999-01-01

    The adhesion forces of cervical carcinoma cells in tissue culture were measured by using the manipulation force microscope, a novel atomic force microscope. The forces were studied as a function of time and temperature for cells cultured on hydrophilic and hydrophobic polystyrene substrates with preadsorbed proteins. The cells attached faster and stronger at 37°C than at 23°C and better on hydrophilic than on hydrophobic substrates, even though proteins adsorb much better to the hydrophobic substrates. Because cell adhesion serves to control several stages in the cell cycle, we anticipate that the manipulation force microscope can help clarify some cell-adhesion related issues. PMID:9892657

  4. Adhesive Contact Sweeper

    NASA Technical Reports Server (NTRS)

    Patterson, Jonathan D.

    1993-01-01

    Adhesive contact sweeper removes hair and particles vacuum cleaner leaves behind, without stirring up dust. Also cleans loose rugs. Sweeper holds commercially available spools of inverted adhesive tape. Suitable for use in environments in which air kept free of dust; optics laboratories, computer rooms, and areas inhabited by people allergic to dust. For carpets, best used in tandem with vacuum cleaner; first pass with vacuum cleaner removes coarse particles, and second pass with sweeper extracts fine particles. This practice extends useful life of adhesive spools.

  5. Controlled Adhesion of Silicone Elastomer Surfaces

    NASA Astrophysics Data System (ADS)

    Owen, Michael

    2000-03-01

    Opportunities exist for controllably enhancing the adhesion of silicone surfaces, ranging from modest enhancement of release force levels of pressure-sensitive adhesive (PSA) release liners by incorporation of adhesion promoters known as high release additives (HRA), to permanent bonding of silicone elastomers using surface modification techniques such as plasma or corona treatment. Although only a part of the complex interaction of factors contributing to adhesion, surface properties such as wettability are a critical component in the understanding and control of release and adhesion phenomena. Surface characterization studies of low-surface-energy silicones before and after various adhesion modification strategies are reviewed. The silicones include polydimethylsiloxane (PDMS) and fluorosiloxane elastomers and coatings. Techniques used include contact angle, the Johnson, Kendall and Roberts (JKR) contact mechanics approach, scanning electron microscopy (SEM), atomic force microscopy (AFM), and x-ray photoelectron spectroscopy (XPS). Topics addressed are: use of HRA in PDMS release liners, the interaction of PDMS PSAs with polytetrafluoroethylene (PTFE), and the effect of plasma treatment on PDMS and fluorosiloxane surfaces.

  6. [Cavitation and boiling of bubbles at the focal region during high intensity focused ultrasound exposure].

    PubMed

    Zhong, Mingsong; Ai, Huijian; Li, Faqi

    2012-10-01

    High intensity focused ultrasound (HIFU) is a very complex transient process and can cause tissue coagulation necrosis. The cavitation and boiling behaviour of bubbles in the focal region play very important roles throughout an injury process. This paper reviews the research done by domestic and foreign scholars on behaviours of bubbles in HIFU irradiation process and summarizes in the focal region bubble cavitation and boiling generation, related detective means and relationships with hyperecho, temperature rise of the focus and injury shape.

  7. Biostatistical evaluation of focal hepatic preneoplasia.

    PubMed

    Kopp-Schneider, Annette

    2003-01-01

    Qualitative analyses of focal hepatic preneoplasia are relatively easy and fast but hypothesis tests based on these analyses often lack statistical power. Evaluating focal hepatic preneoplasia quantitatively, on the other hand, requires more effort but is rewarded by an increased ability to detect differences between treatment groups and by the possibility to investigate the mechanism of a treatment under study. Due to the stereological problems inherent in the data a statistical analysis that concentrates on the evaluation of area fraction will provide clear results whereas the analysis of focal transection density and size distribution can produce misleading results. In addition, the area fraction is a valid variable even in the presence of confluent foci. The number and size distribution of focal transections in liver sections cannot be directly translated to the number and sizes of foci in the liver. As no general statements about the relationship between focal transection density and foci density as well as between focal transection size and foci size distribution can be made, there is need for a parametric mechanistic model to link the number and size distribution of focal transections to those of the underlying foci. The stereological problem therefore can be avoided by introducing a model for foci appearance and change of volume that then can be used to conclude whether the treatment induces foci and whether it changes their volume.

  8. Optical adhesive property study

    SciTech Connect

    Sundvold, P.D.

    1996-01-01

    Tests were performed to characterize the mechanical and thermal properties of selected optical adhesives to identify the most likely candidate which could survive the operating environment of the Direct Optical Initiation (DOI) program. The DOI system consists of a high power laser and an optical module used to split the beam into a number of channels to initiate the system. The DOI requirements are for a high shock environment which current military optical systems do not operate. Five candidate adhesives were selected and evaluated using standardized test methods to determine the adhesives` physical properties. EC2216, manufactured by 3M, was selected as the baseline candidate adhesive based on the test results of the physical properties.

  9. Adhesion of Lunar Dust

    NASA Technical Reports Server (NTRS)

    Walton, Otis R.

    2007-01-01

    This paper reviews the physical characteristics of lunar dust and the effects of various fundamental forces acting on dust particles on surfaces in a lunar environment. There are transport forces and adhesion forces after contact. Mechanical forces (i.e., from rover wheels, astronaut boots and rocket engine blast) and static electric effects (from UV photo-ionization and/or tribo-electric charging) are likely to be the major contributors to the transport of dust particles. If fine regolith particles are deposited on a surface, then surface energy-related (e.g., van der Walls) adhesion forces and static-electric-image forces are likely to be the strongest contributors to adhesion. Some measurement techniques are offered to quantify the strength of adhesion forces. And finally some dust removal techniques are discussed.

