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Sample records for actinic defect characterization

  1. Actinic characterization of EUV bump-type phase defects

    SciTech Connect

    Goldberg, Kenneth A.; Mochi, Iacopo; Liang, Ted

    2011-01-10

    Despite tremendous progress and learning with EUV lithography, quantitative experimental information about the severity of point-like phase defects remains in short supply. We present a study of measured, EUV aerial images from a series of well-characterized, open-field, bump-type programmed phase defects, created on a substrate before multilayer deposition.

  2. Pharmacological characterization of actin-binding (-)-doliculide.

    PubMed

    Foerster, Florian; Braig, Simone; Chen, Tao; Altmann, Karl-Heinz; Vollmar, Angelika M

    2014-09-15

    Natural compounds offer a broad spectrum of potential drug candidates against human malignancies. Several cytostatic drugs, which are in clinical use for decades, derive directly from natural sources or are synthetically optimized derivatives of natural lead structures. An eukaryote target molecule to which many natural derived anti-cancer drugs bind to is the microtubule network. Of similar importance for the cell is the actin cytoskeleton, responsible for cell movements, migration of cells and cytokinesis. Nature provides also a broad range of compounds directed against actin as intracellular target, but none of these actin-targeting compounds has ever been brought to clinical trials. One reason why actin-binding compounds have not yet been considered for further clinical investigations is that little is known about their pharmacological properties in cancer cells. Herein, we focused on the closer characterization of doliculide, an actin binding natural compound of marine origin in the breast cancer cell lines MCF7 and MDA-MB-231. We used fluorescence-recovery-after-photobleaching (FRAP) analysis to determine doliculide's early effects on the actin cytoskeleton and rhodamin-phalloidin staining for long-term effects on the actin CSK. After validating the disruption of the actin network, we further investigated the functional effects of doliculide. Doliculide treatment leads to inhibition of proliferation and impairs the migratory potential. Finally, we could also show that doliculide leads to the induction of apoptosis in both cell lines. Our data for the first time provide a closer characterization of doliculide in breast cancer cells and propagate doliculide for further investigations as lead structure and potential therapeutic option as actin-targeting compound.

  3. Actinic imaging of native and programmed defects on a full-field mask

    SciTech Connect

    Mochi, I.; Goldberg, K. A.; Fontaine, B. La; Tchikoulaeva, A.; Holfeld, C.

    2010-03-12

    We describe the imaging and characterization of native defects on a full field extreme ultraviolet (EUV) mask, using several reticle and wafer inspection modes. Mask defect images recorded with the SEMA TECH Berkeley Actinic Inspection Tool (AIT), an EUV-wavelength (13.4 nm) actinic microscope, are compared with mask and printed-wafer images collected with scanning electron microscopy (SEM) and deep ultraviolet (DUV) inspection tools. We observed that defects that appear to be opaque in the SEM can be highly transparent to EUV light, and inversely, defects that are mostly transparent to the SEM can be highly opaque to EUV. The nature and composition of these defects, whether they appear on the top surface, within the multilayer coating, or on the substrate as buried bumps or pits, influences both their significance when printed, and their detectability with the available techniques. Actinic inspection quantitatively predicts the characteristics of printed defect images in ways that may not be possible with non-EUV techniques. As a quantitative example, we investigate the main structural characteristics of a buried pit defect based on EUV through-focus imaging.

  4. In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins.

    PubMed

    Zimmermann, Dennis; Morganthaler, Alisha N; Kovar, David R; Suarez, Cristian

    2016-01-01

    Characterizing the biochemical and biophysical properties of purified proteins is critical to understand the underlying molecular mechanisms that facilitate complicated cellular processes such as cytokinesis. Here we outline in vitro assays to investigate the effects of cytokinesis actin-binding proteins on actin filament dynamics and organization. We describe (1) multicolor single-molecule TIRF microscopy actin assembly assays, (2) "bulk" pyrene actin assembly/disassembly assays, and (3) "bulk" sedimentation actin filament binding and bundling assays.

  5. EUV actinic defect inspection and defect printability at the sub-32 nm half pitch

    SciTech Connect

    Huh, Sungmin; Kearney, Patrick; Wurm, Stefan; Goodwin, Frank; Han, Hakseung; Goldberg, Kenneth; Mochi, Iacopp; Gullikson, Eric M.

    2009-08-01

    Extreme ultraviolet (EUV) mask blanks with embedded phase defects were inspected with a reticle actinic inspection tool (AIT) and the Lasertec M7360. The Lasertec M7360, operated at SEMA TECH's Mask Blank Development Center (MBDC) in Albany, NY, has a sensitivity to multilayer defects down to 40-45 nm, which is not likely sufficient for mask blank development below the 32 nm half-pitch node. Phase defect printability was simulated to calculate the required defect sensitivity for a next generation blank inspection tool to support reticle development for the sub-32 nm half-pitch technology node. Defect mitigation technology is proposed to take advantage of mask blanks with some defects. This technology will reduce the cost of ownership of EUV mask blanks. This paper will also discuss the kind of infrastructure that will be required for the development and mass production stages.

  6. Myopathy mutations in alpha-skeletal-muscle actin cause a range of molecular defects.

    PubMed

    Costa, Céline F; Rommelaere, Heidi; Waterschoot, Davy; Sethi, Kamaljit K; Nowak, Kristen J; Laing, Nigel G; Ampe, Christophe; Machesky, Laura M

    2004-07-01

    Mutations in the gene encoding alpha-skeletal-muscle actin, ACTA1, cause congenital myopathies of various phenotypes that have been studied since their discovery in 1999. Although much is now known about the clinical aspects of myopathies resulting from over 60 different ACTA1 mutations, we have very little evidence for how mutations alter the behavior of the actin protein and thus lead to disease. We used a combination of biochemical and cell biological analysis to classify 19 myopathy mutants and found a range of defects in the actin. Using in vitro expression systems, we probed actin folding and actin's capacity to interact with actin-binding proteins and polymerization. Only two mutants failed to fold; these represent recessive alleles, causing severe myopathy, indicating that patients produce nonfunctional actin. Four other mutants bound tightly to cyclase-associated protein, indicating a possible instability in the nucleotide-binding pocket, and formed rods and aggregates in cells. Eleven mutants showed defects in the ability to co-polymerize with wild-type actin. Some of these could incorporate into normal actin structures in NIH 3T3 fibroblasts, but two of the three tested also formed aggregates. Four mutants showed no defect in vitro but two of these formed aggregates in cells, indicating functional defects that we have not yet tested for. Overall, we found a range of defects and behaviors of the mutants in vitro and in cultured cells, paralleling the complexity of actin-based muscle myopathy phenotypes.

  7. Enhancing native defect sensitivity for EUV actinic blank inspection: optimized pupil engineering and photon noise study

    NASA Astrophysics Data System (ADS)

    Wang, Yow-Gwo; Neureuther, Andrew; Naulleau, Patrick

    2016-03-01

    In this paper, we discuss the impact of optimized pupil engineering and photon noise on native defect sensitivity in EUV actinic blank inspection. Native defects include phase-dominated defects, absorber defects, and defects with a combination of phase and absorption behavior. First, we extend the idea of the Zernike phase contrast (ZPC) method and study the impact of optimum phase shift in the pupil plane on native defect sensitivity, showing a 23% signal-to-noise ratio (SNR) enhancement compare to bright field (BF) for a phase defect with 20% absorption. We also describe the possibility to increase target defect SNR on target defect sizes at the price of losing the sensitivity on smaller (non-critical) defects. Moreover, we show the advantage of the optimized phase contrast (OZPC) method over BF EUV actinic blank inspection. A single focus scan from OZPC has better inspection efficiency over BF. Second, we make a detailed comparison between the phase contrast with apodization (AZPC) method and dark field (DF) method based on defect sensitivity in the presence of both photon shot noise and camera noise. Performance is compared for a variety of photon levels, mask roughness conditions, and combinations of defect phase and absorption.

  8. 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

  9. 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.

  10. Mechanics of F-actin characterized with microfabricated cantilevers.

    PubMed Central

    Liu, Xiumei; Pollack, Gerald H

    2002-01-01

    In this report we characterized the longitudinal elasticity of single actin filaments manipulated by novel silicon-nitride microfabricated levers. Single actin filaments were stretched from zero tension to maximal physiological tension, P(0). The obtained length-tension relation was nonlinear in the low-tension range (0-50 pN) with a resultant strain of approximately 0.4-0.6% and then became linear at moderate to high tensions (approximately 50-230 pN). In this region, the stretching stiffness of a single rhodamine-phalloidin-labeled, 1-microm-long F-actin is 34.5 +/- 3.5 pN/nm. Such a length-tension relation could be characterized by an entropic-enthalpic worm-like chain model, which ascribes most of the energy consumed in the nonlinear portion to overcoming thermal undulations arising from the filament's interaction with surrounding solution and the linear portion to the intrinsic stretching elasticity. By fitting the experimental data with such a worm-like chain model, an estimation of persistence length of approximately 8.75 microm was derived. These results suggest that F-actin is more compliant than previously thought and that thin filament compliance may account for a substantial fraction of the sarcomere's elasticity. PMID:12414703

  11. Comparison of fast 3D simulation and actinic inspection for EUV masks with buries defects

    SciTech Connect

    Clifford, C. H.; Wiraatmadja, S.; Chan, T. T.; Neureuther, A. R.; Goldberg, K. A.; Mochi, I.; Liang, T.

    2009-02-23

    Aerial images for isolated defects and the interactions of defects with features are compared between the Actinic Inspection Tool (AIT) at Lawrence Berkeley National Laboratory (LBNL) and the fast EUV simulation program RADICAL. Comparisons between AIT images from August 2007 and RADICAL simulations are used to extract aberrations. At this time astigmatism was the dominant aberration with a value of 0.55 waves RMS. Significant improvements in the imaging performance of the AIT were made between August 2007 and December 2008. A good match will be shown between the most recent AIT images and RADICAL simulations without aberrations. These comparisons will demonstrate that a large defect, in this case 7nm tall on the surface, is still printable even if it is centered under the absorber line. These comparisons also suggest that the minimum defect size is between 1.5nm and 0.8nm surface height because a 1.5nm defect was printable but a 0.8nm was not. Finally, the image of a buried defect near an absorber line through focus will demonstrate an inversion in the effect of the defect from a protrusion of the dark line into the space to a protrusion of the space into the line.

  12. Defect Characterization Using Two-Dimensional Arrays

    NASA Astrophysics Data System (ADS)

    Velichko, A.; Wilcox, P. D.

    2011-06-01

    2D arrays are able to `view' a given defect from a range of angles leading to the possibility of obtaining richer characterization detail than possible with 1D arrays. In this paper a quantitative comparison of 2D arrays with different element layouts is performed. A technique for extracting the scattering matrix of a defect from the raw 2D array data is also presented. The method is tested on experimental data for characterization of various volumetric defects.

  13. Characterization of an actin-myosin head interface in the 40-113 region of actin using specific antibodies as probes.

    PubMed Central

    Labbé, J P; Méjean, C; Benyamin, Y; Roustan, C

    1990-01-01

    Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the 'multisite interface model', where the two actin sites could modulate each other during S-1 interaction. Images Fig. 1. Fig. 4. PMID:2146951

  14. Multimode model based defect characterization in composites

    NASA Astrophysics Data System (ADS)

    Roberts, R.; Holland, S.; Gregory, E.

    2016-02-01

    A newly-initiated research program for model-based defect characterization in CFRP composites is summarized. The work utilizes computational models of the interaction of NDE probing energy fields (ultrasound and thermography), to determine 1) the measured signal dependence on material and defect properties (forward problem), and 2) an assessment of performance-critical defect properties from analysis of measured NDE signals (inverse problem). Work is reported on model implementation for inspection of CFRP laminates containing delamination and porosity. Forward predictions of measurement response are presented, as well as examples of model-based inversion of measured data for the estimation of defect parameters.

  15. Identification and Characterization of a Candidate Wolbachia pipientis Type IV Effector That Interacts with the Actin Cytoskeleton

    PubMed Central

    Sheehan, Kathy B.; Martin, MaryAnn; Lesser, Cammie F.; Isberg, Ralph R.

    2016-01-01

    ABSTRACT Many bacteria live as intracellular symbionts, causing persistent infections within insects. One extraordinarily common infection is that of Wolbachia pipientis, which infects 40% of insect species and induces reproductive effects. The bacteria are passed from generation to generation both vertically (through the oocyte) and horizontally (by environmental transmission). Maintenance of the infection within Drosophila melanogaster is sensitive to the regulation of actin, as Wolbachia inefficiently colonizes strains hemizygous for the profilin or villin genes. Therefore, we hypothesized that Wolbachia must depend on the host actin cytoskeleton. In this study, we identify and characterize a Wolbachia protein (WD0830) that is predicted to be secreted by the bacterial parasite. Expression of WD0830 in a model eukaryote (the yeast Saccharomyces cerevisiae) induces a growth defect associated with the appearance of aberrant, filamentous structures which colocalize with rhodamine-phalloidin-stained actin. Purified WD0830 bundles actin in vitro and cosediments with actin filaments, suggesting a direct interaction of the two proteins. We characterized the expression of WD0830 throughout Drosophila development and found it to be upregulated in third-instar larvae, peaking in early pupation, during the critical formation of adult tissues, including the reproductive system. In transgenic flies, heterologously expressed WD0830 localizes to the developing oocyte. Additionally, overexpression of WD0830 results in increased Wolbachia titers in whole flies, in stage 9 and 10 oocytes, and in embryos, compared to controls, suggesting that the protein may facilitate Wolbachia’s replication or transmission. Therefore, this candidate secreted effector may play a role in Wolbachia’s infection of and persistence within host niches. PMID:27381293

  16. Characterization of the ATP-G-actin aggregates formed at low potassium chloride concentration.

    PubMed Central

    Grazi, E; Aleotti, A; Ferri, A

    1984-01-01

    The ATP-G-actin aggregates formed by incubation of ATP-G-actin in 7.5 mM-KCl were characterized by electron-microscopical observation, by high-pressure liquid chromatography and by the study of the 1,N6-etheno-ATP-ATP exchange reaction between the free and the actin-bound nucleotide. In 30 mM-KCl the initial rate of the reduced-viscosity increase is found to be directly related to the amount of the aggregates formed in the course of the preincubation in 7.5 mM-KCl. Images Fig. 1. PMID:6721856

  17. Model based defect characterization in composites

    NASA Astrophysics Data System (ADS)

    Roberts, R.; Holland, S.

    2017-02-01

    Work is reported on model-based defect characterization in CFRP composites. The work utilizes computational models of the interaction of NDE probing energy fields (ultrasound and thermography), to determine 1) the measured signal dependence on material and defect properties (forward problem), and 2) an assessment of performance-critical defect properties from analysis of measured NDE signals (inverse problem). Work is reported on model implementation for inspection of CFRP laminates containing multi-ply impact-induced delamination, with application in this paper focusing on ultrasound. A companion paper in these proceedings summarizes corresponding activity in thermography. Inversion of ultrasound data is demonstrated showing the quantitative extraction of damage properties.

  18. Characterization and regulation of an additional actin-filament-binding site in large isoforms of the stereocilia actin-bundling protein espin.

    PubMed

    Zheng, Lili; Beeler, Dina M; Bartles, James R

    2014-03-15

    The espin actin-bundling proteins, which are produced as isoforms of different sizes from a single gene, are required for the growth of hair cell stereocilia. We have characterized an additional actin-filament-binding site present in the extended amino-termini of large espin isoforms. Constitutively active in espin 2, the site increased the size of actin bundles formed in vitro and inhibited actin fluorescence recovery in microvilli. In espin 1, which has an N-terminal ankyrin repeat domain, the site was autoinhibited by binding between the ankyrin repeat domain and a peptide near the actin-binding site. Deletion of this peptide from espin 1 activated its actin-binding site. The peptide resembled tail homology domain I of myosin III, a ligand of the ankyrin repeat domain localized with espin 1 at the tip of stereocilia. A myosin III tail homology domain I peptide, but not scrambled control peptides, inhibited internal binding of the ankyrin repeat domain and released the espin 1 actin-binding site from autoinhibition. Thus, this regulation could result in local activation of the additional actin-binding site of espin 1 by myosin III in stereocilia.

  19. Actin organization in chick embryo fibroblasts after influenza virus infection. I. Isolation and characterization of actin from chick embryo cells.

    PubMed

    Krizanová, O; Závodská, E; Solariková, L; Ciampor, F; Kocisková, D

    1984-05-01

    Comparison of two starting materials for actin purification has shown that preparation of actin from aceton-dried cytoskeleton was more effective than from native chick embryos (CE). The isolated actin formed a single band of Mr = 42-43000 in SDS-PAGE; less purified samples revealed additional faint bands. G form of actin (non-polymerized) inhibited the activity of DNase I, electron microscopy showed actin filaments and bundles formed upon its polymerization. The freshly purified homogeneous actin has not lost its DNase I-inhibiting activity when incubated for 60 min at 35 degrees or 45 degrees C. Older or less purified actin samples kept under similar conditions showed 18-25% decrease of their DNase I-inhibiting activity and a loss of their polymerization ability. Digestion with trypsin caused a decrease of DNase I-inhibiting activity of fresh as well as for older actin samples.

