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Sample records for actin cable formation

  1. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  2. Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

    SciTech Connect

    Aoyama, Kazumasa; Yuki, Ryuzaburo; Horiike, Yasuyoshi; Kubota, Sho; Yamaguchi, Noritaka; Morii, Mariko; Ishibashi, Kenichi; Nakayama, Yuji; Kuga, Takahisa; Hashimoto, Yuuki; Tomonaga, Takeshi; Yamaguchi, Naoto

    2013-12-10

    The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus. - Highlights: • We show the involvement of c-Abl tyrosine kinase in nuclear actin dynamics. • Nuclear F-actin is formed by nuclear-localized c-Abl and its kinase-dead version. • The c-Abl actin-binding domain is prerequisite for nuclear F-actin formation. • Formation of long nuclear F-actin bundles requires nuclear c-Abl kinase activity. • We discuss a role for nuclear F-actin bundle formation in chromatin regulation.

  3. Effects of recombinant baculovirus AcMNPV-BmK IT on the formation of early cables and nuclear polymerization of actin in Sf9 cells.

    PubMed

    Fu, Yuejun; Lin, Taotao; Liang, Aihua; Hu, Fengyun

    2016-05-01

    Autographa californica nuclearpoly hedrosis virus (AcMNPV) is one of the most important baculoviridae. However, the application of AcMNPV as a biocontrol agent has been limited. Previously, we engineered Buthus martensii Karsch insect toxin (BmK IT) gene into the genome of AcMNPV. The bioassay data indicated that the recombinant baculovirus AcMNPV-BmK IT significantly enhanced the anti-insect efficacy of the virus. The actin cytoskeleton is the major component beneath the surface of eukaryotic cells. In this report, the effects of AcMNPV-BmK IT on the formation of early cables of actin and nuclear filamentous-actin (F-actin) were studied. The results indicated that these baculovirus induced rearrangement of the actin cytoskeleton of host cells during infection and actin might participate in the transportation of baculovirus from cytoplasm to the nuclei. AcMNPV-BmK IT delayed the formation of early cables of actin and nuclear F-actin and accelerated the clearance of actin in the nuclei.

  4. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    PubMed

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-09-30

    Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

  5. Myosin Vs organize actin cables in fission yeast

    PubMed Central

    Lo Presti, Libera; Chang, Fred; Martin, Sophie G.

    2012-01-01

    Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV∆ defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7–Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces. PMID:23051734

  6. Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile, long-lived plaques.

    PubMed

    Meijer, Harold J G; Hua, Chenlei; Kots, Kiki; Ketelaar, Tijs; Govers, Francine

    2014-06-01

    The actin cytoskeleton is a dynamic but well-organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact-eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub-apical region whereas in the extreme apex in growing hyphae, waves of fine F-actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ∼30 s while disassembly was accomplished in ∼10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.

  7. Srv2/CAP is required for polarized actin cable assembly and patch internalization during clathrin-mediated endocytosis.

    PubMed

    Toshima, Junko Y; Horikomi, Chika; Okada, Asuka; Hatori, Makiko N; Nagano, Makoto; Masuda, Atsushi; Yamamoto, Wataru; Siekhaus, Daria Elisabeth; Toshima, Jiro

    2016-01-15

    The dynamic assembly and disassembly of actin filaments is essential for the formation and transport of vesicles during endocytosis. In yeast, two types of actin structures, namely cortical patches and cytoplasmic cables, play a direct role in endocytosis, but how their interaction is regulated remains unclear. Here, we show that Srv2/CAP, an evolutionarily conserved actin regulator, is required for efficient endocytosis owing to its role in the formation of the actin patches that aid initial vesicle invagination and of the actin cables that these move along. Deletion of the SRV2 gene resulted in the appearance of aberrant fragmented actin cables that frequently moved past actin patches, the sites of endocytosis. We find that the C-terminal CARP domain of Srv2p is vitally important for the proper assembly of actin patches and cables; we also demonstrate that the N-terminal helical folded domain of Srv2 is required for its localization to actin patches, specifically to the ADP-actin rich region through an interaction with cofilin. These results demonstrate the in vivo roles of Srv2p in the regulation of the actin cytoskeleton during clathrin-mediated endocytosis.

  8. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

    PubMed Central

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R.; Drubin, David G.

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  9. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-01-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes. PMID:24058157

  10. Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells

    PubMed Central

    Vasicova, Pavla; Rinnerthaler, Mark; Haskova, Danusa; Novakova, Lenka; Malcova, Ivana; Breitenbach, Michael; Hasek, Jiri

    2016-01-01

    Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae. This pattern has been shown to be depolarized in glucose-depleted cells after formaldehyde fixation and staining with rhodamine-tagged phalloidin. Loss of actin cables in mother cells was remarkable. Here we extend our knowledge on actin in live glucose-depleted cells co-expressing the marker of actin patches (Abp1-RFP) with the marker of actin cables (Abp140-GFP). Glucose depletion resulted in appearance of actin patches also in mother cells. However, even after 80 min of glucose deprivation these cells showed a clear network of actin cables labeled with Abp140-GFP in contrast to previously published data. In live cells with a mitochondrial dysfunction (rho0 cells), glucose depletion resulted in almost immediate appearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches in mother cells. Residual actin cables were clustered in patch-associated bundles. A similar overlapping “patchy” pattern of both actin markers was observed upon treatment of glucose-deprived rho+ cells with FCCP (the inhibitor of oxidative phosphorylation) and upon treatment with formaldehyde. While the formaldehyde-targeted process stays unknown, our results indicate that published data on yeast actin cytoskeleton obtained from glucose-depleted cells after fixation should be considered with caution.

  11. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  12. Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

    PubMed Central

    Huang, Junqi; Huang, Yinyi; Yu, Haochen; Subramanian, Dhivya; Padmanabhan, Anup; Thadani, Rahul; Tao, Yaqiong; Tang, Xie; Wedlich-Soldner, Roland

    2012-01-01

    In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly. PMID:23185032

  13. Mitochondrial inheritance: cell cycle and actin cable dependence of polarized mitochondrial movements in Saccharomyces cerevisiae.

    PubMed

    Simon, V R; Karmon, S L; Pon, L A

    1997-01-01

    Asymmetric growth and division of budding yeast requires the vectorial transport of growth components and organelles from mother to daughter cells. Time lapse video microscopy and vital staining were used to study motility events which result in partitioning of mitochondria in dividing yeast. We identified four different stages in the mitochondrial inheritance cycle: (1) mitochondria align along the mother-bud axis prior to bud emergence in G1 phase, following polarization of the actin cytoskeleton; (2) during S phase, mitochondria undergo linear, continuous and polarized transfer from mother to bud; (3) during S and G2 phases, inherited mitochondria accumulate in the bud tip. This event occurs concomitant with accumulation of actin patches in this region; and (4) finally, during M phase prior to cytokinesis, mitochondria are released from the bud tip and redistribute throughout the bud. Previous studies showed that yeast mitochondria colocalize with actin cables and that isolated mitochondria contain actin binding and motor activities on their surface. We find that selective destabilization of actin cables in a strain lacking the tropomyosin 1 gene (TPM1) has no significant effect on the velocity of mitochondrial motor activity in vivo or in vitro. However, tpm1 delta mutants display abnormal mitochondrial distribution and morphology; loss of long distance, directional mitochondrial movement; and delayed transfer of mitochondria from the mother cell to the bud. Thus, cell cycle-linked mitochondrial motility patterns which lead to inheritance are strictly dependent on organized and properly oriented actin cables.

  14. An atomic model of the tropomyosin cable on F-actin.

    PubMed

    Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan; Lehman, William

    2014-08-05

    Tropomyosin regulates a wide variety of actin filament functions and is best known for the role that it plays together with troponin in controlling muscle activity. For effective performance on actin filaments, adjacent 42-nm-long tropomyosin molecules are joined together by a 9- to 10-residue head-to-tail overlapping domain to form a continuous cable that wraps around the F-actin helix. Yet, despite the apparent simplicity of tropomyosin's coiled-coil structure and its well-known periodic association with successive actin subunits along F-actin, the structure of the tropomyosin cable on actin is uncertain. This is because the conformation of the overlap region that joins neighboring molecules is poorly understood, thus leaving a significant gap in our understanding of thin-filament structure and regulation. However, recent molecular-dynamics simulations of overlap segments defined their overall shape and provided unique and sufficient cues to model the whole actin-tropomyosin filament assembly in atomic detail. In this study, we show that these MD structures merge seamlessly onto the ends of tropomyosin coiled-coils. Adjacent tropomyosin molecules can then be joined together to provide a comprehensive model of the tropomyosin cable running continuously on F-actin. The resulting complete model presented here describes for the first time (to our knowledge) an atomic-level structure of αα-striated muscle tropomyosin bound to an actin filament that includes the critical overlap domain. Thus, the model provides a structural correlate to evaluate thin-filament mechanics, self-assembly mechanisms, and the effect of disease-causing mutations.

  15. A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

    PubMed

    Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

    2013-04-01

    The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

  16. Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.

    PubMed

    Spracklen, Andrew J; Kelpsch, Daniel J; Chen, Xiang; Spracklen, Cassandra N; Tootle, Tina L

    2014-02-01

    Prostaglandins (PGs)--lipid signals produced downstream of cyclooxygenase (COX) enzymes--regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton--temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling.

  17. The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

    PubMed Central

    Graziano, Brian R.; Yu, Hoi-Ying E.; Alioto, Salvatore L.; Eskin, Julian A.; Ydenberg, Casey A.; Waterman, David P.; Garabedian, Mikael; Goode, Bruce L.

    2014-01-01

    Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology–Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 “restrains” the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network. PMID:24719456

  18. Ultrastructure and behavior of actin cytoskeleton during cell wall formation in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Takagi, Tomoko; Ishijima, Sanae A; Ochi, Hisako; Osumi, Masako

    2003-01-01

    Fluorescence microscopy has shown that F-actin of the fission yeast Schizosaccharomyces pombe forms patch, cable and ring structures. To study the relationship between cell wall formation and the actin cytoskeleton, the process of cell wall regeneration from the protoplast was investigated by transmission electron microscopy (TEM), immunoelectron microscopy (IEM) and three-dimensional reconstruction analysis. During cell wall regeneration from the protoplast, localization of F-actin patches was similar to that of the newly synthesized cell wall materials, as shown by confocal laser scanning microscopy (CLSM). In serial sectioned TEM images, filasomes were spherical, 100-300 nm in diameter and consisted of a single microvesicle (35-70 nm diameter) surrounded by fine filaments. Filasomes were adjacent to the newly formed glucan fibrils in single, cluster or rosary forms. By IEM analysis, we found that colloidal gold particles indicating actin molecules were present in the filamentous area of filasomes. Three-dimensional reconstruction images of serial sections clarified that the distribution of filasomes corresponded to the distribution of F-actin patches revealed by CLSM. Thus, a filasome is one of the F-actin patch structures appearing in the cytoplasm at the site of the initial formation of the cell wall and it may play an important role in this action.

  19. TorsinA controls TAN line assembly and the retrograde flow of dorsal perinuclear actin cables during rearward nuclear movement.

    PubMed

    Saunders, Cosmo A; Harris, Nathan J; Willey, Patrick T; Woolums, Brian M; Wang, Yuexia; McQuown, Alex J; Schoenhofen, Amy; Worman, Howard J; Dauer, William T; Gundersen, Gregg G; Luxton, G W Gant

    2017-03-06

    The nucleus is positioned toward the rear of most migratory cells. In fibroblasts and myoblasts polarizing for migration, retrograde actin flow moves the nucleus rearward, resulting in the orientation of the centrosome in the direction of migration. In this study, we report that the nuclear envelope-localized AAA+ (ATPase associated with various cellular activities) torsinA (TA) and its activator, the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1), are required for rearward nuclear movement during centrosome orientation in migrating fibroblasts. Both TA and LAP1 contributed to the assembly of transmembrane actin-associated nuclear (TAN) lines, which couple the nucleus to dorsal perinuclear actin cables undergoing retrograde flow. In addition, TA localized to TAN lines and was necessary for the proper mobility of EGFP-mini-nesprin-2G, a functional TAN line reporter construct, within the nuclear envelope. Furthermore, TA and LAP1 were indispensable for the retrograde flow of dorsal perinuclear actin cables, supporting the recently proposed function for the nucleus in spatially organizing actin flow and cytoplasmic polarity. Collectively, these results identify TA as a key regulator of actin-dependent rearward nuclear movement during centrosome orientation.

  20. The Role of Actin Cytoskeleton in Memory Formation in Amygdala

    PubMed Central

    Lamprecht, Raphael

    2016-01-01

    The central, lateral and basolateral amygdala (BLA) nuclei are essential for the formation of long-term memories including emotional and drug-related memories. Studying cellular and molecular mechanisms of memory in amygdala may lead to better understanding of how memory is formed and of fear and addiction-related disorders. A challenge is to identify molecules activated by learning that subserve cellular changes needed for memory formation and maintenance in amygdala. Recent studies show that activation of synaptic receptors during fear and drug-related learning leads to alteration in actin cytoskeleton dynamics and structure in amygdala. Such changes in actin cytoskeleton in amygdala are essential for fear and drug-related memories formation. Moreover, the actin cytoskeleton subserves, after learning, changes in neuronal morphogenesis and glutamate receptors trafficking in amygdala. These cellular events are involved in fear and drug-related memories formation. Actin polymerization is also needed for the maintenance of drug-associated memories in amygdala. Thus, the actin cytoskeleton is a key mediator between receptor activation during learning and cellular changes subserving long-term memory (LTM) in amygdala. The actin cytoskeleton may serve as a target for pharmacological treatment of fear memory associated with fear and anxiety disorders and drug addiction to prevent the debilitating consequences of these diseases. PMID:27065800

  1. Lamellipodial actin mechanically links myosin activity with adhesion site formation

    PubMed Central

    Giannone, Gregory; Dubin-Thaler, Benjamin; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P.

    2013-01-01

    Summary Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  2. Closure of supporting cell scar formations requires dynamic actin mechanisms

    PubMed Central

    Hordichok, Andrew J.; Steyger, Peter S.

    2007-01-01

    In many vertebrate inner ear sensory epithelia, dying sensory hair cells are extruded, and the apices of surrounding supporting cells converge to re-seal the epithelial barrier between the electrochemically-distinct endolymph and perilymph. These cellular mechanisms remain poorly understood. Dynamic microtubular mechanisms have been proposed for hair cell extrusion; while contractile actomyosin-based mechanisms are required for cellular extrusion and closure in epithelial monolayers. The hypothesis that cytoskeletal mechanisms are required for hair cell extrusion and supporting cell scar formation was tested using bullfrog saccules incubated with gentamicin (6 hours), and allowed to recover (18 hours). Explants were then fixed, labeled for actin and cytokeratins, and viewed with confocal microscopy. To block dynamic cytoskeletal processes, disruption agents for microtubules (colchicine, paclitaxel) myosin (Y-27632, ML-9) or actin (cytochalasin D, latrunculin A) were added during treatment and recovery. Microtubule disruption agents had no effect on hair cell extrusion or supporting cell scar formation. Myosin disruption agents appeared to slow down scar formation but not hair cell extrusion. Actin disruption agents blocked scar formation, and largely prevented hair cell extrusion. These data suggest that actin-based cytoskeletal processes are required for hair cell extrusion and supporting cell scar formation in bullfrog saccules. PMID:17716843

  3. Role of actin filaments in fusopod formation and osteoclastogenesis.

    PubMed

    Wang, Yongqiang; Brooks, Patricia Joyce; Jang, Janet Jinyoung; Silver, Alexandra Shade; Arora, Pamma D; McCulloch, Christopher A; Glogauer, Michael

    2015-07-01

    Cell fusion process is a critical, rate-limiting step in osteoclastogenesis but the mechanisms that regulate fusopod formation are not defined. We characterized fusopod generation in cultured pre-osteoclasts derived from cells stably transfected with a plasmid that expressed a short, actin filament binding peptide (Lifeact) fused to mEGFP that enables localization of actin filaments in living cells. Fusion was initiated at fusopods, which are cell extensions of width >2 μm and that are immunostained for myosin-X at the extension tips. Fusopods formed at the leading edge of larger migrating cells and from the tail of adjacent smaller cells, both of which migrated in the same direction. Staining for DC-STAMP was circumferential and did not localize to cell-cell fusion sites. Compared with wild-type cells, monocytes null for Rac1 exhibited 6-fold fewer fusopods and formed 4-fold fewer multinucleated osteoclasts. From time-lapse images we found that fusion was temporally related to the formation of coherent and spatially isolated bands of actin filaments that originated in cell bodies and extended into the fusopods. These bands of actin filaments were involved in cell fusion after approaching cells formed initial contacts. We conclude that the formation of fusopods is regulated by Rac1 to initiate intercellular contact during osteoclastogenesis. This step is followed by the tightly regulated assembly of bands of actin filaments in fusopods, which lead to closure of the intercellular gap and finally, cell fusion. These novel, actin-dependent processes are important for fusion processes in osteoclastogenesis.

  4. Decavanadate interactions with actin: cysteine oxidation and vanadyl formation.

    PubMed

    Ramos, Susana; Duarte, Rui O; Moura, José J G; Aureliano, Manuel

    2009-10-14

    Incubation of actin with decavanadate induces cysteine oxidation and oxidovanadium(IV) formation. The studies were performed combining kinetic with spectroscopic (NMR and EPR) methodologies. Although decavanadate is converted to labile oxovanadates, the rate of deoligomerization can be very slow (half-life time of 5.4 h, at 25 degrees C, with a first order kinetics), which effectively allows decavanadate to exist for some time under experimental conditions. It was observed that decavanadate inhibits F-actin-stimulated myosin ATPase activity with an IC(50) of 0.8 microM V(10) species, whereas 50 microM of vanadate or oxidovanadium(IV) only inhibits enzyme activity up to 25%. Moreover, from these three vanadium forms, only decavanadate induces the oxidation of the so called "fast" cysteines (or exposed cysteine, Cys-374) when the enzyme is in the polymerized and active form, F-actin, with an IC(50) of 1 microM V(10) species. Decavanadate exposition to F- and G-actin (monomeric form) promotes vanadate reduction since a typical EPR oxidovanadium(IV) spectrum was observed. Upon observation that V(10) reduces to oxidovanadium(IV), it is proposed that this cation interacts with G-actin (K(d) of 7.48 +/- 1.11 microM), and with F-actin (K(d) = 43.05 +/- 5.34 microM) with 1:1 and 4:1 stoichiometries, respectively, as observed by EPR upon protein titration with oxidovanadium(IV). The interaction of oxidovanadium(IV) with the protein may occur close to the ATP binding site of actin, eventually with lysine-336 and 3 water molecules.

  5. Formation and Destabilization of Actin Filaments with Tetramethylrhodamine-Modified Actin

    PubMed Central

    Kudryashov, Dmitry S.; Phillips, Martin; Reisler, Emil

    2004-01-01

    Actin labeling at Cys374 with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics. PMID:15298916

  6. Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue.

    PubMed

    Clark, Abbot F; Brotchie, Daniel; Read, A Thomas; Hellberg, Peggy; English-Wright, Sherry; Pang, Iok-Hou; Ethier, C Ross; Grierson, Ian

    2005-02-01

    Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Toxoplasma gondii profilin acts primarily to sequester G-actin while formins efficiently nucleate actin filament formation in vitro.

    PubMed

    Skillman, Kristen M; Daher, Wassim; Ma, Christopher I; Soldati-Favre, Dominique; Sibley, L David

    2012-03-27

    Apicomplexan parasites employ gliding motility that depends on the polymerization of parasite actin filaments for host cell entry. Despite this requirement, parasite actin remains almost entirely unpolymerized at steady state; formation of filaments required for motility relies on a small repertoire of actin-binding proteins. Previous studies have shown that apicomplexan formins and profilin exhibit canonical functions on heterologous actins from higher eukaryotes; however, their biochemical properties on parasite actins are unknown. We therefore analyzed the impact of T. gondii profilin (TgPRF) and FH1-FH2 domains of two formin isoforms in T. gondii (TgFRM1 and TgFRM2) on the polymerization of T. gondii actin (TgACTI). Our findings based on in vitro assays demonstrate that TgFRM1-FH1-FH2 and TgFRM2-FH1-FH2 dramatically enhanced TgACTI polymerization in the absence of profilin, making them the sole protein factors known to initiate polymerization of this normally unstable actin. In addition, T. gondii formin domains were shown to both initiate polymerization and induce bundling of TgACTI filaments; however, they did not rely on TgPRF for these activities. In contrast, TgPRF sequestered TgACTI monomers, thus inhibiting polymerization even in the presence of formins. Collectively, these findings provide insight into the unusual control mechanisms of actin dynamics within the parasite.

  9. Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles.

    PubMed Central

    Miyata, H; Hotani, H

    1992-01-01

    Spherical giant liposomes that had encapsulated skeletal-muscle G-actin were made by swelling a dried lipid mixture of dimyristoyl phosphatidylcholine/cardiolipin, 1:1 (wt/wt), in a solution of G-actin/CaCl2 at 0 degree C. Polymerization of the encapsulated G-actin into actin filaments was achieved by raising the temperature to 30 degrees C. We observed the subsequent shape changes of the liposomes by dark-field and differential interference-contrast light microscopy. After approximately 40 min, which was required for completion of actin polymerization, two shapes of liposome were evident: dumbbell and disk. Elongation of the dumbbell-shaped liposomes was concomitant with actin polymerization. Polarization microscopy showed that actin filaments formed thick bundles in the liposomes and that these filaments lay contiguous to the periphery of the liposome. Localization of actin filaments in the liposomes was confirmed by observation of rhodamine phalloidin-conjugated actin filaments by fluorescence microscopy. Both dumbbell- and disk-shaped liposomes were rigid and kept their shapes as far as actin filaments were stabilized. In contrast, liposomes containing bovine serum albumin were fragile, and their shapes continually fluctuated from Brownian motion, indicating that the actin bundles served as mechanical support for the liposome shapes. Images PMID:1454846

  10. Pattern Formation in Polymerizing Actin Flocks: Spirals, Spots, and Waves without Nonlinear Chemistry

    NASA Astrophysics Data System (ADS)

    Le Goff, T.; Liebchen, B.; Marenduzzo, D.

    2016-12-01

    We propose a model solely based on actin treadmilling and polymerization which describes many characteristic states of actin-wave formation: spots, spirals, and traveling waves. In our model, as in experiments on cells recovering motility following actin depolymerization, we choose an isotropic low-density initial condition; polymerization of actin filaments then raises the density towards the Onsager threshold where they align. We show that this alignment, in turn, destabilizes the isotropic phase and generically induces transient actin spots or spirals as part of the dynamical pathway towards a polarized phase which can either be uniform or consist of a series of actin-wave trains (flocks). Our results uncover a universal route to actin-wave formation in the absence of any system-specific nonlinear biochemistry, and it may help to understand the mechanism underlying the observation of actin spots and waves in vivo. They also suggest a minimal setup to design similar patterns in vitro.

  11. Actin-binding proteins implicated in the formation of the punctate actin foci stimulated by the self-incompatibility response in Papaver.

    PubMed

    Poulter, Natalie S; Staiger, Christopher J; Rappoport, Joshua Z; Franklin-Tong, Vernonica E

    2010-03-01

    The actin cytoskeleton is a key target for signaling networks and plays a central role in translating signals into cellular responses in eukaryotic cells. Self-incompatibility (SI) is an important mechanism responsible for preventing self-fertilization. The SI system of Papaver rhoeas pollen involves a Ca(2+)-dependent signaling network, including massive actin depolymerization as one of the earliest cellular responses, followed by the formation of large actin foci. However, no analysis of these structures, which appear to be aggregates of filamentous (F-)actin based on phalloidin staining, has been carried out to date. Here, we characterize and quantify the formation of F-actin foci in incompatible Papaver pollen tubes over time. The F-actin foci increase in size over time, and we provide evidence that their formation requires actin polymerization. Once formed, these SI-induced structures are unusually stable, being resistant to treatments with latrunculin B. Furthermore, their formation is associated with changes in the intracellular localization of two actin-binding proteins, cyclase-associated protein and actin-depolymerizing factor. Two other regulators of actin dynamics, profilin and fimbrin, do not associate with the F-actin foci. This study provides, to our knowledge, the first insights into the actin-binding proteins and mechanisms involved in the formation of these intriguing structures, which appear to be actively formed during the SI response.

  12. The Differential Formation of the LINC-Mediated Perinuclear Actin Cap in Pluripotent and Somatic Cells

    PubMed Central

    Khatau, Shyam B.; Kusuma, Sravanti; Hanjaya-Putra, Donny; Mali, Prashant; Cheng, Linzhao; Lee, Jerry S. H.; Gerecht, Sharon; Wirtz, Denis

    2012-01-01

    The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape. PMID:22574215

  13. Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

    PubMed Central

    1994-01-01

    Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin. PMID:8163553

  14. Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo.

    PubMed

    Eskin, Julian A; Rankova, Aneliya; Johnston, Adam B; Alioto, Salvatore L; Goode, Bruce L

    2016-03-01

    Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1's functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles.

  15. Cofilin nuclear-cytoplasmic shuttling affects cofilin-actin rod formation during stress.

    PubMed

    Munsie, Lise Nicole; Desmond, Carly R; Truant, Ray

    2012-09-01

    Cofilin protein is involved in regulating the actin cytoskeleton during typical steady state conditions, as well as during cell stress conditions where cofilin saturates F-actin, forming cofilin-actin rods. Cofilin can enter the nucleus through an active nuclear localization signal (NLS), accumulating in nuclear actin rods during stress. Here, we characterize the active nuclear export of cofilin through a leptomycin-B-sensitive, CRM1-dependent, nuclear export signal (NES). We also redefine the NLS of cofilin as a bipartite NLS, with an additional basic epitope required for nuclear localization. Using fluorescence lifetime imaging microscopy (FLIM) and Förster resonant energy transfer (FRET) between cofilin moieties and actin, as well as automated image analysis in live cells, we have defined subtle mutations in the cofilin NLS that allow cofilin to bind actin in vivo and affect cofilin dynamics during stress. We further define the requirement of cofilin-actin rod formation in a system of cell stress by temporal live-cell imaging. We propose that cofilin nuclear shuttling is critical for the cofilin-actin rod stress response with cofilin dynamically communicating between the nucleus and cytoplasm during cell stress.

  16. Myosin di-phosphorylation and peripheral actin bundle formation as initial events during endothelial barrier disruption.

    PubMed

    Hirano, Mayumi; Hirano, Katsuya

    2016-02-11

    The phosphorylation of the 20-kD myosin light chain (MLC) and actin filament formation play a key role in endothelial barrier disruption. MLC is either mono- or di-phosphorylated (pMLC and ppMLC) at T18 or S19. The present study investigated whether there are any distinct roles of pMLC and ppMLC in barrier disruption induced by thrombin. Thrombin induced a modest bi-phasic increase in pMLC and a robust mono-phasic increase in ppMLC. pMLC localized in the perinuclear cytoplasm during the initial phase, while ppMLC localized in the cell periphery, where actin bundles were formed. Later, the actin bundles were rearranged into stress fibers, where pMLC co-localized. Rho-kinase inhibitors inhibited thrombin-induced barrier disruption and peripheral localization of ppMLC and actin bundles. The double, but not single, mutation of phosphorylation sites abolished the formation of peripheral actin bundles and the barrier disruption, indicating that mono-phosphorylation of MLC at either T18 or S19 is functionally sufficient for barrier disruption. Namely, the peripheral localization, but not the degree of phosphorylation, is suggested to be essential for the functional effect of ppMLC. These results suggest that MLC phosphorylation and actin bundle formation in cell periphery are initial events during barrier disruption.

  17. Molecular requirements for actin-based lamella formation in Drosophila S2 cells.

    PubMed

    Rogers, Stephen L; Wiedemann, Ursula; Stuurman, Nico; Vale, Ronald D

    2003-09-15

    Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

  18. Actin Capping Protein is required for Dendritic Spine Development and Synapse Formation

    PubMed Central

    Fan, Yanjie; Tang, Xin; Vitriol, Eric; Chen, Gong; Zheng, James Q.

    2011-01-01

    Dendritic spines serve as the postsynaptic platform for most excitatory synapses in the mammalian brain and their shape and size are tightly correlated with synaptic strength. The actin cytoskeleton plays a crucial role in the spine structure and its modifications during synapse development and plasticity, but the underlying regulatory mechanisms remain to be elucidated. Here we report that actin capping protein (CP), a regulator of actin filament growth, plays an essential role for spine development and synapse formation. We found that CP expression in rat hippocampus is elevated at and after the stage of substantial synapse formation. CP knockdown in hippocampal cultures resulted in a marked decline in the spine density accompanied by increased filopodia-like protrusions. Moreover, the spines of CP knockdown neurons exhibited an altered morphology, highlighted by multiple thin filopodia-like protrusions emerging from the spine head. Finally, the number of functional synapses was reduced by CP knockdown as evidenced by a reduction in the density of paired pre- and postsynaptic markers and in the frequency of miniature excitatory postsynaptic currents. These findings indicate that capping of actin filaments by CP represents an essential step for the remodeling of the actin architecture underlying spine morphogenesis and synaptic formation during development. PMID:21752999

  19. Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation

    PubMed Central

    Yamagishi, Yuka; Abe, Hiroshi

    2015-01-01

    We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation. PMID:26424802

  20. The large GTPase dynamin regulates actin comet formation and movement in living cells

    PubMed Central

    Orth, James D.; Krueger, E. W.; Cao, H.; McNiven, Mark A.

    2002-01-01

    The large GTPase dynamin (Dyn2) has been demonstrated by us and others to interact with several different actin-binding proteins. To define how Dyn2 might participate in actin dynamics in livings cells we have expressed green fluorescent protein (GFP)-tagged Dyn2 in cultured cells and observed labeling of comet-like vesicles and macropinosomes. The comet structures progressed with a constant velocity and were reminiscent of actin comets associated with motile vesicles in cells expressing type I phosphatidylinositol phosphate 5-kinases. Based on these observations we sought to determine whether Dyn2 is an integral component of actin comets. Cells expressing type I phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a prominent colocalization of Dyn2 and actin in comet structures. Interestingly, comet formation and motility were normal in cells expressing wild-type Dyn2-GFP but altered markedly in Dyn2 mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a significant reduction in comet number, length, velocity, and efficiency of movement. In contrast, comets in cells expressing Dyn2ΔPRD-GFP appeared dark and did not incorporate the mutant Dyn2 protein, indicating that the proline-rich domain (PRD) is required for Dyn2 recruitment. Further, these comets were significantly longer and slower than those in control cells. These findings demonstrate a role for Dyn2 in actin-based vesicle motility. PMID:11782546

  1. The actin cytoskeleton participates in the early events of autophagosome formation upon starvation induced autophagy.

    PubMed

    Aguilera, Milton Osmar; Berón, Walter; Colombo, María Isabel

    2012-11-01

    Autophagy is a process by which cytoplasmic material is sequestered in a double-membrane vesicle destined for degradation. Nutrient deprivation stimulates the pathway and the number of autophagosomes in the cell increases in response to such stimulus. In the current report we have demonstrated that actin is necessary for starvation-mediated autophagy. When the actin cytoskeleton is depolymerized, the increase in autophagic vacuoles in response to the starvation stimulus was abolished without affecting maturation of remaining autophagosomes. In addition, actin filaments colocalized with ATG14, BECN1/Beclin1 and PtdIns3P-rich structures, and some of them have a typical omegasome shape stained with the double FYVE domain or ZFYVE1/DFCP1. In contrast, no major colocalization between actin and ULK1, ULK2, ATG5 or MAP1LC3/LC3 was observed. Taken together, our data indicate that actin has a role at very early stages of autophagosome formation linked to the PtdIns3P generation step. In addition, we have found that two members of the Rho family of proteins, RHOA and RAC1 have a regulatory function on starvation-mediated autophagy, but with opposite roles. Indeed, RHOA has an activatory role whereas Rac has an inhibitory one. We have also found that inhibition of the RHOA effector ROCK impaired the starvation-mediated autophagic response. We propose that actin participates in the initial membrane remodeling stage when cells require an enhanced rate of autophagosome formation, and this actin function would be tightly regulated by different members of the Rho family.

  2. The actin cytoskeleton participates in the early events of autophagosome formation upon starvation induced autophagy

    PubMed Central

    Aguilera, Milton Osmar; Berón, Walter; Colombo, María Isabel

    2012-01-01

    Autophagy is a process by which cytoplasmic material is sequestered in a double-membrane vesicle destined for degradation. Nutrient deprivation stimulates the pathway and the number of autophagosomes in the cell increases in response to such stimulus. In the current report we have demonstrated that actin is necessary for starvation-mediated autophagy. When the actin cytoskeleton is depolymerized, the increase in autophagic vacuoles in response to the starvation stimulus was abolished without affecting maturation of remaining autophagosomes. In addition, actin filaments colocalized with ATG14, BECN1/Beclin1 and PtdIns3P-rich structures, and some of them have a typical omegasome shape stained with the double FYVE domain or ZFYVE1/DFCP1. In contrast, no major colocalization between actin and ULK1, ULK2, ATG5 or MAP1LC3/LC3 was observed. Taken together, our data indicate that actin has a role at very early stages of autophagosome formation linked to the PtdIns3P generation step. In addition, we have found that two members of the Rho family of proteins, RHOA and RAC1 have a regulatory function on starvation-mediated autophagy, but with opposite roles. Indeed, RHOA has an activatory role whereas Rac has an inhibitory one. We have also found that inhibition of the RHOA effector ROCK impaired the starvation-mediated autophagic response. We propose that actin participates in the initial membrane remodeling stage when cells require an enhanced rate of autophagosome formation, and this actin function would be tightly regulated by different members of the Rho family. PMID:22863730

  3. The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri.

    PubMed

    Sohn, Hae-Jin; Kim, Jong-Hyun; Shin, Myeong-Heon; Song, Kyoung-Ju; Shin, Ho-Joon

    2010-03-01

    Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

  4. Syndapin promotes pseudocleavage furrow formation by actin organization in the syncytial Drosophila embryo

    PubMed Central

    Sherlekar, Aparna; Rikhy, Richa

    2016-01-01

    Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila. Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. Here we report a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. We conclude that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division. PMID:27146115

  5. The Eps8/IRSp53/VASP Network Differentially Controls Actin Capping and Bundling in Filopodia Formation

    PubMed Central

    Milanesi, Francesca; Di Fiore, Pier Paolo; Menna, Elisabetta; Matteoli, Michela; Gov, Nir S.; Scita, Giorgio; Ciliberto, Andrea

    2011-01-01

    There is a body of literature that describes the geometry and the physics of filopodia using either stochastic models or partial differential equations and elasticity and coarse-grained theory. Comparatively, there is a paucity of models focusing on the regulation of the network of proteins that control the formation of different actin structures. Using a combination of in-vivo and in-vitro experiments together with a system of ordinary differential equations, we focused on a small number of well-characterized, interacting molecules involved in actin-dependent filopodia formation: the actin remodeler Eps8, whose capping and bundling activities are a function of its ligands, Abi-1 and IRSp53, respectively; VASP and Capping Protein (CP), which exert antagonistic functions in controlling filament elongation. The model emphasizes the essential role of complexes that contain the membrane deforming protein IRSp53, in the process of filopodia initiation. This model accurately accounted for all observations, including a seemingly paradoxical result whereby genetic removal of Eps8 reduced filopodia in HeLa, but increased them in hippocampal neurons, and generated quantitative predictions, which were experimentally verified. The model further permitted us to explain how filopodia are generated in different cellular contexts, depending on the dynamic interaction established by Eps8, IRSp53 and VASP with actin filaments, thus revealing an unexpected plasticity of the signaling network that governs the multifunctional activities of its components in the formation of filopodia. PMID:21814501

  6. Antagonistic regulation of F-BAR protein assemblies controls actin polymerization during podosome formation.

    PubMed

    Tsujita, Kazuya; Kondo, Akihiro; Kurisu, Shusaku; Hasegawa, Junya; Itoh, Toshiki; Takenawa, Tadaomi

    2013-05-15

    FBP17, an F-BAR domain protein, has emerged as a crucial factor linking the plasma membrane to WASP-mediated actin polymerization. Although it is well established that FBP17 has a powerful self-polymerizing ability that promotes actin nucleation on membranes in vitro, knowledge of inhibitory factors that counteract this activity in vivo is limited. Here, we demonstrate that the assembly of FBP17 on the plasma membranes is antagonized by PSTPIP2, another F-BAR protein implicated in auto-inflammatory disorder. Knockdown of PSTPIP2 in macrophage promotes the assembly of FBP17 as well as subsequent actin nucleation at podosomes, resulting in an enhancement of matrix degradation. This phenotype is rescued by expression of PSTPIP2 in a manner dependent on its F-BAR domain. Time-lapse total internal reflection fluorescence (TIRF) microscopy observations reveal that the self-assembly of FBP17 at the podosomal membrane initiates actin polymerization, whereas the clustering of PSTPIP2 has an opposite effect. Biochemical analysis and live-cell imaging show that PSTPIP2 inhibits actin polymerization by competing with FBP17 for assembly at artificial as well as the plasma membrane. Interestingly, the assembly of FBP17 is dependent on WASP, and its dissociation by WASP inhibition strongly induces a self-organization of PSTPIP2 at podosomes. Thus, our data uncover a previously unappreciated antagonism between different F-BAR domain assemblies that determines the threshold of actin polymerization for the formation of functional podosomes and may explain how the absence of PSTPIP2 causes auto-inflammatory disorder.

  7. [The reorganization of actin cytoskeleton and microtubule system of human endothelial vein in the intercellular contacts formation].

    PubMed

    Shahov, A S; Dugina, V B; Alieva, I B

    2015-01-01

    Endothelial cells are tightly fitted to each other and lining the interior surface of all vessels of living organism to provide vascular permeability regulation and interchange between the blood circulating in vessels and tissue fluids of those organs in which these vessels are located. In vitro endothelial monolayer conserve it's basic barrier function which is native for vessels endothelium. Based on this fact we used endothelial cells growing in vitro as a model system in experimental studies of cytoskeletal and adhesion cell components interaction. In current paper, cultured human vein endothelial cells monolayer was used to quantify cytoskeleton alterations in the of endothelial cells from spreading and formation of the first cell-cell contacts to confluent monolayer formation. The system of actin filaments formed two different cytoskeletal structures in the cells of venous endothelium: 1) cortical actin network; 2) actin stress fibers (bundles) arranged parallel to the substrate. Two actin isoforms, β- and γ-cytoplasmic (non-muscle) actins, are expressed in endothelial cells. The bundles of actin stress fibers were detected by immunofluorescent staining with antibody against β-actin, whereas antibodies against γ-actin identified cortical and lamellar networks. For assessment of the actin cytoskeleton organization it's fluorescence intensity on the area of 10 μM2 located (1) near the free edge, and (2) in the zone of cell-cell contacts were analyzed. Fluorescence intensity of β-actin structures was higher in the areas of cell-cell contact. The fluorescence of γ-actin structures was more intensive at the leading edges of the lamellae, and was the lowest on the stable edges of the cells with formed cell-cell contacts. The endothelial monolayer formation was accompanied by microtubule system alteration: the number of microtubules increased at the cell edge, and besides the microtubules quantity in the area of already formed cell-cell contact was always

  8. A mechanical model of actin stress fiber formation and substrate elasticity sensing in adherent cells.

    PubMed

    Walcott, Sam; Sun, Sean X

    2010-04-27

    Tissue cells sense and respond to the stiffness of the surface on which they adhere. Precisely how cells sense surface stiffness remains an open question, though various biochemical pathways are critical for a proper stiffness response. Here, based on a simple mechanochemical model of biological friction, we propose a model for cell mechanosensation as opposed to previous more biochemically based models. Our model of adhesion complexes predicts that these cell-surface interactions provide a viscous drag that increases with the elastic modulus of the surface. The force-velocity relation of myosin II implies that myosin generates greater force when the adhesion complexes slide slowly. Then, using a simple cytoskeleton model, we show that an external force applied to the cytoskeleton causes actin filaments to aggregate and orient parallel to the direction of force application. The greater the external force, the faster this aggregation occurs. As the steady-state probability of forming these bundles reflects a balance between the time scale of bundle formation and destruction (because of actin turnover), more bundles are formed when the cytoskeleton time-scale is small (i.e., on stiff surfaces), in agreement with experiment. As these large bundles of actin, called stress fibers, appear preferentially on stiff surfaces, our mechanical model provides a mechanism for stress fiber formation and stiffness sensing in cells adhered to a compliant surface.

  9. Pattern formation in actin gels: A study in the mechanics of gels formed by the important cytoskeletal protein actin, especially as applied to cellular motility

    NASA Astrophysics Data System (ADS)

    Balter, Ariel

    We have studied pattern formation in actin gels to better understand how they function in biological systems, especially in the motility mechanism used by some pathogenic bacteria such as Listeria. By coating themselves with certain enzymes, these bacteria appropriate actin (a protein) from the surrounding host cell's cytoplasm and cause a network or "gel" of actin filaments to grow on their outer surface. As the resulting "comet tail" shaped protrusion grows, it pushes the bacterium away. In experiments, polystyrene beads coated with the same enzymes will also generate comet tails and swim in a very similar manner. However, these bead experiments have also generated anomalous results such as the formation of many comet tails. In some experiments, when two comet tails formed they systematically grew into regular, oppositely handed helices. The formation of any comet tails on a bead poses a physical conundrum. The bacterial enzyme coating is asymmetrical so the comet tail forms in a particular place. But the beads are symmetrical, so comet tails formation constitutes symmetry breaking and spontaneous pattern formation. We have modeled this process as a competition between elastic energy (which favors many tails) and chemical energy (which favors few tails). Our analytical model explains the factors that experimentally determine the number of tails, and numerical simulations confirm these predictions. To understand the helical tails, we did extensive data analysis involving image processing, statistical analysis and mathematical modeling of images of the helical tails. We identified some important features of how the twin tails form. For instance, the tail growth rate is independent of drag force, and bead rotation must accompany helical tail formation. We also created a physical model for helical growth. Numerical simulations of our model show that at very low Reynolds number, a cylindrical object growing under the conditions of an actin comet tail can spontaneously

  10. Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation

    PubMed Central

    Bencsik, Norbert; Szíber, Zsófia; Liliom, Hanna; Tárnok, Krisztián; Borbély, Sándor; Gulyás, Márton; Rátkai, Anikó; Szűcs, Attila; Hazai-Novák, Diána; Ellwanger, Kornelia; Rácz, Bence; Pfizenmaier, Klaus; Hausser, Angelika

    2015-01-01

    Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines. PMID:26304723

  11. Cingulin and actin mediate midbody-dependent apical lumen formation during polarization of epithelial cells

    PubMed Central

    Mangan, Anthony J.; Sietsema, Daniel V.; Li, Dongying; Moore, Jeffrey K.; Citi, Sandra; Prekeris, Rytis

    2016-01-01

    Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis. PMID:27484926

  12. Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions.

    PubMed

    Raval, Manmeet H; Quintero, Omar A; Weck, Meredith L; Unrath, William C; Gallagher, James W; Cui, Runjia; Kachar, Bechara; Tyska, Matthew J; Yengo, Christopher M

    2016-10-21

    Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.