  10. Adhesives for Aerospace

    NASA Technical Reports Server (NTRS)

    Meade, L. E.

    1985-01-01

    The industry is hereby challenged to integrate adhesive technology with the total structure requirements in light of today's drive into automation/mechanization. The state of the art of adhesive technology is fairly well meeting the needs of the structural designers, the processing engineer, and the inspector, each on an individual basis. The total integration of these needs into the factory of the future is the next collective hurdle to be achieved. Improved processing parameters to fit the needs of automation/mechanization will necessitate some changes in the adhesive forms, formulations, and chemistries. Adhesives have, for the most part, kept up with the needs of the aerospace industry, normally leading the rest of the industry in developments. The wants of the aerospace industry still present a challenge to encompass all elements, achieving a totally integrated joined and sealed structural system. Better toughness with hot-wet strength improvements is desired. Lower cure temperatures, longer out times, and improved corrosion inhibition are desired.

  11. Do focal colors look particularly "colorful"?

    PubMed

    Witzel, Christoph; Franklin, Anna

    2014-04-01

    If the most typical red, yellow, green, and blue were particularly colorful (i.e., saturated), they would "jump out to the eye." This would explain why even fundamentally different languages have distinct color terms for these focal colors, and why unique hues play a prominent role in subjective color appearance. In this study, the subjective saturation of 10 colors around each of these focal colors was measured through a pairwise matching task. Results show that subjective saturation changes systematically across hues in a way that is strongly correlated to the visual gamut, and exponentially related to sensitivity but not to focal colors.

  12. Advanced approaches to focal plane integration

    NASA Astrophysics Data System (ADS)

    Nelson, R. D.; Smith, E. C., Jr.

    1980-01-01

    Both visible and infrared focal plane assemblies have common architectural driving parameters which guide their design approaches. The key drivers for advanced focal plane assemblies (FPA) are: the detector type and performance required; the number of detector chips; the packaging density; and the geometry. The impact of these drivers is seen to determine the engineering compromises necessary to establish FPA design approach. Several new designs are discussed which show a range of applications from single detector assemblies to monolithic detector chips with on-chip signal processing. The main objective of many advanced designs is to integrate the focal plane components in order to reduce power and reduce the number of interconnections.

  13. Unilateral Isolated Proximal Femoral Focal Deficiency

    PubMed Central

    Doğer, Emek; Köpük, Şule Y.; Çakıroğlu, Yiğit; Çakır, Özgür; Yücesoy, Gülseren

    2013-01-01

    Objective. To discuss a patient with a prenatal diagnosis of unilateral isolated femoral focal deficiency. Case. Antenatal diagnosis of unilateral isolated femoral focal deficiency was made at 20 weeks of gestation. The length of left femur was shorter than the right, and fetal femur length was below the fifth percentile. Proximal femoral focal deficiency was diagnosed. After delivery, the diagnosis was confirmed with skeletal radiographs and magnetic resonance imaging. In prenatal ultrasonographic examination, the early recognition and exclusion of skeletal dysplasias is important; moreover, treatment plans should be initiated, and valuable information should be provided to the family. PMID:23984135

  14. Focal Cortical Dysplasia in Childhood Epilepsy.

    PubMed

    Shaker, Tarek; Bernier, Anne; Carmant, Lionel

    2016-05-01

    Focal cortical dysplasia is a common cause of medication resistant epilepsy. A better understanding of its presentation, pathophysiology and consequences have helped us improved its treatment and outcome. This paper reviews the most recent classification, pathophysiology and imaging findings in clinical research as well as the knowledge gained from studying genetic and lesional animal models of focal cortical dysplasia. This review of this recently gained knowledge will most likely help develop new research models and new therapeutic targets for patients with epilepsy associated with focal cortical dysplasia. PMID:27544467

  15. A bioinspired wet/dry microfluidic adhesive for aqueous environments.

    PubMed

    Majumder, Abhijit; Sharma, Ashutosh; Ghatak, Animangsu

    2010-01-01

    A pressure-sensitive, nonreacting and nonfouling adhesive which can perform well both in air and underwater is very desirable because of its potential applications in various settings such as biomedical, marine, and automobile. Taking a clue from nature that many natural adhesive pads have complex structures underneath the outer adhesive layer, we have prepared thin elastic adhesive films with subsurface microstructures using PDMS (poly(dimethylsiloxane)) and investigated their performance underwater. The presence of embedded structure enhances the energy of adhesion considerably both in air and underwater. Furthermore, filling the channels with liquid of suitable surface tension modifies the internal stress profile, resulting into significant enhancement in adhesive performance. As this increase in adhesion is mediated by mechanics and not by surface chemistry, the presence of water does not alter its performance much. For the same reason, this adhesion mechanism works with both hydrophobic and hydrophilic surfaces. The adhesive can be reused because of its elastic surface. Moreover, unlike many other present-day adhesives, its performance does not decrease with time. PMID:20038181

  16. Integrin-dependent force transmission to the extracellular matrix by α-actinin triggers adhesion maturation

    PubMed Central

    Roca-Cusachs, Pere; del Rio, Armando; Puklin-Faucher, Eileen; Gauthier, Nils C.; Biais, Nicolas; Sheetz, Michael P.