  20. 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

  1. 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

  2. Defect characterization of silicon dendritic web ribbons

    NASA Technical Reports Server (NTRS)

    Cheng, L. J.

    1985-01-01

    Progress made in the study of defect characterization of silicon dendritic web ribbon is presented. Chemical etching is used combined with optical microscopy, as well as the electron beam induced current (EBIC) technique. Thermal annealing effect on carrier lifetime is examined.

  3. Beta environmental fine structure characterization of defects

    NASA Astrophysics Data System (ADS)

    Benedek, G.; Fiorini, E.; Giuliani, A.; Milani, P.; Monfardini, A.; Nucciotti, A.; Prandoni, M. L.; Sancrotti, M.

    1999-04-01

    The fine structure of beta emission (BEFS) due to the interference with the scattered waves from neighboring atoms, analogous to EXAFS, is known to produce oscillations in the Kurie plot. Here we suggest the use of BEFS for characterizing the lattice environment of β-emitting defects located at a distance from the crystal surface not exceeding the mean free path of β-electrons. Examples of defective structures in semiconductors whose atomic arrangement could be conveniently studied with BEFS are tritium-passivated dangling bonds, β-radioactive ions implanted in the crystal lattice or segregated at extended defects such as dislocations, grain boundaries or radiation damage. Also 14C-doped diamond-like materials and other exotic carbon forms, as well as the atomic environment of ions in metal alloys could be good candidate for BEFS. In this work we have calculated the fractional BEFS modulation for 187Re in its ordinary hcp crystal lattice for which experimental data by Cosulich et al. are available. The good correspondence between theory and experiment permits to conclude that BEFS experiments at low temperature are accessible to the present bolometric detection techniques and can provide an expedient method, as compared to EXAFS, for an accurate structural assessment of extended defects in solids.

  4. Cloning and characterization of an actin gene of Chlamys farreri and the phylogenetic analysis of mollusk actins

    NASA Astrophysics Data System (ADS)

    Ma, Hongming; Mai, Kangsen; Liufu, Zhiguo; Xu, Wei

    2007-07-01

    An actingene (CfACTI) was cloned by using RT-PCR, 3’ and 5’RACE from hemocytes of the sea scallop Chlamys farreri. The full length of the transcript is 1 535 bp, which contains a long 3’ un-translated region of 436bp and 59bp of a 5’ un-translated sequence. The open reading frame encodes a polypeptide of 376 amino acids. Sequence comparisons indicated that CfACTI is more closely related to vertebrate cytoplasmic actins than muscle types. Phylogenetic analysis showed that molluscan actins could be generally divided into two categories: muscle and cytoplasmic, although both are similar to vertebrate cytoplasmic actins. It was also inferred that different isotypes existed in muscle or cytoplasma in mollusks. The genomic sequence of CfACTI was cloned and sequenced. Only one intron was detected: it was located between codons 42 and 43 and different from vertebrate actin genes.

  5. HHF35, a muscle-actin-specific monoclonal antibody. I. Immunocytochemical and biochemical characterization.

    PubMed

    Tsukada, T; Tippens, D; Gordon, D; Ross, R; Gown, A M

    1987-01-01

    A monoclonal antibody to muscle cell actin isotypes was produced and characterized. Immunocytochemical analysis of methanol-Carnoy's-fixed, paraffin-embedded human tissue revealed that this antibody, termed HHF35, reacts with skeletal muscle cells, cardiac muscle cells, smooth muscle cells, pericytes, and myoepithelial cells, but is nonreactive with endothelial, epithelial, neural, or connective tissue cells. When assayed by indirect immunofluorescence, HHF35 reacts with microfilament bundles from various cultured mammalian smooth muscle cells, but does not react with cultured human dermal fibroblasts or various epithelial tumor cell lines. In one-dimensional gel electrophoresis immunoblot experiments this antibody detects a 42-kd polypeptide from tissue extracts of uterus, ileum, aorta, diaphragm, and heart and extract from smooth muscle cells. The antibody also reacts with a comigrating 42-kd band of highly purified rabbit skeletal muscle actin. HHF35 is nonreactive on immunoblots of extracts from all tested nonmuscle cell extracts. Immunoelectrophoresis followed by immunoblotting performed in the presence of urea and reducing agents reveals recognition of the alpha isoelectrophoretic variant of actin from skeletal, cardiac, and smooth muscle sources and of the gamma variant from smooth muscle sources. Because HHF35 reacts with virtually all muscle cells, it will be useful as a marker for muscle and muscle-derived cells.

  6. A Novel Alpha Cardiac Actin (ACTC1) Mutation Mapping to a Domain in Close Contact with Myosin Heavy Chain Leads to a Variety of Congenital Heart Defects, Arrhythmia and Possibly Midline Defects

    PubMed Central

    Augière, Céline; Mégy, Simon; El Malti, Rajae; Boland, Anne; El Zein, Loubna; Verrier, Bernard; Mégarbané, André; Deleuze, Jean-François; Bouvagnet, Patrice

    2015-01-01

    Background A Lebanese Maronite family presented with 13 relatives affected by various congenital heart defects (mainly atrial septal defects), conduction tissue anomalies and midline defects. No mutations were found in GATA4 and NKX2-5. Methods and Results A set of 399 poly(AC) markers was used to perform a linkage analysis which peaked at a 2.98 lod score on the long arm of chromosome 15. The haplotype analysis delineated a 7.7 meganucleotides genomic interval which included the alpha-cardiac actin gene (ACTC1) among 36 other protein coding genes. A heterozygous missense mutation was found (c.251T>C, p.(Met84Thr)) in the ACTC1 gene which changed a methionine residue conserved up to yeast. This mutation was absent from 1000 genomes and exome variant server database but segregated perfectly in this family with the affection status. This mutation and 2 other ACTC1 mutations (p.(Glu101Lys) and p.(Met125Val)) which result also in congenital heart defects are located in a region in close apposition to a myosin heavy chain head region by contrast to 3 other alpha-cardiac actin mutations (p.(Ala297Ser),p.(Asp313His) and p.(Arg314His)) which result in diverse cardiomyopathies and are located in a totally different interaction surface. Conclusions Alpha-cardiac actin mutations lead to congenital heart defects, cardiomyopathies and eventually midline defects. The consequence of an ACTC1 mutation may in part be dependent on the interaction surface between actin and myosin. PMID:26061005

  7. Multiscale experimental characterization of solar cell defects

    NASA Astrophysics Data System (ADS)

    Škarvada, Pavel; Škvarenina, Lubomír.; Tománek, Pavel; Sobola, Dinara; Macků, Robert; Brüstlová, Jitka; Grmela, Lubomír.; Smith, Steve

    2016-12-01

    The search for alternative sources of renewable energy, including novel photovoltaics structures, is one of the principal tasks of 21th century development. In the field of photovoltaics there are three generations of solar cells of different structures going from monocrystalline silicon through thin-films to hybrid and organic cells, moreover using nanostructure details. Due to the diversity of these structures, their complex study requires the multiscale interpretations which common core includes an integrated approach bridging not only the length scales from macroscale to the atomistic, but also multispectral investigation under different working temperatures. The multiscale study is generally applied to theoretical aspects, but is also applied to experimental characterization. We investigate multiscale aspects of electrical, optical and thermal properties of solar cells under illumination and in dark conditions when an external bias is applied. We present the results of a research of the micron and sub-micron defects in a crystalline solar cell structure utilizing scanning probe microscopy and electric noise measurement.

  8. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

    PubMed

    Gottwald, U; Brokamp, R; Karakesisoglou, I; Schleicher, M; Noegel, A A

    1996-02-01

    In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.

  9. Identification and characterization of the actin-binding motif of phostensin.

    PubMed

    Wang, Tzu-Fan; Lai, Ning-Sheng; Huang, Kuang-Yung; Huang, Hsien-Lu; Lu, Ming-Chi; Lin, Yu-Shan; Chen, Chun-Yu; Liu, Su-Qin; Lin, Ta-Hsien; Huang, Hsien-Bin

    2012-11-28

    Phostensin, a protein phosphatase 1 F-actin cytoskeleton-targeting subunit encoded by KIAA1949, consists of 165 amino acids and caps the pointed ends of actin filaments. Sequence alignment analyses suggest that the C-terminal region of phostensin, spanning residues 129 to 155, contains a consensus actin-binding motif. Here, we have verified the existence of an actin-binding motif in the C-terminal domain of phostensin using colocalization, F-actin co-sedimentation and single filament binding assays. Our data indicate that the N-terminal region of phostensin (1-129) cannot bind to actin filaments and cannot retard the pointed end elongation of gelsolin-actin seeds. Furthermore, the C-terminal region of phostensin (125-165) multiply bind to the sides of actin filaments and lacks the ability to block the pointed end elongation, suggesting that the actin-binding motif is located in the C-terminal region of the phostensin. Further analyses indicate that phostensin binding to the pointed end of actin filament requires N-terminal residues 35 to 51. These results suggest that phostensin might fold into a rigid structure, allowing the N-terminus to sterically hinder the binding of C-terminus to the sides of actin filament, thus rendering phostensin binding to the pointed ends of actin filaments.

  10. Advances in defect characterizations of semiconductors using positrons

    SciTech Connect

    Lynn, K.G.; Asoka-Kumar, P.

    1996-12-31

    Positron Annihilation Spectroscopy (PAS) is a sensitive probe for studying the electronic structure of defects in solids. The authors summarize recent developments in defect characterization of semiconductors using depth-resolved PAS. The progress achieved in extending the capabilities of the PAS method is also described.

  11. Cloning and characterization of an abalone (Haliotis discus hannai) actin gene

    NASA Astrophysics Data System (ADS)

    Ma, Hongming; Xu, Wei; Mai, Kangsen; Liufu, Zhiguo; Chen, Hong

    2004-10-01

    An actin encoding gene was cloned by using RT-PCR, 3‧ RACE and 5‧ RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3‧ untranslated region of 307 base pairs and 79 base pairs of 5‧ untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin (β-actin) than to human muscle actin.

  12. Crystallization and preliminary structural characterization of the two actin isoforms of the malaria parasite

    PubMed Central

    Bhargav, Saligram Prabhakar; Vahokoski, Juha; Kumpula, Esa-Pekka; Kursula, Inari

    2013-01-01

    Malaria is a devastating disease caused by apicomplexan parasites of the genus Plasmodium that use a divergent actin-powered molecular motor for motility and invasion. Plasmodium actin differs from canonical actins in sequence, structure and function. Here, the purification, crystallization and secondary-structure analysis of the two Plasmodium actin isoforms are presented. The recombinant parasite actins were folded and could be purified to homogeneity. Plasmodium actins I and II were crystallized in complex with the gelsolin G1 domain; the crystals diffracted to resolutions of 1.19 and 2.2 Å and belonged to space groups P212121 and P21, respectively, each with one complex in the asymmetric unit. PMID:24100575

  13. The Characterization of Crack-Like Defects Using Ultrasonic Images

    NASA Astrophysics Data System (ADS)

    Zhang, J.; Velichko, A.; Drinkwater, B. W.; Wilcox, P. D.

    2010-02-01

    The use of ultrasonic arrays to image and size crack-like defects is an important area in non-destructive evaluation. The features in the ultrasonic data from a crack-like defect provide information about the size and orientation angle of the defect. In this paper, the characteristics of a crack-like defect were measured from its scattering coefficient matrix, when the angular coverage of measurement includes the specular regions of its scattering matrix. Alternatively, the imaged features for a large crack-like defect (its length more than two wavelengths) were directly used to characterize the defect through a rectangular box fitting approach. An efficient hybrid model was used to generate the full matrix of array data from samples with a defect and for a specified inspection configuration. This hybrid model combines far-field scattering coefficient matrix for defects with a ray based forward model. This model offers the potential to compile a look-up table through which defects can be classified and then sized. Good agreement was achieved between simulation and experimental results hence validating this model based approach.

  14. Diffusion and Defect Characterization Studies of Mercury Cadmium Telluride

    DTIC Science & Technology

    1989-08-01

    Mercury Cadmium Telluride" Principal Investigator: D. A. Stevenson Department of Materials Science and Engineering Stanford University Stanford, CA 94305...Include Security Classificatton, Difuson Defect Characterization Studies of Mercury Cadmium Telluride 12 PERSONAL AUTHOR(S) ProfessJL Ddvid A. Stevenson 13a...diffusion and defect chemistry of mercury cadmium telluride (MCT; Hg Cd Te). In this study, we have measured tracer self- diffusion and interdiffusion

  15. Actin evolution in ciliates (Protist, Alveolata) is characterized by high diversity and three duplication events.

    PubMed

    Yi, Zhenzhen; Huang, Lijuan; Yang, Ran; Lin, Xiaofeng; Song, Weibo

    2016-03-01

    Ciliates possess two distinct nuclear genomes and unique genomic features, including highly fragmented chromosomes and extensive chromosomal rearrangements. Recent transcriptomic surveys have revealed that ciliates have several multi-copy genes providing an ideal template to study gene family evolution. Nonetheless, this process remains little studied in ciliated protozoa and consequently, the evolutionary patterns that govern it are not well understood. In this study, we focused on obtaining fine-scale information relative to ciliate species divergence for the first time. A total of 230 actin gene sequences were derived from this study, among which 217 were from four closely related Pseudokeronopsis species and 13 from other hypotrichous ciliates. Our investigation shows that: (1) At least three duplication events occurred in ciliates: diversification of three actin genes (Actin I, II, III) happened after the divergence of ciliate classes but before that of subclasses. And several recent and genus-specific duplications were followed within Actin I (Sterkiella, Oxytricha, Uroleptus, etc.), Actin II (Sterkiella), respectively. (2) Within the genus Pseudokeronopsis, Actin I gene duplication events happened after P. carnea and P. erythrina diverged. In contrast, in the morphologically similar species P. flava and P. rubra, the duplication event preceded diversification of the two species. The Actin II gene duplication events preceded divergence of the genus Pseudokeronopsis. (3) Phylogenetic analyses revealed that actin is suitable for resolving ciliate classes, but may not be used to infer lower taxon relationships.

  16. A mutation in the Cc.arp9 gene encoding a putative actin-related protein causes defects in fruiting initiation and asexual development in the agaricomycete Coprinopsis cinerea.

    PubMed

    Nakazawa, Takehito; Ando, Yuki; Hata, Takeshi; Nakahori, Kiyoshi

    2016-08-01

    Agaricomycetes exhibit a remarkable morphological differentiation from vegetative mycelia to huge fruiting bodies. To investigate the molecular mechanism underlying the fruiting body development, we have isolated and characterized many Coprinopsis cinerea mutant strains defective in fruiting initiation to date. Dikaryon formation in agaricomycetes, which is followed by fruiting development, is governed by the mating type loci, A and B. Recently, mutations in the Cc.snf5 gene, which encodes a putative component of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF), were shown to cause defects in A-regulated clamp cell morphogenesis, as well as in fruiting initiation. Here, we demonstrate that Cc.arp9, which encodes a putative actin-related protein associated with two chromatin remodeling complexes, SWI/SNF and remodels the structure of chromatin (RSC), is also essential for fruiting initiation. In contrast to Cc.snf5 mutants, Cc.arp9 mutants were not defective in clamp cell formation. The effects of mutations in Cc.arp9 and Cc.snf5 on oidia production and the transcriptional expression levels of clp1 and pcc1, which are under the control of the A gene, were also examined. These indicated that Cc.Snf5 is involved in A-regulated pathways, whereas Cc.Arp9 is not apparently. Cc.arp9/Cc.snf5 double-gene disruptants were generated and their phenotypes were analyzed, which suggested a complicated developmental regulation mechanism mediated by chromatin remodeling.

  17. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

    PubMed Central

    Gottwald, U; Brokamp, R; Karakesisoglou, I; Schleicher, M; Noegel, A A

    1996-01-01

    In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner. Images PMID

  18. Adhesion characterization and defect sizing of sandwich honeycomb composites.

    PubMed

    Ndiaye, Elhadji Barra; Maréchal, Pierre; Duflo, Hugues

    2015-09-01

    Defects may appear in composite structures during their life cycle. A 10MHz 128 elements phased array transducer was investigated to characterize join bonds and defects in sandwich honeycomb composite structures. An adequate focal law throughout the composite skin gives the ultrasonic dispersive properties of the composite skin and glue layer behind. The resulting B-scan cartographies allow characterizing locally the honeycomb adhesion. Experimental measurements are compared in good agreement with the Debye Series Method (DSM). In the processed C-scan image, flaws are detectable and measurable, localized both in the scanning plane and in the thickness of the composite skin.