  13. Rapid signaling of estrogen to WAVE1 and moesin controls neuronal spine formation via the actin cytoskeleton.

    PubMed

    Sanchez, Angel Matias; Flamini, Marina Ines; Fu, Xiao-Dong; Mannella, Paolo; Giretti, Maria Silvia; Goglia, Lorenzo; Genazzani, Andrea Riccardo; Simoncini, Tommaso

    2009-08-01

    Estrogens are important regulators of neuronal cell morphology, and this is thought to be critical for gender-specific differences in brain function and dysfunction. Dendritic spine formation is dependent on actin remodeling by the WASP-family verprolin homologous (WAVE1) protein, which controls actin polymerization through the actin-related protein (Arp)-2/3 complex. Emerging evidence indicates that estrogens are effective regulators of the actin cytoskeleton in various cell types via rapid, extranuclear signaling mechanisms. We here show that 17beta-estradiol (E2) administration to rat cortical neurons leads to phosphorylation of WAVE1 on the serine residues 310, 397, and 441 and to WAVE1 redistribution toward the cell membrane at sites of dendritic spine formation. WAVE1 phosphorylation is found to be triggered by a Galpha(i)/Gbeta protein-dependent, rapid extranuclear signaling of estrogen receptor alpha to c-Src and to the small GTPase Rac1. Rac1 recruits the cyclin-dependent kinase (Cdk5) that directly phosphorylates WAVE1 on the three serine residues. After WAVE1 phosphorylation by E2, the Arp-2/3 complex concentrates at sites of spine formation, where it triggers the local reorganization of actin fibers. In parallel, E2 recruits a Galpha(13)-dependent pathway to RhoA and ROCK-2, leading to activation of actin remodeling via the actin-binding protein, moesin. Silencing of WAVE1 or of moesin abrogates the increase in dendritic spines induced by E2 in cortical neurons. In conclusion, our findings indicate that the control of actin polymerization and branching via moesin or WAVE1 is a key function of estrogen receptor alpha in neurons, which may be particularly relevant for the regulation of dendritic spines.

  14. The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

    PubMed Central

    Strale, Pierre-Olivier; Duchesne, Laurence; Peyret, Grégoire; Montel, Lorraine; Nguyen, Thao; Png, Evelyn; Tampé, Robert; Troyanovsky, Sergey; Hénon, Sylvie; Ladoux, Benoit

    2015-01-01

    Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell–cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell–cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell–cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell–cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell–cell contact fluidity. PMID:26195669

  15. Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

    PubMed Central

    Nakano, Kentaro; Satoh, Kazuomi; Morimatsu, Akeshi; Ohnuma, Masaaki; Mabuchi, Issei

    2001-01-01

    We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively. PMID:11694585

  16. Myosin 1b promotes the formation of post-Golgi carriers by regulating actin assembly and membrane remodelling at the trans-Golgi network.

    PubMed

    Almeida, Claudia G; Yamada, Ayako; Tenza, Danièle; Louvard, Daniel; Raposo, Graça; Coudrier, Evelyne

    2011-06-12

    The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.

  17. A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis.

    PubMed

    Brill, J A; Hime, G R; Scharer-Schuksz, M; Fuller, M T

    2000-09-01

    The endgame of cytokinesis can follow one of two pathways depending on developmental context: resolution into separate cells or formation of a stable intercellular bridge. Here we show that the four wheel drive (fwd) gene of Drosophila melanogaster is required for intercellular bridge formation during cytokinesis in male meiosis. In fwd mutant males, contractile rings form and constrict in dividing spermatocytes, but cleavage furrows are unstable and daughter cells fuse together, producing multinucleate spermatids. fwd is shown to encode a phosphatidylinositol 4-kinase (PI 4-kinase), a member of a family of proteins that perform the first step in the synthesis of the key regulatory membrane phospholipid PIP2. Wild-type activity of the fwd PI 4-kinase is required for tyrosine phosphorylation in the cleavage furrow and for normal organization of actin filaments in the constricting contractile ring. Our results suggest a critical role for PI 4-kinases and phosphatidylinositol derivatives during the final stages of cytokinesis.

  18. Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells

    PubMed Central

    Cheng, Catherine; Nowak, Roberta B.; Biswas, Sondip K.; Lo, Woo-Kuen; FitzGerald, Paul G.; Fowler, Velia M.

    2016-01-01

    Purpose To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end–capping protein. Methods We investigated F-actin and F-actin–binding protein localization in interdigitations of Tmod1+/+ and Tmod1−/− single mature lens fibers. Results F-actin–rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1−/− mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1−/− mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1−/− mature fibers. Conclusions These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin–actin network stabilized by Tmod1. α-Actinin–crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin–associated proteins required for the formation of paddles between lens fibers. PMID:27537257

  19. Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss Physcomitrella patens.

    PubMed

    Yamashita, Hiroko; Sato, Yoshikatsu; Kanegae, Takeshi; Kagawa, Takatoshi; Wada, Masamitsu; Kadota, Akeo

    2011-02-01

    Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.

  20. Genome-Wide siRNA Screen Identifies Complementary Signaling Pathways Involved in Listeria Infection and Reveals Different Actin Nucleation Mechanisms during Listeria Cell Invasion and Actin Comet Tail Formation

    PubMed Central

    Kühbacher, Andreas; Emmenlauer, Mario; Rämo, Pauli; Kafai, Natasha; Dehio, Christoph

    2015-01-01

    ABSTRACT Listeria monocytogenes enters nonphagocytic cells by a receptor-mediated mechanism that is dependent on a clathrin-based molecular machinery and actin rearrangements. Bacterial intra- and intercellular movements are also actin dependent and rely on the actin nucleating Arp2/3 complex, which is activated by host-derived nucleation-promoting factors downstream of the cell receptor Met during entry and by the bacterial nucleation-promoting factor ActA during comet tail formation. By genome-wide small interfering RNA (siRNA) screening for host factors involved in bacterial infection, we identified diverse cellular signaling networks and protein complexes that support or limit these processes. In addition, we could precise previously described molecular pathways involved in Listeria invasion. In particular our results show that the requirements for actin nucleators during Listeria entry and actin comet tail formation are different. Knockdown of several actin nucleators, including SPIRE2, reduced bacterial invasion while not affecting the generation of comet tails. Most interestingly, we observed that in contrast to our expectations, not all of the seven subunits of the Arp2/3 complex are required for Listeria entry into cells or actin tail formation and that the subunit requirements for each of these processes differ, highlighting a previously unsuspected versatility in Arp2/3 complex composition and function. PMID:25991686

  1. DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes

    PubMed Central

    Sun, Ming-Hong; Yang, Mo; Xie, Feng-Yun; Wang, Wei; Zhang, Lili; Shen, Wei; Yin, Shen

    2017-01-01

    As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells. PMID:28099474

  2. The yeast gene, MDM20, is necessary for mitochondrial inheritance and organization of the actin cytoskeleton.

    PubMed

    Hermann, G J; King, E J; Shaw, J M

    1997-04-07

    In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament-binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20 delta cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

  3. A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation.

    PubMed Central

    Rosenshine, I; Ruschkowski, S; Stein, M; Reinscheid, D J; Mills, S D; Finlay, B B

    1996-01-01

    Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement. Images PMID:8654358

  4. Correlation of expression of the actin filament-bundling protein espin with stereociliary bundle formation in the developing inner ear.

    PubMed

    Li, Huawei; Liu, Hong; Balt, Steve; Mann, Sabine; Corrales, C Eduardo; Heller, Stefan

    2004-01-01

    The vertebrate hair cell is named for its stereociliary bundle or hair bundle that protrudes from the cell's apical surface. Hair bundles mediate mechanosensitivity, and their highly organized structure plays a critical role in mechanoelectrical transduction and amplification. The prototypical hair bundle is composed of individual stereocilia, 50-300 in number, depending on the animal species and on the type of hair cell. The assembly of stereocilia, in particular, the formation during development of individual rows of stereocilia with descending length, has been analyzed in great morphological detail. Electron microscopic studies have demonstrated that stereocilia are filled with actin filaments that are rigidly cross-linked. The growth of individual rows of stereocilia is associated with the addition of actin filaments and with progressively increasing numbers of cross-bridges between actin filaments. Recently, a mutation in the actin filament-bundling protein espin has been shown to underlie hair bundle degeneration in the deaf jerker mouse, subsequently leading to deafness. Our study was undertaken to investigate the appearance and developmental expression of espin in chicken inner ear sensory epithelia. We found that the onset of espin expression correlates with the initiation and growth of stereocilia bundles in vestibular and cochlear hair cells. Intense espin immunolabeling of stereocilia was colocalized with actin filament staining in all types of hair cells at all developmental stages and in adult animals. Our analysis of espin as a molecular marker for actin filament cross-links in stereocilia is in full accordance with previous morphological studies and implicates espin as an important structural component of hair bundles from initiation of bundle assembly to mature chicken hair cells.

  5. Formation of actin filament bundles in the ring canals of developing Drosophila follicles

    PubMed Central

    1996-01-01

    Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay

  6. Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging

    PubMed Central

    Smith, Benjamin A.; Daugherty-Clarke, Karen; Goode, Bruce L.; Gelles, Jeff

    2013-01-01

    Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ∼1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors. PMID:23292935

  7. The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

    PubMed Central

    Belmont, Lisa D.; Drubin, David G.

    1998-01-01

    Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables. PMID:9732289

  8. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  9. Multi-photon Imaging of Actin Filament Formation and Mitochondrial Energetics of Human ACBT Gliomas

    PubMed Central

    Hwang, Yu-Jer; Kolettis, Nomiki; Yang, Miso; Gillard, Elizabeth R.; Sanchez, Edgar; Sun, Chung-ho; Tromberg, Bruce J.; Krasieva, Tatiana B.; Lyubovitsky, Julia G.

    2011-01-01

    We studied the three-dimensional (3D) distribution of actin filaments and mitochondria in relation to ACBT glioblastoma cells migration. We embedded the cells in the spheroid form within collagen hydrogels and imaged them by in-situ multi-photon microscopy (MPM). The static 3D overlay of the distribution of actin filaments and mitochondria provided a greater understanding of cell-to-cell and cell-to-substrate interactions and morphology. While imaging mitochondria to obtain ratiometric redox index based on cellular fluorescence from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) we observed differential sensitivity of the migrating ACBT glioblastoma cells to femtosecond laser irradiation employed in MPM. We imaged actin-GFP fluorescence in live ACBT glioma cells and for the first time observed dynamic modulation of the pools of actin during migration in 3D. The MPM imaging, which probes cells directly within the 3D cancer models, could potentially aid in working out a link between the functional performance of mitochondria, actin distribution and cancer invasiveness. PMID:21143483

  10. The formin DIAPH1 (mDia1) regulates megakaryocyte proplatelet formation by remodeling the actin and microtubule cytoskeletons.

    PubMed

    Pan, Jiajia; Lordier, Larissa; Meyran, Deborah; Rameau, Philippe; Lecluse, Yann; Kitchen-Goosen, Susan; Badirou, Idinath; Mokrani, Hayat; Narumiya, Shuh; Alberts, Arthur S; Vainchenker, William; Chang, Yunhua

    2014-12-18

    Megakaryocytes are highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rho-associated protein kinase (Rock)/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that 2 major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly, but also in the regulation of microtubule stability and dynamics during PPF.

  11. Why is Actin Patchy?

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2009-03-01

    The intracellular protein actin, by reversibly polymerizing into filaments, generates forces for motion and shape changes of many types of biological cells. Fluorescence imaging studies show that actin often occurs in the form of localized patches of size roughly one micrometer at the cell membrane. Patch formation is most prevalent when the free-actin concentration is low. I investigate possible mechanisms for the formation of actin patches by numerically simulating the ``dendritic nucleation'' model of actin network growth. The simulations include filament growth, capping, branching, severing, and debranching. The attachment of membrane-bound activators to actin filaments, and subsequent membrane diffusion of unattached activators, are also included. It is found that as the actin concentration increases from zero, the actin occurs in patches at lower actin concentrations, and the size of the patches increases with increasing actin concentration. At a critical value of the actin concentration, the system undergoes a transition to complete coverage. The results are interpreted within the framework of reaction-diffusion equations in two dimensions.

  12. The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption.

    PubMed

    Murphy, Kaitlin C; Morgan, Joshua T; Wood, Joshua A; Sadeli, Adeline; Murphy, Christopher J; Russell, Paul

    2014-10-15

    The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 min. We hypothesize that changes in actin morphology correlate with alterations in cell stiffness induced by Lat-B but this is not a general consequence of other cytoskeletal disrupting agents such as Rho kinase inhibitors. We treated HTM cells with 2 µM Lat-B or 100 µM Y-27632 and allowed the cells to recover for 30-270 min. While examining actin morphology in Lat-B treated cells, we observed striking cortical actin arrays (CAAs). The percentage of CAA positive cells (CPCs) was time dependent and exceeded 30% at 90 min and decreased after 270 min. Y-27632 treated cells exhibited few CAAs and no changes in cell stiffness. Together, these data suggest that the increase in cell stiffness after Lat-B treatment is correlated with CAAs.

  13. Memo-RhoA-mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells.

    PubMed

    Zaoui, Kossay; Honoré, Stéphane; Isnardon, Daniel; Braguer, Diane; Badache, Ali

    2008-11-03

    Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as alpha-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo-RhoA-mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.

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

    PubMed Central

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

    2015-01-01

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

  15. Morphogenetic role of F-actin meshwork in chamber formation: immunolabeling results from symbiont bearing benthic foraminifera

    NASA Astrophysics Data System (ADS)

    Tyszka, Jaroslaw; Raitzsch, Markus; Bijma, Jelle; Höher, Nicole; Bickmeyer, Ulf; Rivera-Ingraham, Georginia; Topa, Paweł; Kaczmarek, Karina; Mewes, Antje; Bowser, Samuel; Travis, Jeffrey

    2015-04-01

    Foraminifera are excellent tracers of palaeoceanographic conditions recorded in their shell (test) morphology and chemical composition. Understanding foraminiferal morphology controlled by chamberwise growth can be reduced to processes of chamber formation. However, little is known about how foraminifera control the shape of the chamber wall to be biosynthesized and precipitated. Searching for fundamental morphogenetic features involved in biomineralization, we focused on foraminifers, which belong to the class Globothalamea. The most critical condition to run experiments was to have convenient access to early stages of chamber formation in any species of cultured benthic foraminifers. We have tested small foraminifers collected from the tidal flats of the North Sea. All species, including Ammonia, Haynesina, and Elphidium, turned out to be unsuitable due to their reproduction seasonality and/or unpredictability. The problem was solved by using symbiont bearing Amphistegina lessonii cultured in small aquaria. In well treated cultures, such foraminifera often reproduce on a glass wall surface, serving as a continuous source of juveniles. They tend to regularly construct chambers. Another important point is that symbiont bearing foraminifers usually do not construct opaque protective cysts from detritus that disturb observations. All these features facilitate immunolabeling experiments observed under confocal microscopy. Therefore, for the first time, we managed to label cytoskeleton proteins during the chamber formation in Foraminifera. The results show that the shape of chamber is predefined by a meshwork of F-actin, which acts as a dynamic organic scaffold most likely responsible for distribution and docking of biomineralizing molecules (glycoproteins). The F-actin meshwork interacts with microtubules and all associated proteins, which are involved in the morphogenesis of biomineralized structures. Foraminifera, like other eukaryotic cells, can form active

  16. Tropomyosin-dependent filament formation by a polymerization-defective mutant yeast actin (V266G,L267G).

    PubMed

    Wen, K K; Kuang, B; Rubenstein, P A

    2000-12-22

    A major function of tropomyosin (TPM) in nonmuscle cells may be stabilization of F-actin by binding longitudinally along the actin filament axis. However, no clear evidence exists in vitro that TPM can significantly affect the critical concentration of actin. We previously made a polymerization-defective mutant actin, GG (V266G, L267G). This actin will not polymerize alone at 25 degrees C but will in the presence of phalloidin or beryllium fluoride. With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive. We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25 degrees C with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac tropomyosin), indicating the importance of the ends in polymerization rescue. In the presence of TPM, the apparent critical concentration of actin is 5.5 microm, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 microm). Non N-acetylated TPMs did not rescue GG-actin polymerization. The TPMs did not prevent cold-induced depolymerization of GG F-actin. TPM-dependent GG-actin polymerization did not occur at temperatures below 20 degrees C. Polymerization rescue may depend initially on the capture of unstable GG-F-actin oligomers by the TPM, resulting in the strengthening of actin monomer-monomer contacts along the filament axis.

  17. The Interaction of Arp2/3 Complex with Actin: Nucleation, High Affinity Pointed End Capping, and Formation of Branching Networks of Filaments

    NASA Astrophysics Data System (ADS)

    Dyche Mullins, R.; Heuser, John A.; Pollard, Thomas D.

    1998-05-01

    The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μ M. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

  18. Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation.

    PubMed

    Klieger, Yair; Almogi-Hazan, Osnat; Ish-Shalom, Eliran; Pato, Aviad; Pauker, Maor H; Barda-Saad, Mira; Wang, Lynn; Baniyash, Michal

    2014-01-01

    TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance.

  19. Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice

    PubMed Central

    Morikawa, Yuka; Zhang, Min; Heallen, Todd; Leach, John; Tao, Ge; Xiao, Yang; Bai, Yan; Li, Wei; Willerson, James T.; Martin, James F.

    2015-01-01

    The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signaling-deficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex (DGC), a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near scar tissue of injured Hippo signaling-deficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to the mechanical changes associated with heart injury to promote repair. PMID:25943351

  20. Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

    PubMed Central

    1995-01-01

    The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion. PMID:7490299

  1. Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

    PubMed

    Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

    2015-04-24

    EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein.

  2. Magnet cable manufacturing

    SciTech Connect

    Royet, J.

    1990-10-01

    The cable is the heart of a superconducting accelerator magnet. Since the initial development of the Rutherford Cable more than twenty years ago, many improvements in manufacturing techniques have increased the current carrying capacity. When the Tevatron cable was specified fifteen years ago the current carrying capacity was 1800 A/mm{sup 2} at a field of 5.3T. During the intervening years it has been increased to 3000 A/mm{sup 2}. These improvements were due to refinements in the fabrication of the strands and the formation of the cable from the strands. The metallurgists were able to impart significant gains in performance by improving the homogeneity of the conductor. The engineers and technicians who designed and built the modern cabling machines made an enormous contribution by significantly reducing the degradation of wire performance that occurs when the wire was cabled. The fact that these gains were made while increasing the speed of cabling is one of the technological advances that made accelerators like the SSC possible. This article describes the cabling machines that were built to manufacture the cable for the full scale SSC prototype magnets and the low beta quadrupoles for the Fermilab Tevatron. This article also presents a compendium of the knowledge that was gained in the struggle to make high performance cable to exacting dimensional standards and at the throughput needed for the SSC. The material is an important part of the technology transfer from the Department of energy Laboratories to Industry.

  3. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar); Skin lesion - actinic keratosis ... likely to develop it if you: Have fair skin, blue or green eyes, or blond or red ...

  4. A polar-localized iron-binding protein determines the polar targeting of Burkholderia BimA autotransporter and actin tail formation.

    PubMed

    Lu, Qiuhe; Xu, Yue; Yao, Qing; Niu, Miao; Shao, Feng

    2015-03-01

    Intracellular bacterial pathogens including Shigella, Listeria, Mycobacteria, Rickettsia and Burkholderia spp. deploy a specialized surface protein onto one pole of the bacteria to induce filamentous actin tail formation for directional movement within host cytosol. The mechanism underlying polar targeting of the actin tail proteins is unknown. Here we perform a transposon screen in Burkholderia thailandensis and identify a conserved bimC that is required for actin tail formation mediated by BimA from B. thailandensis and its closely related pathogenic species B. pseudomallei and B. mallei. bimC is located upstream of bimA in the same operon. Loss of bimC results in even distribution of BimA on the outer membrane surface, where actin polymerization still occurs. BimC is targeted to the same bacterial pole independently of BimA. BimC confers polar targeting of BimA prior to BimA translocation across bacterial inner membrane. BimC is an iron-binding protein, requiring a four-cysteine cluster at the carboxyl terminus. Mutation of the cysteine cluster disrupts BimC polar localization. Truncation analyses identify the transmembrane domain in BimA being responsible for its polar targeting. Consistently, BimC can interact with BimA transmembrane domain in an iron binding-dependent manner. Our study uncovers a new mechanism that determines the polar distribution of bacteria-induced actin tail in infected host cells.

  5. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

    PubMed Central

    Brzoska, Anthony J.; Jensen, Slade O.; Barton, Deborah A.; Davies, Danielle S.; Overall, Robyn L.; Skurray, Ronald A.; Firth, Neville

    2016-01-01

    Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments. PMID:27310470

  6. Platelet rich plasma promotes skeletal muscle cell migration in association with up-regulation of FAK, paxillin, and F-Actin formation.

    PubMed

    Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Lin, Mioa-Sui; Tsai, Ting-Ta; Pang, Jong-Hwei S

    2017-02-24

    Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell-substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F-actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small-interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP-mediated promotion of cell migration. Dose-dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up-regulated dose-dependently. F-actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up-regulation of proteins expression of paxillin and FAK as well as increasing F-actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

  7. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  8. RhoA-mediated FMNL1 regulates GM130 for actin assembly and phosphorylates MAPK for spindle formation in mouse oocyte meiosis.

    PubMed

    Wang, Fei; Zhang, Liang; Duan, Xing; Zhang, Guang-Li; Wang, Zhen-Bo; Wang, Qiang; Xiong, Bo; Sun, Shao-Chen

    2015-01-01

    Formin-like 1 (FMNL1) is a member of Formin family proteins which are the actin nucleators. Although FMNL1 activities have been shown to be essential for cell adhesion, cytokinesis, cell polarization and migration in mitosis, the functional roles of mammalian FMNL1 during oocyte meiosis remain uncertain. In this study, we investigated the functions of FMNL1 in mouse oocytes using specific morpholino (MO) microinjection and live cell imaging. Immunofluorescent staining showed that in addition to its cytoplasmic distribution, FMNL1 was primarily localized at the spindle poles after germinal vesicle breakdown (GVBD). FMNL1 knockdown caused the low rate of polar body extrusion and resulted in large polar bodies. Time-lapse microscopic and immunofluorescence intensity analysis indicated that this might be due to the aberrant actin expression levels. Cortical polarity was disrupted as shown by a loss of actin cap and cortical granule free domain (CGFD) formation, which was confirmed by a failure of meiotic spindle positioning. And this might be the reason for the large polar body formation. Spindle formation was also disrupted, which might be due to the abnormal localization of p-MAPK. These results indicated that FMNL1 affected both actin dynamics and spindle formation for the oocyte polar body extrusion. Moreover, FMNL1 depletion resulted in aberrant localization and expression patterns of a cis-Golgi marker protein, GM130. Finally, we found that the small GTPase RhoA might be the upstream regulator of FMNL1. Taken together, our data indicate that FMNL1 is required for spindle organization and actin assembly through a RhoA-FMNL1-GM130 pathway during mouse oocyte meiosis.

  9. Superconductor cable

    DOEpatents

    Allais, Arnaud; Schmidt, Frank; Marzahn, Erik

    2010-05-04

    A superconductor cable is described, having a superconductive flexible cable core (1) , which is laid in a cryostat (2, 3, 4), in which the cable core (1) runs in the cryostat (2, 3, 4) in the form of a wave or helix at room temperature.

  10. Cable Television.

    ERIC Educational Resources Information Center

    Federal Communications Commission, Washington, DC.

    The Federal Communications Commission (FCC) presents a brief description of cable television and explains some basic regulations pertaining to it. The history of cable regulation covers the initial jurisdiction, economic considerations of the regulation, court tests, and the holding of public hearings. The major provisions of new cable rules are…

  11. Cable Economics.

    ERIC Educational Resources Information Center

    Cable Television Information Center, Washington, DC.

    A guide to the economic factors that influence cable television systems is presented. Designed for local officials who must have some familiarity with cable operations in order to make optimum decisions, the guide analyzes the financial framework of a cable system, not only from the operators viewpoint, but also from the perspective of the…

  12. Bacterial nucleators: actin' on actin

    PubMed Central

    Bugalhão, Joana N.; Mota, Luís Jaime; Franco, Irina S.

    2015-01-01

    The actin cytoskeleton is a key target of numerous microbial pathogens, including protozoa, fungi, bacteria and viruses. In particular, bacterial pathogens produce and deliver virulence effector proteins that hijack actin dynamics to enable bacterial invasion of host cells, allow movement within the host cytosol, facilitate intercellular spread or block phagocytosis. Many of these effector proteins directly or indirectly target the major eukaryotic actin nucleator, the Arp2/3 complex, by either mimicking nucleation promoting factors or activating upstream small GTPases. In contrast, this review is focused on a recently identified class of effector proteins from Gram-negative bacteria that function as direct actin nucleators. These effector proteins mimic functional activities of formins, WH2-nucleators and Ena/VASP assembly promoting factors demonstrating that bacteria have coopted the complete set of eukaryotic actin assembly pathways. Structural and functional analyses of these nucleators have revealed several motifs and/or mechanistic activities that are shared with eukaryotic actin nucleators. However, functional effects of these proteins during infection extend beyond plain actin polymerization leading to interference with other host cell functions such as vesicle trafficking, cell cycle progression and cell death. Therefore, their use as model systems could not only help in the understanding of the mechanistic details of actin polymerization but also provide novel insights into the connection between actin dynamics and other cellular pathways. PMID:26416078

  13. Actinous enigma or enigmatic actin

    PubMed Central

    Povarova, Olga I; Uversky, Vladimir N; Kuznetsova, Irina M; Turoverov, Konstantin K

    2014-01-01

    Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins.

  14. The ER Stress Sensor PERK Coordinates ER-Plasma Membrane Contact Site Formation through Interaction with Filamin-A and F-Actin Remodeling.

    PubMed

    van Vliet, Alexander R; Giordano, Francesca; Gerlo, Sarah; Segura, Inmaculada; Van Eygen, Sofie; Molenberghs, Geert; Rocha, Susana; Houcine, Audrey; Derua, Rita; Verfaillie, Tom; Vangindertael, Jeroen; De Keersmaecker, Herlinde; Waelkens, Etienne; Tavernier, Jan; Hofkens, Johan; Annaert, Wim; Carmeliet, Peter; Samali, Afshin; Mizuno, Hideaki; Agostinis, Patrizia

    2017-03-02

    Loss of ER Ca(2+) homeostasis triggers endoplasmic reticulum (ER) stress and drives ER-PM contact sites formation in order to refill ER-luminal Ca(2+). Recent studies suggest that the ER stress sensor and mediator of the unfolded protein response (UPR) PERK regulates intracellular Ca(2+) fluxes, but the mechanisms remain elusive. Here, using proximity-dependent biotin identification (BioID), we identified the actin-binding protein Filamin A (FLNA) as a key PERK interactor. Cells lacking PERK accumulate F-actin at the cell edges and display reduced ER-PM contacts. Following ER-Ca(2+) store depletion, the PERK-FLNA interaction drives the expansion of ER-PM juxtapositions by regulating F-actin-assisted relocation of the ER-associated tethering proteins Stromal Interaction Molecule 1 (STIM1) and Extended Synaptotagmin-1 (E-Syt1) to the PM. Cytosolic Ca(2+) elevation elicits rapid and UPR-independent PERK dimerization, which enforces PERK-FLNA-mediated ER-PM juxtapositions. Collectively, our data unravel an unprecedented role of PERK in the regulation of ER-PM appositions through the modulation of the actin cytoskeleton.

  15. Formation and ingression of division furrow can progress under the inhibitory condition of actin polymerization in ciliate Tetrahymena pyriformis.

    PubMed

    Shimizu, Yuhta; Kushida, Yasuharu; Kiriyama, Shuhei; Nakano, Kentaro; Numata, Osamu

    2013-12-01

    In eukaryotic cells that multiply by binary fission, the interaction of actin filaments with myosin II in the contractile ring is widely recognized to generate force for membrane ingression into the cleavage furrow; however, the expression of myosin II is restricted in animals, yeast, fungi, and amoeba (collectively, unikonts). No corresponding motor protein capable of forming mini-filaments that could exert sufficient tension to cleave the cell body is found in bikonts, consisting of planta, algae, and most protozoa; however, cells in some bikont lineages multiply by binary fission, as do animal cells. Of these, the ciliate Tetrahymena is known to form an actin ring beneath the division furrow in cytokinesis. Here, we investigated the role of filamentous actin in the cytokinesis of Tetrahymena pyriformis by treating synchronized dividing cells with an actin-inhibiting drug, Latrunculin-A. Video microscopic observation of live cells undergoing cytokinesis was performed, and contrary to expectation, we found that initiation of furrow ingression and its progress are not suppressed under the inhibitory condition of actin polymerization in Tetrahymena cells. We suggest that an actin filament-independent mechanism of binary fission may have been acquired during the evolution in this organism.

  16. Role of G protein signaling in the formation of the fibrin(ogen)-integrin αIIbβ3-actin cytoskeleton complex in platelets.

    PubMed

    Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

    2016-09-01

    Effective platelet function requires formation of a physical link between fibrin(ogen), integrin αIIbβ3, and cytoplasmic actin filaments. We investigated the role of the Gαq, Gαi, and Gα12/13 families of heterotrimeric GTP-binding proteins (G proteins) in the assembly of a ligand-αIIbβ3-actin cytoskeleton complex. Selective and combined activation of the G proteins was achieved by using combinations of various platelet agonists and inhibitors. Formation and stability of fibrinogen-αIIbβ3 interaction were evaluated by the extent of platelet aggregation and the rate of eptifibatide-induced platelet disaggregation; association of αIIbβ3 with the cytoskeleton was analyzed by western blot. Formation of the fibrin-αIIbβ3-actin cytoskeleton complex was evaluated by rotational thromboelastometry assay in which clot formation was induced by the mixture of reptilase and factor XIIIa. We demonstrated that involvement of heterotrimeric G proteins in the formation of the ligand-αIIbβ3-cytoskeleton complex depends on whether fibrinogen or fibrin serves as the integrin ligand. Formation of the fibrinogen-αIIbβ3-cytoskeleton complex requires combined activation of at least two G protein pathways while the maximal αIIbβ3-cytoskeleton association and the strongest αIIbβ3-fibrinogen binding supporting irreversible platelet aggregation require combined activation of all three-Gαq, Gαi, and Gα12/13-G protein families. In contrast, formation of the fibrin-αIIbβ3-cytoskeleton complex mediating clot retraction is critically dependent on the activation of the Gαi family, especially on the activation of Gαz.

  17. Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans.

    PubMed

    Ketelaar, Tijs; Meijer, Harold J G; Spiekerman, Marjolein; Weide, Rob; Govers, Francine

    2012-12-01

    The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin

  18. Polycation induced actin bundles.

    PubMed

    Muhlrad, Andras; Grintsevich, Elena E; Reisler, Emil

    2011-04-01

    Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.

  19. Cable compliance

    NASA Technical Reports Server (NTRS)

    Kerley, J.; Eklund, W.; Burkhardt, R.; Rossoni, P.

    1992-01-01

    The object of the investigation was to solve mechanical problems using cable-in-bending and cable-in-torsion. These problems included robotic contacts, targets, and controls using cable compliance. Studies continued in the use of cable compliance for the handicapped and the elderly. These included work stations, walkers, prosthetic knee joints, elbow joints, and wrist joints. More than half of these objects were met, and models were made and studies completed on most of the others. It was concluded that the many different and versatile solutions obtained only opened the door to many future challenges.

  20. ERK and phosphoinositide 3-kinase temporally coordinate different modes of actin-based motility during embryonic wound healing.

    PubMed

    Li, Jingjing; Zhang, Siwei; Soto, Ximena; Woolner, Sarah; Amaya, Enrique

    2013-11-01

    Embryonic wound healing provides a perfect example of efficient recovery of tissue integrity and homeostasis, which is vital for survival. Tissue movement in embryonic wound healing requires two functionally distinct actin structures: a contractile actomyosin cable and actin protrusions at the leading edge. Here, we report that the discrete formation and function of these two structures is achieved by the temporal segregation of two intracellular upstream signals and distinct downstream targets. The sequential activation of ERK and phosphoinositide 3-kinase (PI3K) signalling divides Xenopus embryonic wound healing into two phases. In the first phase, activated ERK suppresses PI3K activity, and is responsible for the activation of Rho and myosin-2, which drives actomyosin cable formation and constriction. The second phase is dominated by restored PI3K signalling, which enhances Rac and Cdc42 activity, leading to the formation of actin protrusions that drive migration and zippering. These findings reveal a new mechanism for coordinating different modes of actin-based motility in a complex tissue setting, namely embryonic wound healing.

  1. Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation

    PubMed Central

    Winans, Amy M; Collins, Sean R; Meyer, Tobias

    2016-01-01

    Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon. DOI: http://dx.doi.org/10.7554/eLife.12387.001 PMID:26836307

  2. Temperature-induced sol-gel transition and microgel formation in α-actinin cross-linked actin networks: A rheological study

    NASA Astrophysics Data System (ADS)

    Tempel, M.; Isenberg, G.; Sackmann, E.

    1996-08-01

    We have studied the sol-gel transition, the viscoelastic and the structural properties of networks constituted of semiflexible actin filaments cross-linked by α-actinin. Cross-linking was regulated in a reversible way by varying the temperature through the association-dissociation equilibrium of the actin-α-actinin system. Viscoelastic parameters [shear storage modulus G'(ω), phase shift tan(Φ)(ω), creep compliance J(t)] were measured as a function of temperature and actin-to-cross-linker ratio by a magnetically driven rotating disc rheometer. G'(ω) and tan(Φ)(ω) were studied at a frequency ω corresponding to the elastic plateau regime of the G'(ω) versus ω spectrum of the purely entangled solution. The microstructure of the networks was viewed by negative staining electron microscopy (EM). The phase shift tan(Φ) (or equivalently the viscosity η) diverges and reaches a maximum when approaching the apparent gel point from lower and higher temperatures, and the maximum defines the gel point (temperature Tg). The elastic plateau modulus G'N diverges at temperatures beyond this gel point TTg. The cross-linking transition (corresponding to a sol-gel transition at zero frequency) is interpreted in terms of a percolation model and the divergence of G'N at TTg), (2) that microscopic segregation takes place at T<=Tg leading to local formation of clusters (a state termed microgel), and (3) that at low actin-α-actinin ratios (rAα<=10) and low temperatures (T<=10 °C) macroscopic segregation into bundles of cross-linked actin filaments and a diluted solution of actin filaments is observed. The three regimes of network structure are represented by an

  3. [Alterations in actin cytoskeleton and rate of reparation of human endothelium (the wound-healing model) under the condition of clinostatting].

    PubMed

    Romanov, Iu A; Kabaeva, N V; Buravkova, L B

    2001-01-01

    Effects of long-term simulation of hypogravity on actin cytoskeleton and cell migration were investigated in cultured human endothelium cells (EC). In control, F-actin resided predominantly on the periphery of cell forming an array of parallel bundles with "dense bodies" along the edge. A small number of actin cable fibers was found in the center. Already after 1-2 hrs of clinostatting at 5 RPM the cell cytoskeleton showed actin filament thinning and displacement toward the cell edges. In subsequent 6-18 hrs, almost all actin fibers had left the center part of EC and had ranged themselves in a continuous F-actin line in the intercellular contact area. In most cases, these changes resulted in the so-called "ruff-edge". Since both the disappearance of cable fibers and formation of the "ruff-edge" add to the cell migration activity, this parameter was studied with the would-healing model. According to our data, 24-48 hrs of exposure to hypogravity stimulates cell migration and expedites 2-3 times reparation of mechanically damaged monolayer. The results suggest that effects of hypogravity on cultured human EC are likely to be consequent to alterations in the activity of protein kinase C and/or adenylate cyclase involving many members of the cellular metabolism.

  4. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.

    PubMed Central

    Kozma, R; Ahmed, S; Best, A; Lim, L

    1995-01-01

    The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology. PMID:7891688

  5. Fascin regulates nuclear actin during Drosophila oogenesis

    PubMed Central

    Kelpsch, Daniel J.; Groen, Christopher M.; Fagan, Tiffany N.; Sudhir, Sweta; Tootle, Tina L.

    2016-01-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved. PMID:27535426

  6. Actinic reticuloid

    SciTech Connect

    Marx, J.L.; Vale, M.; Dermer, P.; Ragaz, A.; Michaelides, P.; Gladstein, A.H.

    1982-09-01

    A 58-year-old man has his condition diagnosed as actinic reticuloid on the basis of clinical and histologic findings and phototesting data. He had clinical features resembling mycosis fungoides in light-exposed areas. Histologic findings disclosed a bandlike infiltrate with atypical mononuclear cells in the dermis and scattered atypical cells in the epidermis. Electron microscopy disclosed mononuclear cells with bizarre, convoluted nuclei, resembling cerebriform cells of Lutzner. Phototesting disclosed a diminished minimal erythemal threshold to UV-B and UV-A. Microscopic changes resembling actinic reticuloid were reproduced in this patient 24 and 72 hours after exposure to 15 minimal erythemal doses of UV-B.

  7. TGFβ2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways

    PubMed Central

    Montecchi-Palmer, Michela; Bermudez, Jaclyn Y.; Webber, Hannah C.; Patel, Gaurang C.; Clark, Abbot F.; Mao, Weiming

    2017-01-01

    Purpose Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation. Methods Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4′,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. Results TGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. Conclusions TGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers. PMID:28241317

  8. 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

  9. G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers.

    PubMed

    Higashida, Chiharu; Suetsugu, Shiro; Tsuji, Takahiro; Monypenny, James; Narumiya, Shuh; Watanabe, Naoki

    2008-10-15

    mDia1 belongs to the formin family of proteins that share FH1 and FH2 domains. Although formins play a critical role in the formation of many actin-based cellular structures, the physiological regulation of formin-mediated actin assembly within the cell is still unknown. Here we show that cells possess an acute actin polymer restoration mechanism involving mDia1. By using single-molecule live-cell imaging, we found that several treatments including low-dose G-actin-sequestering drugs and unpolymerizable actin mutants activate mDia1 to initiate fast directional movement. The FH2 region, the core domain for actin nucleation, is sufficient to respond to latrunculin B (LatB) to increase its actin nucleation frequency. Simulation analysis revealed an unexpected paradoxical effect of LatB that leads to a several fold increase in free G-actin along with an increase in total G-actin. These results indicate that in cells, the actin nucleation frequency of mDia1 is enhanced not only by Rho, but also strongly through increased catalytic efficiency of the FH2 domain. Consistently, frequent actin nucleation by mDia1 was found around sites of vigorous actin disassembly. Another major actin nucleator, the Arp2/3 complex, was not affected by the G-actin increase induced by LatB. Taken together, we propose that transient accumulation of G-actin works as a cue to promote mDia1-catalyzed actin nucleation to execute rapid reassembly of actin filaments.

  10. Superconductor cable

    DOEpatents

    Allais, Arnaud; Schmidt, Frank (Langenhagen, DE

    2009-12-15

    A superconductor cable includes a superconductive cable core (1) and a cryostat (2) enclosing the same. The cable core (1) has a superconductive conductor (3), an insulation (4) surrounding the same and a shielding (5) surrounding the insulation (4). A layer (3b) of a dielectric or semiconducting material is applied to a central element (3a) formed from a normally conducting material as a strand or tube and a layer (3c) of at least one wire or strip of superconductive material is placed helically on top. The central element (3a) and the layer (3c) are connected to each other in an electrically conducting manner at the ends of the cable core (1).

  11. Cable manufacture

    NASA Technical Reports Server (NTRS)

    Gamble, P.

    1972-01-01

    A survey is presented of flat electrical cable manufacturing, with particular reference to patented processes. The economics of manufacture based on an analysis of material and operating costs is considered for the various methods. Attention is given to the competitive advantages of the several processes and their resulting products. The historical area of flat cable manufacture is presented to give a frame of reference for the survey.

  12. Cable tester

    NASA Astrophysics Data System (ADS)

    Rammage, Robert L.

    1990-10-01

    A device for sequentially testing the plurality of connectors in a wiring harness is disclosed. The harness is attached to the tester by means of adapter cables and a rotary switch is used to sequentially, individually test the connectors by passing a current through the connector. If the connector is unbroken, a light will flash to show it is electrically sound. The adapters allow a large number of cable configurations to be tested using a single tester configuration.

  13. miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

    PubMed Central

    Amelio, Ivano; Lena, Anna Maria; Viticchiè, Giuditta; Shalom-Feuerstein, Ruby; Terrinoni, Alessandro; Dinsdale, David; Russo, Giandomenico; Fortunato, Claudia; Bonanno, Elena; Spagnoli, Luigi Giusto; Aberdam, Daniel; Knight, Richard Austen

    2012-01-01

    During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis. PMID:23071155

  14. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

    Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

    2001-05-01

    Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

  15. Modeling actin waves in dictyostelium cells

    NASA Astrophysics Data System (ADS)

    Wasnik, Vaibhav; Mukhopadhyay, Ranjan

    2011-03-01

    Actin networks in living cells demonstrate a high capacity for self-organization and are responsible for the formation of a variety of structures such as lamellopodia, phagocytic cups, and cleavage furrows. Recent experiments have studied actin waves formed on the surface of dictyostelium cells that have been treated with a depolymerizing agent. These waves are believed to be physiologically important, for example, for the formation of phagocytic cups. We propose and study a minimal model, based on the dendritic nucleation of actin polymers, to explain the formation of these waves. This model can be extended to study the dynamics of the coupled actin-membrane system.

  16. Actin dynamics and cofilin-actin rods in Alzheimer disease

    PubMed Central

    Bamburg, James R.; Bernstein, Barbara W.

    2017-01-01

    Cytoskeletal abnormalities and synaptic loss, typical of both familial and sporadic Alzheimer disease (AD), are induced by diverse stresses such as neuroinflammation, oxidative stress, and energetic stress, each of which may be initiated or enhanced by proinflammatory cytokines or amyloid-β (Aβ) peptides. Extracellular Aβ-containing plaques and intracellular phospho-tau-containing neurofibrillary tangles are postmortem pathologies required to confirm AD and have been the focus of most studies. However, AD brain, but not normal brain, also have increased levels of cytoplasmic rod-shaped bundles of filaments composed of ADF/cofilin-actin in a 1:1 complex (rods). Cofilin, the major ADF/cofilin isoform in mammalian neurons, severs actin filaments at low cofilin/actin ratios and stabilizes filaments at high cofilin/actin ratios. It binds cooperatively to ADP-actin subunits in F-actin. Cofilin is activated by dephosphorylation and may be oxidized in stressed neurons to form disulfide-linked dimers, required for bundling cofilin-actin filaments into stable rods. Rods form within neurites causing synaptic dysfunction by sequestering cofilin, disrupting normal actin dynamics, blocking transport, and exacerbating mitochondrial membrane potential loss. Aβ and proinflammatory cytokines induce rods through a cellular prion protein-dependent activation of NADPH oxidase and production of reactive oxygen species. Here we review recent advances in our understanding of cofilin biochemistry, rod formation, and the development of cognitive deficits. We will then discuss rod formation as a molecular pathway for synapse loss that may be common between all three prominent current AD hypotheses, thus making rods an attractive therapeutic target. PMID:26873625

  17. Cable Television.

    ERIC Educational Resources Information Center

    Federal Communications Commission, Washington, DC.

    This report provides information about cable television and the Federal Communications Commission's (FCC) responsibilities in regulating its operation. The initial jurisdiction and rules covered in this report pertain to the court test, public hearing, certificate of compliance, franchising, signal carriage, leapfrogging, access and origination…

  18. Drosophila tensin plays an essential role in cell migration and planar polarity formation during oogenesis by mediating integrin-dependent extracellular signals to actin organization.