    2013-01-01

    Focal adhesions are mechanosensitive elements that enable mechanical communication between cells and the extracellular matrix. Here, we demonstrate a major mechanosensitive pathway in which α-actinin triggers adhesion maturation by linking integrins to actin in nascent adhesions. We show that depletion of the focal adhesion protein α-actinin enhances force generation in initial adhesions on fibronectin, but impairs mechanotransduction in a subsequent step, preventing adhesion maturation. Expression of an α-actinin fragment containing the integrin binding domain, however, dramatically reduces force generation in depleted cells. This behavior can be explained by a competition between talin (which mediates initial adhesion and force generation) and α-actinin for integrin binding. Indeed, we show in an in vitro assay that talin and α-actinin compete for binding to β3 integrins, but cooperate in binding to β1 integrins. Consistently, we find opposite effects of α-actinin depletion and expression of mutants on substrates that bind β3 integrins (fibronectin and vitronectin) versus substrates that only bind β1 integrins (collagen). We thus suggest that nascent adhesions composed of β3 integrins are initially linked to the actin cytoskeleton by talin, and then α-actinin competes with talin to bind β3 integrins. Force transmitted through α-actinin then triggers adhesion maturation. Once adhesions have matured, α-actinin recruitment correlates with force generation, suggesting that α-actinin is the main link transmitting force between integrins and the cytoskeleton in mature adhesions. Such a multistep process enables cells to adjust forces on matrices, unveiling a role of α-actinin that is different from its well-studied function as an actin cross-linker. PMID:23515331

  17. Vinculin tension distributions of individual stress fibers within cell–matrix adhesions

    PubMed Central

    Chang, Ching-Wei; Kumar, Sanjay

    2013-01-01

    Summary Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell–ECM interface. Although it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force. PMID:23687380

  18. T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion

    PubMed Central

    Morriswood, Brooke; Ryzhakov, Grigory; Puri, Claudia; Arden, Susan D.; Roberts, Rhys; Dendrou, Calliope; Kendrick-Jones, John; Buss, Folma

    2009-01-01

    Summary Myosin VI has been implicated in many cellular processes including endocytosis, secretion, membrane ruffling and cell motility. We carried out a yeast two-hybrid screen and identified TRAF6-binding protein (T6BP) and nuclear dot protein 52 (NDP52) as myosin VI binding partners. Myosin VI interaction with T6BP and NDP52 was confirmed in vitro and in vivo and the binding sites on each protein were accurately mapped. Immunofluorescence and electron microscopy showed that T6BP, NDP52 and myosin VI are present at the trans side of the Golgi complex, and on vesicles in the perinuclear region. Although the SKICH domain in T6BP and NDP52 does not mediate recruitment into membrane ruffles, loss of T6BP and NDP52 in RNAi knockdown cells results in reduced membrane ruffling activity and increased stress fibre and focal adhesion formation. Furthermore, we observed in these knockdown cells an upregulation of constitutive secretion of alkaline phosphatase, implying that both proteins act as negative regulators of secretory traffic at the Golgi complex. T6BP was also found to inhibit NF-κB activation, implicating it in the regulation of TRAF6-mediated cytokine signalling. Thus myosin VI-T6BP interactions may link membrane trafficking pathways with cell adhesion and cytokine-dependent cell signalling. PMID:17635994

  19. Primary focal hyperhidrosis: scope of the problem.

    PubMed

    Glaser, Dee Anna; Hebert, Adelaide A; Pariser, David M; Solish, Nowell

    2007-05-01

    Focal hyperhidrosis (HH) can cause debilitating reductions in the physical and emotional quality of life (QOL) of patients, which can result in numerous restrictions of a patient's personal and professional lifestyle and activities. A variety of treatment options are available for primary focal HH, including topical and oral agents, tap water iontophoresis (TWI), botulinum toxin type A (BTX-A), and surgery. Studies evaluating BTX-A (Botox) treatment for palmar, plantar, and facial HH reveal that BTX-A provides effective treatment of primary focal HH, with a reasonable duration of effect, and has a good safety profile. Physicians should understand the impact of focal HH and the need to stay abreast of the available treatment options to provide the best care for patients.

  20. 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

  1. An uncommon focal epithelial hyperplasia manifestation.

    PubMed

    dos Santos-Pinto, Lourdes; Giro, Elisa Maria Aparecida; Pansani, Cyneu Aguiar; Ferrari, Junia; Massucato, Elaine Maria Sgavioli; Spolidório, Luis Carlos

    2009-01-01

    Focal epithelial hyperplasia is a rare, contagious disease associated with infection of the oral mucosa by human papillomavirus types 13 or 32, characterized by multiple soft papules of the same color as the adjacent normal mucosa. It mainly affects the lower lip, buccal mucosa, and tongue. The purpose of this case report was to describe a rare verrucal lesion located in the upper gingiva that was clinically and histologically consistent with focal epithelial hyperplasia. PMID:19941767

  2. Dry adhesives with sensing features

    NASA Astrophysics Data System (ADS)

    Krahn, J.; Menon, C.

