Adenomatous polyposis coli nucleates actin assembly to drive cell migration and microtubule-induced focal adhesion turnover

PubMed Central

Eskin, Julian A.; Jaiswal, Richa

2017-01-01

Cell motility depends on tight coordination between the microtubule (MT) and actin cytoskeletons, but the mechanisms underlying this MT–actin cross talk have remained poorly understood. Here, we show that the tumor suppressor protein adenomatous polyposis coli (APC), which is a known MT-associated protein, directly nucleates actin assembly to promote directed cell migration. By changing only two residues in APC, we generated a separation-of-function mutant, APC (m4), that abolishes actin nucleation activity without affecting MT interactions. Expression of full-length APC carrying the m4 mutation (APC (m4)) rescued cellular defects in MT organization, MT dynamics, and mitochondrial distribution caused by depletion of endogenous APC but failed to restore cell migration. Wild-type APC and APC (m4) localized to focal adhesions (FAs), and APC (m4) was defective in promoting actin assembly at FAs to facilitate MT-induced FA turnover. These results provide the first direct evidence for APC-mediated actin assembly in vivo and establish a role for APC in coordinating MTs and actin at FAs to direct cell migration. PMID:28663347

Nephrin Regulates Lamellipodia Formation by Assembling a Protein Complex That Includes Ship2, Filamin and Lamellipodin

PubMed Central

Venkatareddy, Madhusudan; Cook, Leslie; Abuarquob, Kamal; Verma, Rakesh; Garg, Puneet

2011-01-01

Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5′ inositol phosphatase), Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics. PMID:22194892

Nanoengineered surfaces for focal adhesion guidance trigger mesenchymal stem cell self-organization and tenogenesis.

PubMed

Iannone, Maria; Ventre, Maurizio; Formisano, Lucia; Casalino, Laura; Patriarca, Eduardo J; Netti, Paolo A

2015-03-11

The initial conditions for morphogenesis trigger a cascade of events that ultimately dictate structure and functions of tissues and organs. Here we report that surface nanopatterning can control the initial assembly of focal adhesions, hence guiding human mesenchymal stem cells (hMSCs) through the process of self-organization and differentiation. This process self-sustains, leading to the development of macroscopic tissues with molecular profiles and microarchitecture reminiscent of embryonic tendons. Therefore, material surfaces can be in principle engineered to set off the hMSC program toward tissuegenesis in a deterministic manner by providing adequate sets of initial environmental conditions.

Tunable swelling of polyelectrolyte multilayers in cell culture media for modulating NIH-3T3 cells adhesion.

PubMed

Qi, Wei; Cai, Peng; Yuan, Wenjing; Wang, Hua

2014-11-01

For polyelectrolyte multilayers (PEMs) assembled by the layer-by-layer (LbL) assembly technique, their nanostructure and properties can be governed by many parameters during the building process. Here, it was demonstrated that the swelling of the PEMs containing poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) in cell culture media could be tuned with changing supporting salt solutions during the assembly process. Importantly, the influence of the PEMs assembled in different salt solutions on NIH-3T3 cell adhesion was observable. Specifically, the cells could possess a higher affinity for the films assembled in low salt concentration (i.e. 0.15M NaCl) or no salt, the poorly swelling films in cell culture media, which was manifested by the large cell spreading area and focal adhesions. In contrast, those were assembled in higher salt concentration, highly swelling films in cell culture media, were less attractive for the fibroblasts. As a result, the cell adhesion behaviors may be manipulated by tailoring the physicochemical properties of the films, which could be performed by changing the assembly conditions such as supporting salt concentration. Such a finding might promise a great potential in designing desired biomaterials for tissue engineering and regenerative medicine. © 2014 Wiley Periodicals, Inc.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

PubMed

Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

2015-11-01

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Isolation of integrin-based adhesion complexes.

PubMed

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

2015-03-02

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 signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and 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 via subsequent downstream analysis by western blotting or mass spectrometry. Copyright © 2015 John Wiley & Sons, Inc.

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

PubMed Central

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

2015-01-01

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

Cold plasma selectivity in the interaction with various types of the cells

NASA Astrophysics Data System (ADS)

Volotskova, Olga; Stepp, Mary Ann; Keidar, Michael

2011-10-01

Present research in the area of cold atmospheric plasma (CAP) demonstrates great potential in various areas including medicine and biology. Depending on their configuration they can be used for wound healing, sterilization, targeted cell/tissue removal, and cancer treatments. Here we explore potential mechanisms by which CAP alters cell migration and influences cell adhesion. The migration studies are focused on the CAP interaction with fibroblasts and corneal epithelial cells. Data show that various types of cells have different thresholds (treatment times) required to achieve maximum inhibition of cell migration which is around ~30-40%. Studies to assess the impact of CAP treatment on the activation state of integrins and focal adhesion size by immunofluorescence showed more active b1 integrin on the cell surface and large focal adhesions after CAP treatment. Based on these data, a thermodynamic model is presented to explain how CAP leads to integrin activation and focal adhesion assembly. Also responses of the various types of the cells to the cold plasma treatment on the example of the epithelial keratinocytes, papilloma and carcinoma cells are studied. Cell cycle, migration and cell vitality analysis were performed. The goal of this study is to understand the mechanism by which the CAP jet alters cell migration, influences adhesion and cell survival.

The G Protein-Coupled Estrogen Receptor-1, GPER-1, Promotes Fibrillogenesis via a Shc-Dependent Pathway Resulting in Anchorage-Independent Growth

PubMed Central

Magruder, Hilary T.; Quinn, Jeffrey A.; Schwartzbauer, Jean E.; Reichner, Jonathan; Huang, Allan

2016-01-01

The G protein-coupled estrogen receptor-1, GPER-1, coordinates fibronectin (FN) matrix assembly and release of heparan-bound epidermal growth factor (HB-EGF). This mechanism of action results in the recruitment of FN-engaged integrin α5β1 to fibrillar adhesions and the formation of integrin α5β1-Shc adaptor protein complexes. Here, we show that GPER-1 stimulation of murine 4 T1 or human SKBR3 breast cancer cells with 17β-estradiol (E2β) promotes the formation of focal adhesions and actin stress fibers and results in increased cellular adhesion and haptotaxis on FN, but not collagen. These actions are also induced by the xenoestrogen, bisphenol A, and the estrogen receptor (ER) antagonist, ICI 182, 780, but not the inactive stereoisomer, 17α-estradiol (E2α). In addition, we show that GPER-1 stimulation of breast cancer cells allows for FN-dependent, anchorage-independent growth and FN fibril formation in “hanging drop” assays, indicating that these GPER-1-mediated actions occur independently of adhesion to solid substrata. Stable expression of Shc mutant Y317F lacking its primary tyrosyl phosphorylation site disrupts E2β-induced focal adhesion and actin stress fiber formation and abolishes E2β-enhanced haptotaxis on FN and anchorage-dependent growth. Collectively, these data demonstrate that E2β action via GPER-1 enhances cellular adhesivity and FN matrix assembly and allows for anchorage-independent growth, cellular events that may allow for cellular survival, and tumor progression. PMID:25096985

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

PubMed

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

2015-07-20

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature. © 2015 Treuner-Lange et al.

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

PubMed Central

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

2015-01-01

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

Signal transduction by beta1 integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-I receptor.

PubMed

Shakibaei, M; John, T; De Souza, P; Rahmanzadeh, R; Merker, H J

1999-09-15

We have examined the mechanism by which collagen-binding integrins co-operate with insulin-like growth factor-I (IGF-I) receptors (IGF-IR) to regulate chondrocyte phenotype and differentiation. Adhesion of chondrocytes to anti-beta1 integrin antibodies or collagen type II leads to phosphorylation of cytoskeletal and signalling proteins localized at focal adhesions, including alpha-actinin, vinculin, paxillin and focal adhesion kinase (FAK). These stimulate docking proteins such as Shc (Src-homology collagen). Moreover, exposure of collagen type II-cultured chondrocytes to IGF-I leads to co-immunoprecipitation of Shc protein with the IGF-IR and with beta1, alpha1 and alpha5 integrins, but not with alpha3 integrin. Shc then associates with growth factor receptor-bound protein 2 (Grb2), an adaptor protein and extracellular signal-regulated kinase. The expression of the docking protein Shc occurs only when chondrocytes are bound to collagen type II or integrin antibodies and increases when IGF-I is added, suggesting a collaboration between integrins and growth factors in a common/shared biochemical signalling pathway. Furthermore, these results indicate that focal adhesion assembly may facilitate signalling via Shc, a potential common target for signal integration between integrin and growth-factor signalling regulatory pathways. Thus, the collagen-binding integrins and IGF-IR co-operate to regulate focal adhesion components and these signalling pathways have common targets (Shc-Grb2 complex) in subcellular compartments, thereby linking to the Ras-mitogen-activated protein kinase signalling pathway. These events may play a role during chondrocyte differentiation.

Targeting and transport: How microtubules control focal adhesion dynamics

PubMed Central

Stehbens, Samantha

2012-01-01

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge. PMID:22908306

Focal adhesions and Ras are functionally and spatially integrated to mediate IL-1 activation of ERK

PubMed Central

Wang, Qin; Downey, Gregory P.; McCulloch, Christopher A.

2011-01-01

In connective tissue cells, IL-1-induced ERK activation leading to matrix metalloproteinase (MMP)-3 expression is dependent on cooperative interactions between focal adhesions and the endoplasmic reticulum (ER). As Ras can be activated on the ER, we investigated the role of Ras in IL-1 signaling and focal adhesion formation. We found that constitutively active H-Ras, K-Ras or N-Ras enhanced focal adhesion maturation and β1-integrin activation. IL-1 promoted the accumulation of Ras isoforms in ER and focal adhesion fractions, as shown in cells cotransfected with GFP-tagged Ras isoforms and YFP-ER protein and by analysis of subcellular fractions enriched for ER or focal adhesion proteins. Dominant-negative H-Ras or K-Ras reduced accumulation of H-Ras and K-Ras in focal adhesions induced by IL-1 and also blocked ERK activation and focal adhesion maturation. Ras-GRF was enriched constitutively in focal adhesion fractions and was required for Ras recruitment to focal adhesions. We conclude that Ras activation and IL-1 signaling are interactive processes that regulate the maturation of focal adhesions, which, in turn, is required for ERK activation.—Wang, Q., Downey, G. P., McCulloch, C. A. Focal adhesions and Ras are functionally and spatially integrated to mediate IL-1 activation of ERK. PMID:21719512

Integrin activation by a cold atmospheric plasma jet

NASA Astrophysics Data System (ADS)

Volotskova, Olga; Stepp, Mary Ann; Keidar, Michael

2012-05-01

Current breakthrough research on cold atmospheric plasma (CAP) demonstrates that CAP has great potential in various areas, including medicine and biology, thus providing a new tool for living tissue treatment. In this paper, we explore potential mechanisms by which CAP alters cell migration and influences cell adhesion. We focus on the study of CAP interaction with fibroblasts and corneal epithelial cells. The data show that fibroblasts and corneal epithelial cells have different thresholds (treatment times) required to achieve maximum inhibition of cell migration. Both cell types reduced their migration rates by ˜30-40% after CAP compared to control cells. Also, the impact of CAP treatment on cell migration and persistence of fibroblasts after integrin activation by MnCl2, serum starvation or replating cells onto surfaces coated with integrin ligands is assessed; the results show that activation by MnCl2 or starvation attenuates cells’ responses to plasma. Studies carried out to assess the impact of CAP treatment on the activation state of β1 integrin and focal adhesion size by using immunofluorescence show that fibroblasts have more active β1 integrin on their surface and large focal adhesions after CAP treatment. Based on these data, a thermodynamic model is presented to explain how CAP leads to integrin activation and focal adhesion assembly.

αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin

PubMed Central

Bharadwaj, Mitasha; Strohmeyer, Nico; Colo, Georgina P.; Helenius, Jonne; Beerenwinkel, Niko; Schiller, Herbert B.; Fässler, Reinhard; Müller, Daniel J.

2017-01-01

Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5β1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5β1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5β1 integrins. Once engaged, αV-class integrins signal to α5β1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5β1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix. PMID:28128308

Rho GTPases Control Polarity, Protrusion, and Adhesion during Cell Movement

PubMed Central

Nobes, Catherine D.; Hall, Alan

1999-01-01

Cell movement is essential during embryogenesis to establish tissue patterns and to drive morphogenetic pathways and in the adult for tissue repair and to direct cells to sites of infection. Animal cells move by crawling and the driving force is derived primarily from the coordinated assembly and disassembly of actin filaments. The small GTPases, Rho, Rac, and Cdc42, regulate the organization of actin filaments and we have analyzed their contributions to the movement of primary embryo fibroblasts in an in vitro wound healing assay. Rac is essential for the protrusion of lamellipodia and for forward movement. Cdc42 is required to maintain cell polarity, which includes the localization of lamellipodial activity to the leading edge and the reorientation of the Golgi apparatus in the direction of movement. Rho is required to maintain cell adhesion during movement, but stress fibers and focal adhesions are not required. Finally, Ras regulates focal adhesion and stress fiber turnover and this is essential for cell movement. We conclude that the signal transduction pathways controlled by the four small GTPases, Rho, Rac, Cdc42, and Ras, cooperate to promote cell movement. PMID:10087266

Mechanical Mounting and Adhesive Junction for Large Quartz Optics Operatng at Cryogenic Temperature

NASA Astrophysics Data System (ADS)

Pellizzari, M.; Mosciarello, P.

2012-07-01

Gaia is a global space astrometry mission, with the goal to make the largest, most precise three-dimensional map of our Galaxy. Gaia contains two optical telescopes: in front of their Focal Plane Assembly -FPA- two narrow quartz prisms are mounted for spectrophotometer science: the Blue and Red Photometer Prisms -BPP and RPP-. They are framed in a SiC structure by means of brackets and adhesive junctions between metal parts and quartz optical elements. SELEX GALILEO developed this project as subcontractor of Astrium France. The assembly has to withstand thermoelastic loads due to CTE mismatch at an operative temperature of 120 K. The mechanical mountings design to reduce the stresses due to thermal loads on the adhesive joint is described and the results of the bonding qualification process as well as the flight hardware bonding results are reported.

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

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells. Copyright © 2017 Albecka et al.

In vivo epidermal migration requires focal adhesion targeting of ACF7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yue, Jiping; Zhang, Yao; Liang, Wenguang G.

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

In vivo epidermal migration requires focal adhesion targeting of ACF7

DOE PAGES

Yue, Jiping; Zhang, Yao; Liang, Wenguang G.; ...

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

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

PubMed

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

2015-08-01

The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. © 2014 John Wiley & Sons A/S.

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

PubMed Central

Bjerke, Maureen A.; Dzamba, Bette; Wang, Chong; DeSimone, Douglas W.

2014-01-01

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity. PMID:25127991

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

DOE PAGES

Gao, Zhiwen; Gao, Yanfei

2016-05-14

We report that 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 formore » 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. Finally, 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Zhiwen; Gao, Yanfei

We report that 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 formore » 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. Finally, 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.« less

Tropomyosin Tm5NM1 Spatially Restricts Src Kinase Activity through Perturbation of Rab11 Vesicle Trafficking

PubMed Central

Bach, Cuc T.; Murray, Rachael Z.; Owen, Dylan; Gaus, Kat

2014-01-01

In order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules. PMID:25288639

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

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

PubMed Central

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-01-01

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min−1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics. PMID:27991512

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

NASA Astrophysics Data System (ADS)

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-12-01

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions.

PubMed

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-12-19

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min -1 . The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Localization-based super-resolution imaging of cellular structures.

PubMed

Kanchanawong, Pakorn; Waterman, Clare M

2013-01-01

Fluorescence microscopy allows direct visualization of fluorescently tagged proteins within cells. However, the spatial resolution of conventional fluorescence microscopes is limited by diffraction to ~250 nm, prompting the development of super-resolution microscopy which offers resolution approaching the scale of single proteins, i.e., ~20 nm. Here, we describe protocols for single molecule localization-based super-resolution imaging, using focal adhesion proteins as an example and employing either photoswitchable fluorophores or photoactivatable fluorescent proteins. These protocols should also be easily adaptable to imaging a broad array of macromolecular assemblies in cells whose components can be fluorescently tagged and assemble into high density structures.

Super-resolution links vinculin localization to function in focal adhesions.

PubMed

Giannone, Grégory

2015-07-01

Integrin-based focal adhesions integrate biochemical and biomechanical signals from the extracellular matrix and the actin cytoskeleton. The combination of three-dimensional super-resolution imaging and loss- or gain-of-function protein mutants now links the nanoscale dynamic localization of proteins to their activation and function within focal adhesions.

Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption.

PubMed

Hadzic, Ermin; Catillon, Marie; Halavatyi, Aliaksandr; Medves, Sandrine; Van Troys, Marleen; Moes, Michèle; Baird, Michelle A; Davidson, Michael W; Schaffner-Reckinger, Elisabeth; Ampe, Christophe; Friederich, Evelyne

2015-01-01

Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading.

Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption

PubMed Central

Hadzic, Ermin; Catillon, Marie; Halavatyi, Aliaksandr; Medves, Sandrine; Van Troys, Marleen; Moes, Michèle; Baird, Michelle A.; Davidson, Michael W.; Schaffner-Reckinger, Elisabeth; Ampe, Christophe; Friederich, Evelyne

2015-01-01

Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading. PMID:26509500

TES is a novel focal adhesion protein with a role in cell spreading.

PubMed

Coutts, Amanda S; MacKenzie, Elaine; Griffith, Elen; Black, Donald M

2003-03-01

Previously, we identified TES as a novel candidate tumour suppressor gene that mapped to human chromosome 7q31.1. In this report we demonstrate that the TES protein is localised at focal adhesions, actin stress fibres and areas of cell-cell contact. TES has three C-terminal LIM domains that appear to be important for focal adhesion targeting. Additionally, the N-terminal region is important for targeting TES to actin stress fibres. Yeast two-hybrid and biochemical analyses yielded interactions with several focal adhesion and/or cytoskeletal proteins including mena, zyxin and talin. The fact that TES localises to regions of cell adhesion suggests that it functions in events related to cell motility and adhesion. In support of this, we demonstrate that fibroblasts stably overexpressing TES have an increased ability to spread on fibronectin.

Calpain-Mediated Proteolysis of Talin and FAK Regulates Adhesion Dynamics Necessary for Axon Guidance

PubMed Central

Kerstein, Patrick C.; Patel, Kevin M.

2017-01-01

Guidance of axons to their proper synaptic target sites requires spatially and temporally precise modulation of biochemical signals within growth cones. Ionic calcium (Ca2+) is an essential signal for axon guidance that mediates opposing effects on growth cone motility. The diverse effects of Ca2+ arise from the precise localization of Ca2+ signals into microdomains containing specific Ca2+ effectors. For example, differences in the mechanical and chemical composition of the underlying substrata elicit local Ca2+ signals within growth cone filopodia that regulate axon guidance through activation of the protease calpain. However, how calpain regulates growth cone motility remains unclear. Here, we identify the adhesion proteins talin and focal adhesion kinase (FAK) as proteolytic targets of calpain in Xenopus laevis spinal cord neurons both in vivo and in vitro. Inhibition of calpain increases the localization of endogenous adhesion signaling to growth cone filopodia. Using live cell microscopy and specific calpain-resistant point-mutants of talin (L432G) and FAK (V744G), we find that calpain inhibits paxillin-based adhesion assembly through cleavage of talin and FAK, and adhesion disassembly through cleavage of FAK. Blocking calpain cleavage of talin and FAK inhibits repulsive turning from focal uncaging of Ca2+ within filopodia. In addition, blocking calpain cleavage of talin and FAK in vivo promotes Rohon–Beard peripheral axon extension into the skin. These data demonstrate that filopodial Ca2+ signals regulate axon outgrowth and guidance through calpain regulation of adhesion dynamics through specific cleavage of talin and FAK. SIGNIFICANCE STATEMENT The proper formation of neuronal networks requires accurate guidance of axons and dendrites during development by motile structures known as growth cones. Understanding the intracellular signaling mechanisms that govern growth cone motility will clarify how the nervous system develops and regenerates, and may identify areas of therapeutic intervention in disease or injury. One important signal that controls growth cones is that of local Ca2+ transients, which control the rate and direction of axon outgrowth. We demonstrate here that Ca2+-dependent inhibition axon outgrowth and guidance is mediated by calpain proteolysis of the adhesion proteins talin and focal adhesion kinase. Our findings provide mechanistic insight into Ca2+/calpain regulation of growth cone motility and axon guidance during neuronal development. PMID:28069919

Focal Adhesion-Independent Cell Migration.

PubMed

Paluch, Ewa K; Aspalter, Irene M; Sixt, Michael

2016-10-06

Cell migration is central to a multitude of physiological processes, including embryonic development, immune surveillance, and wound healing, and deregulated migration is key to cancer dissemination. Decades of investigations have uncovered many of the molecular and physical mechanisms underlying cell migration. Together with protrusion extension and cell body retraction, adhesion to the substrate via specific focal adhesion points has long been considered an essential step in cell migration. Although this is true for cells moving on two-dimensional substrates, recent studies have demonstrated that focal adhesions are not required for cells moving in three dimensions, in which confinement is sufficient to maintain a cell in contact with its substrate. Here, we review the investigations that have led to challenging the requirement of specific adhesions for migration, discuss the physical mechanisms proposed for cell body translocation during focal adhesion-independent migration, and highlight the remaining open questions for the future.

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

PubMed

Hocking, D C; Smith, R K; McKeown-Longo, P J

1996-04-01

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

Autophagy promotes degradation of internalized collagen and regulates distribution of focal adhesions to suppress cell adhesion

PubMed Central

Kawano, Shinichi; Esaki, Motohiro; Torisu, Kumiko; Matsuno, Yuichi; Kitazono, Takanari

2017-01-01

ABSTRACT Adhesion of cells to the extracellular matrix (ECM) via focal adhesions (FAs) is crucial for cell survival, migration, and differentiation. Although the regulation of FAs, including by integrins and the ECM, is important to cell behavior, how FAs are regulated is not well known. Autophagy is induced by both cell adhesion and cell detachment. Here, we showed that autophagosomes are located close to internalized collagen and paxillin, which is a well-known marker of FAs. Autophagy-deficient cells showed increased levels of internalized collagen compared with control cells. Moreover, paxillin exhibited a more peripheral distribution and the area of paxillin was increased, and adhesion-induced focal adhesion kinase signaling was impaired and adhesion was enhanced, in autophagy-deficient cells. These results suggest that autophagy suppressed cell adhesion by regulating internalized ECM and FAs. PMID:28970230

Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

PubMed Central

Morgan, Mark R.; Hamidi, Hellyeh; Bass, Mark D.; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J.

2013-01-01

Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. PMID:23453597

Manipulation of the response of human endothelial colony-forming cells by focal adhesion assembly using gradient nanopattern plates.

PubMed

Cui, Long-Hui; Joo, Hyung Joon; Kim, Dae Hwan; Seo, Ha-Rim; Kim, Jung Suk; Choi, Seung-Cheol; Huang, Li-Hua; Na, Ji Eun; Lim, I-Rang; Kim, Jong-Ho; Rhyu, Im Joo; Hong, Soon Jun; Lee, Kyu Back; Lim, Do-Sun

2018-01-01

Nanotopography plays a pivotal role in the regulation of cellular responses. Nonetheless, little is known about how the gradient size of nanostructural stimuli alters the responses of endothelial progenitor cells without chemical factors. Herein, the fabrication of gradient nanopattern plates intended to mimic microenvironment nanotopography is described. The gradient nanopattern plates consist of nanopillars of increasing diameter ranges [120-200 nm (GP 120/200), 200-280 nm (GP 200/280), and 280-360 nm (GP 280/360)] that were used to screen the responses of human endothelial colony-forming cells (hECFCs). Nanopillars with a smaller nanopillar diameter caused the cell area and perimeter of hECFCs to decrease and their filopodial outgrowth to increase. The structure of vinculin (a focal adhesion marker in hECFCs) was also modulated by nanostructural stimuli of the gradient nanopattern plates. Moreover, Rho-associated protein kinase (ROCK) gene expression was significantly higher in hECFCs cultured on GP 120/200 than in those on flat plates (no nanopillars), and ROCK suppression impaired the nanostructural-stimuli-induced vinculin assembly. These results suggest that the gradient nanopattern plates generate size-specific nanostructural stimuli suitable for manipulation of the response of hECFCs, in a process dependent on ROCK signaling. This is the first evidence of size-specific nanostructure-sensing behavior of hECFCs. Nano feature surfaces are of growing interest as materials for a controlled response of various cells. In this study, we successfully fabricated gradient nanopattern plates to manipulate the response of blood-derived hECFCs without any chemical stimulation. Interestingly, we find that the sensitive nanopillar size for manipulation of hECFCs is range between 120 nm and 200 nm, which decreased the area and increased the filopodial outgrowth of hECFCs. Furthermore, we only modulate the nanopillar size to increase ROCK expression can be an attractive method for modulating the cytoskeletal integrity and focal adhesion of hECFCs. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Monitoring in real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus

NASA Astrophysics Data System (ADS)

von Bilderling, Catalina; Caldarola, Martín; Masip, Martín E.; Bragas, Andrea V.; Pietrasanta, Lía I.

2017-01-01

The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.

Monitoring in real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus.

PubMed

von Bilderling, Catalina; Caldarola, Martín; Masip, Martín E; Bragas, Andrea V; Pietrasanta, Lía I

2017-01-01

The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.

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 (periportal areas) in both focal nodular hyperplasia and hepatic adenoma. One case of telangiectatic focal nodular hyperplasia revealed both hepatic adenoma–like and focal nodular hyperplasia–like staining patterns. Distinct cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule staining patterns are seen in hepatic adenoma and focal nodular hyperplasia possibly suggest activation of different subsets of hepatic progenitor/stem cell and can be diagnostically useful. PMID:18602664

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.

Heterogeneity of Focal Adhesions and Focal Contacts in Motile Fibroblasts.

PubMed

Gladkikh, Aleena; Kovaleva, Anastasia; Tvorogova, Anna; Vorobjev, Ivan A

2018-01-01

Cell-extracellular matrix (ECM) adhesion is an important property of virtually all cells in multicellular organisms. Cell-ECM adhesion studies, therefore, are very significant both for biology and medicine. Over the last three decades, biomedical studies resulted in a tremendous advance in our understanding of the molecular basis and functions of cell-ECM adhesion. Based on morphological and molecular criteria, several different types of model cell-ECM adhesion structures including focal adhesions, focal complexes, fibrillar adhesions, podosomes, and three-dimensional matrix adhesions have been described. All the subcellular structures that mediate cell-ECM adhesion are quite heterogeneous, often varying in size, shape, distribution, dynamics, and, to a certain extent, molecular constituents. The morphological "plasticity" of cell-ECM adhesion perhaps reflects the needs of cells to sense, adapt, and respond to a variety of extracellular environments. In addition, cell type (e.g., differentiation status, oncogenic transformation, etc.) often exerts marked influence on the structure of cell-ECM adhesions. Although molecular, genetic, biochemical, and structural studies provide important maps or "snapshots" of cell-ECM adhesions, the area of research that is equally valuable is to study the heterogeneity of FA subpopulations within cells. Recently time-lapse observations on the FA dynamics become feasible, and behavior of individual FA gives additional information on cell-ECM interactions. Here we describe a robust method of labeling of FA using plasmids with fluorescent markers for paxillin and vinculin and quantifying the morphological and dynamical parameters of FA.

Cell type–dependent mechanisms for formin-mediated assembly of filopodia

PubMed Central

Young, Lorna E.; Heimsath, Ernest G.; Higgs, Henry N.

2015-01-01

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex–nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins. PMID:26446836

Cell Adhesion on RGD-Displaying Knottins with Varying Numbers of Tryptophan Amino Acids to Tune the Affinity for Assembly on Cucurbit[8]uril Surfaces.

PubMed

Sankaran, Shrikrishnan; Cavatorta, Emanuela; Huskens, Jurriaan; Jonkheijm, Pascal

2017-09-05

Cell adhesion is studied on multivalent knottins, displaying RGD ligands with a high affinity for integrin receptors, that are assembled on CB[8]-methylviologen-modified surfaces. The multivalency in the knottins stems from the number of tryptophan amino acid moieties, between 0 and 4, that can form a heteroternary complex with cucurbit[8]uril (CB[8]) and surface-tethered methylviologen (MV 2+ ). The binding affinity of the knottins with CB[8] and MV 2+ surfaces was evaluated using surface plasmon resonance spectroscopy. Specific binding occurred, and the affinity increased with the valency of tryptophans on the knottin. Additionally, increased multilayer formation was observed, attributed to homoternary complex formation between tryptophan residues of different knottins and CB[8]. Thus, we were able to control the surface coverage of the knottins by valency and concentration. Cell experiments with mouse myoblast (C2C12) cells on the self-assembled knottin surfaces showed specific integrin recognition by the RGD-displaying knottins. Moreover, cells were observed to elongate more on the supramolecular knottin surfaces with a higher valency, and in addition, more pronounced focal adhesion formation was observed on the higher-valency knottin surfaces. We attribute this effect to the enhanced coverage and the enhanced affinity of the knottins in their interaction with the CB[8] surface. Collectively, these results are promising for the development of biomaterials including knottins via CB[8] ternary complexes for tunable interactions with cells.

Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

PubMed Central

George, Britta; Verma, Rakesh; Soofi, Abdulsalam A.; Garg, Puneet; Zhang, Jidong; Park, Tae-Ju; Giardino, Laura; Ryzhova, Larisa; Johnstone, Duncan B.; Wong, Hetty; Nihalani, Deepak; Salant, David J.; Hanks, Steven K.; Curran, Tom; Rastaldi, Maria Pia; Holzman, Lawrence B.

2012-01-01

The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation — markers of focal adhesion complex–mediated Crk-dependent signaling — was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2–dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases. PMID:22251701

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.

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.

BRMS1 Suppresses Breast Cancer Metastasis to Bone via Its Regulation of microRNA-125b and Downstream Attenuation of TNF-Alpha and HER2 Signaling Pathways

DTIC Science & Technology

2014-04-01

cytoskeleton genes and genes regulating focal adhesion assembly, such as a5 integrin, Tenascin C, Talin-1, Profilin 1, and Actinin [35]. Intravital ...and allowed to adhere for time indicated, at which point cells were fixed and stainedwith crystal violet. Representative images for times 5, 10, 15...matrix milieu and imaged by time-lapse microscopy for 1 h or fixed and stained with crystal violet at times indicated. As shown in Figure 1C and

Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

PubMed Central

Zhang, Min; Maddala, Rupalatha; Rao, Ponugoti Vasantha

2008-01-01

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells. PMID:18799648

Somatostatin, acting at receptor subtype 1, inhibits Rho activity, the assembly of actin stress fibers, and cell migration.

PubMed

Buchan, Alison M J; Lin, Chin-Yu; Choi, Jimmy; Barber, Diane L

2002-08-09

Somatostatin regulates multiple biological functions by acting through a family of five G protein-coupled receptors, somatostatin receptors (SSTRs) 1-5. Although all five receptor subtypes inhibit adenylate cyclase activity and decrease intracellular cAMP levels, specific receptor subtypes also couple to additional signaling pathways. In CCL39 fibroblasts expressing either human SSTR1 or SSTR2, we demonstrate that activation of SSTR1 (but not SSTR2) attenuated both thrombin- and integrin-stimulated Rho-GTP complex formation. The reduction in Rho-GTP formation in the presence of somatostatin was associated with decreased translocation of Rho and LIM kinase to the plasma membrane and fewer focal contacts. Activation of Rho resulted in the formation of intracellular actin stress fibers and cell migration. In CCL39-R1 cells, somatostatin treatment prevented actin stress fiber assembly and attenuated thrombin-stimulated cell migration through Transwell membranes to basal levels. To show that native SSTR1 shares the ability to inhibit Rho activation, we demonstrated that somatostatin treatment of human umbilical vein endothelial cells attenuated thrombin-stimulated Rho-GTP accumulation. These data show for the first time that a G protein-coupled receptor, SSTR1, inhibits the activation of Rho, the assembly of focal adhesions and actin stress fibers, and cell migration.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-12-18

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

p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion.

PubMed

Jafari, Naser; Zheng, Qiaodan; Li, Liqing; Li, Wei; Qi, Lei; Xiao, Jianyong; Gao, Tianyan; Huang, Cai

2016-12-02

Phosphatidylinositol 4-phosphate 5-kinase type I γ (PIPKIγ90) ubiquitination and subsequent degradation regulate focal adhesion assembly, cell migration, and invasion. However, it is unknown how upstream signals control PIPKIγ90 ubiquitination or degradation. Here we show that p70S6K1 (S6K1), a downstream target of mechanistic target of rapamycin (mTOR), phosphorylates PIPKIγ90 at Thr-553 and Ser-555 and that S6K1-mediated PIPKIγ90 phosphorylation is essential for cell migration and invasion. Moreover, PIPKIγ90 phosphorylation is required for the development of focal adhesions and invadopodia, key machineries for cell migration and invasion. Surprisingly, substitution of Thr-553 and Ser-555 with Ala promoted PIPKIγ90 ubiquitination but enhanced the stability of PIPKIγ90, and depletion of S6K1 also enhanced the stability of PIPKIγ90, indicating that PIPKIγ90 ubiquitination alone is insufficient for its degradation. These data suggest that S6K1-mediated PIPKIγ90 phosphorylation regulates cell migration and invasion by controlling PIPKIγ90 degradation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src.

PubMed

Willey, Christopher D; Balasubramanian, Sundaravadivel; Rodríguez Rosas, María C; Ross, Robert S; Kuppuswamy, Dhandapani

2003-06-01

In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src and other signaling proteins which was partially mimicked in vitro using adult feline cardiomyocytes embedded in three-dimensional (3D) collagen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide. In the present study, we improved this model further to activate c-Src and obtain a full assembly of the focal adhesion complex (FAC), and characterized c-Src localization and integrin subtype(s) involved. RGD dose response experiments revealed that c-Src activation occurs subsequent to its cytoskeletal recruitment and is accompanied by p130Cas cytoskeletal binding and focal adhesion kinase (FAK) Tyr925 phosphorylation. When cardiomyocytes expressing hexahistidine-tagged c-Src via adenoviral gene delivery were used for RGD stimulation, the expressed c-Src exhibited relocation: (i) biochemical analysis revealed c-Src movement from the detergent-soluble to the -insoluble cytoskeletal fraction and (ii) confocal microscopic analysis showed c-Src movement from a nuclear/perinuclear to a sarcolemmal region. RGD treatment also caused sarcolemmal co-localization of FAK and vinculin. Characterization of integrin subtypes revealed that beta3, but not beta1, integrin plays a predominant role: (i) expression of cytoplasmic domain of beta1A integrin did not affect the RGD-stimulated FAC formation and (ii) both pressure-overloaded myocardium and RGD-stimulated cardiomyocytes exhibited phosphorylation of beta3 integrin at Tyr773/785 sites but not beta1 integrin at Thr788/789 sites. Together these data indicate that RGD treatment in cardiomyocytes causes beta3 integrin activation and c-Src sarcolemmal localization, that subsequent c-Src activation is accompanied by p130Cas binding and FAK Tyr925 phosphorylation, and that these events might be crucial for growth and remodeling of hypertrophying adult cardiomyocytes.

The cancer glycocalyx mechanically primes integrin-mediated growth and survival

PubMed Central

Paszek, Matthew J.; DuFort, Christopher C.; Rossier, Olivier; Bainer, Russell; Mouw, Janna K.; Godula, Kamil; Hudak, Jason E.; Lakins, Jonathon N.; Wijekoon, Amanda C.; Cassereau, Luke; Rubashkin, Matthew G.; Magbanua, Mark J.; Thorn, Kurt S.; Davidson, Michael W.; Rugo, Hope S.; Park, John W.; Hammer, Daniel A.; Giannone, Grégory; Bertozzi, Carolyn R.; Weaver, Valerie M.

2015-01-01

Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function. PMID:25030168

Suppression of lysyl-tRNA synthetase, KRS, causes incomplete epithelial-mesenchymal transition and ineffective cell‑extracellular matrix adhesion for migration.

PubMed

Nam, Seo Hee; Kang, Minkyung; Ryu, Jihye; Kim, Hye-Jin; Kim, Doyeun; Kim, Dae Gyu; Kwon, Nam Hoon; Kim, Sunghoon; Lee, Jung Weon

2016-04-01

The cell-adhesion properties of cancer cells can be targeted to block cancer metastasis. Although cytosolic lysyl-tRNA synthetase (KRS) functions in protein synthesis, KRS on the plasma membrane is involved in cancer metastasis. We hypothesized that KRS is involved in cell adhesion-related signal transduction for cellular migration. To test this hypothesis, colon cancer cells with modulated KRS protein levels were analyzed for cell-cell contact and cell-substrate adhesion properties and cellular behavior. Although KRS suppression decreased expression of cell-cell adhesion molecules, cells still formed colonies without being scattered, supporting an incomplete epithelial mesenchymal transition. Noteworthy, KRS-suppressed cells still exhibited focal adhesions on laminin, with Tyr397-phopshorylated focal adhesion kinase (FAK), but they lacked laminin-adhesion-mediated extracellular signal-regulated kinase (ERK) and paxillin activation. KRS, p67LR and integrin α6β1 were found to interact, presumably to activate ERK for paxillin expression and Tyr118 phosphorylation even without involvement of FAK, so that specific inhibition of ERK or KRS in parental HCT116 cells blocked cell-cell adhesion and cell-substrate properties for focal adhesion formation and signaling activity. Together, these results indicate that KRS can promote cell-cell and cell-ECM adhesion for migration.

Focal adhesion interactions with topographical structures: a novel method for immuno-SEM labelling of focal adhesions in S-phase cells.

PubMed

Biggs, M J P; Richards, R G; Wilkinson, C D W; Dalby, M J

2008-07-01

Current understanding of the mechanisms involved in osseointegration following implantation of a biomaterial has led to adhesion quantification being implemented as an assay of cytocompatibility. Such measurement can be hindered by intra-sample variation owing to morphological changes associated with the cell cycle. Here we report on a new scanning electron microscopical method for the simultaneous immunogold labelling of cellular focal adhesions and S-phase nuclei identified by BrdU incorporation. Prior to labelling, cellular membranes are removed by tritonization and antigens of non-interest blocked by serum incubation. Adhesion plaque-associated vinculin and S-phase nuclei were both separately labelled with a 1.4 nm gold colloid and visualized by subsequent colloid enhancement via silver deposition. This study is specifically concerned with the effects microgroove topographies have on adhesion formation in S-phase osteoblasts. By combining backscattered electron (BSE) imaging with secondary electron (SE) imaging it was possible to visualize S-phase nuclei and the immunogold-labelled adhesion sites in one energy 'plane' and the underlying nanotopography in another. Osteoblast adhesion to these nanotopographies was ascertained by quantification of adhesion complex formation.

Glycogen Synthase Kinase 3β Dictates Podocyte Motility and Focal Adhesion Turnover by Modulating Paxillin Activity

PubMed Central

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

2015-01-01

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. PMID:25239564

Actin dynamics at focal adhesions: a common endpoint and putative therapeutic target for proteinuric kidney diseases.

PubMed

Sever, Sanja; Schiffer, Mario

2018-06-01

Proteinuria encompasses diverse causes including both genetic diseases and acquired forms such as diabetic and hypertensive nephropathy. The basis of proteinuria is a disturbance in size selectivity of the glomerular filtration barrier, which largely depends on the podocyte: a terminally differentiated epithelial cell type covering the outer surface of the glomerulus. Compromised podocyte structure is one of the earliest signs of glomerular injury. The phenotype of diverse animal models and podocyte cell culture firmly established the essential role of the actin cytoskeleton in maintaining functional podocyte structure. Podocyte foot processes, actin-based membrane extensions, contain 2 molecularly distinct "hubs" that control actin dynamics: a slit diaphragm and focal adhesions. Although loss of foot processes encompasses disassembly of slit diaphragm multiprotein complexes, as long as cells are attached to the glomerular basement membrane, focal adhesions will be the sites in which stress due to filtration flow is counteracted by forces generated by the actin network in foot processes. Numerous studies within last 20 years have identified actin binding and regulatory proteins as well as integrins as essential components of signaling and actin dynamics at focal adhesions in podocytes, suggesting that some of them may become novel, druggable targets for proteinuric kidney diseases. Here we review evidence supporting the idea that current treatments for chronic kidney diseases beneficially and directly target the podocyte actin cytoskeleton associated with focal adhesions and suggest that therapeutic reagents that target the focal adhesion-regulated actin cytoskeleton in foot processes have potential to modernize treatments for chronic kidney diseases. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Nanotopographical Modulation of Cell Function through Nuclear Deformation

PubMed Central

Wang, Kai; Bruce, Allison; Mezan, Ryan; Kadiyala, Anand; Wang, Liying; Dawson, Jeremy; Rojanasakul, Yon; Yang, Yong

2016-01-01

Although nanotopography has been shown to be a potent modulator of cell behavior, it is unclear how the nanotopographical cue, through focal adhesions, affects the nucleus, eventually influencing cell phenotype and function. Thus, current methods to apply nanotopography to regulate cell behavior are basically empirical. We, herein, engineered nanotopographies of various shapes (gratings and pillars) and dimensions (feature size, spacing and height), and thoroughly investigated cell spreading, focal adhesion organization and nuclear deformation of human primary fibroblasts as the model cell grown on the nanotopographies. We examined the correlation between nuclear deformation and cell functions such as cell proliferation, transfection and extracellular matrix protein type I collagen production. It was found that the nanoscale gratings and pillars could facilitate focal adhesion elongation by providing anchoring sites, and the nanogratings could orient focal adhesions and nuclei along the nanograting direction, depending on not only the feature size but also the spacing of the nanogratings. Compared with continuous nanogratings, discrete nanopillars tended to disrupt the formation and growth of focal adhesions and thus had less profound effects on nuclear deformation. Notably, nuclear volume could be effectively modulated by the height of nanotopography. Further, we demonstrated that cell proliferation, transfection, and type I collagen production were strongly associated with the nuclear volume, indicating that the nucleus serves as a critical mechanosensor for cell regulation. Our study delineated the relationships between focal adhesions, nucleus and cell function and highlighted that the nanotopography could regulate cell phenotype and function by modulating nuclear deformation. This study provides insight into the rational design of nanotopography for new biomaterials and the cell–substrate interfaces of implants and medical devices. PMID:26844365

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors.

PubMed

Eberwein, Philipp; Laird, Dougal; Schulz, Simon; Reinhard, Thomas; Steinberg, Thorsten; Tomakidi, Pascal

2015-10-01

Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events. Copyright © 2015 Elsevier B.V. All rights reserved.

BRMS1 Suppresses Breast Cancer Metastasis to Bone via Its Regulation of MicroRNA-125b and Downstream Attenuation of TNF-alpha and HER2 Signaling Pathways

DTIC Science & Technology

2013-10-01

genes regulating focal adhesion assembly, such as a5 integrin, Tenascin C, Talin-1, Profilin 1, and Actinin [35]. Intravital microscopy had shown...adhere for time indicated, at which point cells were fixed and stainedwith crystal violet. Representative images for times 5, 10, 15, 30, and 60 min... imaged by time-lapse microscopy for 1 h or fixed and stained with crystal violet at times indicated. As shown in Figure 1C and quantified in Figure 1D

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

PubMed

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.

A nanoporous titanium surface promotes the maturation of focal adhesions and formation of filopodia with distinctive nanoscale protrusions by osteogenic cells.

PubMed

Guadarrama Bello, Dainelys; Fouillen, Aurélien; Badia, Antonella; Nanci, Antonio

2017-09-15

While topography is a key determinant of the cellular response to biomaterials, the mechanisms implicated in the cell-surface interactions are complex and still not fully elucidated. In this context, we have examined the effect of nanoscale topography on the formation of filopodia, focal adhesions, and gene expression of proteins associated with cell adhesion and sensing. Commercially pure titanium discs were treated by oxidative nanopatterning with a solution of H 2 SO 4 /H 2 O 2 50:50 (v/v). Scanning electron microscopy and atomic force microscopy characterizations showed that this facile chemical treatment efficiently creates a unique nanoporous surface with a root-mean-square roughness of 11.5nm and pore diameter of 20±5nm. Osteogenic cells were cultured on polished (control) and nanotextured discs for periods of 6, 24, and 72h. Immunofluorescence analysis revealed increases in the adhesion formation per cell area, focal adhesion length, and maturity on the nanoporous surface. Gene expression for various focal adhesion markers, including paxillin and talin, and different integrins (e.g. α1, β1, and α5) was also significantly increased. Scanning electron microscopy revealed the presence of more filopodia on cells grown on the nanoporous surface. These cell extensions displayed abundant and distinctive nanoscale lateral protrusions of 10-15nm diameter that molded the nanopore walls. Together the increase in the focal adhesions and abundance of filopodia and associated protrusions could contribute to strengthening the adhesive interaction of cells with the surface, and thereby, alter the nanoscale biomechanical relationships that trigger cellular cascades that regulate cell behavior. Oxidative patterning was exploited to create a unique three-dimensional network of nanopores on titanium surfaces. Our study illustrates how a facile chemical treatment can be advantageously used to modulate cellular behavior. The nanoscale lateral protrusions on filopodia elicited by this surface are novel adhesive structures. Altogether, the increases in focal adhesion, length, maturity, and filopodia with distinctive lateral protrusions could substantially increase the contact area and adhesion strength of cells, thereby promoting the activation of cellular signaling cascades that may explain the positive osteogenic outcomes previously achieved with this surface. Such physicochemical cueing offers a simple attractive alternative to the use of bioactive agents for guiding tissue repair/regeneration around implantable metals. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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.

Distinct roles for paxillin and Hic-5 in regulating breast cancer cell morphology, invasion, and metastasis

PubMed Central

Deakin, Nicholas O.; Turner, Christopher E.

2011-01-01

Individual metastatic tumor cells exhibit two interconvertible modes of cell motility during tissue invasion that are classified as either mesenchymal or amoeboid. The molecular mechanisms by which invasive breast cancer cells regulate this migratory plasticity have yet to be fully elucidated. Herein we show that the focal adhesion adaptor protein, paxillin, and the closely related Hic-5 have distinct and unique roles in the regulation of breast cancer cell lung metastasis by modulating cell morphology and cell invasion through three-dimensional extracellular matrices (3D ECMs). Cells depleted of paxillin by RNA interference displayed a highly elongated mesenchymal morphology, whereas Hic-5 knockdown induced an amoeboid phenotype with both cell populations exhibiting reduced plasticity, migration persistence, and velocity through 3D ECM environments. In evaluating associated signaling pathways, we determined that Rac1 activity was increased in cells devoid of paxillin whereas Hic-5 silencing resulted in elevated RhoA activity and associated Rho kinase–induced nonmuscle myosin II activity. Hic-5 was essential for adhesion formation in 3D ECMs, and analysis of adhesion dynamics and lifetime identified paxillin as a key regulator of 3D adhesion assembly, stabilization, and disassembly. PMID:21148292

Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers.

PubMed

Fee, Timothy; Surianarayanan, Swetha; Downs, Crawford; Zhou, Yong; Berry, Joel

2016-01-01

To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes.

Actomyosin-dependent formation of the mechanosensitive talin–vinculin complex reinforces actin anchoring

PubMed Central

Ciobanasu, Corina; Faivre, Bruno; Le Clainche, Christophe

2014-01-01

The force generated by the actomyosin cytoskeleton controls focal adhesion dynamics during cell migration. This process is thought to involve the mechanical unfolding of talin to expose cryptic vinculin-binding sites. However, the ability of the actomyosin cytoskeleton to directly control the formation of a talin–vinculin complex and the resulting activity of the complex are not known. Here we develop a microscopy assay with pure proteins in which the self-assembly of actomyosin cables controls the association of vinculin to a talin-micropatterned surface in a reversible manner. Quantifications indicate that talin refolding is limited by vinculin dissociation and modulated by the actomyosin network stability. Finally, we show that the activation of vinculin by stretched talin induces a positive feedback that reinforces the actin–talin–vinculin association. This in vitro reconstitution reveals the mechanism by which a key molecular switch senses and controls the connection between adhesion complexes and the actomyosin cytoskeleton. PMID:24452080

Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

DTIC Science & Technology

2009-04-01

Higashi M, Kishi H, Hiwasa T, Koda K, Nakajima N, Harigaya K. CD44 signaling through focal adhesion kinase and its anti-apoptotic effect. FEBS Lett...Nakamura, S., Azuma, K., Ishii, G., Higashi, M., Kishi, H., Hiwasa, T., Koda , K., Nakajima, N. and Harigaya, K.: CD44 signaling through focal adhesion

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

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin*

PubMed Central

Kwak, Tae Kyoung; Lee, Mi-Sook; Ryu, Jihye; Choi, Yoon-Ju; Kang, Minkyung; Jeong, Doyoung; Lee, Jung Weon

2012-01-01

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration. PMID:22761432

Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness

PubMed Central

Le, Lily Thao-Nhi; Cazares, Oscar; Mouw, Janna K.; Chatterjee, Sharmila; Macias, Hector; Moran, Angel; Ramos, Jillian; Keely, Patricia J.; Weaver, Valerie M.

2016-01-01

Breast tumor progression is accompanied by changes in the surrounding extracellular matrix (ECM) that increase stiffness of the microenvironment. Mammary epithelial cells engage regulatory pathways that permit dynamic responses to mechanical cues from the ECM. Here, we identify a SLIT2/ROBO1 signaling circuit as a key regulatory mechanism by which cells sense and respond to ECM stiffness to preserve tensional homeostasis. We observed that Robo1 ablation in the developing mammary gland compromised actin stress fiber assembly and inhibited cell contractility to perturb tissue morphogenesis, whereas SLIT2 treatment stimulated Rac and increased focal adhesion kinase activity to enhance cell tension by maintaining cell shape and matrix adhesion. Further investigation revealed that a stiff ECM increased Robo1 levels by down-regulating miR-203. Consistently, patients whose tumor expressed a low miR-203/high Robo1 expression pattern exhibited a better overall survival prognosis. These studies show that cells subjected to stiffened environments up-regulate Robo1 as a protective mechanism that maintains cell shape and facilitates ECM adherence. PMID:26975850

ADAM12 induction by Twist1 promotes tumor invasion and metastasis via regulation of invadopodia and focal adhesions

PubMed Central

Eckert, Mark A.; Santiago-Medina, Miguel; Lwin, Thinzar M.; Kim, Jihoon; Courtneidge, Sara A.

2017-01-01

ABSTRACT The Twist1 transcription factor promotes tumor invasion and metastasis by inducing epithelial–mesenchymal transition (EMT) and invadopodia-mediated extracellular matrix (ECM) degradation. The critical transcription targets of Twist1 for mediating these events remain to be uncovered. Here, we report that Twist1 strongly induces expression of a disintegrin and metalloproteinase 12 (ADAM12). We observed that the expression levels of Twist1 mRNA and ADAM12 mRNA are tightly correlated in human breast tumors. Knocking down ADAM12 blocked cell invasion in a 3D mammary organoid culture. Suppression of ADAM12 also inhibited Twist1-induced tumor invasion and metastasis in human breast tumor xenografts, without affecting primary tumor formation. Mechanistically, knockdown of ADAM12 in breast cancer cells significantly reduced invadopodia formation and matrix degradation, and simultaneously increased overall cell adhesion to the ECM. Live-imaging analysis showed that knockdown of ADAM12 significantly inhibited focal adhesion turnover. Mechanistically, both the disintegrin and metalloproteinase domains of ADAM12 are required for its function at invadopodia, whereas the metalloproteinase domain is dispensable for its function at focal adhesions. Taken together, these data suggest that ADAM12 plays a crucial role in tumor invasion and metastasis by regulating both invadopodia and focal adhesions. PMID:28468988

The virus-like particles of a braconid endoparasitoid wasp, Meteorus pulchricornis, inhibit hemocyte spreading in its noctuid host, Pseudaletia separata.

PubMed

Suzuki, M; Miura, K; Tanaka, T

2008-06-01

We previously reported that the virus-like particles of Meteorus pulchricornis (MpVLPs) are capable of inducing apoptosis by around 6h in the hemocytes of the host, Pseudaletia separata [Suzuki, M., Tanaka, T., 2006. Virus-like particles in venom of Meteorus pulchricornis induce host hemocyte apoptosis. Journal of Insect Physiology 52, 602-611], thereby protecting the oviposited egg. In the present study, we focused on analyses of the earlier events caused by the MpVLPs upon the host immune response, namely their effects on hemocyte spreading. After recognition and attachment on foreign substance, the granulocytes and plasmatocytes assemble focal complexes and focal adhesions and spread by protruding filopodia/lamellipodia. The well-spread, cultured hemocytes were subjected to MpVLPs exposure, and the morphological changes were observed. The granulocytes lost the focal complexes/adhesions visualized as phosphotyrosine clusters and retracted the filopodia/lamellipodia within 30min after exposure, while the plasmatocytes exhibited similar but distinct responses. The two hemocyte species prepared from either parasitized or MpVLP-injected hosts lost the ability to form both filopodia/lamellipodia and phosphotyrosine clusters. A caspase inhibitor, Z-VAD-FMK, did not affect these MpVLP-induced morphological changes, indicating that these earlier changes found in the hemocytes precede apoptosis. The present study together with our previous data has established that the attenuation of host immune defense by the MpVLPs comprises at least two temporally distinguishable phases: immediate and early inhibition of hemocyte spreading and the eventual induction of hemocyte apoptosis.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakwe, Amos M., E-mail: asakwe@mmc.edu; Koumangoye, Rainelli; Guillory, Bobby

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 collagenmore » 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.« less

Limitation of Cell Adhesion by the Elasticity of the Extracellular Matrix

PubMed Central

Nicolas, Alice; Safran, Samuel. A.

2006-01-01

Cell/matrix adhesions are modulated by cytoskeletal or external stresses and adapt to the mechanical properties of the extracellular matrix. We propose that this mechanosensitivity arises from the activation of a mechanosensor located within the adhesion itself. We show that this mechanism accounts for the observed directional growth of focal adhesions and the reduction or even cessation of their growth when cells adhere to a soft extracellular matrix. We predict quantitatively that both the elasticity and the thickness of the matrix play a key role in the dynamics of focal adhesions. Two different types of dynamics are expected depending on whether the thickness of the matrix is of order of or much larger than the adhesion size. In the latter situation, we predict that the adhesion region reaches a saturation size that can be tuned by the mechanical properties of the matrix. PMID:16581840

The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions.

PubMed

Garvalov, Boyan K; Higgins, Theresa E; Sutherland, James D; Zettl, Markus; Scaplehorn, Niki; Köcher, Thomas; Piddini, Eugenia; Griffiths, Gareth; Way, Michael

2003-04-14

The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.

Structural basis for the interaction between Pyk2-FAT domain and leupaxin LD repeats

DOE PAGES

Vanarotti, Murugendra S.; Finkelstein, David B.; Guibao, Cristina D.; ...

2016-02-11

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase and belongs to the focal adhesion kinase (FAK) family. Like FAK, the C-terminal focal adhesion-targeting (FAT) domain of Pyk2 binds to paxillin, a scaffold protein in focal adhesions; however, the interaction between the FAT domain of Pyk2 and paxillin is dynamic and unstable. Leupaxin is another member in the paxillin family and was suggested to be the native binding partner of Pyk2; Pyk2 gene expression is strongly correlated with that of leupaxin in many tissues including primary breast cancer. Here, we report that leupaxin interacts with Pyk2-FAT. Leupaxin has fourmore » leucine–aspartate (LD) motifs. The first and third LD motifs of leupaxin preferably target the two LD-binding sites on the Pyk2-FAT domain, respectively. Moreover, the full-length leupaxin binds to Pyk2-FAT as a stable one-to-one complex. Together, we propose that there is an underlying selectivity between leupaxin and paxillin for Pyk2, which may influence the differing behavior of the two proteins at focal adhesion sites.« less

Structural basis for the interaction between Pyk2-FAT domain and leupaxin LD repeats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vanarotti, Murugendra S.; Finkelstein, David B.; Guibao, Cristina D.