    PubMed

    Cha, In Jun; Lee, Jang Ho; Cho, Kyoung Sang; Lee, Sung Bae

    2017-03-11

    Oogenesis in Drosophila involves very dynamic cellular changes such as cell migration and polarity formation inside an ovary during short period. Previous studies identified a number of membrane-bound receptors directly receiving certain types of extracellular inputs as well as intracellular signalings to be involved in the regulation of these dynamic cellular changes. However, yet our understanding on exactly how these receptor-mediated extracellular inputs lead to dynamic cellular changes remains largely unclear. Here, we identified Drosophila tensin encoded by blistery (by) as a novel regulator of cell migration and planar polarity formation and characterized the genetic interaction between tensin and integrin during oogenesis. Eggs from by mutant showed decreased hatching rate and morphological abnormality, a round-shape, compared to the wild-type eggs. Further analyses revealed that obvious cellular defects such as defective border cell migration and planar polarity formation might be primarily associated with the decreased hatching rate and the round-shape phenotype of by mutant eggs, respectively. Moreover, by mutation also induced marked defects in F-actin organization closely associated with both cell migration and planar polarity formation during oogenesis of Drosophila. Notably, all these defective phenotypes observed in by mutant eggs became much severer by reduced level of integrin, indicative of a close functional association between integrin and tensin during oogenesis. Collectively, our findings suggest that tensin acts as a crucial regulator of dynamic cellular changes during oogenesis by bridging integrin-dependent extracellular signals to intracellular cytoskeletal organization.

  19. Actinic Prurigo.

    PubMed

    Rodríguez-Carreón, Alma Angélica; Rodríguez-Lobato, Erika; Rodríguez-Gutiérrez, Georgina; Cuevas-González, Juan Carlos; Mancheno-Valencia, Alexandra; Solís-Arias, Martha Patricia; Vega-Memije, María Elisa; Hojyo-Tomoka, María Teresa; Domínguez-Soto, Luciano

    2015-01-01

    Actinic prurigo is an idiopathic photodermatosis that affects the skin, as well as the labial and conjunctival mucosa in indigenous and mestizo populations of Latin America. It starts predominantly in childhood, has a chronic course, and is exacerbated with solar exposure. Little is known of its pathophysiology, including the known mechanisms of the participation of HLA-DR4 and an abnormal immunologic response with increase of T CD4+ lymphocytes. The presence of IgE, eosinophils, and mast cells suggests that it is a hypersensitivity reaction (likely type IVa or b). The diagnosis is clinical, and the presence of lymphoid follicles in the mucosal histopathologic study of mucosa is pathognomonic. The best available treatment to date is thalidomide, despite its secondary effects.

  20. Structural Differences Explain Diverse Functions of Plasmodium Actins

    PubMed Central

    Vahokoski, Juha; Martinez, Silvia Muñico; Ignatev, Alexander; Lepper, Simone; Frischknecht, Friedrich; Sidén-Kiamos, Inga; Sachse, Carsten; Kursula, Inari

    2014-01-01

    Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties. PMID:24743229

  1. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

    2001-01-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

  2. Superconducting Cable

    DOEpatents

    Hughey, Raburn L.; Sinha, Uday K.; Reece, David S.; Muller, Albert C.

    2005-07-22

    In order to provide a flexible oxide superconducting cable which is reduced in AC loss, tape-shaped superconducting wires covered with a stabilizing metal are wound on a flexible former. The superconducting wires are preferably laid on the former at a bending strain of not more than 0.2%. In laying on the former, a number of tape-shaped superconducting wires are laid on a core member in a side-by-side manner, to form a first layer. A prescribed number of tape-shaped superconducting wires are laid on top of the first layer in a side-by-side manner, to form a second layer. The former may be made of a metal, plastic, reinforced plastic, polymer, or a composite and provides flexibility to the superconducting wires and the cable formed therewith.

  3. Superconducting Cable

    DOEpatents

    Hughey, Raburn L.; Sinha, Uday K.; Reece, David S.; Muller, Albert C.

    2005-03-08

    In order to provide a flexible oxide superconducting cable which is reduced in AC loss, tape-shaped superconducting wires covered with a stabilizing metal are wound on a flexible former. The superconducting wires are preferably laid on the former at a bending strain of not more than 0.2%. In laying on the former, a number of tape-shaped superconducting wires are laid on a core member in a side-by-side manner, to form a first layer. A prescribed number of tape-shaped superconducting wires are laid on top of the first layer in a side-by-side manner, to form a second layer. The former may be made of a metal, plastic, reinforced plastic, polymer, or a composite and provides flexibility to the superconducting wires and the cable formed therewith.

  4. Molecular mechanisms underlying skeletal muscle weakness in human cancer: reduced myosin-actin cross-bridge formation and kinetics.

    PubMed

    Toth, Michael J; Miller, Mark S; Callahan, Damien M; Sweeny, Andrew P; Nunez, Ivette; Grunberg, Steven M; Der-Torossian, Hirak; Couch, Marion E; Dittus, Kim

    2013-04-01

    Many patients with cancer experience physical disability following diagnosis, although little is known about the mechanisms underlying these functional deficits. To characterize skeletal muscle adaptations to cancer in humans, we evaluated skeletal muscle structure and contractile function at the molecular, cellular, whole-muscle, and whole-body level in 11 patients with cancer (5 cachectic, 6 noncachectic) and 6 controls without disease. Patients with cancer showed a 25% reduction in knee extensor isometric torque after adjustment for muscle mass (P < 0.05), which was strongly related to diminished power output during a walking endurance test (r = 0.889; P < 0.01). At the cellular level, single fiber isometric tension was reduced in myosin heavy chain (MHC) IIA fibers (P = 0.05) in patients with cancer, which was explained by a reduction (P < 0.05) in the number of strongly bound cross-bridges. In MHC I fibers, myosin-actin cross-bridge kinetics were reduced in patients, as evidenced by an increase in myosin attachment time (P < 0.01); and reductions in another kinetic parameter, myosin rate of force production, predicted reduced knee extensor isometric torque (r = 0.689; P < 0.05). Patients with cancer also exhibited reduced mitochondrial density (-50%; P < 0.001), which was related to increased myosin attachment time in MHC I fibers (r = -0.754; P < 0.01). Finally, no group differences in myofilament protein content or ultrastructure were noted that explained the observed functional alterations. Collectively, our results suggest reductions in myofilament protein function as a potential molecular mechanism contributing to muscle weakness and physical disability in human cancer.

  5. Mediatory role of interleukin-6 in α smooth muscle actin induction and myofibroblast formation around silicone tissue expander.

    PubMed

    Joseph, Josna; Variathu, Kumary Thrikkovil; Mohanty, Mira

    2013-10-01

    Materials used for medical devices are usually tested for their biocompatibility, before use. However, it is known that long-term implantation in the body may lead to degradation of the material leading to an adverse tissue response. The failure of silicone breast implants due to excessive fibrosis and contracture has led to studies to delineate the cause of fibrosis around this material. To detect the biological moieties involved, conditioned media from RAW 264.7 macrophages seeded over commercially available silicone tissue expander material was added to L929 fibroblasts. Ultrahigh-molecular-weight polyethylene and tissue culture grade polystyrene served as the control materials. The gene expression of fibrogenic cytokines, interleukin-6 (IL-6), and transforming growth factor beta (TGFβ) in the RAW macrophages and myofibroblast marker alpha smooth muscle actin (αSMA) in L929 cells were quantitated by real time polymerase chain reaction. Protein expression analysis of αSMA was carried out by immunocytochemical staining and confocal microscopy. An in vitro degradation study of silicone expander material in pseudoextracellular fluid (PECF) and the αSMA expression in fibroblasts incubated with the silicone extract containing PECF has revealed the role of silicone leachants in induction of myofibroblasts. This in vitro expression study revealed the additional profibrotic role of IL-6 in fibroblast to myofibroblast transition and the synergy between material aspects and biomolecules in regulating fibrosis around Silicone implants. These findings may help in targeting newer biological moieties in the profibrotic pathway and in devising better manufacturing processes aiding the life of millions of patients.

  6. CABLE CONNECTOR

    DOEpatents

    Caller, J.M.

    1962-05-01

    An electrical connector is designed for utilization in connection with either round or flat coaxial cables. The connector comprises a bayonet-type coupling arrangement with a splined movable locking sleeve adapted to lock together components of the connector. A compression spring is attached to one of the connector components and functions to forcibly separate mating components when the locking sleeve is in an unlocked condition so as to minimize the possibility of leaving the conductors electrically coupled. (AEC)

  7. Superconductor cable

    DOEpatents

    Smith, Jr., Darrell F.; Lake, Bill L.; Ballinger, Ronald G.

    1988-01-01

    A superconducting cable comprising an in-situ-formed type II superconductor, e.g. Nb.sub.3 Sn, in association with a stabilizing conductor both in heat transfer relationship with at least one passage adapted to carry liquified gaseous refrigerant. The conductor and said at least one passage are enclosed by a sheath comprising an alloy consisting essentially of about 49% nickel, about 4% chromium, about 3% niobium, about 1.4% titanium, about 1% aluminum, balance essentially iron.

  8. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.

    PubMed

    Spracklen, Andrew J; Fagan, Tiffany N; Lovander, Kaylee E; Tootle, Tina L

    2014-09-15

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool

  9. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

    PubMed Central

    Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

    2015-01-01

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling

  10. Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

    PubMed

    Sari-Hassoun, Meryem; Clement, Marie-Jeanne; Hamdi, Imane; Bollot, Guillaume; Bauvais, Cyril; Joshi, Vandana; Toma, Flavio; Burgo, Andrea; Cailleret, Michel; Rosales-Hernández, Martha Cecilia; Macias Pérez, Martha Edith; Chabane-Sari, Daoudi; Curmi, Patrick A

    2016-02-15

    Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research.

  11. Structural Transitions of F-Actin:Espin Bundles

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin; Bartles, James; Wong, Gerard

    2006-03-01

    Espin is an actin bundling protein involved in the formation of the parallel bundles of filamentous actin in hair cell stereocilia. Mutations in espin are implicated in deafness phenotypes in mice and humans. We present measurements of the F-actin structures induced by wild type and by mutated espin obtained via small angle x-ray scattering and fluorescence microscopy. We found that wild type espin induced a paracrystalline hexagonal array of twisted F-actin, whereas the mutated espin only condensed the F-actin into a nematic-like phase. The possibility of coexisting nematic and bundled actin in mixtures containing both mutant and wild type espins was also investigated.

  12. 47 CFR 76.640 - Support for unidirectional digital cable products on digital cable systems.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 4 2012-10-01 2012-10-01 false Support for unidirectional digital cable... information tables: EIT-0, -1, -2 and -3; (C) The format of event information data format shall conform to... Delivered Out-of-Band for Digital Cable Television” (incorporated by reference, see § 76.602) (profiles 4...

  13. Cable Television Service; Cable Television Relay Service.

    ERIC Educational Resources Information Center

    Federal Register, 1972

    1972-01-01

    The rules and regulations of the Federal Communications Commission (FCC) concerning cable television service and cable relay service are presented along with the comments of the National Cable Television Association, the National Association of Broadcasters, the Association of Maximum Service Telecasters, and a major group of program suppliers.…

  14. Motility of Electric Cable Bacteria

    PubMed Central

    Damgaard, Lars Riis; Holm, Simon Agner; Schramm, Andreas; Nielsen, Lars Peter

    2016-01-01

    ABSTRACT Cable bacteria are filamentous bacteria that electrically couple sulfide oxidation and oxygen reduction at centimeter distances, and observations in sediment environments have suggested that they are motile. By time-lapse microscopy, we found that cable bacteria used gliding motility on surfaces with a highly variable speed of 0.5 ± 0.3 μm s−1 (mean ± standard deviation) and time between reversals of 155 ± 108 s. They frequently moved forward in loops, and formation of twisted loops revealed helical rotation of the filaments. Cable bacteria responded to chemical gradients in their environment, and around the oxic-anoxic interface, they curled and piled up, with straight parts connecting back to the source of sulfide. Thus, it appears that motility serves the cable bacteria in establishing and keeping optimal connections between their distant electron donor and acceptors in a dynamic sediment environment. IMPORTANCE This study reports on the motility of cable bacteria, capable of transmitting electrons over centimeter distances. It gives us a new insight into their behavior in sediments and explains previously puzzling findings. Cable bacteria greatly influence their environment, and this article adds significantly to the body of knowledge about this organism. PMID:27084019

  15. Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro

    PubMed Central

    Yahara, I; Harada, F; Sekita, S; Yoshihira, K; Natori, S

    1982-01-01

    To compare the effects of cytochalasins on the cellular level with those on the molecular level, 24 cytochalasins, 20 natural compounds and 4 derivatives, were used. The following effects were tested for each of 24 cytochalasins; (a) four high dose (2-20 muM) effects on the cellular level: rounding up of fibroblastic cells, contraction of actin cables, formation of hairy filaments containing actin, and inhibition of lymphocyte capping; (b) a low dose (0.2-2 muM) effect: inhibition of membrane ruffling; and (c) two in vitro effects: an inhibition of actin filament elongation (the high affinity effect [low dose effect] in vitro) and an effect on viscosity of actin filaments(the low affinity effect [high dose effect] in vitro). These results indicated that there are almost the same hierarchic orders of relative effectiveness of different cytochalasins between low and high dose effects and between cellular and molecular effects. From the data obtained with the 24 cytochalasins, we have calculated correlation coefficients of 0.87 and 0.79 between an effect in vivo, inhibition of capping, and an effect in vitro, inhibition of actin filament elongation, as well as between inhibition of capping and another effect in vitro, effect on viscosity of actin filaments, respectively. Furthermore, a correlation coefficient between the high affinity effect and the low affinity effect determined in vitro was calculated to be 0.90 from the data obtained in this study. The strong positive correlation among low and high dose effects in vivo and those in vitro suggests that most of the effects caused by a cytochalasin, irrespective of doses or affected phenomena, might be attributed to the interaction between the drug and the common target protein, actin. In the course of the immunofluorescence microscope study on cytochalasin-treated cells using actin antibody, we have found that aspochalasin D, a 10-isopropylcytochalasin, strongly induced the formation of rodlets containing actin in

  16. Spatial control of the actin cytoskeleton in Drosophila epithelial cells.

    PubMed

    Baum, B; Perrimon, N

    2001-10-01

    The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.

  17. Flat-cable fabrication

    NASA Astrophysics Data System (ADS)

    Groot, K. J.

    1982-11-01

    Processes, environment, designs, and materials were reviewed to lower the attrition rate and improve the manufacturing ability of flat, flexible cables. Attrition caused by foreign material, damage, and dents was lowered. A new termination was developed for two cables. An alternative design for Kapton insulated cables reduced notch sensitivity. Alternative methods of cable manufacturing and inspection are investigated.

  18. Cable-splice detector

    NASA Technical Reports Server (NTRS)

    Lee, R. D.; Iufer, E. J.; Giovannetti, A.

    1980-01-01

    Detector has possible uses in aerial cable-car systems, equipment handling in mines, boreholes, and undersea operations, and other applications where moving steel cable must be measured, monitored, or controlled. Detector consists of Hall-effect magnetic sensor located close to cable. Magnetic markings on cable are converted to electrical signals. Signals are filtered, amplified, and can actuate alarm.

  19. Cable-Twisting Machine

    NASA Technical Reports Server (NTRS)

    Kurnett, S.

    1982-01-01

    New cable-twisting machine is smaller and faster than many production units. Is useful mainly in production of short-run special cables. Already-twisted cable can be fed along axis of machine. Faster operation than typical industrial cable-twisting machines possible by using smaller spools of wire.

  20. Cable Tensiometer for Aircraft

    NASA Technical Reports Server (NTRS)

    Nunnelee, Mark (Inventor)

    2008-01-01

    The invention is a cable tensiometer that can be used on aircraft for real-time, in-flight cable tension measurements. The invention can be used on any aircraft cables with high precision. The invention is extremely light-weight, hangs on the cable being tested and uses a dual bending beam design with a high mill-volt output to determine tension.

  1. Regulation of actin polymerization by tropomodulin-3 controls megakaryocyte actin organization and platelet biogenesis.

    PubMed

    Sui, Zhenhua; Nowak, Roberta B; Sanada, Chad; Halene, Stephanie; Krause, Diane S; Fowler, Velia M

    2015-07-23

    The actin cytoskeleton is important for platelet biogenesis. Tropomodulin-3 (Tmod3), the only Tmod isoform detected in platelets and megakaryocytes (MKs), caps actin filament (F-actin) pointed ends and binds tropomyosins (TMs), regulating actin polymerization and stability. To determine the function of Tmod3 in platelet biogenesis, we studied Tmod3(-/-) embryos, which are embryonic lethal by E18.5. Tmod3(-/-) embryos often show hemorrhaging at E14.5 with fewer and larger platelets, indicating impaired platelet biogenesis. MK numbers are moderately increased in Tmod3(-/-) fetal livers, with only a slight increase in the 8N population, suggesting that MK differentiation is not significantly affected. However, Tmod3(-/-) MKs fail to develop a normal demarcation membrane system (DMS), and cytoplasmic organelle distribution is abnormal. Moreover, cultured Tmod3(-/-) MKs exhibit impaired proplatelet formation with a wide range of proplatelet bud sizes, including abnormally large proplatelet buds containing incorrect numbers of von Willebrand factor-positive granules. Tmod3(-/-) MKs exhibit F-actin disturbances, and Tmod3(-/-) MKs spreading on collagen fail to polymerize F-actin into actomyosin contractile bundles. Tmod3 associates with TM4 and the F-actin cytoskeleton in wild-type MKs, and confocal microscopy reveals that Tmod3, TM4, and F-actin partially colocalize near the membrane of proplatelet buds. In contrast, the abnormally large proplatelets from Tmod3(-/-) MKs show increased F-actin and redistribution of F-actin and TM4 from the cortex to the cytoplasm, but normal microtubule coil organization. We conclude that F-actin capping by Tmod3 regulates F-actin organization in mouse fetal liver-derived MKs, thereby controlling MK cytoplasmic morphogenesis, including DMS formation and organelle distribution, as well as proplatelet formation and sizing.

  2. Structure and function analysis of the CMS/CIN85 protein family identifies actin-bundling properties and heterotypic-complex formation.

    PubMed

    Gaidos, Gabriel; Soni, Shefali; Oswald, Duane J; Toselli, Paul A; Kirsch, Kathrin H

    2007-07-15

    Members of the CMS/CIN85 protein family participate in clathrin-mediated endocytosis and play a crucial role in maintaining the kidney filtration barrier. The CMS protein structure includes three Src homology 3 (SH3) domains and a proline-rich (PR) region that is connected by a 'linker' sequence to a coiled-coil (CC) domain. We show that CMS is a component of special actin-rich adhesion structures--podosomes--and demonstrate specific actin-binding properties of CMS. We have found that the entire C-terminal half of CMS is necessary for efficient binding to filamentous actin (F-actin). CMS and CIN85 can crosslink F-actin into bundles, a function that depends on the PR region and the CC domain. Removal of these domains reduces migration. CMS can also form heterotypic complexes with CIN85. CIN85 is expressed as multiple isoforms that share the CC domain, suggesting that heterotypic interactions with CMS provides a mechanism to regulate CMS binding to F-actin and thus for modulating dynamic rearrangements of the cytoskeleton.

  3. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks.

  4. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

    Nejedla, Michaela; Sadi, Sara; Sulimenko, Vadym; de Almeida, Francisca Nunes; Blom, Hans; Draber, Pavel; Aspenström, Pontus; Karlsson, Roger

    2016-01-01

    Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element. PMID:27307590

  5. Marine cable location system

    SciTech Connect

    Zachariadis, R.G.

    1984-05-01

    An acoustic positioning system locates a marine cable at an exploration site, such cable employing a plurality of hydrophones at spaced-apart positions along the cable. A marine vessel measures water depth to the cable as the vessel passes over the cable and interrogates the hydrophones with sonar pulses along a slant range as the vessel travels in a parallel and horizontally offset path to the cable. The location of the hydrophones is determined from the recordings of water depth and slant range.

  6. Mechanism of Actin Filament Bundling by Fascin

    SciTech Connect

    Jansen, Silvia; Collins, Agnieszka; Yang, Changsong; Rebowski, Grzegorz; Svitkina, Tatyana; Dominguez, Roberto

    2013-03-07

    Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four {beta}-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in {beta}-trefoil domains 1 and 3. The site in {beta}-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in {beta}-trefoil-3 is related by pseudo-2-fold symmetry to that in {beta}-trefoil-1. The two sites are {approx}5 nm apart, resulting in a distance between actin filaments in the bundle of {approx}8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

  7. Dynamic reorganization of the actin cytoskeleton

    PubMed Central

    Gressin, Laurène; Théry, Manuel; Blanchoin, Laurent

    2015-01-01

    Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations. PMID:26989473

  8. Cable load sensing device

    DOEpatents

    Beus, Michael J.; McCoy, William G.

    1998-01-01

    Apparatus for sensing the magnitude of a load on a cable as the cable is employed to support the load includes a beam structure clamped to the cable so that a length of the cable lies along the beam structure. A spacer associated with the beam structure forces a slight curvature in a portion of the length of cable under a cable "no-load" condition so that the portion of the length of cable is spaced from the beam structure to define a cable curved portion. A strain gauge circuit including strain gauges is secured to the beam structure by welding. As the cable is employed to support a load the load causes the cable curved portion to exert a force normal to the cable through the spacer and on the beam structure to deform the beam structure as the cable curved portion attempts to straighten under the load. As this deformation takes place, the resistance of the strain gauges is set to a value proportional to the magnitude of the normal strain on the beam structure during such deformation. The magnitude of the normal strain is manipulated in a control device to generate a value equal to the magnitude or weight of the load supported by the cable.

  9. Actin-mediated motion of meiotic chromosomes

    PubMed Central

    Koszul, R.; Kim, K. P.; Prentiss, M.; Kleckner, N.; Kameoka, S.

    2008-01-01

    Summary Chromosome movement is prominent during meiosis. Here, using a combination of in vitro and in vivo approaches, we elucidate the basis for dynamic mid-prophase chromosome movement in budding yeast. Diverse finding reveal a process in which, at the pachytene stage, individual telomere/nuclear envelope (NE) ensembles attach passively to, and then move in concert with, nucleus-hugging actin cables that are continuous with the global cytoskeletal actin network. Other chromosomes move in concert with lead chromosome(s). The same process, in modulated form, explains the zygotene "bouquet" configuration in which, immediately preceding pachytene, chromosome ends colocalize dynamically in a restricted region of the NE. Mechanical properties of the system and biological roles of mid-prophase movement for meiosis, including recombination, are discussed. PMID:18585353

  10. Cutting Edge Cable Management.

    ERIC Educational Resources Information Center

    Peach, Roger

    1997-01-01

    Describes how one school district was able to efficiently install fragile telecommunication cabling throughout its high school and save thousands of dollars. Discusses solutions to some common cable-management problems. (GR)

  11. Dendritic Actin Nucleation Causes Traveling Waves and Patches

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2010-03-01

    Reversible polymerization of the intracellular protein actin into semiflexible filaments is crucial for cell motion and environmental sensing. Recent studies have shown that polymerized actin can spontaneously form traveling waves and/or moving patches. I investigate possible mechanisms for such phenomena by numerically simulating the ``dendritic nucleation'' model of actin network growth. The simulations treat the growth of an actin network on a flat portion of a cell membrane, using a stochastic-growth method which calculates an explicit three-dimensional network structure. The calculations treat processes including filament growth, capping, branching, severing, and Brownian motion. The dynamics of membrane proteins stimulating actin polymerization are also included: they diffuse in the membrane, and detach/deactivate in the presence of polymerized actin. The simulations show three types of polymerized-actin behavior: 1) traveling waves, 2) coherently moving patches, and 3) random fluctuations with occasional moving patches. Wave formation is favored at low free-actin concentrations by a long reattachment time for the membrane proteins, and by weakness of the attractive interaction between filaments and the membrane. Raising the free-actin concentration results in a randomly varying distribution of polymerized actin. Lowering the free-actin concentration below the optimal value for waves causes the waves to break up into patches which, however, move coherently. Effects of similar magnitude are predicted when other intracellular protein concentrations are varied. Diffusion of the membrane proteins slows the waves, and, if fast enough, stops them completely, resulting in the formation of a static spot.

  12. Thymosin beta4: actin regulation and more.

    PubMed

    Yarmola, Elena G; Klimenko, Evguenia S; Fujita, Go; Bubb, Michael R

    2007-09-01

    The intracellular function of thymosin beta(4) is not limited to simple sequestration of globular actin. Our recent studies revealed that thymosin beta(4) affects actin critical concentration and forms a ternary complex with actin and profilin. The consequences of this complex formation can be very significant. Our new data demonstrate that it is likely that profilin affects binding of thymosin beta(4) to actin in the ternary complex through allosteric changes in actin rather than through competition for the binding site. The N- and C-terminal thymosin beta(4) helices are known to be unstructured in aqueous solution and to adopt helical conformation in organic solvents or upon binding to actin. Osmolytes stabilize protein structure, and TMAO (trimethylamine N-oxide) specifically stabilizes hydrogen bonds. This increases affinity of intact thymosin beta(4) to actin significantly, but the increase is much less for thymosin beta(4) sulfoxide. Our data show that oxidation does not alter binding of profilin to form a ternary complex, and therefore it is very likely that there is no direct steric interference by methionine 6 of thymosin beta(4). Rather, since TMAO has little effect on thymosin beta(4) sulfoxide, this observation is consistent with the hypothesis that methionine oxidation prevents helix transition. The experiment with truncated versions of thymosin beta(4) also supports this hypothesis. Oxidation and formation of the helices are important for both intra- and extracellular properties of thymosin beta(4). We found that actin and, in lesser extent, profilin-actin complex protect thymosin beta(4) from oxidation.

  13. Vertex Detector Cable Considerations

    SciTech Connect

    Cooper, William E.; /Fermilab

    2009-02-01

    Vertex detector cable requirements are considered within the context of the SiD concept. Cable material should be limited so that the number of radiation lengths represented is consistent with the material budget. In order to take advantage of the proposed accelerator beam structure and allow cooling by flow of dry gas, 'pulsed power' is assumed. Potential approaches to power distribution, cable paths, and cable design for operation in a 5 T magnetic field are described.

  14. In vitro inhibition of incompatible pollen tubes in Nicotiana alata involves the uncoupling of the F-actin cytoskeleton and the endomembrane trafficking system.

    PubMed

    Roldán, Juan A; Rojas, Hernán J; Goldraij, Ariel

    2015-01-01

    In the S-RNase-based self-incompatibility system, subcellular events occurring in the apical region of incompatible pollen tubes during the pollen rejection process are poorly understood. F-actin dynamics and endomembrane trafficking are crucial for polar growth, which is temporally and spatially controlled in the tip region of pollen tubes. Thus, we developed a simple in vitro assay to study the changes in the F-actin cytoskeleton and the endomembrane system at the apical region of incompatible pollen tubes in Nicotiana alata. Growth but not germination of pollen tubes of S c₁₀-, S₇₀-, and S₇₅-haplotypes was selectively inhibited by style extracts carrying the same haplotypes. Pollen F-actin cytoskeleton and endomembrane system, visualized by fluorescent markers, were normal during the initial 60 min of pollen culture in the presence of compatible and incompatible style extracts. Additional culture resulted in complete growth arrest and critical alterations in the integrity of the F-actin cytoskeleton and the endomembrane system of incompatible pollen tubes. The F-actin ring and the V-shaped zone disappeared from the apical region, while distorted F-actin cables and progressive formation of membrane aggregates evolved in the subapical region and the shank. The vacuolar network of incompatible pollen tubes invaded the tip region, but vacuolar membrane integrity remained mostly unaffected. The polar growth machinery of incompatible pollen tubes was uncoupled, as evidenced by the severe disruption of colocalization between the F-actin cytoskeleton and the endomembrane compartments. A model of pollen rejection integrating the main subcellular events occurring in incompatible pollen is discussed.

  15. Cable Television: Franchising Considerations.

    ERIC Educational Resources Information Center

    Baer, Walter S.; And Others

    This volume is a comprehensive reference guide to cable television technology and issues of planning, franchising, and regulating a cable system. It is intended for local government officials and citizens concerned with the development of cable television systems in their communities, as well as for college and university classes in…

  16. Cables and fire hazards

    NASA Technical Reports Server (NTRS)

    Zanelli, C.; Philbrick, S.; Beretta, G.

    1986-01-01

    Besides describing the experiments conducted to develop a nonflammable cable, this article discusses several considerations regarding other hazards which might result from cable fires, particularly the toxicity and opacity of the fumes emitted by the burning cable. In addition, this article examines the effects of using the Oxygen Index as a gauge of quality control during manufacture.

  17. 3D actin network centerline extraction with multiple active contours.

    PubMed

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2014-02-01

    Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we propose a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels.

  18. DC Cable for Railway

    NASA Astrophysics Data System (ADS)

    Tomita, Masaru

    The development of a superconducting cable for railways has commenced, assuming that a DC transmission cable will be used for electric trains. The cable has been fabricated based on the results of current testing of a superconducting wire, and various evaluation tests have been performed to determine the characteristics of the cable. A superconducting transmission cable having zero electrical resistance and suitable for railway use is expected to enhance regeneration efficiency, reduce power losses, achieve load leveling and integration of sub-stations, and reduce rail potential.

  19. Actin as a potential target for decavanadate.

    PubMed

    Ramos, Susana; Moura, José J G; Aureliano, Manuel

    2010-12-01

    ATP prevents G-actin cysteine oxidation and vanadyl formation specifically induced by decavanadate, suggesting that the oxometalate-protein interaction is affected by the nucleotide. The ATP exchange rate is increased by 2-fold due to the presence of decavanadate when compared with control actin (3.1×10(-3) s(-1)), and an apparent dissociation constant (k(dapp)) of 227.4±25.7 μM and 112.3±8.7 μM was obtained in absence or presence of 20 μM V(10), respectively. Moreover, concentrations as low as 50 μM of decameric vanadate species (V(10)) increases the relative G-actin intrinsic fluorescence intensity by approximately 80% whereas for a 10-fold concentration of monomeric vanadate (V(1)) no effects were observed. Upon decavanadate titration, it was observed a linear increase in G-actin hydrophobic surface (2.6-fold), while no changes were detected for V(1) (0-200 μM). Taken together, three major ideas arise: i) ATP prevents decavanadate-induced G-actin cysteine oxidation and vanadate reduction; ii) decavanadate promotes actin conformational changes resulting on its inactivation, iii) decavanadate has an effect on actin ATP binding site. Once it is demonstrated that actin is a new potential target for decavanadate, being the ATP binding site a suitable site for decavanadate binding, it is proposed that some of the biological effects of vanadate can be, at least in part, explained by decavanadate interactions with actin.

  20. Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks.

    PubMed

    Christensen, Jenna R; Hocky, Glen M; Homa, Kaitlin E; Morganthaler, Alisha N; Hitchcock-DeGregori, Sarah E; Voth, Gregory A; Kovar, David R

    2017-03-10

    The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.

  1. PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

    PubMed Central

    Hong, Nan Hyung; Qi, Aidong

    2015-01-01

    Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691

  2. Cable Television for Librarians. Cable Television Primer

    ERIC Educational Resources Information Center

    Briscoe, Wallace C.

    1973-01-01

    The development of cable television, its present state, and future prospects, including a possible role for libraries, are discussed. (Other conference materials are LI 503071 and 503073 through 503084.) (SJ)

  3. Cable Tester Box

    NASA Technical Reports Server (NTRS)

    Lee, Jason H.

    2011-01-01

    Cables are very important electrical devices that carry power and signals across multiple instruments. Any fault in a cable can easily result in a catastrophic outcome. Therefore, verifying that all cables are built to spec is a very important part of Electrical Integration Procedures. Currently, there are two methods used in lab for verifying cable connectivity. (1) Using a Break-Out Box and an ohmmeter this method is time-consuming but effective for custom cables and (2) Commercial Automated Cable Tester Boxes this method is fast, but to test custom cables often requires pre-programmed configuration files, and cables used on spacecraft are often uniquely designed for specific purposes. The idea is to develop a semi-automatic continuity tester that reduces human effort in cable testing, speeds up the electrical integration process, and ensures system safety. The JPL-Cable Tester Box is developed to check every single possible electrical connection in a cable in parallel. This system indicates connectivity through LED (light emitting diode) circuits. Users can choose to test any pin/shell (test node) with a single push of a button, and any other nodes that are shorted to the test node, even if they are in the same connector, will light up with the test node. The JPL-Cable Tester Boxes offers the following advantages: 1. Easy to use: The architecture is simple enough that it only takes 5 minutes for anyone to learn how operate the Cable Tester Box. No pre-programming and calibration are required, since this box only checks continuity. 2. Fast: The cable tester box checks all the possible electrical connections in parallel at a push of a button. If a cable normally takes half an hour to test, using the Cable Tester Box will improve the speed to as little as 60 seconds to complete. 3. Versatile: Multiple cable tester boxes can be used together. As long as all the boxes share the same electrical potential, any number of connectors can be tested together.

  4. MARCKS actin-binding capacity mediates actin filament assembly during mitosis in human hepatic stellate cells.

    PubMed

    Rombouts, Krista; Mello, Tommaso; Liotta, Francesco; Galli, Andrea; Caligiuri, Alessandra; Annunziato, Francesco; Pinzani, Massimo

    2012-08-15

    Cross-linking between the actin cytoskeleton and plasma membrane actin-binding proteins is a key interaction responsible for the mechanical properties of the mitotic cell. Little is known about the identity, the localization, and the function of actin filament-binding proteins during mitosis in human hepatic stellate cells (hHSC). The aim of the present study was to identify and analyze the cross talk between actin and myristoylated alanine-rich kinase C substrate (MARCKS), an important PKC substrate and actin filament-binding protein, during mitosis in primary hHSC. Confocal analysis and chromosomal fraction analysis of mitotic hHSC demonstrated that phosphorylated (P)-MARCKS displays distinct phase-dependent localizations, accumulates at the perichromosomal layer, and is a centrosomal protein belonging to the chromosomal cytosolic fraction. Aurora B kinase (AUBK), an important mitotic regulator, β-actin, and P-MARCKS concentrate at the cytokinetic midbody during cleavage furrow formation. This localization is critical since MARCKS-depletion in hHSC is characterized by a significant loss in cytosolic actin filaments and cortical β-actin that induces cell cycle inhibition and dislocation of AUBK. A depletion of AUBK in hHSC affects cell cycle, resulting in multinucleation. Quantitative live cell imaging demonstrates that the actin filament-binding capacity of MARCKS is key to regulate mitosis since the cell cycle inhibitory effect in MARCKS-depleted cells caused abnormal cell morphology and an aberrant cytokinesis, resulting in a significant increase in cell cycle time. These findings implicate that MARCKS, an important PKC substrate, is essential for proper cytokinesis and that MARCKS and its partner actin are key mitotic regulators during cell cycle in hHSC.

  5. Coaxial cable stripping device facilitates RF cabling fabrication

    NASA Technical Reports Server (NTRS)

    Hughes, R. S.; Tobias, R. A.

    1967-01-01

    Coaxial cable stripping device assures clean, right angled shoulder for RF cable connector fabrication. This method requires minimal skill and creates a low voltage standing wave ratio and mechanical stability in the interconnecting RF Cables.

  6. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

    2016-01-01

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

  7. Actin filament curvature biases branching direction

    NASA Astrophysics Data System (ADS)

    Wang, Evan; Risca, Viviana; Chaudhuri, Ovijit; Chia, Jia-Jun; Geissler, Phillip; Fletcher, Daniel

    2012-02-01

    Actin filaments are key components of the cellular machinery, vital for a wide range of processes ranging from cell motility to endocytosis. Actin filaments can branch, and essential in this process is a protein complex known as the Arp2/3 complex, which nucleate new ``daughter'' filaments from pre-existing ``mother'' filaments by attaching itself to the mother filament. Though much progress has been made in understanding the Arp2/3-actin junction, some very interesting questions remain. In particular, F-actin is a dynamic polymer that undergoes a wide range of fluctuations. Prior studies of the Arp2/3-actin junction provides a very static notion of Arp2/3 binding. The question we ask is how differently does the Arp2/3 complex interact with a straight filament compared to a bent filament? In this study, we used Monte Carlo simulations of a surface-tethered worm-like chain to explore possible mechanisms underlying the experimental observation that there exists preferential branch formation by the Arp2/3 complex on the convex face of a curved filament. We show that a fluctuation gating model in which Arp2/3 binding to the actin filament is dependent upon a rare high-local-curvature shape fluctuation of the filament is consistent with the experimental data.

  8. Aging assessment of cables

    SciTech Connect

    Jacobus, M.J.

    1992-04-01

    This paper summarizes the results of aging, condition monitoring, and accident testing of Class 1E cables used in nuclear power generating stations. Three sets of cables were aged for up to 9 months under simultaneous thermal ({approximately}100{degrees}C) and radiation ({approximately}0.10 kGy/hr) conditions. After the aging, the cables were exposed to a simulated accident consisting of high dose rate irradiation ({approximately}6 kGy/hr) followed by a high temperature steam (up to 400{degrees}C) exposure. A fourth set of cables, which were unaged, was also exposed to the accident conditions. The cables that were aged for 3 months and then accident tested were subsequently exposed to a high temperature steam fragility test (up to 400{degrees}C), while the cables that were aged for 6 months and then accident tested were subsequently exposed to a 1000-hour submergence test in a chemical solution. The results of these tests do not indicate any reason to believe that many popular nuclear power plant cable products cannot inherently be qualified for 60 years of operation for conditions simulated by this testing. Mechanical measurements (primarily elongation, modulus, and density) are more effective than electrical measurements for monitoring age-related degradation. In the high temperature steam test, ethylene propylene rubber (EPR) cable materials generally survived to higher temperatures than crosslinked polyolefin (XLPO) cable materials. In dielectric testing after the submergence testing, the XLPO materials performed better than the EPR materials.

  9. Aging assessment of cables

    SciTech Connect

    Jacobus, M.J.

    1992-01-01

    This paper summarizes the results of aging, condition monitoring, and accident testing of Class 1E cables used in nuclear power generating stations. Three sets of cables were aged for up to 9 months under simultaneous thermal ({approximately}100{degrees}C) and radiation ({approximately}0.10 kGy/hr) conditions. After the aging, the cables were exposed to a simulated accident consisting of high dose rate irradiation ({approximately}6 kGy/hr) followed by a high temperature steam (up to 400{degrees}C) exposure. A fourth set of cables, which were unaged, was also exposed to the accident conditions. The cables that were aged for 3 months and then accident tested were subsequently exposed to a high temperature steam fragility test (up to 400{degrees}C), while the cables that were aged for 6 months and then accident tested were subsequently exposed to a 1000-hour submergence test in a chemical solution. The results of these tests do not indicate any reason to believe that many popular nuclear power plant cable products cannot inherently be qualified for 60 years of operation for conditions simulated by this testing. Mechanical measurements (primarily elongation, modulus, and density) are more effective than electrical measurements for monitoring age-related degradation. In the high temperature steam test, ethylene propylene rubber (EPR) cable materials generally survived to higher temperatures than crosslinked polyolefin (XLPO) cable materials. In dielectric testing after the submergence testing, the XLPO materials performed better than the EPR materials.

  10. Axonal actin in action: Imaging actin dynamics in neurons.

    PubMed

    Ladt, Kelsey; Ganguly, Archan; Roy, Subhojit

    2016-01-01

    Actin is a highly conserved, key cytoskeletal protein involved in numerous structural and functional roles. In neurons, actin has been intensively investigated in axon terminals-growth cones-and dendritic spines, but details about actin structure and dynamics in axon shafts have remained obscure for decades. A major barrier in the field has been imaging actin. Actin exists as soluble monomers (G-actin) as well as actin filaments (F-actin), and labeling actin with conventional fluorescent probes like GFP/RFP typically leads to a diffuse haze that makes it difficult to discern kinetic behaviors. In a recent publication, we used F-actin selective probes to visualize actin dynamics in axons, resolving striking actin behaviors that have not been described before. However, using these probes to visualize actin dynamics is challenging as they can cause bundling of actin filaments; thus, experimental parameters need to be strictly optimized. Here we describe some practical methodological details related to using these probes for visualizing F-actin dynamics in axons.

  11. A structural study of F-actin - filamin networks

    NASA Astrophysics Data System (ADS)

    Ahrens-Braunstein, Ashley; Nguyen, Lam; Hirst, Linda

    2010-03-01

    The cell's ability to move and contract is attributed to the semi-flexible filamentous protein, F -actin, one of the three filaments in the cytoskeleton. Actin bundling can be formed by a cross-linking actin binding protein (ABP) filamin. By examining filamin's cross-linking abilities at different concentrations and molar ratios, we can study the flexibility, structure and multiple network formations created when cross-linking F-actin with this protein. We have studied the phase diagram of this protein system using fluorescence microscopy, analyzing the network structures observed in the context of a coarse grained molecular dynamics simulation carried out by our group.

  12. Why Are Two Different Cross-linkers Necessary for Actin Bundle Formation In Vivo and What Does Each Cross-link Contribute?

    PubMed Central

    Tilney, Lewis G.; Connelly, Patricia S.; Vranich, Kelly A.; Shaw, Michael K.; Guild, Gregory M.

    1998-01-01

    In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus ∼700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur. PMID:9763425

  13. Photodynamic therapy for actinic keratoses.

    PubMed

    Kalisiak, Michal S; Rao, Jaggi

    2007-01-01

    Actinic keratoses (AKs) are one of the most common conditions that are treated by dermatologists and they have the potential to progress to squamous cell carcinoma if left untreated. Photodynamic therapy (PDT) has emerged as a novel and versatile method of treating those lesions. Topical preparations of aminolevulinic acid and methyl aminolevulinate are commercially available photosensitizers, and numerous light sources may be used for photoactivation. This article focuses on practical aspects of PDT in the treatment of AKs, outcomes of relevant clinical trials, and special applications of PDT in transplant recipients and other who are predisposed to AK formation. Step-by-step descriptions of PDT sessions are presented.

  14. A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

    SciTech Connect

    Zhang, Bingyu; Luo, Qing; Mao, Xinjian; Xu, Baiyao; Yang, Li; Ju, Yang; Song, Guanbin

    2014-03-10

    Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway. - Highlights: • Mechano-growth factor E peptide (MGF-C25E) promotes migration of rat tenocytes. • MGF-C25E activates the FAK-ERK1/2 pathway in rat tenocytes. • MGF-C25E induces the actin remodeling and the formation of pseudopodia, and decreases the stiffness in rat tenocytes. • MGF-C25E promotes tenocyte migration via altering stiffness and forming pseudopodia by the activation of the FAK-ERK1

  15. Interaction of calponin with actin and its functional implications.

    PubMed Central

    Kołakowski, J; Makuch, R; Stepkowski, D; Dabrowska, R

    1995-01-01

    Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP. Images Figure 2 PMID:7864810

  16. Cable Diagnostic Focused Initiative

    SciTech Connect

    Hartlein, R.A.; Hampton, R.N.