    2013-08-01

    Geckos are capable of detecting detachment of their feet. Inspired by this basic observation, a novel functional dry adhesive is proposed, which can be used to measure the instantaneous forces and torques acting on an adhesive pad. Such a novel sensing dry adhesive could potentially be used by climbing robots to quickly realize and respond appropriately to catastrophic detachment conditions. The proposed torque and force sensing dry adhesive was fabricated by mixing Carbon Black (CB) and Polydimethylsiloxane (PDMS) to form a functionalized adhesive with mushroom caps. The addition of CB to PDMS resulted in conductive PDMS which, when under compression, tension or torque, resulted in a change in the resistance across the adhesive patch terminals. The proposed design of the functionalized dry adhesive enables distinguishing an applied torque from a compressive force in a single adhesive pad. A model based on beam theory was used to predict the change in resistance across the terminals as either a torque or compressive force was applied to the adhesive patch. Under a compressive force, the sensing dry adhesive was capable of measuring compression stresses from 0.11 Pa to 20.9 kPa. The torque measured by the adhesive patch ranged from 2.6 to 10 mN m, at which point the dry adhesives became detached. The adhesive strength was 1.75 kPa under an applied preload of 1.65 kPa for an adhesive patch with an adhesive contact area of 7.07 cm2.

  3. Calreticulin modulates cell adhesiveness via regulation of vinculin expression

    PubMed Central

    1996-01-01

    Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)- binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell- substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness. PMID:8991101

  4. Polymeric Nanoelectrodes for Investigating Cellular Adhesion

    NASA Astrophysics Data System (ADS)

    Thapa, Prem; Paneru, Govind; Flanders, Bret

    2011-03-01

    Polyethylene dioxythiophene nano-filaments were grown on lithographic electrode arrays by the recently developed directed electrochemical nanowire assembly technique. These filaments are firmly attached to the electrode but are not attached to the glass substrate. Hence, they behave like cantilevered rods (with one free end). Individual cells of the slime mold Dictystolium discoideum initiate contact by extending pseudopods to the nanoelectrodes when cultured on the electrode arrays. Scanning electron micrographs of the interfaces show the contact area to be of the order of 0.1 μ m 2 . Confocal images reveal the focal adhesions in the cell-electrode contact region. Deflection of the nanoelectrode by an individual cell can be used to measure the force exerted by the cell. Recent results on this innovative force sensing approach will be discussed. NSF.

  5. Sighting optics including an optical element having a first focal length and a second focal length

    SciTech Connect

    Crandall, David Lynn

    2011-08-01

    One embodiment of sighting optics according to the teachings provided herein may include a front sight and a rear sight positioned in spaced-apart relation. The rear sight includes an optical element having a first focal length and a second focal length. The first focal length is selected so that it is about equal to a distance separating the optical element and the front sight and the second focal length is selected so that it is about equal to a target distance. The optical element thus brings into simultaneous focus, for a user, images of the front sight and the target.

  6. Non-uniform breaking of molecular bonds, peripheral morphology and releasable adhesion by elastic anisotropy in bio-adhesive contacts

    PubMed Central

    Liu, Yan; Gao, Yanfei

    2015-01-01

    Biological adhesive contacts are usually of hierarchical structures, such as the clustering of hundreds of sub-micrometre spatulae on keratinous hairs of gecko feet, or the clustering of molecular bonds into focal contacts in cell adhesion. When separating these interfaces, releasable adhesion can be accomplished by asymmetric alignment of the lowest scale discrete bonds (such as the inclined spatula that leads to different peeling force when loading in different directions) or by elastic anisotropy. However, only two-dimensional contact has been analysed for the latter method (Chen & Gao 2007 J. Mech. Phys. Solids 55, 1001–1015 (doi:10.1016/j.jmps.2006.10.008)). Important questions such as the three-dimensional contact morphology, the maximum to minimum pull-off force ratio and the tunability of releasable adhesion cannot be answered. In this work, we developed a three-dimensional cohesive interface model with fictitious viscosity that is capable of simulating the de-adhesion instability and the peripheral morphology before and after the onset of instability. The two-dimensional prediction is found to significantly overestimate the maximum to minimum pull-off force ratio. Based on an interface fracture mechanics analysis, we conclude that (i) the maximum and minimum pull-off forces correspond to the largest and smallest contact stiffness, i.e. ‘stiff-adhere and compliant-release’, (ii) the fracture toughness is sensitive to the crack morphology and the initial contact shape can be designed to attain a significantly higher maximum-to-minimum pull-off force ratio than a circular contact, and (iii) since the adhesion is accomplished by clustering of discrete bonds or called bridged crack in terms of fracture mechanics terminology, the above conclusions can only be achieved when the bridging zone is significantly smaller than the contact size. This adhesion-fracture analogy study leads to mechanistic predictions that can be readily used to design biomimetics and

  7. Non-uniform breaking of molecular bonds, peripheral morphology and releasable adhesion by elastic anisotropy in bio-adhesive contacts.

    PubMed

    Liu, Yan; Gao, Yanfei

    2015-01-01

    Biological adhesive contacts are usually of hierarchical structures, such as the clustering of hundreds of sub-micrometre spatulae on keratinous hairs of gecko feet, or the clustering of molecular bonds into focal contacts in cell adhesion. When separating these interfaces, releasable adhesion can be accomplished by asymmetric alignment of the lowest scale discrete bonds (such as the inclined spatula that leads to different peeling force when loading in different directions) or by elastic anisotropy. However, only two-dimensional contact has been analysed for the latter method (Chen & Gao 2007 J. Mech. Phys. Solids 55, 1001-1015 (doi:10.1016/j.jmps.2006.10.008)). Important questions such as the three-dimensional contact morphology, the maximum to minimum pull-off force ratio and the tunability of releasable adhesion cannot be answered. In this work, we developed a three-dimensional cohesive interface model with fictitious viscosity that is capable of simulating the de-adhesion instability and the peripheral morphology before and after the onset of instability. The two-dimensional prediction is found to significantly overestimate the maximum to minimum pull-off force ratio. Based on an interface fracture mechanics analysis, we conclude that (i) the maximum and minimum pull-off forces correspond to the largest and smallest contact stiffness, i.e. 'stiff-adhere and compliant-release', (ii) the fracture toughness is sensitive to the crack morphology and the initial contact shape can be designed to attain a significantly higher maximum-to-minimum pull-off force ratio than a circular contact, and (iii) since the adhesion is accomplished by clustering of discrete bonds or called bridged crack in terms of fracture mechanics terminology, the above conclusions can only be achieved when the bridging zone is significantly smaller than the contact size. This adhesion-fracture analogy study leads to mechanistic predictions that can be readily used to design biomimetics and