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase and belongs to the focal adhesion kinase (FAK) family. Like FAK, the C-terminal focal adhesion-targeting (FAT) domain of Pyk2 binds to paxillin, a scaffold protein in focal adhesions; however, the interaction between the FAT domain of Pyk2 and paxillin is dynamic and unstable. Leupaxin is another member in the paxillin family and was suggested to be the native binding partner of Pyk2; Pyk2 gene expression is strongly correlated with that of leupaxin in many tissues including primary breast cancer. Here, we report that leupaxin interacts with Pyk2-FAT. Leupaxin has fourmore » leucine–aspartate (LD) motifs. The first and third LD motifs of leupaxin preferably target the two LD-binding sites on the Pyk2-FAT domain, respectively. Moreover, the full-length leupaxin binds to Pyk2-FAT as a stable one-to-one complex. Together, we propose that there is an underlying selectivity between leupaxin and paxillin for Pyk2, which may influence the differing behavior of the two proteins at focal adhesion sites.« less

RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisation.

PubMed

Griffith, Elen; Coutts, Amanda S; Black, Donald M

2005-03-01

TES was originally identified as a candidate tumour suppressor gene and has subsequently been found to encode a novel focal adhesion protein. As well as localising to cell-matrix adhesions, TES localises to cell-cell contacts and to actin stress fibres. TES interacts with a variety of cytoskeletal proteins including zyxin, mena, VASP, talin and actin. There is evidence that TES may function in actin-dependent processes as overexpression of TES results in increased cell spreading and decreased cell motility. Together with TES's interacting partners, these data suggest that TES might be involved in regulation of the actin cytoskeleton. Here, for the first time, we have used RNAi to successfully knockdown TES in HeLa cells and we demonstrate that loss of TES from focal adhesions results in loss of actin stress fibres. Similarly, and as previously reported, RNAi-mediated knockdown of zyxin results in loss of actin stress fibres. TES siRNA treated cells show reduced RhoA activity, suggesting that the Rho GTPase pathway may be involved in the TES RNAi-induced loss of stress fibres. We have also used RNAi to examine the requirement of TES and zyxin for each other's localisation at focal adhesions, and we propose a hierarchy of recruitment, with zyxin being first, followed by VASP and then TES. Cell Motil. Copyright 2005 Wiley-Liss, Inc.

Mechanisms underlying the attachment and spreading of human osteoblasts: from transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces.

PubMed

Brun, Paola; Scorzeto, Michele; Vassanelli, Stefano; Castagliuolo, Ignazio; Palù, Giorgio; Ghezzo, Francesca; Messina, Grazia M L; Iucci, Giovanna; Battaglia, Valentina; Sivolella, Stefano; Bagno, Andrea; Polzonetti, Giovanni; Marletta, Giovanni; Dettin, Monica

2013-04-01

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Adhesion to the extracellular matrix is positively regulated by retinoic acid in HepG2 cells.

PubMed

Massimi, Mara; Devirgiliis, Laura Conti

2007-02-01

In this work, we aimed to investigate the possible modulation of cell-matrix interactions by retinoic acid (RA), in view of the well-known role of the extracellular matrix (ECM) and integrins in hepatocyte differentiation and proliferation. For this purpose, we analysed the adhesion ability of HepG2 cells on different substrates in the presence and absence of RA evaluating both the expression and cellular localisation of major proteins involved in focal contacts, using Western blot and confocal microscopy. A positive and substrate-dependent effect of RA on cell-matrix adhesion was observed after long-term culture. The increased adhesiveness in the treated cells was accompanied by an enhanced expression of beta1 and alpha3 integrin subunits, together with a redistribution of beta1 receptors clustered at the basal surface. In contrast, the levels of focal adhesion kinase (FAK), paxillin and alpha-actinin were unchanged, as was the phosphorylation state of FAK. Nonetheless, a stronger association between beta1 integrin and intracytoplasmatic proteins of focal contacts was observed in coimmunoprecipitation experiments after RA treatment, suggesting improved connection with the actin cytoskeleton. These results are consistent with previously described antiproliferative and differentiative effects of RA on transformed hepatocytes, and confirm the hypothesis of a direct influence of RA on specific adhesion molecules.

Invasion of Epithelial Cells and Proteolysis of Cellular Focal Adhesion Components by Distinct Types of Porphyromonas gingivalis Fimbriae

PubMed Central

Nakagawa, Ichiro; Inaba, Hiroaki; Yamamura, Taihei; Kato, Takahiro; Kawai, Shinji; Ooshima, Takashi; Amano, Atsuo

2006-01-01

Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by Nα-p-tosyl-l-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arg-gingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration. PMID:16790749

Bifurcations: Focal Points of Particle Adhesion in Microvascular Networks

PubMed Central

Prabhakarpandian, Balabhaskar; Wang, Yi; Rea-Ramsey, Angela; Sundaram, Shivshankar; Kiani, Mohammad F.; Pant, Kapil

2011-01-01

Objective Particle adhesion in vivo is dependent on microcirculation environment which features unique anatomical (bifurcations, tortuosity, cross-sectional changes) and physiological (complex hemodynamics) characteristics. The mechanisms behind these complex phenomena are not well understood. In this study, we used a recently developed in vitro model of microvascular networks, called Synthetic Microvascular Network, for characterizing particle adhesion patterns in the microcirculation. Methods Synthetic microvascular networks were fabricated using soft lithography processes followed by particle adhesion studies using avidin and biotin-conjugated microspheres. Particle adhesion patterns were subsequently analyzed using CFD based modeling. Results Experimental and modeling studies highlighted the complex and heterogeneous fluid flow patterns encountered by particles in microvascular networks resulting in significantly higher propensity of adhesion (>1.5X) near bifurcations compared to the branches of the microvascular networks. Conclusion Bifurcations are the focal points of particle adhesion in microvascular networks. Changing flow patterns and morphology near bifurcations are the primary factors controlling the preferential adhesion of functionalized particles in microvascular networks. Synthetic microvascular networks provide an in vitro framework for understanding particle adhesion. PMID:21418388

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.

FERMT2 links cortical actin structures, plasma membrane tension and focal adhesion function to stabilize podocyte morphology.

PubMed

Yasuda-Yamahara, M; Rogg, M; Frimmel, J; Trachte, P; Helmstaedter, M; Schroder, P; Schiffer, M; Schell, C; Huber, T B

2018-01-11

Simplification and retraction of podocyte protrusions, generally termed as foot process effacement, is a uniform pathological pattern observed in the majority of glomerular disease, including focal segmental glomerulosclerosis. However, it is still incompletely understood how the interaction of cortical actin structures, actomyosin contractility and focal adhesions, is being orchestrated to control foot process morphology in health and disease. By uncovering the functional role of fermitin family member 2 (FERMT2 or kindlin-2) in podocytes, we provide now evidence, how cell-extracellular matrix (ECM) interactions modulate membrane tension and actomyosin contractility. A genetic modeling approach was applied by deleting FERMT2 in a set of in vivo systems as well as in CRISPR/Cas9 modified human podocytes. Loss of FERMT2 results in altered cortical actin composition, cell cortex destabilization associated with plasma membrane blebbing and a remodeling of focal adhesions. We further show that FERMT2 knockout podocytes have high levels of RhoA activation and concomitantly increased actomyosin contractility. Inhibition of actomyosin tension reverses the membrane blebbing phenotype. Thus, our findings establish a direct link between cell-matrix adhesions, cortical actin structures and plasma membrane tension allowing to better explain cell morphological changes in foot process effacement. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

PubMed Central

Moy, A B; Van Engelenhoven, J; Bodmer, J; Kamath, J; Keese, C; Giaever, I; Shasby, S; Shasby, D M

1996-01-01

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces. PMID:8613524

The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

PubMed

Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

2013-07-01

To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Active cell-matrix coupling regulates cellular force landscapes of cohesive epithelial monolayers

NASA Astrophysics Data System (ADS)

Zhao, Tiankai; Zhang, Yao; Wei, Qiong; Shi, Xuechen; Zhao, Peng; Chen, Long-Qing; Zhang, Sulin

2018-03-01

Epithelial cells can assemble into cohesive monolayers with rich morphologies on substrates due to competition between elastic, edge, and interfacial effects. Here we present a molecularly based thermodynamic model, integrating monolayer and substrate elasticity, and force-mediated focal adhesion formation, to elucidate the active biochemical regulation over the cellular force landscapes in cohesive epithelial monolayers, corroborated by microscopy and immunofluorescence studies. The predicted extracellular traction and intercellular tension are both monolayer size and substrate stiffness dependent, suggestive of cross-talks between intercellular and extracellular activities. Our model sets a firm ground toward a versatile computational framework to uncover the molecular origins of morphogenesis and disease in multicellular epithelia.

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.

Glucosamine Treatment-mediated O-GlcNAc Modification of Paxillin Depends on Adhesion State of Rat Insulinoma INS-1 Cells*

PubMed Central

Kwak, Tae Kyoung; Kim, Hyeonjung; Jung, Oisun; Lee, Sin-Ae; Kang, Minkyung; Kim, Hyun Jeong; Park, Ji-Min; Kim, Sung-Hoon; Lee, Jung Weon

2010-01-01

Protein-protein interactions and/or signaling activities at focal adhesions, where integrin-mediated adhesion to extracellular matrix occurs, are critical for the regulation of adhesion-dependent cellular functions. Although the phosphorylation and activities of focal adhesion molecules have been intensively studied, the effects of the O-GlcNAc modification of their Ser/Thr residues on cellular functions have been largely unexplored. We investigated the effects of O-GlcNAc modification on actin reorganization and morphology of rat insulinoma INS-1 cells after glucosamine (GlcN) treatment. We found that paxillin, a key adaptor molecule in focal adhesions, could be modified by O-GlcNAc in INS-1 cells treated with GlcN and in pancreatic islets from mice treated with streptozotocin. Ser-84/85 in human paxillin appeared to be modified by O-GlcNAc, which was inversely correlated to Ser-85 phosphorylation (Ser-83 in rat paxillin). Integrin-mediated adhesion signaling inhibited the GlcN treatment-enhanced O-GlcNAc modification of paxillin. Adherent INS-1 cells treated with GlcN showed restricted protrusions, whereas untreated cells showed active protrusions for multiple-elongated morphologies. Upon GlcN treatment, expression of a triple mutation (S83A/S84A/S85A) resulted in no further restriction of protrusions. Together these observations suggest that murine pancreatic β cells may have restricted actin organization upon GlcN treatment by virtue of the O-GlcNAc modification of paxillin, which can be antagonized by a persistent cell adhesion process. PMID:20829364

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling

PubMed Central

Hoffman, Laura M.; Jensen, Christopher C.; Kloeker, Susanne; Wang, C.-L. Albert; Yoshigi, Masaaki; Beckerle, Mary C.

2006-01-01

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling. PMID:16505170

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

PubMed Central

Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

2012-01-01

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. PMID:22710062

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

PubMed

Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

2012-09-10

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

Analysis of a vinculin homolog in a sponge (phylum Porifera) reveals that vertebrate-like cell adhesions emerged early in animal evolution.

PubMed

Miller, Phillip W; Pokutta, Sabine; Mitchell, Jennyfer M; Chodaparambil, Jayanth V; Clarke, D Nathaniel; Nelson, William; Weis, William I; Nichols, Scott A

2018-06-07

The evolution of cell adhesion mechanisms in animals facilitated the assembly of organized multicellular tissues. Studies in traditional animal models have revealed two predominant adhesion structures, the adherens junction (AJ) and focal adhesions (FAs), which are involved in the attachment of neighboring cells to each other and to the secreted extracellular matrix (ECM), respectively. The AJ (containing cadherins and catenins) and FAs (comprising integrins, talin, and paxillin) differ in protein composition, but both junctions contain the actin-binding protein vinculin. The near ubiquity of these structures in animals suggests that AJ and FAs evolved early, possibly coincident with multicellularity. However, a challenge to this perspective is that previous studies of sponges-a divergent animal lineage-indicate that their tissues are organized primarily by an alternative, sponge-specific cell adhesion mechanism called "aggregation factor." In this study, we examined the structure, biochemical properties, and tissue localization of a vinculin ortholog in the sponge Oscarella pearsei ( Op ). Our results indicate that Op vinculin localizes to both cell-cell and cell-ECM contacts and has biochemical and structural properties similar to those of vertebrate vinculin. We propose that Op vinculin played a role in cell adhesion and tissue organization in the last common ancestor of sponges and other animals. These findings provide compelling evidence that sponge tissues are indeed organized like epithelia in other animals and support the notion that AJ- and FA-like structures extend to the earliest periods of animal evolution. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Mena binds α5 integrin directly and modulates α5β1 function.

PubMed

Gupton, Stephanie L; Riquelme, Daisy; Hughes-Alford, Shannon K; Tadros, Jenny; Rudina, Shireen S; Hynes, Richard O; Lauffenburger, Douglas; Gertler, Frank B

2012-08-20

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell-cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue "LERER" repeats. In fibroblasts, the Mena-α5 complex was required for "outside-in" α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins.

Mena binds α5 integrin directly and modulates α5β1 function

PubMed Central

Riquelme, Daisy; Hughes-Alford, Shannon K.; Tadros, Jenny; Rudina, Shireen S.; O.Hynes, Richard; Lauffenburger, Douglas

2012-01-01

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell–cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue “LERER” repeats. In fibroblasts, the Mena–α5 complex was required for “outside-in” α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins. PMID:22908313

Deregulation of focal adhesion formation and cytoskeletal tension due to loss of A-type lamins.

PubMed

Corne, Tobias D J; Sieprath, Tom; Vandenbussche, Jonathan; Mohammed, Danahe; Te Lindert, Mariska; Gevaert, Kris; Gabriele, Sylvain; Wolf, Katarina; De Vos, Winnok H

2017-09-03

The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the lamina's major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.

Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

PubMed

Cerecedo, Doris; Martínez-Vieyra, Ivette; Maldonado-García, Deneb; Hernández-González, Enrique; Winder, Steve J

2015-11-01

Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and β-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete β-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for β-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics. © 2015 Wiley Periodicals, Inc.

Self-Assembling Multi-Component Nanofibers for Strong Bioinspired Underwater Adhesives

PubMed Central

Zhong, Chao; Gurry, Thomas; Cheng, Allen A; Downey, Jordan; Deng, Zhengtao; Stultz, Collin M.; Lu, Timothy K

2014-01-01

Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly, and structure-function relationship of those natural amyloid fibers remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibers. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibers have an underwater adhesion energy approaching 20.9 mJ/m2, which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibers taken on their own at all pHs and exhibit better tolerance to auto-oxidation than Mfps at pH ≥7.0. This work establishes a platform for engineering multi-component self-assembling materials inspired by nature. PMID:25240674

MiR-375 inhibits the hepatocyte growth factor-elicited migration of mesenchymal stem cells by downregulating Akt signaling.

PubMed

He, Lihong; Wang, Xianyao; Kang, Naixin; Xu, Jianwei; Dai, Nan; Xu, Xiaojing; Zhang, Huanxiang

2018-04-01

The migration of mesenchymal stem cells (MSCs) is critical for their use in cell-based therapies. Accumulating evidence suggests that microRNAs are important regulators of MSC migration. Here, we report that the expression of miR-375 was downregulated in MSCs treated with hepatocyte growth factor (HGF), which strongly stimulates the migration of these cells. Overexpression of miR-375 decreased the transfilter migration and the migration velocity of MSCs triggered by HGF. In our efforts to determine the mechanism by which miR-375 affects MSC migration, we found that miR-375 significantly inhibited the activation of Akt by downregulating its phosphorylation at T308 and S473, but had no effect on the activity of mitogen-activated protein kinases. Further, we showed that 3'phosphoinositide-dependent protein kinase-1 (PDK1), an upstream kinase necessary for full activation of Akt, was negatively regulated by miR-375 at the protein level. Moreover, miR-375 suppressed the phosphorylation of focal adhesion kinase (FAK) and paxillin, two important regulators of focal adhesion (FA) assembly and turnover, and decreased the number of FAs at cell periphery. Taken together, our results demonstrate that miR-375 inhibits HGF-elicited migration of MSCs through downregulating the expression of PDK1 and suppressing the activation of Akt, as well as influencing the tyrosine phosphorylation of FAK and paxillin and FA periphery distribution.

Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization

PubMed Central

Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel H.; Jedlicka, Sabrina S.; Vavylonis, Dimitrios

2015-01-01

The morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked fibers along the contacting surface. The motor activity and minifilament assembly of non-muscle myosin-II is an important component of cortical cytoskeletal remodeling during mechanosensing. We used experiments and computational modeling to study cortical myosin-II dynamics in adhered cells. Confocal microscopy was used to image the medial cell cortex of HeLa cells stably expressing myosin regulatory light chain tagged with GFP (MRLC-GFP). The distribution of MRLC-GFP fibers and focal adhesions was classified into three types of network morphologies. Time-lapse movies show: myosin foci appearance and disappearance; aligning and contraction; stabilization upon alignment. Addition of blebbistatin, which perturbs myosin motor activity, leads to a reorganization of the cortical networks and to a reduction of contractile motions. We quantified the kinetics of contraction, disassembly and reassembly of myosin networks using spatio-temporal image correlation spectroscopy (STICS). Coarse-grained numerical simulations include bipolar minifilaments that contract and align through specified interactions as basic elements. After assuming that minifilament turnover decreases with increasing contractile stress, the simulations reproduce stress-dependent fiber formation in between focal adhesions above a threshold myosin concentration. The STICS correlation function in simulations matches the function measured in experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. PMID:25641802

Role of PTPα in the Destruction of Periodontal Connective Tissues

PubMed Central

Rajshankar, Dhaarmini; Sima, Corneliu; Wang, Qin; Goldberg, Stephanie R.; Kazembe, Mwayi; Wang, Yongqiang; Glogauer, Michael; Downey, Gregory P.; McCulloch, Christopher A.

2013-01-01

IL-1β contributes to connective tissue destruction in part by up-regulating stromelysin-1 (MMP-3), which in fibroblasts is a focal adhesion-dependent process. Protein tyrosine phosphatase-α (PTPα) is enriched in and regulates the formation of focal adhesions, but the role of PTPα in connective tissue destruction is not defined. We first examined destruction of periodontal connective tissues in adult PTPα+/+ and PTPα−/− mice subjected to ligature-induced periodontitis, which increases the levels of multiple cytokines, including IL-1β. Three weeks after ligation, maxillae were processed for morphometry, micro-computed tomography and histomorphometry. Compared with unligated controls, there was ∼1.5–3 times greater bone loss as well as 3-fold reduction of the thickness of the gingival lamina propria and 20-fold reduction of the amount of collagen fibers in WT than PTPα−/− mice. Immunohistochemical staining of periodontal tissue showed elevated expression of MMP-3 at ligated sites. Second, to examine mechanisms by which PTPα may regulate matrix degradation, human MMP arrays were used to screen conditioned media from human gingival fibroblasts treated with vehicle, IL-1β or TNFα. Although MMP-3 was upregulated by both cytokines, only IL-1β stimulated ERK activation in human gingival fibroblasts plated on fibronectin. TIRF microscopy and immunoblotting analyses of cells depleted of PTPα activity with the use of various mutated constructs or with siRNA or PTPαKO and matched wild type fibroblasts were plated on fibronectin to enable focal adhesion formation and stimulated with IL-1β. These data showed that the catalytic and adaptor functions of PTPα were required for IL-1β-induced focal adhesion formation, ERK activation and MMP-3 release. We conclude that inflammation-induced connective tissue degradation involving fibroblasts requires functionally active PTPα and in part is mediated by IL-1β signaling through focal adhesions. PMID:23940616

Netrin-1 Preserves Blood-Brain Barrier Integrity Through Deleted in Colorectal Cancer/Focal Adhesion Kinase/RhoA Signaling Pathway Following Subarachnoid Hemorrhage in Rats.

PubMed

Xie, Zongyi; Enkhjargal, Budbazar; Reis, Cesar; Huang, Lei; Wan, Weifeng; Tang, Jiping; Cheng, Yuan; Zhang, John H

2017-05-19

Netrin-1 (NTN-1) has been established to be a novel intrinsic regulator of blood-brain barrier (BBB) maintenance. This study was carried out to investigate the potential roles of exogenous NTN-1 in preserving BBB integrity after experimental subarachnoid hemorrhage (SAH) as well as the underlying mechanisms of its protective effects. A total of 309 male Sprague-Dawley rats were subjected to an endovascular perforation model of SAH. Recombinant NTN-1 was administered intravenously 1 hour after SAH induction. NTN-1 small interfering RNA or Deleted in Colorectal Cancer small interfering RNA was administered intracerebroventricular at 48 hours before SAH. Focal adhesion kinase inhibitor was administered by intraperitoneal injection at 1 hour prior to SAH. Neurological scores, brain water content, BBB permeability, RhoA activity, Western blot, and immunofluorescence staining were evaluated. The expression of endogenous NTN-1 and its receptor Deleted in Colorectal Cancer were increased after SAH. Administration of exogenous NTN-1 significantly reduced brain water content and BBB permeability and ameliorated neurological deficits at 24 and 72 hours after SAH. Exogenous NTN-1 treatment significantly promoted phosphorylated focal adhesion kinase activation and inhibited RhoA activity, as well as upregulated the expression of ZO-1 and Occludin. Conversely, depletion of endogenous NTN-1 aggravated BBB breakdown and neurological impairments at 24 hours after SAH. The protective effects of NTN-1 at 24 hours after SAH were also abolished by pretreatment with Deleted in Colorectal Cancer small interfering RNA and focal adhesion kinase inhibitor. NTN-1 treatment preserved BBB integrity and improved neurological functions through a Deleted in Colorectal Cancer/focal adhesion kinase/RhoA signaling pathway after SAH. Thus, NTN-1 may serve as a promising treatment to alleviate early brain injury following SAH. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

PubMed

Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

2012-05-01

The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Yanwu; Wang, Xiaoxia; Hawkins, Cheryl A.

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

Global architecture of the F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction.

PubMed

Shao, Yue; Mann, Jennifer M; Chen, Weiqiang; Fu, Jianping

2014-03-01

Uniaxial stretch is an important biophysical regulator of cell morphology (or shape) and functions of vascular endothelial cells (ECs). However, it is unclear whether and how cell shape can independently regulate EC mechanotransductive properties under uniaxial stretch. Herein, utilizing a novel uniaxial cell-stretching device integrated with micropost force sensors, we reported the first experimental evidence showing cell shape-dependent EC mechanotransduction via cytoskeleton (CSK) contractile forces in response to uniaxial stretch. Combining experiments and theoretical modeling from first principles, we showed that it was the global architecture of the F-actin CSK that instructed the cell shape-dependent EC mechanotransductive process. Furthermore, a cell shape-dependent nature was relayed in EC mechanotransduction via dynamic focal adhesion (FA) assembly. Our results suggested a novel mechanotransductive process in ECs wherein the global architecture of the F-actin CSK, governed by cell shape, controls mechanotransduction via CSK contractile forces and force-dependent FA assembly under uniaxial stretch.

Performance of the QWIP Focal Plane Arrays for NASA's Landsat Data Continuity Mission

NASA Technical Reports Server (NTRS)

Jhabvala, M.; Choi, K.; Waczynski, A.; La, A.; Sundaram, M.; Costard, E.; Jhabvala, C.; Kan, E.; Kahle, D.; Foltz, R.;

Polymeric particles conjugated with a ligand to VCAM-1 exhibit selective, avid, and focal adhesion to sites of atherosclerosis.

PubMed

Deosarkar, Sudhir P; Malgor, Ramiro; Fu, Jie; Kohn, Leonard D; Hanes, Justin; Goetz, Douglas J

2008-10-01

The increased expression of VCAM-1 on endothelial segments within plaque regions could be used as a target to deliver polymeric drug carriers selectively to sites of atherosclerosis. We probed the hypothesis that polymeric particles conjugated with a ligand for VCAM-1 exhibit selective and avid adhesion to sites of atherosclerosis. Particles made from polystyrene or the biodegradable polymer poly(sebacic acid)-block-polyethylene glycol (PSA-PEG) were conjugated with an antibody to VCAM-1 (alpha-VCAM-1) or IgG (negative control). The particles were injected into the jugular vein of ApoE(-/-) (a murine model of atherosclerosis) or wild type mice and their adhesion to the aorta determined. alpha-VCAM-1 particles exhibited significantly greater adhesion to ApoE(-/-) mouse aorta [32 +/- 5 (mean +/- SEM) particles/mm(2) for polystyrene particles and 31 +/- 7 particles/mm(2) for PSA-PEG particles] compared to the level of adhesion to wild type mouse aorta (18 +/- 1 particles/mm(2) for polystyrene particles and 6 +/- 1 particles/mm(2) for PSA-PEG particles). Within ApoE(-/-) mice, the alpha-VCAM-1 particles exhibited significantly greater adhesion to the aorta (32 +/- 5 particles/mm(2) for polystyrene particles and 31 +/- 7 particles/mm(2) for PSA-PEG particles) compared to the adhesion of IgG particles (1 +/- 1 particles/mm(2) for polystyrene particles and 2 +/- 1 particles/mm(2) for PSA-PEG particles). Detailed analysis of the adhesion revealed that alpha-VCAM-1 particles exhibited focal adhesion to plaque regions, in particular the periphery of the plaques, within the ApoE(-/-) mouse aorta. Combined the data demonstrate that polymeric particles conjugated with a ligand to VCAM-1 exhibit selective, avid and focal adhesion to sites of atherosclerosis providing strong evidence that VCAM-1 ligand bearing polymeric particles could be used for targeting drugs selectively to atherosclerotic tissue.

Spatially modulated ephrinA1:EphA2 signaling increases local contractility and global focal adhesion dynamics to promote cell motility.

PubMed

Chen, Zhongwen; Oh, Dongmyung; Biswas, Kabir H; Yu, Cheng-Han; Zaidel-Bar, Ronen; Groves, Jay T

2018-06-19

Recent studies have revealed pronounced effects of the spatial distribution of EphA2 receptors on cellular response to receptor activation. However, little is known about molecular mechanisms underlying this spatial sensitivity, in part due to lack of experimental systems. Here, we introduce a hybrid live-cell patterned supported lipid bilayer experimental platform in which the sites of EphA2 activation and integrin adhesion are spatially controlled. Using a series of live-cell imaging and single-molecule tracking experiments, we map the transmission of signals from ephrinA1:EphA2 complexes. Results show that ligand-dependent EphA2 activation induces localized myosin-dependent contractions while simultaneously increasing focal adhesion dynamics throughout the cell. Mechanistically, Src kinase is activated at sites of ephrinA1:EphA2 clustering and subsequently diffuses on the membrane to focal adhesions, where it up-regulates FAK and paxillin tyrosine phosphorylation. EphrinA1:EphA2 signaling triggers multiple cellular responses with differing spatial dependencies to enable a directed migratory response to spatially resolved contact with ephrinA1 ligands.

Focal adhesion kinase (FAK) perspectives in mechanobiology: implications for cell behaviour.

PubMed

Tomakidi, Pascal; Schulz, Simon; Proksch, Susanne; Weber, Wilfried; Steinberg, Thorsten

2014-09-01

Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.

Cell directional migration and oriented division on three-dimensional laser-induced periodic surface structures on polystyrene.

PubMed

Wang, Xuefeng; Ohlin, Christian A; Lu, Qinghua; Hu, Jun

2008-05-01

The extracellular matrix in animal tissues usually provides a three-dimensional structural support to cells in addition to performing various other important functions. In the present study, wavy submicrometer laser-irradiated periodic surface structures (LIPSS) were produced on a smooth polystyrene film by polarized laser irradiation with a wavelength of 266 nm. Rat C6 glioma cells exhibited directional migration and oriented division on laser-irradiated polystyrene, which was parallel to the direction of LIPSS. However, rat C6 glioma cells on smooth polystyrene moved in a three-step invasion cycle, with faster migration speed than that on laser-irradiated polystyrene. In addition, focal adhesions examined by immunostaining focal adhesion kinase in human epithelial carcinoma HeLa cells were punctuated on smooth polystyrene, whereas dash-like on laser-irradiated polystyrene. We hypothesized that LIPSS on laser-irradiated polystyrene acted as an anisotropic and persistent mechanical stimulus to guide cell anisotropic spreading, migration and division through focal adhesions.

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

PubMed

Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

2001-09-18

It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

Strong underwater adhesives made by self-assembling multi-protein nanofibres.

PubMed

Zhong, Chao; Gurry, Thomas; Cheng, Allen A; Downey, Jordan; Deng, Zhengtao; Stultz, Collin M; Lu, Timothy K

2014-10-01

Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly and structure-function relationships of these natural amyloid fibres remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibres. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibres have an underwater adhesion energy approaching 20.9 mJ m(-2), which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibres taken on their own and exhibit better tolerance to auto-oxidation than Mfps at pH ≥ 7.0.

Robust adhesive precision bonding in automated assembly cells

NASA Astrophysics Data System (ADS)

Müller, Tobias; Haag, Sebastian; Bastuck, Thomas; Gisler, Thomas; Moser, Hansruedi; Uusimaa, Petteri; Axt, Christoph; Brecher, Christian

2014-03-01

Diode lasers are gaining importance, making their way to higher output powers along with improved BPP. The assembly of micro-optics for diode laser systems goes along with the highest requirements regarding assembly precision. Assembly costs for micro-optics are driven by the requirements regarding alignment in a submicron and the corresponding challenges induced by adhesive bonding. For micro-optic assembly tasks a major challenge in adhesive bonding at highest precision level is the fact, that the bonding process is irreversible. Accordingly, the first bonding attempt needs to be successful. Today's UV-curing adhesives inherit shrinkage effects crucial for submicron tolerances of e.g. FACs. The impact of the shrinkage effects can be tackled by a suitable bonding area design, such as minimal adhesive gaps and an adapted shrinkage offset value for the specific assembly parameters. Compensating shrinkage effects is difficult, as the shrinkage of UV-curing adhesives is not constant between two different lots and varies even over the storage period even under ideal circumstances as first test results indicate. An up-to-date characterization of the adhesive appears necessary for maximum precision in optics assembly to reach highest output yields, minimal tolerances and ideal beamshaping results. Therefore, a measurement setup to precisely determine the up-to-date level of shrinkage has been setup. The goal is to provide necessary information on current shrinkage to the operator or assembly cell to adjust the compensation offset on a daily basis. Impacts of this information are expected to be an improved beam shaping result and a first-time-right production.

Lateral assembly of the immunoglobulin protein SynCAM 1 controls its adhesive function and instructs synapse formation.

PubMed

Fogel, Adam I; Stagi, Massimiliano; Perez de Arce, Karen; Biederer, Thomas

2011-09-16

Synapses are specialized adhesion sites between neurons that are connected by protein complexes spanning the synaptic cleft. These trans-synaptic interactions can organize synapse formation, but their macromolecular properties and effects on synaptic morphology remain incompletely understood. Here, we demonstrate that the synaptic cell adhesion molecule SynCAM 1 self-assembles laterally via its extracellular, membrane-proximal immunoglobulin (Ig) domains 2 and 3. This cis oligomerization generates SynCAM oligomers with increased adhesive capacity and instructs the interactions of this molecule across the nascent and mature synaptic cleft. In immature neurons, cis assembly promotes the adhesive clustering of SynCAM 1 at new axo-dendritic contacts. Interfering with the lateral self-assembly of SynCAM 1 in differentiating neurons strongly impairs its synaptogenic activity. At later stages, the lateral oligomerization of SynCAM 1 restricts synaptic size, indicating that this adhesion molecule contributes to the structural organization of synapses. These results support that lateral interactions assemble SynCAM complexes within the synaptic cleft to promote synapse induction and modulate their structure. These findings provide novel insights into synapse development and the adhesive mechanisms of Ig superfamily members.

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

MVL-PLA2, a Snake Venom Phospholipase A2, Inhibits Angiogenesis through an Increase in Microtubule Dynamics and Disorganization of Focal Adhesions

PubMed Central

Bazaa, Amine; Pasquier, Eddy; Defilles, Céline; Limam, Ines; Kessentini-Zouari, Raoudha; Kallech-Ziri, Olfa; Battari, Assou El; Braguer, Diane; Ayeb, Mohamed El; Marrakchi, Naziha; Luis, José

2010-01-01

Integrins are essential protagonists of the complex multi-step process of angiogenesis that has now become a major target for the development of anticancer therapies. We recently reported and characterized that MVL-PLA2, a novel phospholipase A2 from Macrovipera lebetina venom, exhibited anti-integrin activity. In this study, we show that MVL-PLA2 also displays potent anti-angiogenic properties. This phospholipase A2 inhibited adhesion and migration of human microvascular-endothelial cells (HMEC-1) in a dose-dependent manner without being cytotoxic. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrated that MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both in vitro and in vivo. We have also found that the actin cytoskeleton and the distribution of αvβ3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. In order to further investigate the mechanism of action of this protein on endothelial cells, we analyzed the dynamic instability behavior of microtubules in living endothelial cells. Interestingly, we showed that MVL-PLA2 significantly increased microtubule dynamicity in HMEC-1 cells by 40%. We propose that the enhancement of microtubule dynamics may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration. PMID:20405031

A non-canonical role for Rgnef in promoting integrin-stimulated focal adhesion kinase activation

PubMed Central

Miller, Nichol L. G.; Lawson, Christine; Kleinschmidt, Elizabeth G.; Tancioni, Isabelle; Uryu, Sean; Schlaepfer, David D.

2013-01-01

Summary Rgnef (also known as p190RhoGEF or ARHGEF28) is a Rho guanine-nucleotide-exchange factor (GEF) that binds focal adhesion kinase (FAK). FAK is recruited to adhesions and activated by integrin receptors binding to matrix proteins, such as fibronectin (FN). Canonical models place Rgnef downstream of integrin–FAK signaling in regulating Rho GTPase activity and cell movement. Herein, we establish a new, upstream role for Rgnef in enhancing FAK localization to early peripheral adhesions and promoting FAK activation upon FN binding. Rgnef-null mouse embryo fibroblasts (MEFs) exhibit defects in adhesion formation, levels of FAK phosphotyrosine (pY)-397 and FAK localization to peripheral adhesions upon re-plating on FN. Rgnef re-expression rescues these defects, but requires Rgnef–FAK binding. A mutation in the Rgnef pleckstrin homology (PH) domain inhibits adhesion formation, FAK localization, and FAK-Y397 and paxillin-Y118 phosphorylation without disrupting the Rgnef–FAK interaction. A GEF-inactive Rgnef mutant rescues FAK-Y397 phosphorylation and early adhesion localization, but not paxillin-Y118 phosphorylation. This suggests that, downstream of FN binding, paxillin-pY118 requires Rgnef GEF activity through a mechanism distinct from adhesion formation and FAK activation. These results support a scaffolding role for Rgnef in FAK localization and activation at early adhesions in a PH-domain-dependent but GEF-activity-independent manner. PMID:24006257

Force loading explains spatial sensing of ligands by cells

NASA Astrophysics Data System (ADS)

Oria, Roger; Wiegand, Tina; Escribano, Jorge; Elosegui-Artola, Alberto; Uriarte, Juan Jose; Moreno-Pulido, Cristian; Platzman, Ilia; Delcanale, Pietro; Albertazzi, Lorenzo; Navajas, Daniel; Trepat, Xavier; García-Aznar, José Manuel; Cavalcanti-Adam, Elisabetta Ada; Roca-Cusachs, Pere

2017-12-01

Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts. Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin-ligand bonds are separated by more than a few tens of nanometres. It has thus been suggested that a crosslinking ‘adaptor’ protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly. Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model, in which individual integrin-ECM bonds—the molecular clutches—respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow speeds support our model. Our results provide a general framework for how cells sense spatial and physical information at the nanoscale, precisely tuning the range of conditions at which they form adhesions and activate transcriptional regulation.

Hypergravity Stimulates the Extracellular Matrix/Integrin-Signaling Axis and Proliferation in Primary Osteoblasts

NASA Technical Reports Server (NTRS)

Parra, M.; Vercoutere, W.; Roden, C.; Banerjee, I.; Krauser, W.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

2003-01-01

We set out to determine the molecular mechanisms involved in the proliferative response of primary rat osteoblasts to mechanical stimulation using cell culture centrifugation as a model for hypergravity. We hypothesized that this proliferative response is mediated by specific integrin/Extracellular Matrix (ECM) interactions. To investigate this question we developed a cell culture centrifuge and an automated system that performs cell fixation during hypergravity loading. We generated expression vectors for various focal adhesion and cytoskeletal proteins fused to GFP or dsRed and visualized these structures in transfected (or infected) osteoblasts. The actin cytoskeleton was also visualized using rhodamine-phalloidin staining and Focal Adhesion Kinase (FAK) levels were assessed biochemically. We observed that a 24 hour exposure to 50-g stimulated proliferation compared to the 1-g control when cells were plated on fibronectin, collagen Type I , and collagen Type IV, but not on uncoated tissue culture plastic surfaces. This proliferative response was greatest for osteoblasts grown on fibronectin (2-fold increase over 1-g control) and collagen Type I (1.4 fold increase over 1-g control), suggesting that specific matrices and integrins are involved in the signaling pathways required for proliferation. Exposing osteoblasts grown on different matrices to 10-g or 25-g showed that effects on proliferation depended on both matrix type and loading level. We found that osteoblasts exposed to a short pulse of hypergravity during adhesion spread further and had more GFP-FAK containing focal adhesions compared to their 1-g controls. While overall levels of FAK did not change, more FAK was in the active (phosphorylated) form under hypergravity than in the 1-g controls. Cytoskeletal F-actin organization into filaments was also more prominent after brief exposures to hypergravity during the first five minutes of adhesion. These results suggest that specific integrins sense hypergravity and activate distinct matrix-dependent FAK signaling pathways that can enhance proliferation. Our results also imply that brief exposures to hypergravity accelerate cell adhesion and spreading processes via the focal adhesion-signaling axis. These results support the role of the ECM/integrin-signaling axis in osteoblast response to hypergravity loading.

Carbon nanotube multilayered nanocomposites as multifunctional substrates for actuating neuronal differentiation and functions of neural stem cells.

PubMed

Shao, Han; Li, Tingting; Zhu, Rong; Xu, Xiaoting; Yu, Jiandong; Chen, Shengfeng; Song, Li; Ramakrishna, Seeram; Lei, Zhigang; Ruan, Yiwen; He, Liumin

2018-08-01

Carbon nanotubes (CNTs) have shown potential applications in neuroscience as growth substrates owing to their numerous unique properties. However, a key concern in the fabrication of homogeneous composites is the serious aggregation of CNTs during incorporation into the biomaterial matrix. Moreover, the regulation mechanism of CNT-based substrates on neural differentiation remains unclear. Here, a novel strategy was introduced for the construction of CNT nanocomposites via layer-by-layer assembly of negatively charged multi-walled CNTs and positively charged poly(dimethyldiallylammonium chloride). Results demonstrated that the CNT-multilayered nanocomposites provided a potent regulatory signal over neural stem cells (NSCs), including cell adhesion, viability, differentiation, neurite outgrowth, and electrophysiological maturation of NSC-derived neurons. Importantly, the dynamic molecular mechanisms in the NSC differentiation involved the integrin-mediated interactions between NSCs and CNT multilayers, thereby activating focal adhesion kinase, subsequently triggering downstream signaling events to regulate neuronal differentiation and synapse formation. This study provided insights for future applications of CNT-multilayered nanomaterials in neural fields as potent modulators of stem cell behavior. Copyright © 2018 Elsevier Ltd. All rights reserved.

Osteogenic differentiation on DLC-PDMS-h surface.

PubMed

Soininen, Antti; Kaivosoja, Emilia; Sillat, Tarvo; Virtanen, Sannakaisa; Konttinen, Yrjö T; Tiainen, Veli-Matti

2014-10-01

The hypothesis was that anti-fouling diamond-like carbon polydimethylsiloxane hybrid (DLC-PDMS-h) surface impairs early and late cellular adhesion and matrix-cell interactions. The effect of hybrid surface on cellular adhesion and cytoskeletal organization, important for osteogenesis of human mesenchymal stromal cells (hMSC), where therefore compared with plain DLC and titanium (Ti). hMSCs were induced to osteogenesis and followed over time using scanning electron microscopy (SEM), time-of-flight secondary ion mass spectrometry (ToF-SIMS), immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), and hydroxyapatite (HA) staining. SEM at 7.5 hours showed that initial adherence and spreading of hMSC was poor on DLC-PDMS-h. At 5 days some hMSC were undergoing condensation and apoptotic fragmentation, whereas cells on DLC and Ti grew well. DAPI-actin-vinculin triple staining disclosed dwarfed cells with poorly organized actin cytoskeleton-focal complex/adhesion-growth substrate attachments on hybrid coating, whereas spread cells, organized microfilament bundles, and focal adhesions were seen on DLC and in particular on Ti. Accordingly, at day one ToF-SIMS mass peaks showed poor protein adhesion to DLC-PDMS-h compared with DLC and Ti. COL1A1, ALP, OP mRNA levels at days 0, 7, 14, 21, and/or 28 and lack of HA deposition at day 28 demonstrated delayed or failed osteogenesis on DLC-PDMS-h. Anti-fouling DLC-PDMS-h is a poor cell adhesion substrate during the early protein adsorption-dependent phase and extracellular matrix-dependent late phase. Accordingly, some hMSCs underwent anoikis-type apoptosis and failed to complete osteogenesis, due to few focal adhesions and poor cell-to-ECM contacts. DLC-PDMS-h seems to be a suitable coating for non-integrating implants/devices designed for temporary use. © 2014 Wiley Periodicals, Inc.

PREFACE: Cell-substrate interactions Cell-substrate interactions

NASA Astrophysics Data System (ADS)

Gardel, Margaret; Schwarz, Ulrich

2010-05-01

One of the most striking achievements of evolution is the ability to build cellular systems that are both robust and dynamic. Taken by themselves, both properties are obvious requirements: robustness reflects the fact that cells are there to survive, and dynamics is required to adapt to changing environments. However, it is by no means trivial to understand how these two requirements can be implemented simultaneously in a physical system. The long and difficult quest to build adaptive materials is testimony to the inherent difficulty of this goal. Here materials science can learn a lot from nature, because cellular systems show that robustness and dynamics can be achieved in a synergetic fashion. For example, the capabilities of tissues to repair and regenerate are still unsurpassed in the world of synthetic materials. One of the most important aspects of the way biological cells adapt to their environment is their adhesive interaction with the substrate. Numerous aspects of the physiology of metazoan cells, including survival, proliferation, differentiation and migration, require the formation of adhesions to the cell substrate, typically an extracellular matrix protein. Adhesions guide these diverse processes both by mediating force transmission from the cell to the substrate and by controlling biochemical signaling pathways. While the study of cell-substrate adhesions is a mature field in cell biology, a quantitative biophysical understanding of how the interactions of the individual molecular components give rise to the rich dynamics and mechanical behaviors observed for cell-substrate adhesions has started to emerge only over the last decade or so. The recent growth of research activities on cell-substrate interactions was strongly driven by the introduction of new physical techniques for surface engineering into traditional cell biological work with cell culture. For example, microcontact printing of adhesive patterns was used to show that cell fate depends not on the amount of ligand for adhesion receptors, but on its spatial distribution [1]. New protocols for the preparation of soft elastic substrates were essential to show that adhesion structures and cytoskeleton of adherent cells strongly adapt to substrate stiffness [2], with dramatic effects for cellular decision making. For example, it has been shown recently that differentiation of mesenchymal stem cells is strongly influenced by substrate stiffness [3]. Thus, physical factors appear to be equally important as biochemical ones in determining the cellular response to its substrate [4]. The introduction of novel physical techniques not only opened up completely new perspectives regarding biological function, it also introduced a new quantitative element into this field. For example, the availability of soft elastic substrates with controlled stiffness allows us to reconstruct cellular traction forces and to correlate them with other cellular features. This development enables modeling approaches to work in close contact with experimental data, thus opening up the perspective that the field of cell-substrate interactions will become a quantitative and predictive science in the future. Because physical research into cell-substrate interactions has become one of the fastest growing research areas in cellular biophysics and materials science, we believe that it is very timely that this special issue gathers some of the on-going research effort in this field. In contrast to the non-living world, cellular systems usually interact with their environment through specific adhesion, mainly based on adhesion receptors from the integrin family. During recent years, force spectroscopy has emerged as one of the main methods to study the physics of specific adhesion. In this special issue, single cell force spectroscopy is used by Boettiger and Wehrle-Haller to characterize the strength of cell-matrix adhesion and how it is modulated by the glycocalyx [5], while Chirasatitsin and Engler use force spectroscopy mapping to characterize the spatial distribution of adhesive sites on the substrate [6]. Scrimgeour et al describe a new method to adhesively pattern self-assembled monolayers for cell adhesion by a simple photobleaching setup [7] and Stricker et al demonstrate how elastic substrates can be combined with microcontact printing to improve the reconstruction of traction forces [8]. The work by Metzner et al shows that meaningful results on the cell-substrate interactions can be extracted also from experiments in which cells interact with biofunctionalized beads [9]. If cells start to adhere to a substrate, the main rate-limiting step is establishment of close contact between the plasma membrane and the substrate. This process can be followed with high spatial and temporal resolution with reflection interference microscopy, as demonstrated by Ryzhkov et al for mouse embryonic fibroblasts [10] and by Cretel et al for T lymphocytes [11]. Once mature adhesion has been achieved, the integrin-based focal adhesions providing anchorage to the substrate are strongly connected to the actin cytoskeleton, the main determinant of cell shape and structure. Heil and Spatz use microfabricated pillars to perturb the mechanical balance and quantitatively characterize the fast response of the focal adhesions [12]. A similar approach is used by Kirchenbüchler et al, who use deformation of an elastic substrate to demonstrate that the weak link in the mechanical system of substrate, adhesions and actin cytoskeleton is most likely located at the adhesion-cytoskeleton interface [13]. Rather than using external perturbations, Zemel et al quantify and model how cells spontaneously polarize their cytoskeleton in response to the physical properties of the substrate [14]. Quantitative analysis of cellular data has become standard in the field of cell-substrate interactions. Moreover, theoretical models for cell-substrate interactions help us to identify and understand the mechanisms underlying the observed phenomena in these complex systems. Recently, a large effort has been invested into understanding how force transmitted by the actin cytoskeleton changes the state of focal adhesions. In the contribution by Biton and Safran, this issue is addressed for the case that force arises from shear flow over an adhering cell [15]. Another important source for force on focal adhesions is actin retrograde flow, which has been demonstrated before to show variable coupling to the underlying layer of adhesion receptors. Two contributions discuss how stochastic bond dynamics at the cell-substrate interface is modulated by physical factors. The model by Sabass and Schwarz suggests that dissipation in the actin cytoskeleton stabilizes bond dynamics [16] and the model by Li et al suggests that catch bonding and multiple layers are important elements of the way focal adhesions function [17]. If interacting with an elastic environment, the combined system of focal adhesions and actin cytoskeleton can be used by cells to sense its rigidity and to make decisions on its response. Moshayedi et al show that great care has to be taken when preparing soft elastic substrates for cell culture studies and then use their protocols to quantitatively evaluate the mechanosensitive response of astrocytes from the brain [18]. The cellular system used by Lee et al is pericytes from the microvasculature, for which the authors show that they exert sufficient forces to stimulate vascular endothelial cells [19]. Buxboim et al use the technology of soft elastic substrates to measure how far mesenchymal stem cells can mechanically sense into their substrate [20]. The mechanical activity of cells observed in two-dimensional cell culture has significant consequences for both physiological and disease-related situations, including cell migration, tissue maintenance and tumor growth. Jannat et al show that chemotaxis of neutrophils, that is the first line of the immune system, is strongly modulated by mechanosensing on substrates of varying stiffness [21]. Mogilner and Rubinstein present a theoretical systems analysis for the shape of rapidly migrating keratocytes [22]. Saez et al show, with microfabricated pillar assays, how force is distributed within a layer of epithelial cells [23]. For three-dimensional tissue models, new techniques have to be developed to characterize the complex mechanics of hydrogels. Levental et al [24] and Kotlarchyk et al [25] approach this challenge with mechanical and optical methods, respectively. Narayanan et al combine experiments and continuum models to explore how chemo-mechanical interactions influence tumor growth [26]. References [1] Chen C S, Mrksich M, Huang S, Whitesides G M and Ingber D E 1997 Geometric control of cell life and death Science 276 1425 [2] Pelham R J Jr and Wang Y-L 1997 Cell locomotion and focal adhesions are regulated by substrate flexibility Proc. Natl. Acad. Sci. USA 94 13661 [3] Engler A J, Sen S, Sweeney H L and Discher D E 2006 Matrix elasticity directs stem cell lineage specification Cell 126 677-89 [4] Geiger B, Spatz J P and Bershadsky A D 2009 Environmental sensing through focal adhesions Nat. Rev. Mol. Cell Biol. 10 21 [5] Boettiger D and Wehrle-Haller B 2010 Integrin and glycocalyx mediated contributions to cell adhesion identified by single cell force spectroscopy J. Phys.: Condens. Matter 22 194101 [6] Chirasatitsin S and Engler A J 2010 Detecting cell-adhesive sites in extracellular matrix using force spectroscopy mapping J. Phys.: Condens. Matter 22 194102 [7] Scrimgeour J, Kodali V K, Kovari D T and Curtis J E 2010 Photobleaching-activated micropatterning on self-assembled monolayers J. Phys.: Condens. Matter 22 194103 [8] Stricker J, Sabass B, Schwarz U S and Gardel M L 2010 Optimization of traction force microscopy for micron-sized focal adhesions J. Phys.: Condens. Matter 22 194104 [9] Metzner C, Raupach C, Mierke C T and Fabry B 2010 Fluctuations of cytoskeleton-bound microbeads—the effect of bead-receptor binding dynamics J. Phys.: Condens. Matter 22 194105 [10] Ryzhkov P, Prass M, Gummich M, Kühn J-S, Oettmeier C and Döbereiner H-G 2010 Adhesion patterns in early cell spreading J. Phys.: Condens. Matter 22 194106 [11] Cretel E, Touchard D, Benoliel A M, Bongrand P and Pierres A 2010 Early contacts between T lymphocytes and activating surfaces J. Phys.: Condens. Matter 22 194107 [12] Heil P and Spatz J P 2010 Lateral shear forces applied to cells with single elastic micropillars to influence focal adhesion dynamics J. Phys.: Condens. Matter 22 194108 [13] Kirchenbüchler D, Born S, Kirchgeßner N, Houben S, Hoffmann B and Merkel R 2010 Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins J. Phys.: Condens. Matter 22 194109 [14] Zemel A, Rehfeldt F, Brown A E X, Discher D E and Safran S A 2010 Cell shape, spreading symmetry, and the polarization of stress-fibers in cells J. Phys.: Condens. Matter 22 194110 [15] Biton Y Y and Safran S A 2010 Theory of the mechanical response of focal adhesions to shear flow J. Phys.: Condens. Matter 22 194111 [16] Sabass B and Schwarz U S 2010 Modeling cytoskeletal flow over adhesion sites: competition between stochastic bond dynamics and intracellular relaxation J. Phys.: Condens. Matter 22 194112 [17] Li Y, Bhimalapuram P and Dinner A R 2010 Model for how retrograde actin flow regulates adhesion traction stresses J. Phys.: Condens. Matter 22 194113 [18] Moshayedi P, da F Costa L, Christ A, Lacour S P, Fawcett J, Guck J and Franze K 2010 Mechanosensitivity of astrocytes on optimized polyacrylamide gels analyzed by quantitative morphometry J. Phys.: Condens. Matter 22 194114 [19] Lee S, Zeiger A, Maloney J M, Kotecki M, Van Vliet K J and Herman I M 2010 Pericyte contraction at the cell-material interface can modulate the microvascular niche J. Phys.: Condens. Matter 22 194115 [20] Buxboim A, Rajagopal K, Brown A E X and Discher D E 2010 How deeply cells feel: methods for thin gels J. Phys.: Condens. Matter 22 194116 [21] Jannat R A, Robbins G P, Ricart B G, Dembo M and Hammer D A 2010 Neutrophil adhesion and chemotaxis depend on substrate mechanics J. Phys.: Condens. Matter 22 194117 [22] Mogilner A and Rubinstein B 2010 Actin disassembly 'clock' and membrane tension determine cell shape and turning: a mathematical method J. Phys.: Condens. Matter 22 194118 [23] Saez A, Anon E, Ghibaudo M, du Roure O, Di Meglio J-M, Hersen P, Silberzan P, Buguin A, Ladoux B 2010 Traction forces exerted by epithelial cell sheets J. Phys.: Condens. Matter 22 194119 [24] Levental I, Levental K R, Klein E A, Assoian R, Miller R T, Wells R G and Janmey P A 2010 A simple indentation device for measuring micrometer-scale tissue stiffness J. Phys.: Condens. Matter 22 194120 [25] Kotlarchyk M A, Botvinick E L and Putnam A J 2010 Characterization of hydrogel microstructure using laser tweezers particle tracking and confocal reflection imaging J. Phys.: Condens. Matter 22 194121 [26] Narayanan H, Verner S N, Mills K L, Kemkemer R and Garikipati K 2010 In silico estimates of the free energy rates in growing tumor spheroids J. Phys.: Condens. Matter 22 194122

Disrupting the Scaffold to Improve Focal Adhesion Kinase–Targeted Cancer Therapeutics

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-03-26

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

X-rays effects on cytoskeleton mechanics of healthy and tumor cells.

PubMed

Panzetta, Valeria; De Menna, Marta; Musella, Ida; Pugliese, Mariagabriella; Quarto, Maria; Netti, Paolo A; Fusco, Sabato

2017-01-01

Alterations in the cytoskeleton structure are frequently found in several diseases and particularly in cancer cells. It is also through the alterations of the cytoskeleton structure that cancer cells acquire most of their common features such as uncontrolled cell proliferation, cell death evasion, and the gaining of migratory and invasive characteristics. Although radiation therapies currently represent one of the most effective treatments for patients, the effects of X-irradiation on the cytoskeleton architecture are still poorly understood. In this case we investigated the effects, over time of two different doses of X-ray irradiation, on cell cytoskeletons of BALB/c3T3 and Sv40-transformed BALB/c 3T3 cells (SVT2). Biophysical parameters - focal adhesion size, actin bundles organization, and cell mechanical properties - were measured before and after irradiations (1 and 2 Gy) at 24 and 72 h, comparing the cytoskeleton properties of normal and transformed cells. The differences, before and after X-irradiation, were revealed in terms of cell morphology and deformability. Finally, such parameters were correlated to the alterations of cytoskeleton dynamics by evaluating cell adhesion at the level of focal adhesion and cytoskeleton mechanics. X-irradiation modifies the structure and the activity of cell cytoskeleton in a dose-dependent manner. For transformed cells, radiation sensitively increased cell adhesion, as indicated by paxillin-rich focal adhesion, flat morphology, a well-organized actin cytoskeleton, and intracellular mechanics. On the other hand, for normal fibroblasts IR had negligible effects on cytoskeletal and adhesive protein organization. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Inhibiting focal adhesion kinase (FAK) blocks IL-4 induced VCAM-1 expression and eosinophil recruitment in vitro and in vivo.