    2010-12-30

    This report summarizes an extensive effort made to understand how to effectively use the various diagnostic technologies to establish the condition of medium voltage underground cable circuits. These circuits make up an extensive portion of the electric delivery infrastructure in the United States. Much of this infrastructure is old and experiencing unacceptable failure rates. By deploying efficient diagnostic testing programs, electric utilities can replace or repair circuits that are about to fail, providing an optimal approach to improving electric system reliability. This is an intrinsically complex topic. Underground cable systems are not homogeneous. Cable circuits often contain multiple branches with different cable designs and a range of insulation materials. In addition, each insulation material ages differently as a function of time, temperature and operating environment. To complicate matters further, there are a wide variety of diagnostic technologies available for assessing the condition of cable circuits with a diversity of claims about the effectiveness of each approach. As a result, the benefits of deploying cable diagnostic testing programs have been difficult to establish, leading many utilities to avoid the their use altogether. This project was designed to help address these issues. The information provided is the result of a collaborative effort between Georgia Tech NEETRAC staff, Georgia Tech academic faculty, electric utility industry participants, as well as cable system diagnostic testing service providers and test equipment providers. Report topics include: •How cable systems age and fail, •The various technologies available for detecting potential failure sites, •The advantages and disadvantages of different diagnostic technologies, •Different approaches for utilities to employ cable system diagnostics. The primary deliverables of this project are this report, a Cable Diagnostic Handbook (a subset of this report) and an online

  17. Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

    PubMed

    Pang, K M; Lee, E; Knecht, D A

    1998-03-26

    Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

  18. Truncated Cables1 causes agenesis of the corpus callosum in mice.

    PubMed

    Mizuno, Seiya; Tra, Dinh T H; Mizobuchi, Atsushi; Iseki, Hiroyoshi; Mizuno-Iijima, Saori; Kim, Jun-Dal; Ishida, Junji; Matsuda, Yoichi; Kunita, Satoshi; Fukamizu, Akiyoshi; Sugiyama, Fumihiro; Yagami, Ken-ichi

    2014-03-01

    Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are known to be involved in corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear. Here, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis to determine the genomic insertion site. Transgenes were inserted into chromosome 18 ∼12.0 Mb from the centromere. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1(TAS). Interestingly, Cables1(TAS) mRNA consisted of exons 1-3 of Cables1 and part of the transgene that encoded a novel truncated Cables1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1(TAS) in the fetal cerebrum, but not that of wild-type Cables1. To investigate whether a dominant negative effect of Cables1(TAS) or complete loss of function of Cables1 gives rise to ACC, we produced Cables1-null mutant mice. ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of Cables1(TAS) impairs callosal formation. Moreover, ACC frequency in Cables1(+/TAS) mice was significantly lower than that in Cables1(-/TAS) mice, indicating that wild-type Cables1 interfered with the dominant negative effect of Cables1(TAS). This study indicated that truncated Cables1 causes ACC and wild-type Cables1 contributes to callosal formation.

  19. Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast

    PubMed Central

    Boldogh, Istvan R.; Yang, Hyeong-Cheol; Nowakowski, W. Dan; Karmon, Sharon L.; Hays, Lara G.; Yates, John R.; Pon, Liza A.

    2001-01-01

    The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex. PMID:11248049

  20. Multistrand superconductor cable

    DOEpatents

    Borden, Albert R.

    1985-01-01

    Improved multistrand Rutherford-type superconductor cable is produced by using strands which are preformed, prior to being wound into the cable, so that each strand has a variable cross section, with successive portions having a substantially round cross section, a transitional oval cross section, a rectangular cross section, a transitional oval cross section, a round cross section and so forth, in repetitive cycles along the length of the strand. The cable is wound and flattened so that the portions of rectangular cross section extend across the two flat sides of the cable at the strand angle. The portions of round cross section are bent at the edges of the flattened cable, so as to extend between the two flat sides. The rectangular portions of the strands slide easily over one another, so as to facilitate flexing and bending of the cable, while also minimizing the possibility of causing damage to the strands by such flexing or bending. Moreover, the improved cable substantially maintains its compactness and cross-sectional shape when the cable is flexed or bent.

  1. Submarine cable route survey

    SciTech Connect

    Herrouin, G.; Scuiller, T.

    1995-12-31

    The growth of telecommunication market is very significant. From the beginning of the nineties, more and more the use of optical fiber submarine cables is privileged to that of satellites. These submarine telecommunication highways require accurate surveys in order to select the optimum route and determine the cable characteristics. Advanced technology tools used for these surveys are presented along with their implementation.

  2. Chlamydia trachomatis Tarp cooperates with the Arp2/3 complex to increase the rate of actin polymerization.

    PubMed

    Jiwani, Shahanawaz; Ohr, Ryan J; Fischer, Elizabeth R; Hackstadt, Ted; Alvarado, Stephenie; Romero, Adriana; Jewett, Travis J

    2012-04-20

    Actin polymerization is required for Chlamydia trachomatis entry into nonphagocytic host cells. Host and chlamydial actin nucleators are essential for internalization of chlamydiae by eukaryotic cells. The host cell Arp2/3 complex and the chlamydial translocated actin recruiting phosphoprotein (Tarp) are both required for entry. Tarp and the Arp2/3 complex exhibit unique actin polymerization kinetics individually, but the molecular details of how these two actin nucleators cooperate to promote bacterial entry is not understood. In this study we provide biochemical evidence that the two actin nucleators act synergistically by co-opting the unique attributes of each to enhance the dynamics of actin filament formation. This process is independent of Tarp phosphorylation. We further demonstrate that Tarp colocalization with actin filaments is independent of the Tarp phosphorylation domain. The results are consistent with a model in which chlamydial and host cell actin nucleators cooperate to increase the rate of actin filament formation.

  3. Multistrand superconductor cable

    DOEpatents

    Borden, A.R.

    1984-03-08

    Improved multistrand Rutherford-type superconductor cable is produced by using strands which are preformed, prior to being wound into the cable, so that each strand has a variable cross section, with successive portions having a substantially round cross section, a transitional oval cross section, a rectangular cross section, a transitional oval cross section, a round cross section and so forth, in repetitive cycles along the length of the strand. The cable is wound and flattened so that the portions of rectangular cross section extend across the two flat sides of the cable at the strand angle. The portions of round cross section are bent at the edges of the flattened cable, so as to extend between the two flat sides. The rectangular portions of the strands slide easil

  4. Cable and line inspection mechanism

    NASA Technical Reports Server (NTRS)

    Ross, Terence J. (Inventor)

    2003-01-01

    An automated cable and line inspection mechanism visually scans the entire surface of a cable as the mechanism travels along the cable=s length. The mechanism includes a drive system, a video camera, a mirror assembly for providing the camera with a 360 degree view of the cable, and a laser micrometer for measuring the cable=s diameter. The drive system includes an electric motor and a plurality of drive wheels and tension wheels for engaging the cable or line to be inspected, and driving the mechanism along the cable. The mirror assembly includes mirrors that are positioned to project multiple images of the cable on the camera lens, each of which is of a different portion of the cable. A data transceiver and a video transmitter are preferably employed for transmission of video images, data and commands between the mechanism and a remote control station.

  5. Noise performance of magneto-inductive cables

    NASA Astrophysics Data System (ADS)

    Wiltshire, M. C. K.; Syms, R. R. A.

    2014-07-01

    Magneto-inductive (MI) waveguides are metamaterial structures based on periodic arrangements of inductively coupled resonant magnetic elements. They are of interest for power transfer, communications and sensing, and can be realised in a flexible cable format. Signal-to-noise ratio is extremely important in applications involving signals. Here, we present the first experimental measurements of the noise performance of metamaterial cables. We focus on an application involving radiofrequency signal transmission in internal magnetic resonance imaging (MRI), where the subdivision of the metamaterial cable provides intrinsic patient safety. We consider MI cables suitable for use at 300 MHz during 1H MRI at 7 T, and find noise figures of 2.3-2.8 dB/m, together with losses of 3.0-3.9 dB/m, in good agreement with model calculations. These values are high compared to conventional cables, but become acceptable when (as here) the environment precludes the use of continuous conductors. To understand this behaviour, we present arguments for the fundamental performance limitations of these cables.

  6. Noise performance of magneto-inductive cables

    SciTech Connect

    Wiltshire, M. C. K. Syms, R. R. A.

    2014-07-21

    Magneto-inductive (MI) waveguides are metamaterial structures based on periodic arrangements of inductively coupled resonant magnetic elements. They are of interest for power transfer, communications and sensing, and can be realised in a flexible cable format. Signal-to-noise ratio is extremely important in applications involving signals. Here, we present the first experimental measurements of the noise performance of metamaterial cables. We focus on an application involving radiofrequency signal transmission in internal magnetic resonance imaging (MRI), where the subdivision of the metamaterial cable provides intrinsic patient safety. We consider MI cables suitable for use at 300 MHz during {sup 1}H MRI at 7 T, and find noise figures of 2.3–2.8 dB/m, together with losses of 3.0–3.9 dB/m, in good agreement with model calculations. These values are high compared to conventional cables, but become acceptable when (as here) the environment precludes the use of continuous conductors. To understand this behaviour, we present arguments for the fundamental performance limitations of these cables.

  7. Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts.

    PubMed

    Chang, Wakam; Folker, Eric S; Worman, Howard J; Gundersen, Gregg G

    2013-12-01

    In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking emerin. TAN lines formed normally in cells lacking emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with emerin, and emerin depletion prevented myosin IIB localization near nuclei. These results show that emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.

  8. Actin interaction and regulation of cyclin-dependent kinase 5/p35 complex activity.

    PubMed

    Xu, Jiqing; Tsutsumi, Koji; Tokuraku, Kiyotaka; Estes, Katherine A; Hisanaga, Shin-ichi; Ikezu, Tsuneya

    2011-01-01

    Cyclin-dependent kinase 5 (Cdk5) plays a critical role during neurodevelopment, synaptic plasticity, and neurodegeneration. Cdk5 activity depends on association with neuronal proteins p35 and p25, a proteolytic product of p35. Cdk5 regulates the actin cytoskeletal dynamics that are essential for neuronal migration, neuritic growth, and synaptogenesis. However, little is known about the interaction of actin and Cdk5 and its effect on neuronal Cdk5 activity. In a previous study, we observed that Cdk5/p35 activity is negatively correlated with co-immunoprecipitated F-actin (filamentous actin) amounts in the mouse brain, and suggested that F-actin inhibits the formation of the Cdk5/p35 complex [Journal of Neuroscience (2008) vol. 28, p. 14511]. The experiments reported here were undertaken to elucidate the relationship between actin and the formation of the Cdk5/p35 complex and its activity. Instead of an F-actin-mediated inhibition, we propose that G-actin (globular actin) in the F-actin preparations is responsible for inhibiting Cdk5/p35 and Cdk5/p25 kinase activity. We found that F-actin binds to p35 but not p25 or Cdk5. We have shown that G-actin binds directly to Cdk5 without disrupting the formation of the Cdk5/p35 or Cdk5/p25 complexes. G-actin potently suppressed Cdk5/p35 and Cdk5/p25 activity when either histone H1 or purified human tau protein were used as substrates, indicating a substrate-independent inhibitory effect of G-actin on Cdk5 activity. Finally, G-actin suppressed the activity of Cdk5 immunoprecipitated from wild type and p35-deficient mouse brain, suggesting that G-actin suppresses endogenous Cdk5 activity in a p35-independent manner. Together, these results suggest a novel mechanism of actin cytoskeletal regulation of Cdk5/p35 activity.

  9. Serum Calcium-decreasing Factor, Caldecrin, Inhibits Receptor Activator of NF-κB Ligand (RANKL)-mediated Ca2+ Signaling and Actin Ring Formation in Mature Osteoclasts via Suppression of Src Signaling Pathway*

    PubMed Central

    Tomomura, Mineko; Hasegawa, Hiroya; Suda, Naoto; Sakagami, Hiroshi; Tomomura, Akito

    2012-01-01

    Osteoclasts are essential for bone dynamics and calcium homeostasis. Recently, we reported that serum calcium-decreasing factor, caldecrin, which is a secretory-type serine protease isolated from the pancreas, inhibits osteoclast differentiation by suppression of NFATc1 activity regardless of its own protease activity (Hasegawa, H., Kido, S., Tomomura, M., Fujimoto, K., Ohi, M., Kiyomura, M., Kanegae, H., Inaba, A., Sakagami, H., and Tomomura, A. (2010) Serum calcium-decreasing factor, caldecrin, inhibits osteoclast differentiation by suppression of NFATc1 activity. J. Biol. Chem. 285, 25448–25457). Here, we investigated the effects of caldecrin on the function of mature osteoclasts by treatment with receptor activator of NF-κB ligand (RANKL). Caldecrin inhibited the RANKL-stimulated bone resorptive activity of mature osteoclasts. Furthermore, caldecrin inhibited RANKL-mediated sealing actin ring formation, which is associated with RANKL-evoked Ca2+ entry through transient receptor potential vanilloid channel 4. The inhibitors of phospholipase Cγ, Syk, and c-Src suppressed RANKL-evoked Ca2+ entry and actin ring formation of mature osteoclasts. Interestingly, caldecrin significantly inhibited RANKL-stimulated phosphorylation of c-Src, Syk, phospholipase Cγ1 and Cγ2, SLP-76, and Pyk2 but not that of ERK, JNK, or Akt. Caldecrin inhibited RANKL-stimulated c-Src kinase activity and c-Src·Syk association. These results suggest that caldecrin inhibits RANKL-stimulated calcium signaling activation and cytoskeletal organization by suppression of the c-Src·Syk pathway, which may in turn reduce the bone resorptive activity of mature osteoclasts. Thus, caldecrin is capable of acting as a negative regulator of osteoclastogenesis and osteoclast function of bone resorption. PMID:22461633

  10. Bringing Cable into the Classroom.

    ERIC Educational Resources Information Center

    Blubaugh, Donelle

    1999-01-01

    Cable TV may be an educationally and fiscally sound way to inspire active learning. Creative TV applications help teachers address potentially disabling social and emotional factors. The Cable in the Classroom program offers over 80,000 eligible schools free cable connections, free basic monthly cable service, and copyright clearances for off-air…

  11. Cable Television; A Bibliographic Review.

    ERIC Educational Resources Information Center

    Schoenung, James

    This bibliographic review of publications in the field of cable television begins with an introduction to cable television and an outline of the history and development of cable television. Particular attention is given to the regulatory activities of the Federal Communications Commission and the unfulfilled potential of cable television. The…

  12. Correction coil cable

    DOEpatents

    Wang, S.T.

    1994-11-01

    A wire cable assembly adapted for the winding of electrical coils is taught. A primary intended use is for use in particle tube assemblies for the Superconducting Super Collider. The correction coil cables have wires collected in wire array with a center rib sandwiched therebetween to form a core assembly. The core assembly is surrounded by an assembly housing having an inner spiral wrap and a counter wound outer spiral wrap. An alternate embodiment of the invention is rolled into a keystoned shape to improve radial alignment of the correction coil cable on a particle tube in a particle tube assembly. 7 figs.

  13. Directed actin assembly and motility.

    PubMed

    Boujemaa-Paterski, Rajaa; Galland, Rémi; Suarez, Cristian; Guérin, Christophe; Théry, Manuel; Blanchoin, Laurent

    2014-01-01

    The actin cytoskeleton is a key component of the cellular architecture. However, understanding actin organization and dynamics in vivo is a complex challenge. Reconstitution of actin structures in vitro, in simplified media, allows one to pinpoint the cellular biochemical components and their molecular interactions underlying the architecture and dynamics of the actin network. Previously, little was known about the extent to which geometrical constraints influence the dynamic ultrastructure of these networks. Therefore, in order to study the balance between biochemical and geometrical control of complex actin organization, we used the innovative methodologies of UV and laser patterning to design a wide repertoire of nucleation geometries from which we assembled branched actin networks. Using these methods, we were able to reconstitute complex actin network organizations, closely related to cellular architecture, to precisely direct and control their 3D connections. This methodology mimics the actin networks encountered in cells and can serve in the fabrication of innovative bioinspired systems.

  14. Cable equation for general geometry.

    PubMed

    López-Sánchez, Erick J; Romero, Juan M

    2017-02-01

    The cable equation describes the voltage in a straight cylindrical cable, and this model has been employed to model electrical potential in dendrites and axons. However, sometimes this equation might give incorrect predictions for some realistic geometries, in particular when the radius of the cable changes significantly. Cables with a nonconstant radius are important for some phenomena, for example, discrete swellings along the axons appear in neurodegenerative diseases such as Alzheimers, Parkinsons, human immunodeficiency virus associated dementia, and multiple sclerosis. In this paper, using the Frenet-Serret frame, we propose a generalized cable equation for a general cable geometry. This generalized equation depends on geometric quantities such as the curvature and torsion of the cable. We show that when the cable has a constant circular cross section, the first fundamental form of the cable can be simplified and the generalized cable equation depends on neither the curvature nor the torsion of the cable. Additionally, we find an exact solution for an ideal cable which has a particular variable circular cross section and zero curvature. For this case we show that when the cross section of the cable increases the voltage decreases. Inspired by this ideal case, we rewrite the generalized cable equation as a diffusion equation with a source term generated by the cable geometry. This source term depends on the cable cross-sectional area and its derivates. In addition, we study different cables with swelling and provide their numerical solutions. The numerical solutions show that when the cross section of the cable has abrupt changes, its voltage is smaller than the voltage in the cylindrical cable. Furthermore, these numerical solutions show that the voltage can be affected by geometrical inhomogeneities on the cable.

  15. Cable equation for general geometry

    NASA Astrophysics Data System (ADS)

    López-Sánchez, Erick J.; Romero, Juan M.

    2017-02-01

    The cable equation describes the voltage in a straight cylindrical cable, and this model has been employed to model electrical potential in dendrites and axons. However, sometimes this equation might give incorrect predictions for some realistic geometries, in particular when the radius of the cable changes significantly. Cables with a nonconstant radius are important for some phenomena, for example, discrete swellings along the axons appear in neurodegenerative diseases such as Alzheimers, Parkinsons, human immunodeficiency virus associated dementia, and multiple sclerosis. In this paper, using the Frenet-Serret frame, we propose a generalized cable equation for a general cable geometry. This generalized equation depends on geometric quantities such as the curvature and torsion of the cable. We show that when the cable has a constant circular cross section, the first fundamental form of the cable can be simplified and the generalized cable equation depends on neither the curvature nor the torsion of the cable. Additionally, we find an exact solution for an ideal cable which has a particular variable circular cross section and zero curvature. For this case we show that when the cross section of the cable increases the voltage decreases. Inspired by this ideal case, we rewrite the generalized cable equation as a diffusion equation with a source term generated by the cable geometry. This source term depends on the cable cross-sectional area and its derivates. In addition, we study different cables with swelling and provide their numerical solutions. The numerical solutions show that when the cross section of the cable has abrupt changes, its voltage is smaller than the voltage in the cylindrical cable. Furthermore, these numerical solutions show that the voltage can be affected by geometrical inhomogeneities on the cable.

  16. Optimization of cable preloading on cable-stayed bridges

    NASA Astrophysics Data System (ADS)

    Wang, Yang Cheng; Vlahinos, Andreas S.; Shu, HungShan

    1997-05-01

    Generally, geometric nonlinearities of cable-stayed bridges depend on the behaviors of cables, pylons, the bridge deck and their interactions. These are geometry change, cable sag, and the interactions of axial forces, the bending moment and their deformations in the pylons and bridge deck. Therefore, a large cable-stayed bridges system having a large number of cables can be analyzed under different load conditions. In investigating nonlinear behaviors of cable- stayed bridges, the nonlinear behavior of cables needs to be considered because it may cause the nonlinear behavior of whole bridge system. The nonlinear behavior of a cable gained from its sag. With an increasing axial load, the elongation of the cable is increased but the total cable sag is decreased. Cable-stayed bridge uses cables instead of the internal piers to support the bridge deck. Usually, cable- stayed bridge decks are straight with a little camber compared to the total length of the bridge. Keeping the bridge deck in the position where is the designer desired is not only for bridge aesthetics but also for people on the bridge in terms of psychological effect of improving confidence in structure and engineering considerations. To achieve the serviceability and engineering requirements, preloading of the cable is necessary. In this paper, one such a bridge with geometry similarly to an existing cable- stayed bridge. Quincy Bayview Bridge, located in Illinois, USA, has been considered. Quincy Bayview Bridge has 58 cables in the two planes. Four methods have been considered in this paper to make the optimum selection of cable preloading. The objective is to select appropriate method to determine cable prestrains in order to minimize the deformations and stresses due to dead load of the bridge. Thus, it is not a trivial problem since a change in the prestress of a cable influence the deformation every where in the structure. The best method would be determined by comparing the calculated bending and

  17. Infiniband Based Cable Comparison

    SciTech Connect

    Minich, Makia

    2007-07-01

    As Infiniband continues to be more broadly adopted in High Performance Computing (HPC) and datacenter applications, one major challenge still plagues implementation: cabling. With the transition to DDR (double data rate) from SDR (single datarate), currently available Infiniband implementations such as standard CX4/IB4x style copper cables severely constrain system design (10m maximum length for DDR copper cables, thermal management due to poor airflow, etc.). This paper will examine some of the options available and compare performance with the newly released Intel Connects Cables. In addition, we will take a glance at Intel's dual-core and quad-core systems to see if core counts have noticeable effect on expected IO patterns.

  18. The Discrete Hanging Cable

    ERIC Educational Resources Information Center

    Peters, James V.

    2004-01-01

    Using the methods of finite difference equations the discrete analogue of the parabolic and catenary cable are analysed. The fibonacci numbers and the golden ratio arise in the treatment of the catenary.

  19. Robotic Arm Biobarrier Cable

    NASA Technical Reports Server (NTRS)

    2008-01-01

    This image, taken by the Surface Stereo Imager on NASA's Phoenix Mars Lander on the 14th Martian day of the mission (June 7, 2008), shows the cable that held the Robotic Arm's biobarrier in place during flight has snapped. The cable's springs retracted to release the biobarrier right after landing.

    To the lower right of the image a spring is visible. Extending from that spring is a length of cable that snapped during the biobarrier's release. A second spring separated from the cable when it snapped and has been photographed on the ground under the lander near one of the legs.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  20. Amplification of actin polymerization forces

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2016-01-01

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments. PMID:27002174

  1. Amplification of actin polymerization forces.

    PubMed

    Dmitrieff, Serge; Nédélec, François

    2016-03-28

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments.

  2. AC Cable: Yokohama Project

    NASA Astrophysics Data System (ADS)

    Masuda, Takato

    High Temperature Superconducting (HTS) cables can transmit large amounts of electricity in a compact size with minimal losses. Therefore, they are expected to save the construction cost of underground lines in urban areas and decrease transmission losses. Several HTS cables have recently been demonstrated in networks around the world, and full-scale commercialization is expected in the near future. In Japan, the development of compact HTS cables suitable for urban deployment has been underway since the early 1990s. In 2007, a national project was started to verify their operational performance and long-term reliability in the grid. An HTS cable 240 m long was installed at the Asahi substation of the Tokyo Electric Power Company (TEPCO) in Yokohama; then a joint, terminations and cooling system was constructed in 2011. After successful performance tests, the cable was connected to the grid for the first time in Japan, and started to deliver electricity to 70,000 households in October 2012. This trouble-free in-grid service continued for over a year. We can conclude that the HTS cable system performs well and has the stability required for long-term in-grid operations.

  3. Actinic keratosis. Current treatment options.

    PubMed

    Jeffes, E W; Tang, E H

    2000-01-01

    Actinic keratoses are hyperkeratotic skin lesions that represent focal abnormal proliferation of epidermal keratinocytes. Some actinic keratoses evolve into squamous cell carcinoma of the skin, while others resolve spontaneously. The conversion rate of actinic keratosis to squamous cell carcinoma is not accurately known, but appears to be in the range of 0.25 to 1% per year. Although there is a low rate of conversion of actinic keratoses to squamous cell carcinoma, 60% of squamous cell carcinomas of the skin probably arise from actinic keratoses. The main cause of actinic keratoses in otherwise healthy Caucasians appears to be the sun. Therapy for actinic keratoses begins with prevention which starts with sun avoidance and physical protection. Sunprotection with sunscreens actually slows the return of actinic keratoses in patients already getting actinic keratoses. Interestingly, a few studies are available that demonstrate that a high fat diet is associated with the production of more actinic keratoses than is a low fat diet. One of the mainstays of therapy has been local destruction of the actinic keratoses with cryotherapy, and curettage and electrodesiccation. A new addition to this group of therapies to treat individual actinic keratoses is photodynamic therapy with topical aminolevulinic acid and light. In patients who have numerous actinic keratoses in an area of severely sun damaged skin, therapies which are applied to the whole actinic keratosis area are used. The goal of treating such an area of skin is to treat all of the early as well as the numerous clinically evident actinic keratoses at the same time. The classical approaches for treating areas of photodamaged skin without treating actinic keratoses individually include: the use of topically applied fluorouracil cream, dermabrasion, and cutaneous peels with various agents like trichloroacetic acid. Both topically as well as orally administered retinoids have been used to treat actinic keratoses but

  4. Role of Actin Polymerization in Cell Locomotion: Molecules and Models

    PubMed Central

    Bearer, E. L.

    2015-01-01

    Actin filaments forming at the anterior margin of a migrating cell are essential for the formation of filopodia, lamellipodia, and pseudopodia, the “feet” that the cell extends before it. These structures in turn are required for cell locomotion. Yet the molecular nature of the “nucleator” that seeds the polymerization of actin at the leading edge is unknown. Recent advances, including video microscopy of actin dynamics, discovery of proteins unique to the leading edge such as ponticulin, the Mab 2E4 antigen, and ABP 120, and novel experimental models of actin polymerization such as the actin-based movements of intracellular parasites, promise to shed light on this problem in the near future. PMID:8323743

  5. Aluminum modifies the viscosity of filamentous actin solutions as measured by optical displacement microviscometry.

    PubMed

    Arnoys, E J; Schindler, M

    2000-01-01

    A microtechnique has been developed that is capable of measuring the viscosity of filamentous actin (F-actin) solutions. This method, called optical displacement microviscometry (ODM), was utilized to determine the changes in viscosity of solutions of rabbit muscle, human platelet, and maize pollen actin when measured in the absence and presence of aluminum. Measurements demonstrated that the viscosity of the different actin solutions decreased with aluminum concentration. In contrast, increases in viscosity were observed when aluminum was added to F-actin solutions containing filamin (chicken gizzard), a protein that bundles actin filaments. Confocal fluorescence imaging of pure actin solutions in the presence of aluminum showed a disrupted actin network composed of fragmented actin filaments in the form of small aggregates. In contrast, in the presence of filamin, aluminum promoted the formation of thicker actin filaments. These measurements demonstrate that aluminum can affect actin filaments differentially depending on the presence of an actin-binding protein. In addition, a strong correlation is observed between the changes in viscosity as measured by ODM and the thickness and assembled state of bundles of actin filaments.

  6. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  7. IFT88 influences chondrocyte actin organization and biomechanics

    PubMed Central

    Wang, Z.; Wann, A.K.T.; Thompson, C.L.; Hassen, A.; Wang, W.; Knight, M.M.

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. Results IFT88orpk cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88orpk cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88orpk cells. Following membrane blebbing, IFT88orpk cells exhibited slower reformation of the actin cortex. IFT88orpk cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. Conclusions This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. PMID:26493329

  8. Fetal akinesia caused by a novel actin filament aggregate myopathy skeletal muscle actin gene (ACTA1) mutation.

    PubMed

    Stenzel, Werner; Prokop, Stefan; Kress, Wolfram; Huppmann, Stephanie; Loui, Andrea; Sarioglu, Nanette M E; Laing, Nigel G; Sparrow, John C; Heppner, Frank L; Goebel, Hans H

    2010-08-01

    We report a female newborn, diagnosed with fetal akinesia in utero, who died one hour after birth. Post-mortem muscle biopsy demonstrated actin-filament myopathy based on immunolabelling for sarcomeric actin, and large areas of filaments, without rod formation, ultrastructurally. Analysis of DNA extracted from the muscle disclosed a novel de novo heterozygous c.44G>A, GGC>GAC, 'p.Gly15Asp' mutation in the ACTA1 gene. Analysis of the location of the mutated amino-acid in the actin molecule suggests the mutation most likely causes abnormal nucleotide binding, and consequent pathological actin polymerization. This case emphasizes the association of fetal akinesia with actin-filament myopathy.

  9. Actin-Based Motility of Intracellular Microbial Pathogens

    PubMed Central

    Goldberg, Marcia B.

    2001-01-01

    A diverse group of intracellular microorganisms, including Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, utilize actin-based motility to move within and spread between mammalian host cells. These organisms have in common a pathogenic life cycle that involves a stage within the cytoplasm of mammalian host cells. Within the cytoplasm of host cells, these organisms activate components of the cellular actin assembly machinery to induce the formation of actin tails on the microbial surface. The assembly of these actin tails provides force that propels the organisms through the cell cytoplasm to the cell periphery or into adjacent cells. Each of these organisms utilizes preexisting mammalian pathways of actin rearrangement to induce its own actin-based motility. Particularly remarkable is that while all of these microbes use the same or overlapping pathways, each intercepts the pathway at a different step. In addition, the microbial molecules involved are each distinctly different from the others. Taken together, these observations suggest that each of these microbes separately and convergently evolved a mechanism to utilize the cellular actin assembly machinery. The current understanding of the molecular mechanisms of microbial actin-based motility is the subject of this review. PMID:11729265

  10. Recent advances into vanadyl, vanadate and decavanadate interactions with actin.

    PubMed

    Ramos, S; Moura, J J G; Aureliano, M

    2012-01-01

    Although the number of papers about "vanadium" has doubled in the last decade, the studies about "vanadium and actin" are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate (51)V NMR signals, at -516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at -560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1:1 binding stoichiometry and a K(d) of 7.5 μM(-1). Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC(50) of 68 and 300 μM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 μM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Both vanadium species increased the ε-ATP exchange rate (k = 6.5 × 10(-3) s(-1) and 4.47 × 10(-3) s(-1) for decavanadate and vanadyl, respectively). Finally, (1)H NMR spectra of G-actin treated with 0.1 mM decavanadate clearly indicate that major alterations occur in protein structure, which are much less visible in the presence of ATP, confirming the preventive effect of the nucleotide on the decavanadate interaction with the protein. Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl. By affecting actin structure and function, vanadium can

  11. Actin stress in cell reprogramming

    PubMed Central

    Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

    2014-01-01

    Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

  12. An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes.

    PubMed

    Wang, Huei-Jing; Wan, Ai-Ru; Jauh, Guang-Yuh

    2008-08-01

    Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.

  13. Instrumentation Cables Test Plan

    SciTech Connect

    Muna, Alice Baca; LaFleur, Chris Bensdotter

    2016-10-01

    A fire at a nuclear power plant (NPP) has the potential to damage structures, systems, and components important to safety, if not promptly detected and suppressed. At Browns Ferry Nuclear Power Plant on March 22, 1975, a fire in the reactor building damaged electrical power and control systems. Damage to instrumentation cables impeded the function of both normal and standby reactor coolant systems, and degraded the operators’ plant monitoring capability. This event resulted in additional NRC involvement with utilities to ensure that NPPs are properly protected from fire as intended by the NRC principle design criteria (i.e., general design criteria 3, Fire Protection). Current guidance and methods for both deterministic and performance based approaches typically make conservative (bounding) assumptions regarding the fire-induced failure modes of instrumentation cables and those failure modes effects on component and system response. Numerous fire testing programs have been conducted in the past to evaluate the failure modes and effects of electrical cables exposed to severe thermal conditions. However, that testing has primarily focused on control circuits with only a limited number of tests performed on instrumentation circuits. In 2001, the Nuclear Energy Institute (NEI) and the Electric Power Research Institute (EPRI) conducted a series of cable fire tests designed to address specific aspects of the cable failure and circuit fault issues of concern1. The NRC was invited to observe and participate in that program. The NRC sponsored Sandia National Laboratories to support this participation, whom among other things, added a 4-20 mA instrumentation circuit and instrumentation cabling to six of the tests. Although limited, one insight drawn from those instrumentation circuits tests was that the failure characteristics appeared to depend on the cable insulation material. The results showed that for thermoset insulated cables, the instrument reading tended to drift

  14. 54Mn2+ as a tracer of the polymerization of actin. Intermediate oligomers condense to give F-actin.

    PubMed Central

    Grazi, E

    1984-01-01

    Mg2+, at submicromolar concentrations, is needed for the nucleation of actin [Maruyama (1981) J. Biol. Chem. 256, 1060-1062]. I show here that Mn2+ fulfils the same function. It binds to oligomers present in the ATP-G-actin solutions with a ratio of 2-3 Mn2+ ions per 100 actin monomers and with an association constant of 0.66 X 10(10) M-1 at pH 8.2 at 25 degrees C. The time course of the binding of Mn2+ to polymerizing actin is not affected by the initial concentration of the protein. Analysis of the distribution of the binding shows that, both in the large oligomeric species and in the polymers, 1 Mn2+ ion is bound for every 14-25 actin monomers, whereas in the smaller oligomeric species 1 Mn2+ ion is bound for every 4 actin monomers. The proposal is made that Mn2+ stabilizes actin nuclei and decreases the concentration of the monomers at the steady state. It is also proposed that, at least in some experimental conditions, the direct condensation of oligomers of intermediate length is an effective mechanism of F-actin formation. PMID:6508731

  15. Cable-Dispensing Cart

    NASA Technical Reports Server (NTRS)

    Bredberg, Alan S.

    2003-01-01

    A versatile cable-dispensing cart can support as many as a few dozen reels of cable, wire, and/or rope. The cart can be adjusted to accommodate reels of various diameters and widths, and can be expanded, contracted, or otherwise reconfigured by use of easily installable and removable parts that can be carried onboard. Among these parts are dispensing rods and a cable guide that enables dispensing of cables without affecting the direction of pull. Individual reels can be mounted on or removed from the cart without affecting the other reels: this feature facilitates the replacement or reuse of partially depleted reels, thereby helping to reduce waste. Multiple cables, wires, or ropes can be dispensed simultaneously. For maneuverability, the cart is mounted on three wheels. Once it has been positioned, the cart is supported by rubber mounts for stability and for prevention of sliding or rolling during dispensing operations. The stability and safety of the cart are enhanced by a low-center-of-gravity design. The cart can readily be disassembled into smaller units for storage or shipping, then reassembled in the desired configuration at a job site.

  16. Moesin and cortactin control actin-dependent multivesicular endosome biogenesis

    PubMed Central

    Muriel, Olivia; Tomas, Alejandra; Scott, Cameron C.; Gruenberg, Jean

    2016-01-01

    We used in vivo and in vitro strategies to study the mechanisms of multivesicular endosome biogenesis. We found that, whereas annexinA2 and ARP2/3 mediate F-actin nucleation and branching, respectively, the ERM protein moesin supports the formation of F-actin networks on early endosomes. We also found that moesin plays no role during endocytosis and recycling to the plasma membrane but is absolutely required, much like actin, for early-to-late-endosome transport and multivesicular endosome formation. Both actin network formation in vitro and early-to-late endosome transport in vivo also depend on the F-actin–binding protein cortactin. Our data thus show that moesin and cortactin are necessary for formation of F-actin networks that mediate endosome biogenesis or maturation and transport through the degradative pathway. We propose that the primary function of endosomal F-actin is to control the membrane remodeling that accompanies endosome biogenesis. We also speculate that this mechanism helps segregate tubular and multivesicular membranes along the recycling and degradation pathways, respectively. PMID:27605702

  17. Correction coil cable

    DOEpatents

    Wang, Sou-Tien

    1994-11-01

    A wire cable assembly (10, 310) adapted for the winding of electrical coils is taught. A primary intended use is for use in particle tube assemblies (532) for the superconducting super collider. The correction coil cables (10, 310) have wires (14, 314) collected in wire arrays (12, 312) with a center rib (16, 316) sandwiched therebetween to form a core assembly (18, 318 ). The core assembly (18, 318) is surrounded by an assembly housing (20, 320) having an inner spiral wrap (22, 322) and a counter wound outer spiral wrap (24, 324). An alternate embodiment (410) of the invention is rolled into a keystoned shape to improve radial alignment of the correction coil cable (410) on a particle tube (733) in a particle tube assembly (732).

  18. Hypervelocity impact testing of cables

    NASA Technical Reports Server (NTRS)

    Jex, D. W.; Adkinson, A. B.; English, J. E.; Linebaugh, C. E.

    1973-01-01

    The physics and electrical results obtained from simulated micrometeoroid testing of certain Skylab cables are presented. The test procedure, electrical circuits, test equipment, and cable types utilized are also explained.

  19. Mechanical Rope and Cable

    DTIC Science & Technology

    1975-04-01

    Steel, Carbon, High Strength MIL-W-24223 Wire Rope , Aluminized MIL-W-32312 (MO) Vire Rope Assemblies, Single Log; for Medium Cableway MII-W-81002 (WP...II AD-AO13 345 MECHANICAL ROPE AND CABLE National Materials Advisory Board (NAS-NAE) I’I Prepared for: Department of Defense April 1975 DISTRIBUTED...andSubtitle) S. TYPE[ OF REPORT & PERIOD COVERED TITLE(endSubttle)Final Report Mechanical Rope and Cable FnalReport____ 4. PERFORMING ORG. REPORT

  20. Coaxial cable cutter

    DOEpatents

    Hall, Leslie C.; Hedges, Robert S.

    1990-04-10

    A cutting device is provided which is useful in trimming the jackets from semi-rigid coaxial cables and wire having a cutting bit and support attached to movable jaws. A thumbpiece is provided to actuate the opening of the jaws for receiving the cable to be trimmed, and a spring member is provided to actuate the closing of the jaws when thumbpiece is released. The cutting device utilizes one moving part during the cutting operation by using a rolling cut action. The nature of the jaws allows the cutting device to work in space having clearances less than 0.160 inches.

  1. A cable SGEMP tutorial :

    SciTech Connect

    Liscum-Powell, Jennifer L.; Bohnhoff, William J.; Turner, C. David

    2007-05-01

    This tutorial is aimed at guiding a user through the process of performing a cable SGEMP simulation. The tutorial starts with processing a differential photon spectrum obtained from a Monte Carlo code such as ITS into a discrete (multi-group) spectrum used in CEPXS and CEPTRE. Guidance is given in the creation of a nite element mesh of the cable geometry. The set-up of a CEPTRE simulation is detailed. Users are instructed in evaluating the quality of the CEPTRE radiation transport results. The post-processing of CEPTRE results using Exostrip is detailed. And finally, an EMPHASIS/CABANA simulation is detailed including the interpretation of the output.

  2. Cabling design for phased arrays

    NASA Technical Reports Server (NTRS)

    Kruger, I. D.; Turkiewicz, L.

    1972-01-01

    The ribbon-cabling system used for the AEGIS phased array which provides minimum cable bulk, complete EMI shielding, rugged mechanical design, repeatable electrical characteristics, and ease of assembly and maintenance is described. The ribbon cables are 0.040-inch thick, and in widths up to 2 1/2 inches. Their terminations are molded connectors that can be grouped in a three-tier arrangement, with cable branching accomplished by a matrix-welding technique.

  3. Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation.

    PubMed

    Naresh, S; Atreja, S K

    2015-12-01

    In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.

  4. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites.

    PubMed

    Skillman, Kristen M; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L David

    2011-10-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

  5. Actin-dependent propulsion of endosomes and lysosomes byrecruitment of n-wasp

    SciTech Connect

    Taunton J; Rowning BA; Coughlin ML; Wu M; Moon RT; Mitchison TJ; Larabell CA

    2000-02-07

    We examined the spatial and temporal control of actin assembly in living Xenopus eggs. Within minutes of egg activation,dynamic actin-rich comet tails appeared on a subset of cytoplasmic vesicles that were enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo was stimulated by the PKC activator phorbol myristate acetate (PMA),and this process could be reconstituted in a cell-free system. We used this system to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. We found that the protein, N-WASP, was recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP, The motile vesicles accumulated the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails had the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly and moved in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton.

  6. Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

    PubMed Central

    Skillman, Kristen M.; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L. David

    2011-01-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility. PMID:21998582

  7. Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia.

    PubMed

    Cohan, C S; Welnhofer, E A; Zhao, L; Matsumura, F; Yamashiro, S

    2001-02-01

    Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.

  8. Differential Effects of Caldesmon on the Intermediate Conformational States of Polymerizing Actin*

    PubMed Central

    Huang, Renjian; Grabarek, Zenon; Wang, Chih-Lueh Albert

    2010-01-01

    The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton. PMID:19889635

  9. Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments

    PubMed Central

    1995-01-01

    The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin- binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere. PMID:7822423

  10. Direct observation of motion of single F-actin filaments in the presence of myosin

    NASA Astrophysics Data System (ADS)

    Yanagida, Toshio; Nakase, Michiyuki; Nishiyama, Katsumi; Oosawa, Fumio

    1984-01-01

    Actin is found in almost all kinds of non-muscle cells where it is thought to have an important role in cell motility. A proper understanding of that role will only be possible when reliable in vitro systems are available for investigating the interaction of cellular actin and myosin. A start has been made on several systems1-4, most recently by Sheetz and Spudich who demonstrated unidirectional movement of HMM-coated beads along F-actin cables on arrays of chloroplasts exposed by dissection of a Nitella cell5. As an alternative approach, we report here the direct observation by fluorescence microscopy of the movements of single F-actin filaments interacting with soluble myosin fragments energized by Mg2+-ATP.

  11. George W. Cable and Tradition.

    ERIC Educational Resources Information Center

    Trotman, C. James

    There have been no critical studies of Cable's fiction treated for its own sake; and, without such studies, readers may not become aware of these stories or of their possible value. The absence of a critical position on Cable is due to the decline in his reputation as an artist when he became a writer of popular literature. Before 1925 Cable and…

  12. High Temperature Superconducting Underground Cable

    SciTech Connect

    Farrell, Roger, A.

    2010-02-28

    The purpose of this Project was to design, build, install and demonstrate the technical feasibility of an underground high temperature superconducting (HTS) power cable installed between two utility substations. In the first phase two HTS cables, 320 m and 30 m in length, were constructed using 1st generation BSCCO wire. The two 34.5 kV, 800 Arms, 48 MVA sections were connected together using a superconducting joint in an underground vault. In the second phase the 30 m BSCCO cable was replaced by one constructed with 2nd generation YBCO wire. 2nd generation wire is needed for commercialization because of inherent cost and performance benefits. Primary objectives of the Project were to build and operate an HTS cable system which demonstrates significant progress towards commercial progress and addresses real world utility concerns such as installation, maintenance, reliability and compatibility with the existing grid. Four key technical areas addressed were the HTS cable and terminations (where the cable connects to the grid), cryogenic refrigeration system, underground cable-to-cable joint (needed for replacement of cable sections) and cost-effective 2nd generation HTS wire. This was the world’s first installation and operation of an HTS cable underground, between two utility substations as well as the first to demonstrate a cable-to-cable joint, remote monitoring system and 2nd generation HTS.

  13. Power superconducting power transmission cable

    DOEpatents

    Ashworth, Stephen P.

    2003-06-10

    The present invention is for a compact superconducting power transmission cable operating at distribution level voltages. The superconducting cable is a conductor with a number of tapes assembled into a subconductor. These conductors are then mounted co-planarly in an elongated dielectric to produce a 3-phase cable. The arrangement increases the magnetic field parallel to the tapes thereby reducing ac losses.

  14. Power superconducting power transmission cable

    DOEpatents

    Ashworth, Stephen P.

    2003-01-01

    The present invention is for a compact superconducting power transmission cable operating at distribution level voltages. The superconducting cable is a conductor with a number of tapes assembled into a subconductor. These conductors are then mounted co-planarly in an elongated dielectric to produce a 3-phase cable. The arrangement increases the magnetic field parallel to the tapes thereby reducing ac losses.