  19. Multiple focused EMAT designs for improved surface breaking defect characterization

    NASA Astrophysics Data System (ADS)

    Thring, C. B.; Fan, Y.; Edwards, R. S.

    2017-02-01

    Ultrasonic Rayleigh waves can be employed for the detection of surface breaking defects such as rolling contact fatigue and stress corrosion cracking. Electromagnetic Acoustic Transducers (EMATs) are well suited to this technique as they can directly generate Rayleigh waves within the sample without the requirement for wedges, and they are robust and inexpensive compared to laser ultrasonics. Three different EMAT coil types have been developed, and these are compared to assess their ability to detect and characterize small (down to 0.5 mm depth, 1 mm diameter) surface breaking defects in aluminium. These designs are: a pair of linear meander coils used in a pseudo-pulse-echo mode, a pair of focused meander coils also used in pseudo-pulse-echo mode, and a pair of focused racetrack coils used in pitch-catch mode. The linear meander coils are able to detect most of the defects tested, but have a much lower signal to noise ratio and give limited sizing information. The focused meander coils and the focused racetrack coils can detect all defects tested, but have the advantage that they can also characterize the defect sizes on the sample surface, and have a stronger sensitivity at their focal point. Measurements using all three EMAT designs are presented and compared for high resolution imaging of surface-breaking defects.

  20. Isolation and partial characterization of a 110-kD dimer actin-binding protein

    PubMed Central

    1986-01-01

    Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I. PMID:2942552

  1. Characterization of Periodic Cylindrical Subsurface Defects by Pulsed Flash Thermography

    NASA Astrophysics Data System (ADS)

    Dikić, Goran; Tomić, Ljubiša; Damnjanović, Vesna; Milanović, Bojan

    2015-03-01

    A characterization of cylindrical periodic subsurface defects of different sizes by means of pulsed thermography is presented in the paper. To ensure a uniform thermal flux distribution, the test samples were heated in lab conditions using two photographic flashes. Surface temperature was intentionally recorded at an angle to the normal of the sample surface. Recorded temperatures were compared with simulated temperatures and the differences in temperature peak values and temperature peak positions were noted. The thermal image was transformed based on known positions of four noncollinear points, in order to cancel out errors resulting from image recording at an angle. The uniformity of surface heating and the effect of the positions of the defects on the results were tested by means of a simulation model. The positions did not affect defect characterization. It was also found that in spite of nonuniform heating, if the reference points were selected properly, the difference in temperature contrast was negligible.

  2. 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

  3. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    SciTech Connect

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  4. 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 ...

  5. A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity

    PubMed Central

    1981-01-01

    Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre- extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat- denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat- denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X- 100 extraction of isolated D. discoideum membranes results in a Triton- insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane. PMID:6894148

  6. Characterization of point defects in nonlinear optical materials

    NASA Astrophysics Data System (ADS)

    Chirila, Madalina M.

    Thermoluminescence (TL), optical absorption, and electron paramagnetic resonance (EPR) were used to characterize point defects in LiNbO3 and LiTaO3 Crystals. A broad TL emission, peaking at 440 nm, is observed near 94 K from LiNbO3 when the crystal is irradiated at 77 K and then rapidly warmed. From the LiTaO3 crystals two overlapping TL peaks occur at 94 and 98 K, with each showing a 350-nm maximum in spectral emission. These peaks are observed after 77-K exposure of the crystals to x rays or lasers (266, 325, or 355 nm). During excitation of these crystals at 77 K, holes are trapped on oxygen ions adjacent to lithium vacancies and electrons are trapped on niobium and tantalum ions at regular lattice sites. These defects have characteristic EPR spectra, and the trapped electron center has an optical absorption band peaking at 1200 nm in LiNbO3 and 1600 nm in LiTaO3. Upon warming, the electrons become thermally unstable and migrate to the trapped-hole sites where radiative recombination occurs. Optical absorption and EPR were used to characterize the production and thermal decay of point defects in KD2PO4. A crystal was irradiated at 77 K with x rays and then warmed to room temperature. Immediately after the irradiation broad optical absorption bands were formed at 230, 390, and 550 nm. These bands thermally decayed in the 80 to 140 K range. Another absorption band near 450 nm appeared as the three bands disappeared. Correlations with EPR data suggest that the band at 230-nm is associated with interstitial deuterium atoms, the two bands at 390 and 550 nm are associated with self-trapped holes, and the band at 450 nm is associated with holes trapped adjacent to deuterium vacancies. Results from quantum-mechanical calculations performed with Gaussian 98 were correlated with hyperfine data from EPR measurements for several point defects in KH2PO4. The point defects modeled with calculations are: the self-trapped hole, the proton vacancy, the silicon hole, and the

  7. 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

  8. 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.

  9. Evolution of the Actin Gene Family in Testate Lobose Amoebae (Arcellinida) is Characterized by Two Distinct Clades of Paralogs and Recent Independent Expansions

    PubMed Central

    Lahr, Daniel J. G.; Nguyen, Truc B.; Barbero, Erika; Katz, Laura A.

    2011-01-01

    The evolution of actin gene families is characterized by independent expansions and contractions across the eukaryotic tree of life. Here, we assess diversity of actin gene sequences within three lineages of the genus Arcella, a free-living testate (shelled) amoeba in the Arcellinida. We established four clonal lines of two morphospecies, Arcella hemisphaerica and A. vulgaris, and assessed their phylogenetic relationship within the “Amoebozoa” using small subunit ribosomal DNA (SSU-rDNA) genealogy. We determined that the two lines of A. hemisphaerica are identical in SSU-rDNA, while the two A. vulgaris are independent genetic lineages. Furthermore, we characterized multiple actin gene copies from all lineages. Analyses of the resulting sequences reveal numerous diverse actin genes, which differ mostly by synonymous substitutions. We estimate that the actin gene family contains 40–50 paralogous members in each lineage. None of the three independent lineages share the same paralog with another, and divergence between actins reaches 29% in contrast to just 2% in SSU-rDNA. Analyses of effective number of codons (ENC), compositional bias, recombination signatures, and genetic diversity in the context of a gene tree indicate that there are two groups of actins evolving with distinct patterns of molecular evolution. Within these groups, there have been multiple independent expansions of actin genes within each lineage. Together, these data suggest that the two groups are located in different regions of the Arcella genome. Furthermore, we compare the Arcella actin gene family with the relatively well-described gene family in the slime mold Dictyostelium discoideum and other members of the Amoebozoa clade. Overall patterns of molecular evolution are similar in Arcella and Dictyostelium. However, the separation of genes in two distinct groups coupled with recent expansion is characteristic of Arcella and might reflect an unusual pattern of gene family evolution in the

  10. Defect localization, characterization and reliability assessment in emerging photovoltaic devices.

    SciTech Connect

    Yang, Benjamin Bing-Yeh; Cruz-Campa, Jose Luis; Haase, Gad S.; Tangyunyong, Paiboon; Colr, Edward Isaac; Okandan, Murat; Nielson, Gregory N.

    2014-04-01

    Microsystems-enabled photovoltaics (MEPV) can potentially meet increasing demands for light-weight, portable, photovoltaic solutions with high power density and efficiency. The study in this report examines failure analysis techniques to perform defect localization and evaluate MEPV modules. CMOS failure analysis techniques, including electroluminescence, light-induced voltage alteration, thermally-induced voltage alteration, optical beam induced current, and Seabeck effect imaging were successfully adapted to characterize MEPV modules. The relative advantages of each approach are reported. In addition, the effects of exposure to reverse bias and light stress are explored. MEPV was found to have good resistance to both kinds of stressors. The results form a basis for further development of failure analysis techniques for MEPVs of different materials systems or multijunction MEPVs. The incorporation of additional stress factors could be used to develop a reliability model to generate lifetime predictions for MEPVs as well as uncover opportunities for future design improvements.

  11. Functional characterization of skeletal F-actin labeled on the NH2-terminal segment of residues 1-28.

    PubMed

    Bertrand, R; Chaussepied, P; Audemard, E; Kassab, R

    1989-05-15

    Rabbit skeletal alpha-actin was covalently labeled in the filamentous state by the fluorescent nucleophile, N-(5-sulfo-1-naphthyl)ethylenediamine (EDANS) in the presence of the carboxyl group activator 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDC). The coupling reaction was continued until the incorporation of nearly 1 mol EDANS/mol actin. After limited proteolytic digestion of the labeled protein and chromatographic identification of the EDANS-peptides, about 80% of the attached fluorophore was found on the actin segment of residues 1-28, most probably within the N-terminal acidic region of residues 1-7. A minor labeling site was located on the segment that consists of residues 40-113. No label was incorporated into the COOH-terminal moiety consisting of residues 113-375. The isolated EDANS-G-actin undergoes polymerization in the presence of salts but at a rate significantly greater than unlabeled actin. The EDANS-F-actin could be complexed to skeletal chymotryptic myosin subfragment 1 (S-1) and to tropomyosin. The complex formed between EDANS-F-actin and S-1 could not be further crosslinked by EDC but the two proteins were readily joined by glutaraldehyde as observed for native actin-S-1, suggesting that the EDANS-substituted carboxyl site is also involved in the EDC crosslinking of native actin to S-1. Moreover, the EDANS labeling of F-actin resulted in a 20-fold increase in the Km of the actin-activated Mg2+.ATPase of S-1. Thus, this labeling, while it did not much affect the rigor actin-S-1 interaction, changes the actin binding to the S-1-nucleotide complexes significantly. The selective introduction of a variety of spectral probes, like EDANS, or other classes of fluorophores, on the N-terminal region of actin, through the reported carbodiimide coupling reaction, would provide several different derivatives valuable for assessing the functional role of the negatively charged N-terminus of actin during its interaction with myosin and other actin

  12. Identification of Arabidopsis cyclase-associated protein 1 as the first nucleotide exchange factor for plant actin.

    PubMed

    Chaudhry, Faisal; Guérin, Christophe; von Witsch, Matthias; Blanchoin, Laurent; Staiger, Christopher J

    2007-08-01

    The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP-actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP- and ATP-monomeric actin (Kd approximately 1.3 microM). Binding of AtCAP1 to ATP-actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux

  13. Characterization of defective interfering RNAs associated with RNA plant viruses

    SciTech Connect

    Morris, T.J. . School of Biological Sciences); Jackson, A.O. . Dept. of Plant Pathology)

    1993-01-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since the original observation with TBSV, we discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), and many other reports have now appeared characterizing DI and DI-like RNAs in other plant viral infections. We are seeking to improve our understanding of the mechanisms of DI generation and the precise nature of the RNA sequences necessary for DI replication and encapsidation. We also want to address the nature of the DI mediated symptom attenuation and interference effects in plants, and to determine the feasibility of using transgenic plants constitutively expressing DI RNAs for disease control. The progress made on each of these objectives is summarized along with the proposed experiments for the continuation period.

  14. Characterizing and Targeting Replication Stress Response Defects in Breast Cancer

    DTIC Science & Technology

    2013-08-01

    collaboration with our colleague Dr. Chun Li, an outstanding leader in nanotechnology , we aimed to develop nanoparticles that can carry in vivo imaging...Pɘ.001. Task 4b. To develop nanoparticles to kill RSR-defective breast cancer cells through their binding to the RSR-defect-specific membrane...proteins on cancer cells. We have created the nanoparticles that can specifically bind to RSR-defect breast cells. We will continue to optimize

  15. 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.

  16. Characterization of line defects in CVD graphene films with scanning plasmon interferometry

    NASA Astrophysics Data System (ADS)

    Fei, Zhe; Rodin, Aleksandr; Gannett, Will; Dai, Siyuan; Regan, William; McLeod, Alexander; Wagner, Martin; Aleman, Benji; Thiemens, Mark; Dominguez, Gerardo; Castro-Neto, Antonio; Zettle, Alex; Keilmann, Fritz; Fogler, Michael; Basov, Dimitri

    2013-03-01

    Line defects that are omnipresent in graphene films fabricated with chemical vapor deposition method (CVD) were studied with scanning plasmon interferometry (SPI)--a technique capable of convenient nano-characterization of graphene devices in ambient conditions. The characteristic SPI patterns of line defects are plasmonic twin fringes, which are generated due to interference between surface plasmons (SPs) of graphene launched by a scanning probe and reflected by the line defects. The twin fringes allow us to visualize and distinguish various types of line defects including cracks, wrinkles, and even grain boundaries. Further modeling of the twin fringes provides detailed information on the electronic properties associated with these line defects.

  17. Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis

    PubMed Central

    1987-01-01

    We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm- mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells. PMID:3680376

  18. Identification and Characterization of Aspergillus Nidulans Mutants Defective in Cytokinesis

    PubMed Central

    Harris, S. D.; Morrell, J. L.; Hamer, J. E.

    1994-01-01

    Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokinesis mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis. PMID:8150280

  19. A scanning defect mapping system for semiconductor characterization

    NASA Technical Reports Server (NTRS)

    Sopori, Bushnan L.

    1994-01-01

    We have developed an optical scanning system that generates maps of the spatial distributions of defects in single and polycrystalline silicon wafers. This instrument, called Scanning Defect Mapping System, utilizes differences in the scattering characteristics of dislocation etch pits and grain boundaries from a defect-etched sample to identify and count them. This system simultaneously operates in the dislocation mode and the grain boundary (GB) mode. In the 'dislocation mode,' the optical scattering from the etch pits is used to statistically count dislocations, while ignoring the GB's. Likewise, in the 'grain boundary mode' the system only recognizes the local scattering from the GB's to generate grain boundary distributions. The information generated by this instrument is valuable for material quality control, identifying mechanisms of defect generation and the nature of thermal stresses during the crystal growth, and the solar cell process design.

  20. Characterization of Halobacterium halobium mutants defective in taxis.

    PubMed

    Sundberg, S A; Alam, M; Lebert, M; Spudich, J L; Oesterhelt, D; Hazelbauer, G L

    1990-05-01

    Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.

  1. Electronic characterization of defects in narrow gap semiconductors

    NASA Technical Reports Server (NTRS)

    Patterson, James D.

    1993-01-01

    The study of point defects in semiconductors has a long and honorable history. In particular, the detailed understanding of shallow defects in common semiconductors traces back to the classic work of Kohn and Luttinger. However, the study of defects in narrow gap semiconductors represents a much less clear story. Here, both shallow defects (caused by long range potentials) and deep defects (from short range potentials) are far from being completely understood. In this study, all results are calculational and our focus is on the chemical trend of deep levels in narrow gap semiconductors. We study substitutional (including antisite), interstitial and ideal vacancy defects. For substitutional and interstitial impurities, the efects of relaxation are included. For materials like Hg(1-x)Cd(x)Te, we study how the deep levels vary with x, of particular interest is what substitutional and interstitial atoms yield energy levels in the gap i.e. actually produce deep ionized levels. Also, since the main technique utilized is Green's functions, we include some summary of that method.

  2. Effect of roughness on imaging and characterizing rough crack-like defect using ultrasonic arrays

    NASA Astrophysics Data System (ADS)

    Zhang, J.; Drinkwater, B. W.; Wilcox, P. D.

    2012-05-01

    All naturally occurring crack-like defects in solid structures are rough to some degree, which can affect defect inspection and characterization. Based on the simulated array data for various rough cracks and the total focusing method imaging algorithm, the effect of roughness on defect imaging and characterization was discussed. The array data was simulated by using the forward model combining with scattering matrices for various rough cracks. The scattering matrix describes the scattering field of a scatterer from all possible incident and scattering directions. It is shown that roughness can be either beneficial or detrimental to the detectability of a crack-like defect, depending on the defect characteristics such as length, roughness, correlation length, orientation angle, and array inspection configuration. It is also shown that roughness can cause the underestimation of length of rough crack-like defects by using the image-based approach.

  3. Characterization and phylogeny of two beta-cytoskeletal actins from Hemibarbus mylodon (Cyprinidae, Cypriniformes), a threatened fish species in Korea.

    PubMed

    Kim, Keun-Yong; Lee, Sang Yoon; Cho, Young Sun; Bang, In Chul; Kim, Dong Soo; Nam, Yoon Kwon

    2008-04-01

    Complementary DNA and genomic sequences representing two different beta-actins were isolated from a threatened freshwater fish species Hemibarbus mylodon. The beta-actin 1 and 2 encoded an identical number of amino acids (375 aa), and shared 88.8 and 99.7% of identity at coding nucleotide and amino acid levels, respectively. Genomic open reading frame (ORF) sequences of both isoforms contained five translated exons interrupted by four introns with conserved GT/AG exon/intron boundary rule. Semi-quantitative RT-PCR showed that the two isoform mRNAs were ubiquitously detected in all tissues tested, but transcript levels were variable across tissues. Phylogenetic analysis showed that H. mylodon beta-actin 1 and 2 were clustered into two distinct major and minor branches of Cypriniformes, respectively. Comparisons of the 5'-upstream region and 3'-UTR of H. mylodon beta-actin 1 also showed a high degree of homology with those of the major teleost beta-actins and warmblooded vertebrate beta-cytoskeletal actins, suggesting their more recent common origin.