PubMed

Aulakh, Gurpreet K; Petri, Björn; Wojcik, Katarzyna M; Colarusso, Pina; Lee, James J; Patel, Kamala D

2018-04-06

Leukocyte recruitment plays a critical role during both normal inflammation and chronic inflammatory diseases, and ongoing studies endeavor to better understand the complexities of this process. Focal adhesion kinase (FAK) is well known for its role in cancer, yet it also has been shown to regulate aspects of neutrophil and B16 melanoma cell recruitment by rapidly influencing endothelial cell focal adhesion dynamics and junctional opening. Recently, we found that FAK related non-kinase (FRNK), a protein that is often used as a FAK dominant negative, blocked eosinophil transmigration by preventing the transcription of vascular cell adhesion molecule-1 (VCAM-1) and eotaxin-3 (CCL26). Surprisingly, the blocking occurred even in the absence of endogenous FAK. To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vitro and in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment. ©2018 Society for Leukocyte Biology.

Integrin activation and focal complex formation in cardiac hypertrophy.

PubMed

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

2000-11-10

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.

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.

Self-assembled Nano-layering at the Adhesive interface.

PubMed

Yoshida, Y; Yoshihara, K; Nagaoka, N; Hayakawa, S; Torii, Y; Ogawa, T; Osaka, A; Meerbeek, B Van

2012-04-01

According to the 'Adhesion-Decalcification' concept, specific functional monomers within dental adhesives can ionically interact with hydroxyapatite (HAp). Such ionic bonding has been demonstrated for 10-methacryloyloxydecyl dihydrogen phosphate (MDP) to manifest in the form of self-assembled 'nano-layering'. However, it remained to be explored if such nano-layering also occurs on tooth tissue when commercial MDP-containing adhesives (Clearfil SE Bond, Kuraray; Scotchbond Universal, 3M ESPE) were applied following common clinical application protocols. We therefore characterized adhesive-dentin interfaces chemically, using x-ray diffraction (XRD) and energy-dispersive x-ray spectroscopy (EDS), and ultrastructurally, using (scanning) transmission electron microscopy (TEM/STEM). Both adhesives revealed nano-layering at the adhesive interface, not only within the hybrid layer but also, particularly for Clearfil SE Bond (Kuraray), extending into the adhesive layer. Since such self-assembled nano-layering of two 10-MDP molecules, joined by stable MDP-Ca salt formation, must make the adhesive interface more resistant to biodegradation, it may well explain the documented favorable clinical longevity of bonds produced by 10-MDP-based adhesives.

Endosomal-sorting complexes required for transport (ESCRT) pathway-dependent endosomal traffic regulates the localization of active Src at focal adhesions.

PubMed

Tu, Chun; Ortega-Cava, Cesar F; Winograd, Paul; Stanton, Marissa Jo; Reddi, Alagarsamy Lakku; Dodge, Ingrid; Arya, Ranjana; Dimri, Manjari; Clubb, Robert J; Naramura, Mayumi; Wagner, Kay-Uwe; Band, Vimla; Band, Hamid

2010-09-14

Active Src localization at focal adhesions (FAs) is essential for cell migration. How this pool is linked mechanistically to the large pool of Src at late endosomes (LEs)/lysosomes (LY) is not well understood. Here, we used inducible Tsg101 gene deletion, TSG101 knockdown, and dominant-negative VPS4 expression to demonstrate that the localization of activated cellular Src and viral Src at FAs requires the endosomal-sorting complexes required for transport (ESCRT) pathway. Tsg101 deletion also led to impaired Src-dependent activation of STAT3 and focal adhesion kinase and reduced cell migration. Impairment of the ESCRT pathway or Rab7 function led to the accumulation of active Src at aberrant LE/LY compartments followed by its loss. Analyses using fluorescence recovery after photo-bleaching show that dynamic mobility of Src in endosomes is ESCRT pathway-dependent. These results reveal a critical role for an ESCRT pathway-dependent LE/LY trafficking step in Src function by promoting localization of active Src to FAs.

Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through α2β1 integrin

PubMed Central

Bix, Gregory; Fu, Jian; Gonzalez, Eva M.; Macro, Laura; Barker, Amy; Campbell, Shelly; Zutter, Mary M.; Santoro, Samuel A.; Kim, Jiyeun K.; Höök, Magnus; Reed, Charles C.; Iozzo, Renato V.

2004-01-01

Endorepellin, the COOH-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits several aspects of angiogenesis. We provide evidence for a novel biological axis that links a soluble fragment of perlecan protein core to the major cell surface receptor for collagen I, α2β1 integrin, and provide an initial investigation of the intracellular signaling events that lead to endorepellin antiangiogenic activity. The interaction between endorepellin and α2β1 integrin triggers a unique signaling pathway that causes an increase in the second messenger cAMP; activation of two proximal kinases, protein kinase A and focal adhesion kinase; transient activation of p38 mitogen-activated protein kinase and heat shock protein 27, followed by a rapid down-regulation of the latter two proteins; and ultimately disassembly of actin stress fibers and focal adhesions. The end result is a profound block of endothelial cell migration and angiogenesis. Because perlecan is present in both endothelial and smooth muscle cell basement membranes, proteolytic activity during the initial stages of angiogenesis could liberate antiangiogenic fragments from blood vessels' walls, including endorepellin. PMID:15240572

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release*

PubMed Central

Malenczyk, Katarzyna; Jazurek, Magdalena; Keimpema, Erik; Silvestri, Cristoforo; Janikiewicz, Justyna; Mackie, Ken; Di Marzo, Vincenzo; Redowicz, Maria J.; Harkany, Tibor; Dobrzyn, Agnieszka

2013-01-01

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB1 cannabinoid receptors (CB1Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB1R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB1Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB1Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes. PMID:24089517

Micropillar displacements by cell traction forces are mechanically correlated with nuclear dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Qingsen; Makhija, Ekta; Hameed, F.M.

2015-05-29

Cells sense physical cues at the level of focal adhesions and transduce them to the nucleus by biochemical and mechanical pathways. While the molecular intermediates in the mechanical links have been well studied, their dynamic coupling is poorly understood. In this study, fibroblast cells were adhered to micropillar arrays to probe correlations in the physical coupling between focal adhesions and nucleus. For this, we used novel imaging setup to simultaneously visualize micropillar deflections and EGFP labeled chromatin structure at high spatial and temporal resolution. We observed that micropillar deflections, depending on their relative positions, were positively or negatively correlated tomore » nuclear and heterochromatin movements. Our results measuring the time scales between micropillar deflections and nucleus centroid displacement are suggestive of a strong elastic coupling that mediates differential force transmission to the nucleus. - Highlights: • Correlation between focal adhesions and nucleus studied using novel imaging setup. • Micropillar and nuclear displacements were measured at high resolution. • Correlation timescales show strong elastic coupling between cell edge and nucleus.« less

Bacillus cereus Certhrax ADP-ribosylates Vinculin to Disrupt Focal Adhesion Complexes and Cell Adhesion*

PubMed Central

Simon, Nathan C.; Barbieri, Joseph T.

2014-01-01

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. PMID:24573681

Cancer Cell Migration in 3D

NASA Astrophysics Data System (ADS)

Wirtz, Denis

2014-03-01

Two-dimensional (2D) in vitro culture systems have for a number of years provided a controlled and versatile environment for mechanistic studies of cell adhesion, polarization, and migration, three interrelated cell functions critical to cancer metastasis. However, the organization and functions of focal adhesion proteins, protrusion machinery, and microtubule-based polarization in cells embedded in physiologically more relevant 3D extracellular matrices is qualitatively different from their organization and functions on conventional 2D planar substrates. This talk will describe the implications of the dependence of focal adhesion protein-based cell migration on micro-environmental dimensionality (1D vs. 2D vs.. 3D), how cell micromechanics plays a critical role in promoting local cell invasion, and associated validation in mouse models. We will discuss the implications of this work in cancer metastasis.

Inhibition of cell migration by focal adhesion kinase: Time-dependent difference in integrin-induced signaling between endothelial and hepatoblastoma cells.

PubMed

Yu, Hongchi; Gao, Min; Ma, Yunlong; Wang, Lijuan; Shen, Yang; Liu, Xiaoheng

2018-05-01

angiogenesis plays an important role in the development and progression of tumors, and it involves a series of signaling pathways contributing to the migration of endothelial cells for vascularization and to the invasion of cancer cells for secondary tumor formation. Among these pathways, the focal adhesion kinase (FAK) signaling cascade has been implicated in a variety of human cancers in connection with cell adhesion and migration events leading to tumor angiogenesis, metastasis and invasion. Therefore, the inhibition of FAK in endothelial and/or cancer cells is a potential target for anti‑angiogenic therapy. In the present study, a small‑molecule FAK inhibitor, 1,2,4,5-benzenetetramine tetrahydrochloride (Y15), was used to study the effects of FAK inhibition on the adhesion and migration behaviors of vascular endothelial cells (VECs) and human hepatoblastoma cells. Furthermore, the time-dependent differences in proteins associated with the integrin-mediated FAK/Rho GTPases signaling pathway within 2 h were examined. The results indicated that the inhibition of FAK significantly decreased the migration ability of VECs and human hepatoblastoma cells in a dose-dependent manner. Inhibition of FAK promoted cell detachment by decreasing the expression of focal adhesion components, and blocked cell motility by reducing the level of Rho GTPases. However, the expression of crucial proteins involved in integrin-induced signaling in two cell lines exhibited a time-dependent difference with increased duration of FAK inhibitor treatment, suggesting different mechanisms of FAK-mediated cell migration behavior. These results suggest that the mechanism underlying FAK-mediated adhesion and migration behavior differs among various cells, which is expected to provide evidence for future FAK therapy targeted against tumor angiogenesis.

Focal plane transport assembly for the HEAO-B X-ray telescope

NASA Technical Reports Server (NTRS)

Brissette, R.; Allard, P. D.; Keller, F.; Strizhak, E.; Wester, E.

1979-01-01

The High Energy Astronomy Observatory - Mission B (HEAO-B), an earth orbiting X-ray telescope facility capable of locating and imaging celestial X-ray sources within one second of arc in the celestial sphere, is considered. The Focal Plane Transport Assembly (FPTA) is one of the basic structural elements of the three thousand pound HEAO-B experiment payload. The FPTA is a multifunctional assembly which supports seven imaging X-ray detectors circumferentially about a central shaft and accurately positions any particular one into the focus of a high resolution mirror assembly. A drive system, position sensor, rotary coupler, and detent alignment system, all an integral part of the rotatable portion which in turn is supported by main bearings to the stationary focal plane housing are described.

Heat resistant soy adhesives for structural wood products

Treesearch

Christopher G. Hunt; Charles Frihart; Jane O' Dell

2009-01-01

Because load-bearing bonded wood assemblies must support the structure during a fire, the limited softening and depolymerization of biobased polymers at elevated temperatures should be an advantage of biobased adhesives compared to fossil fuel-based adhesives. Because load-bearing bonded wood assemblies must support the structure during a fire, the limited softening...

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. © 2011 Macmillan Publishers Limited. All rights reserved.

Anisotropic forces from spatially constrained focal adhesions mediate contact guidance directed cell migration

PubMed Central

Ray, Arja; Lee, Oscar; Win, Zaw; Edwards, Rachel M.; Alford, Patrick W.; Kim, Deok-Ho; Provenzano, Paolo P.

2017-01-01

Directed migration by contact guidance is a poorly understood yet vital phenomenon, particularly for carcinoma cell invasion on aligned collagen fibres. We demonstrate that for single cells, aligned architectures providing contact guidance cues induce constrained focal adhesion maturation and associated F-actin alignment, consequently orchestrating anisotropic traction stresses that drive cell orientation and directional migration. Consistent with this understanding, relaxing spatial constraints to adhesion maturation either through reduction in substrate alignment density or reduction in adhesion size diminishes the contact guidance response. While such interactions allow single mesenchymal-like cells to spontaneously ‘sense' and follow topographic alignment, intercellular interactions within epithelial clusters temper anisotropic cell–substratum forces, resulting in substantially lower directional response. Overall, these results point to the control of contact guidance by a balance of cell–substratum and cell–cell interactions, modulated by cell phenotype-specific cytoskeletal arrangements. Thus, our findings elucidate how phenotypically diverse cells perceive ECM alignment at the molecular level. PMID:28401884

Stonefish toxin defines an ancient branch of the perforin-like superfamily

PubMed Central

Ellisdon, Andrew M.; Reboul, Cyril F.; Huynh, Kitmun; Oellig, Christine A.; Winter, Kelly L.; Hodgson, Wayne C.; Seymour, Jamie; Dearden, Peter K.; Tweten, Rodney K.; Whisstock, James C.; McGowan, Sheena

2015-01-01

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom. PMID:26627714

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

PubMed Central

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

2013-01-01

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

A multi-targeted natural flavonoid myricetin impedes abnormal glioblastoma cell motility and invasiveness via suppressing lamellipodia and focal adhesions formation.

PubMed

Zhao, Hua-Fu; Wang, Gang; Wu, Chang-Peng; Zhou, Xiu-Ming; Wang, Jing; Chen, Zhong-Ping; To, Shing-Shun Tony; Li, Wei-Ping

2018-06-10

Glioblastoma multiforme (GBM) is the most aggressive and malignant primary brain tumor characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma, which contribute to tumor recurrence and poor prognosis. Myricetin is a natural flavonoid with potent anti-oxidant, anti-inflammatory and anti-cancer activities, which may serve as a potential and harmless agent for GBM treatment. To investigate the anti-glioblastoma effects of myricetin, GBM cells were treated with myricetin alone or in combination with temozolomide. Its effects on GBM cell motility and cytoskeletal structures including lamellipodia, focal adhesions and membrane ruffles were also evaluated. We showed that myricetin alone inhibited glioblastoma U-87 MG cell proliferation, migration and invasion, whereas combination of myricetin and temozolomide did not exhibit any synergistic effect. The inhibitory effect on GBM cell proliferation is independent of PTEN status. Moreover, myricetin showed less cytotoxicity to normal astrocytes than GBM cells. Formation of lamellipodia, focal adhesions, membrane ruffles and vasculogenic mimicry were blocked by myricetin though suppressing ROCK2, paxillin and cortactin phosphorylation. In addition, myricetin could bind to a series of kinases and scaffold proteins including PI3K catalytic isoforms (p110α, p110β and p110δ), PDK1, JNK, c-Jun, ROCK2, paxillin, vinculin and VE-cadherin, leading to inactivation of PI3K/Akt and JNK signaling. In conclusion, myricetin is a multi-targeted drug that has potent anti-migratory and anti-invasive effects on GBM cells via suppressing formation of lamellipodia and focal adhesions, suggesting that it may serve as an alternative option for GBM treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chi; Miller, Darcie J.; Guibao, Cristina D.

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by whichmore » the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ~4.2 μM) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.« less

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. © 2016 The Author(s).

AlphaII-spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts.

PubMed

Rotter, Björn; Bournier, Odile; Nicolas, Gael; Dhermy, Didier; Lecomte, Marie-Christine

2005-06-01

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of alpha- and beta-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent alpha-spectrin subunit in non-erythroid cells, alphaII-spectrin, contains two particular spectrin repeats in its central region, alpha9 and alpha10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the alpha9-alpha10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the alpha10 repeat of alphaII-spectrin whereas EVL interacts with the Src homology 3 domain located within the alpha9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between alphaII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

Various fates of neuronal progenitor cells observed on several different chemical functional groups

NASA Astrophysics Data System (ADS)

Liu, Xi; Wang, Ying; He, Jin; Wang, Xiu-Mei; Cui, Fu-Zhai; Xu, Quan-Yuan

2011-12-01

Neuronal progenitor cells cultured on gold-coated glass surfaces modified by different chemical functional groups, including hydroxyl (-OH), carboxyl (-COOH), amino (-NH2), bromo (-Br), mercapto (-SH), - Phenyl and methyl (-CH3), were studied here to investigate the influence of surface chemistry on the cells' adhesion, morphology, proliferation and functional gene expression. Focal adhesion staining indicated in the initial culture stage cells exhibited morphological changes in response to different chemical functional groups. Cells cultured on -NH2 grafted surface displayed focal adhesion plaque and flattened morphology and had the largest contact area. However, their counter parts on -CH3 grafted surface displayed no focal adhesion and rounded morphology and had the smallest contact area. After 6 days culture, the proliferation trend was as follows: -NH2 > -SH> -COOH> - Phenyl > - Br > -OH> -CH3. To determine the neural functional properties of the cells affected by surface chemistry, the expression of glutamate decarboxylase (GAD67), nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF) were characterized. An increase of GAD67 expression was observed on -NH2, -COOH and -SH grafted surfaces, while no increase in NGF and BDNF expression was observed on any chemical surfaces. These results highlight the importance of surface chemistry in the fate determination of neuronal progenitor cells, and suggest that surface chemistry must be considered in the design of biomaterials for neural tissue engineering.

CD147-targeting siRNA inhibits cell-matrix adhesion of human malignant melanoma cells by phosphorylating focal adhesion kinase.

PubMed

Nishibaba, Rie; Higashi, Yuko; Su, Juan; Furukawa, Tatsuhiko; Kawai, Kazuhiro; Kanekura, Takuro

2012-01-01

CD147/basigin, highly expressed on the surface of malignant tumor cells including malignant melanoma (MM) cells, plays a critical role in the invasiveness and metastasis of MM. Metastasis is an orchestrated process comprised of multiple steps including adhesion and invasion. Integrin, a major adhesion molecule, co-localizes with CD147/basigin on the cell surface. Using the human MM cell line A375 that highly expresses CD147/basigin, we investigated whether CD147/basigin is involved in adhesion in association with integrin. CD147/basigin was knocked-down using siRNA targeting CD147 to elucidate the role of CD147/basigin. Cell adhesion was evaluated by adhesion assay on matrix-coated plates. The localization of integrin was inspected under a confocal microscope and the expression and phosphorylation of focal adhesion kinase (FAK), a downstream kinase of integrin, were examined by western blot analysis. Silencing of CD147/basigin in A375 cells by siRNA induced the phosphorylation of FAK at Y397. Integrin identified on the surface of parental cells was distributed in a speckled fashion in the cytoplasm of CD147 knockdown cells, resulting in morphological changes from a round to a polygonal shape with pseudopodial protrusions. Silencing of CD147/basigin in A375 cells clearly weakened their adhesiveness to collagen I and IV. Our results suggest that CD147/basigin regulates the adhesion of MM cells to extracellular matrices and of integrin β1 signaling via the phosphorylation of FAK. © 2011 Japanese Dermatological Association.

Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis

PubMed Central

Bryan, Chase D.; Chien, Chi-Bin; Kwan, Kristen M.

2016-01-01

The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1UW1) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1UW1 mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis. PMID:27339294

Adhesives for assembly of lightweight wood containers

Treesearch

R. S. Kurtenacker

1964-01-01

This report discusses the screening of various adhesive and mastic systems for possible use in assembling lightweight wood containers. Results showed that dynamic tests of simulated box corners correlated reasonably well with rough handling evaluations of eight selected systems when used to assemble lightweight wood boxes made from a Group I container wood....

Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting.

PubMed

Staquicini, Daniela I; Rangel, Roberto; Guzman-Rojas, Liliana; Staquicini, Fernanda I; Dobroff, Andrey S; Tarleton, Christy A; Ozbun, Michelle A; Kolonin, Mikhail G; Gelovani, Juri G; Marchiò, Serena; Sidman, Richard L; Hajjar, Katherine A; Arap, Wadih; Pasqualini, Renata

2017-06-26

Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jing; Liao, Qian-jin; Zhang, Yi

Highlights: • Silence of TRPM7 in ovarian cancer cells inhibits cell proliferation, migration and invasion. • Silence of TRPM7 decreases phosphorylation levels of Akt, Src and p38 in ovarian cancer cells. • Silence of TRPM7 increases expression of filamentous actin and number of focal adhesions in ovarian cancer cells. - Abstract: Our previous study demonstrated that the melastatin-related transient receptor potential channel 7 (TRPM7) was highly expressed in ovarian carcinomas and its overexpression was significantly associated with poor prognosis in ovarian cancer patients. However, the function of TRPM7 in ovarian cancer is mostly unknown. In this study, we examined themore » roles of TRPM7 in ovarian cancer cell proliferation, migration and invasion. We found that short hairpin RNA interference-mediated silence of TRPM7 significantly inhibited cell proliferation, colony formation, migration and invasion in multiple ovarian cancer cell lines. Mechanistic investigation revealed that silence of TRPM7 decreased phosphorylation levels of Akt, Src and p38 and increased filamentous actin and focal adhesion number in ovarian cancer cells. Thus, our results suggest that TRPM7 is required for proliferation, migration and invasion of ovarian cancer cells through regulating multiple signaling transduction pathways and the formation of focal adhesions.« less

Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development

NASA Astrophysics Data System (ADS)

Wieringa, Paul; Tonazzini, Ilaria; Micera, Silvestro; Cecchini, Marco

2012-07-01

The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds.

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

Transition from Actin-Driven to Water-Driven Cell Migration Depends on External Hydraulic Resistance.

PubMed

Li, Yizeng; Sun, Sean X

2018-06-19

Cells in vivo can reside in diverse physical and biochemical environments. For example, epithelial cells typically live in a two-dimensional (2D) environment, whereas metastatic cancer cells can move through dense three-dimensional matrices. These distinct environments impose different kinds of mechanical forces on cells and thus potentially can influence the mechanism of cell migration. For example, cell movement on 2D flat surfaces is mostly driven by forces from focal adhesion and actin polymerization, whereas in confined geometries, it can be driven by water permeation. In this work, we utilize a two-phase model of the cellular cytoplasm in which the mechanics of the cytosol and the F-actin network are treated on an equal footing. Using conservation laws and simple force balance considerations, we are able to describe the contributions of water flux, actin polymerization and flow, and focal adhesions to cell migration both on 2D surfaces and in confined spaces. The theory shows how cell migration can seamlessly transition from a focal adhesion- and actin-based mechanism on 2D surfaces to a water-based mechanism in confined geometries. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Membrane related dynamics and the formation of actin in cells growing on micro-topographies: a spatial computational model.

PubMed

Bittig, Arne T; Matschegewski, Claudia; Nebe, J Barbara; Stählke, Susanne; Uhrmacher, Adelinde M

2014-09-09

Intra-cellular processes of cells at the interface to an implant surface are influenced significantly by their extra-cellular surrounding. Specifically, when growing osteoblasts on titanium surfaces with regular micro-ranged geometry, filaments are shorter, less aligned and they concentrate at the top of the geometric structures. Changes to the cytoskeleton network, i. e., its localization, alignment, orientation, and lengths of the filaments, as well as the overall concentration and distribution of key-actors are induced. For example, integrin is distributed homogeneously, whereas integrin in activated state and vinculin, both components of focal adhesions, have been found clustered on the micro-ranged geometries. Also, the concentration of Rho, an intracellular signaling protein related to focal adhesion regulation, was significantly lower. To explore whether regulations associated with the focal adhesion complex can be responsible for the changed actin filament patterns, a spatial computational model has been developed using ML-Space, a rule-based model description language, and its associated Brownian-motion-based simulator. The focus has been on the deactivation of cofilin in the vicinity of the focal adhesion complex. The results underline the importance of sensing mechanisms to support a clustering of actin filament nucleations on the micro-ranged geometries, and of intracellular diffusion processes, which lead to spatially heterogeneous distributions of active (dephosphorylated) cofilin, which in turn influences the organization of the actin network. We find, for example, that the spatial heterogeneity of key molecular actors can explain the difference in filament lengths in cells on different micro-geometries partly, but to explain the full extent, further model assumptions need to be added and experimentally validated. In particular, our findings and hypothesis referring to the role, distribution, and amount of active cofilin have still to be verified in wet-lab experiments. Letting cells grow on surface structures is a possibility to shed new light on the intricate mechanisms that relate membrane and actin related dynamics in the cell. Our results demonstrate the need for declarative expressive spatial modeling approaches that allow probing different hypotheses, and the central role of the focal adhesion complex not only for nucleating actin filaments, but also for regulating possible severing agents locally.

Membrane related dynamics and the formation of actin in cells growing on micro-topographies: a spatial computational model

PubMed Central

2014-01-01

Background Intra-cellular processes of cells at the interface to an implant surface are influenced significantly by their extra-cellular surrounding. Specifically, when growing osteoblasts on titanium surfaces with regular micro-ranged geometry, filaments are shorter, less aligned and they concentrate at the top of the geometric structures. Changes to the cytoskeleton network, i. e., its localization, alignment, orientation, and lengths of the filaments, as well as the overall concentration and distribution of key-actors are induced. For example, integrin is distributed homogeneously, whereas integrin in activated state and vinculin, both components of focal adhesions, have been found clustered on the micro-ranged geometries. Also, the concentration of Rho, an intracellular signaling protein related to focal adhesion regulation, was significantly lower. Results To explore whether regulations associated with the focal adhesion complex can be responsible for the changed actin filament patterns, a spatial computational model has been developed using ML-Space, a rule-based model description language, and its associated Brownian-motion-based simulator. The focus has been on the deactivation of cofilin in the vicinity of the focal adhesion complex. The results underline the importance of sensing mechanisms to support a clustering of actin filament nucleations on the micro-ranged geometries, and of intracellular diffusion processes, which lead to spatially heterogeneous distributions of active (dephosphorylated) cofilin, which in turn influences the organization of the actin network. We find, for example, that the spatial heterogeneity of key molecular actors can explain the difference in filament lengths in cells on different micro-geometries partly, but to explain the full extent, further model assumptions need to be added and experimentally validated. In particular, our findings and hypothesis referring to the role, distribution, and amount of active cofilin have still to be verified in wet-lab experiments. Conclusion Letting cells grow on surface structures is a possibility to shed new light on the intricate mechanisms that relate membrane and actin related dynamics in the cell. Our results demonstrate the need for declarative expressive spatial modeling approaches that allow probing different hypotheses, and the central role of the focal adhesion complex not only for nucleating actin filaments, but also for regulating possible severing agents locally. PMID:25200251

Point Relay Scanner Utilizing Ellipsoidal Mirrors

NASA Technical Reports Server (NTRS)

Manhart, Paul K. (Inventor); Pagano, Robert J. (Inventor)

1997-01-01

A scanning system uses a polygonal mirror assembly with each facet of the polygon having an ellipsoidal mirror located thereon. One focal point of each ellipsoidal mirror is located at a common point on the axis of rotation of the polygonal mirror assembly. As the mirror assembly rotates. a second focal point of the ellipsoidal mirrors traces out a scan line. The scanner can be utilized for scanned output display of information or for scanning information to be detected.

DESI focal plate mechanical integration and cooling

NASA Astrophysics Data System (ADS)

Lambert, A. R.; Besuner, R. W.; Claybaugh, T. M.; Silber, J. H.

2016-08-01

The Dark Energy Spectroscopic Instrument (DESI) is under construction to measure the expansion history of the Universe using the Baryon Acoustic Oscillation technique[1]. The spectra of 40 million galaxies over 14000 sq. deg will be measured during the life of the experiment. A new prime focus corrector for the KPNO Mayall telescope will deliver light to 5000 fiber optic positioners. The fibers in turn feed ten broad-band spectrographs. This paper describes the mechanical integration of the DESI focal plate and the thermal system design. The DESI focal plate is comprised of ten identical petal assemblies. Each petal contains 500 robotic fiber positioners. Each petal is a complete, self-contained unit, independent from the others, with integrated power supply, controllers, fiber routing, and cooling services. The major advantages of this scheme are: (1) supports installation and removal of complete petal assemblies in-situ, without disturbing the others, (2) component production, assembly stations, and test procedures are repeated and parallelizable, (3) a complete, full-scale prototype can be built and tested at an early date, (4) each production petal can be surveyed and tested as a complete unit, prior to integration, from the fiber tip at the focal surface to the fiber slit at the spectrograph. The ten petal assemblies will be installed in a single integration ring, which is mounted to the DESI corrector. The aluminum integration ring attaches to the steel corrector barrel via a flexured steel adapter, isolating the focal plate from differential thermal expansions. The plate scale will be kept stable by conductive cooling of the petal assembly. The guider and wavefront sensors (one per petal) will be convectively cooled by forced flow of air. Heat will be removed from the system at ten liquid-cooled cold plates, one per petal, operating at ambient temperature. The entire focal plate structure is enclosed in an insulating shroud, which serves as a thermal barrier between the heat-generating focal plate components and the ambient air of the Mayall dome, to protect the seeing[2].

Focal plane AIT sequence: evolution from HRG-Spot 5 to Pleiades HR

NASA Astrophysics Data System (ADS)

Le Goff, Roland; Pranyies, Pascal; Toubhans, Isabelle

2017-11-01

Optical and geometrical image qualities of Focal Planes, for "push-broom" high resolution remote sensing satellites, require the implementation of specific means and methods for the AIT sequence. Indeed the geometric performances of the focal plane mainly axial focusing and transverse registration, are duly obtained on the basis of adjustment, setting and measurement of optical and CCD components with an accuracy of a few microns. Since the end of the 1970s, EADS-SODERN has developed a series of detection units for earth observation instruments like SPOT and Helios. And EADS-SODERN is now responsible for the development of the Pleiades High Resolution Focal Plane assembly. This paper presents the AIT sequences. We introduce all the efforts, innovative solutions and improvements made on the assembly facilities to match the technical evolutions and breakthrough of the Pleiades HR FP concept in comparison with the previous High Resolution Geometric SPOT 5 Focal Plane. The main evolution drivers are the implementation of strip filters and the realization of 400 mm continuous retinas. For Pleiades HR AIT sequence, three specific integration and measuring benches, corresponding with the different assembly stages, are used: a 3-D non-contact measurement machine for the assembly of detection module, a 3-D measurement machine for mirror integration on the main Focal Plane SiC structure, and a 3-D geometric coordinates control bench to focus detection module lines and to ensure they are well registered together.

Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

PubMed

Yang, Huiying; Ke, Yuehua; Wang, Jian; Tan, Yafang; Myeni, Sebenzile K; Li, Dong; Shi, Qinghai; Yan, Yanfeng; Chen, Hui; Guo, Zhaobiao; Yuan, Yanzhi; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

2011-11-01

A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.

New polymeric materials for designing photoresistors and photodetective assemblies based on CdHgTe

NASA Astrophysics Data System (ADS)

Khitrova, L. M.; Troshkin, Y. S.; Belyaev, V. P.; Popovyan, G. E.; Kiseleva, L. V.

1999-06-01

In order to improve quality of photodetectors and photodetective assemblies two new cryo- and chemically resistant adhesives were developed: epoxy-silico-organic adhesive `(Phi) X-5P' and acrylic `OPHOH-2' adhesive for gluing of CdHgTe wafers to a substrate `XCK-H' vacuum-tight modified adhesive is used for attaching of inlet windows and glass holder elements. `OPUOH-65' vibration damping thixotropic composition was developed for mounting of multi- layer printed circuits.

Utility of Horioka’s and Marra’s models for adhesive failure

Treesearch

Charles R. Frihart

2005-01-01

Bond formation primarily involves adhesive rheology and interface chemistry. Bonded assembly strength, however, primarily involves the viscoelastic dissipation of stress over the entire assembly. Models can aid in the understanding of where and why failure occurs and how to improve the strength of the assembly. Horioka and Marra have both proposed models which define...

LWS design replacement study: Optimum design and tradeoff analysis

NASA Technical Reports Server (NTRS)

1973-01-01

A design for two long-wavelength (LW) focal-plane and cooler assemblies, including associated preamplifiers and post-amplifiers is presented. The focal-planes and associated electronic assemblies are intended as direct replacement hardware to be installed into the existing 24-channel multispectral scanner used with the NASA Earth Observations Aircraft Program. An organization skilled in the art of LWIR systems can fabricate and deliver the two long-wavelength focal-plane assemblies described in this report when provided with the data and drawings developed during the performance of this contract. The concepts developed during the study including the alternative approaches and selection of components are discussed. Modifications to the preliminary design as reported in a preliminary design review meeting have also been included.

Mammary epithelial-specific disruption of focal adhesion kinase retards tumor formation and metastasis in a transgenic mouse model of human breast cancer.

PubMed

Provenzano, Paolo P; Inman, David R; Eliceiri, Kevin W; Beggs, Hilary E; Keely, Patricia J

2008-11-01

Focal adhesion kinase (FAK) is a central regulator of the focal adhesion, influencing cell proliferation, survival, and migration. Despite evidence demonstrating FAK overexpression in human cancer, its role in tumor initiation and progression is not well understood. Using Cre/LoxP technology to specifically knockout FAK in the mammary epithelium, we showed that FAK is not required for tumor initiation but is required for tumor progression. The mechanistic underpinnings of these results suggested that FAK regulates clinically relevant gene signatures and multiple signaling complexes associated with tumor progression and metastasis, such as Src, ERK, and p130Cas. Furthermore, a systems-level analysis identified FAK as a major regulator of the tumor transcriptome, influencing genes associated with adhesion and growth factor signaling pathways, and their cross talk. Additionally, FAK was shown to down-regulate the expression of clinically relevant proliferation- and metastasis-associated gene signatures, as well as an enriched group of genes associated with the G(2) and G(2)/M phases of the cell cycle. Computational analysis of transcription factor-binding sites within ontology-enriched or clustered gene sets suggested that the differentially expressed proliferation- and metastasis-associated genes in FAK-null cells were regulated through a common set of transcription factors, including p53. Therefore, FAK acts as a primary node in the activated signaling network in transformed motile cells and is a prime candidate for novel therapeutic interventions to treat aggressive human breast cancers.

An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization

PubMed Central

Mazaki, Yuichi; Hashimoto, Shigeru; Okawa, Katsuya; Tsubouchi, Asako; Nakamura, Kuniaki; Yagi, Ryohei; Yano, Hajime; Kondo, Akiko; Iwamatsu, Akihiro; Mizoguchi, Akira; Sabe, Hisataka

2001-01-01

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein β-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling. PMID:11251077

Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

PubMed Central

Faundez, Victor; Koval, Michael; Mattheyses, Alexa L.; Kowalczyk, Andrew P.

2014-01-01

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV. PMID:24498201

Method and apparatus for a multibeam beacon laser assembly for optical communications

NASA Technical Reports Server (NTRS)

Biswas, Abhijit (Inventor); Sanji, Babak (Inventor); Wright, Malcolm W. (Inventor); Page, Norman Alan (Inventor)

2005-01-01

An optical beacon is comprised of a telescope having a primary focal plane or Coud? focal plane, a plurality of fiber coupled laser sources for generating a plurality of beams, a collimator for collimating the plurality of beams, and optics for combining and focusing the plurality of collimated beams onto the primary or Coud? focal plane of the telescope. The telescope propagates the optical beacon, which is arranged into a ring of incoherent plurality of collimated beams. The apparatus further comprises fiber splitters coupled to each laser source to provide at least eight beams from at least four laser sources. The optics comprises a prism assembly, a combiner lens, a focusing lens and a field lens for focusing the plurality of collimated beams onto the primary focal plane or Coud? focal plane of the telescope.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hwan; Translational Research Center for Protein Function Control; Kim, Nam Doo

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 turnedmore » 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/migration/invasion of tumor cells.« less

Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier

Highlights: •The three ERM proteins are expressed in vascular smooth muscle cell. •ERM depletion inhibited PDGF-evoked migration redundantly. •ERM depletion increased cell adhesion redundantly. •ERM depletion did not affect PDGF-evoked Ca signal, Rac1 activation, proliferation. •ERM proteins control PDGF-induced migration by regulating adhesion. -- Abstract: Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the rolemore » of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca{sup 2+} signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.« less

Timing is everything: Rac1 controls Net1A localization to regulate cell adhesion.

PubMed

Carr, Heather S; Frost, Jeffrey A

2013-01-01

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion.

RAFTK, a novel member of the focal adhesion kinase family, is phosphorylated and associates with signaling molecules upon activation of mature T lymphocytes.

PubMed

Ganju, R K; Hatch, W C; Avraham, H; Ona, M A; Druker, B; Avraham, S; Groopman, J E

1997-03-17

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

Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes

PubMed Central

Cappello, Renato E; Estrada-Gutierrez, Guadalupe; Irles, Claudine; Giono-Cerezo, Silvia; Bloch, Robert J; Nataro, James P

2011-01-01

Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid encoded toxin (Pet), which elicits cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known if this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, prior to the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephophorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication. PMID:21205005

The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic NMR spectroscopy.

PubMed

Lindfors, Hanna E; Drijfhout, Jan Wouter; Ubbink, Marcellus

2012-06-01

The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signaling processes from focal adhesions. The phosphorylated tyrosine residue 397 in FAK is able to bind the Src SH2 domain. To establish the extent of the FAK binding motif, the binding affinity of the SH2 domain for phosphorylated and unphosphorylated FAK-derived peptides of increasing length was determined and compared with that of the internal Src SH2 binding site. It is shown that the FAK peptides have higher affinity than the internal binding site and that seven negative residues adjacent to the core SH2 binding motif increase the binding constant 30-fold. A rigid spin-label incorporated in the FAK peptides was used to establish on the basis of paramagnetic relaxation enhancement whether the peptide-protein complex is well defined. A large spread of the paramagnetic effects on the surface of the SH2 domain suggests that the peptide-protein complex exhibits dynamics, despite the high affinity of the peptide. The strong electrostatic interaction between the positive side of the SH2 domain and the negative peptide results in a high affinity but may also favor a dynamic interaction. Copyright © 2012 Wiley Periodicals, Inc.

Timing is everything

PubMed Central

Carr, Heather S; Frost, Jeffrey A

2013-01-01

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion. PMID:23792411

Fabrication of biomimetic dry-adhesion structures through nanosphere lithography

NASA Astrophysics Data System (ADS)

Kuo, P. C.; Chang, N. W.; Suen, Y.; Yang, S. Y.

2018-03-01

Components with surface nanostructures suitable for biomimetic dry adhesion have a great potential in applications such as gecko tape, climbing robots, and skin patches. In this study, a nanosphere lithography technique with self-assembly nanospheres was developed to achieve effective and efficient fabrication of dry-adhesion structures. Self-assembled monolayer nanospheres with high regularity were obtained through tilted dip-coating. Reactive-ion etching of the self-assembled nanospheres was used to fabricate nanostructures of different shapes and aspect ratios by varying the etching time. Thereafter, nickel molds with inverse nanostructures were replicated using the electroforming process. Polydimethylsiloxane (PDMS) nanostructures were fabricated through a gas-assisted hot-embossing method. The pulling test was performed to measure the shear adhesion on the glass substrate of a sample, and the static contact angle was measured to verify the hydrophobic property of the structure. The enhancement of the structure indicates that the adhesion force increased from 1.2 to 4.05 N/cm2 and the contact angle increased from 118.6° to 135.2°. This columnar structure can effectively enhance the adhesion ability of PDMS, demonstrating the potential of using nanosphere lithography for the fabrication of adhesive structures.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.

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. Despitemore » 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 focal adhesion kinase activity. • Shb is critical for the long-term maintenance of the hematopoietic stem cell pool.« less

Assembly of Nanorods into Designer Superstructures: The Role of Templating, Capillary Forces, Adhesion, and Polymer Hydration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ciszek, Jacob W.; Huang, Ling; Tsonchev, Stefan

The assembly mechanism by which hundreds of thousands of two-segment gold-polypyrrole nanorods are assembled into kinetically controlled shape-directed superstructures is examined to predict the range of nanoparticle sizes and materials that can be utilized in their formation. Four processes are responsible for assembly: templating, capillary force assembly, adhesion, and polymer hydration. It is shown that templating, where rods are prepositioned for assembly, is scale invariant and that the energy-minimized state after this step is highly disordered. In addition, we predict that superstructures can be made independently from patterns of rods separated by a distance as small as six times themore » inter-rod spacing. Both modeling and experiment show that adhesion and polymer dehydration, which induces curvature in the superstructures, are applicable to other materials. However, the high surface energy and low elastic modulus of polypyrrole are advantageous toward generating three-dimensional structures, inducing curvature at gold/polypyrrole length ratios as large as 7:1.« less

Allosteric regulation of focal adhesion kinase by PIP₂ and ATP.

PubMed

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

2015-02-03

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. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2008-01-01

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

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

PubMed

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

2012-09-01

Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.

Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins

NASA Technical Reports Server (NTRS)

Alenghat, Francis J.; Ingber, Donald E.

2002-01-01

Mechanical stresses modulate cell function by either activating or tuning signal transduction pathways. Mechanotransduction, the process by which cells convert mechanical stimuli into a chemical response, occurs both in cells specialized for sensing mechanical cues and in parenchymal cells whose primary function is not mechanosensory. However, common among the various responses to mechanical stress is the importance of direct or indirect connections between the internal cytoskeleton, the extracellular matrix (ECM), and traditional signal transducing molecules. In many instances, these elements converge at focal adhesions, sites of structural attachment between the cytoskeleton and ECM that are anchored by cell surface integrin receptors. Alenghat and Ingber discuss the accumulating evidence for the central role of cytoskeleton, ECM, and integrin-anchored focal adhesions in several mechanotransduction pathways.

Cell/surface interactions on laser micro-textured titanium-coated silicon surfaces.

PubMed

Mwenifumbo, Steven; Li, Mingwei; Chen, Jianbo; Beye, Aboubaker; Soboyejo, Wolé

2007-01-01

This paper examines the effects of nano-scale titanium coatings, and micro-groove/micro-grid patterns on cell/surface interactions on silicon surfaces. The nature of the cellular attachment and adhesion to the coated/uncoated micro-textured surfaces was elucidated by the visualization of the cells and relevant cytoskeletal & focal adhesion proteins through scanning electron microscopy and immunofluorescence staining. Increased cell spreading and proliferation rates are observed on surfaces with 50 nm thick Ti coatings. The micro-groove geometries have been shown to promote contact guidance, which leads to reduced scar tissue formation. In contrast, smooth surfaces result in random cell orientations and the increased possibility of scar tissue formation. Immunofluorescence cell staining experiments also reveal that the actin stress fibers are aligned along the groove dimensions, with discrete focal adhesions occurring along the ridges, within the grooves and at the ends of the cell extensions. The implications of the observed cell/surface interactions are discussed for possible applications of silicon in implantable biomedical systems.

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

PubMed Central

Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Moore, Travis I.; Koga, Nobuyasu; Baker, David A.; Oldenbourg, Rudolf; Tani, Tomomi; Springer, Timothy A.; Waterman, Clare M.

2017-01-01

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven “retrograde flow” of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues. PMID:29073038

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. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins

PubMed Central

Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A.

2012-01-01

Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis. PMID:22222499

Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

PubMed

Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A; Marciano-Cabral, Francine

2012-03-01

Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase.

PubMed

Aminian, Alieh; Shirzadi, Bahareh; Azizi, Zahra; Maedler, Kathrin; Volkmann, Eike; Hildebrand, Nils; Maas, Michael; Treccani, Laura; Rezwan, Kurosch

2016-12-01

Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials. Copyright © 2016 Elsevier B.V. All rights reserved.

Interaction between mDia1 and ROCK in Rho-induced migration and adhesion of human dental pulp cells.

PubMed

Cheng, L; Xu, J; Qian, Y Y; Pan, H Y; Yang, H; Shao, M Y; Cheng, R; Hu, T

2017-01-01

To investigate the effects of mammalian homologue of Drosophila diaphanous-1(mDia1) and Rho-associated coiled-coil-containing protein kinase (ROCK) on the migration and adhesion of dental pulp cells (DPCs). Lysophosphatidic acid (LPA) was used to activate Rho signalling. mDia1 and ROCK were inhibited by short interfering RNA and the specific inhibitor, Y-27632, respectively. The migration of DPCs was assessed using the transwell migration assay and scratch test. Formation of cytoskeleton and focal adhesions(FAs) was observed by confocal laser scanning microscopy. Cell adhesion and spreading assays were performed. Phosphorylation of focal adhesion kinase (FAK) and paxillin was detected by Western blotting, and the bands were analysed using Adobe Photoshop CS5 software. All experiments were performed at least three times, and data were analysed with one-way anova and a post hoc test. LPA-triggered activation of Rho and inhibition of ROCK significantly increased the cell migration rate. Cell migration was inhibited by silencing mDia1. mDia1 silencing and ROCK inhibition suppressed the LPA-induced formation of the cytoskeleton, FA and phosphorylation of FAK and paxillin. Inhibition of ROCK or mDia1 facilitated early cell adhesion and spreading; by contrast, the combined inhibition of ROCK and mDia1 neutralized these effects. mDia1 promoted RhoA-induced migration of DPCs, but ROCK had an opposite effect. Both mDia1 and ROCK participated in cytoskeleton formation and adhesion of DPCs. The interactions between mDia1 and ROCK might influence dental pulp repair by determining the migration and adhesion of DPCs. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Involvement of rho-gtpases in fibroblast adhesion and fibronectine fibrillogenesis under stretch

NASA Astrophysics Data System (ADS)

Guignandon, A.; Lambert, C.; Rattner, A.; Servotte, S.; Lapiere, C.; Nusgens, B.; Vico, L.

The Rho family small GTPases play a crucial role in mediating cellular adaptation to mechanical stimulation (MS), and possibly to microgravity (μg), through effects on the cytoskeleton and cell adhesion which is, in turn, mainly regulated by fibronectin fibrillogenesis (FnF). It remains unclear how mechanical stimulation is transduced to the Rho signaling pathways and how it impacts on fibronectin (fbn) fibrillogenesis (FnF). μg (2 days, mission STS-095) led to de-adhesion of fibroblasts and modification of the underlying extracellular matrix. To determine whether GTPases modulated FnF, we generated stable cell lines expressing high level of activated RhoA and Rac1 (QL) as compared to wild type (WI26-WT). After MS application [8% deformation, 1Hz, 15 min., 3 times/day for 1-2 days], we quantified focal adhesion (vinculin, paxillin, FAKY397), f-actin stress fibers (Sf) and FnF with home-developed softwares. We reported that after MS, Sf are more rapidly (30min) formed under the nucleus in Wi26-WT (+100%) and Rac1 (+200%) than in RhoA (+20%). Vinculin & paxillin were only restricted to the cell edge in static conditions and homogeneously distributed after MS in WT and Rac1. The relative area of contacts (vinculin & paxillin) was more dramatically enhanced by MS in Rac1 (+80%) than in WT (+40%) and RhoA (+25%) indicating that new focal contacts are formed under MS and supported the presence of Sf. MS Activation of FAK (FAKY397) was clear in WT and Rac1 and reduced in RhoA. FnF was restricted to cell-cell contacts zone without any change in the relative area of fbn after a 2-days MS. However we found more numerous spots of fbn at the cell center in Rac1 as compared with RhoA & WT suggesting that these fibrillar contacts will grow upon maturation and modulate FnF. The results indicate that MS induces formation of Sf and focal adhesions and enhances FF. RhoA has been shown to induce the formation of Sf and focal adhesions, and Rac1 activation decreases Rho activity in some cell types. As the sensitivity to MS is Rac1>WI-26>RhoA we speculate (1) that MS increases RhoA activity, and (2) that this effect is more prominent in Rac1 cells due to an induced lower basal level of RhoA activity. This study is a feasibility work for future space missions allowing evaluating the effect of μg on our models.

Molecular assemblies as protective barriers and adhesion promotion interlayer

DOEpatents

King, David E.; Czanderna, Alvin W.; Kennedy, Cheryl E.

1996-01-01

A protective diffusion barrier having adhesive qualifies for metalized surfaces is provided by a passivating agent having the formula HS--(CH.sub.2).sub.11 --COOH Which forms a very dense, transparent organized molecular assembly or layer that is impervious to water, alkali, and other impurities and corrosive substances that typically attack metal surfaces.

Colocalization of kindlin-1, kindlin-2, and migfilin at keratinocyte focal adhesion and relevance to the pathophysiology of Kindler syndrome.

PubMed

Lai-Cheong, J E; Ussar, S; Arita, K; Hart, I R; McGrath, J A

2008-09-01

Kindler syndrome (KS) results from pathogenic loss-of-function mutations in the KIND1 gene, which encodes kindlin-1, a focal adhesion and actin cytoskeleton-related protein. How and why abnormalities in kindlin-1 disrupt keratinocyte cell biology in KS, however, is not yet known. In this study, we identified two previously unreported binding proteins of kindlin-1: kindlin-2 and migfilin. Co-immunoprecipitation and confocal microscopy studies show that these three proteins bind to each other and colocalize at focal adhesion in HaCaT cells and normal human keratinocytes. Moreover, loss-of-function mutations in KIND1 result in marked variability in kindlin-1 immunolabeling in KS skin, which is mirrored by similar changes in kindlin-2 and migfilin immunoreactivity. Kindlin-1, however, may function independently of kindlin-2 and migfilin, as loss of kindlin-1 expression in HaCaT keratinocytes by RNA interference and in KS keratinocytes does not affect KIND2 or FBLIM1 (migfilin) gene expression or kindlin-2 and migfilin protein localization. In addition to identifying protein-binding partners for kindlin-1, this study also highlights that KIND1 gene expression and kindlin-1 protein labeling are not always reduced in KS, findings that are relevant to the accurate laboratory diagnosis of this genodermatosis by skin immunohistochemistry.

PI(3,4)P2 plays critical roles in the regulation of focal adhesion dynamics of MDA-MB-231 breast cancer cells.

PubMed

Fukumoto, Miki; Ijuin, Takeshi; Takenawa, Tadaomi

2017-05-01

Phosphoinositides play pivotal roles in the regulation of cancer cell phenotypes. Among them, phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ) localizes to the invadopodia, and positively regulates tumor cell invasion. In this study, we examined the effect of PI(3,4)P 2 on focal adhesion dynamics in MDA-MB-231 basal breast cancer cells. Knockdown of SHIP2, a phosphatidylinositol 3,4,5-trisphosphatase (PIP 3 ) 5-phosphatase that generates PI(3,4)P 2 , in MDA-MB-231 breast cancer cells, induced the development of focal adhesions and cell spreading, leading to the suppression of invasion. In contrast, knockdown of PTEN, a 3-phosphatase that de-phosphorylates PIP 3 and PI(3,4)P 2 , induced cell shrinkage and increased cell invasion. Interestingly, additional knockdown of SHIP2 rescued these phenotypes. Overexpression of the TAPP1 PH domain, which binds to PI(3,4)P 2 , and knockdown of Lpd, a downstream effector of PI(3,4)P 2 , resulted in similar phenotypes to those induced by SHIP2 knockdown. Taken together, our results suggest that inhibition of PI(3,4)P 2 generation and/or downstream signaling could be useful for inhibiting breast cancer metastasis. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Micro spectrometer for parallel light and method of use

NASA Technical Reports Server (NTRS)

Park, Yeonjoon (Inventor); Choi, Sang H. (Inventor); King, Glen C. (Inventor); Elliott, James R. (Inventor)

2011-01-01

A spectrometer system includes an optical assembly for collimating light, a micro-ring grating assembly having a plurality of coaxially-aligned ring gratings, an aperture device defining an aperture circumscribing a target focal point, and a photon detector. An electro-optical layer of the grating assembly may be electrically connected to an energy supply to change the refractive index of the electro-optical layer. Alternately, the gratings may be electrically connected to the energy supply and energized, e.g., with alternating voltages, to change the refractive index. A data recorder may record the predetermined spectral characteristic. A method of detecting a spectral characteristic of a predetermined wavelength of source light includes generating collimated light using an optical assembly, directing the collimated light onto the micro-ring grating assembly, and selectively energizing the micro-ring grating assembly to diffract the predetermined wavelength onto the target focal point, and detecting the spectral characteristic using a photon detector.

Direct view zoom scope with single focal plane and adaptable reticle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bagwell, Brett

A direct view telescopic sight includes objective lens, eyepiece, and prism erector assemblies. The objective lens assembly is mounted to receive light of an image from an object direction and direct the light along an optical path. The eyepiece assembly is mounted to receive the light along the optical path and to emit the light of the image along an eye-ward direction. The prism erector assembly is positioned between the objective lens and eyepiece assemblies and includes first and second prism elements through which the optical path passes. The first and second prism elements invert the image. A reticle elementmore » is disposed on or adjacent to a surface of one of the first or second prism elements to combine a reticle on the image. The image is brought into focus at only a single focal plane between the objective lens and eyepiece assemblies at a given time.« less

Sundew adhesive: a naturally occurring hydrogel

PubMed Central

Huang, Yujian; Wang, Yongzhong; Sun, Leming; Agrawal, Richa; Zhang, Mingjun

2015-01-01

Bioadhesives have drawn increasing interest in recent years, owing to their eco-friendly, biocompatible and biodegradable nature. As a typical bioadhesive, sticky exudate observed on the stalked glands of sundew plants aids in the capture of insects and this viscoelastic adhesive has triggered extensive interests in revealing the implied adhesion mechanisms. Despite the significant progress that has been made, the structural traits of the sundew adhesive, especially the morphological characteristics in nanoscale, which may give rise to the viscous and elastic properties of this mucilage, remain unclear. Here, we show that the sundew adhesive is a naturally occurring hydrogel, consisting of nano-network architectures assembled with polysaccharides. The assembly process of the polysaccharides in this hydrogel is proposed to be driven by electrostatic interactions mediated with divalent cations. Negatively charged nanoparticles, with an average diameter of 231.9 ± 14.8 nm, are also obtained from this hydrogel and these nanoparticles are presumed to exert vital roles in the assembly of the nano-networks. Further characterization via atomic force microscopy indicates that the stretching deformation of the sundew adhesive is associated with the flexibility of its fibrous architectures. It is also observed that the adhesion strength of the sundew adhesive is susceptible to low temperatures. Both elasticity and adhesion strength of the sundew adhesive reduce in response to lowering the ambient temperature. The feasibility of applying sundew adhesive for tissue engineering is subsequently explored in this study. Results show that the fibrous scaffolds obtained from sundew adhesive are capable of increasing the adhesion of multiple types of cells, including fibroblast cells and smooth muscle cells, a property that results from the enhanced adsorption of serum proteins. In addition, in light of the weak cytotoxic activity exhibited by these scaffolds towards a variety of mammal cells, evidence is sufficient to propose that sundew adhesive is a promising nanomaterial worth further exploitation in the field of tissue engineering. PMID:25948615

Wood adhesion and adhesives

Treesearch

Charles R. Frihart

2005-01-01

An appreciation of rheology, material science, organic chemistry, polymer science, and mechanics leads to better understanding of the factors controlling the performance of the bonded assemblies. Given the complexity of wood as a substrate, it is hard to understand why some wood adhesives work better than other wood adhesives, especially when under the more severe...

Joining Tubes With Adhesive

NASA Technical Reports Server (NTRS)

Bateman, W. A.

1984-01-01

Cylindrical tubes joined together, end to end, by method employing adhesive, tapered ends, and spacing wires. Tapered joint between tubular structural elements provides pressure between bonding surfaces during adhesive curing. Spacing wires prevent adhesive from being scraped away when one element inserted in other. Method developed for assembling structural elements made of composite materials.

The influence of Pyk2 on the mechanical properties in fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klemm, Anna H.; Kienle, Sandra; Rheinlaender, Johannes

2010-03-19

The cell surface receptor integrin is involved in signaling mechanical stresses via the focal adhesion complex (FAC) into the cell. Within FAC, the focal adhesion kinase (FAK) and Pyk2 are believed to act as important scaffolding proteins. Based on the knowledge that many signal transducing molecules are transiently immobilized within FAC connecting the cytoskeleton with integrins, we applied magnetic tweezer and atomic force microscopic measurements to determine the influence of FAK and Pyk2 in cells mechanically. Using mouse embryonic fibroblasts (MEF; FAK{sup +/+}, FAK{sup -/-}, and siRNA-Pyk2 treated FAK{sup -/-} cells) provided a unique opportunity to describe the function ofmore » FAK and Pyk2 in more detail and to define their influence on FAC and actin distribution.« less

Linker length dependent binding of a focal adhesion kinase derived peptide to the Src SH3-SH2 domains.