  15. Economical Aspects of Superconducting Cable

    NASA Astrophysics Data System (ADS)

    Ohya, Masayoshi

    High-temperature superconducting (HTS) cables are expected to resolve technical problems with power grids because they put large-capacity, low-loss power transmission into a compact package. One problem is replacing old 275-kV oil filled (OF) cables with cross-linked polyethylene insulated vinyl sheath cables (XLPE cables). This is difficult because XLPE cable has a lower transmission capacity than OF cable. In addition, the high concentration of public infrastructure underground makes it extremely difficult to build new ones. However, if 66-kV HTS cables can be installed inside existing underground conduits and can achieve a power capacity equivalent to conventional 275-kV cables, construction costs could be significantly reduced. Moreover, if XLPE cables are used for a 1,000 MVA-class transmission line, then three circuits of nine 275-kV single-core cables would be required, which would incur a transmission loss of 90 W/m/cct. Three circuits of three 66-kV Three-in-One HTS cables, however, with an AC loss of 1 W/m/ph@3 kA, heat invasion of 2 W/m, and cooling system efficiency of 0.1, would reduce transmission loss to less than three-fifths that of XLPE cables.

  16. The transcriptional repressor Sum1p counteracts Sir2p in regulation of the actin cytoskeleton, mitochondrial quality control and replicative lifespan in Saccharomyces cerevisiae

    PubMed Central

    Higuchi-Sanabria, Ryo; Vevea, Jason D.; Charalel, Joseph K.; Sapar, Maria L.; Pon, Liza A.

    2016-01-01

    Increasing the stability or dynamics of the actin cytoskeleton can extend lifespan in C. elegans and S. cerevisiae. Actin cables of budding yeast, bundles of actin filaments that mediate cargo transport, affect lifespan control through effects on mitochondrial quality control. Sir2p, the founding member of the Sirtuin family of lifespan regulators, also affects actin cable dynamics, assembly, and function in mitochondrial quality control. Here, we obtained evidence for novel interactions between Sir2p and Sum1p, a transcriptional repressor that was originally identified through mutations that genetically suppress sir2∆ phenotypes unrelated to lifespan. We find that deletion of SUM1 in wild-type cells results in increased mitochondrial function and actin cable abundance. Furthermore, deletion of SUM1 suppresses defects in actin cables and mitochondria of sir2∆ yeast, and extends the replicative lifespan and cellular health span of sir2∆ cells. Thus, Sum1p suppresses Sir2p function in control of specific aging determinants and lifespan in budding yeast. PMID:28357337

  17. The association of myosin IB with actin waves in dictyostelium requires both the plasma membrane-binding site and actin-binding region in the myosin tail.

    PubMed

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A; Korn, Edward D

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.

  18. The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

    PubMed Central

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

  19. Pediatrics and Cable Television.

    ERIC Educational Resources Information Center

    Wallerstein, Edward; And Others

    The Department of Community Medicine of the Mount Sinai School of Medicine (New York City), in cooperation with the TelePrompTer Corporation and with funding from the Health Services and Mental Health Administration of the Department of Health, Education, and Welfare, has developed a bidirectional television system using coaxial cable which links…

  20. Cable indenter aging monitor

    SciTech Connect

    Shook, T.A.; Gardner, J.B.

    1988-07-01

    This project was undertaken to develop a hand-held, nondestructive test device to assess the aged condition of electrical cable by in situ measurement of mechanical properties of polymeric jackets and insulations. The device is an indenter similar to those used to make hardness measurements. Comparison of measurements made on installed cables with previous measurements serving as baseline aging/mechanical property data will determine the state of aging of the field cables. Such a device will be valuable in nuclear and fossil plant life extension programs. Preliminary laboratory tests on cables covered with ethylene propylene rubber (EPR) and chlorosulfated polyethylene (CSPE) point to the measurement of the rate of force increase resulting from constant rate deformation as having the best correlation with progressive thermal aging. This first phase of the work has demonstrated the technical feasibility of the method. A second phase will include the generation of additional groundwork data and the design of the portable indenter for in situ plant measurements.

  1. Cable TV: Now What?

    ERIC Educational Resources Information Center

    Ohio Educational Library/Media Association, Columbus.

    Designed to aid in planning the best use of cable television in a particular educational situation, this pamphlet was prepared by a joint committee of the Ohio Educational Library Media Association and the Greater Miami Valley Instructional Television Council in order to share their plans, experiences, problems, and solutions with others who are…

  2. Lightweight Electric Power Cable.

    DTIC Science & Technology

    1982-09-01

    8I~ .4 111 162 MICROCi Pi RL’ LUHION TESI CHARI "LIGHTWEIGHT ELECTRIC POWER CABLE" FINAL TECHNICAL REPORT SEPTEMBER 30, 1981 to SEPTEMBER 30, 1982... Vulcanized by heat to crosslink. TPE (Thermoplastic Elastomer) - Polymers having elastomeric proper- ties. Used as thermoplastics - melt formed by

  3. Multilayer flat electrical cable

    NASA Technical Reports Server (NTRS)

    Silverman, P. G.

    1973-01-01

    Flat electrical cable is lightweight, flexible over wide temperature range, withstands continuous exposure to high levels of nuclear radiation, and can carry high currents with minimum of temperature rise. Its magnetic cleanliness is equal to or better than twisted pair of wires, and it can be terminated in conventional electrical connector.

  4. Comparison of cable ageing

    NASA Astrophysics Data System (ADS)

    Plaček, Vít; Kohout, Tomáš

    2010-03-01

    Two cable types, which currently are used in nuclear power plants (NPP) and which are composed by jacket/insulation materials, i.e. PVC/PVC and PVC/PE, were exposed to accelerated ageing conditions, in order to simulate their behavior after 10 years in service. The cables were aged under two different test conditions: With relatively high accelerating ageing speed:Radiation ageing was carried out at room temperature at a dose rate of 2900 Gy/h, followed by thermal ageing at 100 °C. This accelerated ageing condition was fairly fast, but still in compliance with the standards. With moderate ageing speed:The radiation and thermal ageing was performed simultaneously (superimposed) at a dose rate of 2.7-3.7Gy/h and a temperature of 68-70 °C. Such a test condition seems to be very close to the radiation and temperature impact onto the cables in the real NPP service. Finally, mechanical properties were measured to characterize the ageing status of the cables. The purpose of this study was to compare degradation effects, derived from both ageing methods, and to demonstrate that results obtained from high values of accelerating parameters and from fast ageing simulation can be very different from reality. The observed results corroborated this assumption.

  5. Schools and Cable Television.

    ERIC Educational Resources Information Center

    National Education Association, Washington, DC. Educational Technology Div.

    The papers gathered here are designed to provide a foundation of background information for those charged with the responsibility of formulating school district goals regarding cable television (CATV) and of obtaining the necessary cooperation from the local CATV franchise operators to reach these goals. The position of the National Education…

  6. Superconducting Cable Termination

    DOEpatents

    Sinha, Uday K.; Tolbert, Jerry

    2005-08-30

    Disclosed is a termination that connects high temperature superconducting (HTS) cable immersed in pressurized liquid nitrogen to high voltage and neutral (shield) external bushings at ambient temperature and pressure. The termination consists of a splice between the HTS power (inner) and shield (outer) conductors and concentric copper pipes which are the conductors in the termination. There is also a transition from the dielectric tape insulator used in the HTS cable to the insulators used between and around the copper pipe conductors in the termination. At the warm end of the termination the copper pipes are connected via copper braided straps to the conventional warm external bushings which have low thermal stresses. This termination allows for a natural temperature gradient in the copper pipe conductors inside the termination which enables the controlled flashing of the pressurized liquid coolant (nitrogen) to the gaseous state. Thus the entire termination is near the coolant supply pressure and the high voltage and shield cold bushings, a highly stressed component used in most HTS cables, are eliminated. A sliding seal allows for cable contraction as it is cooled from room temperature to ˜72-82 K. Seals, static vacuum, and multi-layer superinsulation minimize radial heat leak to the environment.

  7. Urban Cable Systems.

    ERIC Educational Resources Information Center

    Mason, William F.; And Others

    Analysis of demographic, social, municipal and commercial characteristics of Washington, D.C., indicate that a sophisticated three-stage cable television (CATV) system could be economically viable. The first stage would provide one-way CATV service offering 30 video channels and local program origination at a monthly fee of $3.50. The second stage…

  8. Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium.

    PubMed

    Furukawa, Ruth; Maselli, Andrew; Thomson, Susanne A M; Lim, Rita W L; Stokes, John V; Fechheimer, Marcus

    2003-01-01

    The actin cytoskeleton is sensitive to changes in calcium, which affect contractility, actin-severing proteins, actin-crosslinking proteins and calmodulin-regulated enzymes. To dissect the role of calcium control on the activity of individual proteins from effects of calcium on other processes, calcium-insensitive forms of these proteins were prepared and introduced into living cells to replace a calcium-sensitive form of the same protein. Crosslinking and bundling of actin filaments by the Dictyostelium 34 kDa protein is inhibited in the presence of micromolar free calcium. A modified form of the 34 kDa protein with mutations in the calcium binding EF hand (34 kDa deltaEF2) was prepared using site-directed mutagenesis and expressed in E. coli. Equilibrium dialysis using [(45)Ca]CaCl(2) revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 microM. This calcium binding is absent in the 34 kDa deltaEF2 protein. The actin-binding activity of the 34 kDa deltaEF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa deltaEF2 proteins were expressed in 34-kDa-null and 34 kDa/alpha-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa deltaEF2 failed to supply function of the 34 kDa protein important for control of cell size and for normal growth to either of these 34-kDa-null strains. Furthermore, the distribution of the 34 kDa protein and actin were abnormal in cells expressing 34 kDa deltaEF2. Thus, calcium regulation of the formation and/or dissolution of crosslinked actin structures is required for dynamic behavior of the actin cytoskeleton important for cell structure and growth.

  9. Internal coaxial cable seal system

    DOEpatents

    Hall, David R.; Sneddon, Cameron; Dahlgren, Scott Steven; Briscoe, Michael A.

    2006-07-25

    The invention is a seal system for a coaxial cable and is placed within the coaxial cable and its constituent components. A series of seal stacks including load ring components and elastomeric rings are placed on load bearing members within the coaxial cable sealing the annular space between the coaxial cable and an electrical contact passing there through. The coaxial cable is disposed within drilling components to transmit electrical signals between drilling components within a drill string. The seal system can be used in a variety of downhole components, such as sections of pipe in a drill string, drill collars, heavy weight drill pipe, and jars.

  10. Disposable telemetry cable deployment system

    DOEpatents

    Holcomb, David Joseph

    2000-01-01

    A disposable telemetry cable deployment system for facilitating information retrieval while drilling a well includes a cable spool adapted for insertion into a drill string and an unarmored fiber optic cable spooled onto the spool cable and having a downhole end and a stinger end. Connected to the cable spool is a rigid stinger which extends through a kelly of the drilling apparatus. A data transmission device for transmitting data to a data acquisition system is disposed either within or on the upper end of the rigid stinger.

  11. Flat conductor cable commercialization project

    NASA Technical Reports Server (NTRS)

    Hogarth, P.; Wadsworth, E.

    1977-01-01

    An undercarpet flat conductor cable and a baseboard flat conductor cable system were studied for commercialization. The undercarpet system is designed for use in office and commercial buildings. It employs a flat power cable, protected by a grounded metal shield, that terminates in receptacles mounted on the floor. It is designed to interface with a flat conductor cable telephone system. The baseboard system consists of a flat power cable mounted in a plastic raceway; both the raceway and the receptacles are mounted on the surface of the baseboard. It is designed primarily for use in residential buildings, particularly for renovation and concrete and masonry construction.

  12. Tapping the television cable.

    PubMed

    Clarke, M; Findlay, A; Canac, J F; Vergez, A

    1996-01-01

    Immediate access to patient data is essential to support good clinical decision making and support. However, away from the surgery, the doctor is currently unable to have any access to the clinical database. Solutions exist to support remote access, such as modems or radio data networks, but these are slow, with typical speeds in the 2-10 kbaud region. We propose a novel solution, to use the TV cable already installed in many homes. Using this technology, a suitably equipped computer (RF modern) is capable of connecting at speeds in excess of 500 kbaud and will run applications in exactly the same way as if connected to a surgery network: the cable TV becomes a LAN, but on a metropolitan scale. Brunel University, in collaboration with the Cable Corporation, has been piloting such a network. Issues include not only levels of service, but also security on the network and access, since the data are being effectively received in every home. However, close scrutiny of channel use can create closed networks reserved for specific users. The technology involves use of an RF modem to transmit data on a reverse channel (based at 16 MHz) on each subnet to a router at the head end of the cable network. This frequency translates the packet and retransmits it to all the subnets on a forward channel (based at 178 MHz). Each channel occupies the bandwidth normally allocated to one TV channel. Access is based on a modified CSMA/CD protocol, so treating the cable network as single multiple access network. The modem comes as a standard card installed in a PC and appears much as an ethernet card, but at reduced speed. With an NDIS driver it is quite able to support almost any network software, and has successfully demonstrated Novell and TCP/IP. We describe the HomeWorker network and the results from a pilot study being undertaken to determine the performance of the system and its impact on working practice.

  13. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor.

    PubMed

    Morita, Tsuyoshi; Hayashi, Ken'ichiro

    2013-08-02

    Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin-MRTFs interaction.

  14. RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape

    PubMed Central

    Gay, Olivia; Gilquin, Benoît; Nakamura, Fumihiko; Jenkins, Zandra A.; McCartney, Rosannah; Krakow, Deborah; Deshiere, Alexandre; Assard, Nicole; Hartwig, John H.; Robertson, Stephen P.; Baudier, Jacques

    2011-01-01

    The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actin-binding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial–mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actin-based network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. PMID:21709252

  15. Multiple supramolecular structures formed by interaction of actin with protamine

    PubMed Central

    Grazi, Enrico; Magri, Ermes; Pasquali-Ronchetti, Ivonne

    1982-01-01

    When protamine is added to actin, different supramolecular structures are formed depending on the molar ratio of the two proteins and of the ionic strength of the medium. At low ionic strength, and going from a molar ratio of protamine to G-actin of 4:1, 2:1 and 1:1, globular aggregates are first converted into extended structures and then to long threads in which the constituent ATP–G-actin is rapidly exchangeable with the actin of the medium. At high ionic strength {Tyrode [(1910) Arch. Int. Pharmacodyn. Ther. 20, 205–212] solution}, starting from G-actin and protamine in the 1:1 molar ratio, long ropes are formed that can be resolved into intertwining filaments of 4–5nm diameter. The addition of protamine in a 1:1 molar ratio to a solution of F-actin in Tyrode solution causes the breakage of the actin filaments, which is also revealed by the decrease of the viscosity of the solution and the formation of ordered latero-lateral aggregates. The structures formed by reaction of protamine with G-actin can be separated from free G-actin and protamine by filtration through 0.45μm-pore-size Millipore filters. This technique has been exploited to study the exchange reaction between free actin and the actin–protamine complexes. For these studies the 1:1 actin–protamine complex formed at low ionic strength and the 2:1 actin–protamine complex formed in the presence of 23nm-free Mg2+ have been selected. In the first case the exchange reaction is practically complete in the dead time of the experiment (20s). In the second case, where the complex operates like a true ATPase, the rate of the exchange is initially comparable with the rate of the ATP cleavage. Later on, however, the complex undergoes a change and the rate of the exchange between free actin and the actin bound to protamine becomes lower than the rate of the ATPase reaction. It is proposed that the ATP exchanges for ADP directly on the G-actin bound in the complex. ImagesPLATE 1PLATE 2PLATE 3 PMID

  16. Mechanical force-induced polymerization and depolymerization of F-actin at water/solid interfaces

    NASA Astrophysics Data System (ADS)

    Zhang, Xueqiang; Hu, Xiuyuan; Lei, Haozhi; Hu, Jun; Zhang, Yi

    2016-03-01

    polymerization and depolymerization behaviors at water/solid interfaces using an atomic force microscope (AFM) operated in liquid. By raster scanning an AFM probe on a substrate surface with a certain load, it was found that actin monomers could polymerize into filaments without the help of actin related proteins (ARPs). Further study indicated that actin monomers were inclined to form filaments only under a small scanning load. The polymerized actin filaments would be depolymerized when the mechanical force was stronger. A possible mechanism has been suggested to explain the mechanical force induced actin polymerization. Electronic supplementary information (ESI) available: The height histograms of Fig. 1b-1d, the effect of G-actin concentration on the mechanical-force-induced F-actin formation, and the effect of different mechanical forces on the depolymerization of F-actin. See DOI: 10.1039/c5nr08713a

  17. Periodic actin structures in neuronal axons are required to maintain microtubules

    PubMed Central

    Qu, Yue; Hahn, Ines; Webb, Stephen E.D.; Pearce, Simon P.; Prokop, Andreas

    2017-01-01

    Axons are cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily conserved, ubiquitous, highly ordered feature of axons, the function of PMS is unknown. Here we studied PMS abundance, organization, and function, combining versatile Drosophila genetics with superresolution microscopy and various functional readouts. Analyses with 11 actin regulators and three actin-targeting drugs suggest that PMS contains short actin filaments that are depolymerization resistant and sensitive to spectrin, adducin, and nucleator deficiency, consistent with microscopy-derived models proposing PMS as specialized cortical actin. Upon actin removal, we observed gaps in microtubule bundles, reduced microtubule polymerization, and reduced axon numbers, suggesting a role of PMS in microtubule organization. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilizing protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerization contributes to their maintenance in axons. We discuss potential implications of this novel PMS function along axon shafts for axon maintenance and regeneration. PMID:27881663

  18. The Drosophila javelin Gene Encodes a Novel Actin-Associated Protein Required for Actin Assembly in the Bristle ▿

    PubMed Central

    Shapira, Shira; Bakhrat, Anna; Bitan, Amir; Abdu, Uri

    2011-01-01

    The Drosophila melanogaster bristle is a highly polarized cell that builds specialized cytoskeletal structures. Whereas actin is required for increasing bristle length, microtubules are essential for bristle axial growth. To identify new proteins involved in cytoskeleton organization during bristle development, we focused on identifying and characterizing the javelin (jv) locus. We found that in a jv mutant, the bristle tip is swollen and abnormal organization of bristle grooves is seen over the entire bristle. Using confocal and electron microscopy, we found that in jv mutant bristles, actin bundles do not form properly due to a loss of actin filaments within the bundle. We show that jv is an allele of the predicted CG32397 gene that encodes a protein with no homologs outside insects. Expression of the Jv protein fused to a green fluorescent protein (GFP) shows that the protein is colocalized with actin bundles in the bristle. Moreover, expression of Jv-GFP within the germ line led to the formation of ectopic actin bundles that surround the nucleus of nurse cells. Thus, we report that Jv is a novel actin-associated protein required for actin assembly during Drosophila bristle development. PMID:21930794

  19. Microfluidic devices for the study of actin cytoskeleton in constricted environments: Evidence for podosome formation in endothelial cells exposed to a confined slit.

    PubMed

    Spuul, Pirjo; Chi, Pei-Yin; Billottet, Clotilde; Chou, Chia-Fu; Génot, Elisabeth

    2016-02-01

    The study of cell behavior in constricted environment is particularly relevant to our understanding of the mechanisms of cell invasion. In this regard, microfluidic systems offer promising platforms as microfabricated fluidic chips provide well-controlled physical, chemical and confined environments to study cell phenotype and behavior. Here, we report a fast and effective manufacturing process of user-friendly microfluidic chips ideally suited for quantitative live cell analysis in combination with immunofluorescence microscopy. The chip body, made of polydimethylsiloxane, is composed of two incubation chambers connected by one rectangular intermediate entry channel which provides access to a series of transversal slits where the observation can be made. The height of the slit is designed to be slightly smaller than that of the cells under study. To validate the chip performance, we analyzed the reorganization of the cytoskeleton of endothelial cells under various degree of spatial confinement. We illustrate how the constricted environment affects endothelial cell behavior in inducing the formation of podosomes. Moreover, the process was stimulated further when the surface of the slit was coated with a thin layer of fibronectin. The study demonstrates the suitability of this technological process for cost-effective fabrication of custom-made single-use chips for biological applications.

  20. Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis.

    PubMed

    Stossel, T P; Hartwig, J H

    1976-03-01

    Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.

  1. eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.

    PubMed

    García-Ortiz, Almudena; Martín-Cofreces, Noa B; Ibiza, Sales; Ortega, Ángel; Izquierdo-Álvarez, Alicia; Trullo, Antonio; Victor, Víctor M; Calvo, Enrique; Sot, Begoña; Martínez-Ruiz, Antonio; Vázquez, Jesús; Sánchez-Madrid, Francisco; Serrador, Juan M

    2017-04-01

    The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.

  2. Investigation of Shielded and Unshielded Cables.

    DTIC Science & Technology

    1980-11-11

    increase the yield strength of cables. Table 2 lists the required and measured strengths of the test cables. Both shielded and unshielded Size 2 cables do...selected for evaluation, five shielded and five unshielded, and were examined, dissected and the internal components measured . A list of the cables and...components were measured following the testing parameters shown in Table 17. All complete cables, cables less jacket and cables less jacket and shield

  3. Development of Inspection Robots for Bridge Cables

    PubMed Central

    Kim, Se-Hoon; Lee, Jong-Jae

    2013-01-01

    This paper presents the bridge cable inspection robot developed in Korea. Two types of the cable inspection robots were developed for cable-suspension bridges and cable-stayed bridge. The design of the robot system and performance of the NDT techniques associated with the cable inspection robot are discussed. A review on recent advances in emerging robot-based inspection technologies for bridge cables and current bridge cable inspection methods is also presented. PMID:24459453

  4. Synaptic Clustering of PSD-95 Is Regulated by c-Abl through Tyrosine Phosphorylation

    PubMed Central

    de Arce, Karen Perez; Varela-Nallar, Lorena; Farias, Olivia; Cifuentes, Alejandra; Bull, Paulina; Couch, Brian A.; Koleske, Anthony J.; Inestrosa, Nibaldo C.; Alvarez, Alejandra R.

    2010-01-01

    The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95. PMID:20220006

  5. Ring closure in actin polymers

    NASA Astrophysics Data System (ADS)

    Sinha, Supurna; Chattopadhyay, Sebanti

    2017-03-01

    We present an analysis for the ring closure probability of semiflexible polymers within the pure bend Worm Like Chain (WLC) model. The ring closure probability predicted from our analysis can be tested against fluorescent actin cyclization experiments. We also discuss the effect of ring closure on bend angle fluctuations in actin polymers.

  6. Role of actin in auxin transport and transduction of gravity

    NASA Astrophysics Data System (ADS)

    Hu, S.; Basu, S.; Brady, S.; Muday, G.

    Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

  7. ATP-dependent membrane assembly of F-actin facilitates membrane fusion.

    PubMed

    Jahraus, A; Egeberg, M; Hinner, B; Habermann, A; Sackman, E; Pralle, A; Faulstich, H; Rybin, V; Defacque, H; Griffiths, G

    2001-01-01

    We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.

  8. Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton

    PubMed Central

    Illner, Doris; Scherthan, Harry

    2013-01-01

    Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP–tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3′-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H2O2-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments. PMID:24046368

  9. Method to improve superconductor cable

    DOEpatents

    Borden, A.R.

    1984-03-08

    A method is disclosed of making a stranded superconductor cable having improved flexing and bending characteristics. In such method, a plurality of superconductor strands are helically wound around a cylindrical portion of a mandrel which tapers along a transitional portion to a flat end portion. The helically wound strands form a multistrand hollow cable which is partially flattened by pressure rollers as the cable travels along the transitional portion. The partially flattened cable is impacted with repeated hammer blows as the hollow cable travels along the flat end portion. The hammer blows flatten both the internal and the external surfaces of the strands. The cable is fully flattened and compacted by two sets of pressure rollers which engage the flat sides and the edges of the cable after it has traveled away from the flat end portion of the mandrel. The flattened internal surfaces slide easily over one another when the cable is flexed or bent so that there is very little possibility that the cable will be damaged by the necessary flexing and bending required to wind the cable into magnet coils.

  10. Cable shield connecting device

    DOEpatents

    Silva, Frank A.

    1979-01-01

    A cable shield connecting device for installation on a high voltage cable of the type having a metallic shield, the device including a relatively conformable, looped metal bar for placement around a bared portion of the metallic shield to extend circumferentially around a major portion of the circumference of the metallic shield while being spaced radially therefrom, a plurality of relatively flexible metallic fingers affixed to the bar, projecting from the bar in an axial direction and spaced circumferentially along the bar, each finger being attached to the metallic shield at a portion located remote from the bar to make electrical contact with the metallic shield, and a connecting conductor integral with the bar.

  11. Competition for actin between two distinct F-actin networks defines a bistable switch for cell polarization.

    PubMed

    Lomakin, Alexis J; Lee, Kun-Chun; Han, Sangyoon J; Bui, Duyen A; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-11-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype after relaxation of the actomyosin cytoskeleton. We find that myosin II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. Under low-contractility regimes, epithelial cells polarize in a front-back manner owing to the emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin II from the front to the back of the cell, where the motor locally 'locks' actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high-contractility-driven cell motion is inefficient.

  12. High conductance surge cable

    DOEpatents

    Murray, M.M.; Wilfong, D.H.; Lomax, R.E.

    1998-12-08

    An electrical cable for connecting transient voltage surge suppressors to electrical power panels. A strip of electrically conductive foil defines a longitudinal axis, with a length of an electrical conductor electrically attached to the metallic foil along the longitudinal axis. The strip of electrically conductive foil and the length of an electrical conductor are covered by an insulating material. For impedance matching purposes, triangular sections can be removed from the ends of the electrically conductive foil at the time of installation. 6 figs.

  13. High conductance surge cable

    DOEpatents

    Murray, Matthew M.; Wilfong, Dennis H.; Lomax, Ralph E.

    1998-01-01

    An electrical cable for connecting transient voltage surge suppressers to ectrical power panels. A strip of electrically conductive foil defines a longitudinal axis, with a length of an electrical conductor electrically attached to the metallic foil along the longitudinal axis. The strip of electrically conductive foil and the length of an electrical conductor are covered by an insulating material. For impedance matching purposes, triangular sections can be removed from the ends of the electrically conductive foil at the time of installation.

  14. Non-Intrusive Cable Tester

    NASA Technical Reports Server (NTRS)

    Medelius, Pedro J. (Inventor); Simpson, Howard J. (Inventor)

    1999-01-01

    A cable tester is described for low frequency testing of a cable for faults. The tester allows for testing a cable beyond a point where a signal conditioner is installed, minimizing the number of connections which have to be disconnected. A magnetic pickup coil is described for detecting a test signal injected into the cable. A narrow bandpass filter is described for increasing detection of the test signal. The bandpass filter reduces noise so that a high gain amplifier provided for detecting a test signal is not completely saturate by noise. To further increase the accuracy of the cable tester, processing gain is achieved by comparing the signal from the amplifier with at least one reference signal emulating the low frequency input signal injected into the cable. Different processing techniques are described evaluating a detected signal.

  15. Novel actin depolymerizing macrolide aplyronine A.

    PubMed

    Saito, S; Watabe, S; Ozaki, H; Kigoshi, H; Yamada, K; Fusetani, N; Karaki, H

    1996-09-01

    Aplyronine A is a macrolide isolated from Aplysia kurodai. By monitoring fluorescent intensity of pyrenyl-actin, it was found that aplyronine A inhibited both the velocity and the degree of actin polymerization. Aplyronine A also quickly depolymerized F-actin. The kinetics of depolymerization suggest that aplyronine A severs F-actin. The relationship between the concentration of total actin and F-actin at different concentrations of aplyronine A suggests that aplyronine A forms a 1:1 complex with G-actin. From these results, it is concluded that aplyronine A inhibits actin polymerization and depolymerizes F-actin by nibbling. Comparison of the chemical structure of aplyronine A and another actin-depolymerizing macrolide, mycalolide B, suggests that the side-chain but not the macrolide ring of aplyronine A may account for its actin binding and severing activity.

  16. Bacterial Actins and Their Interactors.

    PubMed

    Gayathri, Pananghat

    2017-01-01

    Bacterial actins polymerize in the presence of nucleotide (preferably ATP), form a common arrangement of monomeric interfaces within a protofilament, and undergo ATP hydrolysis-dependent change in stability of the filament-all of which contribute to performing their respective functions. The relative stability of the filament in the ADP-bound form compared to that of ATP and the rate of addition of monomers at the two ends decide the filament dynamics. One of the major differences between eukaryotic actin and bacterial actins is the variety in protofilament arrangements and dynamics exhibited by the latter. The filament structure and the polymerization dynamics enable them to perform various functions such as shape determination in rod-shaped bacteria (MreB), cell division (FtsA), plasmid segregation (ParM family of actin-like proteins), and organelle positioning (MamK). Though the architecture and dynamics of a few representative filaments have been studied, information on the effect of interacting partners on bacterial actin filament dynamics is not very well known. The chapter reviews some of the structural and functional aspects of bacterial actins, with special focus on the effect that interacting partners exert on the dynamics of bacterial actins, and how these assist them to carry out the functions within the bacterial cell.

  17. Long-range conformational effects of proteolytic removal of the last three residues of actin.

    PubMed Central

    Strzelecka-Gołaszewska, H; Mossakowska, M; Woźniak, A; Moraczewska, J; Nakayama, H

    1995-01-01

    Truncated derivatives of actin devoid of either the last two (actin-2C) or three residues (actin-3C) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critical concentration and polymerization rate increased in the order: intact actin < actin-2C < actin-3C. Conversely, the rate of hydrolysis of actin-bound ATP during spontaneous polymerization of Mg-actin decreased in the same order, so that, for actin-3C, the ATP hydrolysis significantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of the bound nucleotide exchange revealed a similar change upon removal of either the two or three residues from the C-terminus. The C-terminal truncation also resulted in a slight decrease in the rate of subtilisin cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site within this loop, specifically cleaved by a proteinase from Escherichia coli A2 strain, gradually increased upon sequential removal of the two and of the third residue from the C-terminus. From these and other observations made in this work it has been concluded that perturbation of the C-terminal structure in monomeric actin is transmitted to the cleft, where nucleotide and bivalent cation are bound, and to the DNAse-I binding loop on the top of subdomain 2. Further changes at these sites, observed on the polymer level, seem to result from elimination of the intersubunit contact between the C-terminal residues and the DNAse-I binding loop. It is suggested that formation of this contact plays an essential role in regulating the hydrolysis of actin-bound ATP associated with the polymerization process. Images Figure 5 Figure 6 Figure 8 PMID:7733893

  18. Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

  19. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

    PubMed

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q P; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q P

    2016-10-20

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

  20. Control of actin filament dynamics at barbed ends by WH2 domains: from capping to permissive and processive assembly.

    PubMed

    Carlier, Marie-France; Pernier, Julien; Avvaru, Balendu Sankara

    2013-10-01

    WH2 domains are multifunctional regulators of actin assembly that can either sequester G-actin or allow polarized barbed end growth. They all bind similarly to a hydrophobic pocket at the barbed face of actin. Depending on their electrostatic environment, WH2 domains can nucleate actin assembly by facilitating the formation of prenuclei dimers along the canonical spontaneous assembly pathway. They also modulate filament barbed end dynamics in a versatile fashion, acting either as barbed end cappers or assisting barbed end growth like profilin or uncapping barbed ends and potentially mediating processive elongation like formins when they are dimerized. Tandem repeats of WH2 domains can sever filaments and either remain bound to created barbed ends like gelsolin, or strip off an ADP-actin subunit from the severed polymer end, depending on their relative affinity for terminal ADP-F-actin or ADP-G-actin. In summary, WH2 domains recapitulate all known elementary regulatory functions so far found in individual actin-binding proteins. By combining different discrete sets of these multifunctional properties, they acquire specific functions in various actin-based processes, and participate in activities as diverse as filament branching, filopodia extension, or actin remodeling in ciliogenesis and asymmetric meiotic division. They also integrate these functions with other actin-binding motifs present either in the same protein or in a complex with another protein, expanding the range of complexity in actin regulation. The details of their molecular mechanisms and the underlying structural basis provide exciting avenues in actin research.

  1. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

    PubMed Central

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T.; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q. P.; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q. P.

    2016-01-01

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells. PMID:27762277

  2. F-actin distribution and function during sexual development in Eimeria maxima.

    PubMed

    Frölich, Sonja; Wallach, Michael

    2015-06-01

    To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

  3. Actin-myosin network is required for proper assembly of influenza virus particles

    SciTech Connect

    Kumakura, Michiko; Kawaguchi, Atsushi Nagata, Kyosuke

    2015-02-15

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.

  4. Cable structures and lunar environment

    NASA Astrophysics Data System (ADS)

    Ettouney, Mohammed; Benaroya, Haym; Agassi, Nissim

    1992-07-01

    The feasibility of using cable-based structural systems for lunar bases is examined. Small, medium, and long structural spans are considered, and it is shown that each span category dictates the use of a different structural system. It is demonstrated that efficient cable and secondary support systems and foundations are feasible for each span group. The advantages and possibilities offered by cable structures for lunar bases are discussed.

  5. Rotating Connection for Electrical Cables

    NASA Technical Reports Server (NTRS)

    Manges, D. R.

    1986-01-01

    Cable reel provides electrical connections between fixed structure and rotating one. Reel carries power and signal lines while allowing rotating structure to turn up to 360 degrees with respect to fixed structure. Reel replaces sliprings. Can be used to electrically connect arm of robot with body. Reel releases cable to rotating part as it turns and takes up cable as rotating part comes back to its starting position, without tangling, twisting, or kinking.

  6. Monitoring cables for local degradation

    SciTech Connect

    Bustard, L.D.; Sliter, G.E.

    1989-01-01

    Recent experiences in operating nuclear plants in the United States have demonstrated the need for an in situ cable condition monitoring technique that can assess whether installed, low-voltage, unshielded cables have local damage that could compromise their ability to function under normal and accident service conditions. This paper summarizes current US programs that have been initiated to develop a technological basis for monitoring cables with local degradation. 7 refs.

  7. Actin-cytoskeleton rearrangement modulates proton-induced uptake

    SciTech Connect

    Ben-Dov, Nadav; Korenstein, Rafi

    2013-04-15

    Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.

  8. Actin cytoskeleton: putting a CAP on actin polymerization.

    PubMed

    Stevenson, V A; Theurkauf, W E

    2000-10-05

    Two recent studies have identified a Drosophila homolog of cyclase-associated protein (CAP) as a developmentally important negative regulator of actin polymerization that may also directly mediate signal transduction.

  9. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner.

  10. Superconducting flat tape cable magnet

    DOEpatents

    Takayasu, Makoto

    2015-08-11

    A method for winding a coil magnet with the stacked tape cables, and a coil so wound. The winding process is controlled and various shape coils can be wound by twisting about the longitudinal axis of the cable and bending following the easy bend direction during winding, so that sharp local bending can be obtained by adjusting the twist pitch. Stack-tape cable is twisted while being wound, instead of being twisted in a straight configuration and then wound. In certain embodiments, the straight length should be half of the cable twist-pitch or a multiple of it.

  11. In Vivo Imaging and Characterization of Actin Microridges

    PubMed Central

    Lam, Pui-ying; Mangos, Steve; Green, Julie M.; Reiser, Jochen; Huttenlocher, Anna

    2015-01-01

    Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo. PMID:25629723

  12. Self-organizing actin waves that simulate phagocytic cup structures.

    PubMed

    Gerisch, Günther

    2010-03-18

    This report deals with actin waves that are spontaneously generated on the planar, substrate-attached surface of Dictyostelium cells. These waves have the following characteristics. (1) They are circular structures of varying shape, capable of changing the direction of propagation. (2) The waves propagate by treadmilling with a recovery of actin incorporation after photobleaching of less than 10 seconds. (3) The waves are associated with actin-binding proteins in an ordered 3-dimensional organization: with myosin-IB at the front and close to the membrane, the Arp2/3 complex throughout the wave, and coronin at the cytoplasmic face and back of the wave. Coronin is a marker of disassembling actin structures. (4) The waves separate two areas of the cell cortex that differ in actin structure and phosphoinositide composition of the membrane. The waves arise at the border of membrane areas rich in phosphatidylinositol (3,4,5) trisphosphate (PIP3). The inhibition of PIP3 synthesis reversibly inhibits wave formation. (5) The actin wave and PIP3 patterns resemble 2-dimensional projections of phagocytic cups, suggesting that they are involved in the scanning of surfaces for particles to be taken up.PACS Codes: 87.16.Ln, 87.19.lp, 89.75.Fb.

  13. Effect of temperature on the mechanism of actin polymerization.

    PubMed

    Zimmerle, C T; Frieden, C

    1986-10-21

    The rate of the Mg2+-induced polymerization of rabbit skeletal muscle G-actin has been measured as as function of temperature at pH 8 by using various concentrations of Mg2+, Ca2+, and G-actin. A polymerization mechanism similar to that proposed at this pH [Frieden, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6513-6517] was found to fit the data from 10 to 35 degrees C. From the kinetic data, no evidence for actin filament fragmentation was found at any temperature. Dimer formation is the most temperature-sensitive step, with the ratio of forward and reverse rate constants changing 4 orders of magnitude from 10 to 35 degrees C. Over this temperature change, all other ratios of forward and reverse rate constants change 7-fold or less, and the critical concentration remains nearly constant. The reversible Mg2+-induced isomerization of G-actin monomer occurs to a greater extent with increasing temperature, measured either by using N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin or by simulation of the full-time course of the polymerization reaction. This is partially due to Mg2+ binding becoming tighter, and Ca2+ binding becoming weaker, with increasing temperature. Elongation rates from the filament-pointed end, determined by using actin nucleated by plasma gelsolin, show a temperature dependence slightly larger than that expected for a diffusion-limited reaction.

  14. Dual pools of actin at presynaptic terminals.

    PubMed

    Bleckert, Adam; Photowala, Huzefa; Alford, Simon

    2012-06-01

    We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

  15. 5. VIEW OF CABLE SHED AND CABLE TRAY EMANATING FROM ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. VIEW OF CABLE SHED AND CABLE TRAY EMANATING FROM SOUTH FACE OF LAUNCH OPERATIONS BUILDING. MICROWAVE DISH IN FOREGROUND. METEOROLOGICAL TOWER IN BACKGROUND. - Vandenberg Air Force Base, Space Launch Complex 3, Launch Operations Building, Napa & Alden Roads, Lompoc, Santa Barbara County, CA

  16. 51. View of sitdown cable car and cable way for ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    51. View of sit-down cable car and cable way for stream gaging, looking west. Photo by Robin Lee Tedder, Puget Power, 1989. - Puget Sound Power & Light Company, White River Hydroelectric Project, 600 North River Avenue, Dieringer, Pierce County, WA

  17. 52. View of sitdown cable car, cable way, and stream ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    52. View of sit-down cable car, cable way, and stream gaging station, looking southeast. Photo by Robin Lee Tedder, Puget Power, 1989. - Puget Sound Power & Light Company, White River Hydroelectric Project, 600 North River Avenue, Dieringer, Pierce County, WA

  18. Involvement of β- and γ-actin isoforms in actin cytoskeleton organization and migration abilities of bleb-forming human colon cancer cells

    PubMed Central

    Simiczyjew, Aleksandra; Mazur, Antonina Joanna; Dratkiewicz, Ewelina; Nowak, Dorota

    2017-01-01

    Amoeboid movement is characteristic for rounded cells, which do not form strong adhesion contacts with the ECM and use blebs as migratory protrusions. It is well known that actin is the main component of mature forms of these structures, but the exact role fulfilled by non-muscle actin isoforms β- and γ- in bleb formation and migration of these cells is still not fully understood. The aim of this study was to establish the role of β- and γ-actin in migration of bleb-forming cancer cells using isoform-specific antibodies and expression of fluorescently tagged actin isoforms. We observed, after staining with monoclonal antibodies, that both actins are present in these cells in the form of a cortical ring as well as in the area of blebs. Additionally, using simultaneous expression of differentially tagged β- and γ-actin in cells, we observed that the actin isoforms are present together in a single bleb. They were involved during bleb expansion as well as retraction. Also present in the area of these protrusions formed by both isoforms were the bleb markers–ezrin and myosin II. The overexpression of β- or γ-actin led to actin cytoskeletal rearrangement followed by the growth of migration and invasion abilities of examined human colon cancer cells, LS174T line. In summary these data prove that both actin isoforms have an impact on motility of bleb-forming cancer cells. Moreover, we conclude that monoclonal antibodies directed against actin isoforms in combination with the tagged actins are good tools to study their role in important biological processes. PMID:28333953

  19. [Photodynamic therapy for actinic cheilitis].

    PubMed

    Castaño, E; Comunión, A; Arias, D; Miñano, R; Romero, A; Borbujo, J

    2009-12-01

    Actinic cheilitis is a subtype of actinic keratosis that mainly affects the lower lip and has a higher risk of malignant transformation. Its location on the labial mucosa influences the therapeutic approach. Vermilionectomy requires local or general anesthetic and is associated with a risk of an unsightly scar, and the treatment with 5-fluorouracil or imiquimod lasts for several weeks and the inflammatory reaction can be very intense. A number of authors have used photodynamic therapy as an alternative to the usual treatments. We present 3 patients with histologically confirmed actinic cheilitis treated using photodynamic therapy with methyl aminolevulinic acid as the photosensitizer and red light at 630 nm. The clinical response was good, with no recurrences after 3 to 6 months of follow-up. Our experience supports the use of photodynamic therapy as a good alternative for the treatment of actinic cheilitis.

  20. Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.

    PubMed

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament-severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca(2+), in vitro. Analysis of a mutant that bears a point mutation at the Ca(2+) binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca(2+) level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament-severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.

  1. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  2. Self-organized DNA/F-actin gels: entangled networks of nematic domains with tunable density

    NASA Astrophysics Data System (ADS)

    Butler, John; Zribi, Olena; Smalyukh, Ivan; Hwee Lai, Ghee; Golestanian, Ramin; Angelini, Thomas; Wong, Gerard

    2008-03-01

    We examine mixtures of DNA and F-actin as a model system of like-charged rigid rods and flexible chains. Confocal microscopy reveals the formation of elongated nematic F-actin domains reticulated via defect-free vertices into a network, all embedded in a mesh of random DNA. Synchrotron x-ray scattering results indicate that the DNA mesh squeezes the F-actin domains into a nematic state via the osmotic pressure of uncondensed counterions, so that the inter-actin spacing within the domains decreases with increasing DNA concentration. These observations are consistent with arguments based on electrostatics and nematic elasticity.

  3. Universal cable head for a multiconductor logging cable

    SciTech Connect

    Ip, M.

    1989-10-17

    This patent describes a universal cable head. It comprises: an armored logging cable having up to seven electrical conductors therein and incorporating a woven wire rope, and a surrounding armor wrap; stress relieving stinger means on the cable; an encircling housing to transfer axial loads from the cable to the housing; an internal transverse bulkhead within the housing adjacent to an internal cavity therein; electrical feedthrus for connection of conductors in the logging cable; exposed mating connectors electrically connected to the feedthrus; the housing extending past and encircling the mating connectors; and a rotatable, shoulder limited sleeve on the exterior of the housing having threading means thereon for connecting with a mating female connector.

  4. Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons.

    PubMed

    Schneider, Gabriela; Nieznanski, Krzysztof; Jozwiak, Jolanta; Slomnicki, Lukasz P; Redowicz, Maria J; Filipek, Anna

    2010-11-01

    CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.

  5. Contractile basis of ameboid movement. VII. The distribution of fluorescently labeled actin in living amebas

    PubMed Central

    1980-01-01

    The technique of molecular cytochemistry has been used to follow the distribution of fluorescently labeled actin in living Chaos carolinensis and Amoeba proteus during ameboid movement and various cellular processes. The distribution of 5-iodoacetamidofluorescein- labeled actin was compared with that of Lissamine rhodamine B sulfonyl chloride-labeled ovalbumin microinjected into the same cell and recorded with an image intensification microscope system. Actively motile cells demonstrated a rather uniform distribution of actin throughout most of the cytoplasm, except in the tail ectoplasm and in plasma gel sheets, where distinct actin structures were observed. In addition, actin-containing structures were induced in the cortex during wound healing, concanavalin A capping, pinocytosis, and contractions elicited by phalloidin injections. The formation of distinct fluorescent actin structures has been correlated with contractile activities. PMID:6893200

  6. Structural polymorphism of the actin-espin system: a prototypical system of filaments and linkers in stereocilia.

    PubMed

    Purdy, Kirstin R; Bartles, James R; Wong, Gerard C L

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  7. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin R.; Bartles, James R.; Wong, Gerard C. L.