  4. CMOS standard cells characterization for open defects for test pattern generation

    NASA Astrophysics Data System (ADS)

    Wielgus, Andrzej; Pleskacz, Witold

    2016-12-01

    This paper presents an extended method of CMOS standard cells characterization for defect based voltage testing. It allows to estimate the probabilities of physical open defects occurrences in a cell, describes its faulty behavior caused by the defects and finds the test sequences that detect those faults. Finally, the minimal set of test sequences is selected to cover all detectable faults and the optimal complex test sequence is constructed. Experimental results for cells from industrial standard cell library are presented as well.

  5. Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

    PubMed Central

    Lu, Hailong; Fagnant, Patricia M.; Bookwalter, Carol S.; Joel, Peteranne; Trybus, Kathleen M.

    2015-01-01

    Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD. PMID:26153420

  6. Non-Contact Ultrasonic Characterization of Angled Surface Defects

    NASA Astrophysics Data System (ADS)

    Edwards, R. S.; Dutton, B.; Rosli, M. H.; Clough, A. R.

    2011-06-01

    Surface ultrasonic waves have been shown to have many uses in non-destructive testing, in particular for gauging the depth of surface defects. Much of the previous work has assumed that these defects are oriented normal to the surface. However, this is not always the case; for example, rolling contact fatigue in rails propagates at an angle of around 25° to the surface, and this angle may affect the characterisation. We present results using non-contact ultrasonic methods to generate and detect ultrasound on samples with a range of defect angles, and compare these with finite element method (FEM) models. We use both electromagnetic acoustic transducers (EMATs) and laser ultrasound. The depth calibration when measuring ultrasound transmission is considered, and what affect the angle of a defect has. Several other methods of characterising crack depth and angle are also discussed, including the arrival times of reflected and mode-converted waves, the delay in the transmission of the high-frequency Rayleigh wave, and the enhancement of the signal at the defect in both the in-plane and out-of-plane components.

  7. Characterization of the nitrogen split interstitial defect in wurtzite aluminum nitride using density functional theory

    SciTech Connect

    Szállás, A.; Szász, K.; Gali, A.

    2014-09-21

    We carried out Heyd-Scuseria-Ernzerhof hybrid density functional theory plane wave supercell calculations in wurtzite aluminum nitride in order to characterize the geometry, formation energies, transition levels, and hyperfine tensors of the nitrogen split interstitial defect. The calculated hyperfine tensors may provide useful fingerprint of this defect for electron paramagnetic resonance measurement.

  8. Automated objective characterization of visual field defects in 3D

    NASA Technical Reports Server (NTRS)

    Fink, Wolfgang (Inventor)

    2006-01-01

    A method and apparatus for electronically performing a visual field test for a patient. A visual field test pattern is displayed to the patient on an electronic display device and the patient's responses to the visual field test pattern are recorded. A visual field representation is generated from the patient's responses. The visual field representation is then used as an input into a variety of automated diagnostic processes. In one process, the visual field representation is used to generate a statistical description of the rapidity of change of a patient's visual field at the boundary of a visual field defect. In another process, the area of a visual field defect is calculated using the visual field representation. In another process, the visual field representation is used to generate a statistical description of the volume of a patient's visual field defect.

  9. Ion beam nanopatterning in graphite: characterization of single extended defects.

    PubMed

    Mélinon, P; Hannour, A; Bardotti, L; Prével, B; Gierak, J; Bourhis, E; Faini, G; Canut, B

    2008-06-11

    The morphology and the electronic structure of a single focused ion-beam-induced artificial extended defect is probed by several methods including micro-Raman spectroscopy, atomic force and scanning tunneling microscopies and Monte Carlo and/or semi-analytical simulation within standard codes. The efficiency of the artificial defect for deposited metallic cluster pinning is also investigated. We show a correlation between the ion dose, morphology, electronic structure and cluster trapping efficiency. At room temperature, cluster pinning is efficient when the displacement per atom is one or more. Well-ordered patterned cluster networks are considered for potential applications.

  10. Preparation and characterization of low-defect surfaces

    SciTech Connect

    Robinson, T.O.

    1991-12-01

    Silver crystal surfaces with low defect densities were prepared electrochemically from aqueous solutions using capillary-growth techniques. These surfaces had low rates for the nucleation of new silver layers. The impedance of these inert silver/aqueous silver nitrate interfaces was used to determine silver adatom concentration and water dipole reorientation energetics.

  11. 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.

  12. 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.

  13. Characterizing and Targeting Replication Stress Response Defects in Breast Cancer

    DTIC Science & Technology

    2015-08-01

    oncogene activation or loss of tumor suppressor genes induces stalling and collapse of DNA replication forks, which in turn activates the replication...stress response (RSR) to maintain genome integrity [1-4]. RSR is a subset of the DNA damage response that safeguards the replication process [5]; defects...Taiwan, and one poster presentation by my postdoctoral fellow at the Conference of Exploring DNA Repair Pathways as Targets for Cancer at Cancun

  14. Characterizing and Targeting Replication Stress Response Defects in Breast Cancer

    DTIC Science & Technology

    2012-08-01

    Li, an outstanding leader in nanotechnology , we aimed to develop nanoparticles that can carry in vivo imaging agents to target breast cancer cells...and develop RSR-defect-targeting nanoparticles for diagnostic imaging, prevention, and treatment of breast cancer . As demonstrated in our first...antibody to hollow gold nanoparticles (HAuNP). REPORTABLE OUTCOMES Part of the our study has led to a publication in Cancer Research (7 and

  15. Electronic characterization of defects in narrow gap semiconductors

    NASA Technical Reports Server (NTRS)

    Patterson, James D.

    1994-01-01

    We use a Green's function technique to calculate the position of deep defects in narrow gap semiconductors. We consider substitutional (including antisite), vacancy, and interstitial (self and foreign) deep defects. We also use perturbation theory to look at the effect of nonparabolic bands on shallow defect energies and find nonparabolicity can increase the binding by 10 percent or so. We consider mercury cadmium telluride (MCT), mercury zinc telluride (MZT), and mercury zinc selenide (MZS). For substitutional and interstitial defects we look at the situation with and without relaxation. For substitutional impurities in MCT, MZT, and MZS, we consider x (the concentration of Cd or Zn) in the range 0.1 less than x less than 0.3 and also consider appropriate x so E(sub g) = 0.1 eV for each of the three compounds. We consider several cation site s-like deep levels and anion site p-like levels. For E(sub g) = 0.1 eV, we also consider the effects of relaxation. Similar comments apply to the interstitial deep levels whereas no relaxation is considered for the ideal vacancy model. Relaxation effects can be greater for the interstitial than the substitutional cases. Specific results are given in figures and tables and comparison to experiment is made in a limited number of cases. We find, for example, that I, Se, S, Rn, and N are possible cation site, s-like deep levels in MCT and Zn and Mg are for anion site, p-like levels (both levels for substitutional cases). The corresponding cation and anion site levels for interstitial deep defects are (Au, Ag, Hg, Cd, Cu, Zn) and (N, Ar, O, F). For the substitutional cases we have some examples of relaxation moving the levels into the band gap, whereas for the interstitial case we have examples where relaxation moves it out of the band gap. Future work involves calculating the effects of charge state interaction and seeing the effect of relaxation on vacancy levels.

  16. Transport Effects on Capacitance-Frequency Analysis for Defect Characterization in Organic Photovoltaic Devices

    NASA Astrophysics Data System (ADS)

    Xu, Liang; Wang, Jian; Hsu, Julia W. P.

    2016-12-01

    Using capacitance-frequency (C -f ) analysis to characterize the density-of-states (DOS) distribution of defects has been well established for inorganic thin-film photovoltaic devices. While C -f analysis has also been applied to bulk-heterojunction (BHJ) organic photovoltaic (OPV) devices, we show that the low carrier mobility in the BHJ material can severely alter the C -f behaviors and lead to misinterpretations. Because of the complicated nature of disorders in organic materials, artifacts from an erroneous C -f analysis are difficult to identify. Here we compare drift-diffusion simulations with experiments to reveal situations when the validity of C -f analysis for defect characterization breaks down. When a flat-band region is present in the low-mobility active layer, the capacitive response cannot follow the electrical modulation and behaves as if the active layer is a dielectric at frequencies higher than the characteristic frequency determined by carrier mobility and thickness. The transition produces a fictitious shallow defect when defect analysis is applied. Even in fully depleted devices, the defect distributions derived from C -f analysis can appear at spuriously deeper energies if the mobility is too low. Through simulations, we determine the ranges of mobility and thickness for which the C -f analysis can effectively yield credible defect DOS information. Insight from this study also sheds light on transport limitation when using capacitance spectroscopy for defect characterization in general.

  17. A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor

    PubMed Central

    1990-01-01

    Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross- linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays. PMID:1698791

  18. 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

  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. Definitive molecular level characterization of defects in UiO-66 crystals.

    PubMed

    Trickett, Christopher A; Gagnon, Kevin J; Lee, Seungkyu; Gándara, Felipe; Bürgi, Hans-Beat; Yaghi, Omar M

    2015-09-14

    The identification and characterization of defects, on the molecular level, in metal-organic frameworks (MOFs) remain a challenge. With the extensive use of single-crystal X-ray diffraction (SXRD), the missing linker defects in the zirconium-based MOF UiO-66, Zr6 O4 (OH)4 (C8 H4 O4 )6 , have been identified as water molecules coordinated directly to the zirconium centers. Charge balancing is achieved by hydroxide anions, which are hydrogen bonded within the pores of the framework. Furthermore, the precise nature of the defects and their concentration can be manipulated by altering the starting materials, synthesis conditions, and post-synthetic modifications.

  1. Application of DLTS and Laplace-DLTS to defect characterization in high-resistivity semiconductors

    NASA Astrophysics Data System (ADS)

    Makarenko, L. F.; Evans-Freeman, J. H.

    2007-12-01

    This paper shows that the use of conventional analytical procedures to determine defect parameters from DLTS spectra may lead to erroneous results for high-resistivity semiconductors. The effect is observed when the temperature range of a DLTS peak encompasses the temperature at which the equilibrium Fermi level intersects the energy level of defect under study. Based on this Fermi level effect, a procedure is proposed to determine the occupancy levels for defects with a strong temperature dependence of the carrier capture cross-section. It has been found that the procedure can be useful for characterization of positive-U states through to negative-U centers in semiconductors.

  2. Functional characterization of protein 4.1 homolog in amphioxus: defining a cryptic spectrin-actin-binding site.

    PubMed

    Wang, Lixia; Wang, Yuan; Li, Zhaohe; Gao, Zhan; Zhang, Shicui

    2013-10-07

    Vertebrate 4.1 proteins have a spectrin-actin-binding (SAB) domain, which is lacking in all the invertebrate 4.1 proteins indentified so far, and it was therefore proposed that the SAB domain emerged with the advent of vertebrates during evolution. Here we demonstrated for the first time that amphioxus (an invertebrate chordate) protein 4.1, though lacking a recognizable SAB, was able to bind both spectrin and actin, with a binding capacity comparable to that of human protein 4.1. Detailed structure-activity analyses revealed that the unique domain U2/3 was a newly identified SAB-like domain capable of interacting with spectrin and actin, suggesting the presence of a "cryptic" SAB domain in amphioxus 4.1 protein. We also showed that amphioxus 4.1 protein gene was the common ancestor of vertebrate 4.1 protein genes, from which 4.1R, 4.1N, 4.1G, and 4.1B genes originated. This work will encourage further study on the structure-activity of invertebrate 4.1 protein and its interacting proteins.

  3. Selective chemical imaging of static actin in live cells.

    PubMed

    Milroy, Lech-Gustav; Rizzo, Stefano; Calderon, Abram; Ellinger, Bernhard; Erdmann, Silke; Mondry, Justine; Verveer, Peter; Bastiaens, Philippe; Waldmann, Herbert; Dehmelt, Leif; Arndt, Hans-Dieter

    2012-05-23

    We have characterized rationally designed and optimized analogues of the actin-stabilizing natural products jasplakinolide and chondramide C. Efficient actin staining was achieved in fixed permeabilized and non-permeabilized cells using different combinations of dye and linker length, thus highlighting the degree of molecular flexibility of the natural product scaffold. Investigations into synthetically accessible, non-toxic analogues have led to the characterization of a powerful cell-permeable probe to selectively image static, long-lived actin filaments against dynamic F-actin and monomeric G-actin populations in live cells, with negligible disruption of rapid actin dynamics.

  4. Defect chemistry and characterization of (Hg, Cd)Te

    NASA Technical Reports Server (NTRS)

    Vydyanath, H. R.

    1981-01-01

    Single crystal samples of phosphorus doped Hg sub 0.8 Cd sub 0.2 Te were anneald at temperatures varying from 450 C to 600 C in various Hg atmospheres. The samples were quenched to room temperature from the annealing temperatures. Hall effect and mobility measurements were performed at 77 K on all these samples. The results indicate the crystals to be p type for a total phosphorus concentration of 10 to the 19th power/cu cm in all the samples. The hole concentration at 77 K increases with increasing Hg pressures at 450 C and 500 C contrary to the observation in undoped crystals. Also, at low Hg pressures the concentration of holes in the phosphorus doped crystals is lower than in the undoped crystals. The hole concentration in all the samples is lower than the intrinsic carrier concentration at the annealing temperatures. The hole mobility in the doped crystals is similar to that in the undoped crystals. A defect model according to which phosphorus behaves as a single acceptor interstitially, occupying Te lattice sites while it acts as a single donor occupying Hg lattice sites was established. Equilibrum constants established for the incorporation of all the phosphorus species explain the experimental results

  5. Structural Characterization of the Binding of Myosin*ADP*Pi to Actin in Permeabilized Rabbit Psoas Muscle

    SciTech Connect

    Xu,S.; Gu, J.; Belknap, B.; White, H.; Yu, L.

    2006-01-01

    When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A{center_dot}M{center_dot}ADP and A{center_dot}M) and the weakly bound A{center_dot}M{center_dot}ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ('stereospecific' attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A{center_dot}M{center_dot}ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A{center_dot}M{center_dot}ADP{center_dot}P{sub i}, however, is poorly understood. This state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A{center_dot}M{center_dot}ADP{center_dot}P{sub i} state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M{center_dot}ATP, M{center_dot}ADP{center_dot}P{sub i} states and the weakly attached A{center_dot}M{center_dot}ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A{center_dot}M{center_dot}ADP{center_dot}P{sub i}. The series of experiments presented in this article were carried out under relaxing conditions at 25{sup o}C, where {approx}95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in

  6. 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.

  7. Actinic imaging and evaluation of phase structures on EUV lithography masks

    SciTech Connect

    Mochi, Iacopo; Goldberg, Kenneth; Huh, Sungmin

    2010-09-28

    The authors describe the implementation of a phase-retrieval algorithm to reconstruct phase and complex amplitude of structures on EUV lithography masks. Many native defects commonly found on EUV reticles are difficult to detect and review accurately because they have a strong phase component. Understanding the complex amplitude of mask features is essential for predictive modeling of defect printability and defect repair. Besides printing in a stepper, the most accurate way to characterize such defects is with actinic inspection, performed at the design, EUV wavelength. Phase defect and phase structures show a distinct through-focus behavior that enables qualitative evaluation of the object phase from two or more high-resolution intensity measurements. For the first time, phase of structures and defects on EUV masks were quantitatively reconstructed based on aerial image measurements, using a modified version of a phase-retrieval algorithm developed to test optical phase shifting reticles.

  8. CASE REPORT Reconstruction and Characterization of Composite Mandibular Defects Requiring Double Skin Paddle Fibular Free Flaps

    PubMed Central

    Badeau, Austin M.; Deleyiannis, Frederic W.-B.

    2013-01-01

    Objective: Fibular free flaps are the preferred method for reconstruction of composite lateral mandibular defects. This reconstructive technique is limited by the skin paddle's inability to freely rotate when attempting to fill 2 poorly aligned defects. Reconstructive surgeons have been exploring multiple methods of creating 2 independent skin paddles based on the same peroneal blood supply. We present a variation of these techniques. Method: Our patient with a history of squamous cell carcinoma presented with a left retromolar recurrence and osteoradionecrosis of the mandible with a draining anterior sinus tract. The combination of these defects warranted further composite resection with fibular free flap reconstruction. Results: A subperiosteal dissection was performed to create 2 separate septocutaneous skin paddles based on the same peroneal blood supply. This dissection and discard of proximal fibula provided the rotational freedom needed for the 2 skin islands to fill both a lateral oral defect and anterior cutaneous defect. Conclusion: Although similar reconstructive methods have been reported in the literature, the characterization of defects benefiting from these techniques is scarce and unclear. We describe clear and concise characteristics of these defects, which should be meaningful to the reconstructive surgeon when considering operative technique. PMID:23653822

  9. Using Deep Level Transient Spectroscopy (DLTS) to characterize defects in semiconductor devices

    NASA Astrophysics Data System (ADS)

    Lang, David

    2012-02-01

    Deep Level Transient Spectroscopy (DLTS) is a member of the class of instrumentation methods that utilizes the detection of trapped electronic charge to characterize defects in solids. Such methods detect this charge either directly, e.g. via capacitance measurements, or indirectly, e.g. via the current associated with the release of trapped charge. These types of instrumentation have been widely used since the dawn of solid-state physics, particularly for nonradiative defects in semiconductors and insulators. In the case of semiconductor devices, the highly sensitive capacitive detection of trapped charge in the junction depletion layer makes these methods particularly powerful. The DLTS method introduced the concept of time-domain filtering (the so-called ``rate window'') to create a defect spectrum from the transient response of the device versus temperature. This talk will give an overview of DLTS, with particular emphasis on the correlation between defects and device performance.