PubMed

Lindfors, Hanna E; Venkata, Bharat Somireddy; Drijfhout, Jan W; Ubbink, Marcellus

2011-02-18

The interaction between a peptide encompassing the SH3 and SH2 binding motifs of focal adhesion kinase (FAK) and the Src SH3-SH2 domains has been investigated with NMR spectroscopy and calorimetry. The binding to both motifs is anti-cooperative. Reduction of the long linker connecting the motifs does not lead to cooperativity. Short linkers that do not allow simultaneous intramolecular binding of the peptide to both motifs cause peptide-mediated dimerisation, even with a linker of only three amino acids. The role of the SH3 binding motif is discussed in view of the independent nature of the SH interactions. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Biphasic response of cell invasion to matrix stiffness in 3-dimensional biopolymer networks

PubMed Central

Lang, Nadine R.; Skodzek, Kai; Hurst, Sebastian; Mainka, Astrid; Steinwachs, Julian; Schneider, Julia; Aifantis, Katerina E.; Fabry, Ben

2015-01-01

When cells come in contact with an adhesive matrix, they begin to spread and migrate with a speed that depends on the stiffness of the extracellular matrix. On a flat surface, migration speed decreases with matrix stiffness mainly due to an increased stability of focal adhesions. In a 3-dimensional (3D) environment, cell migration is thought to be additionally impaired by the steric hindrance imposed by the surrounding matrix. For porous 3D biopolymer networks such as collagen gels, however, the effect of matrix stiffness on cell migration is difficult to separate from effects of matrix pore size and adhesive ligand density, and is therefore unknown. Here we used glutaraldehyde as a crosslinker to increase the stiffness of self-assembled collagen biopolymer networks independently of collagen concentration or pore size. Breast carcinoma cells were seeded onto the surface of 3D collagen gels, and the invasion depth was measured after 3 days of culture. Cell invasion in gels with pore sizes larger than 5 μm increased with higher gel stiffness, whereas invasion in gels with smaller pores decreased with higher gel stiffness. These data show that 3D cell invasion is enhanced by higher matrix stiffness, opposite to cell behavior in 2D, as long as the pore size does not fall below a critical value where it causes excessive steric hindrance. These findings may be important for optimizing the recellularization of soft tissue implants or for the design of 3D invasion models in cancer research. PMID:25462839

Chronic hyperperfusion and angiogenesis follow subacute hypoperfusion in the thalamus of rats with focal cerebral ischemia

PubMed Central

Hayward, Nick MEA; Yanev, Pavel; Haapasalo, Annakaisa; Miettinen, Riitta; Hiltunen, Mikko; Gröhn, Olli; Jolkkonen, Jukka

2011-01-01

Cerebral blood flow (CBF) is disrupted after focal ischemia in rats. We examined long-term hemodynamic and cerebrovascular changes in the rat thalamus after focal cerebral ischemia. Cerebral blood flow quantified by arterial spin labeling magnetic resonance imaging was decreased in the ipsilateral and contralateral thalamus 2 days after cerebral ischemia. Partial thalamic CBF recovery occurred by day 7, then the ipsilateral thalamus was chronically hyperperfused at 30 days and 3 months compared with its contralateral side. This contrasted with permanent hypoperfusion in the ipsilateral cortex. Angiogenesis was indicated by endothelial cell (RECA-1) immunohistochemistry that showed increased blood vessel branching in the ipsilateral thalamus at the end of the 3-month follow-up. Only transient thalamic IgG extravasation was observed, indicating that the blood–brain barrier was intact after day 2. Angiogenesis was preceded by transiently altered expression levels of cadherin family adhesion molecules, cadherin-7, protocadherin-1, and protocadherin-17. In conclusion, thalamic pathology after focal cerebral ischemia involved long-term hemodynamic changes and angiogenesis preceded by altered expression of vascular adhesion factors. Postischemic angiogenesis in the thalamus represents a novel type of remote plasticity, which may support removal of necrotic brain tissue and aid functional recovery. PMID:21081957

Bone sialoprotein stimulates focal adhesion-related signaling pathways: role in migration and survival of breast and prostate cancer cells.

PubMed

Gordon, Jonathan A R; Sodek, Jaro; Hunter, Graeme K; Goldberg, Harvey A

2009-08-15

Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP-induced migration and tumor survival were examined in breast and prostate cancer cells (MDA-MB-231, Hs578T and PC3). Additionally, the effects of exogenous TGF-beta1 and EGF, cytokines associated with tumor metastasis and present in high-levels in the bone microenvironment, were examined in BSP-expressing cancer cells. Expression of BSP but not an integrin-binding mutant (BSP-KAE) in tumor cell lines resulted in increased levels of alpha(v)-containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP-1-family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP-2, MMP-9, and MMP-14. The BSP-mediated activation of the FAK-associated pathway resulted in increased cancer cell invasion in a Matrigel-coated Boyden-chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF-beta1 to the BSP-expressing cell lines significantly increased ERK phosphorylation, AP-1 activation, MMP-2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF-beta1 and EGF. (c) 2009 Wiley-Liss, Inc.

Molecular assemblies as protective barriers and adhesion promotion interlayer

DOEpatents

King, D.E.; Czanderna, A.W.; Kennedy, C.E.

1996-01-30

A protective diffusion barrier having adhesive qualities for metalized surfaces is provided by a passivating agent having the formula HS--(CH{sub 2}){sub 11}--COOH which forms a very dense, transparent organized molecular assembly or layer that is impervious to water, alkali, and other impurities and corrosive substances that typically attack metal surfaces. 8 figs.

Cellular level robotic surgery: Nanodissection of intermediate filaments in live keratinocytes.

PubMed

Yang, Ruiguo; Song, Bo; Sun, Zhiyong; Lai, King Wai Chiu; Fung, Carmen Kar Man; Patterson, Kevin C; Seiffert-Sinha, Kristina; Sinha, Animesh A; Xi, Ning

2015-01-01

We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Talamás-Lara, Daniel, E-mail: daniel_talamas@hotmail.com; Talamás-Rohana, Patricia, E-mail: ptr@cinvestav.mx; Fragoso-Soriano, Rogelio Jaime, E-mail: rogelio@fis.cinvestav.mx

Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves amore » dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica. - Highlights: • Striking differences in adhesion to FN between E. histolytica and E. dispar. • A greater degree of cell stiffness in E. histolytica with respect to E. dispar. • E. histolytica but not E. dispar forms regions of close contact with FN. • The actin cytoskeleton is involved in the pathogenicity of E. histolytica.« less

Silicon concentrator cell-assembly development

NASA Astrophysics Data System (ADS)

1982-08-01

The purpose was to develop an improved cell assembly design for photovoltaic concentrator receivers. Efforts were concentrated on a study of adhesive/separator systems that might be applied between cell and substrate, because this area holds the key to improved heat transfer, electrical isolation and adhesion. It is also the area in which simpler construction methods offer the greatest benefits for economy and reliability in the manufacturing process. Of the ten most promising designs subjected to rigorous environmental testing, eight designs featuring acrylic and silicon adhesives and fiberglass and polyester separators performed very well.

Quick-sealing design for radiological containment

DOEpatents

Rampolia, Donald S.; Speer, Elmer

1990-01-01

A quick-sealing assembly and method for forming an adhesive seal on opposite sides of a mechanical seal for a flexible containment bag of the type used for working with radioactively contaminated objects. The assembly includes an elongated mechanical fastener having opposing engaging members affixed at a predetermined distance from each of the elongated edges, with an adhesive layer formed between the mechanical fastener and the elongated edge such that upon engagement of the mechanical fastener and adhesive layers to opposing containment fabric, a neat triple hermetic seal is formed.

Quick-sealing design for radiological containment

DOEpatents

Rampolla, Donald S.; Speer, Elmer

1991-01-01

A quick-sealing assembly and method for forming an adhesive seal on opposite sides of a mechanical seal for a flexible containment bag of the type used for working with radioactively contaminated objects. The assembly includes an elongated mechanical fastener having opposing engaging members affixed at a predetermined distance from each of the elongated edges, with an adhesive layer formed between the mechanical fastener and the elongated edge such that upon engagement of the mechanical fastener and adhesive layers to opposing containment fabric, a neat triple hermetic seal is formed.

Prevailing Torque Locking Feature in Threaded Fasteners Using Anaerobic Adhesive

NASA Technical Reports Server (NTRS)

Hernandez, Alan; Hess, Daniel P.

2016-01-01

This paper presents results from tests to assess the use of anaerobic adhesive for providing a prevailing torque locking feature in threaded fasteners. Test procedures are developed and tests are performed on three fastener materials, four anaerobic adhesives, and both unseated assembly conditions. Five to ten samples are tested for each combination. Tests for initial use, reuse without additional adhesive, and reuse with additional adhesive are performed for all samples. A 48-hour cure time was used for all initial use and reuse tests. Test data are presented as removal torque versus removal angle with the specification required prevailing torque range added for performance assessment. Percent specification pass rates for the all combinations of fastener material, adhesive, and assembly condition are tabulated and reveal use of anaerobic adhesive as a prevailing torque locking feature is viable. Although not every possible fastener material and anaerobic adhesive combination provides prevailing torque values within specification, any combination can be assessed using the test procedures presented. Reuse without additional anaerobic adhesive generally provides some prevailing torque, and in some cases within specification. Reuse with additional adhesive often provides comparable removal torque data as in initial use.

The Mechanical Design of a Kinematic Mount for the Mid Infrared Instrument Focal Plane Module on the James Webb Space Telescope

NASA Technical Reports Server (NTRS)

Thelen, Michael P.; Moore, Donald M.

2009-01-01

The detector assembly for the Mid Infrared Instrument (MIRI) of the James Webb Space Telescope (JWST) is mechanically supported in the Focal Plane Module (FPM) Assembly with an efficient hexapod design. The kinematic mount design allows for precision adjustment of the detector boresight to assembly alignment fiducials and maintains optical alignment requirements during flight conditions of launch and cryogenic operations below 7 Kelvin. This kinematic mounting technique is able to be implemented in a variety of optical-mechanical designs and is capable of micron level adjustment control and stability over wide dynamic and temperature ranges.

Mussel-inspired nano-building block assemblies for mimicking extracellular matrix microenvironments with multiple functions.

PubMed

Wang, Zhenming; Jia, Zhanrong; Jiang, Yanan; Li, Pengfei; Han, Lu; Lu, Xiong; Ren, Fuzeng; Wang, Kefeng; Yuan, Huiping

2017-08-03

The assembly of nano-building blocks is an effective way to produce artificial extracellular matrix microenvironments with hierarchical micro/nano structures. However, it is hard to assemble different types of nano-building blocks, to form composite coatings with multiple functions, by traditional layer-by-layer (LbL) self-assembly methods. Inspired by the mussel adhesion mechanism, we developed polydopamine (PDA)-decorated bovine serum albumin microspheres (BSA-MS) and nano-hydroxyapatite (nano-HA), and assembled them to form bioactive coatings with micro/nano structures encapsulating bone morphogenetic protein-2 (BMP-2). First, PDA-decorated nano-HA (nano-pHA) was obtained by oxidative polymerization of dopamine on nano-HA. Second, BMP-2-encapsulated BSA microspheres were prepared through desolvation, and then were also decorated by PDA (pBSA-MS). Finally, the nano-pHA and pBSA-MS were assembled using the adhesive properties of PDA. Bone marrow stromal cell cultures and in vivo implantation, showed that the pHA/pBSA (BMP-2) coatings can promote cell adhesion, proliferation, and benefited for osteoinductivity. PDA decoration was also applied to assemble various functional nanoparticles, such as nano-HA, polystyrene, and Fe 3 O 4 nanoparticles. In summary, this study provides a novel strategy for the assembly of biofunctional nano-building blocks, which surpasses traditional LbL self-assembly of polyelectrolytes, and can find broad applications in bioactive agents delivery or multi-functional coatings.

Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness

PubMed Central

Meller, Julia; Rogozin, Igor B.; Poliakov, Eugenia; Meller, Nahum; Bedanov-Pack, Mark; Plow, Edward F.; Qin, Jun; Podrez, Eugene A.; Byzova, Tatiana V.

2015-01-01

Kindlins are integrin-interacting proteins essential for integrin-mediated cell adhesiveness. In this study, we focused on the evolutionary origin and functional specialization of kindlins as a part of the evolutionary adaptation of cell adhesive machinery. Database searches revealed that many members of the integrin machinery (including talin and integrins) existed before kindlin emergence in evolution. Among the analyzed species, all metazoan lineages—but none of the premetazoans—had at least one kindlin-encoding gene, whereas talin was present in several premetazoan lineages. Kindlin appears to originate from a duplication of the sequence encoding the N-terminal fragment of talin (the talin head domain) with a subsequent insertion of the PH domain of separate origin. Sequence analysis identified a member of the actin filament–associated protein 1 (AFAP1) superfamily as the most likely origin of the kindlin PH domain. The functional divergence between kindlin paralogues was assessed using the sequence swap (chimera) approach. Comparison of kindlin 2 (K2)/kindlin 3 (K3) chimeras revealed that the F2 subdomain, in particular its C-terminal part, is crucial for the differential functional properties of K2 and K3. The presence of this segment enables K2 but not K3 to localize to focal adhesions. Sequence analysis of the C-terminal part of the F2 subdomain of K3 suggests that insertion of a variable glycine-rich sequence in vertebrates contributed to the loss of constitutive K3 targeting to focal adhesions. Thus emergence and subsequent functional specialization of kindlins allowed multicellular organisms to develop additional tissue-specific adaptations of cell adhesiveness. PMID:25540429

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

PubMed

Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Nanoscale TiO2 nanotubes govern the biological behavior of human glioma and osteosarcoma cells

PubMed Central

Tian, Ang; Qin, Xiaofei; Wu, Anhua; Zhang, Hangzhou; Xu, Quan; Xing, Deguang; Yang, He; Qiu, Bo; Xue, Xiangxin; Zhang, Dongyong; Dong, Chenbo

2015-01-01

Cells respond to their surroundings through an interactive adhesion process that has direct effects on cell proliferation and migration. This research was designed to investigate the effects of TiO2 nanotubes with different topographies and structures on the biological behavior of cultured cells. The results demonstrated that the nanotube diameter, rather than the crystalline structure of the coatings, was a major factor for the biological behavior of the cultured cells. The optimal diameter of the nanotubes was 20 nm for cell adhesion, migration, and proliferation in both glioma and osteosarcoma cells. The expression levels of vitronectin and phosphor-focal adhesion kinase were affected by the nanotube diameter; therefore, it is proposed that the responses of vitronectin and phosphor-focal adhesion kinase to the nanotube could modulate cell fate. In addition, the geometry and size of the nanotube coating could regulate the degree of expression of acetylated α-tubulin, thus indirectly modulating cell migration behavior. Moreover, the expression levels of apoptosis-associated proteins were influenced by the topography. In conclusion, a nanotube diameter of 20 nm was the critical threshold that upregulated the expression level of Bcl-2 and obviously decreased the expression levels of Bax and caspase-3. This information will be useful for future biomedical and clinical applications. PMID:25848261

A Novel Role for Integrin-linked Kinase in Epithelial Sheet Morphogenesis

PubMed Central

Vespa, Alisa; D'Souza, Sudhir J.A.; Dagnino, Lina

2005-01-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca2+ triggers formation of cell–cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca2+ treatment of keratinocytes induces rapid (≤1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and β-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell–cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/β-catenin to cell borders, precluding Ca2+-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell–cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell–cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration. PMID:15975904

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

PubMed

Vespa, Alisa; D'Souza, Sudhir J A; Dagnino, Lina

2005-09-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions.

PubMed

Spiess, Matthias; Hernandez-Varas, Pablo; Oddone, Anna; Olofsson, Helene; Blom, Hans; Waithe, Dominic; Lock, John G; Lakadamyali, Melike; Strömblad, Staffan

2018-06-04

Integrins are the core constituents of cell-matrix adhesion complexes such as focal adhesions (FAs) and play key roles in physiology and disease. Integrins fluctuate between active and inactive conformations, yet whether the activity state influences the spatial organization of integrins within FAs has remained unclear. In this study, we address this question and also ask whether integrin activity may be regulated either independently for each integrin molecule or through locally coordinated mechanisms. We used two distinct superresolution microscopy techniques, stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion microscopy (STED), to visualize active versus inactive β1 integrins. We first reveal a spatial hierarchy of integrin organization with integrin molecules arranged in nanoclusters, which align to form linear substructures that in turn build FAs. Remarkably, within FAs, active and inactive β1 integrins segregate into distinct nanoclusters, with active integrin nanoclusters being more organized. This unexpected segregation indicates synchronization of integrin activities within nanoclusters, implying the existence of a coordinate mechanism of integrin activity regulation. © 2018 Spiess et al.

Structure, Function, and Assembly of Type 1 Fimbriae

NASA Astrophysics Data System (ADS)

Knight, Stefan D.; Bouckaert, Julie

Bacterial infections constitute a major global health problem, acutely accentuated by the rapid spread of antibiotic resistant bacterial strains. The widespread need for bacteria to attach - adhere - to target cells before they can initiate an infection may be used to advantage by targeting the bacterial adhesion tools such as pili and fimbriae for development of novel anti-bacterial vaccines and drugs. Type 1 fimbriae are widely expressed by Escherichia coli. and are used by uropathogenic strains to mediate attachment to specific niches in the urinary tract. These fimbriae belong to a class of fibrillar adhesion organelles assembled through the chaperone/usher pathway, one of the terminal branches of the general secretion pathway in Gram-negative bacteria. Our understanding of the assembly, structure and function of these structures has evolved significantly over the last decade. Here, we summarize current understanding of the function and biogenesis of fibrillar adhesion organelles, and provide some examples of recent progress towards interfering with bacterial adhesion as a means to prevent infection.

Biofriendly bonding processes for nanoporous implantable SU-8 microcapsules for encapsulated cell therapy.

PubMed

Nemani, Krishnamurthy; Kwon, Joonbum; Trivedi, Krutarth; Hu, Walter; Lee, Jeong-Bong; Gimi, Barjor

2011-01-01

Mechanically robust, cell encapsulating microdevices fabricated using photolithographic methods can lead to more efficient immunoisolation in comparison to cell encapsulating hydrogels. There is a need to develop adhesive bonding methods which can seal such microdevices under physiologically friendly conditions. We report the bonding of SU-8 based substrates through (i) magnetic self assembly, (ii) using medical grade photocured adhesive and (iii) moisture and photochemical cured polymerization. Magnetic self-assembly, carried out in biofriendly aqueous buffers, provides weak bonding not suitable for long term applications. Moisture cured bonding of covalently modified SU-8 substrates, based on silanol condensation, resulted in weak and inconsistent bonding. Photocured bonding using a medical grade adhesive and of acrylate modified substrates provided stable bonding. Of the methods evaluated, photocured adhesion provided the strongest and most stable adhesion.

Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA.

PubMed

Mauriello, Emilia M F; Mouhamar, Fabrice; Nan, Beiyan; Ducret, Adrien; Dai, David; Zusman, David R; Mignot, Tâm

2010-01-20

Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S-motility powered by type-IV pili and A-motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin-like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S- and A-motility, causing rapid dispersal of S- and A-motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a DeltamglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA-YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A-motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin-like protein and a small GTPase spatial regulator.

Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA

PubMed Central

Mauriello, Emilia M F; Mouhamar, Fabrice; Nan, Beiyan; Ducret, Adrien; Dai, David; Zusman, David R; Mignot, Tâm

2010-01-01

Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S-motility powered by type-IV pili and A-motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin-like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S- and A-motility, causing rapid dispersal of S- and A-motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a ΔmglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA–YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A-motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin-like protein and a small GTPase spatial regulator. PMID:19959988

Epimorphin regulates bile duct formation via effects on mitosis orientation in rat liver epithelial stem-like cells.

PubMed

Zhou, Junnian; Zhao, Lei; Qin, Lipeng; Wang, Jing; Jia, Yali; Yao, Hailei; Sang, Chen; Hu, Qinghua; Shi, Shuangshuang; Nan, Xue; Yue, Wen; Zhuang, Fengyuan; Yang, Chun; Wang, Yunfang; Pei, Xuetao

2010-03-17

Understanding how hepatic precursor cells can generate differentiated bile ducts is crucial for studies on epithelial morphogenesis and for development of cell therapies for hepatobiliary diseases. Epimorphin (EPM) is a key morphogen for duct morphogenesis in various epithelial organs. The role of EPM in bile duct formation (DF) from hepatic precursor cells, however, is not known. To address this issue, we used WB-F344 rat epithelial stem-like cells as model for bile duct formation. A micropattern and a uniaxial static stretch device was used to investigate the effects of EPM and stress fiber bundles on the mitosis orientation (MO) of WB cells. Immunohistochemistry of liver tissue sections demonstrated high EPM expression around bile ducts in vivo. In vitro, recombinant EPM selectively induced DF through upregulation of CK19 expression and suppression of HNF3alpha and HNF6, with no effects on other hepatocytic genes investigated. Our data provide evidence that EPM guides MO of WB-F344 cells via effects on stress fiber bundles and focal adhesion assembly, as supported by blockade EPM, beta1 integrin, and F-actin assembly. These blockers can also inhibit EPM-induced DF. These results demonstrate a new biophysical action of EPM in bile duct formation, during which determination of MO plays a crucial role.

Hot melt adhesive pad surface attachment assembly concept for on-orbit operations

NASA Technical Reports Server (NTRS)

Progar, D. J.; Stein, B. A.

1984-01-01

The use of a hot melt adhesive concept to develop a Surface Attachment Assembly (SAA) for on-orbit attachment and detachment operations for the Manned Maneuvering Unit (MMU) was investigated. The concept involved impregnation of the hot melt adhesive into a fiberglass covered pad which contained electrical heating and thermoelectric cooling devices. The polyamide hot melt adhesive selected can be repeatedly heated to its melting point in a vacuum and provide good adhesion to various surfaces, i.e., reusable surface insulation tiles, metals, and composites, when cooled. After a series of adhesive screening tests, Jet-Melt 3746 was selected from a group of commercially available thermoplastic adhesive candidates which met or exceeded many of the criteria established for the SAA system. The SAA system was designed and fabricted with the goal of proving the concept with a working model rather than attempting to optimize all facets of the system. This system evolved by investigating alternate attachment concepts, designing and fabricating electronic systems to heat and cool the adhesive, and then fabricating electronic systems to heat and cool the adhesive, and then fabricating and testing two prototype full-size units.

Kindler syndrome in mice and men.

PubMed

Duperret, Elizabeth K; Ridky, Todd W

2014-09-01

Kindler syndrome (KS) in humans is a severe skin blistering disease associated with inflammation and increased risk of epidermal squamous cell carcinoma (SCC). This disease is known to be caused by loss-of-function mutations in Kindlin-1, a focal adhesion β-integrin binding protein. Thus far, it has been unclear what specific signaling events occur in KS keratinocytes to promote tumorigenesis, especially since loss of β-integrins and focal adhesion complexes has been previously shown to prevent or delay tumor formation. In the April issue of Nature Medicine, Rognoni and colleagues generate a transgenic mouse lacking Kindlin-1 in the epidermis to model the key features of KS, and show that Kindlin-1 regulates Wnt and TGFβ signaling independent of β-integrins. These β1-integrin-independent functions of Kindlin-1 may contribute to the increased SCC risk in KS patients.

Small interfering RNA targeting focal adhesion kinase prevents cardiac dysfunction in endotoxemia.

PubMed

Guido, Maria C; Clemente, Carolina F; Moretti, Ana I; Barbeiro, Hermes V; Debbas, Victor; Caldini, Elia G; Franchini, Kleber G; Soriano, Francisco G

2012-01-01

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

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.

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

NASA Astrophysics Data System (ADS)

Mierke, Claudia Tanja

2013-12-01

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

Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer*

PubMed Central

Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S.; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

2015-01-01

Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. PMID:26330541

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

PubMed Central

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

2014-01-01

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

Selective fibronectin adsorption against albumin and enhanced stem cell attachment on helium atmospheric pressure glow discharge treated titanium

NASA Astrophysics Data System (ADS)

Han, Inho; Vagaska, Barbora; Joo Park, Bong; Lee, Mi Hee; Jin Lee, Seung; Park, Jong-Chul

2011-06-01

Successful tissue integration of implanted medical devices depends on appropriate initial cellular response. In this study, the effect of helium atmospheric pressure glow discharge (He-APGD) treatment of titanium on selective protein adsorption and the initial attachment processes and focal adhesion formation of osteoprogenitor cells and stem cells were examined. Titanium disks were treated in a self-designed He-APGD system. Initial attachment of MC3T3-E1 mouse pre-osteoblasts and human mesenchymal stem cells (MSCs) was evaluated by MTT assay and plasma membrane staining followed by morphometric analysis. Fibronectin adsorption was investigated by Enzyme-Linked ImmunoSorbant Assay. MSCs cell attachment to treated and non-treated titanium disks coated with different proteins was verified also in serum-free culture. Organization of actin cytoskeleton and focal adhesions was evaluated microscopically. He-APGD treatment effectively modified the titanium surfaces by creating a super-hydrophilic surface, which promoted selectively higher adsorption of fibronectin, a protein of critical importance for cell/biomaterial interaction. In two different types of cells, the He-APGD treatment enhanced the number of attaching cells as well as their attachment area. Moreover, cells had higher organization of actin cytoskeleton and focal adhesions. Faster acceptance of the material by the progenitor cells in the early phases of tissue integration after the implantation may significantly reduce the overall healing time; therefore, titanium treatment with He-APGD seems to be an effective method of surface modification of titanium for improving its tissue inductive properties.

The heel and toe of the cell’s foot: A multifaceted approach for understanding the structure and dynamics of focal adhesions

PubMed Central

Wolfenson, Haguy; Henis, Yoav I.; Geiger, Benjamin; Bershadsky, Alexander D.

2010-01-01

Focal adhesions (FAs) are large clusters of transmembrane receptors of the integrin family and a multitude of associated cytoplasmic “plaque” proteins, which connect the extracellular matrix-bound receptors with the actin cytoskeleton. The formation of nearly stationary focal adhesions defines a boundary between dense and highly dynamic actin network in lamellipodium and the sparser and more diverse cytoskeletal organization in the lamella proper, creating a template for the organization of entire actin network. The major “mechanical” and “sensory” functions of FAs, namely, the nucleation and regulation of the contractile, myosin-II-containing, stress fibers and the mechanosensing of external surfaces depend, to a major extent, on the dynamics of molecular components within FAs. A central element in FA regulation concerns the positive feedback loop, based on the most intriguing feature of FAs, namely, their dependence on mechanical tension developing by the growing stress fibers. FAs grow in response to such tension, and rapidly disassemble upon its relaxation. In this article we address the mechanistic relationships between the process of FA development, maturation and dissociation and the dynamic molecular events, which take place in different regions of the FA, primarily in the distal end of this structure (the “toe”) and the proximal “heel”, and discuss the central role of local mechanical forces in orchestrating the complex interplay between FAs and the actin system. PMID:19598236

Self-assembled monolayer of designed and synthesized triazinedithiolsilane molecule as interfacial adhesion enhancer for integrated circuit

PubMed Central

2011-01-01

Self-assembled monolayer (SAM) with tunable surface chemistry and smooth surface provides an approach to adhesion improvement and suppressing deleterious chemical interactions. Here, we demonstrate the SAM comprising of designed and synthesized 6-(3-triethoxysilylpropyl)amino-1,3,5-triazine-2,4-dithiol molecule, which can enhance interfacial adhesion to inhibit copper diffusion used in device metallization. The formation of the triazinedithiolsilane SAM is confirmed by X-ray photoelectron spectroscopy. The adhesion strength between SAM-coated substrate and electroless deposition copper film was up to 13.8 MPa. The design strategy of triazinedithiolsilane molecule is expected to open up the possibilities for replacing traditional organosilane to be applied in microelectronic industry. PMID:21812994

Enhanced vbasis laser diode package

DOEpatents

Deri, Robert J.; Chen, Diana; Bayramian, Andy; Freitas, Barry; Kotovsky, Jack

2014-08-19

A substrate having an upper surface and a lower surface is provided. The substrate includes a plurality of v-grooves formed in the upper surface. Each v-groove includes a first side and a second side perpendicular to the first side. A laser diode bar assembly is disposed within each of the v-grooves and attached to the first side. The laser diode bar assembly includes a first adhesion layer disposed on the first side of the v-groove, a metal plate attached to the first adhesion layer, a second adhesion layer disposed over the metal plate, and a laser diode bar attached to the second adhesion layer. The laser diode bar has a coefficient of thermal expansion (CTE) substantially similar to that of the metal plate.

Adhesive coated electrical apparatus having sublimable protective covering and an assembly method

DOEpatents

Wootton, Roy E.

1982-01-01

Electrical apparatus including an enclosure, an electrode disposed within the enclosure, and supports for insulatably supporting the electrode within the enclosure has a permanently sticky adhesive material which is disposed on the interior surface of the outer enclosure. A high-vapor-pressure sublimable material is disposed on the permanently sticky adhesive material, with the sublimable material capable of subliming away in the presence of a vacuum. The presence of the sublimable material enables the apparatus to be non-sticky during assembly and handling operations, while being rendered sticky upon commissioning of the apparatus.

Influence of anatomical, physical, and mechanical properties of diffuse-porous hardwoods on moisture durability of bonded assemblies

Treesearch

Daniel J. Yelle; Ashley M. Stirgus

2016-01-01

Studying wood adhesive bond durability is challenging because wood is highly variable and heterogeneous at all length scales. In this study, three North American diffuse-porous hardwoods (hard maple, soft maple, and basswood) and their adhesively bonded as-semblies were exposed to wet and dry cyclic tests. Then, their den-sity differences were related to bond...

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes.

PubMed

Lin, Z X; Holtzer, S; Schultheiss, T; Murray, J; Masaki, T; Fischman, D A; Holtzer, H

1989-06-01

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes

PubMed Central

1989-01-01

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown. PMID:2472405

Thermography Examination of Abdominal Area Skin Temperatures in Individuals With and Without Focal-Onset Epilepsy.

PubMed

King, Hollis H; Cayce, Charles Thomas; Herrin, Jeph

Early osteopathic theory and practice, and the work of the medical intuitive Edgar Cayce suggested that the abdominal areas of individuals with epilepsy would manifest "cold spots." The etiology for this phenomenon was thought to be abdominal adhesions caused by inflammation and viscero-somatic reflexes caused by adhesions or injury to visceral or musculoskeletal system structures. Indeed, until that advent of electroencephalography in the 1930s, medical practice regarding epilepsy focused on abdominal neural and visceral structures. Following two hypotheses were formulated to evaluate any abdominal temperature phenomena: (1) an abdominal quadrant division analysis would find one or more quadrants "colder" in the focal-onset epilepsy group (ICD9-CM 345.4 and 345.5) compared to controls. (2) Total abdominal areas of individuals with focal-onset epilepsy wound be colder than a control group. Overall, 50 patients with the diagnosis of focal-onset epilepsy were recruited from the office of the Epilepsy Foundation of Florida and 50 control subjects with no history of epilepsy were recruited through advertising to the public. Under controlled room conditions all subjects had infrared thermographic images made and recorded by Med-Hot Model MH-731 FLIR equipment. There were no significant demographic difference between experimental patients and control subjects, though the control group tended to be younger and more often male; however, these were controlled for in all analyses. In the quadrant analysis, there were significant differences in that more epileptic patients had colder left upper abdominal quadrant temperatures than the control group (66.8% versus 44.9%; P = .030). In the total abdominal analysis, however, there were no significant differences. The results support the hypothesis that individuals with focal-onset epilepsy have colder abdominal areas. If substantiated in further research, present study results will require further examination of the mechanisms of action for epilepsy, and suggest the need for re-examination of older formulations of abdominal epilepsy, including the place of abdominal injury, inflammation, and adhesions in epileptic pathology. The concept of somato-visceral and viscero-somatic neurological interactions is one of the possible mechanisms underlying the "cold spot" findings and warrants further consideration. Copyright © 2017 Elsevier Inc. All rights reserved.

The design and application of large area intensive lens array focal spots measurement system

NASA Astrophysics Data System (ADS)

Chen, Bingzhen; Yao, Shun; Yang, Guanghui; Dai, Mingchong; Wang, Zhiyong

2014-12-01

Concentrating Photovoltaic (CPV) modules are getting thinner and using smaller cells now days. Correspondingly, large area intensive lens arrays with smaller unit dimension and shorter focal length are wanted. However, the size and power center of lens array focal spots usually differ from the design value and are hard to measure, especially under large area situation. It is because the machining error and deformation of material of the lens array are hard to simulate in the optical design process. Thus the alignment error between solar cells and focal spots in the module assembly process will be hard to control. Under this kind of situation, the efficiency of CPV module with thinner body and smaller cells is much lower than expected. In this paper, a design of large area lens array focal spots automatic measurement system is presented, as well as its prototype application results. In this system, a four-channel parallel light path and its corresponding image capture and process modules are designed. These modules can simulate focal spots under sunlight and have the spots image captured and processed using charge coupled devices and certain gray level algorithm. Thus the important information of focal spots such as spot size and location will be exported. Motion control module based on grating scale signal and interval measurement method are also employed in this system in order to get test results with high speed and high precision on large area lens array no less than 1m×0.8m. The repeatability of the system prototype measurement is +/-10μm with a velocity of 90 spot/min. Compared to the original module assembled using coordinates from optical design, modules assembled using data exported from the prototype is 18% higher in output power, reaching a conversion efficiency of over 31%. This system and its design can be used in the focal spot measurement of planoconvex lens array and Fresnel lens array, as well as other kinds of large area lens array application with small focal spots.

TiO2 nanoparticles disrupt cell adhesion and the architecture of cytoskeletal networks of human osteoblast-like cells in a size dependent manner.

PubMed

Ibrahim, Mohamed; Schoelermann, Julia; Mustafa, Kamal; Cimpan, Mihaela R

2018-04-30

Human exposure to titanium dioxide nanoparticles (nano-TiO 2 ) is increasing. An internal source of nano-TiO 2 is represented by titanium-based orthopedic and dental implants can release nanoparticles (NPs) upon abrasion. Little is known about how the size of NPs influences their interaction with cytoskeletal protein networks and the functional/homeostatic consequences that might follow at the implant-bone interface with regard to osteoblasts. We investigated the effects of size of anatase nano-TiO 2 on SaOS-2 human osteoblast-like cells exposed to clinically relevant concentrations (0.05, 0.5, 5 mg/L) of 5 and 40 nm spherical nano-TiO 2 . Cell viability and proliferation, adhesion, spread and migration were assessed, as well as the orientation of actin and microtubule cytoskeletal networks. The phosphorylation of focal adhesion kinase (p-FAK Y397 ) and the expression of vinculin in response to nano-TiO 2 were also assessed. Treatment with nano-TiO 2 disrupted the actin and microtubule cytoskeletal networks leading to morphological modifications of SaOS-2 cells. The phosphorylation of p-FAK Y397 and the expression of vinculin were also modified depending on the particle size, which affected cell adhesion. Consequently, the cell migration was significantly impaired in the 5 nm-exposed cells compared to unexposed cells. The present work shows that the orientation of cytoskeletal networks and the focal adhesion proteins and subsequently the adhesion, spread and migration of SaOS-2 cells were affected by the selected nano-TiO 2 in a size dependent manner. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Suppressor of cytokine signalling (SOCS) 1 and 3 enhance cell adhesion and inhibit migration towards the chemokine eotaxin/CCL11.

PubMed

Stevenson, Nigel J; McFarlane, Cheryl; Ong, Seow Theng; Nahlik, Krystyna; Kelvin, Alyson; Addley, Mark R; Long, Aideen; Greaves, David R; O'Farrelly, Cliona; Johnston, James A

2010-11-05

Suppressors of cytokine signalling (SOCS) proteins regulate signal transduction, but their role in responses to chemokines remains poorly understood. We report that cells expressing SOCS1 and 3 exhibit enhanced adhesion and reduced migration towards the chemokine CCL11. Focal adhesion kinase (FAK) and the GTPase RhoA, control cell adhesion and migration and we show the presence of SOCS1 or 3 regulates expression and tyrosine phosphorylation of FAK, while also enhancing activation of RhoA. Our novel findings suggest that SOCS1 and 3 may control chemotaxis and adhesion by significantly enhancing both FAK and RhoA activity, thus localizing immune cells to the site of allergic inflammation. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Human Papillomavirus E6 Oncogene Represses a Cell Adhesion Pathway and Disrupts Focal Adhesion through Degradation of TAp63β upon Transformation

PubMed Central

Ben Khalifa, Youcef; Teissier, Sébastien; Tan, Meng-Kwang Marcus; Phan, Quang Tien; Daynac, Mathieu; Wong, Wei Qi; Thierry, Françoise

2011-01-01

Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical pathway in human carcinogenesis. PMID:21980285

A new organofunctional ethoxysilane self-assembly monolayer for promoting adhesion of rubber to aluminum.

PubMed

Wang, Fang; Xu, Juan; Luo, Heyi; Wang, Jinggang; Wang, Qian

2009-10-12

Practical adhesion of rubber to aluminum is measured for various aluminum silanization treatments. In this study, 6-(3-triethoxysilylpropylamino)-1,3,5-triazine-2,4-dithiol (TES) was used as the coupling agent for preparing self-assembly monolayers (SAMs) on an aluminum surface. The structure and chemical composition of the SAMs were analyzed using Fourier transform infra-red spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The changes in the surface features of the aluminum surface due to TES treatment were investigated by atomic force microscopy (AFM). The adhesive properties of the silanized aluminum surface and EPDM rubber have been evaluated by a T-peel strength test. The results suggested that the Si-O-Al bonding at aluminum TES interface existed and a TES self-assembly monolayer was formed on the aluminum surface. More than 6.0 KN/m adhesion strength is obtained when the aluminum is silanized with 2.5 mmol/dm(3) TES, cured at 160 degrees C and vulcanized with EPDM rubber at 160 degrees C for 30 min. It is suggested that the TES self-assembly monolayer is bound to aluminum through its ethoxysilyl functional group, and the thiol function group is strongly crosslinked to EPDM rubber, respectively.

Magnesium modification of a calcium phosphate cement alters bone marrow stromal cell behavior via an integrin-mediated mechanism.

PubMed

Zhang, Jing; Ma, Xiaoyu; Lin, Dan; Shi, Hengsong; Yuan, Yuan; Tang, Wei; Zhou, Huanjun; Guo, Han; Qian, Jiangchao; Liu, Changsheng

2015-06-01

The chemical composition, structure and surface characteristics of biomaterials/scaffold can affect the adsorption of proteins, and this in turn influences the subsequent cellular response and tissue regeneration. With magnesium/calcium phosphate cements (MCPC) as model, the effects of magnesium (Mg) on the initial adhesion and osteogenic differentiation of bone marrow stromal cells (BMSCs) as well as the underlying mechanism were investigated. A series of MCPCs with different magnesium phosphate cement (MPC) content (0∼20%) in calcium phosphate cement (CPC) were synthesized. MCPCs with moderate proportion of MPC (5% and 10%, referred to as 5MCPC and 10MCPC) were found to effectively modulate the orientation of the adsorbed fibronectin (Fn) to exhibit enhanced receptor binding affinity, and to up-regulate integrin α5β1 expression of BMSCs, especially for 5MCPC. As a result, the attachment, morphology, focal adhesion formation, actin filaments assembly and osteogenic differentiation of BMSCs on 5MCPC were strongly enhanced. Further in vivo experiments confirmed that 5MCPC induced promoted osteogenesis in comparison to ot her CPC/MCPCs. Our results also suggested that the Mg on the underlying substrates but not the dissolved Mg ions was the main contributor to the above positive effects. Based on these results, it can be inferred that the specific interaction of Fn and integrin α5β1 had predominant effect on the MCPC-induced enhanced cellular response of BMSCs. These results provide a new strategy to regulate BMSCs adhesion and osteogenic differentiation by adjusting the Mg/Ca content and distribution in CPC, guiding the development of osteoinductive scaffolds for bone tissue regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Flexible organic TFT bio-signal amplifier using reliable chip component assembly process with conductive adhesive.

PubMed

Yoshimoto, Shusuke; Uemura, Takafumi; Akiyama, Mihoko; Ihara, Yoshihiro; Otake, Satoshi; Fujii, Tomoharu; Araki, Teppei; Sekitani, Tsuyoshi

2017-07-01

This paper presents a flexible organic thin-film transistor (OTFT) amplifier for bio-signal monitoring and presents the chip component assembly process. Using a conductive adhesive and a chip mounter, the chip components are mounted on a flexible film substrate, which has OTFT circuits. This study first investigates the assembly technique reliability for chip components on the flexible substrate. This study also specifically examines heart pulse wave monitoring conducted using the proposed flexible amplifier circuit and a flexible piezoelectric film. We connected the amplifier to a bluetooth device for a wearable device demonstration.

Thematic mapper flight model preshipment review data package. Volume 2, part B: Subsystem data

NASA Technical Reports Server (NTRS)

1982-01-01

Summarized performance data are presented for the following major subsystems of the thematic mapper: the focal plane assembly, the radiative cooler, the radiative cooler door assembly, the top optical assembly, and the telescope assembly. Reference lists of the configurations status and of nonconforming material reports, failure reports, and requests for deviation/waiver are included.

Genetic interactions between Drosophila melanogaster Atg1 and paxillin reveal a role for paxillin in autophagosome formation.

PubMed

Chen, Guang-Chao; Lee, Janice Y; Tang, Hong-Wen; Debnath, Jayanta; Thomas, Sheila M; Settleman, Jeffrey

2008-01-01

Autophagy is a conserved cellular process of macromolecule recycling that involves vesicle-mediated degradation of cytoplasmic components. Autophagy plays essential roles in normal cell homeostasis and development, the response to stresses such as nutrient starvation, and contributes to disease processes including cancer and neurodegeneration. Although many of the autophagy components identified from genetic screens in yeast are well conserved in higher organisms, the mechanisms by which this process is regulated in any species are just beginning to be elucidated. In a genetic screen in Drosophila melanogaster, we have identified a link between the focal adhesion protein paxillin and the Atg1 kinase, which has been previously implicated in autophagy. In mammalian cells, we find that paxillin is redistributed from focal adhesions during nutrient deprivation, and paxillin-deficient cells exhibit defects in autophagosome formation. Together, these findings reveal a novel evolutionarily conserved role for paxillin in autophagy.

A Phenomenlogical Model of Durotaxis

NASA Astrophysics Data System (ADS)

Yu, Guangyuan; Feng, Jingchen; Levine, Herbert; CenterTheoretical Biological Physics Collaboration

Cells exhibit qualitatively different behaviors on substrates with different rigidities. The fact that cells are more polarized on the stiffer substrate motivates us to construct a two-dimensional cell with the distribution of focal adhesions dependent on substrate rigidities. Our model reproduces the experimental observation that the persistence time is higher on the stiffer substrate. We show that stiffness dependent polarization will lead to the so-called durotaxis, the preference in moving towards stiffer substrates. This propensity is then characterized by the durotactic index first defined in experiments. We also derive and validate the 2D corresponding Fokker-Planck equation associated with our model. Our model highlights the role of focal adhesion arrangement in durotaxis. It may be applied to manipulate the movement of cells for clinical purposes. This work was supported by the National Science Foundation Center for Theoretical Biological Physics (Grant NSF PHY-1427654). HL was also supported by the CPRIT Scholar program of the State of Texas.

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

PubMed

Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

2017-09-01

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

78 FR 37785 - Foreign-Trade Zone (FTZ) 196-Fort Worth, Texas; Notification of Proposed Production Activity...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-24

... carrying cases, wrist straps, screws, power supplies, nickel/ cadmium batteries, lithium/ion batteries, other batteries, antenna assemblies, audio flex assemblies, bridge flex assemblies, interplex assembly... components and materials sourced from abroad include: labels, battery adhesives, decals, Kevlar protective...

Segmental jejunal entrapment, volvulus, and strangulation secondary to intra-abdominal adhesions in a dog.

PubMed

Di Cicco, Michael F; Bennett, R Avery; Ragetly, Chantal; Sippel, Kate M

2011-01-01

A 4 yr old, castrated male dachshund was presented for lethargy, restlessness, a "hunched" posture, and a painful abdomen. A gastric foreign body had been surgically removed 24 mo previously. Exploratory celiotomy revealed a devitalized segment of jejunum with twisted mesentery. Several adhesions and fibrous bands were present within the abdomen, presumptively from the previous gastric foreign body surgery. Histopathology determined that a fibrous tissue band caused entrapment of the segment of intestine and its mesentery resulting in volvulus and ischemic necrosis of the intestine. This case is unique because it involved a focal area of the jejunum that was incarcerated in fibrous adhesions.

Using RNA interference to knock down the adhesion protein TES.

PubMed

Griffith, Elen

2007-01-01

RNA interference (RNAi) is a specific and efficient method to knock down protein levels using small interfering RNAs (siRNAs), which target mRNA degradation. RNAi can be used in mammalian cell culture systems to target any protein of interest, and several studies have used this method to knock down adhesion proteins. We used siRNAs to knock down the levels of TES, a focal adhesion protein, in HeLa cells. We demonstrated knockdown of both TES mRNA and TES protein. Although total knockdown of TES was not achieved, the observed reduction in TES protein was sufficient to result in a cellular phenotype of reduced actin stress fibers.

Impacts of icodextrin on integrin-mediated wound healing of peritoneal mesothelial cells.

PubMed

Matsumoto, Mika; Tamura, Masahito; Miyamoto, Tetsu; Furuno, Yumi; Kabashima, Narutoshi; Serino, Ryota; Shibata, Tatsuya; Kanegae, Kaori; Takeuchi, Masaaki; Abe, Haruhiko; Okazaki, Masahiro; Otsuji, Yutaka

2012-06-14

Exposure to glucose and its metabolites in peritoneal dialysis fluid (PDF) results in structural alterations of the peritoneal membrane. Icodextrin-containing PDF eliminates glucose and reduces deterioration of peritoneal membrane function, but direct effects of icodextrin molecules on peritoneal mesothelial cells have yet to be elucidated. We compared the impacts of icodextrin itself with those of glucose under PDF-free conditions on wound healing processes of injured mesothelial cell monolayers, focusing on integrin-mediated cell adhesion mechanisms. Regeneration processes of the peritoneal mesothelial cell monolayer were investigated employing an in vitro wound healing assay of cultured rat peritoneal mesothelial cells treated with icodextrin powder- or glucose-dissolved culture medium without PDF, as well as icodextrin- or glucose-containing PDF. The effects of icodextrin on integrin-mediated cell adhesions were examined by immunocytochemistry and Western blotting against focal adhesion kinase (FAK). Cell migration over fibronectin was inhibited in conventional glucose-containing PDF, while icodextrin-containing PDF exerted no significant inhibitory effects. Culture medium containing 1.5% glucose without PDF also inhibited wound healing of mesothelial cells, while 7.5% icodextrin-dissolved culture medium without PDF had no inhibitory effects. Glucose suppressed cell motility by inhibiting tyrosine phosphorylation of FAK, formation of focal adhesions, and cell spreading, while icodextrin had no effects on any of these mesothelial cell functions. Our results demonstrate icodextrin to have no adverse effects on wound healing processes of peritoneal mesothelial cells. Preservation of integrin-mediated cell adhesion might be one of the molecular mechanisms accounting for the superior biocompatibility of icodextrin-containing PDF. Copyright © 2012 Elsevier Inc. All rights reserved.

Suspension state increases reattachment of breast cancer cells by up-regulating lamin A/C.

PubMed

Zhang, Xiaomei; Lv, Yonggang

2017-12-01

Extravasation is a rate-limiting step of tumor metastasis, for which adhesion to endothelium of circulating tumor cells (CTCs) is the prerequisite. The suspension state of CTCs undergoing detachment from primary tumor is a persistent biomechanical cue, which potentially regulates the biophysical characteristics and cellular behaviors of tumor cells. In this study, breast tumor cells MDA-MB-231 in suspension culture condition were used to investigate the effect of suspension state on reattachment of CTCs. Our study demonstrated that suspension state significantly increased the adhesion ability of breast tumor cells. In addition, suspension state markedly promoted the formation of stress fibers and focal adhesions and reduced the motility in reattached breast cancer cells. Moreover, lamin A/C was reversibly accumulated at posttranscriptional level under suspension state, improving the cell stiffness of reattached breast cancer cells. Disruption of actin cytoskeleton by cytochalasin D caused lamin A/C accumulation. Conversely, decreasing actomyosin contraction by ROCK inhibitor Y27632 reduced lamin A/C level. Knocking down lamin A/C weakened the suspension-induced increase of adhesion, and also abolished the suspension-induced decrease of motility and increase of stress fibers and focal adhesion in reattaching tumor cells, suggesting a crucial role of lamin A/C. In conclusion, it was demonstrated that suspension state promoted the reattachment of breast tumor cells by up-regulating lamin A/C via cytoskeleton disruption. These findings highlight the important role of suspension state for tumor cells in tumor metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Single molecule force spectroscopy reveals the adhesion mechanism of hydrophobins

NASA Astrophysics Data System (ADS)

Cao, Yi; Li, Bing; Qin, Meng; Wang, Wei

Hydrophobins are a special class of amphiphilic proteins produced by filamentous fungi. They show outstanding interfacial self-assembly and adhesion properties, which are critical to their biological function. Such feature also inspires their broad applications in bio-engineering, surface modification, and nanotechnology. However, the biophysical properties of hydrophobins are not well understood. We combined atomic force microscopy based single molecule force spectroscopy and protein engineering to directly quantify the adhesion strength of a hydorphobin (HFB1) to various surfaces in both the monomer and oligomer states to reveal the molecular determinant of the adhesion strength of hydrophobins. We found that the monomer HFB1 showed distinct adhesion properties towards hydrophobic and hydrophilic surfaces. The adhesion to hydrophobic surfaces (i.e. graphite and gold) was significantly higher than that to the hydrophilic ones (e.g. mica and silicon). However, when self-assembled monolayers were formed, the adhesion strengths to various surfaces were similar and were ubiquitously stronger than the monomer cases. We hypothesized that the interactions among hydrophobins in the monolayer played significant roles for the enhance adhesion strengths. Extracting any single hydrophobin monomers from the surface required the break of interactions not only with the surface but also with the neighboring units. We proposed that such a mechanism may be widely explored in nature for many biofilms for surface adhesion. May also inspire the design of novel adhesives.

Downregulation of SS18-SSX1 expression in synovial sarcoma by small interfering RNA enhances the focal adhesion pathway and inhibits anchorage-independent growth in vitro and tumor growth in vivo.

PubMed

Takenaka, Satoshi; Naka, Norifumi; Araki, Nobuhito; Hashimoto, Nobuyuki; Ueda, Takafumi; Yoshioka, Kiyoko; Yoshikawa, Hideki; Itoh, Kazuyuki

2010-04-01

Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by a unique t(X;18) translocation resulting in expression of SS18-SSX fusion protein. In order to investigate the biological function of this fusion protein and to develop a novel therapeutic option, we examined downregulation of SS18-SSX1 expression by small interfering RNA targeting SS18-SSX1 in three human SS cell lines. Microarray analysis comparing SS18-SSX1-silenced cells with control cells in three SS cell lines showed that SS18-SSX1 mainly affected the focal adhesion pathway. In accord with the array data, silencing of SS18-SSX1 enhances adhesion to the extracellular matrix through the induction of expression of myosin light-chain kinase. Furthermore, the silencing of SS18-SSX1 inhibits anchorage-independent growth in vitro and systemic delivery of siRNA against SS18-SSX1 using a nanoparticle system inhibited tumor growth in a nude mouse xenograft model. Our results demonstrate that siRNA targeting of SS18-SSX1 has therapeutic potential for the treatment of SS.

Characterisation of chicken TES and its role in cell spreading and motility.

PubMed

Griffith, Elen; Coutts, Amanda S; Black, Donald M

2004-03-01

Previously we identified TES as a candidate tumour suppressor gene that is located at human chromosome 7q31.1. More recently, we and others have shown TES to encode a novel LIM domain protein that localises to focal adhesions. Here, we present the cloning and functional analysis of the chicken orthologue of TES, cTES. The TES proteins are highly conserved between chicken and human, showing 89% identity at the amino acid level. We show that the cTES protein localised at focal adhesions, actin stress fibres, and sites of cell-cell contact, and GST-cTES can pull-down zyxin and actin. To investigate a functional role for cTES, we looked at the effect of its overexpression on cell spreading and cell motility. Cells overexpressing cTES showed increased cell spreading on fibronectin, and decreased cell motility, compared to RCAS vector transfected control cells. The data from our studies with cTES support our previous findings with human TES and further implicate TES as a member of a complex of proteins that function together to regulate cell adhesion and additionally demonstrate a role for TES in cell motility. Copyright 2004 Wiley-Liss, Inc.

Kindler syndrome: a focal adhesion genodermatosis.

PubMed

Lai-Cheong, J E; Tanaka, A; Hawche, G; Emanuel, P; Maari, C; Taskesen, M; Akdeniz, S; Liu, L; McGrath, J A

2009-02-01

Kindler syndrome (OMIM 173650) is an autosomal recessive genodermatosis characterized by trauma-induced blistering, poikiloderma, skin atrophy, mucosal inflammation and varying degrees of photosensitivity. Although Kindler syndrome is classified as a subtype of epidermolysis bullosa, it has distinct clinicopathological and molecular abnormalities. The molecular pathology of Kindler syndrome involves loss-of-function mutations in a newly recognized actin cytoskeleton-associated protein, now known as fermitin family homologue 1, encoded by the gene FERMT1. This protein mediates anchorage between the actin cytoskeleton and the extracellular matrix via focal adhesions, and thus the structural pathology differs from other forms of epidermolysis bullosa in which there is a disruption of the keratin intermediate filament-hemidesmosome network and the extracellular matrix. In the skin, fermitin family homologue 1 is mainly expressed in basal keratinocytes and binds to the cytoplasmic tails of beta1 and beta3 integrins as well as to fermitin family homologue 2 and filamin-binding LIM protein 1. It also plays a crucial role in keratinocyte migration, proliferation and adhesion. In this report, we review the clinical, cellular and molecular pathology of Kindler syndrome and discuss the role of fermitin family homologue 1 in keratinocyte biology.

Automated segmentation and tracking for large-scale analysis of focal adhesion dynamics.

PubMed

Würflinger, T; Gamper, I; Aach, T; Sechi, A S

2011-01-01

Cell adhesion, a process mediated by the formation of discrete structures known as focal adhesions (FAs), is pivotal to many biological events including cell motility. Much is known about the molecular composition of FAs, although our knowledge of the spatio-temporal recruitment and the relative occupancy of the individual components present in the FAs is still incomplete. To fill this gap, an essential prerequisite is a highly reliable procedure for the recognition, segmentation and tracking of FAs. Although manual segmentation and tracking may provide some advantages when done by an expert, its performance is usually hampered by subjective judgement and the long time required in analysing large data sets. Here, we developed a model-based segmentation and tracking algorithm that overcomes these problems. In addition, we developed a dedicated computational approach to correct segmentation errors that may arise from the analysis of poorly defined FAs. Thus, by achieving accurate and consistent FA segmentation and tracking, our work establishes the basis for a comprehensive analysis of FA dynamics under various experimental regimes and the future development of mathematical models that simulate FA behaviour. © 2010 The Authors Journal of Microscopy © 2010 The Royal Microscopical Society.