  8. Magnetic field switchable dry adhesives.

    PubMed

    Krahn, Jeffrey; Bovero, Enrico; Menon, Carlo

    2015-02-01

    A magnetic field controllable dry adhesive device is manufactured. The normal adhesion force can be increased or decreased depending on the presence of an applied magnetic field. If the magnetic field is present during the entire normal adhesion test cycle which includes both applying a preloading force and measuring the pulloff pressure, a decrease in adhesion is observed when compared to when there is no applied magnetic field. Similarly, if the magnetic field is present only during the preload portion of the normal adhesion test cycle, a decrease in adhesion is observed because of an increased stiffness of the magnetically controlled dry adhesive device. When the applied magnetic field is present during only the pulloff portion of the normal adhesion test cycle, either an increase or a decrease in normal adhesion is observed depending on the direction of the applied magnetic field.

  9. Adhesive particle shielding

    DOEpatents

    Klebanoff, Leonard Elliott; Rader, Daniel John; Walton, Christopher; Folta, James

    2009-01-06

    An efficient device for capturing fast moving particles has an adhesive particle shield that includes (i) a mounting panel and (ii) a film that is attached to the mounting panel wherein the outer surface of the film has an adhesive coating disposed thereon to capture particles contacting the outer surface. The shield can be employed to maintain a substantially particle free environment such as in photolithographic systems having critical surfaces, such as wafers, masks, and optics and in the tools used to make these components, that are sensitive to particle contamination. The shield can be portable to be positioned in hard-to-reach areas of a photolithography machine. The adhesive particle shield can incorporate cooling means to attract particles via the thermophoresis effect.

  10. Actin Foci Adhesion of D. discoideum

    NASA Astrophysics Data System (ADS)

    Flanders, Bret; Paneru, Govind

    2014-03-01

    Amoeboid migration is a fast (10 μm min-1) integrin-independent mode of migration that is important with D. discoideum, leukocytes, and breast cancer cells. It is poorly understood, but depends on the establishment of adhesive contacts to the substrate where the cell transmits traction forces. In pre-aggregative D. discoideum, a model system for learning about amoeboid migration, these adhesive contacts are discrete complexes that are known as actin-foci. They have an area of ~ 0.5 μm2 and a lifetime of ~ 20 s. This talk will present measurements of the adhesive character of actin foci that have been obtained using a submicron force transducer that was designed for this purpose. Results on the rupture stresses and lifetimes of individual acting foci under nano-newton level forces will be described in the context of a general theory for cellular adhesion. This theory depends on, essentially, three cellular properties: the membrane-medium surface tension, the number density of adhesion receptors in the membrane, and the receptor-substrate potential energy surface. Therefore, the use of the transducer to determine the surface tension will be presented, as well.

  11. NMDA receptor binding in focal epilepsies

    PubMed Central

    McGinnity, C J; Koepp, M J; Hammers, A; Riaño Barros, D A; Pressler, R M; Luthra, S; Jones, P A; Trigg, W; Micallef, C; Symms, M R; Brooks, D J; Duncan, J S

    2015-01-01

    Objective To demonstrate altered N-methyl-d-aspartate (NMDA) receptor availability in patients with focal epilepsies using positron emission tomography (PET) and [18F]GE-179, a ligand that selectively binds to the open NMDA receptor ion channel, which is thought to be overactive in epilepsy. Methods Eleven patients (median age 33 years, 6 males) with known frequent interictal epileptiform discharges had an [18F]GE-179 PET scan, in a cross-sectional study. MRI showed a focal lesion but discordant EEG changes in two, was non-localising with multifocal EEG abnormalities in two, and was normal in the remaining seven patients who all had multifocal EEG changes. Individual patient [18F]GE-179 volume-of-distribution (VT) images were compared between individual patients and a group of 10 healthy controls (47 years, 7 males) using Statistical Parametric Mapping. Results Individual analyses revealed a single cluster of focal VT increase in four patients; one with a single and one with multifocal MRI lesions, and two with normal MRIs. Post hoc analysis revealed that, relative to controls, patients not taking antidepressants had globally increased [18F]GE-179 VT (+28%; p<0.002), and the three patients taking an antidepressant drug had globally reduced [18F]GE-179 VT (−29%; p<0.002). There were no focal abnormalities common to the epilepsy group. Conclusions In patients with focal epilepsies, we detected primarily global increases of [18F]GE-179 VT consistent with increased NMDA channel activation, but reduced availability in those taking antidepressant drugs, consistent with a possible mode of action of this class of drugs. [18F]GE-179 PET showed focal accentuations of NMDA binding in 4 out of 11 patients, with difficult to localise and treat focal epilepsy. PMID:25991402

  12. Elastomer toughened polyimide adhesives

    NASA Technical Reports Server (NTRS)

    St.clair, A. K.; St.clair, T. L. (Inventor)

    1983-01-01

    A rubber-toughened addition-type polyimide composition is disclosed which has excellent high temperature bonding characteristics in the fully cured state, and improved peel strength and adhesive fracture resistance physical property characteristics. The process for making the improved adhesive involves preparing the rubber containing amic acid prepolymer by chemically reacting an amine-terminated elastomer and an aromatic diamine with an aromatic dianhydride with which a reactive chain stopper anhydride was mixed, and utilizing solvent or mixture of solvents for the reaction.