  10. Two-stage neural algorithm for defect detection and characterization uses an active thermography

    NASA Astrophysics Data System (ADS)

    Dudzik, Sebastian

    2015-07-01

    In the paper a two-stage neural algorithm for defect detection and characterization is presented. In order to estimate the defect depth two neural networks trained on data obtained using an active thermography were employed. The first stage of the algorithm is developed to detect the defect by a classification neural network. Then the defects depth is estimated using a regressive neural network. In this work the results of experimental investigations and simulations are shown. Further, the sensitivity analysis of the presented algorithm was conducted and the impacts of emissivity error and the ambient temperature error on the depth estimation errors were studied. The results were obtained using a test sample made of material with a low thermal diffusivity.

  11. Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity.

    PubMed Central

    Lazzarino, D A; Boldogh, I; Smith, M G; Rosand, J; Pon, L A

    1994-01-01

    Sedimentation assays were used to demonstrate and characterize binding of isolated yeast mitochondria to phalloidin-stabilized yeast F-actin. These actin-mitochondrial interactions are ATP sensitive, saturable, reversible, and do not depend upon mitochondrial membrane potential. Protease digestion of mitochondrial outer membrane proteins or saturation of myosin-binding sites on F-actin with the S1 subfragment of skeletal myosin block binding. These observations indicate that a protein (or proteins) on the mitochondrial surface mediates ATP-sensitive, reversible binding of mitochondria to the lateral surface of microfilaments. Actin copurifies with mitochondria during subcellular fractionation and is released from the organelle upon treatment with ATP. Thus, actin-mitochondrial interactions resembling those observed in vitro may also exist in intact yeast cells. Finally, a yeast mutant bearing a temperature-sensitive mutation in the actin-encoding ACT1 gene (act1-3) displays temperature-dependent defects in transfer of mitochondria from mother cells to newly developed buds during yeast cell mitosis. Images PMID:7812049

  12. 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

  13. Integrated electrical and SEM-based defect characterization for rapid yield ramp

    NASA Astrophysics Data System (ADS)

    Orbon, Jacob; Levin, Lior; Bokobza, Ofer; Shimshi, Rinat; Dutta, Manjari; Zhang, Brian; Ciplickas, Dennis; Pham, Teri; Jensen, Jim

    2004-04-01

    Challenges of the new nanometer processes have complicated the yield enhancement process. The systematic yield loss component is increasing, due to the complexity and density of the new processes and the designs that are developed for them. High product yields can now only be achieved when process failure rates are on the order of a few parts per billion structures. Traditional yield ramping techniques cannot ramp yields to these levels and new methods are required. This paper presents a new systematic approach to yield loss pareto generation. The approach uses a sophisticated Design-of-Experiments (DOE) approach to characterize systematic and random yield loss mechanisms in the Back End Of the Line (BEOL). Sophisticated Characterization Vehicle (CV)TM test chips, fast electrical test and Automatic Defect Localization (ADL) are critical components of the method. Advanced statistical analysis and visualization of the detected and localized electrical defects provides a comprehensive view of the yield loss mechanisms. In situations where the defects are not visible in a SEM of the structure surface, automated FIB and imaging is used to characterize the defect. The combined approach provides the required resolution to appropriately characterize parts per billion failure rates.

  14. High expression of Lifeact in Arabidopsis thaliana reduces dynamic reorganization of actin filaments but does not affect plant development.

    PubMed

    van der Honing, Hannie S; van Bezouwen, Laura S; Emons, Anne Mie C; Ketelaar, Tijs

    2011-10-01

    Lifeact is a novel probe that labels actin filaments in a wide range of organisms. We compared the localization and reorganization of Lifeact:Venus-labeled actin filaments in Arabidopsis root hairs and root epidermal cells of lines that express different levels of Lifeact: Venus with that of actin filaments labeled with GFP:FABD2, a commonly used probe in plants. Unlike GFP:FABD2, Lifeact:Venus labeled the highly dynamic fine F-actin in the subapical region of tip-growing root hairs. Lifeact:Venus expression at varying levels was not observed to affect plant development. However, at expression levels comparable to those of GFP:FABD2 in a well-characterized marker line, Lifeact:Venus reduced reorganization rates of bundles of actin filaments in root epidermal cells. Reorganization rates of cytoplasmic strands, which reflect the reorganization of the actin cytoskeleton, were also reduced in these lines. Moreover, in the same line, Lifeact:Venus-decorated actin filaments were more resistant to depolymerization by latrunculin B than those in an equivalent GFP:FABD2-expressing line. In lines where Lifeact: Venus is expressed at lower levels, these effects are less prominent or even absent. We conclude that Lifeact: Venus reduces remodeling of the actin cytoskeleton in Arabidopsis in a concentration-dependent manner. Since this reduction occurs at expression levels that do not cause defects in plant development, selection of normally growing plants is not sufficient to determine optimal Lifeact expression levels. When correct expression levels of Lifeact have been determined, it is a valuable probe that labels dynamic populations of actin filaments such as fine F-actin, better than FABD2 does.

  15. Defect characterization using an ultrasonic array to measure the scattering coefficient matrix.

    PubMed

    Zhang, Jie; Drinkwater, Bruce W; Wilcox, Paul D

    2008-10-01

    Ultrasonic nondestructive evaluation is used for detection, characterization, and sizing of defects. The accurate sizing of defects that are of similar or less size than the ultrasonic wavelength is of particular importance in assessing structural integrity. In this paper, we demonstrate how measurement of the scattering coefficient matrix of a cracklike defect can be used to obtain its size, shape, and orientation. The scattering coefficient matrix describes the far field amplitude of scattered signals from a scatterer as a function of incident and scattering angles. A finite element (FE) modeling procedure is described that predicts the scattering coefficient matrix of various cracklike defects. Experimental results are presented using a commercial 64-element, 5 MHz array on 2 aluminum test samples that contain several machined slots and through thickness circular holes. To minimize the interference from the reflections of neighboring defects, a subarray approach is used to focus ultrasound on each target defect in turn and extract its scattering coefficient matrices. A circular hole and a fine slot can be clearly distinguished by their different scattering coefficient matrices over a specific range of incident angles and scattering angles. The orientation angles of slots directly below the array are deduced from the measured scattering coefficient matrix to an accuracy of a few degrees, and their lengths are determined with an error of 10%.

  16. Defect Characterization of 4H-SiC Wafers for Power Electronic Device Applications.

    NASA Astrophysics Data System (ADS)

    Cicero, G.; Ferrero, S.; Cocuzza, M.; Giorgis, F.; Mandracci, P.; Ricciardi, C.; Scaltrito, L.; Pirri, C. F.; Richieri, G.; Sgorlon, C.

    2002-03-01

    Silicon carbide is a wide band gap semiconductor, interesting for its physical properties such as high breakdown field, high saturated drift velocity and high thermal conductivity, which has been intensively studied in the last years. Although the high potentiality of this material, the SiC technology shows at the moment some limitations and requires further study in order to obtain electronic devices with the same quality standards of the Si technology. Indeed, the reliability of SiC-based devices is strictly correlated to the defects present in the crystalline structure. We have focused our investigation on 4H-SiC wafers and on 4H epitaxial layers in order to determine in both the situations the different type of defects. A preliminary investigation has been performed by optical microscopy and Scanning Electron Microscopy with the aim to evidence the defect morphology on large scale. A deeper insight on the defects typology has been obtained by Atomic Force Microscopy, Profilometer technique, Micro-Raman and Micro-Photoluminescence spectroscopies. Different types of defects such as micropipes, comets, super dislocations, etch pits and so on, have been characterized finding particular physical finger-prints. This investigation is aimed at correlating the defects and the electrical properties of SiC for power electronic device applications.

  17. Actinic Mask Inspection at the ALS Initial Design Review

    SciTech Connect

    Barty, A; Chapman, H; Sweeney, D; Levesque, R; Bokor, J; Gullikson, E; Jong, S; Liu, Y; Yi, M; Denbeaux, G; Goldberg, K; Naulleau, P; Denham, P; Rekawa, S; Baston, P; Tackaberry, R; Barale, P

    2003-03-05

    This report is the first milestone report for the actinic mask blank inspection project conducted at the VNL, which forms sub-section 3 of the Q1 2003 mask blank technology transfer program at the VNL. Specifically this report addresses deliverable 3.1.1--design review and preliminary tool design. The goal of this project is to design an actinic mask inspection tool capable of operating in two modes: high-speed scanning for the detection of multilayer defects (inspection mode), and a high-resolution aerial image mode in which the image emulates the imaging illumination conditions of a stepper system (aerial image or AIM mode). The purpose and objective of these two modes is as follows: (1) Defect inspection mode--This imaging mode is designed to scan large areas of the mask for defects EUV multilayer coatings. The goal is to detect the presence of multilayer defects on a mask blank and to store the co-ordinates for subsequent review in AIM mode, thus it is not essential that the illumination and imaging conditions match that of a production stepper. Potential uses for this imaging mode include: (a) Correlating the results obtained using actinic inspection with results obtained using other non-EUV defect inspection systems to verify that the non-EUV scanning systems are detecting all critical defects; (b) Gaining sufficient information to associate defects with particular processes, such as various stages of the multilayer deposition or different modes of operation of the deposition tool; and (c) Assessing the density and EUV impact of surface and multilayer anomalies. Because of the low defect density achieved using current multilayer coating technology it is necessary to be able to efficiently scan large areas of the mask in order to obtain sufficient statistics for use in cross-correlation experiments. Speed of operation as well as sensitivity is therefore key to operation in defect inspection mode. (2) Aerial Image Microscope (AIM) mode--In AIM mode the tool is

  18. Detection and characterization of defects in moving parts of wind turbines

    NASA Astrophysics Data System (ADS)

    Forero, E.; Tibaduiza, D.; Anaya, M.; Castro, R.

    2016-07-01

    The detection, localization and characterization of defects in a material or a part that conform a structure is possible by using the transmission and reception of ultrasonic signals. Different strategies are used to achieve extract information from the part under evaluation. For this, it is then possible to use a distributed sensors arrays on the surface of the material and using scanning techniques such as are A-scan or B-scan, where it is possible to increase the level of detail regarding location, orientation and size of defects found, according to the strategy used. However, the systems and inspection techniques are often limited by the geometries and access to different types of structures. Due to these reasons, the acquisition of the returned signals, for identification and attenuation time, can suppress valuable information for accurate characterization of imperfections found in shape and location. In this paper, the use of spectral analysis of the collected signals is proposed as a tool for detection and characterization of defects in a structure. This analysis allows to determining the power distribution in a frequency range. This methodology is useful in non-destructive evaluation when it is not possible to have full access to the structure under inspection. In this case it is applied on a wind turbine operating to make the study of different echoes captured according to the geometry of the part and comparing said conducting analysis with previously established patterns of shapes, orientations, and sizes of defects found.

  19. Microstructural and Defect Characterization in Ceramic Composites Using an Ultrasonic Guided Wave Scan System

    NASA Technical Reports Server (NTRS)

    Roth, D. J.; Cosgriff, L. M.; Martin, R. E.; Verrilli, M. J.; Bhatt, R. T.

    2003-01-01

    In this study, an ultrasonic guided wave scan system was used to characterize various microstructural and flaw conditions in two types of ceramic matrix composites, SiC/SiC and C/SiC. Rather than attempting to isolate specific lamb wave modes to use for characterization (as is desired for many types of guided wave inspection problems), the guided wave scan system utilizes the total (multi-mode) ultrasonic response in its inspection analysis. Several time and frequency-domain parameters are calculated from the ultrasonic guided wave signal at each scan location to form images. Microstructural and defect conditions examined include delamination, density variation, cracking, and pre/ post-infiltration. Results are compared with thermographic imaging methods. Although the guided wave technique is commonly used so scanning can be eliminated, applying the technique in the scanning mode allows a more precise characterization of defect conditions.

  20. Characterization and comparison of defects detection limits of three ultrasonic non destructive methods

    NASA Astrophysics Data System (ADS)

    Péronnet, E.; Eyma, F.; Welemane, H.; Pescay, C.

    2010-06-01

    This work deals with the Liquid Resin Infusion (LRI) process developed within the research program “FUSelage COMPosite” of DAHER SOCATA. This manufacturing process enables the realization of complex composite structures or fuselage elements in a single phase (mono-material), which considerably reduce connections and relative difficulties. The concern here is the investigation of non destructive testing (NDT) methods that can be applied to LRI-structures in order to define their capacities for defect detection, and especially their associated critical defect size. In aviation industry, the AITM standards require the ultrasonic testing as NDT for composite materials. Therefore the aim of this work is to characterize and compare three different and complementary ultrasonic techniques on composite specimens. Such analysis allows to define the NDT application field of each method in term of defect detection.

  1. Synthetic actin-binding domains reveal compositional constraints for function.

    PubMed

    Lorenzi, Maria; Gimona, Mario

    2008-01-01

    The actin-binding domains of many proteins consist of a canonical type 1/type 2 arrangement of the structurally conserved calponin homology domain. Using the actin-binding domain of alpha-actinin-1 as a scaffold we have generated synthetic actin-binding domains by altering position and composition of the calponin homology domains. We show that the presence of two calponin homology domains alone and in the context of an actin-binding domain is not sufficient for actin-binding, and that both single and homotypic type 2 calponin homology domain tandems fail to bind to actin in vitro and in transfected cells. In contrast, single and tandem type 1 calponin homology domain arrays bind actin directly but result in defective turnover rates on actin filaments, and in aberrant actin bundling when introduced into the full-length alpha-actinin molecule. An actin-binding domain harboring the calponin homology domains in an inverted position, however, functions both in isolation and in the context of the dimeric alpha-actinin molecule. Our data demonstrate that the dynamics and specificity of actin-binding via actin-binding domains requires both the filament binding properties of the type 1, and regulation by type 2 calponin homology domains, and appear independent of their position.

  2. Neutron induced defects in silicon detectors characterized by DLTS and TSC methods

    NASA Astrophysics Data System (ADS)

    Fretwurst, E.; Dehn, C.; Feick, H.; Heydarpoor, P.; Lindström, G.; Moll, M.; Schütze, C.; Schulz, T.

    1996-02-01

    Neutron induced defects in silicon detectors fabricated from n-type float zone material of different resistivity (100-6000Ω cm) have been studied using the C-DLTS (Capacitance-Deep Level Transient Spectroscopy) and TSC (Thermally Stimulated Current) method. While the application of the C-DLTS technique for high resistivity material is limited to neutron fluences below about 10 11 cm -2 the TSC method remains a powerful tool for the defect characterization even at high fluences. Up to 5 defect levels were observed in some of the unirradiated samples. These partly are due to thermal treatments during the fabrication process. After neutron irradiation defect levels at Ec - 0.17, -0.23 and -0.42 eV and at Ev + 0.36 eV were found. A detailed analysis of the predominant peak at about -0.42 eV has shown that it is a superposition of two levels at -0.39 and -0.42 eV. For these defect levels introduction rates, annealing effects and a comparison between the DLTS and TSC technique are presented. Possible correlations of these results with macroscopic detector properties are discussed.

  3. Electrical characterization of defects introduced in n-Ge during electron beam deposition or exposure

    SciTech Connect

    Coelho, S. M. M.; Auret, F. D.; Janse van Rensburg, P. J.; Nel, J. M.

    2013-11-07

    Schottky barrier diodes prepared by electron beam deposition (EBD) on Sb-doped n-type Ge were characterized using deep level transient spectroscopy (DLTS). Pt EBD diodes manufactured with forming gas in the chamber had two defects, E{sub 0.28} and E{sub 0.31}, which were not previously observed after EBD. By shielding the samples mechanically during EBD, superior diodes were produced with no measureable deep levels, establishing that energetic ions created in the electron beam path were responsible for the majority of defects observed in the unshielded sample. Ge samples that were first exposed to the conditions of EBD, without metal deposition (called electron beam exposure herein), introduced a number of new defects not seen after EBD with only the E-center being common to both processes. Substantial differences were noted when these DLTS spectra were compared to those obtained using diodes irradiated by MeV electrons or alpha particles indicating that very different defect creation mechanisms are at play when too little energy is available to form Frenkel pairs. These observations suggest that when EBD ions and energetic particles collide with the sample surface, inducing intrinsic non-localised lattice excitations, they modify defects deeper in the semiconductor thus rendering them observable.