    2007-02-01

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system’s phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  8. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    SciTech Connect

    Purdy, Kirstin R.; Wong, Gerard C. L.; Bartles, James R.

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  9. Rho GTPases, phosphoinositides, and actin

    PubMed Central

    Croisé, Pauline; Estay-Ahumada, Catherine; Gasman, Stéphane; Ory, Stéphane

    2014-01-01

    Rho GTPases are well known regulators of the actin cytoskeleton that act by binding and activating actin nucleators. They are therefore involved in many actin-based processes, including cell migration, cell polarity, and membrane trafficking. With the identification of phosphoinositide kinases and phosphatases as potential binding partners or effectors, Rho GTPases also appear to participate in the regulation of phosphoinositide metabolism. Since both actin dynamics and phosphoinositide turnover affect the efficiency and the fidelity of vesicle transport between cell compartments, Rho GTPases have emerged as critical players in membrane trafficking. Rho GTPase activity, actin remodeling, and phosphoinositide metabolism need to be coordinated in both space and time to ensure the progression of vesicles along membrane trafficking pathways. Although most molecular pathways are still unclear, in this review, we will highlight recent advances made in our understanding of how Rho-dependent signaling pathways organize actin dynamics and phosphoinositides and how phosphoinositides potentially provide negative feedback to Rho GTPases during endocytosis, exocytosis and membrane exchange between intracellular compartments. PMID:24914539

  10. Apical domain polarization localizes actin-myosin activity to drive ratchet-like apical constriction.

    PubMed

    Mason, Frank M; Tworoger, Michael; Martin, Adam C

    2013-08-01

    Apical constriction promotes epithelia folding, which changes tissue architecture. During Drosophila gastrulation, mesoderm cells exhibit repeated contractile pulses that are stabilized such that cells apically constrict like a ratchet. The transcription factor Twist is required to stabilize cell shape. However, it is unknown how Twist spatially coordinates downstream signals to prevent cell relaxation. We find that during constriction, Rho-associated kinase (Rok) is polarized to the middle of the apical domain (medioapical cortex), separate from adherens junctions. Rok recruits or stabilizes medioapical myosin II (Myo-II), which contracts dynamic medioapical actin cables. The formin Diaphanous mediates apical actin assembly to suppress medioapical E-cadherin localization and form stable connections between the medioapical contractile network and adherens junctions. Twist is not required for apical Rok recruitment, but instead polarizes Rok medioapically. Therefore, Twist establishes radial cell polarity of Rok/Myo-II and E-cadherin and promotes medioapical actin assembly in mesoderm cells to stabilize cell shape fluctuations.

  11. Cable Television and Public Safety.

    ERIC Educational Resources Information Center

    Cranberg, Gilbert

    One of the most promising applications of cable television (CATV) is municipal surveillance of public areas for protection against crime, fire detection, control of air pollution, and traffic. Thus far, however, the CATV industry has made minimal efforts to realize the potential of CATV for community protection--the use of cable for public safety…

  12. What Belongs On the Cable.

    ERIC Educational Resources Information Center

    Ward, John E.

    Many papers and articles over the past few years have suggested that the coaxial cable television (CATV) cable carries sufficient bandwidth into and out of the home that it can serve almost every conceivable communications need--providing many television viewing channels; two-way data, audio and data services; the functions of the present…

  13. Cable Television in Sedalia, Missouri.

    ERIC Educational Resources Information Center

    Lamkin, Kathryn Janel

    A field study was conducted of the status of cable television in Sedalia, Missouri. Based on interviews of city council members and staff members of Cablevision, the Sedalia cable franchise holder, the following issues were investigated: (1) subscription rates; (2) franchise negotiations; (3) quality of existing services; and (4) possible…

  14. Cable Television: Notebook Number Five.

    ERIC Educational Resources Information Center

    Notebook, 1973

    1973-01-01

    Cable television has been introduced to the public as a revolutionary development in communications, but its history, evolving structure, and present operation indicate otherwise. A few large industrial conglomerates have come to dominate the field of cable television and studies by private institutions and the regulatory activities of the Federal…

  15. Distribution of actin of the human erythrocyte membrane cytoskeleton after interaction with radiographic contrast media.

    PubMed

    Franke, R P; Scharnweber, T; Fuhrmann, R; Krüger, A; Wenzel, F; Mrowietz, C; Jung, F

    2013-01-01

    A type-dependent chemotoxic effect of radiographic contrast media on erythrocytes and endothelial cells was reported several times. While mechanisms of toxicity are still unclear the cellular reactions e.g. echinocyte formation in erythrocytes and the buckling of endothelial cells coincided with deterioration of capillary perfusion (in patients with coronary artery disease) and tissue oxygen tension (in the myocardium of pigs). Whether the shape changes in erythrocytes coincide with changes in the arrangement of actin, the core of the actin-spectrin cytoskeletal network and possible actor in membrane stresses and deformation is not known until now. To get specific informations actin was stained using two different staining methods (antibodies to β-actin staining oligomeric G-actin and polymeric F-actin and Phalloidin-Rhodamin staining polymeric F-actin only). In addition, an advanced version of confocal laser scanning microscopes was used enabling the display of the actin arrangement near substrate surfaces. Blood smears were produced after erythrocyte suspension in autologous plasma or in two different plasma/RCM mixtures. In this study an even homogenous distribution of fine grained globular actin in the normal human erythrocyte could be demonstrated. After suspension of erythrocytes in a plasma/Iodixanol mixture an increased number of membrane protrusions appeared densely filled with intensely stained actin similar to cells suspended in autologous plasma, however, there in less numbers. Suspension in Iopromide, in contrast, induced a complete reorganization of the cytoskeletal actin: the fine grained globular actin distribution disappeared and only few, long and thick actin filaments bundled and possibly polymerized appeared, instead, shown here for the first time.

  16. Nuclear factor of activated T cells c1 mediates p21-activated kinase 1 activation in the modulation of chemokine-induced human aortic smooth muscle cell F-actin stress fiber formation, migration, and proliferation and injury-induced vascular wall remodeling.

    PubMed

    Kundumani-Sridharan, Venkatesh; Singh, Nikhlesh K; Kumar, Sanjay; Gadepalli, Ravisekhar; Rao, Gadiparthi N

    2013-07-26

    Recent literature suggests that cyclin-dependent kinases (CDKs) mediate cell migration. However, the mechanisms were not known. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a delayed time-dependent manner. MCP1 also stimulated F-actin stress fiber formation in a delayed manner in HASMCs, as well as the migration and proliferation of these cells. Inhibition of Pak1 suppressed MCP1-induced HASMC F-actin stress fiber formation, migration, and proliferation. MCP1 induced cyclin D1 expression as well as CDK6 and CDK4 activities, and these effects were dependent on activation of NFATc1. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1-induced Pak1 phosphorylation/activation and resulted in decreased HASMC F-actin stress fiber formation, migration, and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. MCP1 also activated Rac1 in a time-dependent manner, and depletion/inhibition of its levels/activation abrogated MCP1-induced NFATc1-cyclin D1-CDK6-CDK4-Pak1 signaling and, thereby, decreased HASMC F-actin stress fiber formation, migration, and proliferation. In addition, smooth muscle-specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities, resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1-dependent, NFATc1-mediated cyclin D1 expression and CDK6 activity mediate this effect. In addition, smooth muscle-specific deletion of NFATc1 prevented the capacity of vascular smooth muscle cells for MCP-1-induced activation of the cyclin D1-CDK6-CDK4-Pak1 signaling axis, affecting

  17. Photonic-powered cable assembly

    DOEpatents

    Sanderson, Stephen N.; Appel, Titus James; Wrye, IV, Walter C.

    2013-01-22

    A photonic-cable assembly includes a power source cable connector ("PSCC") coupled to a power receive cable connector ("PRCC") via a fiber cable. The PSCC electrically connects to a first electronic device and houses a photonic power source and an optical data transmitter. The fiber cable includes an optical transmit data path coupled to the optical data transmitter, an optical power path coupled to the photonic power source, and an optical feedback path coupled to provide feedback control to the photonic power source. The PRCC electrically connects to a second electronic device and houses an optical data receiver coupled to the optical transmit data path, a feedback controller coupled to the optical feedback path to control the photonic power source, and a photonic power converter coupled to the optical power path to convert photonic energy received over the optical power path to electrical energy to power components of the PRCC.

  18. Photonic-powered cable assembly

    DOEpatents

    Sanderson, Stephen N; Appel, Titus James; Wrye, IV, Walter C

    2014-06-24

    A photonic-cable assembly includes a power source cable connector ("PSCC") coupled to a power receive cable connector ("PRCC") via a fiber cable. The PSCC electrically connects to a first electronic device and houses a photonic power source and an optical data transmitter. The fiber cable includes an optical transmit data path coupled to the optical data transmitter, an optical power path coupled to the photonic power source, and an optical feedback path coupled to provide feedback control to the photonic power source. The PRCC electrically connects to a second electronic device and houses an optical data receiver coupled to the optical transmit data path, a feedback controller coupled to the optical feedback path to control the photonic power source, and a photonic power converter coupled to the optical power path to convert photonic energy received over the optical power path to electrical energy to power components of the PRCC.

  19. The effects of collapsing factors on F-actin content and microtubule distribution of Helisoma growth cones.

    PubMed

    Torreano, Paul J; Waterman-Storer, Clare M; Cohan, Christopher S

    2005-03-01

    Growth cone collapsing factors induce growth cone collapse or repulsive growth cone turning by interacting with membrane receptors that induce alterations in the growth cone cytoskeleton. A common change induced by collapsing factors in the cytoskeleton of the peripheral domain, the thin lamellopodial area of growth cones, is a decline in the number of radially aligned F-actin bundles that form the core of filopodia. The present study examined whether ML-7, a myosin light chain kinase inhibitor, serotonin, a neurotransmitter and TPA, an activator of protein kinase C, which induce growth cone collapse of Helisoma growth cones, depolymerized or debundled F-actin. We report that these collapsing factors had different effects. ML-7 induced F-actin reorganization consistent with debundling whereas serotonin and TPA predominately depolymerized and possibly debundled F-actin. Additionally, these collapsing factors induced the formation of a dense actin-ring around the central domain, the thicker proximal area of growth cones [Zhou and Cohan, 2001: J. Cell Biol. 153:1071-1083]. The formation of the actin-ring occurred subsequent to the loss of actin bundles. The ML-7-induced actin-ring was found to inhibit microtubule extension into the P-domain. Thus, ML-7, serotonin, and TPA induce growth cone collapse associated with a decline in radially aligned F-actin bundles through at least two mechanisms involving debundling of actin filaments and/or actin depolymerization.

  20. Nucleus-associated actin in Amoeba proteus.

    PubMed

    Berdieva, Mariia; Bogolyubov, Dmitry; Podlipaeva, Yuliya; Goodkov, Andrew

    2016-10-01

    The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.

  1. Avalanches, hardening and softening in dense cross-linked actin networks

    NASA Astrophysics Data System (ADS)

    Astrom, Jan; Kumar, Sunil; Vattulainen, Ilpo; Karttunen, Mikko

    2008-03-01

    Actin filament networks enable the cytoskeleton to adjust to internal and external forcing. These active networks can adapt to changes by dynamically adjusting their crosslinks. Here, we study actin filaments as elastic fibers having finite dimensions. We employ a full three-dimensional model to study the elastic properties of actin networks by computer simulations. We model a dense actin network with the crosslinks being approximately 1μm apart. The results show that dense actin networks, without any pre-straining, are characterized by (a) strain hardening without entropic elasticity, (b) 'viscotic' hysteresis in the case of strong crosslinks, (c) avalanches of crosslink slippage leading to strain softening in the case of breakable crosslinks, and (d) spontaneous formation of stress fibers in the case of active crosslink formation and destruction. We will discuss the relation to recent experimental observations.

  2. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  3. Optogenetics to target actin-mediated synaptic loss in Alzheimer's

    NASA Astrophysics Data System (ADS)

    Zahedi, Atena; DeFea, Kathryn; Ethell, Iryna

    2013-03-01

    Numerous studies in Alzheimer's Disease (AD) animal models show that overproduction of Aβ peptides and their oligomerization can distort dendrites, damage synapses, and decrease the number of dendritic spines and synapses. Aβ may trigger synapse loss by modulating activity of actin-regulating proteins, such as Rac1 and cofilin. Indeed, Aβ1-42 oligomers can activate actin severing protein cofilin through calcineurin-mediated activation of phosphatase slingshot and inhibit an opposing pathway that suppresses cofilin phosphorylation through Rac-mediated activation of LIMK1. Excessive activation of actin-severing protein cofilin triggers the formation of a non-dynamic actin bundles, called rods that are found in AD brains and cause loss of synapses. Hence, regulation of these actin-regulating proteins in dendritic spines could potentially provide useful tools for preventing the synapse/spine loss associated with earlier stages of AD neuropathology. However, lack of spatiotemporal control over their activity is a key limitation. Recently, optogenetic advancements have provided researchers with convenient light-activating proteins such as photoactivatable Rac (PARac). Here, we transfected cultured primary hippocampal neurons and human embryonic kidney (HEK) cells with a PARac/ mCherry-containing plasmid and the mCherry-positive cells were identified and imaged using an inverted fluorescence microscope. Rac1 activation was achieved by irradiation with blue light (480nm) and live changes in dendritic spine morphology were observed using mCherry (587nm). Rac activation was confirmed by immunostaining for phosphorylated form of effector proteinP21 protein-activated kinase 1 (PAK1) and reorganization of actin. Thus, our studies confirm the feasibility of using the PA-Rac construct to trigger actin re-organization in the dendritic spines.

  4. Competition in the Pay Cable Industry.

    ERIC Educational Resources Information Center

    Albarran, Alan B.

    This paper analyzes the state of competition in the pay cable industry. The analysis conceptualizes competition in pay cable and discusses the current structure of the pay cable industry and the competition for subscribers and programming. The competition for audiences that pay cable faces from both pay-per-view services and the video cassette…

  5. Cable Television: Citizen Participation in Planning.

    ERIC Educational Resources Information Center

    Yin, Robert K.

    The historical background of citizen participation in local affairs and its relevance at the onset of community concern about cable television are briefly discussed in this report. The participation of citizens, municipal officials, and cable operators in laying the groundwork for a cable system as well as the pros and cons of cable television as…

  6. Cable in Connecticut; a Citizen's Handbook.

    ERIC Educational Resources Information Center

    Cleland, Margaret

    This handbook for Connecticut cable television consumers addresses a variety of topics, including: (1) a definition of cable television services; (2) the public stake in cable television; (3) program variety; (4) pay cable service; (5) public satellites; (6) government regulation; (7) proposed regulation; (8) role of the Connecticut Public…

  7. 14 CFR 27.1365 - Electric cables.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Electric cables. 27.1365 Section 27.1365... STANDARDS: NORMAL CATEGORY ROTORCRAFT Equipment Electrical Systems and Equipment § 27.1365 Electric cables. (a) Each electric connecting cable must be of adequate capacity. (b) Each cable that would...

  8. 14 CFR 27.1365 - Electric cables.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Electric cables. 27.1365 Section 27.1365... STANDARDS: NORMAL CATEGORY ROTORCRAFT Equipment Electrical Systems and Equipment § 27.1365 Electric cables. (a) Each electric connecting cable must be of adequate capacity. (b) Each cable that would...

  9. 14 CFR 27.1365 - Electric cables.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Electric cables. 27.1365 Section 27.1365... STANDARDS: NORMAL CATEGORY ROTORCRAFT Equipment Electrical Systems and Equipment § 27.1365 Electric cables. (a) Each electric connecting cable must be of adequate capacity. (b) Each cable that would...

  10. 14 CFR 27.1365 - Electric cables.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Electric cables. 27.1365 Section 27.1365... STANDARDS: NORMAL CATEGORY ROTORCRAFT Equipment Electrical Systems and Equipment § 27.1365 Electric cables. (a) Each electric connecting cable must be of adequate capacity. (b) Each cable that would...

  11. 14 CFR 27.1365 - Electric cables.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Electric cables. 27.1365 Section 27.1365... STANDARDS: NORMAL CATEGORY ROTORCRAFT Equipment Electrical Systems and Equipment § 27.1365 Electric cables. (a) Each electric connecting cable must be of adequate capacity. (b) Each cable that would...

  12. Cable Modem Technology Implementation: Challenges and Prospects.

    ERIC Educational Resources Information Center

    Littman, Marlyn Kemper

    1998-01-01

    Describes cable modem technology (i.e., an external device that facilitates high-speed access to the Internet via the same network configuration employed for cable television). Examples of cable field trials carried out in collaboration with educational user communities are presented, and cable technical capabilities, advantages, and constraints…

  13. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  14. 105. VIEW NORTH FROM SLC3W CABLE TUNNEL INTO CABLE VAULT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    105. VIEW NORTH FROM SLC-3W CABLE TUNNEL INTO CABLE VAULT AND SLC-3E CABLE TUNNEL. NOTE WOODEN PLANKING ON FLOOR OF TUNNEL AND CABLE TRAYS LINING TUNNEL WALLS. STAIRS ON EAST WALL OF CABLE VAULT LEAD INTO LANDLINE INSTRUMENTATION ROOM. - Vandenberg Air Force Base, Space Launch Complex 3, Launch Operations Building, Napa & Alden Roads, Lompoc, Santa Barbara County, CA

  15. Fully synthetic taped insulation cables

    DOEpatents

    Forsyth, E.B.; Muller, A.C.

    1983-07-15

    The present invention is a cable which, although constructed from inexpensive polyolefin tapes and using typical impregnating oils, furnishes high voltage capability up to 765 kV, and has such excellent dielectric characteristics and heat transfer properties that it is capable of operation at capacities equal to or higher than presently available cables at a given voltage. This is accomplished by using polyethylene, polybutene or polypropylene insulating tape which has been specially processed to attain properties which are not generally found in these materials, but are required for their use in impregnated electrical cables. Chief among these properties is compatibility with impregnating oil.

  16. IntelliCable Interface Specification

    DTIC Science & Technology

    2016-07-14

    Specification Page 9 Layer 1 – Physical Each IntelliCable shall identify its capabilities and needed output using a 1- Wire storage and signaling...device. Some informative sites are listed below:  Dallas Semi / Maxim – http://www.maxim-ic.com/products/1- wire /  1- Wire industry standards – http...www.1wire.org A typical IntelliCable will use a simple memory device, such as a DS2431. Other cables may use 1- Wire devices with built-in sense

  17. Robot cable-compliant devices

    NASA Technical Reports Server (NTRS)

    Kerley, James J., Jr. (Inventor)

    1990-01-01

    A cable compliant robotic joint includes two U configuration cross section brackets with their U cross sections lying in different planes, one of their brackets being connected to a robot arm and the other to a tool. Additional angle brackets are displaced from the other brackets at corners of the robotic joint. All the brackets are connected by cable segments which lie in one or more planes which are perpendicular to the direction of tool travel as it approaches a work object. The compliance of the joint is determined by the cable segment characteristics, such as their length, material, angle, stranding, pretwisting, and prestressing.

  18. Mechanism of Deep-Sea Fish α-Actin Pressure Tolerance Investigated by Molecular Dynamics Simulations

    PubMed Central

    Wakai, Nobuhiko; Takemura, Kazuhiro; Morita, Takami; Kitao, Akio

    2014-01-01

    The pressure tolerance of monomeric α-actin proteins from the deep-sea fish Coryphaenoides armatus and C. yaquinae was compared to that of non-deep-sea fish C. acrolepis, carp, and rabbit/human/chicken actins using molecular dynamics simulations at 0.1 and 60 MPa. The amino acid sequences of actins are highly conserved across a variety of species. The actins from C. armatus and C. yaquinae have the specific substitutions Q137K/V54A and Q137K/L67P, respectively, relative to C. acrolepis, and are pressure tolerant to depths of at least 6000 m. At high pressure, we observed significant changes in the salt bridge patterns in deep-sea fish actins, and these changes are expected to stabilize ATP binding and subdomain arrangement. Salt bridges between ATP and K137, formed in deep-sea fish actins, are expected to stabilize ATP binding even at high pressure. At high pressure, deep-sea fish actins also formed a greater total number of salt bridges than non-deep-sea fish actins owing to the formation of inter-helix/strand and inter-subdomain salt bridges. Free energy analysis suggests that deep-sea fish actins are stabilized to a greater degree by the conformational energy decrease associated with pressure effect. PMID:24465747

  19. Mechanism of deep-sea fish α-actin pressure tolerance investigated by molecular dynamics simulations.

    PubMed

    Wakai, Nobuhiko; Takemura, Kazuhiro; Morita, Takami; Kitao, Akio

    2014-01-01

    The pressure tolerance of monomeric α-actin proteins from the deep-sea fish Coryphaenoides armatus and C. yaquinae was compared to that of non-deep-sea fish C. acrolepis, carp, and rabbit/human/chicken actins using molecular dynamics simulations at 0.1 and 60 MPa. The amino acid sequences of actins are highly conserved across a variety of species. The actins from C. armatus and C. yaquinae have the specific substitutions Q137K/V54A and Q137K/L67P, respectively, relative to C. acrolepis, and are pressure tolerant to depths of at least 6000 m. At high pressure, we observed significant changes in the salt bridge patterns in deep-sea fish actins, and these changes are expected to stabilize ATP binding and subdomain arrangement. Salt bridges between ATP and K137, formed in deep-sea fish actins, are expected to stabilize ATP binding even at high pressure. At high pressure, deep-sea fish actins also formed a greater total number of salt bridges than non-deep-sea fish actins owing to the formation of inter-helix/strand and inter-subdomain salt bridges. Free energy analysis suggests that deep-sea fish actins are stabilized to a greater degree by the conformational energy decrease associated with pressure effect.

  20. Bacterial Actins? An Evolutionary Perspective

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

    According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

  1. Actin polymerization is stimulated by actin cross-linking protein palladin.

    PubMed

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G; Orlova, Albina; Egelman, Edward H; Beck, Moriah R

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes.

  2. Active Chemical Thermodynamics promoted by activity of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Rao, Madan

    2011-03-01

    The spatial distribution and dynamics of formation and breakup of the nanoclusters of cell surface proteins is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we have proposed a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. We study the consequences of such active actin-based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that the active remodeling of cortical actin, can give rise to a dramatic increase in efficiency and extent of conformational spread, even at low levels of expression at the cell surface. We define a activity temperature (τa) arising due to actin activities which can be used to describe chemical thermodynamics of the system. We plot TTT (time-temparature-transformation) curves and compute the Arrhenius factors which depend on τa . With this, the active asters can be treated as enzymes whose enzymatic reaction rate can be related to the activity.

  3. Optimal treatment of actinic keratoses

    PubMed Central

    Uhlenhake, Elizabeth E

    2013-01-01

    The most compelling reason and primary goal of treating actinic keratoses is to prevent malignant transformation into invasive squamous cell carcinoma, and although there are well established guidelines outlining treatment modalities and regimens for squamous cell carcinoma, the more commonly encountered precancerous actinic lesions have no such standard. Many options are available with variable success and patient compliance rates. Prevention of these lesions is key, with sun protection being a must in treating aging patients with sun damage as it is never too late to begin protecting the skin. PMID:23345970

  4. Process of modifying a cable end

    DOEpatents

    Roose, L.D.

    1995-08-01

    End moldings for high-voltage cables are described wherein the dielectric insulator of the cable is heated and molded to conform to a desired shape. As a consequence, high quality substantially bubble-free cable connectors suitable for mating to premanufactured fittings are made. Disclosed are a method for making the cable connectors either in the field or in a factory, molds suitable for use with the method, and the molded cable connectors, themselves. 5 figs.

  5. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking.

    PubMed

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

    The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.

  6. Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells.

    PubMed

    Estes, Kathleen A; Szumowski, Suzannah C; Troemel, Emily R

    2011-09-01

    The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo.

  7. Cable Television in Extension Education.

    ERIC Educational Resources Information Center

    Waldron, Mark W.

    1978-01-01

    The experience of producing and presenting two continuing education courses on cable television proved that this technique can be a valuable additional means of presenting courses, complementing classroom discussions, and efficiently extending the university into the community. (Author)

  8. Cable SGEMP Code Validation Study

    SciTech Connect

    Ballard, William Parker

    2013-05-01

    This report compared data taken on the Modular Bremsstrahlung Simulator using copper jacketed (cujac) cables with calculations using the RHSD-RA Cable SGEMP analysis tool. The tool relies on CEPXS/ONBFP to perform radiation transport in a series of 1D slices through the cable, and then uses a Green function technique to evaluate the expected current drive on the center conductor. The data were obtained in 2003 as part of a Cabana verification and validation experiment using 1-D geometries, but were not evaluated until now. The agreement between data and model is not adequate unless gaps between the dielectric and outer conductor (ground) are assumed, and these gaps are large compared with what is believed to be in the actual cable.

  9. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

    PubMed

    Niederman, R; Amrein, P C; Hartwig, J

    1983-05-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

  10. Fully synthetic taped insulation cables

    DOEpatents

    Forsyth, Eric B.; Muller, Albert C.

    1984-01-01

    A high voltage oil-impregnated electrical cable with fully polymer taped insulation operable to 765 kV. Biaxially oriented, specially processed, polyethylene, polybutene or polypropylene tape with an embossed pattern is wound in multiple layers over a conductive core with a permeable screen around the insulation. Conventional oil which closely matches the dielectric constant of the tape is used, and the cable can be impregnated after field installation because of its excellent impregnation characteristics.

  11. Nuclear instrumentation cable end seal

    DOEpatents

    Cannon, Collins P.; Brown, Donald P.

    1979-01-01

    An improved coaxial end seal for hermetically sealed nuclear instrumentation cable exhibiting an improved breakdown pulse noise characteristic under high voltage, high temperature conditions. A tubular insulator body has metallized interior and exterior surface portions which are braze sealed to a center conductor and an outer conductive sheath. The end surface of the insulator body which is directed toward the coaxial cable to which it is sealed has a recessed surface portion within which the braze seal material terminates.

  12. Novel cable coupling technique for improved shallow distributed acoustic sensor VSPs

    NASA Astrophysics Data System (ADS)

    Munn, Jonathan D.; Coleman, Thomas I.; Parker, Beth L.; Mondanos, Michael J.; Chalari, Athena

    2017-03-01

    Vertical seismic profiles (VSPs) collected using fiber optic distributed acoustic sensors (DAS) are becoming increasingly common; yet, ensuring good cable coupling with the borehole wall remains a persistent challenge. Traditional cable deployment techniques used in the petroleum industry are either not possible or do not provide data of sufficient quality for shallow applications. Additionally, no direct field comparison of coupling techniques in the same borehole exists to determine the impacts of poor coupling on DAS VSP data quality. This paper addresses these issues by: (1) presenting a novel cable coupling solution using a removable and relatively inexpensive FLUTe™ flexible borehole liner; and (2) presenting field examples of DAS VSPs under different coupling conditions. The proposed coupling technique is analogous to a fully cemented deployment in that the cable is continuously coupled directly to the formation. Field experiments conducted to assess and validate the technique demonstrate a marked improvement in VSP data quality when the cable is coupled with a flexible borehole liner. Without the liner, seismic profiles are dominated by a high-amplitude cable wave and the p-wave arrival is not observed; however, with cable coupling provided by a borehole liner inflated using hydrostatic pressure, the cable wave is suppressed and clear p-wave arrivals are visible. Additional tests examining the influence of fiber optic cable structure on seismic responses demonstrate that tight buffered fibers are more sensitive to dynamic strain than loose tube fibers making them potentially better suited for certain DAS applications.

  13. Antibodies to Actin in Autoimmune Neutropenia

    DTIC Science & Technology

    1990-02-01

    protein as actin. Purified Acanthamoeba actin by anti-neutrophil antibodies in autoimmune neutropenia, comigrated with the protein and was specifically...anti-rabbit IgG were obtained from ICN Immunobiolog- formed using purified Acanthamoeba actin (gift of Dr Blair Bowers. icals, Naperville, IL. Cells...preparations𔃼 1 - was the protein recognized by these anti-neutrophil antibody 6 .2- positive sera, lgG, and F(ab’) 2. Purified Acanthamoeba actin

  14. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    PubMed Central

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. PMID:21444821

  15. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

    PubMed

    Paredez, Alexander R; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C; Wang, Chung-Ju Rachel; Cande, W Z

    2011-04-12

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

  16. Actinic cheilitis in dental practice.

    PubMed

    Savage, N W; McKay, C; Faulkner, C

    2010-06-01

    Actinic cheilitis is a potentially premalignant condition involving predominantly the vermilion of the lower lip. The aim of the current paper was to review the clinical presentation of actinic cheilitis and demonstrate the development of management plans using a series of cases. These are designed to provide immediate treatment where required but also to address the medium and long-term requirements of the patient. The authors suggest that the clinical examination of lips and the assessment of actinic cheilitis and other lip pathology become a regular part of the routine soft tissue examination undertaken as a part of the periodic examination of dental patients. Early recognition of actinic cheilitis can allow the development of strategies for individual patients that prevent progression. These are based on past sun exposure, future lifestyle changes and the daily use of emollient sunscreens, broad-brimmed hats and avoidance of sun exposure during the middle of the day. This is a service that is not undertaken as a matter of routine in general medical practice as patients are not seen with the regularity of dental patients and generally not under the ideal examination conditions available in the dental surgery.

  17. Actin crosslinkers: repairing the sense of touch.

    PubMed

    Sun, Sean X; Walcott, Sam

    2010-10-26

    Cells use actin bundles infused with myosin to exert contractile forces on the extracellular environment. This active tension is essential for cellular mechanosensation. Now, the role of actin crosslinkers in stabilizing and repairing the actin bundles is coming into clearer view.

  18. Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils

    SciTech Connect

    Eberle, M.; Traynor-Kaplan, A.E.; Sklar, L.A.; Norgauer, J. )

    1990-10-05

    Stimulation of human neutrophils with the chemoattractant N-formyl peptide caused rapid polymerization of F-actin as detected by right angle light scatter and 7-nitrobenz-2-oxa-1,3-diazol (NBD)-phallacidin staining of F-actin. After labeling neutrophils with 32P, exposure to N-formyl peptide induced a fast decrease of phosphatidylinositol 4-bisphosphate (PIP)2, a slow increase of phosphatidic acid, and a rapid rise of phosphatidylinositol 4-trisphosphate (PIP3). Formation of PIP3 as well as actin polymerization was near maximal at 10 s after stimulation. Half-maximal response and PIP3 formation at early time points resulted from stimulation of neutrophils with 0.01 nM N-formyl peptide or occupation of about 200 receptors. Sustained elevation of PIP3, prolonged right angle light scatter response, and F-actin formation required higher concentrations of N-formyl peptide, occupation of thousands of receptors, and high binding rates. When ligand binding was interrupted with an antagonist, F-actin rapidly depolymerized, transient light scatter response recovered immediately, and elevated (32P)PIP3 levels decayed toward initial values. However, recovery of (32P)PIP2 was not influenced by the antagonist. Based on the parallel time courses and dose response of (32P) PIP3, the right angle light scatter response, and F-actin polymerization, PIP3 is more likely than PIP2 to be involved in modulation of actin polymerization and depolymerization in vivo.

  19. Collapsin Response Mediator Protein-1 Regulates Arp2/3-dependent Actin Assembly*

    PubMed Central

    Yu-Kemp, Hui-Chia; Brieher, William M.

    2016-01-01

    Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3-dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions, indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3-dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own, nor does it directly activate the Arp2/3 complex. Rather, CRMP-1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation. PMID:26598519

  20. Comparison Actin- and Glass-Supported Phospholipid Bilayer Diffusion Coefficients

    PubMed Central

    Sterling, Sarah M.; Dawes, Ryan; Allgeyer, Edward S.; Ashworth, Sharon L.; Neivandt, David J.

    2015-01-01

    The formation of biomimetic lipid membranes has the potential to provide insights into cellular lipid membrane dynamics. The construction of such membranes necessitates not only the utilization of appropriate lipids, but also physiologically relevant substrate/support materials. The substrate materials employed have been shown to have demonstrable effects on the behavior of the overlying lipid membrane, and thus must be studied before use as a model cushion support. To our knowledge, we report the formation and investigation of a novel actin protein-supported lipid membrane. Specifically, inner leaflet lateral mobility of globular actin-supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers, deposited via the Langmuir-Blodgett/Langmuir Schaefer methodology, was investigated by z-scan fluorescence correlation spectroscopy across a temperature range of 20–44°C. The actin substrate was found to decrease the diffusion coefficient when compared to an identical membrane supported on glass. The depression of the diffusion coefficient occurred across all measured temperatures. These results indicated that the actin substrate exerted a direct effect on the fluidity of the lipid membrane and highlighted the fact that the choice of substrate/support is critical in studies of model lipid membranes. PMID:25902434

  1. Spatiotemporal regulation of chemical reaction kinetics of cell surface molecules by active remodeling of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Mayor, Satyajit; Rao, Madan

    2010-03-01

    Cell surface proteins such as lipid tethered GPI-anchored proteins and Ras-proteins are distributed as monomers and nanoclusters on the surface of living cells. Recent work from our laboratory suggests that the spatial distribution and dynamics of formation and breakup of these nanoclusters is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we propose a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. Here we study the consequences of such active actin based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that active remodeling of cortical actin, can give rise to a dramatic increase in the reaction efficiency and output levels. In general, such actin driven clustering of membrane proteins could be a cellular mechanism to spatiotemporally regulate and amplify local chemical reaction rates, in the context of signalling and endocytosis.

  2. S-NO-actin: S-nitrosylation kinetics and the effect on isolated vascular smooth muscle.

    PubMed

    Dalle-Donne, I; Milzani, A; Giustarini, D; Di Simplicio, P; Colombo, R; Rossi, R

    2000-02-01

    We describe the modification of reactive actin sulfhydryls by S-nitrosoglutathione. Kinetics of S-nitrosylation and denitrosylation suggest that only one cysteine of actin is involved in the reactions. By using the bifunctional sulfhydryl cross-linking reagent N,N'-1,4-phenylenebismaleimide and the monofunctional reagent N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine, we identified this residue as Cys374. The time course of filament formation followed by high-shear viscosity changes revealed that S-nitrosylated G-actin polymerizes less efficiently than native monomers. The observed decrease in specific viscosity at steady state is due mainly to a marked inhibition of filament end-to-end annealing and, partially, to a reduction in F-actin concentration. Finally, S-nitrosylated actin acts as nitric oxide donor showing a fast, potent vasodilating activity at unusually low concentrations, being comparable with that of low molecular weight nitrosothiols.

  3. Self-organized Gels in DNA/F-Actin mixtures without Crosslinkers

    NASA Astrophysics Data System (ADS)

    Butler, John; Hwee Lai, Ghee; Zribi, Olena; Smalyukh, Ivan; Angelini, Thomas; Purdy, Kirstin; Golestanian, Ramin; Wong, Gerard C. L.

    2009-03-01

    Interactions between flexible chains and rigid rods govern a broad range of soft matter systems. As a model system of like-charged rigid rods and flexible chains, we examine mixtures of DNA and filamentous actin (F-actin). Confocal microscopy reveals the formation of elongated nematic F-actin domains reticulated via defect-free vertices into a network embedded in a mesh of random DNA. Synchrotron small-angle x-ray scattering (SAXS) indicates that the DNA mesh squeezes the F-actin domains into a nematic state with an inter-actin spacing that decreases with increasing DNA concentration. Salt strongly influences the domain sizes and transitions the system from a counterion-controlled regime to a depletion-controlled regime, both mechanisms of which are entropic in origin.

  4. Visualizing Actin Architectures in Cells Incubated with Cell-Penetrating Peptides.

    PubMed

    He, Lin; Watson, Peter D; Jones, Arwyn T

    2015-01-01

    Defining the exact role of the actin cytoskeleton in mediating endocytosis through different pathways is a significant challenge. The general consensus is that actin has an important role in organizing the early stages of endocytosis but there is still much to learn. Actin has also been implicated in cell internalization of cell-penetrating peptides (CPPs). It is suggested that CPP variants such as octaarginine (R8) and the HIV Tat peptide induce actin-dependent plasma membrane perturbation and enter via macropinocytosis. Here, we describe confocal microscopy techniques that allow for high-resolution spatial characterization of the actin cytoskeleton in untreated mammalian cells and those incubated with actin-disrupting agents and CPPs. By performing X-Y-Z projection images through different regions of cells to show basal and apical profiles, we initially highlight how these techniques can be used to reveal major differences in cortical and filamentous actin organization between different cell lines. Using these techniques, we demonstrate that the actin-disrupting agent cytochalasin D rapidly changes this framework at concentrations significantly lower than is normally used. Experiments are also performed to highlight that serum starvation significantly sensitizes cells to the effects of R8 on actin-induced ruffling and lamellapodia formation. The techniques described here can be used to gain a higher level of knowledge of the organization of the actin network in individual model cell systems, how this is perturbed using commonly used actin inhibitors, and how plasma membrane reorganization can be induced by the addition of drug delivery vectors such as CPPs.

  5. Tracer diffusion through F-actin: effect of filament length and cross-linking.

    PubMed Central

    Jones, J D; Luby-Phelps, K

    1996-01-01

    We have determined diffusion coefficients for small (50- to 70-nm diameter) fluorescein-thiocarbamoyl-labeled Ficoll tracers through F-actin as a function of filament length and cross-linking. fx45 was used to regulate filament length and avidin/biotinylated actin or ABP-280 was used to prepare cross-linked actin gels. We found that tracer diffusion was generally independent of filament length in agreement with theoretical predictions for diffusion through solutions of rods. However, in some experiments diffusion was slower through short (< or = 1.0 micron) filaments, although this result was not consistently reproducible. Measured diffusion coefficients through unregulated F-actin and filaments of lengths > 1.0 micron were more rapid than predicted by theory for tracer diffusion through rigid, random networks, which was consistent with some degree of actin bundling. Avidin-induced cross-linking of biotinylated F-actin did not affect diffusion through unregulated F-actin, but in cases where diffusion was slower through short filaments this cross-linking method resulted in enhanced tracer diffusion rates indistinguishable from unregulated F-actin. This finding, in conjunction with increased turbidity of 1.0-micron filaments upon avidin cross-linking, indicated that this cross-linking method induces F-actin bundling. By contrast, ABP-280 cross-linking retarded diffusion through unregulated F-actin and decreased turbidity. Tracer diffusion under these conditions was well approximated by the diffusion theory. Both cross-linking procedures resulted in gel formation as determined by falling ball viscometry. These results demonstrate that network microscopic geometry is dependent on the cross-linking method, although both methods markedly increase F-actin macroscopic viscosity. PMID:8913611

  6. Disease-associated mutant alpha-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity.

    PubMed

    Weins, Astrid; Schlondorff, Johannes S; Nakamura, Fumihiko; Denker, Bradley M; Hartwig, John H; Stossel, Thomas P; Pollak, Martin R

    2007-10-09

    Alpha-actinin-4 is a widely expressed protein that employs an actin-binding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca(2+)-sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the alpha-actinin-4 actin-binding domain. Here we show that alpha-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant alpha-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca(2+) regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant alpha-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT alpha-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of alpha-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of alpha-actinin and possibly other calponin homology domain proteins under physiological conditions.

  7. Flat conductor cable design, manufacture, and installation

    NASA Technical Reports Server (NTRS)

    Angele, W.; Hankins, J. D.

    1973-01-01

    Pertinent information for hardware selection, design, manufacture, and quality control necessary for flat conductor cable interconnecting harness application is presented. Comparisons are made between round wire cable and flat conductor cable. The flat conductor cable interconnecting harness systems show major cost, weight, and space savings, plus increased system performance and reliability. The design application section includes electrical characteristics, harness design and development, and a full treatise on EMC considerations. Manufacturing and quality control sections pertain primarily to the developed conductor-contact connector system and special flat conductor cable to round wire cable transitions.

  8. Contractile properties of thin (actin) filament-reconstituted muscle fibers.

    PubMed

    Ishiwata, S; Funatsu, T; Fujita, H

    1998-01-01

    Selective removal and reconstitution of the components of muscle fibers (fibrils) is a useful means of examining the molecular mechanism underlying the formation of the contractile apparatus. In addition, this approach is powerful for examining the structure-function relationship of a specific component of the contractile system. In previous studies, we have achieved the partial structural and functional reconstitution of thin filaments in the skeletal contractile apparatus and full reconstitution in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with plasma gelsolin, an actin filament-severing protein. Under these conditions, no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted by polymerization on the short actin fragments remaining at the Z line, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached as high as 30% of the original level in skeletal muscle, while it reached 140% in cardiac muscle. The augmentation of tension in cardiac muscle is mainly attributable to the elongation of reconstituted filaments, longer than the average length of thin filaments in an intact muscle. These results indicate that a muscle contractile apparatus with a high order structure and function can be constructed by the self-assembly of constituent proteins. Recently, we applied this reconstitution system to the study of the mechanism of spontaneous oscillatory contraction (SPOC) in thin (actin) filament-reconstituted cardiac muscle fibers. As a result, we found that SPOC occurs even in regulatory protein-free actin filament-reconstituted fibers (Fujita & Ishiwata, manuscript submitted), although the SPOC conditions were slightly different from the standard SPOC conditions. This result strongly suggests that spontaneous oscillation

  9. Nuclear Actin in Development and Transcriptional Reprogramming.

    PubMed

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin's roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation.

  10. Femtosecond pump-probe studies of actinic-wavelength dependence in aqueous chlorine dioxide photochemistry

    SciTech Connect

    Bixby, Teresa J.; Bolinger, Joshua C.; Patterson, Joshua D.; Reid, Philip J.

    2009-04-21

    The actinic or photolysis-wavelength dependence of aqueous chlorine dioxide (OClO) photochemistry is investigated using femtosecond pump-probe spectroscopy. Following photoexcitation at 310, 335, and 410 nm the photoinduced evolution in optical density is measured from the UV to the near IR. Analysis of the optical-density evolution illustrates that the quantum yield for atomic chlorine production ({Phi}{sub Cl}) increases with actinic energy, with {Phi}{sub Cl}=0.16{+-}0.02 for 410 nm excitation and increasing to 0.25{+-}0.01 and 0.54{+-}0.10 for 335 and 310 nm excitations, respectively. Consistent with previous studies, the production of Cl occurs through two channels, with one channel corresponding to prompt (<5 ps) Cl formation and the other corresponding to the thermal decomposition of ClOO formed by OClO photoisomerization. The partitioning between Cl production channels is dependent on actinic energy, with prompt Cl production enhanced with an increase in actinic energy. Limited evidence is found for enhanced ClO production with an increase in actinic energy. Stimulated emission and excited-state absorption features associated with OClO populating the optically prepared {sup 2}A{sub 2} surface decrease with an increase in actinic energy suggesting that the excited-state decay dynamics are also actinic energy dependent. The studies presented here provide detailed information on the actinic-wavelength dependence of OClO photochemistry in aqueous solution.

  11. Femtosecond pump-probe studies of actinic-wavelength dependence in aqueous chlorine dioxide photochemistry

    NASA Astrophysics Data System (ADS)

    Bixby, Teresa J.; Bolinger, Joshua C.; Patterson, Joshua D.; Reid, Philip J.