Quantification of focal adhesion dynamics of cell movement based on cell-induced collagen matrix deformation using second-harmonic generation microscopy.

PubMed

Kang, Yong Guk; Jang, Hwanseok; Yang, Taeseok Daniel; Notbohm, Jacob; Choi, Youngwoon; Park, Yongdoo; Kim, Beop-Min

2018-06-01

Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell-extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell-ECM interaction during angiogenesis, wound healing, and metastasis. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Topography on a subcellular scale modulates cellular adhesions and actin stress fiber dynamics in tumor associated fibroblasts

NASA Astrophysics Data System (ADS)

Azatov, Mikheil; Sun, Xiaoyu; Suberi, Alexandra; Fourkas, John T.; Upadhyaya, Arpita

2017-12-01

Cells can sense and adapt to mechanical properties of their environment. The local geometry of the extracellular matrix, such as its topography, has been shown to modulate cell morphology, migration, and proliferation. Here we investigate the effect of micro/nanotopography on the morphology and cytoskeletal dynamics of human pancreatic tumor-associated fibroblast cells (TAFs). We use arrays of parallel nanoridges with variable spacings on a subcellular scale to investigate the response of TAFs to the topography of their environment. We find that cell shape and stress fiber organization both align along the direction of the nanoridges. Our analysis reveals a strong bimodal relationship between the degree of alignment and the spacing of the nanoridges. Furthermore, focal adhesions align along ridges and form preferentially on top of the ridges. Tracking actin stress fiber movement reveals enhanced dynamics of stress fibers on topographically patterned surfaces. We find that components of the actin cytoskeleton move preferentially along the ridges with a significantly higher velocity along the ridges than on a flat surface. Our results suggest that a complex interplay between the actin cytoskeleton and focal adhesions coordinates the cellular response to micro/nanotopography.

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.

The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells.

PubMed

Itou, Junji; Tanaka, Sunao; Li, Wenzhao; Iida, Atsuo; Sehara-Fujisawa, Atsuko; Sato, Fumiaki; Toi, Masakazu

2017-01-01

During metastasis, cancer cell migration is enhanced. However, the mechanisms underlying this process remain elusive. Here, we addressed this issue by functionally analyzing the transcription factor Sal-like 4 (SALL4) in basal-like breast cancer cells. Loss-of-function studies of SALL4 showed that this transcription factor is required for the spindle-shaped morphology and the enhanced migration of cancer cells. SALL4 also up-regulated integrin gene expression. The impaired cell migration observed in SALL4 knockdown cells was restored by overexpression of integrin α6 and β1. In addition, we clarified that integrin α6 and β1 formed a heterodimer. At the molecular level, loss of the SALL4 - integrin α6β1 network lost focal adhesion dynamics, which impairs cell migration. Over-activation of Rho is known to inhibit focal adhesion dynamics. We observed that SALL4 knockdown cells exhibited over-activation of Rho. Aberrant Rho activation was suppressed by integrin α6β1 expression, and pharmacological inhibition of Rho activity restored cell migration in SALL4 knockdown cells. These results indicated that the SALL4 - integrin α6β1 network promotes cell migration via modulation of Rho activity. Moreover, our zebrafish metastasis assays demonstrated that this gene network enhances cell migration in vivo. Our findings identify a potential new therapeutic target for the prevention of metastasis, and provide an improved understanding of cancer cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2012-01-01

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

Assembly processes comparison for a miniaturized laser used for the Exomars European Space Agency mission

NASA Astrophysics Data System (ADS)

Ribes-Pleguezuelo, Pol; Inza, Andoni Moral; Basset, Marta Gilaberte; Rodríguez, Pablo; Rodríguez, Gemma; Laudisio, Marco; Galan, Miguel; Hornaff, Marcel; Beckert, Erik; Eberhardt, Ramona; Tünnermann, Andreas

2016-11-01

A miniaturized diode-pumped solid-state laser (DPSSL) designed as part of the Raman laser spectrometer (RLS) instrument for the European Space Agency (ESA) Exomars mission 2020 is assembled and tested for the mission purpose and requirements. Two different processes were tried for the laser assembling: one based on adhesives, following traditional laser manufacturing processes; another based on a low-stress and organic-free soldering technique called solderjet bumping technology. The manufactured devices were tested for the processes validation by passing mechanical, thermal cycles, radiation, and optical functional tests. The comparison analysis showed a device improvement in terms of reliability of the optical performances from the soldered to the assembled by adhesive-based means.

Design of Decorated Self-Assembling Peptide Hydrogels as Architecture for Mesenchymal Stem Cells

PubMed Central

Zamuner, Annj; Cavo, Marta; Scaglione, Silvia; Messina, Grazia Maria Lucia; Russo, Teresa; Gloria, Antonio; Marletta, Giovanni; Dettin, Monica

2016-01-01

Hydrogels from self-assembling ionic complementary peptides have been receiving a lot of interest from the scientific community as mimetic of the extracellular matrix that can offer three-dimensional supports for cell growth or can become vehicles for the delivery of stem cells, drugs or bioactive proteins. In order to develop a 3D “architecture” for mesenchymal stem cells, we propose the introduction in the hydrogel of conjugates obtained by chemoselective ligation between a ionic-complementary self-assembling peptide (called EAK) and three different bioactive molecules: an adhesive sequence with 4 Glycine-Arginine-Glycine-Aspartic Acid-Serine-Proline (GRGDSP) motifs per chain, an adhesive peptide mapped on h-Vitronectin and the growth factor Insulin-like Growth Factor-1 (IGF-1). The mesenchymal stem cell adhesion assays showed a significant increase in adhesion and proliferation for the hydrogels decorated with each of the synthesized conjugates; moreover, such functionalized 3D hydrogels support cell spreading and elongation, validating the use of this class of self-assembly peptides-based material as very promising 3D model scaffolds for cell cultures, at variance of the less realistic 2D ones. Furthermore, small amplitude oscillatory shear tests showed that the presence of IGF-1-conjugate did not alter significantly the viscoelastic properties of the hydrogels even though differences were observed in the nanoscale structure of the scaffolds obtained by changing their composition, ranging from long, well-defined fibers for conjugates with adhesion sequences to the compact and dense film for the IGF-1-conjugate. PMID:28773852

Friction-Controlled Traction Force in Cell Adhesion

PubMed Central

Pompe, Tilo; Kaufmann, Martin; Kasimir, Maria; Johne, Stephanie; Glorius, Stefan; Renner, Lars; Bobeth, Manfred; Pompe, Wolfgang; Werner, Carsten

2011-01-01

The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo. PMID:22004739

Surface nanoporosity has a greater influence on osteogenic and bacterial cell adhesion than crystallinity and wettability

NASA Astrophysics Data System (ADS)

Rodriguez-Contreras, Alejandra; Guadarrama Bello, Dainelys; Nanci, Antonio

2018-07-01

There has been much emphasis on the influence of crystallinity and wettability for modulating cell activity, particularly for bone biomaterials. In this context, we have generated titanium oxide layers with similar mesoporous topography and surface roughness but with amorphous or crystalline oxide layers and differential wettability. We then investigated their influence on the behavior of MC3T3 osteoblastic and bacterial cells. There was no difference in cell adhesion, spreading and growth on amorphous and crystalline surfaces. The number of focal adhesions was similar, however, cells on the amorphous surface exhibited a higher frequency of mature adhesions. The crystallinity of the surface layers also had no bearing on bacterial adhesion. While it cannot be excluded that surface crystallinity, roughness and wettability contribute to some degree to determining cell behavior, our data suggest that physical characteristics of surfaces represent the major determinant.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 frommore » 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.« less

Assembly of tissue engineered blood vessels with spatially-controlled heterogeneities.

PubMed

Strobel, Hannah A; Hookway, Tracy; Piola, Marco; Fiore, Gianfranco Beniamino; Soncini, Monica; Alsberg, Eben; Rolle, Marsha

2018-05-04

Tissue-engineered human blood vessels may enable in vitro disease modeling and drug screening to accelerate advances in vascular medicine. Existing methods for tissue engineered blood vessel (TEBV) fabrication create homogenous tubes not conducive to modeling the focal pathologies characteristic of vascular disease. We developed a system for generating self-assembled human smooth muscle cell ring-units, which were fused together into TEBVs. The goal of this study was to assess the feasibility of modular assembly and fusion of ring building units to fabricate spatially-controlled, heterogeneous tissue tubes. We first aimed to enhance fusion and reduce total culture time, and determined that reducing ring pre-culture duration improved tube fusion. Next, we incorporated electrospun polymer ring units onto tube ends as reinforced extensions, which allowed us to cannulate tubes after only 7 days of fusion, and culture tubes with luminal flow in a custom bioreactor. To create focal heterogeneities, we incorporated gelatin microspheres into select ring units during self-assembly, and fused these rings between ring units without microspheres. Cells within rings maintained their spatial position within tissue tubes after fusion. This work describes a platform approach for creating modular TEBVs with spatially-defined structural heterogeneities, which may ultimately be applied to mimic focal diseases such as intimal hyperplasia or aneurysm.

The use of biomaterials for cell function enhancement: acceleration of fibroblast migration and promotion of stem cell proliferation

NASA Astrophysics Data System (ADS)

Qin, Sisi

Wound healing and tissue regeneration proceed via fibroblast migration along three dimensional scaffolds composed of fibers with different diameters, spacing, and junction angles. In order to understand how each of these factors influences fibroblast migration, a technique for preparation of three dimensional fibrillar scaffolds was developed where the fiber diameters and the angles between adjacent fiber layers could be precisely controlled. In order to study the en-mass migration process, the agarose droplet method was chosen since it enabled accurate determinations of the dependence of the migration speed, focal adhesion distribution, and nuclear deformation on the fiber structures. Results showed that on oriented single fiber layers, if the fiber diameters exceeded 1microm, large focal adhesion complexes formed in a linear arrangement along the fiber axis and cell motion was highly correlated. For fibers 1microm or less, some cell alignment along the fiber direction was measured, but no correlation between the distribution of focal adhesion points and fiber orientation was found. On multi layered scaffolds the focal adhesion sites were found to concentrate at the junction points and the migration speed followed a parabolic function with a distinct minimum at 35°. When compared to fibroblasts plated on 90° fibers, fibroblasts plated on 30° fibers showed a decrease of 25% in the degree of nuclear deformation and an increase of 25% in the number of focal adhesion sites, indicating that cell migration speed was correlated to the angle and distance of approach to the junction point. The time dependence of the migration velocity on oriented fibers was measured for four days and compared to the value measured on flat surfaces. After the initial 24 hour incubation period, the cells on both the 8microm fibers and flat surfaces migrated with a similar speed. During the next three days the migration speed for the cells on the fibrillar surfaces doubled in magnitude, while remained constant for the cells on the flat surfaces. The increased speed on the 8microm fiber surfaces could be correlated with a 20% increase in the nuclear deformation, and a decrease around 30% in the number of focal adhesion during the same observation period. RNA expression of Myosin IIA, a protein which complexes to the actin and is responsible for exertion of traction forces during migration was not upregulated during this process. On the other hand, histochemical staining of Myosin IIA showed that the protein had re-organized into large fibers which spanned the length of the cells. Observation of the cell morphology indicated that a new mode of motion had been established. Rather than the classical retraction of the cytoplasm followed by center of mass translation, which was observed on the flat surfaces, the cells were now moving by a contractile motion around the nucleus similar to that found in muscular motion. This mode was found to be more efficient when undergoing oriented motion. In addition to orientation, surface mechanics are also important in the tissue regeneration process. This study demonstrated that mechanical factors are important for the maintenance of pluripotency and control of proliferation rates. CD34+ hematopoietic stem cells (HSCs) were transduced with ICD (intracellular domain)-Notch and plated on gelatin hydrogels, whose moduli were controlled by the crosslinking ratio. On the softer hydrogel, a synergy was achieved which resulted in more than a five-fold increase in proliferation and a four-fold increase in the preservation of stemness, while HSCs maintained their ability to differentiate into multiple blood cell lineages. These results point the way for achieving clinically significant expansion of human HSCs.

Development of a qualification standard for adhesives used in hybrid microcircuits

NASA Technical Reports Server (NTRS)

Licari, J. J.; Weigand, B. L.; Soykin, C. A.

1981-01-01

Improved qualification standards and test procedures for adhesives used in microelectronic packaging are developed. The test methods in specification for the Selection and Use of Organic Adhesives in Hybrid Microcircuits are reevaluated versus industry and government requirements. Four electrically insulative and four electrically conductive adhesives used in the assembly of hybrid microcircuits are selected to evaluate the proposed revised test methods. An estimate of the cost to perform qualification testing of an adhesive to the requirements of the revised specification is also prepared.

Design guidelines for use of adhesives in hybrid microcircuits. [for electronic equipment in space applications

NASA Technical Reports Server (NTRS)

Caruso, S. V.; Perkins, K. L.; Licari, J. J.

1973-01-01

Although it is generally accepted that the use of adhesives in the assembly of hybrid microcircuits offers advantages over other bonding methods, there currently does not exist a set of guidelines for the selection of adhesives which will insure that hybrid microcircuits assembled with them will meet the long use-life, high-reliability requirements of electronic equipment for space applications. This study was directed to the identification of the properties of electrically insulative adhesives that potentially could cause problems in such an application, and to the development of evaluation tests to quantify these properties and thus form the basis for establishing suitable guidelines and, ultimately, specifications. Bond strength, outgassing after cure, and corrosivity were selected for detailed attention since they are considered to be especially critical. Introductory discussion includes enumeration and brief comments on the properties of adhesives considered to be important for the proposed application, a general review of polymeric types of adhesives, and identification of the major types of adhesives commercially available and specifically designed for microelectronic use. The specific tests developed to evaluate bond strength, outgassing after cure, and corrosivity are discussed in detail, and comparative results obtained for selected adhesives representative of the major types are given.

Adhesive Dimerization of Human P-Cadherin Catalyzed by a Chaperone-like Mechanism.

PubMed

Kudo, Shota; Caaveiro, Jose M M; Tsumoto, Kouhei

2016-09-06

Orderly assembly of classical cadherins governs cell adhesion and tissue maintenance. A key event is the strand-swap dimerization of the extracellular ectodomains of two cadherin molecules from apposing cells. Here we have determined crystal structures of P-cadherin in six different conformational states to elaborate a motion picture of its adhesive dimerization at the atomic level. The snapshots revealed that cell-adhesive dimerization is facilitated by several intermediate states collectively termed X-dimer in analogy to other classical cadherins. Based on previous studies and on the combined structural, kinetic, thermodynamic, biochemical, and cellular data reported herein, we propose that the adhesive dimerization of human P-cadherin is achieved by a stepwise mechanism analogous to that of assembly chaperones. This mechanism, applicable to type I classical cadherins, confers high specificity and fast association rates. We expect these findings to guide innovative therapeutic approaches targeting P-cadherin in cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Corrosion resistance and adhesion strength of a spin-assisted layer-by-layer assembled coating on AZ31 magnesium alloy

NASA Astrophysics Data System (ADS)

Zhao, Yan-Bin; Liu, Han-Peng; Li, Chang-Yang; Chen, Yong; Li, Shuo-Qi; Zeng, Rong-Chang; Wang, Zhen-Lin

2018-03-01

A polyvinylpyrrolidone (PVP)/polyacrylic acid (PAA) layer-by-layer (LbL) assembled composite coating with a multilayer structure for the corrosion protection of AZ31 magnesium alloy was prepared by a novel spin-casting method. The microstructure and composition of this coating were investigated by means of SEM, XRD and FT-IR measurements. Moreover, electrochemical, immersion and scratch tests in vitro were performed to measure the corrosion performance and the adhesion strength. These results indicated that the (PVP/PAA)10 composite coating with defect-free, dense and uniform morphologies could be successfully deposited on the surface of magnesium alloy. The coating had excellent corrosion resistance and adhesion strength.

Design of a nanocomposite substrate inducing adult stem cell assembly and progression toward an Epiblast-like or Primitive Endoderm-like phenotype via mechanotransduction.

PubMed

Morena, Francesco; Armentano, Ilaria; Montanucci, Pia; Argentati, Chiara; Fortunati, Elena; Montesano, Simona; Bicchi, Ilaria; Pescara, Teresa; Pennoni, Ilaria; Mattioli, Samantha; Torre, Luigi; Latterini, Loredana; Emiliani, Carla; Basta, Giuseppe; Calafiore, Riccardo; Kenny, Josè Maria; Martino, Sabata

2017-11-01

This work shows that the active interaction between human umbilical cord matrix stem cells and Poly (l-lactide)acid (PLLA) and PLLA/Multi Walled Carbon Nanotubes (MWCNTs) nanocomposite films results in the stem cell assembly as a spheroid conformation and affects the stem cell fate transition. We demonstrated that spheroids directly respond to a tunable surface and the bulk properties (electric, dielectric and thermal) of plain and nanocomposite PLLA films by triggering a mechanotransduction axis. This stepwise process starts from tethering of the cells' focal adhesion proteins to the surface, together with the adherens junctions between cells. Both complexes transmit traction forces to F-Actin stress fibres that link Filamin-A and Myosin-IIA proteins, generating a biological scaffold, with increased stiffening conformation from PLLA to PLLA/MWCNTs, and enable the nucleoskeleton proteins to boost chromatin reprogramming processes. Herein, the opposite expression of NANOG and GATA6 transcription factors, together with other lineage specification related proteins, steer spheroids toward an Epiblast-like or Primitive Endoderm-like lineage commitment, depending on the absence or presence of 1 wt% MWCNTs, respectively. This work represents a pioneering effort to create a stem cell/material interface that can model the stem cell fate transition under growth culture conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Kindler syndrome. A new bullous dermatosis].

PubMed

Has, C

2009-08-01

The Kindler syndrome is a new form of inherited epidermolysis bullosa and the first genodermatosis caused by a defect of the focal adhesions. Kindlin-1, the deficient protein, plays an essential role in integrin activation and in the adhesion of keratinocytes to the extracellular matrix. The adhesion defect leads to skin blistering which begins at birth and ameliorates with age, and to mucosal fragility which leads to scarring and stricture formation. Skin atrophy and poikiloderma develop progressively. Photosensitivity is rather mild, but squamous cell carcinomas develop on sun-exposed areas mainly after the age of 40 years. The most important differential diagnoses are epidermolysis bullosa with mottled pigmentation and dystrophic epidermolysis bullosa. Management aims to treat the symptoms and prevent complications.

Protein adsorption and cell adhesion controlled by the surface chemistry of binary perfluoroalkyl/oligo(ethylene glycol) self-assembled monolayers.

PubMed

Li, Shanshan; Yang, Dingyun; Tu, Haiyang; Deng, Hongtao; Du, Dan; Zhang, Aidong

2013-07-15

This work reports a study of protein adsorption and cell adhesion on binary self-assembled monolayers (SAMs) of alkanethiols with terminal perfluoroalkyl (PFA) and oligo(ethylene glycol) (OEG) chains in varying ratios. The surface chemistry of the SAMs was characterized by contact angle measurement, grazing angle infrared spectroscopy (GIR), X-ray photoelectron spectroscopy, and the effect on protein adsorption was investigated by surface plasmon resonance, GIR, and immunosorbent assay. Hela cell adhesion on these surfaces was also studied by fluorescence microscopy. Results reveal that, compared to OEG, PFA tended to be a higher fraction of the composition in SAM than in the assembly solution. More interestingly, the nearly 38% PFA SAM had a strong antifouling property whereas the 74% PFA SAM showed a high adsorption capacity to protein and cell. The binary PFA/OEG SAMs were favorable for maintaining the fibrinogen conformation, hence its high activity. The findings may have important implications for constructing PFA-containing surfaces with the distinct properties that is highly resistant or highly favorable toward protein adsorption and cell adhesion. Copyright © 2013 Elsevier Inc. All rights reserved.

Membrane-Mediated Cooperativity of Proteins

NASA Astrophysics Data System (ADS)

Weikl, Thomas R.

2018-04-01

Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.

Microstructured elastomeric surfaces with reversible adhesion and examples of their use in deterministic assembly by transfer printing

PubMed Central

Kim, Seok; Wu, Jian; Carlson, Andrew; Jin, Sung Hun; Kovalsky, Anton; Glass, Paul; Liu, Zhuangjian; Ahmed, Numair; Elgan, Steven L.; Chen, Weiqiu; Ferreira, Placid M.; Sitti, Metin; Huang, Yonggang; Rogers, John A.

2010-01-01

Reversible control of adhesion is an important feature of many desired, existing, and potential systems, including climbing robots, medical tapes, and stamps for transfer printing. We present experimental and theoretical studies of pressure modulated adhesion between flat, stiff objects and elastomeric surfaces with sharp features of surface relief in optimized geometries. Here, the strength of nonspecific adhesion can be switched by more than three orders of magnitude, from strong to weak, in a reversible fashion. Implementing these concepts in advanced stamps for transfer printing enables versatile modes for deterministic assembly of solid materials in micro/nanostructured forms. Demonstrations in printed two- and three-dimensional collections of silicon platelets and membranes illustrate some capabilities. An unusual type of transistor that incorporates a printed gate electrode, an air gap dielectric, and an aligned array of single walled carbon nanotubes provides a device example. PMID:20858729

PGE(2) inhibition of TGF-beta1-induced myofibroblast differentiation is Smad-independent but involves cell shape and adhesion-dependent signaling.

PubMed

Thomas, Peedikayil E; Peters-Golden, Marc; White, Eric S; Thannickal, Victor J; Moore, Bethany B

2007-08-01

Myofibroblasts are pathogenic in pulmonary fibrotic disease due to their exuberant production of matrix rich in collagen that interferes with gas exchange and the ability of these cells to contract and distort the alveolar space. Transforming growth factor-beta1 (TGF-beta1) is a well-known inducer of myofibroblast differentiation. TGF-beta1-induced transformation of fibroblasts to apoptosis-resistant myofibroblasts is adhesion-dependent and focal adhesion kinase (FAK)-mediated. Prostaglandin E(2) (PGE(2)) inhibits this differentiation via E prostanoid receptor 2 (EP2) signaling and cAMP elevation, but whether PGE(2) does so by interfering with TGF-beta1 signaling is unknown. Thus we examined the effects of PGE(2) in the presence and absence of TGF-beta1 stimulation on candidate signaling pathways in human lung fibroblasts. We now demonstrate that PGE(2) does not interfere with TGF-beta1-induced Smad phosphorylation or its translocation to the nucleus. Rather, PGE(2) has dramatic effects on cell shape and cytoskeletal architecture and disrupts the formation of appropriate focal adhesions. PGE(2) treatment diminishes TGF-beta1-induced phosphorylation of paxillin, STAT-3, and FAK and, in turn, limits activation of the protein kinase B (PKB/Akt) pathway. These alterations do not, however, result in increased apoptosis within the first 24 h of treatment. Interestingly, the effects of PGE(2) stimulation alone do not always mirror the effects of PGE(2) in the presence of TGF-beta1, indicating that the context for EP2 signaling is different in the presence of TGF-beta1. Taken together, our results demonstrate that PGE(2) has the potential to limit TGF-beta1-induced myofibroblast differentiation via adhesion-dependent, but Smad-independent, pathways.

Actin grips: circular actin-rich cytoskeletal structures that mediate the wrapping of polymeric microfibers by endothelial cells.

PubMed

Jones, Desiree; Park, DoYoung; Anghelina, Mirela; Pécot, Thierry; Machiraju, Raghu; Xue, Ruipeng; Lannutti, John J; Thomas, Jessica; Cole, Sara L; Moldovan, Leni; Moldovan, Nicanor I

2015-06-01

Interaction of endothelial-lineage cells with three-dimensional substrates was much less studied than that with flat culture surfaces. We investigated the in vitro attachment of both mature endothelial cells (ECs) and of less differentiated EC colony-forming cells to poly-ε-capro-lactone (PCL) fibers with diameters in 5-20 μm range ('scaffold microfibers', SMFs). We found that notwithstanding the poor intrinsic adhesiveness to PCL, both cell types completely wrapped the SMFs after long-term cultivation, thus attaining a cylindrical morphology. In this system, both EC types grew vigorously for more than a week and became increasingly more differentiated, as shown by multiplexed gene expression. Three-dimensional reconstructions from multiphoton confocal microscopy images using custom software showed that the filamentous (F) actin bundles took a conspicuous ring-like organization around the SMFs. Unlike the classical F-actin-containing stress fibers, these rings were not associated with either focal adhesions or intermediate filaments. We also demonstrated that plasma membrane boundaries adjacent to these circular cytoskeletal structures were tightly yet dynamically apposed to the SMFs, for which reason we suggest to call them 'actin grips'. In conclusion, we describe a particular form of F-actin assembly with relevance for cytoskeletal organization in response to biomaterials, for endothelial-specific cell behavior in vitro and in vivo, and for tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

Thematic mapper flight model preshipment review data package. Volume 4: Appendix. Part D: Focal plane assembly data

NASA Technical Reports Server (NTRS)

1982-01-01

The data obtained for the Band 1 thematic mapper flight full band assembly (P/N 50797) are summarized. The data were collected from half band, post amplifier, and full band acceptance test data records.

78 FR 60248 - Foreign-Trade Zone (FTZ) 183-Austin, Texas; Notification of Proposed Production Activity...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-10-01

...; input/output assemblies; electromagnetic interference support walls; bumpers; adhesives; sleeves; rubber... shroud assemblies; mechanism bases; storage; busbars; button dim links; electromagnetic interference fans...

Novel Mechanistic Link between Focal Adhesion Remodeling and Glucose-stimulated Insulin Secretion*

PubMed Central

Rondas, Dieter; Tomas, Alejandra; Soto-Ribeiro, Martinho; Wehrle-Haller, Bernhard; Halban, Philippe A.

2012-01-01

Actin cytoskeleton remodeling is well known to be positively involved in glucose-stimulated pancreatic β cell insulin secretion. We have observed glucose-stimulated focal adhesion remodeling at the β cell surface and have shown this to be crucial for glucose-stimulated insulin secretion. However, the mechanistic link between such remodeling and the insulin secretory machinery remained unknown and was the major aim of this study. MIN6B1 cells, a previously validated model of primary β cell function, were used for all experiments. Total internal reflection fluorescence microscopy revealed the glucose-responsive co-localization of focal adhesion kinase (FAK) and paxillin with integrin β1 at the basal cell surface after short term stimulation. In addition, blockade of the interaction between β1 integrins and the extracellular matrix with an anti-β1 integrin antibody (Ha2/5) inhibited short term glucose-induced phosphorylation of FAK (Tyr-397), paxillin (Tyr-118), and ERK1/2 (Thr-202/Tyr-204). Pharmacological inhibition of FAK activity blocked glucose-induced actin cytoskeleton remodeling and glucose-induced disruption of the F-actin/SNAP-25 association at the plasma membrane as well as the distribution of insulin granules to regions in close proximity to the plasma membrane. Furthermore, FAK inhibition also completely blocked short term glucose-induced activation of the Akt/AS160 signaling pathway. In conclusion, these results indicate 1) that glucose-induced activation of FAK, paxillin, and ERK1/2 is mediated by β1 integrin intracellular signaling, 2) a mechanism whereby FAK mediates glucose-induced actin cytoskeleton remodeling, hence allowing docking and fusion of insulin granules to the plasma membrane, and 3) a possible functional role for the Akt/AS160 signaling pathway in the FAK-mediated regulation of glucose-stimulated insulin secretion. PMID:22139838

Constrained Adherable Area of Nanotopographic Surfaces Promotes Cell Migration through the Regulation of Focal Adhesion via Focal Adhesion Kinase/Rac1 Activation.

PubMed

Lim, Jiwon; Choi, Andrew; Kim, Hyung Woo; Yoon, Hyungjun; Park, Sang Min; Tsai, Chia-Hung Dylan; Kaneko, Makoto; Kim, Dong Sung

2018-05-02

Cell migration is crucial in physiological and pathological processes such as embryonic development and wound healing; such migration is strongly guided by the surrounding nanostructured extracellular matrix. Previous studies have extensively studied the cell migration on anisotropic nanotopographic surfaces; however, only a few studies have reported cell migration on isotropic nanotopographic surfaces. We herein, for the first time, propose a novel concept of adherable area on cell migration using isotropic nanopore surfaces with sufficient nanopore depth by adopting a high aspect ratio. As the pore size of the nanopore surface was controlled to 200, 300, and 400 nm in a fixed center-to-center distance of 480 nm, it produced 86, 68, and 36% of adherable area, respectively, on the fabricated surface. A meticulous investigation of the cell migration in response to changes in the constrained adherable area of the nanotopographic surface showed 1.4-, 1.5-, and 1.6-fold increase in migration speeds and a 1.4-, 2-, and 2.5-fold decrease in the number of focal adhesions as the adherable area was decreased to 86, 68, and 36%, respectively. Furthermore, a strong activation of FAK/Rac1 signaling was observed to be involved in the promoted cell migration. These results suggest that the reduced adherable area promotes cell migration through decreasing the FA formation, which in turn upregulates FAK/Rac1 activation. The findings in this study can be utilized to control the cell migration behaviors, which is a powerful tool in the research fields involving cell migration such as promoting wound healing and tissue repair.

Inhibition of glycogen synthase kinase-3β prevents activation of focal adhesion kinase after ischemia/reperfusion of the rat lung.

PubMed

Waldow, Thomas; Witt, Wolfgang; Matschke, Klaus

2010-01-01

Recent studies on the mechanisms of ischemic preconditioning in myocardial tissue have presented convincing evidence that multiple protective pathways converge on inhibition of glycogen synthase kinase-3β (GSK-3β). To directly address the role of GSK-3β in ischemia and reperfusion (I/R) of the lung, a rat model of left lung in situ ischemia was used. The specific non-competitive inhibitor of GSK-3β, TDZD-8, was injected (3 mg/kg, vehicle in controls) 5 min before the left lung hilum was occluded for 60 min. Animals in the ischemia group underwent the same treatment, but without administration of TDZD-8. Lung functional and biochemical parameters were determined at time points 15 min and 60 min reperfusion. Treatment with TDZD-8 improved gas exchange (arterial pO2), but I/R-induced inflammation (plasma interleukin-6, leukocyte invasion) was not affected. The I/R cycle induced a rapid (15 min reperfusion) increase of protein tyrosine phosphorylation, including the activating phosphorylation of focal adhesion kinase at Tyr397, Tyr407, Tyr577, and Tyr861, and the non-receptor kinase Src at Tyr416. The phosphorylation was blocked by the GSK inhibitor. This effect may be related to the reduced plasma level of the strong effector of focal adhesion kinase, transforming growth factor-β1, in the TDZD group. The underlying mechanisms are elusive, but they deserve further investigation, especially in relation to the early increase of lung permeability in this rat model of I/R injury. In conclusion, the results suggest that inhibition of GSK-3β improves rat lung function during an I/R cycle, but only during the early reperfusion phase.

Early storage lesions in apheresis platelets are induced by the activation of the integrin αIIbβ₃ and focal adhesion signaling pathways.

PubMed

Thiele, Thomas; Iuga, Cristina; Janetzky, Susann; Schwertz, Hansjorg; Gesell Salazar, Manuela; Fürll, Birgit; Völker, Uwe; Greinacher, Andreas; Steil, Leif

2012-12-05

Production and storage of platelet concentrates (PC) induce protein changes in platelets leading to impaired platelet function. This study aimed to identify signaling pathways involved in the development of early platelet storage lesions in apheresis-PCs stored in plasma or additive solution (PAS). Apheresis-PCs from four donors were stored in plasma or in PAS at 22°C (n=4 each). Platelets were analyzed at day 0 (production day) and after 1, 6 and 9 days of storage. Platelet response to agonists (TRAP, collagen, ADP) and to hypotonic shock decreased, CD62P expression increased in both storage media over time. Using DIGE 1550 protein spots were monitored and compared to baseline values at day 0. Platelets in plasma displayed changes in 352 spots (166/day 1, 263/day 6 and 201/day 9); in PAS 325 spots changed (202/day 1, 221/day 6, 200/day 9). LC-ESI-MS/MS analysis of 405 platelet proteins revealed 32 proteins changed during storage in plasma (9/day 1, 15/day 6 and 26/day 9) and 28 in PAS (5/day 1, 20/day 6, 26/day 9). Ingenuity pathway analysis found integrin-αII(b)β(3) and focal adhesion signaling pathways involved in early alterations, being confirmed by Western blotting. Corresponding mRNAs in platelets were identified by next generation sequencing for 84 changed proteins. Integrin-αII(b)β(3) and focal adhesion signaling cause irreversible early storage lesions in apheresis platelets. This article is part of a Special Issue entitled: Integrated omics. Copyright © 2012 Elsevier B.V. All rights reserved.

Tumor Necrosis Factor-α (TNFα)-induced Ceramide Generation via Ceramide Synthases Regulates Loss of Focal Adhesion Kinase (FAK) and Programmed Cell Death.

PubMed

Hernández-Corbacho, María José; Canals, Daniel; Adada, Mohamad M; Liu, Mengling; Senkal, Can E; Yi, Jae Kyo; Mao, Cungui; Luberto, Chiara; Hannun, Yusuf A; Obeid, Lina M

2015-10-16

Ceramide synthases (CerS1-CerS6), which catalyze the N-acylation of the (dihydro)sphingosine backbone to produce (dihydro)ceramide in both the de novo and the salvage or recycling pathway of ceramide generation, have been implicated in the control of programmed cell death. However, the regulation of the de novo pathway compared with the salvage pathway is not fully understood. In the current study, we have found that late accumulation of multiple ceramide and dihydroceramide species in MCF-7 cells treated with TNFα occurred by up-regulation of both pathways of ceramide synthesis. Nevertheless, fumonisin B1 but not myriocin was able to protect from TNFα-induced cell death, suggesting that ceramide synthase activity is crucial for the progression of cell death and that the pool of ceramide involved derives from the salvage pathway rather than de novo biosynthesis. Furthermore, compared with control cells, TNFα-treated cells exhibited reduced focal adhesion kinase and subsequent plasma membrane permeabilization, which was blocked exclusively by fumonisin B1. In addition, exogenously added C6-ceramide mimicked the effects of TNFα that lead to cell death, which were inhibited by fumonisin B1. Knockdown of individual ceramide synthases identified CerS6 and its product C16-ceramide as the ceramide synthase isoform essential for the regulation of cell death. In summary, our data suggest a novel role for CerS6/C16-ceramide as an upstream effector of the loss of focal adhesion protein and plasma membrane permeabilization, via the activation of caspase-7, and identify the salvage pathway as the critical mechanism of ceramide generation that controls cell death. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cortactin as a Target for FAK in the Regulation of Focal Adhesion Dynamics

PubMed Central

Ghassemian, Majid; Schlaepfer, David D.

2012-01-01

Background Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear. Principal Findings Using FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin. Conclusions Our results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement. PMID:22952866

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

PubMed

Berk, B C; Corson, M A; Peterson, T E; Tseng, H

1995-12-01

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.

Mitomycin C retardation of corneal fibroblast migration via sustained dephosphorylation of paxillin at tyrosine 118.

PubMed

Chen, Tsan-Chi; Lai, Chien-Hsueh; Chang, Jie-Ling; Chang, Shu-Wen

2012-03-21

To investigate how mitomycin C (MMC) modulates corneal fibroblast migration and its molecular mechanisms in the wound healing process. After treatment with 0 and 0.2 mg · mL(-1) MMC for 5 minutes, effect of MMC on cell migration of human corneal fibroblasts (HCFs) was examined with a cell migration assay. Both focal adhesion kinase (FAK) and paxillin (PXN) expressions in HCFs were analyzed by semiquantitative real-time PCR, immunoblotting, and immunofluorescence confocal microscopy. Using gene silencing or gene overexpression with lentiviral-based pseudovirion infection, the phosphorylation level of FAK, PXN, and mutated PXNs at tyrosine sites 31 (Y31F-EGFP) and 118 (Y118F-EGFP) were verified in HCFs. MMC retarded HCF migration at 1 and 2 days posttreatment (dpt). MMC reduced levels of FAK transcript and FAK protein, but increased both transcript and protein expression of PXN at 1 and 2 dpt. Furthermore, MMC upregulated FAK-pY397, which subsequently enhanced PXN-pY31 in a dose-dependent manner at 1 dpt. Concurrently, MMC downregulated PXN-pY118 at 1 dpt. However, MMC treatment resulted in dephosphorylation of FAK-pY397, PXN-pY31, and PXN-pY118 at 2 dpt. The FAK/PXN complex in MMC-treated HCFs was detected at focal adhesion sites more than at the leading edge at 1 and 2 dpt, contributing to retardation of HCF migration. Y118F-EGFP-expressing HCFs exhibited lower mobility than that of PXN-EGFP- or Y31F-EGFP-expressing HCFs. The sustained PXN-pY118 dephosphorylation resulted in steadfastness of an incompletely active FAK/PXN complex at focal adhesion sites and played a pivotal role in MMC-retarded HCF migration.

Tissue transglutaminase regulates focal adhesion kinase/AKT activation by modulating PTEN expression in pancreatic cancer cells.

PubMed

Verma, Amit; Guha, Sushovan; Wang, Huamin; Fok, Jansina Y; Koul, Dimpy; Abbruzzese, James; Mehta, Kapil

2008-04-01

Pancreatic ductal adenocarcinoma (PDAC) progresses rapidly and exhibits profound resistance to treatment. We recently reported that a great majority of PDAC tumors and tumor cell lines express elevated levels of tissue transglutaminase (TG2). Here, we provide first evidence that TG2 expression in PDAC cells results in constitutive activation of focal adhesion kinase/AKT by modulating the expression of the tumor suppressor phosphatase PTEN. Using PDAC cell lines, we determined the effect of TG2 overexpression on PTEN stability and functions. We confirmed the correlation between TG2 expression and PTEN levels in a few (n=51) PDAC tumor samples. We observed that expression of TG2 is inversely correlated with PTEN expression in PDAC cells. Ectopic expression of TG2 inhibited PTEN phosphorylation and promoted its degradation by ubiquitin-proteasomal pathway. Conversely, down-regulation of TG2 by small interfering RNA up-regulated PTEN expression. Clinical relevance of these results was evident in an athymic nude mouse model where down-regulation of endogenous TG2 caused a significant retardation in PDAC xenograft growth. Importantly, the analysis of 51 tumor samples from patients with stage II PDAC revealed that overexpression of TG2 was associated with loss of PTEN expression (P=0.023; odds ratio, 4.1). In multivariate analysis, TG2-mediated loss of PTEN was a prognostic factor for overall survival in patients with stage II pancreatic ductal carcinoma independent of tumor stage/lymph node status and tumor differentiation (P=0.01). TG2 expression in PDAC promotes degradation of PTEN by ubiquitin-proteasomal pathway and results in constitutive activation of focal adhesion kinase/AKT cell survival signaling.

Stress relaxing hyaluronic acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion formation in 3D cell culture.

PubMed

Lou, Junzhe; Stowers, Ryan; Nam, Sungmin; Xia, Yan; Chaudhuri, Ovijit

2018-02-01

The physical and architectural cues of the extracellular matrix (ECM) play a critical role in regulating important cellular functions such as spreading, migration, proliferation, and differentiation. Natural ECM is a complex viscoelastic scaffold composed of various distinct components that are often organized into a fibrillar microstructure. Hydrogels are frequently used as synthetic ECMs for 3D cell culture, but are typically elastic, due to covalent crosslinking, and non-fibrillar. Recent work has revealed the importance of stress relaxation in viscoelastic hydrogels in regulating biological processes such as spreading and differentiation, but these studies all utilize synthetic ECM hydrogels that are non-fibrillar. Key mechanotransduction events, such as focal adhesion formation, have only been observed in fibrillar networks in 3D culture to date. Here we present an interpenetrating network (IPN) hydrogel system based on HA crosslinked with dynamic covalent bonds and collagen I that captures the viscoelasticity and fibrillarity of ECM in tissues. The IPN hydrogels exhibit two distinct processes in stress relaxation, one from collagen and the other from HA crosslinking dynamics. Stress relaxation in the IPN hydrogels can be tuned by modulating HA crosslinker affinity, molecular weight of the HA, or HA concentration. Faster relaxation in the IPN hydrogels promotes cell spreading, fiber remodeling, and focal adhesion (FA) formation - behaviors often inhibited in other hydrogel-based materials in 3D culture. This study presents a new, broadly adaptable materials platform for mimicking key ECM features of viscoelasticity and fibrillarity in hydrogels for 3D cell culture and sheds light on how these mechanical and structural cues regulate cell behavior. Copyright © 2017 Elsevier Ltd. All rights reserved.

Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

PubMed

Baldo, C; Lopes, D S; Faquim-Mauro, E L; Jacysyn, J F; Niland, S; Eble, J A; Clissa, P B; Moura-da-Silva, A M

2015-12-15

Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2β1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cross talk between the TM4SF5/focal adhesion kinase and the interleukin-6/STAT3 pathways promotes immune escape of human liver cancer cells.

PubMed

Ryu, Jihye; Kang, Minkyung; Lee, Mi-Sook; Kim, Hye-Jin; Nam, Seo Hee; Song, Haeng Eun; Lee, Doohyung; Lee, Jung Weon

2014-08-01

TM4SF5 overexpressed in hepatocellular carcinoma activates focal adhesion kinase (FAK) during tumor cell migration. However, it remains unknown how TM4SF5 in hepatocellular carcinoma cells compromises with immune actions initiated by extracellular cytokines. Normal and cancerous hepatocytes with or without TM4SF5 expression were analyzed for the effects of cytokine signaling activity on TM4SF5/FAK signaling and metastatic potential. We found that interleukin-6 (IL-6) was differentially expressed in hepatocytes depending on cancerous malignancy and TM4SF5 expression. IL-6 treatment activated FAK and STAT3 and enhanced focal adhesion (FA) formation in TM4SF5-null cells, but it decreased TM4SF5-dependent FAK activity and FA formation in SNU761-TM4SF5 cells. STAT3 suppression abolished the IL-6-mediated effects in normal Chang cells, but it did not recover the TM4SF5-dependent FAK activity that was inhibited by IL-6 treatment in cancerous SNU761-TM4SF5 cells. In addition, modulation of FAK activity did not change the IL-6-mediated STAT3 activity in either the Chang or SNU761 cell system. TM4SF5 expression in SNU761 cells caused invasive extracellular matrix degradation negatively depending on IL-6/IL-6 receptor (IL-6R) signaling. Thus, it is likely that hepatic cancer cells adopt TM4SF5-dependent FAK activation and metastatic potential by lowering IL-6 expression and avoiding its immunological action through the IL-6-STAT3 pathway. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Resveratrol Regulates Colorectal Cancer Cell Invasion by Modulation of Focal Adhesion Molecules

PubMed Central

Buhrmann, Constanze; Shayan, Parviz; Goel, Ajay; Shakibaei, Mehdi

2017-01-01

Resveratrol, a safe and multi-targeted agent, has been associated with suppression of survival, proliferation and metastasis of cancer, however, the underlying mechanisms for its anti-cancer activity, particularly on cellular signaling during cancer cell migration still remain poorly understood. We investigated the invasion response of two human colorectal cancer (CRC) cells (HCT116 and SW480) to resveratrol and studied the effect of specific pharmacological inhibitors, cytochalasin D (CytD) and focal adhesion kinase-inhibitor (FAK-I) on FAK, cell viability and migration in CRC. We found that resveratrol altered cell phenotype of both CRC cells, reduced cell viability and the results were comparable to FAK-I and CytD. These effects of resveratrol were associated with marked Sirt1 up-regulation, FAK down-regulation, inhibition of focal adhesion and potentiation of effects by combinatorial treatment of resveratrol and inhibitors. Interestingly, inhibition of FAK with FAK-I or treatment with CytD suppressed resveratrol-induced Sirt1 up-regulation and markedly down-regulated FAK expression. Resveratrol or combination treatment with inhibitors significantly activated caspase-3 and potentiated apoptosis. Moreover, resveratrol suppressed invasion and colony forming capacity, cell proliferation, β1-Integrin expression and activation of FAK of cells in alginate tumor microenvironment, similar to FAK-I or CytD. Finally, we demonstrated that resveratrol, FAK-I or CytD inhibited activation of NF-κB, suppressed NF-κB-dependent gene end-products involved in invasion, metastasis, and apoptosis; and these effects of resveratrol were potentiated by combination treatment with FAK-I or CytD. Our data illustrated that the anti-invasion effect of resveratrol by inhibition of FAK activity has a potential beneficial role in disease prevention and therapeutic management of CRC. PMID:28953264

Resveratrol Regulates Colorectal Cancer Cell Invasion by Modulation of Focal Adhesion Molecules.

PubMed

Buhrmann, Constanze; Shayan, Parviz; Goel, Ajay; Shakibaei, Mehdi

2017-09-27

Resveratrol, a safe and multi-targeted agent, has been associated with suppression of survival, proliferation and metastasis of cancer, however, the underlying mechanisms for its anti-cancer activity, particularly on cellular signaling during cancer cell migration still remain poorly understood. We investigated the invasion response of two human colorectal cancer (CRC) cells (HCT116 and SW480) to resveratrol and studied the effect of specific pharmacological inhibitors, cytochalasin D (CytD) and focal adhesion kinase-inhibitor (FAK-I) on FAK, cell viability and migration in CRC. We found that resveratrol altered cell phenotype of both CRC cells, reduced cell viability and the results were comparable to FAK-I and CytD. These effects of resveratrol were associated with marked Sirt1 up-regulation, FAK down-regulation, inhibition of focal adhesion and potentiation of effects by combinatorial treatment of resveratrol and inhibitors. Interestingly, inhibition of FAK with FAK-I or treatment with CytD suppressed resveratrol-induced Sirt1 up-regulation and markedly down-regulated FAK expression. Resveratrol or combination treatment with inhibitors significantly activated caspase-3 and potentiated apoptosis. Moreover, resveratrol suppressed invasion and colony forming capacity, cell proliferation, β1-Integrin expression and activation of FAK of cells in alginate tumor microenvironment, similar to FAK-I or CytD. Finally, we demonstrated that resveratrol, FAK-I or CytD inhibited activation of NF-κB, suppressed NF-κB-dependent gene end-products involved in invasion, metastasis, and apoptosis; and these effects of resveratrol were potentiated by combination treatment with FAK-I or CytD. Our data illustrated that the anti-invasion effect of resveratrol by inhibition of FAK activity has a potential beneficial role in disease prevention and therapeutic management of CRC.

Dissecting the Impact of Matrix Anchorage and Elasticity in Cell Adhesion

PubMed Central

Pompe, Tilo; Glorius, Stefan; Bischoff, Thomas; Uhlmann, Ina; Kaufmann, Martin; Brenner, Sebastian; Werner, Carsten

2009-01-01

Abstract Extracellular matrices determine cellular fate decisions through the regulation of intracellular force and stress. Previous studies suggest that matrix stiffness and ligand anchorage cause distinct signaling effects. We show herein how defined noncovalent anchorage of adhesion ligands to elastic substrates allows for dissection of intracellular adhesion signaling pathways related to matrix stiffness and receptor forces. Quantitative analysis of the mechanical balance in cell adhesion using traction force microscopy revealed distinct scalings of the strain energy imparted by the cells on the substrates dependent either on matrix stiffness or on receptor force. Those scalings suggested the applicability of a linear elastic theoretical framework for the description of cell adhesion in a certain parameter range, which is cell-type-dependent. Besides the deconvolution of biophysical adhesion signaling, site-specific phosphorylation of focal adhesion kinase, dependent either on matrix stiffness or on receptor force, also demonstrated the dissection of biochemical signaling events in our approach. Moreover, the net contractile moment of the adherent cells and their strain energy exerted on the elastic substrate was found to be a robust measure of cell adhesion with a unifying power-law scaling exponent of 1.5 independent of matrix stiffness. PMID:19843448

Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers.

PubMed

Rodrigues, Sofia N; Gonçalves, Inês C; Martins, M C L; Barbosa, Mário A; Ratner, Buddy D

2006-11-01

The effect of surface wettability on fibrinogen adsorption, platelet adhesion and platelet activation was investigated using self-assembled monolayers (SAMs) containing different ratios of longer chain methyl- and shorter chain hydroxyl-terminated alkanethiols (C15CH3 vs. C11OH) on gold. Protein adsorption studies were performed using radiolabeled human fibrinogen (HFG). Platelet adhesion and activation studies with and without pre-adsorbed fibrinogen, albumin and plasma were assessed using scanning electron microscopy (SEM) and a glutaraldehyde-induced fluorescence technique (GIFT). Results demonstrated a linear decrease of HFG adsorption with the increase of OH groups on the monolayer (increase of the hydrophilicity). Platelet adhesion and activation also decrease with increase of hydrophilicity of surface. Concerning SAMs pre-immersed in proteins, fibrinogen adsorption was related with high platelet adhesion and activation. The passivant effect of albumin on platelet adhesion and activation was only demonstrated on SAMs contained C11OH. When all the blood proteins are present (plasma) platelet adhesion was almost absent on SAMs with 65% and 100% C11OH. This could be explained by the higher albumin affinity of the SAMs with 65% C11OH and the lower total protein adsorption associated with SAMs with 100% C11OH.

Elasticity-dependent fast underwater adhesion demonstrated by macroscopic supramolecular assembly.

PubMed

Ju, Guannan; Cheng, Mengjiao; Guo, Fengli; Zhang, Qian; Shi, Feng

2018-05-30

Macroscopic supramolecular assembly (MSA) is a recent progress in supramolecular chemistry to associate visible building blocks through non-covalent interactions in a multivalent manner. Although various substrates (e. g. hydrogels, rigid materials) have been used, a general design rule of building blocks in MSA systems and interpretation of the assembly mechanism are still lacking and urgently in demand. Here we design three model systems with varied modulus and correlated the MSA probability with the elasticity. Based on the effects of substrate deformability on multivalency, we have proposed an elastic-modulus-dependent rule that building blocks below a critical modulus of 2.5 MPa can achieve MSA for the used host/guest system. Moreover, this MSA rule applies well to the design of materials applicable for fast underwater adhesion: Soft substrates (0.5 MPa) can achieve underwater adhesion within 10 s with one magnitude higher strength than that of rigid substrates (2.5 MPa). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

System integration and demonstration of adhesive bonded high temperature aluminum alloys for aerospace structure, phase 2

NASA Technical Reports Server (NTRS)

Falcone, Anthony; Laakso, John H.

1993-01-01

Adhesive bonding materials and processes were evaluated for assembly of future high-temperature aluminum alloy structural components such as may be used in high-speed civil transport aircraft and space launch vehicles. A number of candidate high-temperature adhesives were selected and screening tests were conducted using single lap shear specimens. The selected adhesives were then used to bond sandwich (titanium core) test specimens, adhesive toughness test specimens, and isothermally aged lap shear specimens. Moderate-to-high lap shear strengths were obtained from bonded high-temperature aluminum and silicon carbide particulate-reinforced (SiC(sub p)) aluminum specimens. Shear strengths typically exceeded 3500 to 4000 lb/in(sup 2) and flatwise tensile strengths exceeded 750 lb/in(sup 2) even at elevated temperatures (300 F) using a bismaleimide adhesive. All faceskin-to-core bonds displayed excellent tear strength. The existing production phosphoric acid anodize surface preparation process developed at Boeing was used, and gave good performance with all of the aluminum and silicon carbide particulate-reinforced aluminum alloys investigated. The results of this program support using bonded assemblies of high-temperature aluminum components in applications where bonding is often used (e.g., secondary structures and tear stoppers).

Clonorchis sinensis excretory-secretory products promote the migration and invasion of cholangiocarcinoma cells by activating the integrin β4-FAK/Src signaling pathway.

PubMed

Pak, Jhang Ho; Bashir, Qudsia; Kim, In Ki; Hong, Sung-Jong; Maeng, Sejung; Bahk, Young Yil; Kim, Tong-Soo

2017-06-01

Cholangiocarcinoma (CCA) is a slow-growing but highly metastatic cancer. Its metastatic potential largely explains its high mortality rate. A recognized risk factor for CCA development is infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis. We previously reported that the excretory-secretory products (ESPs) of C. sinensis promoted the three-dimensional aggregation and invasion of CCA cells. In the present study, a quantitative real-time PCR array of extracellular matrix (ECM) and adhesion molecules was used to examine the regulatory mechanism of ESP-mediated CCA cell migration and invasion. In particular, the expression levels of integrin α isoforms and β4 were upregulated in response to ESPs. Increased expression of integrin β4 was probably correlated with activation of focal adhesion kinase (FAK) and the steroid receptor coactivator (Src) family kinase and the subsequent activation of two downstream focal adhesion molecules, paxillin and vinculin. Moreover, inhibition of FAK/Src activation reduced paxillin and vinculin phosphorylation and attenuated ESP-induced CCA cell migration and invasion. These findings suggest that the integrin β4-FAK/Src signaling axis may play a crucial role in clonorchiasis-associated CCA metastasis during tumor progression. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Preparation and characterization of vinculin-targeted polymer-lipid nanoparticle as intracellular delivery vehicle.

PubMed

Wang, Junping; Ornek-Ballanco, Ceren; Xu, Jiahua; Yang, Weiguo; Yu, Xiaojun

2013-01-01

Intracellular delivery vehicles have been extensively investigated as these can serve as an effective tool in studying the cellular mechanism, by delivering functional protein to specific locations of the cells. In the current study, a polymer-lipid nanoparticle (PLN) system was developed as an intracellular delivery vehicle specifically targeting vinculin, a focal adhesion protein associated with cellular adhesive structures, such as focal adhesions and adherens junctions. The PLNs possessed an average size of 106 nm and had a positively charged surface. With a lower encapsulation efficiency 32% compared with poly(lactic-co-glycolic) acid (PLGA) nanoparticles (46%), the PLNs showed the sustained release profile of model drug BSA, while PLGA nanoparticles demonstrated an initial burst-release property. Cell-uptake experiments using mouse embryonic fibroblasts cultured in fibrin-fibronectin gels observed, under confocal microscope, that the anti-vinculin conjugated PLNs could successfully ship the cargo to the cytoplasm of fibroblasts, adhered to fibronectin-fibrin. With the use of cationic lipid, the unconjugated PLNs were shown to have high gene transfection efficiency. Furthermore, the unconjugated PLNs had nuclear-targeting capability in the absence of nuclear-localization signals. Therefore, the PLNs could be manipulated easily via different type of targeting ligands and could potentially be used as a powerful tool for cellular mechanism study, by delivering drugs to specific cellular organelles.

Protein kinase A-dependent increase in WAVE2 expression induced by the focal adhesion protein vinexin.

PubMed

Mitsushima, Masaru; Sezaki, Takuhito; Akahane, Rie; Ueda, Kazumitsu; Suetsugu, Shiro; Takenawa, Tadaomi; Kioka, Noriyuki

2006-03-01

The focal adhesion protein vinexin is a member of a family of adaptor proteins that are thought to participate in the regulation of cell adhesion, cytoskeletal reorganization, and growth factor signaling. Here, we show that vinexin beta increases the amount of and reduces the mobility on SDS-PAGE of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 protein, which is a key factor modulating actin polymerization in migrating cells. This mobility retardation disappeared after in vitro phosphatase treatment. Co-immunoprecipitation assays revealed the interaction of vinexin beta with WAVE2 as well as WAVE1 and N-WASP. Vinexin beta interacts with the proline-rich region of WAVE2 through the first and second SH3 domains of vinexin beta. Mutations disrupting the interaction impaired the ability of vinexin beta to increase the amount of WAVE2 protein. Treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin beta. Inhibition of protein kinase A (PKA) activity suppressed the vinexin-induced increase in WAVE2 protein, while activation of PKA increased WAVE2 expression without vinexin beta. These results suggest that vinexin beta regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner.