  13. Adhesion in hydrogel contacts

    NASA Astrophysics Data System (ADS)

    Torres, J. R.; Jay, G. D.; Kim, K.-S.; Bothun, G. D.

    2016-05-01

    A generalized thermomechanical model for adhesion was developed to elucidate the mechanisms of dissipation within the viscoelastic bulk of a hyperelastic hydrogel. Results show that in addition to the expected energy release rate of interface formation, as well as the viscous flow dissipation, the bulk composition exhibits dissipation due to phase inhomogeneity morphological changes. The mixing thermodynamics of the matrix and solvent determines the dynamics of the phase inhomogeneities, which can enhance or disrupt adhesion. The model also accounts for the time-dependent behaviour. A parameter is proposed to discern the dominant dissipation mechanism in hydrogel contact detachment.

  14. Detecting cell-adhesive sites in extracellular matrix using force spectroscopy mapping

    PubMed Central

    Chirasatitsin, Somyot; Engler, Adam J

    2010-01-01

    The cell microenvironment is composed of extracellular matrix (ECM), which contains specific binding sites that allow the cell to adhere to its surroundings. Cells employ focal adhesion proteins, which must be able to resist a variety of forces to bind to ECM. Current techniques for detecting the spatial arrangement of these adhesions, however, have limited resolution and those that detect adhesive forces lack sufficient spatial characterization or resolution. Using a unique application of force spectroscopy, we demonstrate here the ability to determine local changes in the adhesive property of a fibronectin substrate down to the resolution of the fibronectin antibody-functionalized tip diameter, ~20 nm. To verify the detection capabilities of force spectroscopy mapping (FSM), changes in loading rate and temperature were used to alter the bond dynamics and change the adhesion force. Microcontact printing was also used to pattern fluorescein isothiocyanate-conjugated fibronectin in order to mimic the discontinuous adhesion domains of native ECM. Fluorescent detection was used to identify the pattern while FSM was used to map cell adhesion sites in registry with the initial fluorescent image. The results show that FSM can be used to detect the adhesion domains at high resolution and may subsequently be applied to native ECM with randomly distributed cell adhesion sites. PMID:21152375

  15. Topographic cell instructive patterns to control cell adhesion, polarization and migration

    PubMed Central

    Ventre, Maurizio; Natale, Carlo Fortunato; Rianna, Carmela; Netti, Paolo Antonio

    2014-01-01

    Topographic patterns are known to affect cellular processes such as adhesion, migration and differentiation. However, the optimal way to deliver topographic signals to provide cells with precise instructions has not been defined yet. In this work, we hypothesize that topographic patterns may be able to control the sensing and adhesion machinery of cells when their interval features are tuned on the characteristic lengths of filopodial probing and focal adhesions (FAs). Features separated by distance beyond the length of filopodia cannot be readily perceived; therefore, the formation of new adhesions is discouraged. If, however, topographic features are separated by a distance within the reach of filopodia extension, cells can establish contact between adjacent topographic islands. In the latter case, cell adhesion and polarization rely upon the growth of FAs occurring on a specific length scale that depends on the chemical properties of the surface. Topographic patterns and chemical properties may interfere with the growth of FAs, thus making adhesions unstable. To test this hypothesis, we fabricated different micropatterned surfaces displaying feature dimensions and adhesive properties able to interfere with the filopodial sensing and the adhesion maturation, selectively. Our data demonstrate that it is possible to exert a potent control on cell adhesion, elongation and migration by tuning topographic features’ dimensions and surface chemistry. PMID:25253035

  16. Focal Plane Metrology for the LSST Camera

    SciTech Connect

    A Rasmussen, Andrew P.; Hale, Layton; Kim, Peter; Lee, Eric; Perl, Martin; Schindler, Rafe; Takacs, Peter; Thurston, Timothy; /SLAC

    2007-01-10

    Meeting the science goals for the Large Synoptic Survey Telescope (LSST) translates into a demanding set of imaging performance requirements for the optical system over a wide (3.5{sup o}) field of view. In turn, meeting those imaging requirements necessitates maintaining precise control of the focal plane surface (10 {micro}m P-V) over the entire field of view (640 mm diameter) at the operating temperature (T {approx} -100 C) and over the operational elevation angle range. We briefly describe the hierarchical design approach for the LSST Camera focal plane and the baseline design for assembling the flat focal plane at room temperature. Preliminary results of gravity load and thermal distortion calculations are provided, and early metrological verification of candidate materials under cold thermal conditions are presented. A detailed, generalized method for stitching together sparse metrology data originating from differential, non-contact metrological data acquisition spanning multiple (non-continuous) sensor surfaces making up the focal plane, is described and demonstrated. Finally, we describe some in situ alignment verification alternatives, some of which may be integrated into the camera's focal plane.

  17. Rapid Development of Wet Adhesion between Carboxymethylcellulose Modified Cellulose Surfaces Laminated with Polyvinylamine Adhesive.