  4. Structural defects in cubic semiconductors characterized by aberration-corrected scanning transmission electron microscopy.

    PubMed

    Arroyo Rojas Dasilva, Yadira; Kozak, Roksolana; Erni, Rolf; Rossell, Marta D

    2016-09-28

    The development of new electro-optical devices and the realization of novel types of transistors require a profound understanding of the structural characteristics of new semiconductor heterostructures. This article provides a concise review about structural defects which occur in semiconductor heterostructures on the basis of micro-patterned Si substrates. In particular, one- and two-dimensional crystal defects are being discussed which are due to the plastic relaxation of epitaxial strain caused by the misfit of crystal lattices. Besides a few selected examples from literature, we treat in particular crystal defects occurring in GaAs/Si, Ge/Si and β-SiC/Si structures which are studied by high-resolution annular dark-field scanning transmission electron microscopy. The relevance of this article is twofold; firstly, it should provide a collection of data which are of help for the identification and characterization of defects in cubic semiconductors by means of atomic-resolution imaging, and secondly, the experimental data shall provide a basis for advancing the understanding of device characteristics with the aid of theoretical modelling by considering the defective nature of strained semiconductor heterostructures.

  5. Concentration of point defects in 4H-SiC characterized by a magnetic measurement

    NASA Astrophysics Data System (ADS)

    Peng, B.; Jia, R. X.; Wang, Y. T.; Dong, L. P.; Hu, J. C.; Zhang, Y. M.

    2016-09-01

    A magnetic method is presented to characterize the concentration of point defects in silicon carbide. In this method, the concentration of common charged point defects, which is related to the density of paramagnetic centers, is determined by fitting the paramagnetic component of the specimen to the Brillouin function. Several parameters in the Brillouin function can be measured such as: the g-factor can be obtained from electron spin resonance spectroscopy, and the magnetic moment of paramagnetic centers can be obtained from positron lifetime spectroscopy combined with a first-principles calculation. To evaluate the characterization method, silicon carbide specimens with different concentrations of point defects are prepared with aluminum ion implantation. The fitting results of the densities of paramagnetic centers for the implanted doses of 1 × 1014 cm-2, 1 × 1015 cm-2 and 1 × 1016 cm-2 are 6.52 × 1014/g, 1.14 × 1015/g and 9.45 × 1014/g, respectively. The same trends are also observed for the S-parameters in the Doppler broadening spectra. It is shown that this method is an accurate and convenient way to obtain the concentration of point defects in 4H-SiC.

  6. Defect Characterization in SiGe/SOI Epitaxial Semiconductors by Positron Annihilation

    NASA Astrophysics Data System (ADS)

    Ferragut, R.; Calloni, A.; Dupasquier, A.; Isella, G.

    2010-12-01

    The potential of positron annihilation spectroscopy (PAS) for defect characterization at the atomic scale in semiconductors has been demonstrated in thin multilayer structures of SiGe (50 nm) grown on UTB (ultra-thin body) SOI (silicon-on-insulator). A slow positron beam was used to probe the defect profile. The SiO2/Si interface in the UTB-SOI was well characterized, and a good estimation of its depth has been obtained. The chemical analysis indicates that the interface does not contain defects, but only strongly localized charged centers. In order to promote the relaxation, the samples have been submitted to a post-growth annealing treatment in vacuum. After this treatment, it was possible to observe the modifications of the defect structure of the relaxed film. Chemical analysis of the SiGe layers suggests a prevalent trapping site surrounded by germanium atoms, presumably Si vacancies associated with misfit dislocations and threading dislocations in the SiGe films.

  7. Characterization of defects situated in a fresco by stimulated infrared thermography

    NASA Astrophysics Data System (ADS)

    Candoré, J. C.; Bodnar, J. L.; Detalle, V.; Grossel, P.

    2012-01-01

    The objective of this work is to approach the possibilities of stimulated infrared thermography in dimensional characterization of defects situated in mural paintings. Towards this end, we have proceeded in two stages. Initially, we have developed, with the help of a point source photothermal analysis, an in situ measurement of the longitudinal thermal diffusivity parameter. Then, we have proceeded to the characterization of the depth of the studied defect, by means of a wide photothermal analysis and of a confrontation between theory and experiment. In this article, we present these two measurement techniques and show that the approach allows a good estimation of the depth of an inclusion of plastazote in a copy of the "Saint Christophe" of the "Campana" collection of the "Louvre Museum".

  8. Characterization of Defects on MOCVD Grown Gallium Nitride Using Transient Analysis Techniques

    NASA Astrophysics Data System (ADS)

    Kasani, Sujan Phani Kumar

    Since the invention of the first visible spectrum (red) LED by Holonyak in 1962, there has been a need for more efficient, more reliable and less expensive LEDs. The III-nitrides revolutionized semiconductor technology with their applications in the blue LED's. However the internal quantum efficiency of LED's are limited by the deep level traps in GaN substrate. Traps are defects in the crystal lattice, which depends on growth parameters. These traps act as non-radiative centers where non-radiative recombination occurs without conversion of available energy into light. Characterization of these traps in a material is necessary for better understanding of the material growth quality and resulting device performance. In this work Capacitance-Voltage (C-V) and Deep Level Transient Spectroscopy (DLTS) are conducted which provide electronic properties of trap centers like activation energy, doping concentration and capture cross-section. In n-GaN grown by Metalorganic Chemical Vapor Deposition (MOCVD) on Sapphire two defects types are detected and are characterized by Capacitance-Voltage and Deep Level Transient Spectroscopy. Two deep levels E1 and E2 are typically observed in n-GaN with the activation energies of 0.21eV and 0.53eV at 125°K and 325°K, respectively. The deep level E1 is caused by linear line defects along dislocation cores while deep level E2 is related to point defects. The characterization techniques, experimental systems and preliminary characterization results are discussed in detail.

  9. Burkholderia mallei Cluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages

    DTIC Science & Technology

    2010-01-01

    horses are the only known reservoir of this host-adapted pathogen (35). Disease in equines presents as chronic or acute illnesses characterized by lung...appropriate, antibiotics were added at the following concentrations: 25 g/ml kanamycin (Km), 50 g/ml zeocin (Zeo), or 15 g/ml polymyxin B (Pm) for E. coli...10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Invitrogen) and a standard mixture of antibiotics (100 U/ml penicillin, 100 g/ml

  10. Detection and Characterization of Package Defects and Integrity Failure using Dynamic Scanning Infrared Thermography (DSIRT).

    PubMed

    Morris, Scott A

    2016-02-01

    A dynamic scanning infrared thermography (DSIRT) system developed at the Univ. of Illinois Urbana-Champaign (UIUC) Packaging Lab relies on variation in transient thermal artifacts to indicate defects, and offers the possibility of characterization of many types of materials and structures. These include newer polymer and laminate-based structures for shelf-stable foods that lack a reliable, nondestructive method for inspection, which is a continuing safety issue. Preliminary trials were conducted on a polyester/aluminum foil/polypropylene retort pouch laminate containing artificially-induced failed seal and insulating inclusion defects ranging from 1 to 10 mm wide in the plane of the seal. The samples were placed in relative motion to a laterally positioned infrared laser, inducing heating through the plane of the seal. The emergent thermal artifact on the obverse side was sensed using either a bolometer camera or a thermopile sensor, with thermal anomalies indicating potential defects and the results of each sensors were compared. The bolometer camera detected defects to the limit of its measured optical resolution-approximately 1 mm at 20 cm-although the lower-resolution thermopile sensors were only capable of detecting 5 mm defects even at closer distances of approximately 5 mm. In addition, a supplementary magnification system was fitted to the bolometer camera which increased resolution but reduced field of view and would require a much higher frame rate to be useful. Automatic processing of the image data rapidly detected the model defects and can lead to development of an automated inspection system.  Much higher material throughput speeds are feasible using faster instruments, and the system is scalable.

  11. Characterization of as-grown and annealed GaAs: Structural defects and electrical properties

    SciTech Connect

    Lee, B.T.

    1988-07-01

    Structural defects in GaAs related to excess As were characterized and their behavior upon heat treatments studied. The observed defects included precipitates and dislocations. Results showed most of the precipitates in As-rich GaAs to the rhombohedral arsenic. Two exceptions were observed in an In-doped LEC (liguid encapsulated Czochralski) GaAs, which were As-rich but could not be further identified. Some of the observed As precipitates showed a simple orientation relationship with the matrix which yields structural coherence between As precipitates and GaAs matrix. Other As precipitates showed less coherent orientation. The dislocation loops in As-rich GaAs consisted a faulted loop with Shockley type Burgers vector and a perfect loop associated with an extra /l brace/111/r brace/ plane. It was proposed that these loops were formed as a result of dual condensation of both excess As interstitials and Ga vacancies, followed by generation and movement of Shockley partial dislocations. These precipitates and dislocation loops disappear after annealing, indicating a solvus temperature between 600--700/degree/C. The EL2 concentration increased as the defects dissolved, showing the defects to be the source of the excess As required to form EL2. The implication is that the As interstitial and Ga vacancies coexist in GaAs at high temperatures, which indicates that these point defects are responsible for the formation of arsenic antisites by direct combination. During the cooling period, they freeze into the matrix as point defects during a rapid cooling and condense as dislocation loops and precipitates during very slow cooling, in the dislocation-free region of the crystals. Around dislocations, the excess As precipitates heterogeneously even during rapid cooling. 217 refs.

  12. 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.

  13. Silicon defects characterization for low temperature ion implantation and RTA process

    NASA Astrophysics Data System (ADS)

    Martirani Paolillo, Diego; Margutti, Giovanni; De Biase, Marco; Barozzi, Mario; Giubertoni, Damiano; Spaggiari, Claudio

    2015-12-01

    In the last years a lot of effort has been directed in order to reduce silicon defects eventually formed during the ion implantation/anneal sequence used in the fabrication of CMOS devices. In this work we explored the effect of ion implant dose rate and temperature on the formation of silicon defects for high fluence 49BF2 implantations. The considered processes (implantation and annealing) conditions are those typically used to form the source/drain regions of p-channel transistors in the submicron technology node and will be detailed in the document. Characterization of implant damage and extended silicon defects left after anneal has been performed by TEM. Dopant distribution and dopant activation has been investigated by SIMS and SRP analysis. We have verified that implant dose rate and temperature modulate the thickness of the amorphous silicon observed after implant, as well as the concentrations of silicon defects left after anneal. Effect of high dose rate low temperature implantation on product device was also evaluated, showing a reduction of leakage current on p-channel transistors. Experimental set up, results and possible explanation will be reported and discussed in the paper.

  14. Routine characterization of 3-D profiles of SRF cavity defects using replica techniques

    SciTech Connect

    Ge, M.; Wu, G.; Burk, D.; Ozelis, J.; Harms, E.; Sergatskov, D.; Hicks, D.; Cooley, L.D.; /Fermilab

    2010-09-01

    Recent coordination of thermometry with optical images has shown that obvious defects at specific locations produce heat or even quench superconducting radio frequency (SRF) cavities, imposing a significant limit on the overall accelerating gradient produced by the cavity. Characterization of the topography at such locations provides clues about how the defects originated, from which schemes for their prevention might be devised. Topographic analyses also provide understanding of the electromagnetic mechanism by which defects limit cavity performance, from which viability of repair techniques might be assessed. In this article we discuss how a variety of two-component silicone-based room-temperature vulcanizing agents can be routinely used to make replicas of the cavity surface and extract topographic details of cavity defects. Previously, this level of detail could only be obtained by cutting suspect regions from the cavity, thus destroying the cavity. We show 3-D profiles extracted from several different 1.3 GHz cavities. The defect locations, which were all near cavity welds, compelled us to develop extraction techniques for both equator and iris welds as well as from deep inside long 9-cell cavities. Profilometry scans of the replicas yield micrometer-scale information, and we describe various curious features, such as small peaks at the bottom of pits, which were not apparent in previous optical inspections. We also discuss contour information in terms of electromagnetic mechanisms proposed by others for local cavity heating. We show that production of the replica followed by high-pressure rinsing dose not adversely affect the cavity RF performance.

  15. Point defect determination by photoluminescence and capacitance—voltage characterization in a GaN terahertz Gunn diode

    NASA Astrophysics Data System (ADS)

    Li, Liang; Yang, Lin-An; Zhou, Xiao-Wei; Zhang, Jin-Cheng; Hao, Yue

    2013-08-01

    Photoluminescence (PL) measurement is used to study the point defect distribution in a GaN terahertz Gunn diode, which is able to the degrade high-field transport characteristic during further device operation. PL, secondary ion mass spectroscopy (SIMS), transmission electron microscope (TEM), and capacitance—voltage (C—V) measurements are used to discuss the origin of point defects responsible for the yellow luminescence in structures. The point defect densities of about 1011 cm-2 in structures are extracted by analysis of C—V characterization. After thermal annealing treatment, diminishments of point defect densities in structures are efficiently demonstrated by PL and C—V results.

  16. Characterization of nodular and thermal defects in hafnia/silica multilayer coatings using optical, photothermal, and atomic force microscopy

    SciTech Connect

    Stolz, C.J.; Yoshiyama, J.M.; Salleo, A.; Wu, Z.L.; Green, J.; Krupka, R.

    1997-12-24

    Multilayer coatings manufactured from metallic hafnium and silica sources by reactive electron beam deposition, are being developed for high fluence optics in a fusion laser with a wavelength of 1053 nm and a 3 ns pulse length. Damage threshold studies have revealed a correlation between laser damage and nodular defects, but interestingly laser damage is also present in nodule-free regions. Photothermal studies of optical coatings reveal the existence of defects with strong optical absorption in nodule-free regions of the coating. A variety of microscopic techniques were employed to characterize the effects for a better understanding of the thermal properties of nodular defects and role of thermal defects in laser damage. Photothermal microscopy, utilizing the surface thermal lensing technique, was used to map the thermal characteristics of 3 mm x 3 mm areas of the coatings. High resolution subaperture scans, with a 1 pm step size and a 3 um pump beam diameter, W= conducted on the defects to characterize their photothermal properties. Optical and atomic force microscopy was used to visually identify defects and characterize their topography. The defects were then irradiated to determine the role of nodular and thermal defects in limiting the damage threshold of the multilayer.

  17. Actinic Granuloma with Focal Segmental Glomerulosclerosis

    PubMed Central

    Phasukthaworn, Ruedee; Chanprapaph, Kumutnart; Vachiramon, Vasanop

    2016-01-01

    Actinic granuloma is an uncommon granulomatous disease, characterized by annular erythematous plaque with central clearing predominately located on sun-damaged skin. The pathogenesis is not well understood, ultraviolet radiation is recognized as precipitating factor. We report a case of a 52-year-old woman who presented with asymptomatic annular erythematous plaques on the forehead and both cheeks persisting for 2 years. The clinical presentation and histopathologic findings support the diagnosis of actinic granuloma. During that period of time, she also developed focal segmental glomerulosclerosis. The association between actinic granuloma and focal segmental glomerulosclerosis needs to be clarified by further studies. PMID:27293392

  18. 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

  19. PROGRESS IN CHARACTERIZATION OF PRECIPITATES AND DEFECT STRUCTURES IN Mg+ ION IMPLANTED CUBIC SILICON CARBIDE

    SciTech Connect

    Jiang, Weilin; Zhang, Jiandong; Zhu, Zihua; Roosendaal, Timothy J.; Hu, Shenyang Y.; Henager, Charles H.; Kurtz, Richard J.; Wang, Yongqiang

    2015-09-01

    This report describes the progress of our current experimental effort on Mg+ ion implanted 3C-SiC. Following our initial study [ ] that suggests possible formation of Mg2Si and MgC2 precipitates as well as tetrahedral voids in 24Mg+ ion implanted 3C-SiC, we have designed specific experiments to confirm the results and examine the inclusions and defects. Relatively low fluence (5.0×1015 24Mg+/cm2) implantation in 3C-SiC was performed to reduce defect concentrations and isolate individual defect features for characterization. In addition, 25Mg+ isotope was implanted in 3C-SiC to the same previously applied ion fluence (9.6×1016 ions/cm2) for atom probe tomography (APT) study of precipitates. Each set of the samples was annealed at 1573 K for 2, 6 and 12 h, respectively. The depth profiles of the implanted Mg were measured using secondary ion mass spectrometry (SIMS) before and after the annealing steps. The samples are currently being analyzed using transmission electron microscopy (TEM) and APT.