Tunable smart digital structure (SDS) to modularly assemble soft actuators with layered adhesive bonding

NASA Astrophysics Data System (ADS)

Jin, Hu; Dong, Erbao; Xu, Min; Xia, Qirong; Liu, Shuai; Li, Weihua; Yang, Jie

2018-01-01

Many shape memory alloy (SMA)-based soft actuators have specific composite structures and manufacture processes, and are therefore unique. However, these exclusive characteristics limit their capabilities and applications, so in this article a soft and smart digital structure (SDS) is proposed that acts like a modular unit to assemble soft actuators by a layered adhesive bonding process. The SDS is a fully soft structure that encapsulates a digital skeleton consisting of four groups of parallel and independently actuated SMA wires capable of outputting a four-channel tunable force. The layered adhesive bonding process modularly bonds several SDSs with an elastic backbone to fabricate a layered soft actuator where the elastic backbone is used to recover the SDSs in a cooling process using the SMA wires. Two kinds of SDS-based soft actuators were modularly assembled, an actuator, SDS-I, with a two-dimensional reciprocal motion, and an actuator, SDS-II, capable of bi-directional reciprocal motion. The thermodynamics and phase transformation modeling of the SDS-based actuator were analyzed. Several extensional soft actuators were also assembled by bonding the SDS with an anomalous elastic backbone or modularly assembling the SDS-Is and SDS-IIs. These modularly assembled soft actuators delivered more output channels and a complicated motion, e.g., an actinomorphic soft actuator with four SDS-Is jumps in a series of hierarchical heights and directional movement by tuning the input channels of the SDSs. This result showed that the SDS can modularly assemble multifarious soft actuators with diverse capabilities, steerability and tunable outputs.

Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

PubMed Central

Tsai, Cheng-Fang; Yeh, Wei-Lan; Chen, Jia-Hong; Lin, Chingju; Huang, Shiang-Suo; Lu, Dah-Yuu

2014-01-01

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK) is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP)-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells. PMID:24599080

Magnolol Inhibits Human Glioblastoma Cell Migration by Regulating N-Cadherin.

PubMed

Cheng, Yu-Chen; Tsao, Min-Jen; Chiu, Chen-Yang; Kan, Po-Chieh; Chen, Ying

2018-06-01

Glioblastoma is a primary malignant brain tumor with a poor prognosis. An effective treatment for glioblastoma is needed. Magnolol is a natural compound from Magnolia officinalis suggested to have antiproliferative activity. The aim of this research was to investigate the anticancer effects of magnolol in glioma, with an emphasis on migration and the underlying mechanism. Magnolol decreased the expression of focal adhesion-related proteins and inhibited LN229 and U87MG glioma cell migration. The levels of phosphorylated myosin light chain (p-MLC), phosphorylated myosin light chain kinase and myosin phosphatase target subunit 1 were reduced in response to magnolol treatment. In addition, immunostaining and membrane fractionation showed that the distribution of N-cadherin at the glioma cell membrane was decreased by magnolol. In an orthotropic xenograft animal model, magnolol treatment not only inhibited tumor progression but also reduced p-MLC and N-cadherin protein expression. In conclusion, magnolol reduces cell migration, potentially through regulating focal adhesions and N-cadherin in glioma cells. Magnolol is a potential candidate for glioma treatment.

The CAMSAP3-ACF7 Complex Couples Noncentrosomal Microtubules with Actin Filaments to Coordinate Their Dynamics.

PubMed

Ning, Wenxiu; Yu, Yanan; Xu, Honglin; Liu, Xiaofei; Wang, Daiwei; Wang, Jing; Wang, Yingchun; Meng, Wenxiang

2016-10-10

For adaptation to complex cellular functions, dynamic cytoskeletal networks are required. There are two major components of the cytoskeleton, microtubules and actin filaments, which form an intricate network maintaining an exquisite cooperation to build the physical basis for their cellular function. However, little is known about the molecular mechanism underlying their synergism. Here, we show that in Caco2 epithelial cells, noncentrosomal microtubules crosstalk with F-actin through their minus ends and contribute to the regulation of focal adhesion size and cell migration. We demonstrate that ACF7, a member of the spectraplakin family of cytoskeletal crosslinking proteins, interacts with Nezha (also called CAMSAP3) at the minus ends of noncentrosomal microtubules and anchors them to actin filaments. Those noncentrosomal microtubules cooperate with actin filaments through retrograde flow to keep their length and orientation perpendicular to the cell edge as well as regulate focal adhesion size and cell migration. Copyright © 2016 Elsevier Inc. All rights reserved.

ACF7 regulates cytoskeletal-focal adhesion dynamics and migration and has ATPase activity.

PubMed

Wu, Xiaoyang; Kodama, Atsuko; Fuchs, Elaine

2008-10-03

Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and actin filaments (F-actin) directly. To elucidate the physiological significance and functions of mammalian spectraplakin ACF7, we've conditionally targeted it in skin epidermis. Intriguingly, ACF7 deficiency compromises the targeting of microtubules along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration. Exploring underlying mechanisms, we show that ACF7's binding domains for F-actin, MTs, and MT plus-end proteins are not sufficient to rescue the defects in FA-cytoskeletal dynamics and migration functions of ACF7 null keratinocytes. We've uncovered an intrinsic actin-regulated ATPase domain in ACF7 and demonstrate that it is both functional and essential for these roles. Our findings provide insight into the functions of this important cytoskeletal crosslinking protein in regulating dynamic interactions between MTs and F-actin to sustain directional cell movement.

Focal adhesion kinase (FAK) regulates polymerase activity of multiple influenza A virus subtypes.

PubMed

Elbahesh, Husni; Bergmann, Silke; Russell, Charles J

2016-12-01

Influenza A viruses (IAVs) cause numerous pandemics and yearly epidemics resulting in ~500,000 annual deaths globally. IAV modulates cellular signaling pathways at every step of the infection cycle. Focal adhesion kinase (FAK) has been shown to play a critical role in endosomal trafficking of influenza A viruses, yet it is unclear how FAK kinase activity regulates IAV replication. Using mini-genomes derived from H1N1, H5N1 and H7N9 viruses, we dissected RNA replication by IAVs independent of viral entry or release. Our results show FAK activity promotes efficient IAV polymerase activity and inhibiting FAK activity with a chemical inhibitor or a kinase-dead mutant significantly reduces IAV polymerase activity. Using co-immunoprecipitations and proximity ligation assays, we observed interactions between FAK and the viral nucleoprotein, supporting a direct role of FAK in IAV replication. Altogether, the data indicates that FAK kinase activity is important in promoting IAV replication by regulating its polymerase activity. Copyright © 2016 Elsevier Inc. All rights reserved.

A systematic analysis of the PARP protein family identifies new functions critical for cell physiology

PubMed Central

Vyas, Sejal; Chesarone-Cataldo, Melissa; Todorova, Tanya; Huang, Yun-Han; Chang, Paul

2013-01-01

The poly(ADP-ribose) polymerase (PARP) family of proteins use NAD+ as their substrate to modify acceptor proteins with adenosine diphosphate-ribose (ADPr) modifications. The function of most PARPs under physiological conditions is unknown. Here, to better understand this protein family, we systematically analyze the cell cycle localization of each PARP and of poly(ADP-ribose), a product of PARP activity, then identify the knock-down phenotype of each protein and perform secondary assays to elucidate function. We show that most PARPs are cytoplasmic, identify cell cycle differences in the ratio of nuclear to cytoplasmic poly(ADP-ribose), and identify four phenotypic classes of PARP function. These include the regulation of membrane structures, cell viability, cell division, and the actin cytoskeleton. Further analysis of PARP14 shows that it is a component of focal adhesion complexes required for proper cell motility and focal adhesion function. In total, we show that PARP proteins are critical regulators of eukaryotic physiology. PMID:23917125

Molecular dynamics simulation analysis of Focal Adhesive Kinase (FAK) docked with solanesol as an anti-cancer agent

PubMed Central

Daneial, Betty; Joseph, Jacob Paul Vazhappilly; Ramakrishna, Guruprasad

2017-01-01

Focal adhesion kinase (FAK) plays a primary role in regulating the activity of many signaling molecules. Increased FAK expression has been associated in a series of cellular processes like cell migration and survival. FAK inhibition by an anti cancer agent is critical. Therefore, it is of interest to identify, modify, design, improve and develop molecules to inhibit FAK. Solanesol is known to have inhibitory activity towards FAK. However, the molecular principles of its binding with FAK is unknown. Solanesol is a highly flexible ligand (25 rotatable bonds). Hence, ligand-protein docking was completed using AutoDock with a modified contact based scoring function. The FAK-solanesol complex model was further energy minimized and simulated in GROMOS96 (53a6) force field followed by post simulation analysis such as Root mean square deviation (RMSD), root mean square fluctuations (RMSF) and solvent accessible surface area (SASA) calculations to explain solanesol-FAK binding. PMID:29081606

A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

PubMed Central

Wolfenson, Haguy; Lubelski, Ariel; Regev, Tamar; Klafter, Joseph; Henis, Yoav I.; Geiger, Benjamin

2009-01-01

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization. PMID:19172999

Focal Adhesion Kinase Is Required for Intestinal Regeneration and Tumorigenesis Downstream of Wnt/c-Myc Signaling

PubMed Central

Ashton, Gabrielle H.; Morton, Jennifer P.; Myant, Kevin; Phesse, Toby J.; Ridgway, Rachel A.; Marsh, Victoria; Wilkins, Julie A.; Athineos, Dimitris; Muncan, Vanesa; Kemp, Richard; Neufeld, Kristi; Clevers, Hans; Brunton, Valerie; Winton, Douglas J.; Wang, Xiaoyan; Sears, Rosalie C.; Clarke, Alan R.; Frame, Margaret C.; Sansom, Owen J.

2012-01-01

SUMMARY The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis. Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high risk of developing colorectal cancer. PMID:20708588

Molecular dynamics simulation analysis of Focal Adhesive Kinase (FAK) docked with solanesol as an anti-cancer agent.

PubMed

Daneial, Betty; Joseph, Jacob Paul Vazhappilly; Ramakrishna, Guruprasad

2017-01-01

Focal adhesion kinase (FAK) plays a primary role in regulating the activity of many signaling molecules. Increased FAK expression has been associated in a series of cellular processes like cell migration and survival. FAK inhibition by an anti cancer agent is critical. Therefore, it is of interest to identify, modify, design, improve and develop molecules to inhibit FAK. Solanesol is known to have inhibitory activity towards FAK. However, the molecular principles of its binding with FAK is unknown. Solanesol is a highly flexible ligand (25 rotatable bonds). Hence, ligand-protein docking was completed using AutoDock with a modified contact based scoring function. The FAK-solanesol complex model was further energy minimized and simulated in GROMOS96 (53a6) force field followed by post simulation analysis such as Root mean square deviation (RMSD), root mean square fluctuations (RMSF) and solvent accessible surface area (SASA) calculations to explain solanesol-FAK binding.

Regulation of Adhesion and Migration by the Rsu1- and PINCH1-mediated Inhibition of Focal Adhesion Formation and Actin Polymerization

DTIC Science & Technology

2012-06-08

RT-PCR and sequencing of the Rsu1 open reading frame revealed that exon 8 is missing in the alternatively spliced Rsu1 RNA in human gliomas and...concentration of 75 nM. Cells were collected 72-96 hours post-transfection. 16 Western blotting Cell lysates were collected in RIPA buffer...cell lysates were collected and bound to the glutathione beads loaded with the GST-fusion of the Rac1/cdc42 binding domain of Pak. The bound Rac1

Reelin promotes the adhesion and drug resistance of multiple myeloma cells via integrin β1 signaling and STAT3

PubMed Central

Lv, Meng; Liang, Xiaodong; Dai, Hui; Qin, Xiaodan; Zhang, Yan; Hao, Jie; Sun, Xiuyuan; Yin, Yanhui; Huang, Xiaojun; Zhang, Jun; Lu, Jin; Ge, Qing

2016-01-01

Reelin is an extracellular matrix (ECM) protein that is essential for neuron migration and positioning. The expression of reelin in multiple myeloma (MM) cells and its association with cell adhesion and survival were investigated. Overexpression, siRNA knockdown, and the addition of recombinant protein of reelin were used to examine the function of reelin in MM cells. Clinically, high expression of reelin was negatively associated with progression-free survival and overall survival. Functionally, reelin promoted the adhesion of MM cells to fibronectin via activation of α5β1 integrin. The resulting phosphorylation of Focal Adhesion Kinase (FAK) led to the activation of Src/Syk/STAT3 and Akt, crucial signaling molecules involved in enhancing cell adhesion and protecting cells from drug-induced cell apoptosis. These findings indicate reelin's important role in the activation of integrin-β1 and STAT3/Akt pathways in multiple myeloma and highlight the therapeutic potential of targeting reelin/integrin/FAK axis. PMID:26848618

Reelin promotes the adhesion and drug resistance of multiple myeloma cells via integrin β1 signaling and STAT3.

PubMed

Lin, Liang; Yan, Fan; Zhao, Dandan; Lv, Meng; Liang, Xiaodong; Dai, Hui; Qin, Xiaodan; Zhang, Yan; Hao, Jie; Sun, Xiuyuan; Yin, Yanhui; Huang, Xiaojun; Zhang, Jun; Lu, Jin; Ge, Qing

2016-03-01

Reelin is an extracellular matrix (ECM) protein that is essential for neuron migration and positioning. The expression of reelin in multiple myeloma (MM) cells and its association with cell adhesion and survival were investigated. Overexpression, siRNA knockdown, and the addition of recombinant protein of reelin were used to examine the function of reelin in MM cells. Clinically, high expression of reelin was negatively associated with progression-free survival and overall survival. Functionally, reelin promoted the adhesion of MM cells to fibronectin via activation of α5β1 integrin. The resulting phosphorylation of Focal Adhesion Kinase (FAK) led to the activation of Src/Syk/STAT3 and Akt, crucial signaling molecules involved in enhancing cell adhesion and protecting cells from drug-induced cell apoptosis. These findings indicate reelin's important role in the activation of integrin-β1 and STAT3/Akt pathways in multiple myeloma and highlight the therapeutic potential of targeting reelin/integrin/FAK axis.

Adhesive bonding and the use of corrosion resistant primers. [for metal surface preparation

NASA Technical Reports Server (NTRS)

Hockridge, R. R.; Thibault, H. G.

1972-01-01

The use of an anti-corrosive primer has been shown to be essential to assure survival of a bonded structure in a hostile environment, particularly if a stress is to be applied to the adhesively bonded joint during the environmental exposure. For example, the Lockheed L-1011 TriStar assembly, after exhaustive evaluation tests specifies use of chromate filled inhibitive polysulfide sealants, and use of corrosion inhibiting adhesive primers prior to structural bonding with film adhesive.

A rigid and thermally stable all ceramic optical support bench assembly for the LSST Camera

NASA Astrophysics Data System (ADS)

Kroedel, Matthias; Langton, J. Brian; Wahl, Bill

2017-09-01

This paper will present the ceramic design, fabrication and metrology results and assembly plan of the LSST camera optical bench structure which is using the unique manufacturing features of the HB-Cesic technology. The optical bench assembly consists of a rigid "Grid" fabrication supporting individual raft plates mounting sensor assemblies by way of a rigid kinematic support system to meet extreme stringent requirements for focal plane planarity and stability.

Hot melt recharge system. [repairing damaged or missing tiles on space shuttle orbiter

NASA Technical Reports Server (NTRS)

Progar, D. J. (Inventor)

1983-01-01

A package assembly is described for pecisely positioning a charge of hot melt adhesive onto an attachment pad or point of use. The adhesive is heated to softening or melt temperature (280 F to 325 F) and thereafter cooled to resolidifying temperature. A single sided pressure sensitive polyimide film tape serves with another film strip to protect a sandwiched adhesive strip until use and to hold the adhesive in precise position until thermally bonded to its point of use. Tab ends serve as aids in stripping tapes and from the adhesive charge.

Hot melt adhesive attachment pad

NASA Technical Reports Server (NTRS)

Fox, R. L.; Frizzill, A. W.; Little, B. D.; Progar, D. J.; Coultrip, R. H.; Couch, R. H.; Gleason, J. R.; Stein, B. A.; Buckley, J. D.; St.clair, T. L. (Inventor)

1984-01-01

A hot melt adhesive attachment pad for releasably securing distinct elements together is described which is particularly useful in the construction industry or a spatial vacuum environment. The attachment pad consists primarily of a cloth selectively impregnated with a charge of hot melt adhesive, a thermo-foil heater, and a thermo-cooler. These components are securely mounted in a mounting assembly. In operation, the operator activates the heating cycle transforming the hot melt adhesive to a substantially liquid state, positions the pad against the attachment surface, and activates the cooling cycle solidifying the adhesive and forming a strong, releasable bond.

Radil controls neutrophil adhesion and motility through β2-integrin activation

PubMed Central

Liu, Lunhua; Aerbajinai, Wulin; Ahmed, Syed M.; Rodgers, Griffin P.; Angers, Stephane; Parent, Carole A.

2012-01-01

Integrin activation is required to facilitate multiple adhesion-dependent functions of neutrophils, such as chemotaxis, which is critical for inflammatory responses to injury and pathogens. However, little is known about the mechanisms that mediate integrin activation in neutrophils. We show that Radil, a novel Rap1 effector, regulates β1- and β2-integrin activation and controls neutrophil chemotaxis. On activation and chemotactic migration of neutrophils, Radil quickly translocates from the cytoplasm to the plasma membrane in a Rap1a-GTP–dependent manner. Cells overexpressing Radil show a substantial increase in cell adhesion, as well as in integrin/focal adhesion kinase (FAK) activation, and exhibit an elongated morphology, with severe tail retraction defects. This phenotype is effectively rescued by treatment with either β2-integrin inhibitory antibodies or FAK inhibitors. Conversely, knockdown of Radil causes severe inhibition of cell adhesion, β2-integrin activation, and chemotaxis. Furthermore, we found that inhibition of Rap activity by RapGAP coexpression inhibits Radil-mediated integrin and FAK activation, decreases cell adhesion, and abrogates the long-tail phenotype of Radil cells. Overall, these studies establish that Radil regulates neutrophil adhesion and motility by linking Rap1 to β2-integrin activation. PMID:23097489

Radil controls neutrophil adhesion and motility through β2-integrin activation.

PubMed

Liu, Lunhua; Aerbajinai, Wulin; Ahmed, Syed M; Rodgers, Griffin P; Angers, Stephane; Parent, Carole A

2012-12-01

Integrin activation is required to facilitate multiple adhesion-dependent functions of neutrophils, such as chemotaxis, which is critical for inflammatory responses to injury and pathogens. However, little is known about the mechanisms that mediate integrin activation in neutrophils. We show that Radil, a novel Rap1 effector, regulates β1- and β2-integrin activation and controls neutrophil chemotaxis. On activation and chemotactic migration of neutrophils, Radil quickly translocates from the cytoplasm to the plasma membrane in a Rap1a-GTP-dependent manner. Cells overexpressing Radil show a substantial increase in cell adhesion, as well as in integrin/focal adhesion kinase (FAK) activation, and exhibit an elongated morphology, with severe tail retraction defects. This phenotype is effectively rescued by treatment with either β2-integrin inhibitory antibodies or FAK inhibitors. Conversely, knockdown of Radil causes severe inhibition of cell adhesion, β2-integrin activation, and chemotaxis. Furthermore, we found that inhibition of Rap activity by RapGAP coexpression inhibits Radil-mediated integrin and FAK activation, decreases cell adhesion, and abrogates the long-tail phenotype of Radil cells. Overall, these studies establish that Radil regulates neutrophil adhesion and motility by linking Rap1 to β2-integrin activation.

Influence of airway wall compliance on epithelial cell injury and adhesion during interfacial flows

PubMed Central

Higuita-Castro, Natalia; Mihai, Cosmin; Hansford, Derek J.

2014-01-01

Interfacial flows during cyclic airway reopening are an important source of ventilator-induced lung injury. However, it is not known how changes in airway wall compliance influence cell injury during airway reopening. We used an in vitro model of airway reopening in a compliant microchannel to investigate how airway wall stiffness influences epithelial cell injury. Epithelial cells were grown on gel substrates with different rigidities, and cellular responses to substrate stiffness were evaluated in terms of metabolic activity, mechanics, morphology, and adhesion. Repeated microbubble propagations were used to simulate cyclic airway reopening, and cell injury and detachment were quantified via live/dead staining. Although cells cultured on softer gels exhibited a reduced elastic modulus, these cells experienced less plasma membrane rupture/necrosis. Cells on rigid gels exhibited a minor, but statistically significant, increase in the power law exponent and also exhibited a significantly larger height-to-length aspect ratio. Previous studies indicate that this change in morphology amplifies interfacial stresses and, therefore, correlates with the increased necrosis observed during airway reopening. Although cells cultured on stiff substrates exhibited more plasma membrane rupture, these cells experienced significantly less detachment and monolayer disruption during airway reopening. Western blotting and immunofluorescence indicate that this protection from detachment and monolayer disruption correlates with increased focal adhesion kinase and phosphorylated paxillin expression. Therefore, changes in cell morphology and focal adhesion structure may govern injury responses during compliant airway reopening. In addition, these results indicate that changes in airway compliance, as occurs during fibrosis or emphysema, may significantly influence cell injury during mechanical ventilation. PMID:25213636

Regulation of human cerebro-microvascular endothelial baso-lateral adhesion and barrier function by S1P through dual involvement of S1P1 and S1P2 receptors.

PubMed

Wiltshire, Rachael; Nelson, Vicky; Kho, Dan Ting; Angel, Catherine E; O'Carroll, Simon J; Graham, E Scott

2016-01-27

Herein we show that S1P rapidly and acutely reduces the focal adhesion strength and barrier tightness of brain endothelial cells. xCELLigence biosensor technology was used to measure focal adhesion, which was reduced by S1P acutely and this response was mediated through both S1P1 and S1P2 receptors. S1P increased secretion of several pro-inflammatory mediators from brain endothelial cells. However, the magnitude of this response was small in comparison to that mediated by TNFα or IL-1β. Furthermore, S1P did not significantly increase cell-surface expression of any key cell adhesion molecules involved in leukocyte recruitment, included ICAM-1 and VCAM-1. Finally, we reveal that S1P acutely and dynamically regulates microvascular endothelial barrier tightness in a manner consistent with regulated rapid opening followed by closing and strengthening of the barrier. We hypothesise that the role of the S1P receptors in this process is not to cause barrier dysfunction, but is related to controlled opening of the endothelial junctions. This was revealed using real-time measurement of barrier integrity using ECIS ZΘ TEER technology and endothelial viability using xCELLigence technology. Finally, we show that these responses do not occur simply though the pharmacology of a single S1P receptor but involves coordinated action of S1P1 and S1P2 receptors.

Pv-Thermal Solar Power Assembly

DOEpatents

Ansley, Jeffrey H.; Botkin, Jonathan D.; Dinwoodie, Thomas L.

2001-10-02

A flexible solar power assembly includes a flexible photovoltaic device attached to a flexible thermal solar collector. The solar power assembly can be rolled up for transport and then unrolled for installation on a surface, such as the roof or side wall of a building or other structure, by use of adhesive and/or other types of fasteners.

40 CFR 63.4581 - What definitions apply to this subpart?

Code of Federal Regulations, 2014 CFR

2014-07-01

... adhesives under this subpart. Assembled on-road vehicle coating means any coating operation in which coating... intended for on-road use including, but not limited to, components or surfaces on automobiles and light...). Assembled on-road vehicle coating includes the concurrent coating of parts of the assembled on-road vehicle...

40 CFR 63.4581 - What definitions apply to this subpart?

Code of Federal Regulations, 2012 CFR

2012-07-01

... adhesives under this subpart. Assembled on-road vehicle coating means any coating operation in which coating... intended for on-road use including, but not limited to, components or surfaces on automobiles and light...). Assembled on-road vehicle coating includes the concurrent coating of parts of the assembled on-road vehicle...

40 CFR 63.4581 - What definitions apply to this subpart?

Code of Federal Regulations, 2013 CFR

2013-07-01

... adhesives under this subpart. Assembled on-road vehicle coating means any coating operation in which coating... intended for on-road use including, but not limited to, components or surfaces on automobiles and light...). Assembled on-road vehicle coating includes the concurrent coating of parts of the assembled on-road vehicle...

Supramolecular Lego assembly towards three-dimensional multi-responsive hydrogels.

PubMed

Ma, Chunxin; Li, Tiefeng; Zhao, Qian; Yang, Xuxu; Wu, Jingjun; Luo, Yingwu; Xie, Tao

2014-08-27

Inspired by the assembly of Lego toys, hydrogel building blocks with heterogeneous responsiveness are assembled utilizing macroscopic supramolecular recognition as the adhesion force. The Lego hydrogel provides 3D transformation upon pH variation. After disassembly of the building blocks by changing the oxidation state, they can be re-assembled into a completely new shape. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Indirect evaporative coolers with enhanced heat transfer

DOEpatents

Kozubal, Eric; Woods, Jason; Judkoff, Ron

2015-09-22

A separator plate assembly for use in an indirect evaporative cooler (IEC) with an air-to-air heat exchanger. The assembly includes a separator plate with a first surface defining a dry channel and a second surface defining a wet channel. The assembly includes heat transfer enhancements provided on the first surface for increasing heat transfer rates. The heat transfer enhancements may include slit fins with bodies extending outward from the first surface of separator plate or may take other forms including vortex generators, offset strip fins, and wavy fins. In slit fin implementations, the separator plate has holes proximate to each of the slit fins, and the separator plate assembly may include a sealing layer applied to the second surface of the separator plate to block air flow through the holes. The sealing layer can be a thickness of adhesive, and a layer of wicking material is applied to the adhesive.

Space telescope optical telescope assembly/scientific instruments. Phase B: Preliminary design and program definition study. Volume 2A. focal plane camera

NASA Technical Reports Server (NTRS)

1976-01-01

Trade studies were conducted to ensure the overall feasibility of the focal plane camera in a radial module. The primary variable in the trade studies was the location of the pickoff mirror, on axis versus off-axis. Two alternatives were: (1) the standard (electromagnetic focus) SECO submodule, and (2) the MOD 15 permanent magnet focus SECO submodule. The technical areas of concern were the packaging affected parameters of thermal dissipation, focal plane obscuration, and image quality.

EVALUATION AND PERFORMANCE ASSESSMENT OF INNOVATIVE LOW-VOC CONTACT ADHESIVES IN WOOD LAMINATING OPERATIONS

EPA Science Inventory

The report gives results of an evaluation and assessment of the perfor-mance, economics, and emission reduction potential upon application of low-volatile organic compound (VOC) waterborne contact adhesive formulations specifically ina manual laminating operation for assembling s...

The QWIP Focal Plane Assembly for NASA's Landsat Data Continuity Mission

NASA Technical Reports Server (NTRS)

Jhabvala, M; Choi, K.; Reuter, D.; Sundaram, M.; Jhabvala, C; La, Anh; Waczynski, Augustyn; Bundas, Jason

2010-01-01

The Thermal Infrared Sensor (TIRS) is a QWIP based instrument intended to supplement the Operational Land Imager (OLI) for the Landsat Data Continuity Mission (LDCM). The TIRS instrument is a dual channel far infrared imager with the two bands centered at 10.8[mu]m and 12.0[mu]m. The focal plane assembly (FPA) consists of three 640x512 GaAs Quantum Well Infrared Photodetector (QWIP) arrays precisely mounted to a silicon carrier substrate that is mounted on an invar baseplate. The two spectral bands are defined by bandpass filters mounted in close proximity to the detector surfaces. The focal plane operating temperature is 43K. The QWIP arrays are hybridized to Indigo ISC9803 readout integrated circuits (ROICs). Two varieties of QWIP detector arrays are being developed for this project, a corrugated surface structure QWIP and a grating surface structure QWIP. This paper will describe the TIRS system noise equivalent temperature difference sensitivity as it affects the QWIP focal plane performance requirements: spectral response, dark current, conversion efficiency, read noise, temperature stability, pixel uniformity, optical crosstalk and pixel yield. Additional mechanical constraints as well as qualification through Technology Readiness Level 6 (TRL 6) will also be discussed.

The QWIP Focal Plane Assembly for NASA's Landsat Data Continuity Mission

NASA Technical Reports Server (NTRS)

Jhabvala, M.; Reuter, D.; Choi, K.; Sundaram, M.; Jhabvala, C.; La, A.; Waczynski, A.; Bundas, J.

2011-01-01

The Thermal Infrared Sensor (TIRS) is a QWIP based instrument intended to supplement the Operational Land Imager (OLI) for the Landsat Data Continuity Mission (LDCM). The TIRS instrument is a dual channel far infrared imager with the two bands centered at 10.8 m and 12.0 m. The focal plane assembly (FPA) consists of three 640x512 GaAs Quantum Well Infrared Photodetector (QWIP) arrays precisely mounted to a silicon carrier substrate that is mounted on an invar baseplate. The two spectral bands are defined by bandpass filters mounted in close proximity to the detector surfaces. The focal plane operating temperature is 43K. The QWIP arrays are hybridized to Indigo ISC9803 readout integrated circuits (ROICs). Two varieties of QWIP detector arrays are being developed for this project, a corrugated surface structure QWIP and a grating surface structure QWIP. This paper will describe the TIRS system noise equivalent temperature difference sensitivity as it affects the QWIP focal plane performance requirements: spectral response, dark current, conversion efficiency, read noise, temperature stability, pixel uniformity, optical crosstalk and pixel yield. Additional mechanical constraints as well as qualification through Technology Readiness Level 6 (TRL 6) will also be discussed.

Flight motor set 360L002 (STS-27R). Volume 5: Nozzle component

NASA Technical Reports Server (NTRS)

Meyer, S. A.

1990-01-01

A review of the performance and post-flight condition of the STS-27 Redesigned Solid Rocket Motor (RSRM) nozzles is presented. Thermal/Structural instrumentation data is reviewed, and applicable Discrepancy Reports (DRs) and Process Departures (PDs) are presented. The Nozzle Component Program Team (NCPT) performance evaluation and the Redesign Program Review Board (RPRB) assessment is included. The STS-27 nozzle assemblies were flown on the RSRM Second Flight (Space Shuttle Atlantis) on 2 December 1988. The nozzles were a partially submerged convergent and/or divergent movable design with an aft pivot point flexible bearing. The nozzle assemblies incorporated the following features: RSRM forward exit cone with snubber assembly, RSRM fixed housing, Structural backup Outer Boot Ring (OBR), RSRM cowl ring, RSRM nose inlet assembly, RSRM throat assembly, RSRM aft exit cone assembly with Linear-Shaped Charge (LSC), RTV backfill in Joints 1, 3, and 4, Use of EA913 NA adhesive in place of EA913 adhesive, Redesigned nozzle plug, and Carbon Cloth Phenolic (CCP) with 750 ppm sodium content. The CCP material usage for the STS-27 forward nozzle and aft exit cone assemblies is shown.

Quantum-Well Infrared Photodetector (QWIP) Focal Plane Assembly

NASA Technical Reports Server (NTRS)

Jhabvala, Murzy; Jhabvala, Christine A.; Ewin, Audrey J.; Hess, Larry A.; Hartmann, Thomas M.; La, Anh T.

2012-01-01

A paper describes the Thermal Infrared Sensor (TIRS), a QWIP-based instrument intended to supplement the Operational Land Imager (OLI) for the Landsat Data Continuity Mission (LDCM). The TIRS instrument is a far-infrared imager operating in the pushbroom mode with two IR channels: 10.8 and 12 microns. The focal plane will contain three 640x512 QWIP arrays mounted on a silicon substrate. The silicon substrate is a custom-fabricated carrier board with a single layer of aluminum interconnects. The general fabrication process starts with a 4-in. (approx.10-cm) diameter silicon wafer. The wafer is oxidized, a single substrate contact is etched, and aluminum is deposited, patterned, and alloyed. This technology development is aimed at incorporating three large-format infrared detecting arrays based on GaAs QWIP technology onto a common focal plane with precision alignment of all three arrays. This focal plane must survive the rigors of flight qualification and operate at a temperature of 43 K (-230 C) for five years while orbiting the Earth. The challenges presented include ensuring thermal compatibility among all the components, designing and building a compact, somewhat modular system and ensuring alignment to very tight levels. The multi-array focal plane integrated onto a single silicon substrate is a new application of both QWIP array development and silicon wafer scale integration. The Invar-based assembly has been tested to ensure thermal reliability.

Receptor clustering drives polarized assembly of ankyrin.

PubMed

Jefford, G; Dubreuil, R R

2000-09-08

Expression of the L1 family cell adhesion molecule neuroglian in Drosophila S2 cells leads to cell aggregation and polarized ankyrin accumulation at sites of cell-cell contact. Thus neuroglian adhesion generates a spatial cue for polarized assembly of ankyrin and the spectrin cytoskeleton. Here we characterized a chimera of the extracellular and transmembrane domains of rat CD2 fused to the cytoplasmic domain of neuroglian. The chimera was used to test the hypothesis that clustering of neuroglian at sites of adhesion generates the signal that activates ankyrin binding. Abundant expression of the chimera at the plasma membrane was not a sufficient cue to drive ankyrin assembly, since ankyrin remained diffusely distributed throughout the cytoplasm of CD2-neuroglian-expressing cells. However, ankyrin became highly enriched at sites of antibody-induced capping of CD2-neuroglian. Spectrin codistributed with ankyrin at capped sites. A green fluorescent protein-tagged ankyrin was used to monitor ankyrin distribution in living cells. Enhanced green fluorescent protein-ankyrin behaved identically to antibody-stained endogenous ankyrin, proving that the polarized accumulation of ankyrin was not an artifact of fixing and staining cells. We propose a model in which clustering of neuroglian induces a conformational change in the cytoplasmic domain that drives polarized assembly of the spectrin cytoskeleton.

Development of the anode bipolar plate/membrane assembly unit for air breathing PEMFC stack using silicone adhesive bonding

NASA Astrophysics Data System (ADS)

Kim, Minkook; Lee, Dai Gil

2016-05-01

Polymer electrolyte membrane fuel cells (PEMFC) exhibit a wide power range, low operating temperature, high energy density and long life time. These advantages favor PEMFC for applications such as vehicle power sources, portable power, and backup power applications. With the push towards the commercialization of PEMFC, especially for portable power applications, the overall balance of plants (BOPs) of the systems should be minimized. To reduce the mass and complexity of the systems, air-breathing PEMFC stack design with open cathode channel configuration is being developed. However, the open cathode channel configuration incurs hydrogen leakage problem. In this study, the bonding strength of a silicon adhesive between the Nafion membrane and the carbon fiber/epoxy composite bipolar plate was measured. Then, an anode bipolar plate/membrane assembly unit which was bonded with the silicone adhesive was developed to solve the hydrogen leakage problem. The reliability of the anode bipolar plate/membrane assembly unit was estimated under the internal pressure of hydrogen by the FE analysis. Additionally, the gas sealability of the developed air breathing PEMFC unit cell was experimentally measured. Finally, unit cell performance of the developed anode bipolar plate/membrane assembly unit was tested and verified under operating conditions without humidity and temperature control.

Molybdenum protective coatings adhesion to steel substrate

NASA Astrophysics Data System (ADS)

Blesman, A. I.; Postnikov, D. V.; Polonyankin, D. A.; Teplouhov, A. A.; Tyukin, A. V.; Tkachenko, E. A.

2017-06-01

Protection of the critical parts, components and assemblies from corrosion is an urgent engineering problem and many other industries. Protective coatings’ forming on surface of metal products is a promising way of corrosionprevention. The adhesion force is one of the main characteristics of coatings’ durability. The paper presents theoretical and experimental adhesion force assessment for coatings formed by molybdenum magnetron sputtering ontoa steel substrate. Validity and reliability of results obtained by simulation and sclerometry method allow applying the developed model for adhesion force evaluation in binary «steel-coating» systems.

The Sentinel-4 UVN focal plane assemblies

NASA Astrophysics Data System (ADS)

Hinger, Jürgen; Hohn, Rüdiger; Gebhardt, Eyk; Reichardt, Jörg

2017-09-01

The Sentinel-4 UVN Instrument is a dispersive imaging spectrometer covering the UV-VIS and the NIR wavelength. It is developed and built under an ESA contract by an industrial consortium led by Airbus Defence and Space. It will be accommodated on board of the MTG-S (Meteosat Third Generation - Sounder) satellite that will be placed in a geostationary orbit over Europe sampling data for generating two-dimensional maps of a number of atmospheric trace gases. The incoming light is dispersed by reflective gratings and detected by the two (UVVIS and NIR) CCDs mounted inside the focal plane assemblies. Both CCD detectors acquire spectral channels and spatial sampling in two orthogonal directions and will be operated at about 215 K mainly to minimize random telegraph signal effects and to reduce dark current. Stringent detector temperature as well as alignment stability requirements of less than +/-0.1 K per day respectively of less than 2 micrometers/2 arcseconds from ground to orbit are driving the FPA thermo-mechanical design. A specific FPA design feature is the redundant LED-calibration system for bad pixel detection as well as pixel gain and linearity monitoring. This paper reports on the design and qualification of the Focal Plane Assemblies with emphasis on thermo-mechanical as well as alignment stability verification.

Effects of substrate conductivity on cell morphogenesis and proliferation using tailored, atomic layer deposition-grown ZnO thin films

PubMed Central

Choi, Won Jin; Jung, Jongjin; Lee, Sujin; Chung, Yoon Jang; Yang, Cheol-Soo; Lee, Young Kuk; Lee, You-Seop; Park, Joung Kyu; Ko, Hyuk Wan; Lee, Jeong-O

2015-01-01

We demonstrate that ZnO films grown by atomic layer deposition (ALD) can be employed as a substrate to explore the effects of electrical conductivity on cell adhesion, proliferation, and morphogenesis. ZnO substrates with precisely tunable electrical conductivity were fabricated on glass substrates using ALD deposition. The electrical conductivity of the film increased linearly with increasing duration of the ZnO deposition cycle (thickness), whereas other physical characteristics, such as surface energy and roughness, tended to saturate at a certain value. Differences in conductivity dramatically affected the behavior of SF295 glioblastoma cells grown on ZnO films, with high conductivity (thick) ZnO films causing growth arrest and producing SF295 cell morphologies distinct from those cultured on insulating substrates. Based on simple electrostatic calculations, we propose that cells grown on highly conductive substrates may strongly adhere to the substrate without focal-adhesion complex formation, owing to the enhanced electrostatic interaction between cells and the substrate. Thus, the inactivation of focal adhesions leads to cell proliferation arrest. Taken together, the work presented here confirms that substrates with high conductivity disturb the cell-substrate interaction, producing cascading effects on cellular morphogenesis and disrupting proliferation, and suggests that ALD-grown ZnO offers a single-variable method for uniquely tailoring conductivity. PMID:25897486

Preparation and characterization of vinculin-targeted polymer–lipid nanoparticle as intracellular delivery vehicle

PubMed Central

Wang, Junping; Örnek-Ballanco, Ceren; Xu, Jiahua; Yang, Weiguo; Yu, Xiaojun

2013-01-01

Intracellular delivery vehicles have been extensively investigated as these can serve as an effective tool in studying the cellular mechanism, by delivering functional protein to specific locations of the cells. In the current study, a polymer–lipid nanoparticle (PLN) system was developed as an intracellular delivery vehicle specifically targeting vinculin, a focal adhesion protein associated with cellular adhesive structures, such as focal adhesions and adherens junctions. The PLNs possessed an average size of 106 nm and had a positively charged surface. With a lower encapsulation efficiency 32% compared with poly(lactic-co-glycolic) acid (PLGA) nanoparticles (46%), the PLNs showed the sustained release profile of model drug BSA, while PLGA nanoparticles demonstrated an initial burst-release property. Cell-uptake experiments using mouse embryonic fibroblasts cultured in fibrin–fibronectin gels observed, under confocal microscope, that the anti-vinculin conjugated PLNs could successfully ship the cargo to the cytoplasm of fibroblasts, adhered to fibronectin–fibrin. With the use of cationic lipid, the unconjugated PLNs were shown to have high gene transfection efficiency. Furthermore, the unconjugated PLNs had nuclear-targeting capability in the absence of nuclear-localization signals. Therefore, the PLNs could be manipulated easily via different type of targeting ligands and could potentially be used as a powerful tool for cellular mechanism study, by delivering drugs to specific cellular organelles. PMID:23293518

Focal Adhesion Kinase-Dependent Role of the Soluble Form of Neurotensin Receptor-3/Sortilin in Colorectal Cancer Cell Dissociation.

PubMed

Béraud-Dufour, Sophie; Devader, Chistelle; Massa, Fabienne; Roulot, Morgane; Coppola, Thierry; Mazella, Jean

2016-11-08

The aim of the present review is to unravel the mechanisms of action of the soluble form of the neurotensin (NT) receptor-3 (NTSR3), also called Sortilin, in numerous physiopathological processes including cancer development, cardiovascular diseases and depression. Sortilin/NTSR3 is a transmembrane protein thought to exert multiple functions both intracellularly and at the level of the plasma membrane. The Sortilin/NTSR3 extracellular domain is released by shedding from all the cells expressing the protein. Although the existence of the soluble form of Sortilin/NTSR3 (sSortilin/NTSR3) has been evidenced for more than 10 years, the studies focusing on the role of this soluble protein at the mechanistic level remain rare. Numerous cancer cells, including colonic cancer cells, express the receptor family of neurotensin (NT), and particularly Sortilin/NTSR3. This review aims to summarize the functional role of sSortilin/NTSR3 characterized in the colonic cancer cell line HT29. This includes mechanisms involving signaling cascades through focal adhesion kinase (FAK), a key pathway leading to the weakening of cell-cell and cell-extracellular matrix adhesions, a series of events which could be responsible for cancer metastasis. Finally, some future approaches targeting the release of sNTSR3 through the inhibition of matrix metalloproteases (MMPs) are suggested.

Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

PubMed

Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

2013-08-01

Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

Adhesion by pathogenic corynebacteria.

PubMed

Rogers, Elizabeth A; Das, Asis; Ton-That, Hung

2011-01-01

Pathogenic members of the genus Corynebacterium cause a wide range of serious infections in humans including diphtheria. Adhesion to host cells is a crucial step during infection. In Corynebacterium diphtheriae, adhesion is mediated primarily by filamentous structures called pili or fimbriae that are covalently attached to the bacterial cell wall. C. diphtheriae produces three distinct pilus structures, SpaA-, SpaD- and SpaH-type pili. Similar to other types, the prototype SpaA pilus consists of SpaA forming the pilus shaft and two minor pilins SpaB and SpaC located at the base and at the tip, respectively. The minor pilins SpaB/SpaC are critical for bacterial binding to human pharyngeal cells, and thus represent the major adhesins of corynebacteria. Like pili of many other gram-positive microbes, the assembly of corynebacterial pili occurs by a two-step mechanism, whereby pilins are covalently polymerized by a transpeptidase enzyme named pilin-specific sortase and the generated pilus polymer is subsequently anchored to the cell wall peptidoglycan via the base pilin by the housekeeping sortase or a non-polymerizing sortase. This chapter reviews the current knowledge of corynebacterial adhesion, with a specific focus on pilus structures, their assembly, and the mechanism of adhesion mediated by pili.

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.

Engineering emergent multicellular behavior through synthetic adhesion

NASA Astrophysics Data System (ADS)

Glass, David; Riedel-Kruse, Ingmar

In over a decade, synthetic biology has developed increasingly robust gene networks within single cells, but constructed very few systems that demonstrate multicellular spatio-temporal dynamics. We are filling this gap in synthetic biology's toolbox by developing an E. coli self-assembly platform based on modular cell-cell adhesion. We developed a system in which adhesive selectivity is provided by a library of outer membrane-displayed peptides with intra-library specificities, while affinity is provided by consistent expression across the entire library. We further provide a biophysical model to help understand the parameter regimes in which this tool can be used to self-assemble into cellular clusters, filaments, or meshes. The combined platform will enable future development of synthetic multicellular systems for use in consortia-based metabolic engineering, in living materials, and in controlled study of minimal multicellular systems. Stanford Bio-X Bowes Fellowship.

Non-destructive component separation using infrared radiant energy

DOEpatents

Simandl, Ronald F [Knoxville, TN; Russell, Steven W [Knoxville, TN; Holt, Jerrid S [Knoxville, TN; Brown, John D [Harriman, TN

2011-03-01

A method for separating a first component and a second component from one another at an adhesive bond interface between the first component and second component. Typically the method involves irradiating the first component with infrared radiation from a source that radiates substantially only short wavelengths until the adhesive bond is destabilized, and then separating the first component and the second component from one another. In some embodiments an assembly of components to be debonded is placed inside an enclosure and the assembly is illuminated from an IR source that is external to the enclosure. In some embodiments an assembly of components to be debonded is simultaneously irradiated by a multi-planar array of IR sources. Often the IR radiation is unidirectional. In some embodiments the IR radiation is narrow-band short wavelength infrared radiation.

Lamination of Hardwood Composite Framing With an Emulsion Polymer-lsocyanate Adhesive and Radio-Frequency Curing

Treesearch

Charles B. Vick

1987-01-01

Composite framing msde from yellow-poplar and sweetgum parallel-laminated veneer and oriented flakeboard was effectively laminated with an emulsion polymer/isocyanate adhesive and radio-frequency curing at an assumed but typical range of material surface characteristics and factory assembly conditions.

Neuron-like PC12 cell patterning on a photoactive self-assembled monolayer.

PubMed

Cheng, Nan; Cao, Xudong

2013-11-01

A new approach to pattern cells using photochemistry and self-assembled monolayer (SAM) was described in this study. Photocleavable 4,5-dimethoxy-2-nitrobenzyl chloroformate (NVOC) protected amine on an alkanethiol-gold SAM was developed for cell patterning. The cleavage of NVOC and the deprotection of amines on the SAM were controlled spatially by two sequential UV exposures with a photomask. Biomolecule patterning was achieved by introducing cell nonadhesive poly(ethylene glycol) after the first exposure and subsequently cell adhesive protein laminin after the second exposure to create surface cell adhesiveness differential for cell patterning. UV-Vis spectrophotometry was used to determine the photolysis of caged self-assembled molecules; in addition, water contact angle, atomic force microscopy, cyclic voltammetry, and X-ray photoelectron spectroscopy were used to characterize properties of different surfaces. To test the efficacy of resulting surfaces in patterning cells, a neuron-like cell line, PC12 cell line, was used. The in vitro cell studies showed successful PC12 cell patterns on the photoactive SAM surfaces. This patterning technique is unique in that it does not rely on cell adhesive or nonadhesive properties of the starting base material as both cell adhesive and cell nonadhesive molecules were individually introduced onto the base material surface through photo-uncaging at preselected regions for the ultimate cell patterning. Copyright © 2013 Wiley Periodicals, Inc.

Do Deregulated Cas Proteins Induce Genomic Instability In Early Stage Ovarian Cancer?

DTIC Science & Technology

2007-12-01

3457–3467. Ezratty, E.J., Partridge, M.A., and Gundersen, G.G. (2005). Microtu- bule -induced focal adhesion disassembly is mediated by dynamin and... bule -associated deacetylase. Nature 417, 455–458. Iomini, C., Tejada, K., Mo, W., Vaananen, H., and Piperno, G. (2004). Primary cilia of human

Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

NASA Technical Reports Server (NTRS)

Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

1995-01-01

The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

NCAM (CD56) expression in keratin-producing odontogenic cysts: aberrant expression in KCOT.

PubMed

Vera-Sirera, Beatriz; Forner-Navarro, Leopoldo; Vera-Sempere, Francisco

2015-02-12

To investigate immunohistochemically the expression of neural cell adhesion molecule (NCAM), which has been identified as a signaling receptor with frequent reactivity in ameloblastomas (AB), in a series of keratin-producing odontogenic cysts (KPOCs). Immunohistochemical expression of NCAM, using a monoclonal antibody, was determined in a series of 58 KPOCs comprising 12 orthokeratinized odontogenic cysts (OOCs) and 46 keratocystic odontogenic tumors (KCOTs), corresponding to 40 non-syndromic KCOT (NS-KCOTs) and 6 syndromic KCOT (S-KCOTs), associated with nevic basocellular syndrome (NBCS). NCAM expression was negative in all OOCs, but 36.45% of KCOTs exhibited focal and heterogeneous expression at the basal cell level, as well as in basal budding areas and the basal cells of daughter cysts. The latter two locations were especially applicable to S-KCOTs, with focal NCAM reactivity occurring in 66.66% of cases. Aberrant NCAM expression, in KCOTs but especially in S-KCOTs, together with its immunomorphological location, suggests that this adhesion molecule and signaling receptor plays a role in the pathogenesis of KCOTs, with a probable impact on lesional recurrence.

Low intensity shear stress increases endothelial ELR+ CXC chemokine production via a focal adhesion kinase-p38{beta} MAPK-NF-{kappa}B pathway.

PubMed

Shaik, Sadiq S; Soltau, Thomas D; Chaturvedi, Gaurav; Totapally, Balagangadhar; Hagood, James S; Andrews, William W; Athar, Mohammad; Voitenok, Nikolai N; Killingsworth, Cheryl R; Patel, Rakesh P; Fallon, Michael B; Maheshwari, Akhil

2009-02-27

CXC chemokines with a glutamate-leucine-arginine (ELR) tripeptide motif (ELR(+) CXC chemokines) play an important role in leukocyte trafficking into the tissues. For reasons that are not well elucidated, circulating leukocytes are recruited into the tissues mainly in small vessels such as capillaries and venules. Because ELR(+) CXC chemokines are important mediators of endothelial-leukocyte interaction, we compared chemokine expression by microvascular and aortic endothelium to investigate whether differences in chemokine expression by various endothelial types could, at least partially, explain the microvascular localization of endothelial-leukocyte interaction. Both in vitro and in vivo models indicate that ELR(+) CXC chemokine expression is higher in microvascular endothelium than in aortic endothelial cells. These differences can be explained on the basis of the preferential activation of endothelial chemokine production by low intensity shear stress. Low shear activated endothelial ELR(+) CXC chemokine production via cell surface heparan sulfates, beta(3)-integrins, focal adhesion kinase, the mitogen-activated protein kinase p38beta, mitogen- and stress-associated protein kinase-1, and the transcription factor.

PKD1/PKCmu promotes alphavbeta3 integrin recycling and delivery to nascent focal adhesions.

PubMed

Woods, Alison J; White, Dominic P; Caswell, Patrick T; Norman, Jim C

2004-07-07

To identify kinases that regulate integrin recycling, we have immunoprecipitated alphavbeta3 integrin from NIH 3T3 fibroblasts in the presence and absence of primaquine (a drug that inhibits receptor recycling and leads to accumulation of integrins in endosomes) and screened for co-precipitating kinases. Primaquine strongly promoted association of alphavbeta3 integrin with PKD1, and fluorescence microscopy indicated that integrin and PKD1 associate at a vesicular compartment that is downstream of a Rab4-dependent transport step. PKD1 association was mediated by the C-terminal region of the beta3 integrin cytodomain, and mutants of beta3 that were unable to recruit PKD1 did not recycle in a PDGF-dependent fashion. Furthermore, suppression of endogenous PKD1 levels by RNAi, or overexpression of catalytically inactive PKD1 inhibited PDGF-dependent recycling of alphavbeta3 from early endosomes to the plasma membrane and blocked recruitment of alphavbeta3 to newly formed focal adhesions during cell spreading. These data indicate that PKD1 influences cell migration by directing vesicular transport of the alphavbeta3 integrin heterodimer.

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

2016-07-01

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. Copyright © 2016 John Wiley & Sons, Inc.

The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dougherty, Gerard W.; Section on Structural Cell Biology, National Institute on Deafness and Communication Disorders; Chopp, Treasa

2005-05-15

Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5more » domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.« less

Gingival Fibroblasts Display Reduced Adhesion and Spreading on Extracellular Matrix: A Possible Basis for Scarless Tissue Repair?

PubMed Central

Guo, Fen; Carter, David E.; Mukhopadhyay, Anuradha; Leask, Andrew

2011-01-01

Unlike skin, oral gingiva do not scar in response to injury. The basis of this difference is likely to be revealed by comparing the responses of dermal and gingival fibroblasts to fibrogenic stimuli. Previously, we showed that, compared to dermal fibroblasts, gingival fibroblasts are less responsive to the potent pro-fibrotic cytokine TGFβ, due to a reduced production of endothelin-1 (ET-1). In this report, we show that, compared to dermal fibroblasts, human gingival fibroblasts show reduced expression of pro-adhesive mRNAs and proteins including integrins α2 and α4 and focal adhesion kinase (FAK). Consistent with these observations, gingival fibroblasts are less able to adhere to and spread on both fibronectin and type I collagen. Moreover, the enhanced production of ET-1 mRNA and protein in dermal fibroblasts is reduced by the FAK/src inhibitor PP2. Given our previous observations suggesting that fibrotic fibroblasts display elevated adhesive properties, our data suggest that scarring potential may be based, at least in part, on differences in adhesive properties among fibroblasts resident in connective tissue. Controlling adhesive properties may be of benefit in controlling scarring in response to tissue injury. PMID:22073262

Collagen remodeling by phagocytosis is determined by collagen substrate topology and calcium-dependent interactions of gelsolin with nonmuscle myosin IIA in cell adhesions

PubMed Central

Arora, P. D.; Wang, Y.; Bresnick, A.; Dawson, J.; Janmey, P. A.; McCulloch, C. A.

2013-01-01

We examine how collagen substrate topography, free intracellular calcium ion concentration ([Ca2+]i, and the association of gelsolin with nonmuscle myosin IIA (NMMIIA) at collagen adhesions are regulated to enable collagen phagocytosis. Fibroblasts plated on planar, collagen-coated substrates show minimal increase of [Ca2+]i, minimal colocalization of gelsolin and NMMIIA in focal adhesions, and minimal intracellular collagen degradation. In fibroblasts plated on collagen-coated latex beads there are large increases of [Ca2+]i, time- and Ca2+-dependent enrichment of NMMIIA and gelsolin at collagen adhesions, and abundant intracellular collagen degradation. NMMIIA knockdown retards gelsolin recruitment to adhesions and blocks collagen phagocytosis. Gelsolin exhibits tight, Ca2+-dependent binding to full-length NMMIIA. Gelsolin domains G4–G6 selectively require Ca2+ to interact with NMMIIA, which is restricted to residues 1339–1899 of NMMIIA. We conclude that cell adhesion to collagen presented on beads activates Ca2+ entry and promotes the formation of phagosomes enriched with NMMIIA and gelsolin. The Ca2+ -dependent interaction of gelsolin and NMMIIA in turn enables actin remodeling and enhances collagen degradation by phagocytosis. PMID:23325791

Single and collective cell migration: the mechanics of adhesions

PubMed Central

De Pascalis, Chiara; Etienne-Manneville, Sandrine

2017-01-01

Chemical and physical properties of the environment control cell proliferation, differentiation, or apoptosis in the long term. However, to be able to move and migrate through a complex three-dimensional environment, cells must quickly adapt in the short term to the physical properties of their surroundings. Interactions with the extracellular matrix (ECM) occur through focal adhesions or hemidesmosomes via the engagement of integrins with fibrillar ECM proteins. Cells also interact with their neighbors, and this involves various types of intercellular adhesive structures such as tight junctions, cadherin-based adherens junctions, and desmosomes. Mechanobiology studies have shown that cell–ECM and cell–cell adhesions participate in mechanosensing to transduce mechanical cues into biochemical signals and conversely are responsible for the transmission of intracellular forces to the extracellular environment. As they migrate, cells use these adhesive structures to probe their surroundings, adapt their mechanical properties, and exert the appropriate forces required for their movements. The focus of this review is to give an overview of recent developments showing the bidirectional relationship between the physical properties of the environment and the cell mechanical responses during single and collective cell migration. PMID:28684609

Self-folding and aggregation of amyloid nanofibrils

NASA Astrophysics Data System (ADS)

Paparcone, Raffaella; Cranford, Steven W.; Buehler, Markus J.