    PubMed

    Gustafsson, Emil; Pelton, Robert; Wågberg, Lars

    2016-09-14

    The surface of regenerated cellulose membranes was modified by irreversible adsorption of carboxymethylcellulose (CMC). Pairs of wet CMC-modified membranes were laminated with polyvinylamine (PVAm) at room temperature, and the delamination force for wet membranes was measured for both dried and never-dried laminates. The wet adhesion was studied as a function of PVAm molecular weight, amine content, and deposition pH of the polyelectrolyte. Surprisingly the PVAm-CMC system gave substantial wet adhesion that exceeded that of TEMPO-oxidized membranes with PVAm for both dried and never-dried laminates. The greatest wet adhesion was achieved for fully hydrolyzed high molecular weight PVAm. Bulk carboxymethylation of cellulose membranes gave inferior wet adhesion combined with PVAm as compared to CMC adsorption which indicates that a CMC layer of the order of 10 nm was necessary. There are no obvious covalent cross-linking reactions between CMC and PVAm at room temperature, and on the basis of our results, we are instead attributing the wet adhesion to complex formation between the PVAm and the irreversibly adsorbed CMC at the cellulose surface. We propose that interdigitation of PVAm chains into the CMC layer is responsible for the high wet adhesion values. PMID:27552256

  18. Intercellular adhesion molecule 1 is the major adhesion molecule expressed during schistosome granuloma formation.

    PubMed Central

    Ritter, D M; McKerrow, J H

    1996-01-01

    Endothelial cell adhesion molecules play a key role in inflammation by initiating leukocyte trafficking. One of the most complex inflammatory responses is the formation of a cellular granuloma. Expression of adhesion molecules during granuloma formation was investigated by using the murine host reaction to schistosome parasite eggs deposited in the liver as a model. By both immunohistochemistry and lymphocyte adhesion assays, the predominant interaction identified was between intercellular adhesion molecule 1 (ICAM-1) and its cognate integrin, leukocyte functional antigen 1 (LFA-1). ICAM-1 expression on sinusoidal endothelium was induced when eggs were first deposited in the liver, peaked in parallel with granuloma size, and was downregulated with modulation of the granuloma. Polyacrylamide beads coated with soluble parasite egg antigens could induce ICAM-1 expression on endothelial cells in vitro only in the presence of tumor necrosis factor alpha, a cytokine previously shown to be key to granuloma formation. A role for ICAM-1 in recruiting lymphocytes to the hepatic granuloma was also supported by the observation that lymphocytes preincubated with anti-LFA-1 antibody did not bind to granulomas in tissue sections. While ICAM-1 is the predominant adhesion molecule in schistosome egg granuloma formation in wild-type mice, when the ICAM-1 gene is knocked out, vascular cell adhesion molecule 1 is upregulated and granuloma formation is preserved. PMID:8890229

  19. Cell adhesion in zebrafish myogenesis: distribution of intermediate filaments, microfilaments, intracellular adhesion structures and extracellular matrix.

    PubMed

    Costa, Manoel L; Escaleira, Roberta C; Jazenko, Fernanda; Mermelstein, Claudia S

    2008-10-01

    To overcome the limitations of in vitro studies, we have been studying myogenesis in situ in zebrafish embryos, at a sub-cellular level. While in previous works we focused on myofibrillogenesis and some aspects of adhesion structures, here we describe in more detail cell adhesion structures and interactions among cytoskeletal components, membrane and extracellular matrix during zebrafish muscle development. We studied the intermediate filaments, and we describe the full range of desmin distribution in zebrafish development, from perinuclear to striated, until its deposition around the intersomite septa of older somites. This adhesion structure, positive for desmin and actin, has not been previously observed in myogenesis in vitro. We also show that actin is initially located in the intersomite septum region whereas it is confined to the myofibrils later on. While actin localization changes during development, the adhesion complex proteins vinculin, paxillin, talin, dystrophin, laminin and fibronectin always appear exclusively at the intersomite septa, and appear to be co-distributed, even though the extracellular proteins accumulates before the intracellular ones. Contrary to the adhesion proteins, that are continuously distributed, desmin and sarcomeric actin form triangular aggregates among the septa and the cytoskeleton. We studied the cytoskeletal linker plectin as well, and we show that it has a distribution similar to desmin and not to actin. We conclude that the in situ adhesion structures differ from their in vitro counterparts, and that the actual zebrafish embryo myogenesis is quite different than that which occurs in in vitro systems.

  20. Switchable Adhesion in Vacuum Using Bio-Inspired Dry Adhesives.

    PubMed

    Purtov, Julia; Frensemeier, Mareike; Kroner, Elmar

    2015-11-01

    Suction based attachment systems for pick and place handling of fragile objects like glass plates or optical lenses are energy-consuming and noisy and fail at reduced air pressure, which is essential, e.g., in chemical and physical vapor deposition processes. Recently, an alternative approach toward reversible adhesion of sensitive objects based on bioinspired dry adhesive structures has emerged. There, the switching in adhesion is achieved by a reversible buckling of adhesive pillar structures. In this study, we demonstrate that these adhesives are capable of switching adhesion not only in ambient air conditions but also in vacuum. Our bioinspired patterned adhesive with an area of 1 cm(2) provided an adhesion force of 2.6 N ± 0.2 N in air, which was reduced to 1.9 N ± 0.2 N if measured in vacuum. Detachment was induced by buckling of the structures due to a high compressive preload and occurred, independent of air pressure, at approximately 0.9 N ± 0.1 N. The switch in adhesion was observed at a compressive preload between 5.6 and 6.0 N and was independent of air pressure. The difference between maximum adhesion force and adhesion force after buckling gives a reasonable window of operation for pick and place processes. High reversibility of the switching behavior is shown over 50 cycles in air and in vacuum, making the bioinspired switchable adhesive applicable for handling operations of fragile objects.