  20. Molecular characterization of the new defective P(brescia) alpha1-antitrypsin allele.

    PubMed

    Medicina, Daniela; Montani, Nadia; Fra, Anna M; Tiberio, Laura; Corda, Luciano; Miranda, Elena; Pezzini, Alessandro; Bonetti, Fausta; Ingrassia, Rosaria; Scabini, Roberta; Facchetti, Fabio; Schiaffonati, Luisa

    2009-08-01

    Alpha1-antitrypsin (alpha(1)AT) deficiency is a hereditary disorder associated with reduced alpha(1)AT serum level, predisposing adults to pulmonary emphysema. Among the known mutations of the alpha(1)AT gene (SERPINA1) causing alpha(1)AT deficiency, a few alleles, particularly the Z allele, may also predispose adults to liver disease. We have characterized a new defective alpha(1)AT allele (c.745G>C) coding for a mutant alpha(1)AT (Gly225Arg), named P(brescia). The P(brescia) alpha(1)AT allele was first identified in combination with the rare defective M(würzburg) allele in an 11-year-old boy showing significantly reduced serum alpha(1)AT level. Subsequently, the P(brescia) allele was found in the heterozygous state with the normal M or the defective Z allele in nine and three adults respectively. In cellular models of the disease, we show that the P(brescia) mutant is retained in the endoplasmic reticulum as ordered polymers and is secreted more slowly than the normal M alpha(1)AT. This behaviour recapitulates the abnormal cellular handling and fate of the Z alpha(1)AT and suggests that the mutation present in the P(brescia) alpha(1)AT causes a conformational change of the protein which, by favouring polymer formation, is etiologic to both severe alpha(1)AT deficiency in the plasma and toxic protein-overload in the liver.

  1. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner*

    PubMed Central

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-01-01

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. PMID:26747606

  2. 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).

  3. Eddy Current Pulsed Thermography with Different Excitation Configurations for Metallic Material and Defect Characterization.

    PubMed

    Tian, Gui Yun; Gao, Yunlai; Li, Kongjing; Wang, Yizhe; Gao, Bin; He, Yunze

    2016-06-08

    This paper reviews recent developments of eddy current pulsed thermography (ECPT) for material characterization and nondestructive evaluation (NDE). Due to the fact that line-coil-based ECPT, with the limitation of non-uniform heating and a restricted view, is not suitable for complex geometry structures evaluation, Helmholtz coils and ferrite-yoke-based excitation configurations of ECPT are proposed and compared. Simulations and experiments of new ECPT configurations considering the multi-physical-phenomenon of hysteresis losses, stray losses, and eddy current heating in conjunction with uniform induction magnetic field have been conducted and implemented for ferromagnetic and non-ferromagnetic materials. These configurations of ECPT for metallic material and defect characterization are discussed and compared with conventional line-coil configuration. The results indicate that the proposed ECPT excitation configurations can be applied for different shapes of samples such as turbine blade edges and rail tracks.

  4. Eddy Current Pulsed Thermography with Different Excitation Configurations for Metallic Material and Defect Characterization

    PubMed Central

    Tian, Gui Yun; Gao, Yunlai; Li, Kongjing; Wang, Yizhe; Gao, Bin; He, Yunze

    2016-01-01

    This paper reviews recent developments of eddy current pulsed thermography (ECPT) for material characterization and nondestructive evaluation (NDE). Due to the fact that line-coil-based ECPT, with the limitation of non-uniform heating and a restricted view, is not suitable for complex geometry structures evaluation, Helmholtz coils and ferrite-yoke-based excitation configurations of ECPT are proposed and compared. Simulations and experiments of new ECPT configurations considering the multi-physical-phenomenon of hysteresis losses, stray losses, and eddy current heating in conjunction with uniform induction magnetic field have been conducted and implemented for ferromagnetic and non-ferromagnetic materials. These configurations of ECPT for metallic material and defect characterization are discussed and compared with conventional line-coil configuration. The results indicate that the proposed ECPT excitation configurations can be applied for different shapes of samples such as turbine blade edges and rail tracks. PMID:27338389

  5. Modeling and experimental characterization of stepped and v-shaped (311) defects in silicon

    SciTech Connect

    Marqués, Luis A. Aboy, María; Dudeck, Karleen J.; Botton, Gianluigi A.; Knights, Andrew P.; Gwilliam, Russell M.

    2014-04-14

    We propose an atomistic model to describe extended (311) defects in silicon. It is based on the combination of interstitial and bond defect chains. The model is able to accurately reproduce not only planar (311) defects but also defect structures that show steps, bends, or both. We use molecular dynamics techniques to show that these interstitial and bond defect chains spontaneously transform into extended (311) defects. Simulations are validated by comparing with precise experimental measurements on actual (311) defects. The excellent agreement between the simulated and experimentally derived structures, regarding individual atomic positions and shape of the distinct structural (311) defect units, provides strong evidence for the robustness of the proposed model.

  6. Electron Paramagnetic Resonance and Electron-Nuclear Double Resonance Characterization of Point Defects in Titanium dioxide Crystals

    NASA Astrophysics Data System (ADS)

    Brant, Adam

    Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) are used to characterize several point defects in titanium dioxide (TiO2) single crystals in the rutile phase. A defect reported in 1961 by P. F. Chester called the “A Center” is assigned to a neutral hydrogen donor. Many researchers believe that the model for this S = 1/2 defect is an interstitial titanium ion (Ti3+) and that Ti3+ interstitials are the most dominant shallow donor in TiO 2. I show that the model for the A center is a neutral hydrogen donor and suggest that the Ti3+ interstitial model is not the most prevalent shallow donor defect in TiO2. Substitutional Cu2+ defects that are unintentionally introduced to TiO2 (rutile) during growth are characterized and assigned to a Cu2+ ion with an adjacent oxygen vacancy. Exact matrix diagonalization is used here to compute accurate values for the nuclear quadrupole parameter. The reduced intensity of the Cu2+ EPR signal when the sample is illuminated with 442 nm laser light as well as the appearance of photoinduced EPR signals due to singly and doubly ionized oxygen vacancies provide evidence that the Cu2+ defect has an adjacent oxygen vacancy. Interstitial lithium ions (Li+) adjacent to Ti 3+ ions and substitutional Fe3+ defects (Fe 3+ - Li+) are also characterized. These defects were introduced to the rutile crystal by heating at 450 °C in LiOH powder for times on the order of several hours. Principal values and principal axis directions of the g matrix are calculated for the interstitial Li+ ion adjacent to a Ti3+ ion and photoinduced effects of the Fe 3+ - Li+ defect are examined.

  7. 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

  8. Isolation and characterization of Pichia heedii mutants defective in xylose uptake

    SciTech Connect

    Does, A.L.; Bisson, L.F. )

    1990-11-01

    To investigate the role of xylose uptake in xylose metabolism in yeasts, we isolated a series of mutated strains of the yeast Pichia heedii which are defective in xylose utilization. Four of these demonstrated defects in xylose uptake. Overlaps between the functional or regulatory mechanisms for glucose and xylose uptake may exist in this yeast since some of the mutants defective in xylose uptake were also defective in glucose transport. None of the mutants were defective in xylose reductase or xylitol dehydrogenase activities.

  9. 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

  10. 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

  11. Profilin Regulates Apical Actin Polymerization to Control Polarized Pollen Tube Growth.

    PubMed

    Liu, Xiaonan; Qu, Xiaolu; Jiang, Yuxiang; Chang, Ming; Zhang, Ruihui; Wu, Youjun; Fu, Ying; Huang, Shanjin

    2015-12-07

    Pollen tube growth is an essential step during flowering plant reproduction, whose growth depends on a population of dynamic apical actin filaments. Apical actin filaments were thought to be involved in the regulation of vesicle fusion and targeting in the pollen tube. However, the molecular mechanisms that regulate the construction of apical actin structures in the pollen tube remain largely unclear. Here, we identify profilin as an important player in the regulation of actin polymerization at the apical membrane in the pollen tube. Downregulation of profilin decreased the amount of filamentous actin and induced disorganization of apical actin filaments, and reduced tip-directed vesicle transport and accumulation in the pollen tube. Direct visualization of actin dynamics revealed that the elongation of actin filaments originating at the apical membrane decreased in profilin mutant pollen tubes. Mutant profilin that is defective in binding poly-L-proline only partially rescues the actin polymerization defect in profilin mutant pollen tubes, although it fully rescues the actin turnover phenotype. We propose that profilin controls the construction of actin structures at the pollen tube tip, presumably by favoring formin-mediated actin polymerization at the apical membrane.

  12. 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

  13. Characterization of 3 MeV H + irradiation induced defects in nuclear grade graphite

    NASA Astrophysics Data System (ADS)

    Kim, Eung-Seon; Kim, Yong-Wan

    2010-09-01

    Atomistic structure change in a nuclear grade graphite irradiated at 353 K to 3.4×10 17 ion/cm 2 with 3 MeV H + was characterized by measuring positron lifetime and Raman spectrum at room temperature. It is evident from the positron lifetime results that the pre-existing structural defect is disoriented crystalline boundaries, and vacancy clusters ranging from di- to quadruple-vacancies were newly formed after ion irradiation. The relative intensity ratio of the Raman D and G peaks increased from 0.25 to 0.67 after ion irradiation. The concentration of radiation-induced vacancies was reasonably estimated by the Raman intensity ratio.

  14. Active and passive infrared thermography applied to the detection and characterization of hidden defects in structure

    NASA Astrophysics Data System (ADS)

    Dumoulin, Jean

    2013-04-01

    Infrared thermography for Non Destructive Testing (NDT) has encountered a wide spreading this last 2 decades, in particular thanks to emergence on the market of low cost uncooled infrared camera. So, infrared thermography is not anymore a measurement technique limited to laboratory application. It has been more and more involved in civil engineering and cultural heritage applications, but also in many other domains, as indicated by numerous papers in the literature. Nevertheless, laboratory, measurements are done as much as possible in quite ideal conditions (good atmosphere conditions, known properties of materials, etc.), while measurement on real site requires to consider the influence of not controlled environmental parameters and additional unknown thermal properties. So, dedicated protocol and additional sensors are required for measurement data correction. Furthermore, thermal excitation is required to enhance the signature of defects in materials. Post-processing of data requires to take into account the protocol used for the thermal excitation and sometimes its nature to avoid false detection. This analysis step is based on signal and image processing tool and allows to carry out the detection. Characterization of anomalies detected at the previous step can be done by additional signal processing in particular for manufactured objects. The use of thermal modelling and inverse method allows to determine properties of the defective area. The present paper will first address a review of some protocols currently in use for field measurement with passive and/or active infrared measurements. Illustrations in various experiments carried out on civil engineering structure will be shown and discussed. In a second part, different post-processing approaches will be presented and discussed. In particular, a review of the most standard processing methods like Fast Fourier Analysis, Principal Components Analysis, Polynomial Decomposition, defect characterization using

  15. Characterization of convection related defects in II-VI compound semiconductors

    NASA Technical Reports Server (NTRS)

    Witt, August F.

    1993-01-01

    The research carried out under NAG8-913, 'Characterization of Convection Related Defects in II-VI Compound Semiconductors', was aimed at exploration of the potential of axial magnetic fields for melt stabilization when applied in Bridgman geometry to the growth of HgMnTe. The thrust of the work was directed at the experimental establishment of the limits of magnetic melt stabilization during crystal growth and at the analytical verification of the effects of stabilization on critical materials properties. The data obtained indicate noticeable stabilization effects, particularly as far as the formation of microscopic compositional inhomogeneities is concerned. The effects of magnetic fields on precipitate formation are found to be minor. Magnetic field effects were investigated for both 'Bridgman' and 'travelling heater' geometries. The research was conducted during the period from May 22 to September 30, 1992.

  16. Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells.

    PubMed

    Bertling, Enni; Hotulainen, Pirta; Mattila, Pieta K; Matilainen, Tanja; Salminen, Marjo; Lappalainen, Pekka

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

  17. 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.

  18. Actin isoform specificity is required for the maintenance of lactation

    PubMed Central

    Weymouth, Nate; Shi, Zengdun; Rockey, Don C.

    2014-01-01

    Smooth muscle α-actin (Acta2) is one of six highly conserved mammalian actin isoforms that appear to exhibit functional redundancy. Nonetheless, we have postulated a specific functional role for the smooth muscle specific isoform. Here, we show that Acta2 deficient mice have a remarkable mammary phenotype such that dams lacking Acta2 are unable to nurse their offspring effectively. The phenotype was rescued in cross fostering experiments with wild type mice, excluding a developmental defect in Acta2 null pups. The mechanism for the underlying phenotype is due to myoepithelial dysfunction postpartum resulting in precocious involution. Further, we demonstrate a specific defect in myoepithelial cell contractility in Acta2 null mammary glands, despite normal expression of cytoplasmic actins. We conclude that Acta2 specifically mediates myoepithelial cell contraction during lactation and that this actin isoform therefore exhibits functional specificity. PMID:22123032

  19. 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.

  20. Structure and function of a G-actin sequestering protein with a vital role in malaria oocyst development inside the mosquito vector.

    PubMed

    Hliscs, Marion; Sattler, Julia M; Tempel, Wolfram; Artz, Jennifer D; Dong, Aiping; Hui, Raymond; Matuschewski, Kai; Schüler, Herwig

    2010-04-09

    Cyclase-associated proteins (CAPs) are evolutionary conserved G-actin-binding proteins that regulate microfilament turnover. CAPs have a modular structure consisting of an N-terminal adenylate cyclase binding domain, a central proline-rich segment, and a C-terminal actin binding domain. Protozoan parasites of the phylum Apicomplexa, such as Cryptosporidium and the malaria parasite Plasmodium, express small CAP orthologs with homology to the C-terminal actin binding domain (C-CAP). Here, we demonstrate by reverse genetics that C-CAP is dispensable for the pathogenic Plasmodium blood stages. However, c-cap(-) parasites display a complete defect in oocyst development in the insect vector. By trans-species complementation we show that the Cryptosporidium parvum ortholog complements the Plasmodium gene functions. Purified recombinant C. parvum C-CAP protein binds actin monomers and prevents actin polymerization. The crystal structure of C. parvum C-CAP shows two monomers with a right-handed beta-helical fold intercalated at their C termini to form the putative physiological dimer. Our results reveal a specific vital role for an apicomplexan G-actin-binding protein during sporogony, the parasite replication phase that precedes formation of malaria transmission stages. This study also exemplifies how Plasmodium reverse genetics combined with biochemical and structural analyses of orthologous proteins can offer a fast track toward systematic gene characterization in apicomplexan parasites.

  1. EUV Dark-Field Microscopy for Defect Inspection

    SciTech Connect

    Juschkin, L.; Maryasov, A.; Herbert, S.; Aretz, A.; Bergmann, K.; Lebert, R.

    2011-09-09

    An actinic EUV microscope for defect detection on mask blanks for operation in dark field using a table-top discharge-produced plasma source has been developed. Several test structures (pits and bumps) on multilayer mirrors were investigated by our Schwarzschild objective-based EUV microscope at 13.5-nm wavelength and then characterized with an atomic force microscope. Possible defect-detection limits with large field of view and moderate magnification are discussed in terms of required irradiation dose and system performance.

  2. Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle-alpha-actin gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Smooth muscle a actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells(vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a ...

  3. Characterization of defective interfering RNAs associated with RNA plant viruses. Progress report

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1993-04-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since the original observation with TBSV, we discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), and many other reports have now appeared characterizing DI and DI-like RNAs in other plant viral infections. We are seeking to improve our understanding of the mechanisms of DI generation and the precise nature of the RNA sequences necessary for DI replication and encapsidation. We also want to address the nature of the DI mediated symptom attenuation and interference effects in plants, and to determine the feasibility of using transgenic plants constitutively expressing DI RNAs for disease control. The progress made on each of these objectives is summarized along with the proposed experiments for the continuation period.

  4. Characterization of defect growth structures in ion plated films by scanning electron microscopy

    NASA Technical Reports Server (NTRS)

    Spalvins, T.

    1979-01-01

    Gold and copper films (0.2-2 micron thick) are ion plated on very smooth stainless steel 304 and mica surfaces. The deposited films are examined by SEM to identify the morphological growth of defects. Three types of coating defects are distinguished: nodular growth, abnormal or runaway growth, and spits. The potential nucleation sites for defect growth are analyzed to determine the cause of defect formation. It is found that nuclear growth is due to inherent surface microdefects, abnormal or runaway growth is due to external surface inclusions, and spits are due to nonuniform evaporation and ejection of droplets. All these defects have adverse effects on the coatings.

  5. Automated Guided-Wave Scanning Developed to Characterize Materials and Detect Defects

    NASA Technical Reports Server (NTRS)

    Martin, Richard E.; Gyekenyeski, Andrew L.; Roth, Don J.