2011-04-01

Amyloids are highly organized protein filaments, rich in β-sheet secondary structures that self-assemble to form dense plaques in brain tissues affected by severe neurodegenerative disorders (e.g. Alzheimer's Disease). Identified as natural functional materials in bacteria, in addition to their remarkable mechanical properties, amyloids have also been proposed as a platform for novel biomaterials in nanotechnology applications including nanowires, liquid crystals, scaffolds and thin films. Despite recent progress in understanding amyloid structure and behavior, the latent self-assembly mechanism and the underlying adhesion forces that drive the aggregation process remain poorly understood. On the basis of previous full atomistic simulations, here we report a simple coarse-grain model to analyze the competition between adhesive forces and elastic deformation of amyloid fibrils. We use simple model system to investigate self-assembly mechanisms of fibrils, focused on the formation of self-folded nanorackets and nanorings, and thereby address a critical issue in linking the biochemical (Angstrom) to micrometre scales relevant for larger-scale states of functional amyloid materials. We investigate the effect of varying the interfibril adhesion energy on the structure and stability of self-folded nanorackets and nanorings and demonstrate that these aggregated amyloid fibrils are stable in such states even when the fibril-fibril interaction is relatively weak, given that the constituting amyloid fibril length exceeds a critical fibril length-scale of several hundred nanometres. We further present a simple approach to directly determine the interfibril adhesion strength from geometric measures. In addition to providing insight into the physics of aggregation of amyloid fibrils our model enables the analysis of large-scale amyloid plaques and presents a new method for the estimation and engineering of the adhesive forces responsible of the self-assembly process of amyloidnanostructures, filling a gap that previously existed between full atomistic simulations of primarily ultra-short fibrils and much larger micrometre-scale amyloid aggregates. Via direct simulation of large-scale amyloid aggregates consisting of hundreds of fibrils we demonstrate that the fibril length has a profound impact on their structure and mechanical properties, where the critical fibril length-scale derived from our analysis of self-folded nanorackets and nanorings defines the structure of amyloid aggregates. A multi-scale modeling approach as used here, bridging the scales from Angstroms to micrometres, opens a wide range of possible nanotechnology applications by presenting a holistic framework that balances mechanical properties of individual fibrils, hierarchical self-assembly, and the adhesive forces determining their stability to facilitate the design of de novoamyloid materials.

PV/thermal solar power assembly

DOEpatents

Ansley, Jeffrey H.; Botkin, Jonathan D.; Dinwoodie, Thomas L.

2004-01-13

A flexible solar power assembly (2) includes a flexible photovoltaic device (16) attached to a flexible thermal solar collector (4). The solar power assembly can be rolled up for transport and then unrolled for installation on a surface, such as the roof (20, 25) or side wall of a building or other structure, by use of adhesive and/or other types of fasteners (23).

Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling.

PubMed

Nasreen, Najmunnisa; Khodayari, Nazli; Sriram, Peruvemba S; Patel, Jawaharlal; Mohammed, Kamal A

2014-06-15

Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although the role of Eph receptors and their ligand Ephrins is well studied in developmental processes, their function in tobacco smoke (TS)-induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction, and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly upregulated EphA2 and EphrinA1 expression, disrupted the actin filaments, decreased E-cadherin expression, and increased protein permeability, whereas the focal adhesion protein paxillin was unaffected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS-induced hyperpermeability in BAEpCs. In addition, when BAEpC monolayer was transfected with EphA2-expressing plasmid and treated with recombinant EphrinA1, the transepithelial electrical resistance decreased significantly. Furthermore, TS downregulated E-cadherin expression and induced hyperpermeability across BAEpC monolayer in a Erk1/Erk2, p38, and JNK MAPK-dependent manner. TS induced hyperpermeability in BAEpC monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were unaffected. The present data suggest that TS causes significant damage to the BAEpCs via induction of EphA2 and downregulation of E-cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.

Natural and bio-inspired underwater adhesives: Current progress and new perspectives

NASA Astrophysics Data System (ADS)

Cui, Mengkui; Ren, Susu; Wei, Shicao; Sun, Chengjun; Zhong, Chao

2017-11-01

Many marine organisms harness diverse protein molecules as underwater adhesives to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Natural underwater adhesion phenomena thus provide inspiration for engineering adhesive materials that can perform in water or high-moisture settings for biomedical and industrial applications. Here we review examples of biological adhesives to show the molecular features of natural adhesives and discuss how such knowledge serves as a heuristic guideline for the rational design of biologically inspired underwater adhesives. In view of future bio-inspired research, we propose several potential opportunities, either in improving upon current L-3, 4-dihydroxyphenylalanine-based and coacervates-enabled adhesives with new features or engineering conceptually new types of adhesives that recapitulate important characteristics of biological adhesives. We underline the importance of viewing natural adhesives as dynamic materials, which owe their outstanding performance to the cellular coordination of protein expression, delivery, deposition, assembly, and curing of corresponding components with spatiotemporal control. We envision that the emerging synthetic biology techniques will provide great opportunities for advancing both fundamental and application aspects of underwater adhesives.

Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.

PubMed

Schlaepfer, D D; Hunter, T

1996-10-01

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.

Substrate micropatterns produced by polymer demixing regulate focal adhesions, actin anisotropy, and lineage differentiation of stem cells.

PubMed

Vega, Sebastián L; Arvind, Varun; Mishra, Prakhar; Kohn, Joachim; Sanjeeva Murthy, N; Moghe, Prabhas V

2018-06-12

Stem cells are adherent cells whose multipotency and differentiation can be regulated by numerous microenvironmental signals including soluble growth factors and surface topography. This study describes a simple method for creating distinct micropatterns via microphase separation resulting from polymer demixing of poly(desaminotyrosyl-tyrosine carbonate) (PDTEC) and polystyrene (PS). Substrates with co-continuous (ribbons) or discontinuous (islands and pits) PDTEC regions were obtained by varying the ratio of PDTEC and sacrificial PS. Human mesenchymal stem cells (MSCs) cultured on co-continuous PDTEC substrates for 3 days in bipotential adipogenic/osteogenic (AD/OS) induction medium showed no change in cell morphology but exhibited increased anisotropic cytoskeletal organization and larger focal adhesions when compared to MSCs cultured on discontinuous micropatterns. After 14 days in bipotential AD/OS induction medium, MSCs cultured on co-continuous micropatterns exhibited increased expression of osteogenic markers, whereas MSCs on discontinuous PDTEC substrates showed a low expression of adipogenic and osteogenic differentiation markers. Substrates with graded micropatterns were able to reproduce the influence of local underlying topography on MSC differentiation, thus demonstrating their potential for high throughput analysis. This work presents polymer demixing as a simple, non-lithographic technique to produce a wide range of micropatterns on surfaces with complex geometries to influence cellular and tissue regenerative responses. Gaining a better understanding of how engineered microenvironments influence stem cell differentiation is integral to increasing the use of stem cells and materials in a wide range of tissue engineering applications. In this study, we show the range of topography obtained by polymer demixing is sufficient for investigating how surface topography affects stem cell morphology and differentiation. Our findings show that co-continuous topographies favor early (3-day) cytoskeletal anisotropy and focal adhesion maturation as well as long-term (14-day) expression of osteogenic differentiation markers. Taken together, this study presents a simple approach to pattern topographies that induce divergent responses in stem cell morphology and differentiation. Copyright © 2018. Published by Elsevier Ltd.

The design and assembly of aluminum mirrors of a three-mirror-anastigmat telescope

NASA Astrophysics Data System (ADS)

Chang, Shenq-Tsong; Lin, Yu-Chuan; Wu, Kun-Huan; Lien, Chun-Chieh; Huang, Ting-Ming; Tsay, Ho-Lin; Chan, Chia-Yen

2017-09-01

Better ground sampling distance (GSD) has been a trend for earth observation satellites. A long-focal-length telescope is required accordingly in systematic point of view. On the other hand, there is size constraint for such long-focal-length telescope especially in space projects. Three-mirror-anastigmat (TMA) was proven to have excellent features of correcting aberrations, wide spectral range and shorter physical requirement [1-3].

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

Optomechanical design concept for GMACS: a wide-field multi-object moderate resolution optical spectrograph for the Giant Magellan Telescope (GMT)

NASA Astrophysics Data System (ADS)

Smee, Stephen A.; Prochaska, Travis; Shectman, Stephen A.; Hammond, Randolph P.; Barkhouser, Robert H.; DePoy, D. L.; Marshall, J. L.

2012-09-01

We describe the conceptual optomechanical design for GMACS, a wide-field, multi-object, moderate-resolution optical spectrograph for the Giant Magellan Telescope (GMT). GMACS is a candidate first-light instrument for the GMT and will be one of several instruments housed in the Gregorian Instrument Rotator (GIR) located at the Gregorian focus. The instrument samples a 9 arcminute x 18 arcminute field of view providing two resolution modes (i.e, low resolution, R ~ 2000, and moderate resolution, R ~ 4000) over a 3700 Å to 10200 Å wavelength range. To minimize the size of the optics, four fold mirrors at the GMT focal plane redirect the full field into four individual "arms", that each comprises a double spectrograph with a red and blue channel. Hence, each arm samples a 4.5 arcminute x 9 arcminute field of view. The optical layout naturally leads to three separate optomechanical assemblies: a focal plane assembly, and two identical optics modules. The focal plane assembly contains the last element of the telescope's wide-field corrector, slit-mask, tent-mirror assembly, and slit-mask magazine. Each of the two optics modules supports two of the four instrument arms and houses the aft-optics (i.e. collimators, dichroics, gratings, and cameras). A grating exchange mechanism, and articulated gratings and cameras facilitate multiple resolution modes. In this paper we describe the details of the GMACS optomechanical design, including the requirements and considerations leading to the design, mechanism details, optics mounts, and predicted flexure performance.

Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

NASA Astrophysics Data System (ADS)

Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

2015-10-01

Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

Low-temperature resistant, elastic adhesives and sealants for gas tank insulation

NASA Astrophysics Data System (ADS)

Karrer, R.

The leading European insulating firms in the domain of liquid natural gas (LNG)/liquid petroleum gas (LPG) carriers have developed special sandwich elements for the insulation of liquid gas tanks. The trend to increasing tank volumes and, at the same time, to reducing the number of cargo tanks in modern liquid gas carriers with loading capacities of up to 135,000 m 3 has in some cases entailed major changes with respect to tank design (Kaefer-Isoliertechnik, Hansa Schiffahrt-Schiffbau-Hafen, 133rd year, 1996, 2, 20-22). These changes have equally influenced both the design and the assembly of the panels used for insulation, as well as the adhesives and sealants applied for this purpose. This article describes the requirement profile and the possible applications of solvent-free two-component polyurethane adhesives (2-K PU) and recently developed polyurethane hot-melt adhesives (PU-HM) for the manufacture and/or assembly of panels. Moreover, it deals with the role of the advanced solvent-free, silane-modified polymers (MS polymers) in the pointing of panels (seam-sealing) exposed to low temperatures.

Micro-masonry for 3D additive micromanufacturing.

PubMed

Keum, Hohyun; Kim, Seok

2014-08-01

Transfer printing is a method to transfer solid micro/nanoscale materials (herein called 'inks') from a substrate where they are generated to a different substrate by utilizing elastomeric stamps. Transfer printing enables the integration of heterogeneous materials to fabricate unexampled structures or functional systems that are found in recent advanced devices such as flexible and stretchable solar cells and LED arrays. While transfer printing exhibits unique features in material assembly capability, the use of adhesive layers or the surface modification such as deposition of self-assembled monolayer (SAM) on substrates for enhancing printing processes hinders its wide adaptation in microassembly of microelectromechanical system (MEMS) structures and devices. To overcome this shortcoming, we developed an advanced mode of transfer printing which deterministically assembles individual microscale objects solely through controlling surface contact area without any surface alteration. The absence of an adhesive layer or other modification and the subsequent material bonding processes ensure not only mechanical bonding, but also thermal and electrical connection between assembled materials, which further opens various applications in adaptation in building unusual MEMS devices.

Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein

PubMed Central

1996-01-01

Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton- insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells. PMID:8647901

Structure, Function, and Assembly of Adhesive Organelles by Uropathogenic Bacteria

PubMed Central

Chahales, Peter; Thanassi, David G.

2015-01-01

Bacteria assemble a wide range of adhesive proteins, termed adhesins, to mediate binding to receptors and colonization of surfaces. For pathogenic bacteria, adhesins are critical for early stages of infection, allowing the bacteria to initiate contact with host cells, colonize different tissues, and establish a foothold within the host. The adhesins expressed by a pathogen are also critical for bacterial-bacterial interactions and the formation of bacterial communities such as biofilms. The ability to adhere to host tissues is particularly important for bacteria that colonize sites such as the urinary tract, where the flow of urine functions to maintain sterility by washing away non-adherent pathogens. Adhesins vary from monomeric proteins that are directly anchored to the bacterial surface to polymeric, hairlike fibers that extend out from the cell surface. These latter fibers are termed pili or fimbriae, and were among the first identified virulence factors of uropathogenic Escherichia coli. Studies since then have identified a range of both pilus and non-pilus adhesins that contribute to bacterial colonization of the urinary tract, and have revealed molecular details of the structures, assembly pathways, and functions of these adhesive organelles. In this review, we describe the different types of adhesins expressed by both Gram-negative and Gram-positive uropathogens, what is known about their structures, how they are assembled on the bacterial surface, and the functions of specific adhesins in the pathogenesis of urinary tract infections. PMID:26542038

Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells.

PubMed

Liu, Yan-Jun; Le Berre, Maël; Lautenschlaeger, Franziska; Maiuri, Paolo; Callan-Jones, Andrew; Heuzé, Mélina; Takaki, Tohru; Voituriez, Raphaël; Piel, Matthieu

2015-02-12

The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration--one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confinement, adhesion, and contractility. Copyright © 2015 Elsevier Inc. All rights reserved.

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rossier, Olivier; Giannone, Grégory; CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements andmore » interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.« less

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

PubMed

Rossier, Olivier; Giannone, Grégory

2016-04-10

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. Copyright © 2015. Published by Elsevier Inc.

Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate

NASA Astrophysics Data System (ADS)

Dalby, Matthew J.; Gadegaard, Nikolaj; Oreffo, Richard O. C.

2014-06-01

Stem cells respond to nanoscale surface features, with changes in cell growth and differentiation mediated by alterations in cell adhesion. The interaction of nanotopographical features with integrin receptors in the cells' focal adhesions alters how the cells adhere to materials surfaces, and defines cell fate through changes in both cell biochemistry and cell morphology. In this Review, we discuss how cell adhesions interact with nanotopography, and we provide insight as to how materials scientists can exploit these interactions to direct stem cell fate and to understand how the behaviour of stem cells in their niche can be controlled. We expect knowledge gained from the study of cell-nanotopography interactions to accelerate the development of next-generation stem cell culture materials and implant interfaces, and to fuel discovery of stem cell therapeutics to support regenerative therapies.

A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

PubMed Central

1995-01-01

To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947

Integrin alphaIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of beta3-subunits in actin cytoskeletal organization.

PubMed

Yamodo, Innocent H; Blystone, Scott D

2004-01-01

Using truncated or mutated alphaIIb integrin cytoplasmic domains fused to the alphaV extracellular domain and expressed with the beta3 integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alphaIIb integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta3 integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alphaIIb-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alphaIIb did not affect beta3 tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta3 phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta3 integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta3 integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta3 phosphorylation is demonstrated without mutation of the beta3 subunit. In support of our previous hypothesis of a role for beta3 phosphorylation in adhesion, these studies demonstrate a strong correlation between beta3 tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.

Parietal Epithelial Cells Participate in the Formation of Sclerotic Lesions in Focal Segmental Glomerulosclerosis

PubMed Central

Smeets, Bart; Kuppe, Christoph; Sicking, Eva-Maria; Fuss, Astrid; Jirak, Peggy; van Kuppevelt, Toin H.; Endlich, Karlhans; Wetzels, Jack F.M.; Gröne, Hermann-Josef; Floege, Jürgen

2011-01-01

The pathogenesis of the development of sclerotic lesions in focal segmental glomerulosclerosis (FSGS) remains unknown. Here, we selectively tagged podocytes or parietal epithelial cells (PECs) to determine whether PECs contribute to sclerosis. In three distinct models of FSGS (5/6-nephrectomy + DOCA-salt; the murine transgenic chronic Thy1.1 model; or the MWF rat) and in human biopsies, the primary injury to induce FSGS associated with focal activation of PECs and the formation of cellular adhesions to the capillary tuft. From this entry site, activated PECs invaded the affected segment of the glomerular tuft and deposited extracellular matrix. Within the affected segment, podocytes were lost and mesangial sclerosis developed within the endocapillary compartment. In conclusion, these results demonstrate that PECs contribute to the development and progression of the sclerotic lesions that define FSGS, but this pathogenesis may be relevant to all etiologies of glomerulosclerosis. PMID:21719782

Organization and PprB-dependent control of the Pseudomonas aeruginosa tad Locus, involved in Flp pilus biology.

PubMed

Bernard, Christophe S; Bordi, Christophe; Termine, Elise; Filloux, Alain; de Bentzmann, Sophie

2009-03-01

Bacterial attachment to the substratum involves several cell surface organelles, including various types of pili. The Pseudomonas aeruginosa Tad machine assembles type IVb pili, which are required for adhesion to abiotic surfaces and to eukaryotic cells. Type IVb pili consist of a major subunit, the Flp pilin, processed by the FppA prepilin peptidase. In this study, we investigated the regulatory mechanism of the tad locus. We showed that the flp gene is expressed late in the stationary growth phase in aerobic conditions. We also showed that the tad locus was composed of five independent transcriptional units. We used transcriptional fusions to show that tad gene expression was positively controlled by the PprB response regulator. We subsequently showed that PprB bound to the promoter regions, directly controlling the expression of these genes. We then evaluated the contribution of two genes, tadF and rcpC, to type IVb pilus assembly. The deletion of these two genes had no effect on Flp production, pilus assembly, or Flp-mediated adhesion to abiotic surfaces in our conditions. However, our results suggest that the putative RcpC protein modifies the Flp pilin, thereby promoting Flp-dependent adhesion to eukaryotic cells.

The role of actin networks in cellular mechanosensing

NASA Astrophysics Data System (ADS)

Azatov, Mikheil

Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis. In addition to stiffness, the local geometry or topography of the surface has been shown to modulate the movement, morphology, and cytoskeletal organization of cells. However, the effect of topography on fluctuations of intracellular structures, which arise from motor driven activity on a viscoelastic actin network are not known. I have used nanofabricated substrates with parallel ridges to show that the cell shape, the actin cytoskeleton and focal adhesions all align along the direction of the ridges, exhibiting a biphasic dependence on the spacing between ridges. I further demonstrated that palladin bands along actin stress fibers undergo a complex diffusive motion with velocities aligned along the direction of ridges. These results provide insight into the mechanisms of cellular mechanosensing of the environment, suggesting a complex interplay between the actin cytoskeleton and cellular adhesions in coordinating cellular response to surface topography. Overall, this work has advanced our understanding of mechanisms that govern cellular responses to their physical environment.

Adhesion characterization and defect sizing of sandwich honeycomb composites.

PubMed

Ndiaye, Elhadji Barra; Maréchal, Pierre; Duflo, Hugues

2015-09-01

Defects may appear in composite structures during their life cycle. A 10MHz 128 elements phased array transducer was investigated to characterize join bonds and defects in sandwich honeycomb composite structures. An adequate focal law throughout the composite skin gives the ultrasonic dispersive properties of the composite skin and glue layer behind. The resulting B-scan cartographies allow characterizing locally the honeycomb adhesion. Experimental measurements are compared in good agreement with the Debye Series Method (DSM). In the processed C-scan image, flaws are detectable and measurable, localized both in the scanning plane and in the thickness of the composite skin. Copyright © 2015 Elsevier B.V. All rights reserved.

Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

PubMed Central

Zheng, Yanhua; Lu, Zhimin

2013-01-01

Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

Wigner function and the successive measurement of position and momentum

NASA Astrophysics Data System (ADS)

Beller, Daniel A.; Gharbi, Mohamed A.; Honglawan, Apiradee; Stebe, Kathleen J.; Yang, Shu; Kamien, Randall D.

2013-10-01

Focal conic domains (FCDs) in smectic-A liquid crystals have drawn much attention, both for their exquisitely structured internal form and for their ability to direct the assembly of micromaterials and nanomaterials in a variety of patterns. A key to directing FCD assembly is control over the eccentricity of the domain. Here, we demonstrate a new paradigm for creating spatially varying FCD eccentricity by confining a hybrid-aligned smectic with curved interfaces. In particular, we manipulate interface behavior with colloidal particles in order to experimentally produce two examples of what has recently been dubbed the flower texture [C. Meyer , Focal Conic Stacking in Smectic A Liquid Crystals: Smectic Flower and Apollonius Tiling, Materials 2, 499, 2009MATEG91996-194410.3390/ma2020499], where the focal hyperbolæ diverge radially outward from the center of the texture, rather than inward as in the canonical éventail or fan texture. We explain how this unconventional assembly can arise from appropriately curved interfaces. Finally, we present a model for this system that applies the law of corresponding cones, showing how FCDs may be embedded smoothly within a “background texture” of large FCDs and concentric spherical layers, in a manner consistent with the qualitative features of the smectic flower. Such understanding could potentially lead to disruptive liquid-crystal technologies beyond displays, including patterning, smart surfaces, microlens arrays, sensors, and nanomanufacturing.

The PAKs come of age

PubMed Central

2012-01-01

Protein kinases are versatile signaling molecules that are involved in the regulation most physiological responses. The p21-activated kinases (PAKs) can be activated directly by the small GTPases Rac and Cdc42 and are among the best characterized downstream effectors of these Rho proteins. The structure, substrate specificity and functional role of PAKS are evolutionarily conserved from protozoa to mammals. Vertebrate PAKs are particularly important for cytoskeletal remodeling and focal adhesion assembly, thereby contributing to dynamic processes such as cell migration and synaptic plasticity. This issue of Cellular Logistics focuses on the PAK family of kinases, with ten reviews written by researchers currently working in the field. Here in this introductory overview we highlight some of the most interesting recent discoveries regarding PAK biochemistry and biology. The reviews in this issue cover a range of topics including the atomic structures of PAK1 and PAK4, their role in animals as assessed by knockout studies, and how PAKs are likely to contribute to cancer and neurodegenerative diseases. The promise remains that PAK inhibitors will emerge that validate current pre-clinical studies suggesting that blocking PAK activity will positively contribute to human health. PMID:23125949

Thermoplastic Adhesives based on polyolefin and olefinic copolymers

NASA Astrophysics Data System (ADS)

Paul, Rituparna

2014-03-01

H.B. Fuller has been a leading global industrial adhesive manufacturer for over 125 years. It is a company with a rich history of consistently delivering adhesive innovations for enhancing product performance in the market place. H.B. Fuller technologies/products find application in several markets including packaging, personal hygiene and nonwovens, durable assembly and electronics. In this presentation, H. B. Fuller's technology innovation journey will be shared with emphasis on groundbreaking technologies/products based on polyolefin and olefin copolymers.

High Resolution Imager (HRI) for the Roentgen Satellite (ROSAT) definition study

NASA Technical Reports Server (NTRS)

1983-01-01

The design of the high resolution imager (HRI) on HEAO 2 was modified for use in the instrument complement of the Roentgen Satellite (ROSAT). Mechanical models of the front end assembly, central electronics assembly, and detector assembly were used to accurately represent the HRI envelope for both fit checks and focal plane configuration studies. The mechanical and electrical interfaces were defined and the requirements for electrical ground support equipment were established. A summary description of the ROSAT telescope and mission is included.

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination.

PubMed

Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-Ichirou; Kimura, Tadashi

2017-10-27

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer.

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

PubMed Central

Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-ichirou; Kimura, Tadashi

2017-01-01

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer. PMID:29163796

Adhesion promoters for large scale fabrication of dielectric elastomer stack transducers (DESTs) made of pre-fabricated dielectric films

NASA Astrophysics Data System (ADS)

Grotepaß, T.; Förster-Zügel, F.; Mößinger, H.; Schlaak, H. F.

2015-04-01

Multilayer dielectric elastomer stack transducers (DESTs) are a promising new transducer technology with many applications in different industry sectors, like medical devices, human-machine-interaction, etc. Stacked dielectric elastomer transducers show larger thickness contraction driven by lower voltages than transducers made from a single dielectric layer. Traditionally multilayered DESTs are produced by repeatedly cross-linking a liquid elastomeric pre-polymer into the required shape. Our recent research focusses on a novel fabrication method for large scale stack transducers with a surface area over 200 x 300 mm by processing pre-fabricated elastomeric thin films of less than 50 μm thicknesses. The thin films are provided as two- or three-layer composites, where the elastomer is sandwiched between one or two sacrificial liners. Separating the elastomeric film from the residual layers and assembling them into dielectric elastomer stack transducers poses many challenges concerning adhesion, since the dielectric film merely separates from the liner if the adhesive forces between them are overcome. Conversely, during the assembly of a dielectric elastomer stack transducer, adhesive forces have to be established between two elastomeric layers or between the dielectric and the electrode layer. The very low Young's modulus of at least one adhesion partner requires suitable means of increasing the adhesive forces between the different adhesive layers of a dielectric elastomer stack transducer to prevent a delamination of the transducer during its lifetime. This work evaluates different surface activation treatments - corona, low-pressure plasma and UV-light - and their applicability in the production of large scale DESTs made from pre-fabricated elastomeric films.

Optomechanical stability design of space optical mapping camera

NASA Astrophysics Data System (ADS)

Li, Fuqiang; Cai, Weijun; Zhang, Fengqin; Li, Na; Fan, Junjie

2018-01-01

According to the interior orientation elements and imaging quality requirements of mapping application to mapping camera and combined with off-axis three-mirror anastigmat(TMA) system, high optomechanical stability design of a space optical mapping camera is introduced in this paper. The configuration is a coaxial TMA system used in off-axis situation. Firstly, the overall optical arrangement is described., and an overview of the optomechanical packaging is provided. Zerodurglass, carbon fiber composite and carbon-fiber reinforced silicon carbon (C/SiC) are widely used in the optomechanical structure, because their low coefficient of thermal expansion (CTE) can reduce the thermal sensitivity of the mirrors and focal plane. Flexible and unloading support are used in reflector and camera supporting structure. Epoxy structural adhesives is used for bonding optics to metal structure is also introduced in this paper. The primary mirror is mounted by means of three-point ball joint flexures system, which is attach to the back of the mirror. Then, In order to predict flexural displacements due to gravity, static finite element analysis (FEA) is performed on the primary mirror. The optical performance peak-to-valley (PV) and root-mean-square (RMS) wavefront errors are detected before and after assemble. Also, the dynamic finite element analysis(FEA) of the whole optical arrangement is carried out as to investigate the performance of optomechanical. Finally, in order to evaluate the stability of the design, the thermal vacuum test and vibration test are carried out and the Modulation Transfer Function (MTF) and elements of interior orientation are presented as the evaluation index. Before and after the thermal vacuum test and vibration test, the MTF, focal distance and position of the principal point of optical system are measured and the result is as expected.

16 CFR 1512.16 - Requirements for reflectors.

Code of Federal Regulations, 2011 CFR

2011-01-01

..., preferred assembly method that shall insure that the reflector meets the optical requirements of this...(m)(2). The reflectors and/or mounts shall incorporate a distinct, preferred assembly method that... on the rim. (2) If the retroreflective material is applied to the rim in the form of a self-adhesive...

The development of autoclave processable, thermally stable adhesives for titanium alloy and graphite composite structures

NASA Technical Reports Server (NTRS)

Vaughan, R. W.; Jones, R. J.

1971-01-01

The A-type polyimide adhesive resin P11B was modified by use of mixed diamines (thio-dianiline and meta phenylene diamine) which provided the desired autoclave processability. This new resin was termed P11BA. It was shown that copolymeric blends of P11BA and Amoco AI-1137 amide-imide resin provided improved adhesive properties when autoclave processed over the properties obtained previously by press bonding with P11B based copolymeric blended adhesives. Properties of bonded assemblies are presented for long-term aging at both elevated and low temperatures, and also stress-rupture tests at elevated temperature.

Modeling cell-substrate de-adhesion dynamics under fluid shear

NASA Astrophysics Data System (ADS)

Maan, Renu; Rani, Garima; Menon, Gautam I.; Pullarkat, Pramod A.

2018-07-01

Changes in cell-substrate adhesion are believed to signal the onset of cancer metastasis, but such changes must be quantified against background levels of intrinsic heterogeneity between cells. Variations in cell-substrate adhesion strengths can be probed through biophysical measurements of cell detachment from substrates upon the application of an external force. Here, we investigate, theoretically and experimentally, the detachment of cells adhered to substrates when these cells are subjected to fluid shear. We present a theoretical framework within which we calculate the fraction of detached cells as a function of shear stress for fast ramps as well as the decay in this fraction at fixed shear stress as a function of time. Using HEK and 3T3 fibroblast cells as experimental model systems, we extract characteristic force scales for cell adhesion as well as characteristic detachment times. We estimate force-scales of  ∼500 pN associated to a single focal contact, and characteristic time-scales of s representing cell-spread-area dependent mean first passage times to the detached state at intermediate values of the shear stress. Variations in adhesion across cell types are especially prominent when cell detachment is probed by applying a time-varying shear stress. These methods can be applied to characterizing changes in cell adhesion in a variety of contexts, including metastasis.

Quantifying the effect of electric current on cell adhesion studied by single-cell force spectroscopy.

PubMed

Jaatinen, Leena; Young, Eleanore; Hyttinen, Jari; Vörös, János; Zambelli, Tomaso; Demkó, László

2016-03-20

This study presents the effect of external electric current on the cell adhesive and mechanical properties of the C2C12 mouse myoblast cell line. Changes in cell morphology, viability, cytoskeleton, and focal adhesion structure were studied by standard staining protocols, while single-cell force spectroscopy based on the fluidic force microscopy technology provided a rapid, serial quantification and detailed analysis of cell adhesion and its dynamics. The setup allowed measurements of adhesion forces up to the μN range, and total detachment distances over 40 μm. Force-distance curves have been fitted with a simple elastic model including a cell detachment protocol in order to estimate the Young's modulus of the cells, as well as to reveal changes in the dynamic properties as functions of the applied current dose. While the cell spreading area decreased monotonously with increasing current doses, small current doses resulted only in differences related to cell elasticity. Current doses above 11 As/m(2), however, initiated more drastic changes in cell morphology, viability, cellular structure, as well as in properties related to cell adhesion. The observed differences, eventually leading to cell death toward higher doses, might originate from both the decrease in pH and the generation of reactive oxygen species.

Ramifications of kinetic partitioning on usher-mediated pilus biogenesis.

PubMed Central

Saulino, E T; Thanassi, D G; Pinkner, J S; Hultgren, S J

1998-01-01

The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperone-adhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone-adhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly. PMID:9545231

Computational simulation of formin-mediated actin polymerization predicts homologue-dependent mechanosensitivity.

PubMed

Bryant, Derek; Clemens, Lara; Allard, Jun

2017-01-01

Many actin structures are nucleated and assembled by the barbed-end tracking polymerase formin family, including filopodia, focal adhesions, the cytokinetic ring and cell cortex. These structures respond to forces in distinct ways. Formins typically have profilin-actin binding sites embedded in highly flexible disordered FH1 domains, hypothesized to diffusively explore space to rapidly capture actin monomers for delivery to the barbed end. Recent experiments demonstrate that formin-mediated polymerization accelerates when under tension. The acceleration has been attributed to modifying the state of the FH2 domain of formin. Intriguingly, the same acceleration is reported when tension is applied to the FH1 domains, ostensibly pulling monomers away from the barbed end. Here we develop a mesoscale coarse-grain model of formin-mediated actin polymerization, including monomer capture and delivery by FH1, which sterically interacts with actin along its entire length. The binding of actin monomers to their specific sites on FH1 is entropically disfavored by the high disorder. We find that this penalty is attenuated when force is applied to the FH1 domain by revealing the binding site, increasing monomer capture efficiency. Overall polymerization rates can decrease or increase with increasing force, depending on the length of FH1 domain and location of binding site. Our results suggest that the widely varying FH1 lengths and binding site locations found in known formins could be used to differentially respond to force, depending on the actin structure being assembled. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Integrative systems and synthetic biology of cell-matrix adhesion sites.

PubMed

Zamir, Eli

2016-09-02

The complexity of cell-matrix adhesion convolves its roles in the development and functioning of multicellular organisms and their evolutionary tinkering. Cell-matrix adhesion is mediated by sites along the plasma membrane that anchor the actin cytoskeleton to the matrix via a large number of proteins, collectively called the integrin adhesome. Fundamental challenges for understanding how cell-matrix adhesion sites assemble and function arise from their multi-functionality, rapid dynamics, large number of components and molecular diversity. Systems biology faces these challenges in its strive to understand how the integrin adhesome gives rise to functional adhesion sites. Synthetic biology enables engineering intracellular modules and circuits with properties of interest. In this review I discuss some of the fundamental questions in systems biology of cell-matrix adhesion and how synthetic biology can help addressing them.

Surface wettability of plasma SiOx:H nanocoating-induced endothelial cells' migration and the associated FAK-Rho GTPases signalling pathways

PubMed Central

Shen, Yang; Wang, Guixue; Huang, Xianliang; Zhang, Qin; Wu, Jiang; Tang, Chaojun; Yu, Qingsong; Liu, Xiaoheng

2012-01-01

Vascular endothelial cell (EC) adhesion and migration are essential processes in re-endothelialization of implanted biomaterials. There is no clear relationship and mechanism between EC adhesion and migration behaviour on surfaces with varying wettabilities. As model substrates, plasma SiOx:H nanocoatings with well-controlled surface wettability (with water contact angles in the range of 98.5 ± 2.3° to 26.3 ± 4.0°) were used in this study to investigate the effects of surface wettability on cell adhesion/migration and associated protein expressions in FAK-Rho GTPases signalling pathways. It was found that EC adhesion/migration showed opposite behaviour on the hydrophilic and hydrophobic surfaces (i.e. hydrophobic surfaces promoted EC migration but were anti-adhesions). The number of adherent ECs showed a maximum on hydrophilic surfaces, while cells adhered to hydrophobic surfaces exhibited a tendency for cell migration. The focal adhesion kinase (FAK) inhibitor targeting the Y-397 site of FAK could significantly inhibit cell adhesion/migration, suggesting that EC adhesion and migration on surfaces with different wettabilities involve (p)FAK and its downstream signalling pathways. Western blot results suggested that the FAK-Rho GTPases signalling pathways were correlative to EC migration on hydrophobic plasma SiOx:H surfaces, but uncertain to hydrophilic surfaces. This work demonstrated that surface wettability could induce cellular behaviours that were associated with different cellular signalling events. PMID:21715399

Perfluorocarbon induces alveolar epithelial cell response through structural and mechanical remodeling.

PubMed

André Dias, Sofia; Planus, Emmanuelle; Angely, Christelle; Lotteau, Luc; Tissier, Renaud; Filoche, Marcel; Louis, Bruno; Pelle, Gabriel; Isabey, Daniel

2018-02-15

During total liquid ventilation, lung cells are exposed to perfluorocarbon (PFC) whose chemophysical properties highly differ from standard aqueous cell feeding medium (DMEM). We herein perform a systematic study of structural and mechanical properties of A549 alveolar epithelial cells in order to characterize their response to PFC exposure, using DMEM as control condition. Changes in F-actin structure, focal adhesion density and glycocalyx distribution are evaluated by confocal fluorescent microscopy. Changes in cell mechanics and adhesion are measured by multiscale magnetic twisting cytometry (MTC). Two different microrheological models (single Voigt and power law) are used to analyze the cell mechanics characterized by cytoskeleton (CSK) stiffness and characteristic relaxation times. Cell-matrix adhesion is analyzed using a stochastic multibond deadhesion model taking into account the non-reversible character of the cell response, allowing us to quantify the adhesion weakness and the number of associated bonds. The roles of F-actin structure and glycocalyx layer are evaluated by depolymerizing F-actin and degrading glycocalyx, respectively. Results show that PFC exposure consistently induces F-actin remodeling, CSK softening and adhesion weakening. These results demonstrate that PFC triggers an alveolar epithelial cell response herein evidenced by a decay in intracellular CSK tension, an adhesion weakening and a glycocalyx layer redistribution. These PFC-induced cell adjustments are consistent with the hypothesis that cells respond to a decrease in adhesion energy at cell surface. This adhesion energy can be even further reduced in the presence of surfactant adsorbed at the cell surface.

Collagen triple helix repeat containing-1 promotes pancreatic cancer progression by regulating migration and adhesion of tumor cells.

PubMed

Park, Eun Hye; Kim, Seokho; Jo, Ji Yoon; Kim, Su Jin; Hwang, Yeonsil; Kim, Jin-Man; Song, Si Young; Lee, Dong-Ki; Koh, Sang Seok

2013-03-01

Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein involved in vascular remodeling, bone formation and developmental morphogenesis. CTHRC1 has recently been shown to be expressed in human cancers such as breast cancer and melanoma. In this study, we show that CTHRC1 is highly expressed in human pancreatic cancer tissues and plays a role in the progression and metastasis of the disease. CTHRC1 promoted primary tumor growth and metastatic spread of cancer cells to distant organs in orthotopic xenograft tumor mouse models. Overexpression of CTHRC1 in cancer cells resulted in increased motility and adhesiveness, whereas these cellular activities were diminished by down-regulation of the protein. CTHRC1 activated several key signaling molecules, including Src, focal adhesion kinase, paxillin, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase and Rac1. Treatment with chemical inhibitors of Src, MEK or Rac1 and expression of dominant-negative Rac1 attenuated CTHRC1-induced cell migration and adhesion. Collectively, our results suggest that CTHRC1 has a role in pancreatic cancer progression and metastasis by regulating migration and adhesion activities of cancer cells.

Hypoxia-inducible factor regulates alphavbeta3 integrin cell surface expression.

PubMed

Cowden Dahl, Karen D; Robertson, Sarah E; Weaver, Valerie M; Simon, M Celeste

2005-04-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of alpha and beta aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt(-/-) and Hifalpha(-/-) TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin alphavbeta3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O(2)). Culturing B16F0 melanoma cells at 1.5% O(2) resulted in increased alphavbeta3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O(2) tension influence placental invasion and tumor migration by increasing cell surface expression of alphavbeta3 integrin.

Hypoxia-inducible Factor Regulates αvβ3 Integrin Cell Surface Expression

PubMed Central

Cowden Dahl, Karen D.; Robertson, Sarah E.; Weaver, Valerie M.; Simon, M. Celeste

2005-01-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of α and β aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt-/- and Hifα-/- TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin αvβ3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O2). Culturing B16F0 melanoma cells at 1.5% O2 resulted in increased αvβ3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O2 tension influence placental invasion and tumor migration by increasing cell surface expression of αvβ3 integrin. PMID:15689487

Computational Modeling of Tissue Self-Assembly

NASA Astrophysics Data System (ADS)

Neagu, Adrian; Kosztin, Ioan; Jakab, Karoly; Barz, Bogdan; Neagu, Monica; Jamison, Richard; Forgacs, Gabor

As a theoretical framework for understanding the self-assembly of living cells into tissues, Steinberg proposed the differential adhesion hypothesis (DAH) according to which a specific cell type possesses a specific adhesion apparatus that combined with cell motility leads to cell assemblies of various cell types in the lowest adhesive energy state. Experimental and theoretical efforts of four decades turned the DAH into a fundamental principle of developmental biology that has been validated both in vitro and in vivo. Based on computational models of cell sorting, we have developed a DAH-based lattice model for tissues in interaction with their environment and simulated biological self-assembly using the Monte Carlo method. The present brief review highlights results on specific morphogenetic processes with relevance to tissue engineering applications. Our own work is presented on the background of several decades of theoretical efforts aimed to model morphogenesis in living tissues. Simulations of systems involving about 105 cells have been performed on high-end personal computers with CPU times of the order of days. Studied processes include cell sorting, cell sheet formation, and the development of endothelialized tubes from rings made of spheroids of two randomly intermixed cell types, when the medium in the interior of the tube was different from the external one. We conclude by noting that computer simulations based on mathematical models of living tissues yield useful guidelines for laboratory work and can catalyze the emergence of innovative technologies in tissue engineering.

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

Apigenin Attenuates Melanoma Cell Migration by Inducing Anoikis through Integrin and Focal Adhesion Kinase Inhibition.

PubMed

Hasnat, Md Abul; Pervin, Mehnaz; Lim, Ji Hong; Lim, Beong Ou

2015-11-27

Apigenin, a nonmutagenic flavonoid, has been found to have antitumor properties and is therefore particularly relevant for the development of chemotherapeutic agents for cancers. In this study, time- and dose-dependent cell viability and cytotoxicity were assessed to determine the effects of apigenin on A2058 and A375 melanoma cells. Melanoma cells were pretreated with different concentrations of apigenin and analyzed for morphological changes, anoikis induction, cell migration, and levels of proteins associated with apoptosis. Apigenin reduced integrin protein levels and inhibited the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK1/2), which induce anoikis in human cutaneous melanoma cells. Apigenin exhibited dose-dependent inhibition of melanoma cell migration, unlike untreated controls. Furthermore, apigenin treatment increased apoptotic factors such as caspase-3 and cleaved poly(ADP-ribose) polymerase in a dose-dependent manner, demonstrating the metastasis of melanoma cells. Our results provide a new insight into the mechanisms by which apigenin prevents melanoma metastasis by sensitizing anoikis induced by the loss of integrin proteins in the FAK/ERK1/2 signaling pathway. These findings elucidate the related mechanisms and suggest the potential of apigenin in developing clinical treatment strategies against malignant melanoma.

c-Src activity is differentially required by cancer cell motility modes.

PubMed

Logue, Jeremy S; Cartagena-Rivera, Alexander X; Chadwick, Richard S

2018-04-01

Cancer cell migration requires that cells respond and adapt to their surroundings. In the absence of extracellular matrix cues, cancer cells will undergo a mesenchymal to ameboid transition, whereas a highly confining space will trigger a switch to "leader bleb-based" migration. To identify oncogenic signaling pathways mediating these transitions, we undertook a targeted screen using clinically useful inhibitors. Elevated Src activity was found to change actin and focal adhesion dynamics, whereas inhibiting Src triggered focal adhesion disassembly and blebbing. On non-adherent substrates and in collagen matrices, amoeboid-like, blebbing cells having high Src activity formed protrusions of the plasma membrane. To evaluate the role of Src in confined cells, we use a novel approach that places cells under a slab of polydimethylsiloxane (PDMS), which is held at a defined height. Using this method, we find that leader bleb-based migration is resistant to Src inhibition. High Src activity was found to markedly change the architecture of cortical actomyosin, reduce cell mechanical properties, and the percentage of cells that undergo leader bleb-based migration. Thus, Src is a signal transducer that can potently influence transitions between migration modes with implications for the rational development of metastasis inhibitors.

Atomic force-multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells.

PubMed

Trache, Andreea; Meininger, Gerald A

2005-01-01

A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.

Decoy receptor 3 promotes cell adhesion and enhances endometriosis development.

PubMed

Tsai, Hsiao-Wen; Huang, Ming-Ting; Wang, Peng-Hui; Huang, Ben-Shian; Chen, Yi-Jen; Hsieh, Shie-Liang

2018-02-01

Endometriosis is a multifactorial inflammatory disease with persistent activation of the nuclear factor-κB (NF-κB) signalling pathway. Aberrant adhesion of endometrium is the essential step in the progression of endometriosis, but the molecular mechanism of ectopic growth of endometrium is still unclear. Decoy receptor 3 (DcR3)/TNFRSF6B, a pleiotropic immunomodulator regulated by oestrogen, is able to activate focal adhesion kinase to promote cell adhesion. We found that DcR3 is upregulated in human ectopic endometrial cells via activation of the Akt-NF-κB signalling pathway, and its expression level correlates positively with that of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and homing cell adhesion molecule (HCAM; CD44). In a multivariate regression model, DcR3 expression level was the most significant parameter associated with endometriosis severity. Knockdown of DcR3 not only downregulated the expression of ICAM-1 and HCAM, but also reduced cell adhesion and migration. In vivo investigation further showed that DcR3 promoted the growth and spread of endometrium, whereas knockdown of DcR3 by lentivirus-delivered short hairpin RNA inhibited ectopic adhesion of endometrium and abrogated endometriosis progression. These observations are in support of DcR3 playing a critical role in the pathogenesis of endometriosis, and the inhibition of DcR3 expression being a promising approach for the treatment of endometriosis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Simultaneous bond degradation and bond formation during phenol-formaldehyde curing with wood

Treesearch

Daniel J. Yelle; John Ralph

2016-01-01

Bonding of wood using phenol–formaldehyde adhesive develops highly durable bonds. Phenol– formaldehyde is believed to form primary bonds with wood cell wall polymers (e.g., lignin). However, it is unclear how this adhesive interacts and bonds to lignin. Through wood solubilisation methodologies, earlywood and latewood bonded assemblies were characterized using two-...

Methods and Devices for Space Optical Communications Using Laser Beams

NASA Technical Reports Server (NTRS)

Goorjian, Peter M. (Inventor)

2018-01-01

Light is used to communicate between objects separated by a large distance. Light beams are received in a telescopic lens assembly positioned in front of a cat's-eye lens. The light can thereby be received at various angles to be output by the cat's-eye lens to a focal plane of the cat's-eye lens, the position of the light beams upon the focal plane corresponding to the angle of the beam received. Lasers and photodetectors are distributed along this focal plane. A processor receives signals from the photodetectors, and selectively signal lasers positioned proximate the photodetectors detecting light, in order to transmit light encoding data through the cat's-eye lens and also through a telescopic lens back in the direction of the received light beams, which direction corresponds to a location upon the focal plane of the transmitting lasers.

An Extraordinary Sulfonated-Graphenal-Polymer-Based Electrolyte Separator for All-Solid-State Supercapacitors.

PubMed

Liu, Xubo; Men, Chuanling; Zhang, Xiaohua; Li, Qingwen

2016-09-01

Sulfonated graphenal polymers can be assembled up by poly(vinyl alcohol) adhesion. The porous assembly structure results in a remarkably improved ionic conductivity and thus enhances electrochemical performances such as specific capacitance, capacitance retention, and cycling stability. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

16 CFR 1631.1 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

..., adhesives, and which has no dimension greater than 1.83 m. (6 ft.) and an area not greater than 2.23 m. 2 (24 sq. ft.). Products such as “Carpet Squares” with dimensions smaller than these but intended to be assembled, upon installation, into assemblies which may have dimensions greater than these, are excluded...

Force Generation via β-Cardiac Myosin, Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions.

PubMed

Chopra, Anant; Kutys, Matthew L; Zhang, Kehan; Polacheck, William J; Sheng, Calvin C; Luu, Rebeccah J; Eyckmans, Jeroen; Hinson, J Travis; Seidman, Jonathan G; Seidman, Christine E; Chen, Christopher S

2018-01-08

Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that β-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from β-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Micro-masonry for 3D Additive Micromanufacturing

PubMed Central

Keum, Hohyun; Kim, Seok

2014-01-01

Transfer printing is a method to transfer solid micro/nanoscale materials (herein called ‘inks’) from a substrate where they are generated to a different substrate by utilizing elastomeric stamps. Transfer printing enables the integration of heterogeneous materials to fabricate unexampled structures or functional systems that are found in recent advanced devices such as flexible and stretchable solar cells and LED arrays. While transfer printing exhibits unique features in material assembly capability, the use of adhesive layers or the surface modification such as deposition of self-assembled monolayer (SAM) on substrates for enhancing printing processes hinders its wide adaptation in microassembly of microelectromechanical system (MEMS) structures and devices. To overcome this shortcoming, we developed an advanced mode of transfer printing which deterministically assembles individual microscale objects solely through controlling surface contact area without any surface alteration. The absence of an adhesive layer or other modification and the subsequent material bonding processes ensure not only mechanical bonding, but also thermal and electrical connection between assembled materials, which further opens various applications in adaptation in building unusual MEMS devices. PMID:25146178

A method to screen and evaluate tissue adhesives for joint repair applications

PubMed Central

2012-01-01

Background Tissue adhesives are useful means for various medical procedures. Since varying requirements cause that a single adhesive cannot meet all needs, bond strength testing remains one of the key applications used to screen for new products and study the influence of experimental variables. This study was conducted to develop an easy to use method to screen and evaluate tissue adhesives for tissue engineering applications. Method Tissue grips were designed to facilitate the reproducible production of substrate tissue and adhesive strength measurements in universal testing machines. Porcine femoral condyles were used to generate osteochondral test tissue cylinders (substrates) of different shapes. Viability of substrates was tested using PI/FDA staining. Self-bonding properties were determined to examine reusability of substrates (n = 3). Serial measurements (n = 5) in different operation modes (OM) were performed to analyze the bonding strength of tissue adhesives in bone (OM-1) and cartilage tissue either in isolation (OM-2) or under specific requirements in joint repair such as filling cartilage defects with clinical applied fibrin/PLGA-cell-transplants (OM-3) or tissues (OM-4). The efficiency of the method was determined on the basis of adhesive properties of fibrin glue for different assembly times (30 s, 60 s). Seven randomly generated collagen formulations were analyzed to examine the potential of method to identify new tissue adhesives. Results Viability analysis of test tissue cylinders revealed vital cells (>80%) in cartilage components even 48 h post preparation. Reuse (n = 10) of test substrate did not significantly change adhesive characteristics. Adhesive strength of fibrin varied in different test settings (OM-1: 7.1 kPa, OM-2: 2.6 kPa, OM-3: 32.7 kPa, OM-4: 30.1 kPa) and was increasing with assembly time on average (2.4-fold). The screening of the different collagen formulations revealed a substance with significant higher adhesive strength on cartilage (14.8 kPa) and bone tissue (11.8 kPa) compared to fibrin and also considerable adhesive properties when filling defects with cartilage tissue (23.2 kPa). Conclusion The method confirmed adhesive properties of fibrin and demonstrated the dependence of adhesive properties and applied settings. Furthermore the method was suitable to screen for potential adhesives and to identify a promising candidate for cartilage and bone applications. The method can offer simple, replicable and efficient evaluation of adhesive properties in ex vivo specimens and may be a useful supplement to existing methods in clinical relevant settings. PMID:22984926

In Silico Analysis of Usher Encoding Genes in Klebsiella pneumoniae and Characterization of Their Role in Adhesion and Colonization

PubMed Central

Khater, Fida; Balestrino, Damien; Charbonnel, Nicolas; Dufayard, Jean François; Brisse, Sylvain; Forestier, Christiane

2015-01-01

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiella pneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and ϭ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches. PMID:25751658

TNFα pathway blockade ameliorates toxic effects of FSGS plasma on podocyte cytoskeleton and β3 integrin activation.

PubMed

Bitzan, Martin; Babayeva, Sima; Vasudevan, Anil; Goodyer, Paul; Torban, Elena

2012-12-01

In the absence of mutant genes encoding components of the podocyte slit diaphragm, about 30-50 % of children with primary glucocorticoid-resistant focal segmental glomerulosclerosis (FSGS) develop recurrent proteinuria and slowly progressive FSGS lesions following renal transplantation. Recurrence of FSGS in the allograft strongly suggests a circulating factor that disturbs normal podocyte biology. To date, the nature of the circulating factor is unclear, and there is no cure for the recurrent form of FSGS (R-FSGS). Cultured differentiated human podocytes were exposed to the plasmapheresis effluent or blood plasma samples from pediatric patients with recurrent or primary FSGS; in some cases, podocytes were pre-incubated with specific antibodies to block the tumor necrosis factor-alpha (TNFα) signaling pathway. Integrity of focal adhesion complexes and actin cytoskeleton were investigated by immunofluorescent microscopy. Plasmapheresis effluent from an R-FSGS child or fresh plasma from two children with primary FSGS rapidly disturbed the cytoskeleton of normal human podocytes in vitro. Plasma from a child with R-FSGS also activated β3 integrin and dispersed focal adhesion complexes. The effects were reversed by pre-incubation with antibodies against TNFα or either of the two TNFα receptors. When our patient with R-FSGS became resistant to plasmapheresis, we initiated treatment with twice weekly etanercept injections and then infliximab. Within 3 weeks of regular anti-TNFα therapy, the patient achieved sustained partial remission of proteinuria, allowing us to wean her off plasmapheresis completely. We suggest that in some FSGS patients, disruption of the podocyte cytoskeleton and β3 integrin-mediated podocyte attachment are driven by the TNFα pathway.

Pulse-echo NDT of adhesively bonded joints in automotive assemblies.

PubMed

Titov, Sergey A; Maev, Roman Gr; Bogachenkov, Alexey N

2008-11-01

A new method for the detection of void-disbonds at the interfaces of adhesively bonded joins is considered. Based on a simple plane wave model, the output waveform is presented as a sum of two responses associated with the reflection of the ultrasonic wave at the first metal-adhesive interface and the second metal-adhesive interface, respectively. The strong response produced by the wave reverberating in the first metal sheet is eliminated through comparison between the pulse-echo signal measured at the area under the test and reference waveform recorded for the bare first metal sheet outside of the joint. The developed decomposition algorithm has been applied to the study of steel and aluminum samples having various adhesive layer thicknesses in a range of 0.1-1mm.

In vivo quantitative analysis of Talin turnover in response to force

PubMed Central

Hákonardóttir, Guðlaug Katrín; López-Ceballos, Pablo; Herrera-Reyes, Alejandra Donají; Das, Raibatak; Coombs, Daniel; Tanentzapf, Guy

2015-01-01

Cell adhesion to the extracellular matrix (ECM) allows cells to form and maintain three-dimensional tissue architecture. Cell–ECM adhesions are stabilized upon exposure to mechanical force. In this study, we used quantitative imaging and mathematical modeling to gain mechanistic insight into how integrin-based adhesions respond to increased and decreased mechanical forces. A critical means of regulating integrin-based adhesion is provided by modulating the turnover of integrin and its adhesion complex (integrin adhesion complex [IAC]). The turnover of the IAC component Talin, a known mechanosensor, was analyzed using fluorescence recovery after photobleaching. Experiments were carried out in live, intact flies in genetic backgrounds that increased or decreased the force applied on sites of adhesion. This analysis showed that when force is elevated, the rate of assembly of new adhesions increases such that cell–ECM adhesion is stabilized. Moreover, under conditions of decreased force, the overall rate of turnover, but not the proportion of adhesion complex components undergoing turnover, increases. Using point mutations, we identify the key functional domains of Talin that mediate its response to force. Finally, by fitting a mathematical model to the data, we uncover the mechanisms that mediate the stabilization of ECM-based adhesion during development. PMID:26446844

Neuron-like differentiation of mesenchymal stem cells on silicon nanowires

NASA Astrophysics Data System (ADS)

Kim, Hyunju; Kim, Ilsoo; Choi, Heon-Jin; Kim, So Yeon; Yang, Eun Gyeong

2015-10-01

The behavior of mammalian cells on vertical nanowire (NW) arrays, including cell spreading and the dynamic distribution of focal adhesions and cytoskeletal proteins, has been intensively studied to extend the implications for cellular manipulations in vitro. Prompted by the result that cells on silicon (Si) NWs showed morphological changes and reduced migration rates, we have explored the transition of mesenchymal stem cells into a neuronal lineage by using SiNWs with varying lengths. When human mesenchymal stem cells (hMSCs) were cultured on the longest SiNWs for 3 days, most of the cells exhibited elongated shapes with neurite-like extensions and dot-like focal adhesions that were prominently observed along with actin filaments. Under these circumstances, the cell motility analyzed by live cell imaging was found to decrease due to the presence of SiNWs. In addition, the slowed growth rate, as well as the reduced population of S phase cells, suggested that the cell cycle was likely arrested in response to the differentiation process. Furthermore, we measured the mRNA levels of several lineage-specific markers to confirm that the SiNWs actually induced neuron-like differentiation of the hMSCs while hampering their osteogenic differentiation. Taken together, our results implied that SiNWs were capable of inducing active reorganization of cellular behaviors, collectively guiding the fate of hMSCs into the neural lineage even in the absence of any inducing reagent.The behavior of mammalian cells on vertical nanowire (NW) arrays, including cell spreading and the dynamic distribution of focal adhesions and cytoskeletal proteins, has been intensively studied to extend the implications for cellular manipulations in vitro. Prompted by the result that cells on silicon (Si) NWs showed morphological changes and reduced migration rates, we have explored the transition of mesenchymal stem cells into a neuronal lineage by using SiNWs with varying lengths. When human mesenchymal stem cells (hMSCs) were cultured on the longest SiNWs for 3 days, most of the cells exhibited elongated shapes with neurite-like extensions and dot-like focal adhesions that were prominently observed along with actin filaments. Under these circumstances, the cell motility analyzed by live cell imaging was found to decrease due to the presence of SiNWs. In addition, the slowed growth rate, as well as the reduced population of S phase cells, suggested that the cell cycle was likely arrested in response to the differentiation process. Furthermore, we measured the mRNA levels of several lineage-specific markers to confirm that the SiNWs actually induced neuron-like differentiation of the hMSCs while hampering their osteogenic differentiation. Taken together, our results implied that SiNWs were capable of inducing active reorganization of cellular behaviors, collectively guiding the fate of hMSCs into the neural lineage even in the absence of any inducing reagent. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05787f

Biophysics of cadherin adhesion.