    2009-04-01

    The actinic or photolysis-wavelength dependence of aqueous chlorine dioxide (OClO) photochemistry is investigated using femtosecond pump-probe spectroscopy. Following photoexcitation at 310, 335, and 410 nm the photoinduced evolution in optical density is measured from the UV to the near IR. Analysis of the optical-density evolution illustrates that the quantum yield for atomic chlorine production (ΦCl) increases with actinic energy, with ΦCl=0.16±0.02 for 410 nm excitation and increasing to 0.25±0.01 and 0.54±0.10 for 335 and 310 nm excitations, respectively. Consistent with previous studies, the production of Cl occurs through two channels, with one channel corresponding to prompt (<5 ps) Cl formation and the other corresponding to the thermal decomposition of ClOO formed by OClO photoisomerization. The partitioning between Cl production channels is dependent on actinic energy, with prompt Cl production enhanced with an increase in actinic energy. Limited evidence is found for enhanced ClO production with an increase in actinic energy. Stimulated emission and excited-state absorption features associated with OClO populating the optically prepared A22 surface decrease with an increase in actinic energy suggesting that the excited-state decay dynamics are also actinic energy dependent. The studies presented here provide detailed information on the actinic-wavelength dependence of OClO photochemistry in aqueous solution.

  12. Nuclear actin depolymerization in transcriptionally active avian and amphibian oocytes leads to collapse of intranuclear structures

    PubMed Central

    Maslova, Antonina; Krasikova, Alla

    2012-01-01

    Actin, which is normally depleted in the nuclei of somatic cells, accumulates in high amounts in giant nuclei of amphibian oocytes. The supramolecular organization and functions of this nuclear pool of actin in growing vertebrate oocyte are controversial. Here, we investigated the role of nuclear actin in the maintenance of the spatial architecture of intranuclear structures in avian and amphibian growing oocytes. A meshwork of filamentous actin was not detected in freshly isolated or fixed oocyte nuclei of Xenopus, chicken or quail. We found that the actin meshwork inside the oocyte nucleus could be induced by phalloidin treatment. Actin polymerization is demonstrated to be required to stabilize the specific spatial organization of nuclear structures in avian and amphibian growing oocytes. In experiments with the actin depolymerizing drugs cytochalasin D and latrunculin A, we showed that disassembly of nuclear actin polymers led to chromosome condensation and their transportation to a limited space within the oocyte nucleus. Experimentally induced “collapsing” of chromosomes and nuclear bodies, together with global inhibition of transcription, strongly resembled the process of karyosphere formation during oocyte growth. PMID:22572951

  13. A posttranslational modification of beta-actin contributes to the slow dissociation of the spectrin-protein 4.1-actin complex of irreversibly sickled cells

    PubMed Central

    1995-01-01

    Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37 degrees C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in beta-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control beta-actin is the inaccessibility of two cysteine residues in ISC beta-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin, or alternatively another modification of cysteine284 and cysteine373 which is reversible with DTT and adds less than 100 D to the molecular weight of beta-actin. PMID:7876306

  14. Actin Dynamics: From Nanoscale to Microscale

    PubMed Central

    Carlsson, Anders E.

    2010-01-01

    The dynamic nature of actin in cells manifests itself in many ways: Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of the cell periphery are frequently seen. I discuss how mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes. The rate of actin incorporation at the leading edge of a moving cell is roughly consistent with existing theories, and the factors determining the characteristic time of actin polymerization are fairly well understood. However, our understanding of actin disassembly is limited, in particular the interplay between severing and depolymerization and the role of specific combinations of proteins in implementing disassembly events. The origins of cell-edge oscillations, and their possible relation to actin waves, are a fruitful area of future research. PMID:20462375

  15. Treponema denticola Major Outer Sheath Protein Induces Actin Assembly at Free Barbed Ends by a PIP2-Dependent Uncapping Mechanism in Fibroblasts

    PubMed Central

    Visser, Michelle B.; Koh, Adeline; Glogauer, Michael; Ellen, Richard P.

    2011-01-01

    The major outer sheath protein (Msp) of Treponema denticola perturbs actin dynamics in fibroblasts by inducing actin reorganization, including subcortical actin filament assembly, leading to defective calcium flux, diminished integrin engagement of collagen, and retarded cell migration. Yet, its mechanisms of action are unknown. We challenged Rat-2 fibroblasts with enriched native Msp. Msp activated the small GTPases Rac1, RhoA and Ras, but not Cdc42, yet only Rac1 localized to areas of actin rearrangement. We used Rac1 dominant negative transfection and chemical inhibition of phosphatidylinositol-3 kinase (PI3K) to show that even though Rac1 activation was PI3K-dependent, neither was required for Msp-induced actin rearrangement. Actin free barbed end formation (FBE) by Msp was also PI3K-independent. Immunoblotting experiments showed that gelsolin and CapZ were released from actin filaments, whereas cofilin remained in an inactive state. Msp induced phosphatidylinositol (4,5)-bisphosphate (PIP2) formation through activation of a phosphoinositide 3-phosphatase and its recruitment to areas of actin assembly at the plasma membrane. Using a PIP2 binding peptide or lipid phosphatase inhibitor, PIP2 was shown to be required for Msp-mediated actin uncapping and FBE formation. Evidently, Msp induces actin assembly in fibroblasts by production and recruitment of PIP2 and release of the capping proteins CapZ and gelsolin from actin barbed ends. PMID:21901132

  16. Cortical flow aligns actin filaments to form a furrow

    PubMed Central

    Reymann, Anne-Cecile; Staniscia, Fabio; Erzberger, Anna; Salbreux, Guillaume; Grill, Stephan W

    2016-01-01

    Cytokinesis in eukaryotic cells is often accompanied by actomyosin cortical flow. Over 30 years ago, Borisy and White proposed that cortical flow converging upon the cell equator compresses the actomyosin network to mechanically align actin filaments. However, actin filaments also align via search-and-capture, and to what extent compression by flow or active alignment drive furrow formation remains unclear. Here, we quantify the dynamical organization of actin filaments at the onset of ring assembly in the C. elegans zygote, and provide a framework for determining emergent actomyosin material parameters by the use of active nematic gel theory. We characterize flow-alignment coupling, and verify at a quantitative level that compression by flow drives ring formation. Finally, we find that active alignment enhances but is not required for ring formation. Our work characterizes the physical mechanisms of actomyosin ring formation and highlights the role of flow as a central organizer of actomyosin network architecture. DOI: http://dx.doi.org/10.7554/eLife.17807.001 PMID:27719759

  17. Early nucleation events in the polymerization of actin, probed by time-resolved small-angle x-ray scattering

    PubMed Central

    Oda, Toshiro; Aihara, Tomoki; Wakabayashi, Katsuzo

    2016-01-01

    Nucleators generating new F-actin filaments play important roles in cell activities. Detailed information concerning the events involved in nucleation of actin alone in vitro is fundamental to understanding these processes, but such information has been hard to come by. We addressed the early process of salt-induced polymerization of actin using the time-resolved synchrotron small-angle X-ray scattering (SAXS). Actin molecules in low salt solution maintain a monomeric state by an electrostatic repulsive force between molecules. On mixing with salts, the repulsive force was rapidly screened, causing an immediate formation of many of non-polymerizable dimers. SAXS kinetic analysis revealed that tetramerization gives the highest energetic barrier to further polymerization, and the major nucleation is the formation of helical tetramers. Filaments start to grow rapidly with the formation of pentamers. These findings suggest an acceleration mechanism of actin assembly by a variety of nucleators in cells. PMID:27775032

  18. Characterization of hyaluronan cable structure and function in renal proximal tubular epithelial cells.

    PubMed

    Selbi, W; de la Motte, C A; Hascall, V C; Day, A J; Bowen, T; Phillips, A O

    2006-10-01

    Alteration in the glycosaminoglycan hyaluronan (HA) has been demonstrated in numerous renal diseases. We have demonstrated that renal proximal tubular epithelial cells (PTCs) surround themselves in vitro with HA in an organized pericellular matrix or 'coat', which is associated with cell migration, and also form pericellular HA cable-like structures which modulate PTC-mononuclear leukocytes interactions. The aim of this study was to characterize potential regulatory mechanism in the assembly of PTC-HA into pericellular cables. HA cables are generated by PTCs in the absence of serum. Immunohistochemical analysis demonstrates the incorporation of components of the inter-alpha-inhibitor (IalphaI) family of proteins and versican into HA cables. Addition of an antibody to IalphaI/PalphaI (pre-alpha-inhibitor) inhibits cable formation. In contrast, inhibition of tumor necrosis factor-alpha-stimulated gene 6 (TSG-6) has no effect on cable formation, suggesting that their generation is independent of the known heavy-chain transfer activity of TSG-6. Overexpression of HAS3 is associated with induction of HA cable formation, and also increased incorporation of HA into pericellular coats. Functionally, this resulted in enhanced HA-dependent monocyte binding and cell migration, respectively. Cell surface expression of CD44 and trypsin-released cell-associated HA were increased in HAS3-overexpressing cells. In addition, hyaluronidase (hyal1 and hyal2) and bikunin mRNA expression were increased, whereas PalphaI HC3 mRNA expression was unchanged in the transfected cells. The data demonstrate the importance of IalphaI/PalphaI in cable formation and suggest that expression of HAS3 may be critical for HA cable assembly.

  19. Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments[W

    PubMed Central

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca2+, in vitro. Analysis of a mutant that bears a point mutation at the Ca2+ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca2+ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth. PMID:24424096

  20. Rapid severing and motility of chloroplast-actin filaments are required for the chloroplast avoidance response in Arabidopsis.

    PubMed

    Kong, Sam-Geun; Arai, Yoshiyuki; Suetsugu, Noriyuki; Yanagida, Toshio; Wada, Masamitsu

    2013-02-01

    Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light-induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light's intensity and position.

  1. Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments.

    PubMed

    Jiwani, Shahanawaz; Alvarado, Stephenie; Ohr, Ryan J; Romero, Adriana; Nguyen, Brenda; Jewett, Travis J

    2013-02-01

    All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells.

  2. Umbilical cable recovery load analysis

    NASA Astrophysics Data System (ADS)

    Yan, Shu-wang; Jia, Zhao-lin; Feng, Xiao-wei; Li, Shi-tao

    2013-06-01

    Umbilical cable is a kind of integrated subsea cable widely used in the exploration and exploitation of oil and gas field. The severe ocean environment makes great challenges to umbilical maintenance and repair work. Damaged umbilical is usually recovered for the regular operation of the offshore production system. Analysis on cables in essence is a two-point boundary problem. The tension load at the mudline must be known first, and then the recovery load and recovery angle on the vessel can be solved by use of catenary equation. The recovery analysis also involves umbilical-soil interaction and becomes more complicated. Calculation methods for recovery load of the exposed and buried umbilical are established and the relationship between the position of touch down point and the recovery load as well as the recovery angle and recovery load are analyzed. The analysis results provide a theoretical reference for offshore on-deck operation.

  3. A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton

    PubMed Central

    Janssen, Erin; Tohme, Mira; Hedayat, Mona; Leick, Marion; Kumari, Sudha; Ramesh, Narayanaswamy; Massaad, Michel J.; Ullas, Sumana; Azcutia, Veronica; Goodnow, Christopher C.; Randall, Katrina L.; Qiao, Qi; Wu, Hao; Al-Herz, Waleed; Cox, Dianne; Hartwig, John; Irvine, Darrell J.; Luscinskas, Francis W.; Geha, Raif S.

    2016-01-01

    Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor–driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency. PMID:27599296

  4. Small GTPase Rab21 mediates fibronectin induced actin reorganization in Entamoeba histolytica: implications in pathogen invasion.

    PubMed

    Emmanuel, Merlyn; Nakano, Yumiko Saito; Nozaki, Tomoyoshi; Datta, Sunando

    2015-03-01

    The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM) plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as 'invadosomes', promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots.

  5. CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization.

    PubMed

    Shabtay, Ortal; Breitbart, Haim

    2016-07-01

    In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR.

  6. WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

    PubMed Central

    Havrylenko, Svitlana; Noguera, Philippe; Abou-Ghali, Majdouline; Manzi, John; Faqir, Fahima; Lamora, Audrey; Guérin, Christophe; Blanchoin, Laurent; Plastino, Julie

    2015-01-01

    The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly. PMID:25355952

  7. The interaction between the adaptor protein APS and Enigma is involved in actin organisation.

    PubMed

    Barrès, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2005-08-15

    APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.

  8. Cable Television: From Here to Where?

    ERIC Educational Resources Information Center

    Canadian Cable Television Association, Ottawa (Ontario).

    A brief description is presented of the present uses, regulatory structure, and future potentials of cable television for Canada. Some 30% of Canada is already wired for cable, as opposed to 9% in the United States. (RH)

  9. Debris protection cover assembly for cable connectors

    NASA Technical Reports Server (NTRS)

    Yovan, Roger D. (Inventor)

    1999-01-01

    A protective cover assembly for an end of a cable connector having a cable housing that encloses a plurality of connective pins or sockets and that satisfies all requirements for space applications. A connector body flange is formed at the extremity of a cable and is positioned so that it may register with a corresponding connector body flange on the end of a companion cable to which a connection is to be made, one cable end having cable lead pins and the companion cable end having lead sockets with which the pins register. A latch mechanism having a latch housing is received in the connector body flange and a crank connected to a manually rotatable cap actuates a spring-loaded latch element that is engageable with a connector body flange to secure or to release the cover assembly with the simple twisting motion of the cap, thereby simplifying the task of effecting coupling and decoupling of the cable ends.

  10. Ames Lab 101: Reinventing the Power Cable

    SciTech Connect

    Russell, Alan

    2013-09-27

    Ames Laboratory researchers are working to develop new electrical power cables that are stronger and lighter than the cables currently used in the nation's power grid. Nano Tube animation by Iain Goodyear

  11. 30 CFR 18.45 - Cable reels.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ..., hydraulically, or electrically driven reel upon which to wind the portable cable. (b) The enclosure for moving... of travel of a machine when receiving power through a portable (trailing) cable shall not exceed...

  12. 30 CFR 18.45 - Cable reels.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., hydraulically, or electrically driven reel upon which to wind the portable cable. (b) The enclosure for moving... of travel of a machine when receiving power through a portable (trailing) cable shall not exceed...

  13. Folding tools for flat conductor cable harnesses

    NASA Technical Reports Server (NTRS)

    Loggins, R.

    1971-01-01

    Vise grip pliers have detachable metal gripping plates which are changed to accommodate cables from 1 to 3 in. wide and to form any desired fold angle. A second tool squeezes cable along crease to complete the fold.

  14. New sensitive seismic cable with imbedded geophones

    NASA Astrophysics Data System (ADS)

    Pakhomov, Alex; Pisano, Dan; Goldburt, Tim

    2005-10-01

    Seismic detection systems for homeland security applications are an important additional layer to perimeter and border protection and other security systems. General Sensing Systems has been developing low mass, low cost, highly sensitive geophones. These geophones are being incorporated within a seismic cable. This article reports on the concept of a seismic sensitive cable and seismic sensitive ribbon design. Unlike existing seismic cables with sensitivity distributed along their lengths, the GSS new cable and ribbon possesses high sensitivity distributed in several points along the cable/ribbon with spacing of about 8-12 to 100 meters between geophones. This cable/ribbon is highly suitable for design and installation in extended perimeter protection systems. It allows the use of a mechanical cable layer for high speed installation. We show that any installation mistakes in using the GSS seismic sensitive cable/ribbon have low impact on output seismic signal value and detection range of security systems.

  15. Ames Lab 101: Reinventing the Power Cable

    ScienceCinema

    Russell, Alan

    2016-07-12

    Ames Laboratory researchers are working to develop new electrical power cables that are stronger and lighter than the cables currently used in the nation's power grid. Nano Tube animation by Iain Goodyear

  16. 30 CFR 7.407 - Test for flame resistance of electric cables and cable splices.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) at an ambient temperature of 104 °F (40 °C). (8) Monitor the electric current through the power... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Test for flame resistance of electric cables... Electric Cables, Signaling Cables, and Cable Splice Kits § 7.407 Test for flame resistance of...

  17. 30 CFR 7.407 - Test for flame resistance of electric cables and cable splices.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...) at an ambient temperature of 104 °F (40 °C). (8) Monitor the electric current through the power... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Test for flame resistance of electric cables... Electric Cables, Signaling Cables, and Cable Splice Kits § 7.407 Test for flame resistance of...

  18. 30 CFR 7.407 - Test for flame resistance of electric cables and cable splices.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...) at an ambient temperature of 104 °F (40 °C). (8) Monitor the electric current through the power... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Test for flame resistance of electric cables... Electric Cables, Signaling Cables, and Cable Splice Kits § 7.407 Test for flame resistance of...

  19. 30 CFR 7.407 - Test for flame resistance of electric cables and cable splices.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Test for flame resistance of electric cables... Electric Cables, Signaling Cables, and Cable Splice Kits § 7.407 Test for flame resistance of electric... to reduce contact resistance. (7) Energize all power conductors of the test specimen with...

  20. 30 CFR 7.407 - Test for flame resistance of electric cables and cable splices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Test for flame resistance of electric cables... Electric Cables, Signaling Cables, and Cable Splice Kits § 7.407 Test for flame resistance of electric... to reduce contact resistance. (7) Energize all power conductors of the test specimen with...

  1. 47 CFR 76.640 - Support for unidirectional digital cable products on digital cable systems.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 4 2013-10-01 2013-10-01 false Support for unidirectional digital cable products on digital cable systems. 76.640 Section 76.640 Telecommunication FEDERAL COMMUNICATIONS... Standards § 76.640 Support for unidirectional digital cable products on digital cable systems. (a)...

  2. 47 CFR 76.640 - Support for unidirectional digital cable products on digital cable systems.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 4 2014-10-01 2014-10-01 false Support for unidirectional digital cable products on digital cable systems. 76.640 Section 76.640 Telecommunication FEDERAL COMMUNICATIONS... Standards § 76.640 Support for unidirectional digital cable products on digital cable systems. (a)...

  3. PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins

    PubMed Central

    Leyton-Puig, Daniela; Kedziora, Katarzyna M.; Isogai, Tadamoto; van den Broek, Bram; Jalink, Kees

    2016-01-01

    ABSTRACT Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible. Here, we show that proper paraformaldehyde (PFA)-based sample preparation preserves the architecture of the actin cytoskeleton almost as faithfully as gold-standard glutaraldehyde fixation. We show that this fixation is essential for proper immuno-based localization of actin-binding and actin-regulatory proteins involved in the formation of lamellipodia and ruffles, such as mDia1, WAVE2 and clathrin heavy chain, and provide detailed guidelines for the execution of our method. In summary, proper PFA-based sample preparation increases the multi-color possibilities and the reproducibility of SRM of the actin cytoskeleton and its associated proteins. PMID:27378434

  4. 47 CFR 76.990 - Small cable operators.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 4 2010-10-01 2010-10-01 false Small cable operators. 76.990 Section 76.990... CABLE TELEVISION SERVICE Cable Rate Regulation § 76.990 Small cable operators. (a) Effective February 8, 1996, a small cable operator is exempt from rate regulation on its cable programming services tier,...

  5. 47 CFR 76.990 - Small cable operators.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 4 2011-10-01 2011-10-01 false Small cable operators. 76.990 Section 76.990... CABLE TELEVISION SERVICE Cable Rate Regulation § 76.990 Small cable operators. (a) Effective February 8, 1996, a small cable operator is exempt from rate regulation on its cable programming services tier,...

  6. Online Cable Tester and Rerouter

    NASA Technical Reports Server (NTRS)

    Lewis, Mark; Medelius, Pedro

    2012-01-01

    Hardware and algorithms have been developed to transfer electrical power and data connectivity safely, efficiently, and automatically from an identified damaged/defective wire in a cable to an alternate wire path. The combination of online cable testing capabilities, along with intelligent signal rerouting algorithms, allows the user to overcome the inherent difficulty of maintaining system integrity and configuration control, while autonomously rerouting signals and functions without introducing new failure modes. The incorporation of this capability will increase the reliability of systems by ensuring system availability during operations.

  7. Equalization of data transmission cable

    NASA Technical Reports Server (NTRS)

    Zobrist, G. W.

    1975-01-01

    The paper describes an equalization approach utilizing a simple RLC network which can obtain a maximum slope of -12dB/octave for reshaping the frequency characteristics of a data transmission cable, so that data may be generated and detected at the receiver. An experimental procedure for determining equalizer design specifications using distortion analysis is presented. It was found that for lengths of 16 PEV-L cable of up to 5 miles and data transmission rates of up to 1 Mbs, the equalization scheme proposed here is sufficient for generation of the data with acceptable error rates.

  8. Membrane Tension Acts Through PLD2 and mTORC2 to Limit Actin Network Assembly During Neutrophil Migration

    PubMed Central

    Diz-Muñoz, Alba; Thurley, Kevin; Chintamen, Sana; Altschuler, Steven J.; Fletcher, Daniel A.; Weiner, Orion D.

    2016-01-01

    For efficient polarity and migration, cells need to regulate the magnitude and spatial distribution of actin assembly. This process is coordinated by reciprocal interactions between the actin cytoskeleton and mechanical forces. Actin polymerization-based protrusion increases tension in the plasma membrane, which in turn acts as a long-range inhibitor of actin assembly. These interactions form a negative feedback circuit that limits the magnitude of membrane tension in neutrophils and prevents expansion of the existing front and the formation of secondary fronts. It has been suggested that the plasma membrane directly inhibits actin assembly by serving as a physical barrier that opposes protrusion. Here we show that efficient control of actin polymerization-based protrusion requires an additional mechanosensory feedback cascade that indirectly links membrane tension with actin assembly. Specifically, elevated membrane tension acts through phospholipase D2 (PLD2) and the mammalian target of rapamycin complex 2 (mTORC2) to limit actin nucleation. In the absence of this pathway, neutrophils exhibit larger leading edges, higher membrane tension, and profoundly defective chemotaxis. Mathematical modeling suggests roles for both the direct (mechanical) and indirect (biochemical via PLD2 and mTORC2) feedback loops in organizing cell polarity and motility—the indirect loop is better suited to enable competition between fronts, whereas the direct loop helps spatially organize actin nucleation for efficient leading edge formation and cell movement. This circuit is essential for polarity, motility, and the control of membrane tension. PMID:27280401

  9. Hawaii Deep Water Cable Program: Executive Summary

    SciTech Connect

    1990-09-01

    The Hawaii Deep Water Cable Program has succeeded unequivocally in determining the feasibility of deploying a submarine power cable system between the islands of Hawaii and Oahu. Major accomplishments of the program include designing, fabricating and testing an appropriate power cable, developing an integrated system to control all aspects of the cable laying operation, and testing all deployment systems at sea in the most challenging sections of the route.

  10. NEMA wire and cable standards development programs

    NASA Technical Reports Server (NTRS)

    Baird, Robert W.

    1994-01-01

    The National Electrical Manufacturers Association (NEMA) is the nation's largest trade association for manufacturers of electrical equipment. Its member companies produce components, end-use equipment and systems for the generation, transmission, distribution, control and use of electricity. The wire and cable division is presented in 6 sections: building wire and cable, fabricated conductors, flexible cords, high performance wire and cable, magnet wire, and power and control cable. Participating companies are listed.

  11. Open Source Cable Models for EMI Simulations

    NASA Astrophysics Data System (ADS)

    Greedy, S.; Smartt, C.; Thomas, D. W. P.

    2016-05-01

    This paper describes the progress of work towards an Open Source software toolset suitable for developing Spice based multi-conductor cable models. The issues related to creating a transmission line model for implementation in Spice which include the frequency dependent properties of real cables are presented and the viability of spice cable models is demonstrated through application to a three conductor crosstalk model. Development of the techniques to include models of shielded cables and incident field excitation has been demonstrated.

  12. Labeling F-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones.

    PubMed

    Marsick, Bonnie M; Letourneau, Paul C

    2011-03-17

    The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant. Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells. When rhodamine-actin is added at a concentration above the critical concentration

  13. Nuclear Actin in Development and Transcriptional Reprogramming

    PubMed Central

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin’s roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation. PMID:28326098

  14. Supervillin Reorganizes the Actin Cytoskeleton and Increases Invadopodial Efficiency

    PubMed Central

    Crowley, Jessica L.; Smith, Tara C.; Fang, Zhiyou; Takizawa, Norio

    2009-01-01

    Tumor cells use actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II, reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV induces redistribution of lamellipodial cortactin and lamellipodin/RAPH1/PREL1 away from the cell periphery to internal sites and concomitantly increases the numbers of F-actin punctae. Most punctae are highly dynamic and colocalize with the podosome/invadopodial proteins, cortactin, Tks5, and cdc42. Cortactin binds SV sequences in vitro and contributes to the formation of enhanced green fluorescent protein (EGFP)-SV induced punctae. SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases average numbers of ECM holes per cell; RNA interference-mediated knockdown of SV decreases these numbers. Although SV knockdown alone has no effect, simultaneous down-regulation of SV and the closely related protein gelsolin reduces invasion through ECM. Together, our results show that SV is a component of podosomes and invadopodia and that SV plays a role in invadopodial function, perhaps as a mediator of cortactin localization, activation state, and/or dynamics of metalloproteinases at the ventral cell surface. PMID:19109420

  15. Actin, microvilli, and the fertilization cone of sea urchin eggs

    PubMed Central

    1980-01-01

    Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges. PMID:6893988

  16. Coronin 3 involvement in F-actin-dependent processes at the cell cortex

    SciTech Connect

    Rosentreter, Andre; Hofmann, Andreas; Xavier, Charles-Peter; Stumpf, Maria; Noegel, Angelika A.; Clemen, Christoph S. . E-mail: christoph.clemen@uni-koeln.de

    2007-03-10

    The actin interaction of coronin 3 has been mainly documented by in vitro experiments. Here, we discuss coronin 3 properties in the light of new structural information and focus on assays that reflect in vivo roles of coronin 3 and its impact on F-actin-associated functions. Using GFP-tagged coronin 3 fusion proteins and RNAi silencing we show that coronin 3 has roles in wound healing, protrusion formation, cell proliferation, cytokinesis, endocytosis, axonal growth, and secretion. During formation of cell protrusions actin accumulation precedes the focal enrichment of coronin 3 suggesting a role for coronin 3 in events that follow the initial F-actin assembly. Moreover, we show that coronin 3 similar to other coronins interacts with the Arp2/3-complex and cofilin indicating that this family in general is involved in regulating Arp2/3-mediated events.

  17. Dynamic Termination On Radiating Coaxial Cable

    NASA Technical Reports Server (NTRS)

    Lombardi, Robert; Stern, Jon; Rassweiler, George

    1993-01-01

    Radiation pattern dithered to reduce adverse effect of nulls. In improved system for radio communication between base station and portable units within building, tunnel, ship, or other large structure, radiating or "leaky" coaxial cable serves as base-station antenna, and radiation pattern of cable dithered by dithering impedance of termination at end of cable remote from base station.

  18. Your Personal Genie in the Cable.

    ERIC Educational Resources Information Center

    Schlafly, Hubert J.

    The technology necessary for the use of cable television (TV) has been invented; it simply must be put to use. By the 1970's, cable TV should be commonplace in this country. Its rapid growth was caused in part by its appearance at a time of explosive expansion of related technologies like data theory and computer design. The coaxial cable system…

  19. Cable: Report to the President, 1974.

    ERIC Educational Resources Information Center

    Office of Telecommunications Policy, Washington, DC.

    A comprehensive, new national policy for cable communications is recommended by the Cabinet Committee on Cable Communications. The goal of the policy is to achieve the orderly integration of cable with other existing communications media so that information may flow freely, protected from both private and governmental barriers. The first two…

  20. 47 CFR 32.2422 - Underground cable.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Underground cable. 32.2422 Section 32.2422 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES UNIFORM SYSTEM OF ACCOUNTS..., Buried Cable. (d) The cost of cables leading from the main distributing frame or equivalent to...

  1. 47 CFR 32.2421 - Aerial cable.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... the original cost of single or paired conductor cable, wire and other associated material used in... cable or aerial wire as well as the cost of other material used in construction of such plant... cost of optical fiber cable and other associated material used in constructing a physical path for...

  2. The putative pocket protein binding site of Autographa californica nucleopolyhedrovirus BV/ODV-C42 is required for virus-induced nuclear actin polymerization.

    PubMed

    Li, Kun; Wang, Yun; Bai, Huimin; Wang, Qian; Song, Jianhua; Zhou, Yuan; Wu, Chunchen; Chen, Xinwen

    2010-08-01

    Nuclear filamentous actin (F-actin) is essential for nucleocapsid morphogenesis of lepidopteran nucleopolyhedroviruses. Previously, we had demonstrated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-C42 (C42) is involved in nuclear actin polymerization by recruiting P78/83, an AcMNPV orf9-encoded N-WASP homology protein that is capable of activating an actin-related-protein 2/3 (Arp2/3) complex to initiate actin polymerization, to the nucleus. To further investigate the role of C42 in virus-induced actin polymerization, the recombinant bacmid vAc(p78/83nls-gfp), with a c42 knockout, p78/83 tagged with a nuclear localization signal coding sequence, and egfp as a reporter gene under the control of the Pp10 promoter, was constructed and transfected to Sf9 cells. In the nuclei of vAc(p78/83nls-gfp)-transfected cells, polymerized F-actin filaments were absent, whereas other actin polymerization elements (i.e., P78/83, G-actin, and Arp2/3 complex) were present. This in vivo evidence indicated that C42 actively participates in the nuclear actin polymerization process as a key element, besides its role in recruiting P78/83 to the nucleus. In order to collect in vitro evidence for the participation of C42 in actin polymerization, an anti-C42 antibody was used to neutralize the viral nucleocapsid, which is capable of initiating actin polymerization in vitro. Both the kinetics of pyrene-actin polymerization and F-actin-specific staining by phalloidin indicated that anti-C42 can significantly attenuate the efficiency of F-actin formation compared to that with control antibodies. Furthermore, we have identified the putative pocket protein binding sequence (PPBS) on C42 that is essential for C42 to exert its function in nuclear actin polymerization.

  3. Actin dynamics in mouse fibroblasts in microgravity

    NASA Astrophysics Data System (ADS)

    Moes, Maarten J. A.; Bijvelt, Jose J.; Boonstra, Johannes

    2007-09-01

    After stimulating with the growth factor PDGF, cells exhibit abundant membrane ruffling and other morphological changes under normal gravity conditions. These morphological changes are largely determined by the actin microfilament system. Now these actin dynamics were studied under microgravity conditions in mouse fibroblasts during the DELTA mission. The aim of the present study was to describe the actin morphology in detail, to establish the effect of PDGF on actin morphology and to study the role of several actin-interacting proteins involved in introduced actin dynamics in microgravity. Identical experiments were conducted at 1G on earth as a reference. No results in microgravity were obtained due to a combination of malfunctioning hardware and unfulfilled temperature requirements.

  4. The actin cytoskeleton in endothelial cell phenotypes

    PubMed Central

    Prasain, Nutan; Stevens, Troy

    2009-01-01

    Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell’s interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but interrelated structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes. PMID:19028505

  5. Polymerization of actin by positively charged liposomes

    PubMed Central

    1988-01-01

    By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N- pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts. PMID:3360852

  6. Cable Television: Developing Community Services.

    ERIC Educational Resources Information Center

    Carpenter-Huffman, Polly; And Others

    The final volume of a four-volume study focuses on community use of cable television systems. Four separate aspects are discussed extensively: the possibilities of public access, use in municipal service applications, uses in education, and a guide for education planners. Each section contains several appendixes and the education sections include…

  7. Educational Uses of Cable Television.

    ERIC Educational Resources Information Center

    Cable Television Information Center, Washington, DC.

    The different educational uses of cable television as well as the methods and problems of that use are described in a state of the art review. The Federal Communications Commission regulations and related franchise activity are described, and the methods of using the educational channel as open or closed circuit TV or pay TV are indicated for…

  8. Optical Fiber Communications Cable Connector.

    DTIC Science & Technology

    1981-07-01

    incorp- oration of the TRW Cinch Optalign 4 double elbow " fiber alignment guide concept. Means for connecting either Siecor or ITT six fiber cable were...the guide, and forced toward the top cusp by the double elbow con- figuration. The geometry of the guide is such that normal tolerances of molded or

  9. Cable Television and Satellite Broadcasting

    DTIC Science & Technology

    1990-05-31

    a TV set from manufacturer A of the interconnection agreement negotiated with Mer- can talk to a VCR from manufacturer B and a compact cury or...Broadcasting Structure for the Next Decade, sion of cable’s unique programing. The expansion began Madame Catherine Tasca, Ministre Delegue Charge de with

  10. Selecting a Cable System Operator.

    ERIC Educational Resources Information Center

    Cable Television Information Center, Washington, DC.

    Intended to assist franchising authorities with the process of selecting a cable television system operator from franchise applicants, this document provides a framework for analysis of individual applications. Section 1 deals with various methods which can be used to select an operator. The next section covers the application form, the vehicle a…

  11. Interactive Cable Television. Final Report.

    ERIC Educational Resources Information Center

    Active Learning Systems, Inc., Minneapolis, MN.

    This report describes an interactive video system developed by Active Learning Systems which utilizes a cable television (TV) network as its delivery system to transmit computer literacy lessons to high school and college students. The system consists of an IBM PC, Pioneer LDV 4000 videodisc player, and Whitney Supercircuit set up at the head end…

  12. 300 Area signal cable study

    SciTech Connect

    Whattam, J.W.

    1994-09-15

    This report was prepared to discuss the alternatives available for removing the 300 Area overhead signal cable system. This system, installed in 1969, has been used for various monitoring and communication signaling needs throughout the 300 Area. Over the years this cabling system has deteriorated, has been continually reconfigured, and has been poorly documented to the point of nonreliability. The first step was to look at the systems utilizing the overhead signal cable that are still required for operation. Of the ten systems that once operated via the signal cable, only five are still required; the civil defense evacuation alarms, the public address (PA) system, the criticality alarms, the Pacific Northwest Laboratory Facilities Management Control System (FMCS), and the 384 annunciator panel. Of these five, the criticality alarms and the FMCS have been dealt with under other proposals. Therefore, this study focused on the alternatives available for the remaining three systems (evacuation alarms, PA system, and 384 panel) plus the accountability aid phones. Once the systems to be discussed were determined, then three alternatives for providing the signaling pathway were examined for each system: (1) re-wire using underground communication ducts, (2) use the Integrated Voice/Data Telecommunications System (IVDTS) already installed and operated by US West, and (3) use radio control. Each alternative was developed with an estimated cost, advantages, and disadvantages. Finally, a recommendation was provided for the best alternative for each system.

  13. COUPLER FOR TOOL AND CABLE

    DOEpatents

    Cawley, W.E.; Frantz, C.E.

    1962-02-27

    A two-part device is designed for pulling a splitting tool through a fuel tube. The device can be readily disconnected by unthreading the parts by means of a movable head carrying a transverse key which fits into a slot in the threaded part attached to the cable. (AEC)

  14. Development of modular cable mesh deployable antenna

    NASA Astrophysics Data System (ADS)

    Meguro, Akira; Mitsugi, Jin; Andou, Kazuhide

    1993-03-01

    This report describes a concept and key technologies for the modular mesh deployable antenna. The antenna reflector composed of independently manufactured and tested modules is presented. Each module consists of a mesh surface, a cable network, and a deployable truss structure. The cable network comprises three kinds of cables, surface, tie, and back cables. Adjustment of tie cable lengths improves the surface accuracy. Synchronous deployment truss structures are considered as a supporting structure. Their design method, BBM's (Bread Board Model) and deployment analysis are also explained.

  15. Aeolic vibration of aerial electricity transmission cables

    NASA Astrophysics Data System (ADS)

    Avila, A.; Rodriguez-Vera, Ramon; Rayas, Juan A.; Barrientos, Bernardino

    2005-02-01

    A feasibility study for amplitude and frequency vibration measurement in aerial electricity transmission cable has been made. This study was carried out incorporating a fringe projection method for the experimental part and horizontal taut string model for theoretical one. However, this kind of model ignores some inherent properties such as cable sag and cable inclination. Then, this work reports advances on aeolic vibration considering real cables. Catenary and sag are considered in our theoretical model in such a way that an optical theodolite for measuring has been used. Preliminary measurements of the catenary as well as numerical simulation of a sagged cable vibration are given.

  16. The GEOS-20 m Cable Boom Mechanism

    NASA Technical Reports Server (NTRS)

    Schmidt, G. K.; Suttner, K.

    1977-01-01

    The GEOS Cable Boom Mechanism which allows the controlled deployment of a 20 m long cable in a centrifugal force field is described. In launch configuration the flat cable is reeled on a 240 mm diameter drum. The electrical connection between the rotating drum and the stationary housing is accomplished via a flexlead positioned inside the drum. Active motion control of this drum is achieved by a self locking worm gear, driven by a stepper motor. The deployment length of the cable is monitored by an optical length indicator, sensing black bars engraved on the cable surface.

  17. GEOS-20 m cable boom mechanism

    NASA Technical Reports Server (NTRS)

    Schmidt, B. K.; Suttner, K.

    1977-01-01

    The GEOS cable boom mechanism allows the controlled deployment of a 20 m long cable in a centrifugal force field. In launch configuration the flat cable is reeled on a 240 mm diameter drum. The electrical connection between the rotating drum and the stationary housing is accomplished via a flexlead positioned inside the drum. Active motion control of this drum is achieved by a self locking worm gear, driven by a stepper motor. The deployment length of the cable is monitored by an optical length indicator, sensing black bars engraved on the cable surface.

  18. Actin dynamics in the regulation of endothelial barrier functions and neutrophil recruitment during endotoxemia and sepsis.

    PubMed

    Schnoor, Michael; García Ponce, Alexander; Vadillo, Eduardo; Pelayo, Rosana; Rossaint, Jan; Zarbock, Alexander

    2017-02-02

    Sepsis is a leading cause of death worldwide. Increased vascular permeability is a major hallmark of sepsis. Dynamic alterations in actin fiber formation play an important role in the regulation of endothelial barrier functions and thus vascular permeability. Endothelial integrity requires a delicate balance between the formation of cortical actin filaments that maintain endothelial cell contact stability and the formation of actin stress fibers that generate pulling forces, and thus compromise endothelial cell contact stability. Current research has revealed multiple molecular pathways that regulate actin dynamics and endothelial barrier dysfunction during sepsis. These include intracellular signaling proteins of the small GTPases family (e.g., Rap1, RhoA and Rac1) as well as the molecules that are directly acting on the actomyosin cytoskeleton such as myosin light chain kinase and Rho kinases. Another hallmark of sepsis is an excessive recruitment of neutrophils that also involves changes in the actin cytoskeleton in both endothelial cells and neutrophils. This review focuses on the available evidence about molecules that control actin dynamics and regulate endothelial barrier functions and neutrophil recruitment. We also discuss treatment strategies using pharmaceutical enzyme inhibitors to target excessive vascular permeability and leukocyte recruitment in septic patients.

  19. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  20. GPCRs and actin-cytoskeleton dynamics.

    PubMed

    Vázquez-Victorio, Genaro; González-Espinosa, Claudia; Espinosa-Riquer, Zyanya P; Macías-Silva, Marina

    2016-01-01

    A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.

  1. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

    Ennomani, Hajer; Letort, Gaëlle; Guérin, Christophe; Martiel, Jean-Louis; Cao, Wenxiang; Nédélec, François; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2016-03-07

    Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of "network connectivity" and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions.

  2. Bioinformatics study of the mangrove actin genes

    NASA Astrophysics Data System (ADS)

    Basyuni, M.; Wasilah, M.; Sumardi

    2017-01-01

    This study describes the bioinformatics methods to analyze eight actin genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, subcellular localization, similarity, and phylogenetic. The physical and chemical properties of eight mangroves showed variation among the genes. The percentage of the secondary structure of eight mangrove actin genes followed the order of a helix > random coil > extended chain structure for BgActl, KcActl, RsActl, and A. corniculatum Act. In contrast to this observation, the remaining actin genes were random coil > extended chain structure > a helix. This study, therefore, shown the prediction of secondary structure was performed for necessary structural information. The values of chloroplast or signal peptide or mitochondrial target were too small, indicated that no chloroplast or mitochondrial transit peptide or signal peptide of secretion pathway in mangrove actin genes. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove actin genes. To clarify the relationship among the mangrove actin gene, a phylogenetic tree was constructed. Three groups of mangrove actin genes were formed, the first group contains B. gymnorrhiza BgAct and R. stylosa RsActl. The second cluster which consists of 5 actin genes the largest group, and the last branch consist of one gene, B. sexagula Act. The present study, therefore, supported the previous results that plant actin genes form distinct clusters in the tree.

  3. Ampacity test of 28 AWG ribbon cables

    SciTech Connect

    Drennan, E.

    1990-04-01

    This report contains the results of testing ribbon cables in order to determine the amount of current they can carry. In some experiments ribbon cables are used to power circuit boards and carry several amperes of current. These tests were required to establish safe operating current limits for electrical safety reviews. The basic principle behind these tests was to increase the amount of current passed through the ribbon cables until the insulation melted and the cable clearly became a fire hazard. As the current through the cable was increased the copper temperature was determined from the increased conductor resistance. The tests were done on two different types of ribbon cables. One was the flat 26 conductor 28AWG and the other was the twist and flat 26 conductor 28AWG ribbon cable, both of which are commonly used in the experiments. The flat cable was tested for current carrying capacity through three conductors and through a single conductor. The twist and flat cable was tested for current carrying capacity through a single conductor. It was found that the maximum single conductor ampacity for both cables is approximately 3A and the three conductor ampacity for the flat cable is about 10A. 4 figs., 3 tabs.

  4. Self-healing cable apparatus and methods

    NASA Technical Reports Server (NTRS)

    Huston, Dryver (Inventor); Esser, Brian (Inventor)

    2007-01-01

    Self-healing cable apparatus and methods are disclosed. The cable has a central core surrounded by an adaptive cover that can extend over the entire length of the cable or just one or more portions of the cable. The adaptive cover includes a protective layer having an initial damage resistance, and a reactive layer. When the cable is subjected to a localized damaging force, the reactive layer responds by creating a corresponding localized self-healed region. The self-healed region provides the cable with enhanced damage resistance as compared to the cable's initial damage resistance. Embodiments of the invention utilize conventional epoxies or foaming materials in the reactive layer that are released to form the self-healed region when the damaging force reaches the reactive layer.

  5. Reconstitution of a Minimal Actin Cortex by Coupling Actin Filaments to Reconstituted Membranes.

    PubMed

    Vogel, Sven K

    2016-01-01

    A thin layer of actin filaments in many eukaryotic cell types drives pivotal aspects of cell morphogenesis and is generally cited as the actin cortex. Myosin driven contractility and actin cytoskeleton membrane interactions form the basis of fundamental cellular processes such as cytokinesis, cell migration, and cortical flows. How the interplay between the actin cytoskeleton, the membrane, and actin binding proteins drives these processes is far from being understood. The complexity of the actin cortex in living cells and the hardly feasible manipulation of the omnipotent cellular key players, namely actin, myosin, and the membrane, are challenging in order to gain detailed insights about the underlying mechanisms. Recent progress in developing bottom-up in vitro systems where the actin cytoskeleton is combined with reconstituted membranes may provide a complementary route to reveal general principles underlying actin cortex properties. In this chapter the reconstitution of a minimal actin cortex by coupling actin filaments to a supported membrane is described. This minimal system may be very well suited to study for example protein interactions on membrane bound actin filaments in a very controlled and quantitative manner as it may be difficult to perform in living systems.