  1. Switchable Adhesion in Vacuum Using Bio-Inspired Dry Adhesives

    PubMed Central

    2015-01-01

    Suction based attachment systems for pick and place handling of fragile objects like glass plates or optical lenses are energy-consuming and noisy and fail at reduced air pressure, which is essential, e.g., in chemical and physical vapor deposition processes. Recently, an alternative approach toward reversible adhesion of sensitive objects based on bioinspired dry adhesive structures has emerged. There, the switching in adhesion is achieved by a reversible buckling of adhesive pillar structures. In this study, we demonstrate that these adhesives are capable of switching adhesion not only in ambient air conditions but also in vacuum. Our bioinspired patterned adhesive with an area of 1 cm2 provided an adhesion force of 2.6 N ± 0.2 N in air, which was reduced to 1.9 N ± 0.2 N if measured in vacuum. Detachment was induced by buckling of the structures due to a high compressive preload and occurred, independent of air pressure, at approximately 0.9 N ± 0.1 N. The switch in adhesion was observed at a compressive preload between 5.6 and 6.0 N and was independent of air pressure. The difference between maximum adhesion force and adhesion force after buckling gives a reasonable window of operation for pick and place processes. High reversibility of the switching behavior is shown over 50 cycles in air and in vacuum, making the bioinspired switchable adhesive applicable for handling operations of fragile objects. PMID:26457864

  2. Switchable Adhesion in Vacuum Using Bio-Inspired Dry Adhesives.

    PubMed

    Purtov, Julia; Frensemeier, Mareike; Kroner, Elmar

    2015-11-01

    Suction based attachment systems for pick and place handling of fragile objects like glass plates or optical lenses are energy-consuming and noisy and fail at reduced air pressure, which is essential, e.g., in chemical and physical vapor deposition processes. Recently, an alternative approach toward reversible adhesion of sensitive objects based on bioinspired dry adhesive structures has emerged. There, the switching in adhesion is achieved by a reversible buckling of adhesive pillar structures. In this study, we demonstrate that these adhesives are capable of switching adhesion not only in ambient air conditions but also in vacuum. Our bioinspired patterned adhesive with an area of 1 cm(2) provided an adhesion force of 2.6 N ± 0.2 N in air, which was reduced to 1.9 N ± 0.2 N if measured in vacuum. Detachment was induced by buckling of the structures due to a high compressive preload and occurred, independent of air pressure, at approximately 0.9 N ± 0.1 N. The switch in adhesion was observed at a compressive preload between 5.6 and 6.0 N and was independent of air pressure. The difference between maximum adhesion force and adhesion force after buckling gives a reasonable window of operation for pick and place processes. High reversibility of the switching behavior is shown over 50 cycles in air and in vacuum, making the bioinspired switchable adhesive applicable for handling operations of fragile objects. PMID:26457864

  3. Towards a chemistry of cohesion and adhesion

    NASA Astrophysics Data System (ADS)

    Eberhart, M. E.; Donovan, M. M.; MacLaren, J. M.; Clougherty, D. P.

    Modern chemistry frequently describes the structure and reaction dynamics of molecules in terms of the general principle of “competition for bonds”; consequently, bonding forms the basis of the language of chemistry. The actual models used to represent these bonds are frequently system specific. Organic reactions are described in terms of bonds based on pairs of atomic valence electrons. Reactions of inorganic coordination complexes are described in terms of bonds based on a molecular orbital representation. In analogy to those chemistries, a representation for a bond and bond strength, suitable for describing the cohesive and adhesive properties of all classes of materials, is introduced. This representation proves to yield an explanation for the observed cohesive properties of a specific class of materials (cleavage in bcc metals), and it also provides a framework for exploring and analyzing the more complex phenomena of cohesion and adhesion, such as environmentally-induced embrittlement. A complete chemistry of cohesion and adhesion will require the demonstration that the specific bonding model used can form the basis for consistent interpretations for a wealth of experimental phenomena beyond environmentally-induced embrittlement; thus, as presented, this model does not provide a complete chemistry of cohesion and adhesion, but does embody the first steps in that direction.

  4. Wood Composite Adhesives

    NASA Astrophysics Data System (ADS)

    Gomez-Bueso, Jose; Haupt, Robert

    The global environment, in which phenolic resins are being used for wood composite manufacture, has changed significantly during the last decade. This chapter reviews trends that are driving the use and consumption of phenolic resins around the world. The review begins with recent data on volume usage and regional trends, followed by an analysis of factors affecting global markets. In a section on environmental factors, the impact of recent formaldehyde emission regulations is discussed. The section on economics introduces wood composite production as it relates to the available adhesive systems, with special emphasis on the technical requirement to improve phenolic reactivity. Advances in composite process technology are introduced, especially in regard to the increased demands the improvements place upon adhesive system performance. The specific requirements for the various wood composite families are considered in the context of adhesive performance needs. The results of research into current chemistries are discussed, with a review of recent findings regarding the mechanisms of phenolic condensation and acceleration. Also, the work regarding alternate natural materials, such as carbohydrates, lignins, tannins, and proteinaceous materials, is presented. Finally, new developments in alternative adhesive technologies are reported.