PubMed

Leckband, Deborah; Sivasankar, Sanjeevi

2012-01-01

Since the identification of cadherins and the publication of the first crystal structures, the mechanism of cadherin adhesion, and the underlying structural basis have been studied with a number of different experimental techniques, different classical cadherin subtypes, and cadherin fragments. Earlier studies based on biophysical measurements and structure determinations resulted in seemingly contradictory findings regarding cadherin adhesion. However, recent experimental data increasingly reveal parallels between structures, solution binding data, and adhesion-based biophysical measurements that are beginning to both reconcile apparent differences and generate a more comprehensive model of cadherin-mediated cell adhesion. This chapter summarizes the functional, structural, and biophysical findings relevant to cadherin junction assembly and adhesion. We emphasize emerging parallels between findings obtained with different experimental approaches. Although none of the current models accounts for all of the available experimental and structural data, this chapter discusses possible origins of apparent discrepancies, highlights remaining gaps in current knowledge, and proposes challenges for further study.

Preparation and regulating cell adhesion of anion-exchangeable layered double hydroxide micropatterned arrays.

PubMed

Yao, Feng; Hu, Hao; Xu, Sailong; Huo, Ruijie; Zhao, Zhiping; Zhang, Fazhi; Xu, Fujian

2015-02-25

We describe a reliable preparation of MgAl-layered double hydroxide (MgAl-LDH) micropatterned arrays on gold substrate by combining SO3(-)-terminated self-assembly monolayer and photolithography. The synthesis route is readily extended to prepare LDH arrays on the SO3(-)-terminated polymer-bonded glass substrate amenable for cell imaging. The anion-exchangeable MgAl-LDH micropattern can act both as bioadhesive region for selective cell adhesion and as nanocarrier for drug molecules to regulate cell behaviors. Quantitative analysis of cell adhesion shows that selective HepG2 cell adhesion and spreading are promoted by the micropatterned MgAl-LDH, and also suppressed by methotrexate drug released from the LDH interlayer galleries.

Epoxy Adhesives for Stator Magnet Assembly in Stirling Radioisotope Generators (SRG)

NASA Technical Reports Server (NTRS)

Cater, George M.

2004-01-01

As NASA seeks to fulfill its goals of exploration and understanding through missions planned to visit the moons of Saturn and beyond, a number of challenges arise from the idea of deep space flight. One of the first problems associated with deep space travel is electrical power production for systems on the spacecraft. Conventional methods such as solar power are not practical because efficiency decreases substantially as the craft moves away from the Sun. The criterion for power generation during deep space missions are very specific, the main points requiring high reliability, low mass, minimal vibration and a long lifespan. A Stirling generator, although fairly old in concept, is considered to be a potential solution for electrical power generation for deep space flight. A Stirling generator works on the same electromagnetic principles of a standard generator, using the linear motion of the alternator through the stationary stator which produces electric induction. The motion of the alternator, however, is produced by the heating and cooling dynamics of pressurized gases. Essentially heating one end and cooling another of a contained gas will cause a periodic expansion and compression of the gas from one side to the other, which a displacer translates into linear mechanical motion. NASA needs to confirm that the materials used in the generator will be able to withstand the rigors of space and the life expectancy of the mission. I am working on the verification of the epoxy adhesives used to bond magnets to the steel lamination stack to complete the stator; in terms of in-service performance and durability under various space environments. Understanding the proper curing conditions, high temperature properties, and degassing problems as well as production difficulties are crucial to the long term success of the generator. system and steel substrate used in the stator. To optimize the curing conditions of the epoxies, modulated differential scanning calorimetry analysis was done as a function of cure time and temperatures. Adhesion bond strength was tested at various temperatures with lap shear samples using Hiperco 50 substrate to ensure that the proper adhesive is being used. To try and solve the problem of bondline thickness, micro glass beads of 0.0017" in diameter were investigated to see if any other physical properties of the epoxy were affected. Efforts will be made to develop a standard, optimized, fabrication process/procedure of sub-scale magnet-stator assemblies for various adhesive performance evaluation studies under simulated generator conditions. Also, accelerated aging testing will be done in a pressurized canister with stator assembly samples for three years to verify if any degassing or thermal degradation of the epoxy occurs. The necessity of verifying the correct epoxy adhesive system for the stator magnet in the SRG is crucial because failure of the stator assembly would jeopardize the electrical system, and thereby the entire mission itself. My work involves specimen fabrications, testing, and data analyses of the epoxy adhesive system for the stator magnet in the SRG is crucial because failure of the stator assembly would jeopardize the electrical system, and thereby the entire mission itself.

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

PubMed

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

2009-10-01

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

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

PubMed Central

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

2014-01-01

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

Lasp1 gene disruption is linked to enhanced cell migration and tumor formation Address for reprint requests and other correspondence: C. S. Chew, Inst. of Molecular Medicine and Genetics, Sanders R&E Bldg., Rm. CB 2803, Medical College of Georgia, Augusta, GA 30912-3175 (e-mail: cchew@mcg.edu).

PubMed Central

Zhang, Han; Chen, Xunsheng; Bollag, Wendy B.; Bollag, Roni J.; Sheehan, Daniel J.; Chew, Catherine S.

2009-01-01

Lasp1 is an actin-binding, signaling pathway-regulated phosphoprotein that is overexpressed in several cancers. siRNA knockdown in cell lines retards cell migration, suggesting the possibility that Lasp1 upregulation influences cancer metastasis. Herein, we utilized a recently developed gene knockout model to assess the role of Lasp1 in modulating nontransformed cell functions. Wound healing and tumor initiation progressed more rapidly in Lasp1−/− mice compared with Lasp1+/+ controls. Embryonic fibroblasts (MEFs) derived from Lasp1−/− mice also migrated more rapidly in vitro. These MEFs characteristically possessed increased focal adhesion numbers and displayed more rapid attachment compared with wild-type MEFs. Differential microarray analyses revealed alterations in message expression for proteins implicated in cell migration, adhesion, and cytoskeletal organization. Notably, the focal adhesion protein, lipoma preferred partner (LPP), a zyxin family member and putative Lasp1 binding protein, was increased about twofold. Because LPP gene disruption reduces cell migration, we hypothesize that LPP plays a role in enhancing the migratory capacity of Lasp1−/− MEFs, perhaps by modifying the subcellular localization of other motility-associated proteins. The striking contrast in the functional effects of loss of Lasp1 in innate cells compared with cell lines reveals distinct differences in mechanisms of motility and attachment in these models. PMID:19531578

Biomagnetics and Cell-Based Biochips

NASA Astrophysics Data System (ADS)

Ingber, Donald

2005-03-01

This presentation will review various micro- and nanotechnologies that we have developed over the past decade in our efforts to manipulate and probe living cells. In early studies, we used magnetic micro-particles to apply controlled mechanical forces to surface membrane receptors. We did this to probe cellular mechanical properties, and to investigate the molecular basis of mechanotransduction -- how mechanical forces are transduced into changes in intracellular biochemistry. The magnetic beads were coated with ligands for adhesion receptors, such as synthetic RGD (arginine-glycine-aspartate) peptides or antibodies that bind to membrane integrin receptors. Controlled twisting (torque) or pulling (tension) forces were exerted on the integrin-bound beads using magnetic twisting or pulling cytometry. To investigate the cellular response to dynamic forces, and to increase the level of stress applied, an electromagnetic needle was developed to apply a temporally varying magnetic field controlled by a user-defined solenoidal current; the end of the needle also was electropolished to produce a nanoscale pole tip. Magnetic forces applied to integrin receptors, but not other cell-surface receptors, induced force-dependent recruitment of cytoskeletal linker (focal adhesion) proteins to the site of bead binding, resulting in assembly and mechanical strengthening of the adhesions. Stress application to integrins also resulted in force-dependent increases in cAMP signaling and induction of gene transcription. These experiments revealed that integrins and the cytoskeleton play a central role in cellular mechanotransduction.studies in collaboration with George Whitesides (Harvard U.), we used microcontact printing techniques with self- assembled monolayers of alkanethiols to microfabricate extracellular matrix-coated adhesive islands of defined size, shape, and position on the micrometer scale. When cells were plated on these islands, the spread to take on the form of the island. These studies revealed that cells can be switched between growth, differentiation, and death (apoptosis) by varying the degree to which a cell physically can distend. When cells grown on islands with corners (e.g., squares, triangles) were stimulated with motility factors, the cells preferentially extended new motile processes from the corner regions, whereas cells on circular islands showed no bias. These findings demonstrated that much of cell behavior is controlled through physical interactions between cells and their adhesive substrate, and that microfabrication methods may be useful for tissue engineering, as well as creation of ``laboratories on a chip'' or biosensor devices that incorporate living mammalian cells. addition, in experiments with Bob Westervelt and Donhee Ham (Harvard U.), we have demonstrated the feasilibility of using microelectromagnetic circuits and CMOS technology to physically pull cells out from medium magnetically, and to move them in a directed manner. This approach may have great value for cell separation applications. Finally, with Whitesides group, we also demonstrated that microfluidics technologies may be used to deliver chemicals or probes to different regions of the same living cell under flow conditions. This provides a novel way to create chemical gradients at the subcellular scale and thereby probe the relation between cell structure and function. We also are currently exploring novel uses of microfluidics technologies, including their application for clinical cell separation applications. Taken together, this body of his work clearly demonstrates the great value of microsystem and microfluidic approaches for the analysis and manipulation of living cells. These approaches may have great value, both for fundamental scientific research and for clinical applications.

Tuning cell adhesive properties via layer-by-layer assembly of chitosan and alginate

PubMed Central

Silva, Joana M.; García, José R.; Reis, Rui L.; García, Andrés J.; Mano, João F.

2017-01-01

Understanding the mechanisms controlling cell-multilayer film interactions is crucial to the successful engineering of these coatings for biotechnological and biomedical applications. Herein, we present a strategy to tune the cell adhesive properties of multilayers based on marine polysaccharides with and without cross-linking and/or coating with extracellular matrix proteins. Chemical cross-linking of multilayers improved mechanical properties of the coatings but also elicited changes in surface chemistry that alter the adhesion of human umbilical vein endothelial cells. We evaluated a strategy to decouple the mechanical and chemical properties of these films, enabling the transition from cell-adhesive to cell-resistant multilayers. Addition of chitosan/alginate multilayers on top of cross-linked films decreased endothelial cell adhesion, spreading, and proliferation to similar levels as uncross-linked films. Our findings highlight the key role of surface chemistry in cell-multilayer film interactions, and these engineered nanocoatings represent a tunable model of cell adhesive and non-adhesive multilayered films. PMID:28126597

Cell-cell adhesion in the cnidaria: insights into the evolution of tissue morphogenesis.

PubMed

Magie, Craig R; Martindale, Mark Q

2008-06-01

Cell adhesion is a major aspect of cell biology and one of the fundamental processes involved in the development of a multicellular animal. Adhesive mechanisms, both cell-cell and between cell and extracellular matrix, are intimately involved in assembling cells into the three-dimensional structures of tissues and organs. The modulation of adhesive complexes could therefore be seen as a central component in the molecular control of morphogenesis, translating information encoded within the genome into organismal form. The availability of whole genomes from early-branching metazoa such as cnidarians is providing important insights into the evolution of adhesive processes by allowing for the easy identification of the genes involved in adhesion in these organisms. Discovery of the molecular nature of cell adhesion in the early-branching groups, coupled with comparisons across the metazoa, is revealing the ways evolution has tinkered with this vital cellular process in the generation of the myriad forms seen across the animal kingdom.

Enterolactone alters FAK-Src signaling and suppresses migration and invasion of lung cancer cell lines.

PubMed

Chikara, Shireen; Lindsey, Kaitlin; Borowicz, Pawel; Christofidou-Solomidou, Melpo; Reindl, Katie M

2017-01-09

Systemic toxicity of chemotherapeutic agents and the challenges associated with targeting metastatic tumors are limiting factors for current lung cancer therapeutic approaches. To address these issues, plant-derived bioactive components have been investigated for their anti-cancer properties because many of these agents are non-toxic to healthy tissues. Enterolactone (EL) is a flaxseed-derived mammalian lignan that has demonstrated anti-migratory properties for various cancers, but EL has not been investigated in the context of lung cancer, and its anticancer mechanisms are ill-defined. We hypothesized that EL could inhibit lung cancer cell motility by affecting the FAK-Src signaling pathway. Non-toxic concentrations of EL were identified for A549 and H460 human lung cancer cells by conducting 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Dephenyltetrazolium Bromide (MTT) assays. The anti-migratory and anti-invasive potential of EL for lung cancer cell lines was determined by scratch wound healing and Matrigel® invasion assays. Changes in filamentous actin (F-actin) fiber density and length in EL-treated cells were determined using phalloidin-conjugated rhodamine dye and fluorescent microscopy. Vinculin expression in focal adhesions upon EL treatment was determined by immunocytochemistry. Gene and protein expression levels of FAK-Src signaling molecules in EL-treated lung cancer cells were determined using PCR arrays, qRT-PCR, and western blotting. Non-toxic concentrations of EL inhibited lung cancer cell migration and invasion in a concentration- and time-dependent manner. EL treatment reduced the density and number of F-actin fibers in lung cancer cell lines, and reduced the number and size of focal adhesions. EL decreased phosphorylation of FAK and its downstream targets, Src, paxillin, and decreased mRNA expression of cell motility-related genes, RhoA, Rac1, and Cdc42 in lung cancer cells. Our data suggest that EL suppresses lung cancer cell motility and invasion by altering FAK activity and subsequent activation of downstream proteins needed for focal adhesion formation and cytoskeletal rearrangement. Therefore, administration of EL may serve as a safe and complementary approach for inhibiting lung tumor cell motility, invasion, and metastasis.

16 CFR 1630.1 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... means such as nails, tacks, barbs, staples, adhesives, and which has one dimension greater than 1.83 m...”, with one dimension less than 1.83 m. (6 ft.) and a surface area less than 2.23 m.2 (24 sq. ft.), but intended to be assembled upon installation into assemblies which may have one dimension greater than 1.83 m...

Mesenchymal stem cells cultured on magnetic nanowire substrates

NASA Astrophysics Data System (ADS)

Perez, Jose E.; Ravasi, Timothy; Kosel, Jürgen

2017-02-01

Stem cells have been shown to respond to extracellular mechanical stimuli by regulating their fate through the activation of specific signaling pathways. In this work, an array of iron nanowires (NWs) aligned perpendicularly to the surface was fabricated by pulsed electrodepositon in porous alumina templates followed by a partial removal of the alumina to reveal 2-3 μm of the NWs. This resulted in alumina substrates with densely arranged NWs of 33 nm in diameter separated by 100 nm. The substrates were characterized by scanning electron microscopy (SEM) energy dispersive x-ray analysis and vibrating sample magnetometer. The NW array was then used as a platform for the culture of human mesenchymal stem cells (hMSCs). The cells were stained for the cell nucleus and actin filaments, as well as immuno-stained for the focal adhesion protein vinculin, and then observed by fluorescence microscopy in order to characterize their spreading behavior. Calcein AM/ethidium homodimer-1 staining allowed the determination of cell viability. The interface between the cells and the NWs was studied using SEM. Results showed that hMSCs underwent a re-organization of actin filaments that translated into a change from an elongated to a spherical cell shape. Actin filaments and vinculin accumulated in bundles, suggesting the attachment and formation of focal adhesion points of the cells on the NWs. Though the overall number of cells attached on the NWs was lower compared to the control, the attached cells maintained a high viability (>90%) for up to 6 d. Analysis of the interface between the NWs and the cells confirmed the re-organization of F-actin and revealed the adhesion points of the cells on the NWs. Additionally, a net of filopodia surrounded each cell, suggesting the probing of the array to find additional adhesion points. The cells maintained their round shape for up to 6 d of culture. Overall, the NW array is a promising nanostructured platform for studying and influencing hMSCs differentiation.

The molecular basis of leukocyte adhesion involving phosphatidic acid and phospholipase D.

PubMed

Speranza, Francis; Mahankali, Madhu; Henkels, Karen M; Gomez-Cambronero, Julian

2014-10-17

Defining how leukocytes adhere to solid surfaces, such as capillary beds, and the subsequent migration through the extracellular matrix, is a central biological issue. We show here that phospholipase D (PLD) and its enzymatic reaction product, phosphatidic acid (PA), regulate cell adhesion of immune cells (macrophages and neutrophils) to collagen and have defined the underlying molecular mechanism in a spatio-temporal manner that coincides with PLD activity timing. A rapid (t½ = 4 min) and transient activation of the PLD1 isoform occurs upon adhesion, and a slower (t½ = 7.5 min) but prolonged (>30 min) activation occurs for PLD2. Importantly, PA directly binds to actin-related protein 3 (Arp3) at EC50 = 22 nm, whereas control phosphatidylcholine did not bind. PA-activated Arp3 hastens actin nucleation with a kinetics of t½ = 3 min at 300 nm (compared with controls of no PA, t½ = 5 min). Thus, PLD and PA are intrinsic components of cell adhesion, which reinforce each other in a positive feedback loop and react from cues from their respective solid substrates. In nascent adhesion, PLD1 is key, whereas a sustained adhesion in mature or established focal points is dependent upon PLD2, PA, and Arp3. A prolonged adhesion could effectively counteract the reversible intrinsic nature of this cellular process and constitute a key player in chronic inflammation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Nanowell-Trapped Charged Ligand-Bearing Nanoparticle Surfaces – A Novel Method of Enhancing Flow-Resistant Cell Adhesion

PubMed Central

Tran, Phat L.; Gamboa, Jessica R.; McCracken, Katherine E.; Riley, Mark R.

2014-01-01

Assuring cell adhesion to an underlying biomaterial surface is vital in implant device design and tissue engineering, particularly under circumstances where cells are subjected to potential detachment from overriding fluid flow. Cell-substrate adhesion is a highly regulated process involving the interplay of mechanical properties, surface topographic features, electrostatic charge, and biochemical mechanisms. At the nanoscale level the physical properties of the underlying substrate are of particular importance in cell adhesion. Conventionally, natural, pro-adhesive, and often thrombogenic, protein biomaterials are frequently utilized to facilitate adhesion. In the present study nanofabrication techniques are utilized to enhance the biological functionality of a synthetic polymer surface, polymethymethacrylate, with respect to cell adhesion. Specifically we examine the effect on cell adhesion of combining: 1. optimized surface texturing, 2. electrostatic charge and 3. cell adhesive ligands, uniquely assembled on the substrata surface, as an ensemble of nanoparticles trapped in nanowells. Our results reveal that the ensemble strategy leads to enhanced, more than simply additive, endothelial cell adhesion under both static and flow conditions. This strategy may be of particular utility for enhancing flow-resistant endothelialization of blood-contacting surfaces of cardiovascular devices subjected to flow-mediated shear. PMID:23225491

Apparatus for installing condition-sensing means in subterranean earth formations

DOEpatents

Shuck, Lowell Z.

1981-01-01

The present invention is directed to an apparatus for installing strain gages or other sensors-transducers in wellbores penetrating subterranean earth formations. The subject apparatus comprises an assembly which is lowered into the wellbore, secured in place, and then actuated to sequentially clean the wellbore or casing surface at a selected location with suitable solvents, etchants and neutralizers, grind the surface to a relatively smooth finish, apply an adhesive to the surface, and attach the strain gages or the like to the adhesive-bearing surface. After installing the condition-sensing gages to the casing or earth formation the assembly is withdrawn from the wellbore leaving the sensing gages securely attached to the casing or the subterranean earth formation.

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

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering

DOE PAGES

Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K.; ...

2015-01-08

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin, which links the CD44 assembled receptor signaling complexes to the cytoskeletal actin and organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered and adopts an autoinhibited conformation, which prevents CD44ct from binding directly to activated Ezrin in solution. Binding to the signaling lipid phosphatidylinositol 4,5-biphosphlate (PIP2) disrupts autoinhibition in CD44ct, and activates CD44ct to associate with Ezrin.more » Further, using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific hetero-tetramer complex of CD44ct with Ezrin. This study reveals a novel autoregulation mechanism in the cytoplasmic tail of CD44 and the role of PIP2 in mediating the assembly of multimeric CD44ct-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of multimeric PIP2-CD44-Ezrin complexes.« less

Basic components of connective tissues and extracellular matrix: elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins.

PubMed

Halper, Jaroslava; Kjaer, Michael

2014-01-01

Collagens are the most abundant components of the extracellular matrix and many types of soft tissues. Elastin is another major component of certain soft tissues, such as arterial walls and ligaments. Many other molecules, though lower in quantity, function as essential components of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its muldomain structure plays a role of "master organizer" in matrix assembly as it forms a bridge between cell surface receptors, e.g., integrins, and compounds such collagen, proteoglycans and other focal adhesion molecules. It also plays an essential role in the assembly of fibrillin-1 into a structured network. Laminins contribute to the structure of the extracellular matrix (ECM) and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin by thrombin, it also binds to a variety of compounds, particularly to various growth factors, and as such fibrinogen is a player in cardiovascular and extracellular matrix physiology. Elastin, an insoluble polymer of the monomeric soluble precursor tropoelastin, is the main component of elastic fibers in matrix tissue where it provides elastic recoil and resilience to a variety of connective tissues, e.g., aorta and ligaments. Elastic fibers regulate activity of TGFβs through their association with fibrillin microfibrils. Elastin also plays a role in cell adhesion, cell migration, and has the ability to participate in cell signaling. Mutations in the elastin gene lead to cutis laxa. Fibrillins represent the predominant core of the microfibrils in elastic as well as non-elastic extracellular matrixes, and interact closely with tropoelastin and integrins. Not only do microfibrils provide structural integrity of specific organ systems, but they also provide a scaffold for elastogenesis in elastic tissues. Fibrillin is important for the assembly of elastin into elastic fibers. Mutations in the fibrillin-1 gene are closely associated with Marfan syndrome. Fibulins are tightly connected with basement membranes, elastic fibers and other components of extracellular matrix and participate in formation of elastic fibers. Tenascins are ECM polymorphic glycoproteins found in many connective tissues in the body. Their expression is regulated by mechanical stress both during development and in adulthood. Tenascins mediate both inflammatory and fibrotic processes to enable effective tissue repair and play roles in pathogenesis of Ehlers-Danlos, heart disease, and regeneration and recovery of musculo-tendinous tissue. One of the roles of thrombospondin 1 is activation of TGFβ. Increased expression of thrombospondin and TGFβ activity was observed in fibrotic skin disorders such as keloids and scleroderma. Cartilage oligomeric matrix protein (COMP) or thrombospondin-5 is primarily present in the cartilage. High levels of COMP are present in fibrotic scars and systemic sclerosis of the skin, and in tendon, especially with physical activity, loading and post-injury. It plays a role in vascular wall remodeling and has been found in atherosclerotic plaques as well.

Improved Scanners for Microscopic Hyperspectral Imaging

NASA Technical Reports Server (NTRS)

Mao, Chengye

2009-01-01

Improved scanners to be incorporated into hyperspectral microscope-based imaging systems have been invented. Heretofore, in microscopic imaging, including spectral imaging, it has been customary to either move the specimen relative to the optical assembly that includes the microscope or else move the entire assembly relative to the specimen. It becomes extremely difficult to control such scanning when submicron translation increments are required, because the high magnification of the microscope enlarges all movements in the specimen image on the focal plane. To overcome this difficulty, in a system based on this invention, no attempt would be made to move either the specimen or the optical assembly. Instead, an objective lens would be moved within the assembly so as to cause translation of the image at the focal plane: the effect would be equivalent to scanning in the focal plane. The upper part of the figure depicts a generic proposed microscope-based hyperspectral imaging system incorporating the invention. The optical assembly of this system would include an objective lens (normally, a microscope objective lens) and a charge-coupled-device (CCD) camera. The objective lens would be mounted on a servomotor-driven translation stage, which would be capable of moving the lens in precisely controlled increments, relative to the camera, parallel to the focal-plane scan axis. The output of the CCD camera would be digitized and fed to a frame grabber in a computer. The computer would store the frame-grabber output for subsequent viewing and/or processing of images. The computer would contain a position-control interface board, through which it would control the servomotor. There are several versions of the invention. An essential feature common to all versions is that the stationary optical subassembly containing the camera would also contain a spatial window, at the focal plane of the objective lens, that would pass only a selected portion of the image. In one version, the window would be a slit, the CCD would contain a one-dimensional array of pixels, and the objective lens would be moved along an axis perpendicular to the slit to spatially scan the image of the specimen in pushbroom fashion. The image built up by scanning in this case would be an ordinary (non-spectral) image. In another version, the optics of which are depicted in the lower part of the figure, the spatial window would be a slit, the CCD would contain a two-dimensional array of pixels, the slit image would be refocused onto the CCD by a relay-lens pair consisting of a collimating and a focusing lens, and a prism-gratingprism optical spectrometer would be placed between the collimating and focusing lenses. Consequently, the image on the CCD would be spatially resolved along the slit axis and spectrally resolved along the axis perpendicular to the slit. As in the first-mentioned version, the objective lens would be moved along an axis perpendicular to the slit to spatially scan the image of the specimen in pushbroom fashion.

High performance large infrared and visible astronomy arrays for low background applications: instruments performance data and future developments at Raytheon

NASA Astrophysics Data System (ADS)

Beuville, Eric; Acton, David; Corrales, Elizabeth; Drab, John; Levy, Alan; Merrill, Michael; Peralta, Richard; Ritchie, William

2007-09-01

Raytheon Vision Systems (RVS) has developed a family of high performance large format infrared detector arrays for astronomy and civil space applications. RVS offers unique off-the-shelf solutions to the astronomy community. This paper describes mega-pixel arrays, based on multiple detector materials, developed for astronomy and low-background applications. New focal plane arrays under development at RVS for the astronomy community will also be presented. Large Sensor Chip Assemblies (SCAs) using various detector materials like Si:PIN, HgCdTe, InSb, and Si:As IBC, covering a detection range from visible to large wavelength infrared (LWIR) have been demonstrated with an excellent quantum efficiency and very good uniformity. These focal plane arrays have been assembled using state-of-the-art low noise, low power, readout integrated circuits (ROIC) designed at RVS. Raytheon packaging capabilities address reliability, precision alignment and flatness requirements for both ground-based and space applications. Multiple SCAs can be packaged into even larger focal planes. The VISTA telescope, for example, contains sixteen 2k × 2k infrared focal plane arrays. RVS astronomical arrays are being deployed world-wide in ground-based and space-based applications. A summary of performance data for each of these array types from instruments in operation will be presented (VIRGO Array for large format SWIR, the ORION and VISTA Arrays, NEWFIRM and other solutions for MWIR spectral ranges).

Resin Dermatitis in a Car Factory

PubMed Central

Engel, H. O.; Calnan, C. D.

1966-01-01

An outbreak of dermatitis in a car assembly factory is described; it affected 50 workers who handled rubber weatherstrips coated with an adhesive. The adhesive was found to contain para-tertiary butyl phenol (P.T.B.P.) formaldehyde resin. Of those patch tested 70% gave positive reactions to the adhesive and 65% to the resin. Improved methods of handling and personal protection succeeded in arresting the occurrence of dermatitis. Barrier creams gave no protection in these circumstances. The episode illustrates the different preventive control methods which have to be tried when dealing with a simple skin hazard which cannot be abolished. Images PMID:5904100

nDEP-driven cell patterning and bottom-up construction of cell aggregates using a new bioelectronic chip.

PubMed

Menad, S; Franqueville, L; Haddour, N; Buret, F; Frenea-Robin, M

2015-04-01

Creating cell aggregates of controlled size and shape and patterning cells on substrates using a bottom-up approach constitutes important challenges for tissue-engineering applications and studies of cell-cell interactions. In this paper, we report nDEP (negative dielectrophoresis) driven assembly of cells as compact aggregates or onto defined areas using a new bioelectronic chip. This chip is composed of a quadripolar electrode array obtained using coplanar electrodes partially covered with a thin, micropatterned PDMS membrane. This thin PDMS layer was coated with poly-L-lysine and played the role of adhesive substrate for cell patterning. For the formation of detachable cell aggregates, the PDMS was not pretreated and cells were simply immobilized into assemblies maintained by cell-cell adhesion after the electric field removal. Cell viability after exposition to DEP buffer was also assessed, as well as cell spreading activity following DEP-driven assembly. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Enhanced Cellular Adhesion on Titanium by Silk Functionalized with titanium binding and RGD peptides

PubMed Central

Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J.; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L.; Egles, Christophe

2012-01-01

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. Quartz Crystal Microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived RGD peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by Scanning Electron Microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk-peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required. PMID:22975628

Phosphatase of regenerating liver-3 is expressed in acute lymphoblastic leukemia and mediates leukemic cell adhesion, migration and drug resistance

PubMed Central

Hjort, Magnus A.; Abdollahi, Pegah; Vandsemb, Esten N.; Fenstad, Mona H.; Lund, Bendik; Slørdahl, Tobias S.; Børset, Magne; Rø, Torstein B.

2018-01-01

Phosphatase of regenerating liver-3 (PRL-3/PTP4A3) is upregulated in multiple cancers, including BCR-ABL1- and ETV6-RUNX-positive acute lymphoblastic leukemia (ALL). With this study, we aim to characterize the biological role of PRL-3 in B cell ALL (B-ALL). Here, we demonstrate that PRL-3 expression at mRNA and protein level was higher in B-ALL cells than in normal cells, as measured by qRT-PCR or flow cytometry. Further, we demonstrate that inhibition of PRL-3 using shRNA or a small molecular inhibitor reduced cell migration towards an SDF-1α gradient in the preB-ALL cell lines Reh and MHH-CALL-4. Knockdown of PRL-3 also reduced cell adhesion towards fibronectin in Reh cells. Mechanistically, PRL-3 mediated SDF-1α stimulated calcium release, and activated focal adhesion kinase (FAK) and Src, important effectors of migration and adhesion. Finally, PRL-3 expression made Reh cells more resistance to cytarabine treatment. In conclusion, the expression level of PRL-3 was higher in B-ALL cells than in normal cells. PRL-3 promoted adhesion, migration and resistance to cytarabine. PRL-3 may represent a novel target in the treatment of B-ALL. PMID:29423065

Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells.

PubMed

Acerbi, Irene; Luque, Tomás; Giménez, Alícia; Puig, Marta; Reguart, Noemi; Farré, Ramon; Navajas, Daniel; Alcaraz, Jordi

2012-01-01

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm(2) cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

Ability of finger-jointed lumber to maintain load at elevated temperatures

Treesearch

Douglas R. Rammer; Samuel L. Zelinka; Laura E Hasburgh; Steven T. Craft

2018-01-01

This article presents a test method that was developed to screen adhesive formulations for finger-jointed lumber. The goal was to develop a small-scale test that could be used to predict whether an adhesive would pass a full-scale ASTM E119 wall assembly test. The method involved loading a 38-mm square finger-jointed sample in a four-point bending test inside of an...

Understanding the effects of PEMFC contamination from balance of plant assembly aids materials: In situ studies

DOE PAGES

Opu, Md.; Bender, G.; Macomber, Clay S.; ...

2015-06-29

In this study, in situ performance data were measured to assess the degree of contamination from leachates of five families of balance of plant (BOP) materials (i.e., 2-part adhesive, grease, thread lock/seal, silicone adhesive/seal and urethane adhesive/seal) broadly classified as assembly aids that may be used as adhesives and lubricants in polymer electrolyte membrane fuel cell (PEMFC) systems. Leachate solutions, derived from soaking the materials in deionized (DI) water at elevated temperature, were infused into the fuel cell to determine the effect of the leachates on fuel cell performance. During the contamination phase of the experiments, leachate solution was introducedmore » through a nebulizer into the cathode feed stream of a 50 cm 2 PEMFC operating at 0.2 A/cm 2 at 80°C and 32%RH. Voltage loss and high frequency resistance (HFR) were measured as a function of time and electrochemical surface area (ECA) before and after contamination were compared. Two procedures of recovery, one self-induced recovery with DI water and one driven recovery through cyclic voltammetry (CV) were investigated. Finally, performance results measured before and after contamination and after CV recovery are compared and discussed.« less

Understanding the effects of PEMFC contamination from balance of plant assembly aids materials: In situ studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Opu, Md.; Bender, G.; Macomber, Clay S.

In this study, in situ performance data were measured to assess the degree of contamination from leachates of five families of balance of plant (BOP) materials (i.e., 2-part adhesive, grease, thread lock/seal, silicone adhesive/seal and urethane adhesive/seal) broadly classified as assembly aids that may be used as adhesives and lubricants in polymer electrolyte membrane fuel cell (PEMFC) systems. Leachate solutions, derived from soaking the materials in deionized (DI) water at elevated temperature, were infused into the fuel cell to determine the effect of the leachates on fuel cell performance. During the contamination phase of the experiments, leachate solution was introducedmore » through a nebulizer into the cathode feed stream of a 50 cm 2 PEMFC operating at 0.2 A/cm 2 at 80°C and 32%RH. Voltage loss and high frequency resistance (HFR) were measured as a function of time and electrochemical surface area (ECA) before and after contamination were compared. Two procedures of recovery, one self-induced recovery with DI water and one driven recovery through cyclic voltammetry (CV) were investigated. Finally, performance results measured before and after contamination and after CV recovery are compared and discussed.« less

Influence of Application Time and Etching Mode of Universal Adhesives on Enamel Adhesion.

PubMed

Sai, Keiichi; Takamizawa, Toshiki; Imai, Arisa; Tsujimoto, Akimasa; Ishii, Ryo; Barkmeier, Wayne W; Latta, Mark A; Miyazaki, Masashi

2018-01-01

To investigate the influence of application time and etching mode of universal adhesives on enamel adhesion. Five universal adhesives, Adhese Universal, Bondmer Lightless, Clearfil Universal Bond Quick, G-Premio Bond, and Scotchbond Universal, were used. Bovine incisors were prepared and divided into four groups of ten teeth each. SBS, Ra, and SFE were determined after the following procedures: 1. self-etch mode with immediate air blowing after application (IA); 2. self-etch mode with prolonged application time (PA); 3. etch-and-rinse mode with IA; 4. etch-and-rinse mode with PA. After 24-h water storage, the bonded assemblies were subjected to shear bond strength (SBS) tests. For surface roughness (Ra) and surface free energy (SFE) measurements, the adhesives were simply applied to the enamel and rinsed with acetone and water before the measurements were carried out. Significantly higher SBS and Ra values were obtained with etch-and-rinse mode than with self-etch mode regardless of the application time or type of adhesive. Although most adhesives showed decreased SFE values with increased application time in self-etch mode, SFE values in etch-and-rinse mode were dependent on the adhesive type and application time. Etching mode, application time, and type of adhesive significantly influenced the SBS, Ra, and SFE values.

PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling.

PubMed

Mansour, Mariam; Nievergall, Eva; Gegenbauer, Kristina; Llerena, Carmen; Atapattu, Lakmali; Hallé, Maxime; Tremblay, Michel L; Janes, Peter W; Lackmann, Martin

2016-01-15

Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling. © 2016. Published by The Company of Biologists Ltd.

Laser beam soldering of micro-optical components

NASA Astrophysics Data System (ADS)

Eberhardt, R.

2003-05-01

MOTIVATION Ongoing miniaturisation and higher requirements within optical assemblies and the processing of temperature sensitive components demands for innovative selective joining techniques. So far adhesive bonding has primarily been used to assemble and adjust hybrid micro optical systems. However, the properties of the organic polymers used for the adhesives limit the application of these systems. In fields of telecommunication and lithography, an enhancement of existing joining techniques is necessary to improve properties like humidity resistance, laserstability, UV-stability, thermal cycle reliability and life time reliability. Against this background laser beam soldering of optical components is a reasonable joining technology alternative. Properties like: - time and area restricted energy input - energy input can be controlled by the process temperature - direct and indirect heating of the components is possible - no mechanical contact between joining tool and components give good conditions to meet the requirements on a joining technology for sensitive optical components. Additionally to the laser soldering head, for the assembly of optical components it is necessary to include positioning units to adjust the position of the components with high accuracy before joining. Furthermore, suitable measurement methods to characterize the soldered assemblies (for instance in terms of position tolerances) need to be developed.

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

PubMed

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

2015-11-27

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.

Controlled Delivery of a Focal Adhesion Kinase Inhibitor Results in Accelerated Wound Closure with Decreased Scar Formation.

PubMed

Ma, Kun; Kwon, Sun Hyung; Padmanabhan, Jagannath; Duscher, Dominik; Trotsyuk, Artem A; Dong, Yixiao; Inayathullah, Mohammed; Rajadas, Jayakumar; Gurtner, Geoffrey C

2018-05-15

Formation of scars following wounding or trauma represents a significant healthcare burden costing the economy billions of dollars every year. Activation of focal adhesion kinase (FAK) has been shown to play a pivotal role in transducing mechanical signals to elicit fibrotic responses and scar formation during wound repair. We have previously shown that inhibition of FAK using local injections of a small molecule FAK inhibitor (FAKI) can attenuate scar development in a hypertrophic scar model. Clinical translation of FAKI therapy has been challenging, however, due to the lack of an effective drug delivery system for extensive burn injuries, blast injuries, and large excisional injuries. To address this issue, we have developed a pullulan collagen-based hydrogel to deliver FAKI to excisional and burn wounds in mice. Specifically, two distinct drug-laden hydrogels were developed for rapid or sustained release of FAKI for treatment of burn wounds and excisional wounds, respectively. Controlled delivery of FAKI via pullulan collagen hydrogels accelerated wound healing, reduced collagen deposition and activation of scar forming myofibroblasts in both wound healing models. Our study highlights a biomaterial-based drug delivery approach for wound and scar management that has significant translational implications. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of Clinically Relevant CAD/CAM Zirconia Polishing on Gingival Fibroblast Proliferation and Focal Adhesions

PubMed Central

Fischer, Nicholas G.; Wong, Jeffrey; Cerutis, D. Roselyn

2017-01-01

Mucosal seal formation around dental abutments is critical to the successful integration of dental implants into the human oral cavity. No information exists for how clinically relevant polishing procedures for computer-aided design and computer-aided manufactured (CAD/CAM) zirconia abutments affects cellular responses important to mucosal seal formation. CAD/CAM zirconia was divided into four groups for clinically relevant polishing utilizing commercial polishing heads: control, coarse, coarse plus medium, and coarse plus medium plus fine. Surfaces were analyzed with scanning electron microscopy (SEM), atomic force microscopy (AFM), and optical profilometry (OP). Subsequently, human gingival fibroblasts (HGFs) were seeded onto the zirconia surfaces. Proliferation was measured via a quantitative SEM technique and focal adhesion kinase (FAK) phosphorylation status was measured by an enzyme-linked immunosorbent assay (ELISA). Results showed an increase in proliferation on all polished surfaces as compared to the control. Phosphorylation of FAK at tyrosine 397 (Y397) was up-modulated on the control surfaces. The associated cell adaptation is discussed. In all cases, FAK phosphorylation was greater at 24 h than 48 h. These results suggest that clinicians should be mindful of the effects of abutment polishing methodology, as this may have an impact on early mucosal seal formation. PMID:29186907

MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

PubMed

Van Itallie, Christina M; Tietgens, Amber Jean; Aponte, Angel; Gucek, Marjan; Cartagena-Rivera, Alexander X; Chadwick, Richard S; Anderson, James M

2018-02-02

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells. © 2018. Published by The Company of Biologists Ltd.

Regulation of Mesenchymal Stem Cell Differentiation by Nanopatterning of Bulk Metallic Glass.

PubMed

Loye, Ayomiposi M; Kinser, Emily R; Bensouda, Sabrine; Shayan, Mahdis; Davis, Rose; Wang, Rui; Chen, Zheng; Schwarz, Udo D; Schroers, Jan; Kyriakides, Themis R

2018-06-08

Mesenchymal stem cell (MSC) differentiation is regulated by surface modification including texturing, which is applied to materials to enhance tissue integration. Here, we used Pt 57.5 Cu 14.7 Ni 5.3 P 22.5 bulk metallic glass (Pt-BMG) with nanopatterned surfaces achieved by thermoplastic forming to influence differentiation of human MSCs. Pt-BMGs are a unique class of amorphous metals with high strength, elasticity, corrosion resistance, and an unusual plastic-like processability. It was found that flat and nanopattened Pt-BMGs induced osteogenic and adipogenic differentiation, respectively. In addition, osteogenic differentiation on flat BMG exceeded that observed on medical grade titanium and was associated with increased formation of focal adhesions and YAP nuclear localization. In contrast, cells on nanopatterned BMGs exhibited rounded morphology, formed less focal adhesions and had mostly cytoplasmic YAP. These changes were preserved on nanopatterns made of nanorods with increased stiffness due to shorter aspect ratios, suggesting that MSC differentiation was primarily influenced by topography. These observations indicate that both elemental composition and nanotopography can modulate biochemical cues and influence MSCs. Moreover, the processability and highly tunable nature of Pt-BMGs enables the creation of a wide range of surface topographies that can be reproducibly and systematically studied, leading to the development of implants capable of engineering MSC functions.

The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds

PubMed Central

Sequeira, Sharon J.; Soscia, David A.; Oztan, Basak; Mosier, Aaron P.; Jean-Gilles, Riffard; Gadre, Anand; Cady, Nathaniel C.; Yener, Bülent; Castracane, James; Larsen, Melinda

2012-01-01

Nanofiber scaffolds have been useful for engineering tissues derived from mesenchymal cells, but few studies have investigated their applicability for epithelial cell-derived tissues. In this study, we generated nanofiber (250 nm) or microfiber (1200 nm) scaffolds via electrospinning from the polymer, poly-L-lactic-co-glycolic acid (PLGA). Cell-scaffold contacts were visualized using fluorescent immunocytochemistry and laser scanning confocal microscopy. Focal adhesion (FA) proteins, such as phosphorylated FAK (Tyr397), paxillin (Tyr118), talin and vinculin were localized to FA complexes in adult cells grown on planar surfaces but were reduced and diffusely localized in cells grown on nanofiber surfaces, similar to the pattern observed in adult mouse salivary gland tissues. Significant differences in epithelial cell morphology and cell clustering were also observed and quantified, using image segmentation and computational cell-graph analyses. No statistically significant differences in scaffold stiffness between planar PLGA film controls compared to nanofibers scaffolds were detected using nanoindentation with atomic force microscopy, indicating that scaffold topography rather than mechanical properties accounts for changes in cell attachments and cell structure. Finally, PLGA nanofiber scaffolds could support the spontaneous self-organization and branching of dissociated embryonic salivary gland cells. Nanofiber scaffolds may therefore have applicability in the future for engineering an artificial salivary gland. PMID:22285464

Focal adhesion kinase regulates neuronal growth, synaptic plasticity and hippocampus-dependent spatial learning and memory.

PubMed

Monje, Francisco J; Kim, Eun-Jung; Pollak, Daniela D; Cabatic, Maureen; Li, Lin; Baston, Arthur; Lubec, Gert

2012-01-01

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase abundantly expressed in the mammalian brain and highly enriched in neuronal growth cones. Inhibitory and facilitatory activities of FAK on neuronal growth have been reported and its role in neuritic outgrowth remains controversial. Unlike other tyrosine kinases, such as the neurotrophin receptors regulating neuronal growth and plasticity, the relevance of FAK for learning and memory in vivo has not been clearly defined yet. A comprehensive study aimed at determining the role of FAK in neuronal growth, neurotransmitter release and synaptic plasticity in hippocampal neurons and in hippocampus-dependent learning and memory was therefore undertaken using the mouse model. Gain- and loss-of-function experiments indicated that FAK is a critical regulator of hippocampal cell morphology. FAK mediated neurotrophin-induced neuritic outgrowth and FAK inhibition affected both miniature excitatory postsynaptic potentials and activity-dependent hippocampal long-term potentiation prompting us to explore the possible role of FAK in spatial learning and memory in vivo. Our data indicate that FAK has a growth-promoting effect, is importantly involved in the regulation of the synaptic function and mediates in vivo hippocampus-dependent spatial learning and memory. Copyright © 2011 S. Karger AG, Basel.

Altered focal adhesion regulation correlates with cardiomyopathy in mice expressing constitutively active rac1

PubMed Central

Sussman, Mark A.; Welch, Sara; Walker, Angela; Klevitsky, Raisa; Hewett, Timothy E.; Price, Robert L.; Schaefer, Erik; Yager, Karen

2000-01-01

The ras family of small GTP-binding proteins exerts powerful effects upon cell structure and function. One member of this family, rac, induces actin cytoskeletal reorganization in nonmuscle cells and hypertrophic changes in cultured cardiomyocytes. To examine the effect of rac1 activation upon cardiac structure and function, transgenic mice were created that express constitutively activated rac1 specifically in the myocardium. Transgenic rac1 protein was expressed at levels comparable to endogenous rac levels, with activation of the rac1 signaling pathway resulting in two distinct cardiomyopathic phenotypes: a lethal dilated phenotype associated with neonatal activation of the transgene and a transient cardiac hypertrophy seen among juvenile mice that resolved with age. Neither phenotype showed myofibril disarray and hypertrophic hearts were hypercontractilein working heart analyses. The rac1 target p21-activated kinase translocated from a cytosolic to a cytoskeletal distribution, suggesting that rac1 activation was inducing focal adhesion reorganization. Corroborating results showed altered localizations of src in dilated cardiomyopathy and paxillin in both cardiomyopathic phenotypes. This study, the first examination of rac1-mediated cardiac effects in vivo, demonstrates that dilation and hypertrophy can share a common molecular origin and presents evidence that both timing and concurrent signaling from multiple pathways can influence cardiac remodeling. PMID:10749567

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.

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

The effects of focal adhesion kinase on the motility, proliferation and apoptosis of Caco2 and SMMC-7721 cells.

PubMed

Gao, Ning; He, Huimin; Xiao, Li; Gao, Xiaojie; Shi, Hongyan; Wu, Qiong; Xu, Nan; Lei, Yuqing; Song, Xigui; Hou, Yingchun

2015-04-01

Focal adhesion kinase (FAK) plays important roles in cancer development. However, the significance of FAK expression in colorectal carcinoma and hepatocellular carcinoma has not been clarified. This study aims to explore the roles FAK played in the progression of colorectal carcinoma and hepatocellular carcinoma. RNAi method was used to inhibit the expression of FAK in Caco2 and SMMC-7721 cells. Reverse transcriptase polymerase chain reaction analysis and Western blot analysis were used to examine mRNA and protein expression of FAK. Then, the proliferation, motility and apoptosis of both types of cells were detected using MTT assay, wound healing/transwell assay and nuclear staining assay. The microstructure changes (F-actin, β-tubulin and lamin B1) of SMMC-7721 cells were visualized by immunofluorescence. FAK was overexpressed in both cell lines and down-regulation of FAK resulted in suppression of cell proliferation, inhibition of cell migration and invasion. The apoptosis of cells was increased significantly following the FAK expression inhibition. Moreover, actin polymerization, β-tubulin and lamin B1 expression of cells were significantly decreased. The results highlight the role of FAK in the progression of cancers. These findings suggest FAK serve as a potential therapeutic target for cancer therapy.

Solexa-Sequencing Based Transcriptome Study of Plaice Skin Phenotype in Rex Rabbits (Oryctolagus cuniculus)

PubMed Central

Pan, Lei; Liu, Yan; Wei, Qiang; Xiao, Chenwen; Ji, Quanan; Bao, Guolian; Wu, Xinsheng

2015-01-01

Background Fur is an important genetically-determined characteristic of domestic rabbits; rabbit furs are of great economic value. We used the Solexa sequencing technology to assess gene expression in skin tissues from full-sib Rex rabbits of different phenotypes in order to explore the molecular mechanisms associated with fur determination. Methodology/Principal Findings Transcriptome analysis included de novo assembly, gene function identification, and gene function classification and enrichment. We obtained 74,032,912 and 71,126,891 short reads of 100 nt, which were assembled into 377,618 unique sequences by Trinity strategy (N50=680 nt). Based on BLAST results with known proteins, 50,228 sequences were identified at a cut-off E-value ≥ 10-5. Using Blast to Gene Ontology (GO), Clusters of Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we obtained several genes with important protein functions. A total of 308 differentially expressed genes were obtained by transcriptome analysis of plaice and un-plaice phenotype animals; 209 additional differentially expressed genes were not found in any database. These genes included 49 that were only expressed in plaice skin rabbits. The novel genes may play important roles during skin growth and development. In addition, 99 known differentially expressed genes were assigned to PI3K-Akt signaling, focal adhesion, and ECM-receptor interactin, among others. Growth factors play a role in skin growth and development by regulating these signaling pathways. We confirmed the altered expression levels of seven target genes by qRT-PCR. And chosen a key gene for SNP to found the differentially between plaice and un-plaice phenotypes rabbit. Conclusions/Significance The rabbit transcriptome profiling data provide new insights in understanding the molecular mechanisms underlying rabbit skin growth and development. PMID:25955442

Synthesis and characterization of designed BMHP1-derived self-assembling peptides for tissue engineering applications

NASA Astrophysics Data System (ADS)

Silva, Diego; Natalello, Antonino; Sanii, Babak; Vasita, Rajesh; Saracino, Gloria; Zuckermann, Ronald N.; Doglia, Silvia Maria; Gelain, Fabrizio

2012-12-01

The importance of self-assembling peptides (SAPs) in regenerative medicine is becoming increasingly recognized. The propensity of SAPs to form nanostructured fibers is governed by multiple forces including hydrogen bonds, hydrophobic interactions and π-π aromatic interactions among side chains of the amino acids. Single residue modifications in SAP sequences can significantly affect these forces. BMHP1-derived SAPs is a class of biotinylated oligopeptides, which self-assemble in β-structured fibers to form a self-healing hydrogel. In the current study, selected modifications in previously described BMHP1-derived SAPs were designed in order to investigate the influence of modified residues on self-assembly kinetics and scaffold formation properties. The Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis demonstrated the secondary structure (β-sheet) formation in all modified SAP sequences, whereas atomic force microscopy (AFM) analysis further confirmed the presence of nanofibers. Furthermore, the fiber shape and dimension analysis by AFM showed flattened and twisted fiber morphology ranging from ~8 nm to ~70 nm. The mechanical properties of the pre-assembled and post assembled solution were investigated by rheometry. The shear-thinning behavior and rapid re-healing properties of the pre-assembled solutions make them a preferable choice for injectable scaffolds. The wide range of stiffnesses (G') -from ~1000 to ~27 000 Pa - exhibited by the post-assembled scaffolds demonstrated their potential for a variety of tissue engineering applications. The extra cellular matrix (ECM) mimicking (physically and chemically) properties of SAP scaffolds enhanced cell adhesion and proliferation. The capability of the scaffold to facilitate murine neural stem cell (mNSC) proliferation was evaluated in vitro: the increased mNSCs adhesion and proliferation demonstrated the potential of newly synthesized SAPs for regenerative medicine approaches.The importance of self-assembling peptides (SAPs) in regenerative medicine is becoming increasingly recognized. The propensity of SAPs to form nanostructured fibers is governed by multiple forces including hydrogen bonds, hydrophobic interactions and π-π aromatic interactions among side chains of the amino acids. Single residue modifications in SAP sequences can significantly affect these forces. BMHP1-derived SAPs is a class of biotinylated oligopeptides, which self-assemble in β-structured fibers to form a self-healing hydrogel. In the current study, selected modifications in previously described BMHP1-derived SAPs were designed in order to investigate the influence of modified residues on self-assembly kinetics and scaffold formation properties. The Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis demonstrated the secondary structure (β-sheet) formation in all modified SAP sequences, whereas atomic force microscopy (AFM) analysis further confirmed the presence of nanofibers. Furthermore, the fiber shape and dimension analysis by AFM showed flattened and twisted fiber morphology ranging from ~8 nm to ~70 nm. The mechanical properties of the pre-assembled and post assembled solution were investigated by rheometry. The shear-thinning behavior and rapid re-healing properties of the pre-assembled solutions make them a preferable choice for injectable scaffolds. The wide range of stiffnesses (G') -from ~1000 to ~27 000 Pa - exhibited by the post-assembled scaffolds demonstrated their potential for a variety of tissue engineering applications. The extra cellular matrix (ECM) mimicking (physically and chemically) properties of SAP scaffolds enhanced cell adhesion and proliferation. The capability of the scaffold to facilitate murine neural stem cell (mNSC) proliferation was evaluated in vitro: the increased mNSCs adhesion and proliferation demonstrated the potential of newly synthesized SAPs for regenerative medicine approaches. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32656f

Nanosurface design of dental implants for improved cell growth and function

NASA Astrophysics Data System (ADS)

Pan, Hsu-An; Hung, Yao-Ching; Chiou, Jin-Chern; Tai, Shih-Ming; Chen, Hsin-Hung; Huang, G. Steven

2012-08-01

A strategy was proposed for the topological design of dental implants based on an in vitro survey of optimized nanodot structures. An in vitro survey was performed using nanodot arrays with dot diameters ranging from 10 to 200 nm. MG63 osteoblasts were seeded on nanodot arrays and cultured for 3 days. Cell number, percentage undergoing apoptotic-like cell death, cell adhesion and cytoskeletal organization were evaluated. Nanodots with a diameter of approximately 50 nm enhanced cell number by 44%, minimized apoptotic-like cell death to 2.7%, promoted a 30% increase in microfilament bundles and maximized cell adhesion with a 73% increase in focal adhesions. An enhancement of about 50% in mineralization was observed, determined by von Kossa staining and by Alizarin Red S staining. Therefore, we provide a complete range of nanosurfaces for growing osteoblasts to discriminate their nanoscale environment. Nanodot arrays present an opportunity to positively and negatively modulate cell behavior and maturation. Our results suggest a topological approach which is beneficial for the design of dental implants.