  6. Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization

    PubMed Central

    Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T.

    2016-01-01

    Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

  7. p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging

    PubMed Central

    Jorques, María; Rada, Patricia; Ramirez, Lorena; Valverde, Ángela M.; Nebreda, Ángel R.; Sastre, Juan

    2017-01-01

    Background Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. Methods Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. Results We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. Conclusions p38α MAPK is essential for actin dynamics with age in hepatocytes. PMID:28166285

  8. Selective staining of actin in live human dermal fibroblast cells using quantum dots

    NASA Astrophysics Data System (ADS)

    Adurkar, Udita S.; Agrawal, Amit; Nie, Shuming

    2005-03-01

    Semiconductor quantum dots (QD) are nanometer size fluorophores with improved brightness, resistance against photobleaching and narrow emission bands. These properties make QDs ideal for ultrasensitive imaging of biomolecules in living cells, in multiplexed format. By conjugating QDs with a delivery agent such as TAT peptide and a target-recognition element such as an antibody, we have delivered and imaged target-specific fluorescent probes in living cells. In this work, we demonstrate staining of actin filaments in living Human Dermal Fibroblast (HDF) cells using QD probes functionalized with monoclonal actin antibody. Actin probes were developed by coupling streptavidin coated QDs (λem = 605 nm, QDC Corp.) to biotinylated monoclonal β-actin antibody. Antibody molecules on QDs were conjugated with the TAT peptide. Finally, HDF cells were incubated with the QD-actin antibody-TAT construct. As expected, the characteristic fine streaks of actin filaments were observed in the cells and on the periphery of the cells, similar to phalloidin staining of actin filaments in fixed cells. Using a similar approach, one may image cellular components, proteins or nucleic acids, in a living cell, in real time.

  9. Regulation of the actin cytoskeleton by the Ndel1-Tara complex is critical for cell migration

    PubMed Central

    Hong, Ji-Ho; Kwak, Yongdo; Woo, Youngsik; Park, Cana; Lee, Seol-Ae; Lee, Haeryun; Park, Sung Jin; Suh, Yeongjun; Suh, Bo Kyoung; Goo, Bon Seong; Mun, Dong Jin; Sanada, Kamon; Nguyen, Minh Dang; Park, Sang Ki

    2016-01-01

    Nuclear distribution element-like 1 (Ndel1) plays pivotal roles in diverse biological processes and is implicated in the pathogenesis of multiple neurodevelopmental disorders. Ndel1 function by regulating microtubules and intermediate filaments; however, its functional link with the actin cytoskeleton is largely unknown. Here, we show that Ndel1 interacts with TRIO-associated repeat on actin (Tara), an actin-bundling protein, to regulate cell movement. In vitro wound healing and Boyden chamber assays revealed that Ndel1- or Tara-deficient cells were defective in cell migration. Moreover, Tara overexpression induced the accumulation of Ndel1 at the cell periphery and resulted in prominent co-localization with F-actin. This redistribution of Ndel1 was abolished by deletion of the Ndel1-interacting domain of Tara, suggesting that the altered peripheral localization of Ndel1 requires a physical interaction with Tara. Furthermore, co-expression of Ndel1 and Tara in SH-SY5Y cells caused a synergistic increase in F-actin levels and filopodia formation, suggesting that Tara facilitates cell movement by sequestering Ndel1 at peripheral structures to regulate actin remodeling. Thus, we demonstrated that Ndel1 interacts with Tara to regulate cell movement. These findings reveal a novel role of the Ndel1-Tara complex in actin reorganization during cell movement. PMID:27546710

  10. Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2.

    PubMed

    Kumar, Atul; Paeger, Lars; Kosmas, Kosmas; Kloppenburg, Peter; Noegel, Angelika A; Peche, Vivek S

    2016-01-01

    Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2(gt/gt) mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2(gt/gt) with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2(gt/gt) neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics.

  11. Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2

    PubMed Central

    Kumar, Atul; Paeger, Lars; Kosmas, Kosmas; Kloppenburg, Peter; Noegel, Angelika A.; Peche, Vivek S.

    2016-01-01

    Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2gt/gt mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2gt/gt with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2gt/gt neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics. PMID:27507934

  12. KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability.

    PubMed

    Yeyati, Patricia L; Schiller, Rachel; Mali, Girish; Kasioulis, Ioannis; Kawamura, Akane; Adams, Ian R; Playfoot, Christopher; Gilbert, Nick; van Heyningen, Veronica; Wills, Jimi; von Kriegsheim, Alex; Finch, Andrew; Sakai, Juro; Schofield, Christopher J; Jackson, Ian J; Mill, Pleasantine

    2017-02-28

    Cilia assembly and disassembly are coupled to actin dynamics, ensuring a coherent cellular response during environmental change. How these processes are integrated remains undefined. The histone lysine demethylase KDM3A plays important roles in organismal homeostasis. Loss-of-function mouse models of Kdm3a phenocopy features associated with human ciliopathies, whereas human somatic mutations correlate with poor cancer prognosis. We demonstrate that absence of KDM3A facilitates ciliogenesis, but these resulting cilia have an abnormally wide range of axonemal lengths, delaying disassembly and accumulating intraflagellar transport (IFT) proteins. KDM3A plays a dual role by regulating actin gene expression and binding to the actin cytoskeleton, creating a responsive "actin gate" that involves ARP2/3 activity and IFT. Promoting actin filament formation rescues KDM3A mutant ciliary defects. Conversely, the simultaneous depolymerization of actin networks and IFT overexpression mimics the abnormal ciliary traits of KDM3A mutants. KDM3A is thus a negative regulator of ciliogenesis required for the controlled recruitment of IFT proteins into cilia through the modulation of actin dynamics.

  13. KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability

    PubMed Central

    Schiller, Rachel; Kawamura, Akane; Gilbert, Nick; Wills, Jimi; von Kriegsheim, Alex

    2017-01-01

    Cilia assembly and disassembly are coupled to actin dynamics, ensuring a coherent cellular response during environmental change. How these processes are integrated remains undefined. The histone lysine demethylase KDM3A plays important roles in organismal homeostasis. Loss-of-function mouse models of Kdm3a phenocopy features associated with human ciliopathies, whereas human somatic mutations correlate with poor cancer prognosis. We demonstrate that absence of KDM3A facilitates ciliogenesis, but these resulting cilia have an abnormally wide range of axonemal lengths, delaying disassembly and accumulating intraflagellar transport (IFT) proteins. KDM3A plays a dual role by regulating actin gene expression and binding to the actin cytoskeleton, creating a responsive “actin gate” that involves ARP2/3 activity and IFT. Promoting actin filament formation rescues KDM3A mutant ciliary defects. Conversely, the simultaneous depolymerization of actin networks and IFT overexpression mimics the abnormal ciliary traits of KDM3A mutants. KDM3A is thus a negative regulator of ciliogenesis required for the controlled recruitment of IFT proteins into cilia through the modulation of actin dynamics. PMID:28246120

  14. Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

    PubMed Central

    Grikscheit, Katharina; Frank, Tanja; Wang, Ying

    2015-01-01

    Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1. PMID:25963818

  15. Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly.

    PubMed

    Freeman, N L; Field, J

    2000-02-01

    Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.

  16. Force of an actin spring

    NASA Astrophysics Data System (ADS)

    Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul

    2003-03-01

    The acrosomal process of the horseshoe crab sperm is a novel mechanochemical molecular spring that converts its elastic stain energy to mechanical work upon the chemical activation by Ca2+. Twisted and bent, the initial state of the acrosomal bundle features a high degree of complexity in its structure and the energy is believed to be stored in the highly strained actin filaments as an elastic potential energy. When activated, the bundle relaxes from the coil of the highly twisted and bent filaments to its straight conformation at a mean velocity of 15um/s. The mean extension velocity increases dramatically from 3um/s to 27um/s when temperature of the medium is changed from 9.6C to 32C (respective viscosities of 1.25-0.75cp), yet it exhibits a very weak dependence on changes in the medium viscosity (1cp-33cp). These experiments suggest that the uncoiling of the actin spring should be limited not by the viscosity of the medium but by the unlatching events of involved proteins at a molecular level. Unlike the viscosity-limited processes, where force is directly related to the rate of the reaction, a direct measurement is required to obtain the spring force of the acrosomal process. The extending acrosomal bundle is forced to push against a barrier and its elastic buckling response is analyzed to measure the force generated during the uncoiling.

  17. High Voltage Cable Splicing and Cable Termination Techniques

    DTIC Science & Technology

    1976-08-01

    solid dielectric insulations currently used are high-molecular-weight polyethylene (EP) and cross-linked poly- ethylene ( XLPE ). The EP insulation is...rated for 750 C a maximum temperature, and the XLPE insulation is rated for 900C maximum temperature. These insulation materials provide the conductor...The cost of installing PILC with copper conductors is compared to the cost of installing XLPE cable with aluminum conductors in Table 1. The

  18. Actin-Regulator Feedback Interactions during Endocytosis

    PubMed Central

    Wang, Xinxin; Galletta, Brian J.; Cooper, John A.; Carlsson, Anders E.

    2016-01-01

    Endocytosis mediated by clathrin, a cellular process by which cells internalize membrane receptors and their extracellular ligands, is an important component of cell signaling regulation. Actin polymerization is involved in endocytosis in varying degrees depending on the cellular context. In yeast, clathrin-mediated endocytosis requires a pulse of polymerized actin and its regulators, which recruit and activate the Arp2/3 complex. In this article, we seek to identify the main protein-protein interactions that 1) cause actin and its regulators to appear in pulses, and 2) determine the effects of key mutations and drug treatments on actin and regulator assembly. We perform a joint modeling/experimental study of actin and regulator dynamics during endocytosis in the budding yeast Saccharomyces cerevisiae. We treat both a stochastic model that grows an explicit three-dimensional actin network, and a simpler two-variable Fitzhugh-Nagumo type model. The models include a negative-feedback interaction of F-actin onto the Arp2/3 regulators. Both models explain the pulse time courses and the effects of interventions on actin polymerization: the surprising increase in the peak F-actin count caused by reduced regulator branching activity, the increase in F-actin resulting from slowing of actin disassembly, and the increased Arp2/3 regulator lifetime resulting from latrunculin treatment. In addition, they predict that decreases in the regulator branching activity lead to increases in accumulation of regulators, and we confirmed this prediction with experiments on yeast harboring mutations in the Arp2/3 regulators, using quantitative fluorescence microscopy. Our experimental measurements suggest that the regulators act quasi-independently, in the sense that accumulation of a particular regulator is most strongly affected by mutations of that regulator, as opposed to the others. PMID:27028652

  19. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  20. Fluorescence studies of the carboxyl-terminal domain of smooth muscle calponin effects of F-actin and salts.

    PubMed

    Bartegi, A; Roustan, C; Kassab, R; Fattoum, A

    1999-06-01

    The fluorescence parameters of the environment-sensitive acrylodan, selectively attached to Cys273 in the C-terminal domain of smooth muscle calponin, were studied in the presence of F-actin and using varying salt concentrations. The formation of the F-actin acrylodan labeled calponin complex at 75 mm NaCl resulted in a 21-nm blue shift of the maximum emission wavelength from 496 nm to 474 nm and a twofold increase of the fluorescent quantum yield at 460 nm. These spectral changes were observed at the low ionic strengths (< 110 mm) where the calponin : F-actin stoichiometry is 1 : 1 as well as at the high ionic strengths (> 110 mm) where the binding stoichiometry is a 1 : 2 ratio of calponin : actin monomers. On the basis of previous three-dimensional reconstruction and chemical crosslinking of the F-actin-calponin complex, the actin effect is shown to derive from the low ionic strength interaction of calponin with the bottom of subdomain-1 of an upper actin monomer in F-actin and not from its further association with the subdomain-1 of the adjacent lower monomer which occurs at the high ionic strength. Remarkably, the F-actin-dependent fluorescence change of acrylodan is qualitatively but not quantitatively similar to that earlier reported for the complexes of calponin and Ca2+-calmodulin or Ca2+-caltropin. As the three calponin ligands bind to the same segment of the protein, encompassing residues 145-182, the acrylodan can be considered as a sensitive probe of the functioning of this critical region. A distance of 29 A was measured by fluorescence resonance energy transfer between Cys273 of calponin and Cys374 of actin in the 1 : 1 F-actin-calponin complex suggesting that the F-actin effect was allosteric reflecting a global conformational change in the C-terminal domain of calponin.

  1. Test plan and report for Space Shuttle launch environment testing of Bergen cable technology safety cable

    NASA Technical Reports Server (NTRS)

    Ralph, John

    1992-01-01

    Bergen Cable Technology (BCT) has introduced a new product they refer to as 'safety cable'. This product is intended as a replacement for lockwire when installed per Aerospace Standard (AS) 4536 (included in Appendix D of this document). Installation of safety cable is reportedly faster and more uniform than lockwire. NASA/GSFC proposes to use this safety cable in Shuttle Small Payloads Project (SSPP) applications on upcoming Shuttle missions. To assure that BCT safety cable will provide positive locking of fasteners equivalent to lockwire, the SSPP will conduct vibration and pull tests of the safety cable.

  2. Synthetic peptides that cause F-actin bundling and block actin depolymerization

    DOEpatents

    Sederoff, Heike [Raleigh, NC; Huber, Steven C [Savoy, IL; Larabell, Carolyn A [Berkeley, CA

    2011-10-18

    Synthetic peptides derived from sucrose synthase, and having homology to actin and actin-related proteins, sharing a common motif, useful for causing acting bundling and preventing actin depolymerization. Peptides exhibiting the common motif are described, as well as specific synthetic peptides which caused bundled actin and inhibit actin depolymerization. These peptides can be useful for treating a subject suffering from a disease characterized by cells having neoplastic growth, for anti-cancer therapeutics, delivered to subjects solely, or concomitantly or sequentially with other known cancer therapeutics. These peptides can also be used for stabilizing microfilaments in living cells and inhibiting growth of cells.

  3. New Life For The Cable Cars

    NASA Technical Reports Server (NTRS)

    1979-01-01

    NASA-Arnes' major recommendations involved ways of extending cable life in the interests of safety and economy. Other recommendations included redesign of the cablegripping device, substitution of modern braking mechanisms, improvements in cable pulleys and other components, and new inspection and repair procedures. Ames followed up by designing and installing new equipment to lengthen cable life, which averages only about two months. These cables-four of them for four different car routes--are endless belts, like ski lift cables, running from the downtown car barn to the end of each line. When a cable is installed, the loop is closed by splicing the ends together in a 72-footlong splice. The splice is the weakest part of the cable and a source of problems. When the car operator applies his grip while over a splice, the resulting friction sometimes causes the splice to "unbraid" and fail; this means shutting down the line until the splice can be repaired. Even when unbraiding does not occur, gripping a splice shortens cable life by friction wear. Worn cables are a safety hazard and must be replaced, which is expensive at $1.60 a foot for 10,000 to 20,000 feet of cable.

  4. Growing an actin gel on spherical surfaces.

    PubMed Central

    Noireaux, V; Golsteyn, R M; Friederich, E; Prost, J; Antony, C; Louvard, D; Sykes, C

    2000-01-01

    Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement. On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a "comet-like" tail of F-actin is produced. We show experimentally that the stationary thickness of the gel depends on the radius of the beads. Moreover, the actin gel is not formed if the ActA surface density is too low. To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel. Our model also takes into account treadmilling of actin. We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN. Finally, we estimate from our theoretical model possible conditions for developing actin comet tails. PMID:10692348

  5. Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection.

    PubMed Central

    Valster, A. H.; Pierson, E. S.; Valenta, R.; Hepler, P. K.; Emons, AMC.

    1997-01-01

    We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. Injection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability to act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive to microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells. PMID:12237348

  6. Tubing and cable cutting tool

    NASA Technical Reports Server (NTRS)

    Mcsmith, D. D.; Richardson, J. I. (Inventor)

    1984-01-01

    A hand held hydraulic cutting tool was developed which is particularly useful in deactivating ejection seats in military aircraft rescue operations. The tool consists primarily of a hydraulic system composed of a fluid reservoir, a pumping piston, and an actuator piston. Mechanical cutting jaws are attached to the actuator piston rod. The hydraulic system is controlled by a pump handle. As the pump handle is operated the actuator piston rod is forced outward and thus the cutting jaws are forced together. The frame of the device is a flexible metal tubing which permits easy positioning of the tool cutting jaws in remote and normally inaccessible locations. Bifurcated cutting edges ensure removal of a section of the tubing or cable to thereby reduce the possibility of accidental reactivation of the tubing or cable being severed.

  7. F-actin-binding protein drebrin regulates CXCR4 recruitment to the immune synapse.

    PubMed

    Pérez-Martínez, Manuel; Gordón-Alonso, Mónica; Cabrero, José Román; Barrero-Villar, Marta; Rey, Mercedes; Mittelbrunn, María; Lamana, Amalia; Morlino, Giulia; Calabia, Carmen; Yamazaki, Hiroyuki; Shirao, Tomoaki; Vázquez, Jesús; González-Amaro, Roberto; Veiga, Esteban; Sánchez-Madrid, Francisco

    2010-04-01

    The adaptive immune response depends on the interaction of T cells and antigen-presenting cells at the immune synapse. Formation of the immune synapse and the subsequent T-cell activation are highly dependent on the actin cytoskeleton. In this work, we describe that T cells express drebrin, a neuronal actin-binding protein. Drebrin colocalizes with the chemokine receptor CXCR4 and F-actin at the peripheral supramolecular activation cluster in the immune synapse. Drebrin interacts with the cytoplasmic tail of CXCR4 and both proteins redistribute to the immune synapse with similar kinetics. Drebrin knockdown in T cells impairs the redistribution of CXCR4 and inhibits actin polymerization at the immune synapse as well as IL-2 production. Our data indicate that drebrin exerts an unexpected and relevant functional role in T cells during the generation of the immune response.

  8. The ATP binding cassette transporter, ABCG1, localizes to cortical actin filaments

    PubMed Central

    Pandzic, Elvis; Gelissen, Ingrid C.; Whan, Renee; Barter, Philip J.; Sviridov, Dmitri; Gaus, Katharina; Rye, Kerry-Anne; Cochran, Blake J.

    2017-01-01

    The ATP-binding cassette sub-family G member 1 (ABCG1) exports cellular cholesterol to high-density lipoproteins (HDL). However, a number of recent studies have suggested ABCG1 is predominantly localised to intracellular membranes. In this study, we found that ABCG1 was organized into two distinct cellular pools: one at the plasma membrane and the other associated with the endoplasmic reticulum (ER). The plasma membrane fraction was organized into filamentous structures that were associated with cortical actin filaments. Inhibition of actin polymerization resulted in complete disruption of ABCG1 filaments. Cholesterol loading of the cells increased the formation of the filamentous ABCG1, the proximity of filamentous ABCG1 to actin filaments and the diffusion rate of membrane associated ABCG1. Our findings suggest that the actin cytoskeleton plays a critical role in the plasma membrane localization of ABCG1. PMID:28165022

  9. Mena-GRASP65 interaction couples actin polymerization to Golgi ribbon linking.

    PubMed

    Tang, Danming; Zhang, Xiaoyan; Huang, Shijiao; Yuan, Hebao; Li, Jie; Wang, Yanzhuang

    2016-01-01

    In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.

  10. Activation of tyrosine kinase c-Abl contributes to α-synuclein–induced neurodegeneration

    PubMed Central

    Lee, Su Hyun; Kim, Donghoon; Karuppagounder, Senthilkumar S.; Kumar, Manoj; Mao, Xiaobo; Shin, Joo Ho; Lee, Yunjong; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.; Ko, Han Seok

    2016-01-01

    Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein–induced neuropathology. In mice expressing a human α-synucleinopathy–associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein–induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies. PMID:27348587

  11. Structural health monitoring approach for detecting ice accretion on bridge cable using the Haar Wavelet Transform

    NASA Astrophysics Data System (ADS)

    Andre, Julia; Kiremidjian, Anne; Liao, Yizheng; Georgakis, Christos; Rajagopal, Ram

    2016-04-01

    Ice accretion on cables of bridge structures poses serious risk to the structure as well as to vehicular traffic when the ice falls onto the road. Detection of ice formation, quantification of the amount of ice accumulated, and prediction of icefalls will increase the safety and serviceability of the structure. In this paper, an ice accretion detection algorithm is presented based on the Continuous Wavelet Transform (CWT). In the proposed algorithm, the acceleration signals obtained from bridge cables are transformed using wavelet method. The damage sensitive features (DSFs) are defined as a function of the wavelet energy at specific wavelet scales. It is found that as ice accretes on the cables, the mass of cable increases, thus changing the wavelet energies. Hence, the DSFs can be used to track the change of cables mass. To validate the proposed algorithm, we use the data collected from a laboratory experiment conducted at the Technical University of Denmark (DTU). In this experiment, a cable was placed in a wind tunnel as ice volume grew progressively. Several accelerometers were installed at various locations along the testing cable to collect vibration signals.

  12. Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network

    PubMed Central

    Luo, Weiwei; Lieu, Zi Zhao; Manser, Ed; Bershadsky, Alexander D.; Sheetz, Michael P.

    2016-01-01

    A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. PMID:27760153

  13. F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins.

    PubMed

    Higashida, Chiharu; Kiuchi, Tai; Akiba, Yushi; Mizuno, Hiroaki; Maruoka, Masahiro; Narumiya, Shuh; Mizuno, Kensaku; Watanabe, Naoki

    2013-04-01

    Physical force evokes rearrangement of the actin cytoskeleton. Signalling pathways such as tyrosine kinases, stretch-activated Ca(2+) channels and Rho GTPases are involved in force sensing. However, how signals are transduced to actin assembly remains obscure. Here we show mechanosensitive actin polymerization by formins (formin homology proteins). Cells overexpressing mDia1 increased the amount of F-actin on release of cell tension. Fluorescence single-molecule speckle microscopy revealed rapid induction of processive actin assembly by mDia1 on cell cortex deformation. mDia1 lacking the Rho-binding domain and other formins exhibited mechanosensitive actin nucleation, suggesting Rho-independent activation. Mechanosensitive actin nucleation by mDia1 required neither Ca(2+) nor kinase signalling. Overexpressing LIM kinase abrogated the induction of processive mDia1. Furthermore, s-FDAPplus (sequential fluorescence decay after photoactivation) analysis revealed a rapid actin monomer increase on cell cortex deformation. Our direct visualization of the molecular behaviour reveals a mechanosensitive actin filament regeneration mechanism in which G-actin released by actin remodelling plays a pivotal role.

  14. The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model

    PubMed Central

    1991-01-01

    contributes to the outer part of the massive base. Quantitative evaluation of successive crossover spacings along individual F-actin filaments revealed the deviations from the mean repeat to be compensatory, i.e., short crossovers frequently followed long ones and vice versa. The variable crossover spacings and diameter of the F-actin filament together with the local unraveling of the two long-pitch helical strands are explained in terms of varying amounts of compensatory "lateral slipping" of the two strands past each other roughly perpendicular to the filament axis. This intrinsic disorder of the actin filament may enable the actin moiety to play a more active role in actin-myosin-based force generation than merely act as a rigid passive cable as has hitherto been assumed. PMID:1918159

  15. High current pulse transmission cable

    SciTech Connect

    Parsons, W.M.

    1990-09-28

    This invention is comprised of a transmission cable for carrying high current pulses in which an even numbered plurality of electrical conductors surrounds a central ground conductor. Each electrical conductor is connected so that it at any instant in time it will carry current of opposite polarity to the polarity carried by adjacent conductors. This arrangement cancels practically all of the external fields generated by current in the conductors.

  16. Lifeact: a versatile marker to visualize F-actin.

    PubMed

    Riedl, Julia; Crevenna, Alvaro H; Kessenbrock, Kai; Yu, Jerry Haochen; Neukirchen, Dorothee; Bista, Michal; Bradke, Frank; Jenne, Dieter; Holak, Tad A; Werb, Zena; Sixt, Michael; Wedlich-Soldner, Roland

    2008-07-01

    Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

  17. Synthetic mimetics of actin-binding macrolides: rational design of actin-targeted drugs.

    PubMed

    Perrins, Richard D; Cecere, Giuseppe; Paterson, Ian; Marriott, Gerard

    2008-03-01

    Actin polymerization and dynamics are involved in a wide range of cellular processes such as cell division and migration of tumor cells. At sites of cell lysis, such as those occurring during a stroke or inflammatory lung diseases, actin is released into the serum where it polymerizes, leading to problems with clot dissolution and sputum viscosity. Therefore, drugs that target these actin-mediated processes may provide one mechanism to treat these conditions. Marine-organism-derived macrolides, such as reidispongiolide A, can bind to, sever, and inhibit polymerization of actin. Our studies show that the function of these complex macrolides resides in their tail region, whereas the head group stabilizes the actin-drug complex. Synthetic compounds derived from this tail region could therefore be used as a mimetic of the natural product, providing a range of designer compounds to treat actin-associated diseases or as probes to study actin polymerization.

  18. F-actin aggregates in transformed cells

    PubMed Central

    1981-01-01

    Polymerized actin has been found aggregated into distinctive patches inside transformed cells in culture. The F-actin-specific fluorescent probe, nitrobenzoxadiazole-phallacidin, labels these F-actin aggregates near the ventral cell surface of cells transformed by RNA or DNA tumor viruses, or by chemical mutagens, or spontaneously. Their appearance in all eight transformed cell types studied suggests their ubiquity and involvement in transformation morphology. Actin patches developed in normal rat kidney (NRK) cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (LA23-NRK) within 30 min after a shift from the nonpermissive (39 degrees C) to the permissive temperature (32 degrees C). Patch appearance paralleling viral src gene expression tends to implicate pp60src kinase activity in destabilizing the cytoskeleton. However, appearance of the actin aggregates in cells not transformed by retrovirus calls for alternative mechanisms, perhaps involving an endogenous kinase, for this apparently common trait. PMID:6270163

  19. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  20. Signalling Pathways Controlling Cellular Actin Organization.

    PubMed

    Steffen, Anika; Stradal, Theresia E B; Rottner, Klemens

    2017-01-01

    The actin cytoskeleton is essential for morphogenesis and virtually all types of cell shape changes. Reorganization is per definition driven by continuous disassembly and re-assembly of actin filaments, controlled by major, ubiquitously operating machines. These are specifically employed by the cell to tune its activities in accordance with respective environmental conditions or to satisfy specific needs.Here we sketch some fundamental signalling pathways established to contribute to the reorganization of specific actin structures at the plasma membrane. Rho-family GTPases are at the core of these pathways, and dissection of their precise contributions to actin reorganization in different cell types and tissues will thus continue to improve our understanding of these important signalling nodes. Furthermore, we will draw your attention to the emerging theme of actin reorganization on intracellular membranes, its functional relation to Rho-GTPase signalling, and its relevance for the exciting phenomenon autophagy.

  1. Actin dynamics shape microglia effector functions.

    PubMed

    Uhlemann, Ria; Gertz, Karen; Boehmerle, Wolfgang; Schwarz, Tobias; Nolte, Christiane; Freyer, Dorette; Kettenmann, Helmut; Endres, Matthias; Kronenberg, Golo

    2016-06-01

    Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion.

  2. Actin-Interacting Protein 1 Contributes to Intranuclear Rod Assembly in Dictyostelium discoideum

    PubMed Central

    Ishikawa-Ankerhold, Hellen C.; Daszkiewicz, Wioleta; Schleicher, Michael; Müller-Taubenberger, Annette

    2017-01-01

    Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation. PMID:28074884

  3. Biophysical model of the role of actin remodeling on dendritic spine morphology

    PubMed Central

    Miermans, C. A.; Kusters, R. P. T.; Hoogenraad, C. C.; Storm, C.

    2017-01-01

    Dendritic spines are small membranous structures that protrude from the neuronal dendrite. Each spine contains a synaptic contact site that may connect its parent dendrite to the axons of neighboring neurons. Dendritic spines are markedly distinct in shape and size, and certain types of stimulation prompt spines to evolve, in fairly predictable fashion, from thin nascent morphologies to the mushroom-like shapes associated with mature spines. It is well established that the remodeling of spines is strongly dependent upon the actin cytoskeleton inside the spine. A general framework that details the precise role of actin in directing the transitions between the various spine shapes is lacking. We address this issue, and present a quantitative, model-based scenario for spine plasticity validated using realistic and physiologically relevant parameters. Our model points to a crucial role for the actin cytoskeleton. In the early stages of spine formation, the interplay between the elastic properties of the spine membrane and the protrusive forces generated in the actin cytoskeleton propels the incipient spine. In the maturation stage, actin remodeling in the form of the combined dynamics of branched and bundled actin is required to form mature, mushroom-like spines. Importantly, our model shows that constricting the spine-neck aids in the stabilization of mature spines, thus pointing to a role in stabilization and maintenance for additional factors such as ring-like F-actin structures. Taken together, our model provides unique insights into the fundamental role of actin remodeling and polymerization forces during spine formation and maturation. PMID:28158194

  4. 47 CFR 76.802 - Disposition of cable home wiring.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Cable Inside Wiring § 76.802 Disposition of cable home wiring... (technical standards) of the Commission's Cable Television Service rules (47 CFR 76.605(a)(13) and 76.610... standards) of the Commission's Cable Television Service rules (47 CFR 76.605(a)(13) and 76.610 through...

  5. Helical grip for the cable cars of San Francisco

    NASA Technical Reports Server (NTRS)

    Peyran, R. J.

    1979-01-01

    A helical cable car grip to minimize high maintenance costs of San Francisco's cable car operation is presented. The grip establishes a rolling contact between the cable and grip to reduce sliding friction and associated cable wear. The design, development, and testing of the helical cable car grip are described.

  6. Demonstration of actin filament stress fibers in microvascular endothelial cells in situ.

    PubMed

    Nehls, V; Drenckhahn, D

    1991-07-01

    We have developed a method for immunostaining the microvascular tree of rat mesenteric windows in situ. The procedure consists of three steps, i.e., mild fixation with formaldehyde, controlled proteolytic digestion of the mesothelial layer, and permeabilization with acetone. Discrimination between different microvascular segments was possible by double-fluorescent staining with antibodies to the smooth muscle isoform of alpha-actin and to nonmuscle myosin from platelets. Antibodies to nonmuscle myosin labeled numerous longitudinally oriented cables in endothelial cells of all microvascular segments (arterioles, metarterioles, pre-, mid-, and postcapillaries, small venules). Occasionally, the myosin-containing cables displayed the interrupted sarcomere-like staining pattern that is diagnostic for stress fibers. In contrast, staining of actin filaments with phalloidin-rhodamin resulted in a noninterrupted, continuous fluorescence of the stress fibers. A possible functional role of microvascular endothelial stress fibers is to serve as a tensile cytoskeletal scaffold that stabilizes the tubular, three-dimensional geometry of microvessels and, in addition, to help the endothelium resist the shear forces created by blood flow and by collision with red and white blood cells.

  7. Dynamics of an actin spring

    NASA Astrophysics Data System (ADS)

    Riera, Christophe; Mahadevan, L.; Shin, Jennifer; Matsudaira, Paul

    2003-03-01

    The acrosome of the sperm of the horseshoe crab (Limulus Polyphemus) is an unusual actin based system that shows a spectacular dynamical transition in the presence of Ca++ that is present in abundance in the neighborhood of the egg. During this process, the bundle, which is initially bent and twisted uncoils and becomes straight in a matter of a few seconds. Based on microstructural data, we propose a model for the dynamics of uncoiling that is best represented by a triple-well potential corresponding to the different structural arrangements of the supertwisted filaments. Each of the false, true and coiled states corresponds to a local minimum of the energy, with the true state being the one with the lowest energy. Using an evolution equation derived by balancing torques, we investigate the nucleation and propagation of the phase transition and compare the results with those of experiments. Our model quantifies the hypothesis that the acrosomal bundle behaves like a mechano-chemical spring.

  8. Initial stem cell adhesion on porous silicon surface: molecular architecture of actin cytoskeleton and filopodial growth

    NASA Astrophysics Data System (ADS)

    Collart-Dutilleul, Pierre-Yves; Panayotov, Ivan; Secret, Emilie; Cunin, Frédérique; Gergely, Csilla; Cuisinier, Frédéric; Martin, Marta

    2014-10-01

    The way cells explore their surrounding extracellular matrix (ECM) during development and migration is mediated by lamellipodia at their leading edge, acting as an actual motor pulling the cell forward. Lamellipodia are the primary area within the cell of actin microfilaments (filopodia) formation. In this work, we report on the use of porous silicon (pSi) scaffolds to mimic the ECM of mesenchymal stem cells from the dental pulp (DPSC) and breast cancer (MCF-7) cells. Our atomic force microscopy (AFM), fluorescence microscopy, and scanning electron microscopy (SEM) results show that pSi promoted the appearance of lateral filopodia protruding from the DPSC cell body and not only in the lamellipodia area. The formation of elongated lateral actin filaments suggests that pores provided the necessary anchorage points for protrusion growth. Although MCF-7 cells displayed a lower presence of organized actin network on both pSi and nonporous silicon, pSi stimulated the formation of extended cell protrusions.

  9. Semiflex cable wires airborne microwave systems

    NASA Astrophysics Data System (ADS)

    Winkler, Martin; Gochenour, Tim; Turner, Gary

    1991-01-01

    The SiO2 semiflexible cable is made of a solid-copper center conductor, a high-purity low-density silicon dioxide ceramic dielectric, and an outer sheath of stainless-steel tubing over a thin copper inner lining. The high-temperature properties of the ceramic insulation make possible the full annealing of the outer sheath in the final processing step. This annealing step is responsible for the flexibility of the cable and is the main distinction between semiflexible SiO2 cable and semirigid Teflon cable. A simulatied application is presented, and considerations of phase stability and proper use are examined. It is pointed out that the semiflexible SiO2 cable's simple construction can result in a weight savings of 20 to 40 percent and a 25 percent reduction in diameter compared with equivalent-performance flexible Teflon cables.

  10. An industrial cabling machine for the SSC

    SciTech Connect

    Royet, J.; Armer, R.; Hannaford, R.; Scanlan, R.

    1989-02-01

    The SSC project will need the manufacturing of some 25,000 kilometers of keystoned flat cable. The technical specifications of the various cables to be produced are the result of five years of research and development work at LBL. An experimental cable machine was built and run in the laboratory; many improvements were implemented and tested. Semi-industrial production of the various cables was performed, and the resulting cables were used and tested in the one-meter model magnets and 17.5 meter dipole prototypes. From these experiments an industrial cabler specification was generated and used for an international RFQ. The winner of the contract is Dour Metal, a Belgium company that built the first industrial prototype which is now in a production line at New England Electric Wire Company. In this paper we describe the main characteristics of the machine and give the first industrial production results of superconducting keystoned cable for the SSC project. 4 refs.

  11. Cable force monitoring system of cable stayed bridges using accelerometers inside mobile smart phone

    NASA Astrophysics Data System (ADS)

    Zhao, Xuefeng; Yu, Yan; Hu, Weitong; Jiao, Dong; Han, Ruicong; Mao, Xingquan; Li, Mingchu; Ou, Jinping

    2015-03-01

    Cable force is one of the most important parameters in structural health monitoring system integrated on cable stayed bridges for safety evaluation. In this paper, one kind of cable force monitoring system scheme was proposed. Accelerometers inside mobile smart phones were utilized for the acceleration monitoring of cable vibration. Firstly, comparative tests were conducted in the lab. The test results showed that the accelerometers inside smartphones can detect the cable vibration, and then the cable force can be obtained. Furthermore, there is good agreement between the monitoring results of different kinds of accelerometers. Finally, the proposed cable force monitoring system was applied on one cable strayed bridge structure, the monitoring result verified the feasibility of the monitoring system.

  12. Long-life cable development. Cable-processing survey. Final report

    SciTech Connect

    Mangaraj, D.; Preston, J.R.

    1985-09-01

    A survey of cable manufacturers in North America, Europe, and Japan identified state-of-the-art techniques for processing extruded dielectric cables. The review highlights optimal approaches to such process operations as materials handling, extrusion, and vulcanization.

  13. Vincennes University: Pioneer in Cable TV

    ERIC Educational Resources Information Center

    Beckes, Isaac K.

    1972-01-01

    The development of cable educational television at Vincennes University (Indiana) is discussed in terms of the financing, securing of franchises, educational goals and plans for future expansion. (RN)

  14. Capacitor discharge process for welding braided cable

    DOEpatents

    Wilson, Rick D.

    1995-01-01

    A capacitor discharge process for welding a braided cable formed from a plurality of individual cable strands to a solid metallic electrically conductive member comprises the steps of: (a) preparing the electrically conductive member for welding by bevelling one of its end portions while leaving an ignition projection extending outwardly from the apex of the bevel; (b) clamping the electrically conductive member in a cathode fixture; (c) connecting the electrically conductive member clamped in the cathode fixture to a capacitor bank capable of being charged to a preselected voltage value; (d) preparing the braided cable for welding by wrapping one of its end portions with a metallic sheet to form a retaining ring operable to maintain the individual strands of the braided cable in fixed position within the retaining ring; (e) clamping the braided cable and the retaining ring as a unit in an anode fixture so that the wrapped end portion of the braided cable faces the ignition projection of the electrically conductive member; and (f) moving the cathode fixture towards the anode fixture until the ignition projection of the electrically conductive member contacts the end portion of the braided cable thereby allowing the capacitor bank to discharge through the electrically conductive member and through the braided cable and causing the electrically conductive member to be welded to the braided cable via capacitor discharge action.

  15. Proinflammatory cytokines provoke oxidative damage to actin in neuronal cells mediated by Rac1 and NADPH oxidase.

    PubMed

    Barth, Brian M; Stewart-Smeets, Shelli; Kuhn, Thomas B

    2009-06-01

    The proinflammatory cytokines TNFalpha and Il-1beta orchestrate the progression of CNS inflammation, which substantially contributes to neurodegeneration in many CNS pathologies. TNFalpha and Il-1beta stimulate actin filament reorganization in non-neuronal cells often accompanied by the formation of reactive oxygen species (ROS). Actin filament dynamics is vital for cellular plasticity, mitochondrial function, and gene expression despite being highly susceptible to oxidative damage. We demonstrated that, in neuronal cells, TNFalpha and Il-1beta stimulate a transient, redox-dependent reorganization of the actin cytoskeleton into lamellipodia under the regulation of Rac1 and a neuronal NADPH oxidase as the source of ROS. The persistent presence of intracellular ROS provoked oxidative damage (carbonylation) to actin coinciding with the loss of lamellipodia and arrest of cellular plasticity. Inhibition of NADPH oxidase activity or Rac1 abolished the adverse effects of cytokines. These findings suggest that oxidative damage to the neuronal actin cytoskeleton could represent a key step in CNS neurodegeneration.

  16. Self-organized gels in DNA/F-actin mixtures without crosslinkers: networks of induced nematic domains with tunable density.

    PubMed

    Lai, Ghee Hwee; Butler, John C; Zribi, Olena V; Smalyukh, Ivan I; Angelini, Thomas E; Purdy, Kirstin R; Golestanian, Ramin; Wong, Gerard C L

    2008-11-21

    We examine mixtures of DNA and filamentous actin (F-actin) as a model system of like-charged rigid rods and flexible chains. Confocal microscopy reveals the formation of elongated nematic F-actin domains reticulated via defect-free vertices into a network embedded in a mesh of random DNA. Synchrotron x-ray scattering results indicate that the DNA mesh squeezes the F-actin domains into a nematic state with an interactin spacing that decreases with increasing DNA concentration as d(actin) proportional, variantrho(DNA)(-1/2). Interestingly, the system changes from a counterion-controlled regime to a depletion-controlled regime with added salt, with drastic consequences for the osmotic pressure induced phase behavior.

  17. Actin from pig and rat uterus.

    PubMed Central

    Elce, J S; Elbrecht, A S; Middlestadt, M U; McIntyre, E J; Anderson, P J

    1981-01-01

    Smooth-muscle actin was isolated from pig uterus and from pregnant-rat uterus. Methods involving acetone-dried powders were unsuccessful, and a column-chromatographic procedure was developed, with proteinase inhibitors and avoiding polymerization as a purification step. The yield of pure actin was 0.8--1.5 mg/g wet wt. of uterus, which should be compared with an expected yield of actin from skeletal muscle of 2--4 mg/g wet wt. The actin was pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and exhibited alpha-, beta-, and gamma-forms on isoelectric focusing. It possessed a blocked N-terminal amino acid residue, and its amino acid analysis conformed to those of other actins. The rat uterine actin was available only in small amounts (5--10 mg) and did not polymerize. The pig uterine actin could be obtained in amounts up to 30 mg, polymerized reversibly, and activated a skeletal myosin Mg2+-dependent ATPase. Images Fig. 2. Fig. 4. PMID:6458278

  18. Reversible stress softening of actin networks

    PubMed Central

    Chaudhuri, Ovijit; Parekh, Sapun H.; Fletcher, Daniel A.

    2011-01-01

    The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes1–3. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension4–8. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks. PMID:17230186

  19. Erbium laser resurfacing for actinic cheilitis.

    PubMed

    Cohen, Joel L

    2013-11-01

    Actinic cheilitis is a precancerous condition characterized by grayish-whitish area(s) of discoloration on the mucosal lip, often blunting the demarcation between mucosa and cutaneous lip. Actinic cheilitis is considered to be an early part of the spectrum of squamous cell carcinoma. Squamous cell carcinoma specifically of the lip has a high rate of recurrence and metastasis through the oral cavity leading to a poor overall survival. Risk factors for the development of actinic cheilitis include chronic solar irradiation, increasing age, male gender, light skin complexion, immunosuppression, and possibly tobacco and alcohol consumption. Treatment options include topical pharmacotherapy (eg, fluorouracil, imiquimod) or procedural interventions (eg, cryotherapy, electrosurgery, surgical vermillionectomy, laser resurfacing), each with their known advantages and disadvantages. There is little consensus as to which treatment options offer the most clinical utility given the paucity of comparative clinical data. In my practice, laser resurfacing has become an important tool for the treatment of actinic cheilitis owing to its ease of use and overall safety, tolerability, and cosmetic acceptability. Herein the use of erbium laser resurfacing is described for three actinic cheilitis presentations for which I find it particularly useful: clinically prominent actinic cheilitis, biopsy-proven actinic cheilitis, and treatment of the entire lip following complete tumor excision of squamous cell carcinoma. All patients were treated with a 2940-nm erbium laser (Sciton Profile Contour Tunable Resurfacing Laser [TRL], Sciton, Inc., Palo Alto, CA).

  20. Actin dynamics: old friends with new stories.

    PubMed

    Staiger, Christopher J; Blanchoin, Laurent

    2006-12-01

    Actin dynamics, or the rapid turnover of actin filaments, play a central role in numerous cellular processes. A large and diverse cast of characters, accessory proteins known as actin-binding proteins, modulate actin dynamics. They do this by binding to the monomer pool, interacting with the side and ends of filaments, creating breaks along a filament, and generating new filaments de novo. Recent biochemical and single-filament imaging analyses of several conserved classes of plant actin-binding proteins reveal unusual and unexpected properties. Examples that are highlighted in this review include: an abundant monomer-binding protein that catalyzes nucleotide exchange; a barbed-end capping protein that is dissociated from filament ends by the signaling lipid, phosphatidic acid; a villin-like bundling protein that lacks all Ca(2+)-regulated activities; and a formin family member that is non-processive and is sufficient to generate actin filament bundles. These and other stories motivate a careful description of the properties of plant proteins in vitro as a prelude to greater insight into the molecular mechanism(s) underlying the regulation of actin dynamics in vivo.