    2004-01-01

    The Nondestructive Evaluation (NDE) Group of the Optical Instrumentation Technology Branch at the NASA Glenn Research Center has developed a scanning system that uses guided waves to characterize materials and detect defects. The technique uses two ultrasonic transducers to interrogate the condition of a material. The sending transducer introduces an ultrasonic pulse at a point on the surface of the specimen, and the receiving transducer detects the signal after it has passed through the material. The aim of the method is to correlate certain parameters in both the time and frequency domains of the detected waveform to characteristics of the material between the two transducers. The scanning system is shown. The waveform parameters of interest include the attenuation due to internal damping, waveform shape parameters, and frequency shifts due to material changes. For the most part, guided waves are used to gauge the damage state and defect growth of materials subjected to various mechanical or environmental loads. The technique has been applied to polymer matrix composites, ceramic matrix composites, and metal matrix composites as well as metallic alloys. Historically, guided wave analysis has been a point-by-point, manual technique with waveforms collected at discrete locations and postprocessed. Data collection and analysis of this type limits the amount of detail that can be obtained. Also, the manual movement of the sensors is prone to user error and is time consuming. The development of an automated guided-wave scanning system has allowed the method to be applied to a wide variety of materials in a consistent, repeatable manner. Experimental studies have been conducted to determine the repeatability of the system as well as compare the results obtained using more traditional NDE methods. The following screen capture shows guided-wave scan results for a ceramic matrix composite plate, including images for each of nine calculated parameters. The system can

  6. Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

    PubMed

    Cunningham, L; Williams, H D

    1995-01-01

    The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme

  7. Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

    PubMed Central

    Rodal, Avital A.; Kozubowski, Lukasz; Goode, Bruce L.; Drubin, David G.; Hartwig, John H.

    2005-01-01

    Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells. PMID:15525671

  8. 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.

  9. Method for characterizing mask defects using image reconstruction from X-ray diffraction patterns

    DOEpatents

    Hau-Riege, Stefan Peter

    2007-05-01

    The invention applies techniques for image reconstruction from X-ray diffraction patterns on the three-dimensional imaging of defects in EUVL multilayer films. The reconstructed image gives information about the out-of-plane position and the diffraction strength of the defect. The positional information can be used to select the correct defect repair technique. This invention enables the fabrication of defect-free (since repaired) X-ray Mo--Si multilayer mirrors. Repairing Mo--Si multilayer-film defects on mask blanks is a key for the commercial success of EUVL. It is known that particles are added to the Mo--Si multilayer film during the fabrication process. There is a large effort to reduce this contamination, but results are not sufficient, and defects continue to be a major mask yield limiter. All suggested repair strategies need to know the out-of-plane position of the defects in the multilayer.

  10. Characterization of a defective interfering RNA that contains a mosaic of a plant viral genome

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1991-01-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since then, we have also discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), a virus structurally related to TBSV. We proposed a thorough characterization of this unique class of symptom modulating RNAs with the overall objective of identifying viral RNA nucleotide, sequences involved in such fundamental processes as virus replication and encapsidation as well as the degree of symptom expression resulting from the viral-DI-host interaction. The proposed research focused on the molecular characterization of the DI RNAs and the helper virus. We had demonstrated that the DIs were collinear deletion mutants of the genome of a cherry strain of tomato bushy stunt virus (TBSV). We had also shown that these low molecular weight RNAs interfered with the helper plant virus and modulated disease expression by preventing the development of a lethal necrotic disease in susceptible host plants. We also suggested that by exploring the mechanisms associated with the symptom attenuation effect, we might be able to devise novel strategies useful for engineering viral disease resistance.

  11. Computational mask defect review for contamination and haze inspections

    NASA Astrophysics Data System (ADS)

    Morgan, Paul; Rost, Daniel; Price, Daniel; Corcoran, Noel; Satake, Masaki; Hu, Peter; Peng, Danping; Yonenaga, Dean; Tolani, Vikram; Wolf, Yulian; Shah, Pinkesh

    2013-09-01

    As optical lithography continues to extend into sub-0.35 k1 regime, mask defect inspection and subsequent review has become tremendously challenging, and indeed the largest component to mask manufacturing cost. The routine use of various resolution enhancement techniques (RET) have resulted in complex mask patterns, which together with the need to detect even smaller defects due to higher MEEFs, now requires an inspection engineer to use combination of inspection modes. This is achieved in 193nm AeraTM mask inspection systems wherein masks are not only inspected at their scanner equivalent aerial exposure conditions, but also at higher Numerical Aperture resolution, and special reflected-light, and single-die contamination modes, providing better coverage over all available patterns, and defect types. Once the required defects are detected by the inspection system, comprehensively reviewing and dispositioning each defect then becomes the Achilles heel of the overall mask inspection process. Traditionally, defects have been reviewed manually by an operator, which makes the process error-prone especially given the low-contrast in the convoluted aerial images. Such manual review also limits the quality and quantity of classifications in terms of the different types of characterization and number of defects that can practically be reviewed by a person. In some ways, such manual classification limits the capability of the inspection tool itself from being setup to detect smaller defects since it often results in many more defects that need to be then manually reviewed. Paper 8681-109 at SPIE Advanced Lithography 2013 discussed an innovative approach to actinic mask defect review using computational technology, and focused on Die-to-Die transmitted aerial and high-resolution inspections. In this approach, every defect is characterized in two different ways, viz., quantitatively in terms of its print impact on wafer, and qualitatively in terms of its nature and origin in

  12. 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.

  13. The Effects of Disease Models of Nuclear Actin Polymerization on the Nucleus

    PubMed Central

    Serebryannyy, Leonid A.; Yuen, Michaela; Parilla, Megan; Cooper, Sandra T.; de Lanerolle, Primal

    2016-01-01

    Actin plays a crucial role in regulating multiple processes within the nucleus, including transcription and chromatin organization. However, the polymerization state of nuclear actin remains controversial, and there is no evidence for persistent actin filaments in a normal interphase nucleus. Further, several disease pathologies are characterized by polymerization of nuclear actin into stable filaments or rods. These include filaments that stain with phalloidin, resulting from point mutations in skeletal α-actin, detected in the human skeletal disease intranuclear rod myopathy, and cofilin/actin rods that form in response to cellular stressors like heatshock. To further elucidate the effects of these pathological actin structures, we examined the nucleus in both cell culture models as well as isolated human tissues. We find these actin structures alter the distribution of both RNA polymerase II and chromatin. Our data suggest that nuclear actin filaments result in disruption of nuclear organization, which may contribute to the disease pathology. PMID:27774069

  14. Temperature-Dependent Photoluminescence Imaging and Characterization of a Multi-Crystalline Silicon Solar Cell Defect Area: Preprint

    SciTech Connect

    Johnston, S.; Yan, F.; Li, J.; Romero, M. J.; Al-Jassim, M.; Zaunbrecher, K.; Sidelkheir, O.; Blosse, A.

    2011-07-01

    Photoluminescence (PL) imaging is used to detect areas in multi-crystalline silicon that appear dark in band-to-band imaging due to high recombination. Steady-state PL intensity can be correlated to effective minority-carrier lifetime, and its temperature dependence can provide additional lifetime-limiting defect information. An area of high defect density has been laser cut from a multi-crystalline silicon solar cell. Both band-to-band and defect-band PL imaging have been collected as a function of temperature from ~85 to 350 K. Band-to-band luminescence is collected by an InGaAs camera using a 1200-nm short-pass filter, while defect band luminescence is collected using a 1350-nm long pass filter. The defect band luminescence is characterized by cathodo-luminescence. Small pieces from adjacent areas within the same wafer are measured by deep-level transient spectroscopy (DLTS). DLTS detects a minority-carrier electron trap level with an activation energy of 0.45 eV on the sample that contained defects as seen by imaging.

  15. Observation results of actual phase defects using micro coherent EUV scatterometry microscope

    NASA Astrophysics Data System (ADS)

    Hashimoto, Hiraku; Harada, Tetsuo; Watanabe, Takeo

    2016-10-01

    One of the critical issue of EUV lithography is fabrication of defect-free mask. The origin of the defect is a particle inside the multilayer and bump or pit on glass substrate. This type of defect is called a phase defect. If there is a phase defect, the reflection phase is disordered. As a result, the phase structure is printed as a defect on a wafer. Thus, we have developed micro coherent EUV scatterometry microscope (we called micro-CSM) for phase defect characterization. Micro-CSM records scattering signal from a defect directly exposed by focused coherent EUV having a spot size of φ140-nm in diameter. An off-axis-type Fresnel zone plate was employed as a focusing optics. Phase distribution of the defect is reconstructed with the scattering image by the coherent-diffraction-imaging method. We observed actual phase defects in this work. Actual phase defects were on a mask blanks which was the same grade of the pre-production mask of the semiconductor devices. The positions of actual phase defects have been already inspected by the actinic blank inspection tool. And, the actual phase defects have been already observed using an atomic force microscope. A purpose of this work is observation of these actual defects using micro-CSM and comparison of the results.

  16. Defect characterization of proton irradiated GaAs pn-junction diodes with layers of InAs quantum dots

    NASA Astrophysics Data System (ADS)

    Sato, Shin-ichiro; Schmieder, Kenneth J.; Hubbard, Seth M.; Forbes, David V.; Warner, Jeffrey H.; Ohshima, Takeshi; Walters, Robert J.

    2016-05-01

    In order to expand the technology of III-V semiconductor devices with quantum structures to both terrestrial and space use, radiation induced defects as well as native defects generated in the quantum structures should be clarified. Electrically active defects in GaAs p+n diodes with embedded ten layers of InAs quantum dots (QDs) are investigated using Deep Level Transient Fourier Spectroscopy. Both majority carrier (electron) and minority carrier (hole) traps are characterized. In the devices of this study, GaP layers are embedded in between the QD layers to offset the compressive stress introduced during growth of InAs QDs. Devices are irradiated with high energy protons for three different fluences at room temperature in order to characterize radiation induced defects. Seven majority electron traps and one minority hole trap are found after proton irradiation. It is shown that four electron traps induced by proton irradiation increase in proportion to the fluence, whereas the EL2 trap, which appears before irradiation, is not affected by irradiation. These defects correspond to electron traps previously identified in GaAs. In addition, a 0.53 eV electron trap and a 0.14 eV hole trap are found in the QD layers before proton irradiation. It is shown that these native traps are also unaffected by irradiation. The nature of the 0.14 eV hole trap is thought to be Ga-vacancies in the GaP strain balancing layers.

  17. Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

    PubMed

    Schnoor, Michael

    2015-04-15

    The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

  18. Identification and characterization of point defects in aluminum nitride and zinc oxide crystals

    NASA Astrophysics Data System (ADS)

    Evans, Sean M.

    Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) studies have been performed on single crystals of aluminum nitride (AlN) and zinc oxide (ZnO), two wide-band-gap semiconductors having the wurtzite crystal structure. These studies were used to characterize point defects in each material. In the first study in AlN, new EPR and ENDOR spectra were acquired from a deep donor. Although observed in as-grown crystals, exposure to x rays significantly increased the concentration of this center. ENDOR identified a strong hyperfine interaction with one aluminum neighbor along the c axis and weaker equivalent hyperfine interactions with three additional aluminum neighbors in the basal plane. These aluminum interactions indicate that the responsible center was located at a nitrogen site. The observed paramagnetic defect is either an oxygen substituting for nitrogen or a nitrogen vacancy. An analysis of the hyperfine data suggests that substitutional oxygen is the most likely candidate. The second point defect studied in AlN was silicon substituting for aluminum. Silicon is a shallow donor in AlN, and its neutral charge state is paramagnetic. Two samples containing silicon were studied. Only one of the samples was intentionally doped with silicon. The silicon-related EPR signals from these two samples had different behaviors. The signal from the doped sample had behavior similar to that described in previous studies where the silicon was explained as a DX center. The undoped sample had behavior that was inconsistent with a DX center. In ZnO, EPR was used to monitor oxygen vacancies and zinc vacancies in a ZnO crystal irradiated near room temperature with 1.5 MeV electrons. Out-of-phase detection at 30 K greatly enhanced the EPR signals from these vacancies. Following the electron irradiation, but before illumination, Fe3+ ions and nonaxial singly ionized zinc vacancies were observed. Illumination with 325 nm laser light at low temperature

  19. Characterization of Neutron-Induced Defects in Isotopically Enriched Lithium Tetraborate

    DTIC Science & Technology

    2011-03-01

    Electron paramagnetic resonance, electron-nuclear double resonance, pulsed anneal, and thermoluminescence studies prior to neutron irradiation concluded...that Ag doped Li2B4O7 crystals contain Ag point defects that trap both electrons and holes. Pulsed anneal and thermoluminescence studies of all...crystal types prior to neutron irradiation suggest neutron induced defects are significantly more stable than as-grown defects. Thermoluminescence may

  20. A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    PubMed Central

    Mansuri, M. Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-01-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics. PMID:25299184

  1. A novel alpha kinase EhAK1 phosphorylates actin and regulates phagocytosis in Entamoeba histolytica.

    PubMed

    Mansuri, M Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-10-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics.

  2. Microphthalmia with linear skin defects (MLS) syndrome: Clinical, cytogenetic, and molecular characterization

    SciTech Connect

    Lindsay, E.A.; Grillo, A.; Ferrero, G.B.; Baldini, A.; Ballabio, A.; Zoghbi, H.Y.; Roth, E.J.; Magenis, E.; Grompe, M.; Hulten, M.

    1994-01-15

    The microphthalmia with linear skin defects (MLS) syndrome (MIM309801) is a severe developmental disorder observed in XX individuals with distal Xp segmental monosomy. The phenotype of this syndrome overlaps with that of both Aicardi (MIM 305050) and Goltz (MIM 305600) syndromes, two X-linked dominant, male-lethal disorders. Here the authors report the clinical, cytogenetic, and molecular characterization of 3 patients with this syndrome. Two of these patients are females with a terminal Xpter-p22.2 deletion. One of these 2 patients had an aborted fetus with anencephaly and the same chromosome abnormality. The third patient is an XX male with Xp/Yp exchange spanning the SRY gene which results in distal Xp monosomy. The extensive clinical variability observed in these patients and the results of the molecular analysis suggest that X-inactivation plays an important role in determining the phenotype of the MLS syndrome. The authors propose that the MLS, Aicardi, and Goltz syndromes are due to the involvement of the same gene(s), and that different patterns of X-inactivation are responsible for the phenotypic differences observed in these 3 disorders. However, they cannot rule out that each component of the MLS phenotype is caused by deletion of a different gene (a contiguous gene syndrome). 24 refs., 4 figs., 1 tab.

  3. Characterization of structural defects in nuclear graphite IG-110 and NBG-18

    SciTech Connect

    Guiqiu Zheng; Peng Xu; Kumar Sridharan; Todd Allen

    2014-03-01

    Nuclear graphite IG-110 and NBG-18 were examined using X-ray diffraction (XRD), Raman spectroscopy, scanning electron microscope (SEM) and high resolution transmission electron microscope (HR-TEM) to understand the structure and microstructure of nuclear graphite. The lattice parameter (a), degree of graphitization ( ), crystallite size parallel and perpendicular to c-direction (Lc and L ), anisotropy (B), as well as in-plane crystallite size (La) were calculated and compared based on XRD patterns and Raman spectra. Results indicate that IG-110 has a larger crystallite size and higher degree of graphitization, but lower anisotropy than NBG-18. These differences are attributed to the properties of coke source and manufacturing processes. Additionally, the shape of the pores and crystallized filler particles, the interface between binders and fillers, Mrozowski cracks and nano-cracks, and the defects of disclination were observed and characterized from SEM and HR-TEM images. The similarities and differences in microstructure between IG-110 and NBG-18 are discussed. The results in this work provide useful information to guide selection of nuclear graphite for the design of next generation nuclear plants (NGNP).

  4. Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.

    PubMed Central

    Gruenert, D C; Basbaum, C B; Welsh, M J; Li, M; Finkbeiner, W E; Nadel, J A

    1988-01-01

    To facilitate understanding of the mechanisms underlying pulmonary diseases, including lung cancer and cystic fibrosis, we have transformed and characterized cultures of human tracheal epithelial cells. Cells were transfected by calcium phosphate precipitation with a plasmid containing a replication-defective simian virus 40 (SV40) genome. Colonies of cells with enhanced growth potential were isolated and analyzed for transformation- and epithelial-specific characteristics. Precrisis cells were observed to express the SV40 large tumor antigen, produce cytokeratins, have microvilli, and form tight junctions. After crisis, cells continued to express the SV40 large tumor antigen as well as epithelial-specific cytokeratins and to display the apical membrane microvilli. Apical membrane Cl channels were opened in postcrisis cells exposed to 50 microM forskolin. These channels showed electrical properties similar to those observed in primary cultures. The postcrisis cells have been in culture for greater than 250 generations and are potentially "immortal." In addition to providing a useful in vitro model for the study of ion transport by human airway epithelial cells, the cells can be used to examine stages of neoplastic progression. Images PMID:2457904

  5. Defect occurrence, detection, location and characterization; essential variables of the LBB concept application to primary piping

    SciTech Connect

    Crutzen, S.; Koble, T.D.; Lemaitre, P.

    1997-04-01

    Applications of the Leak Before Break (LBB) concept involve the knowledge of flaw presence and characteristics. In Service Inspection is given the responsibility of detecting flaws of a determined importance to locate them precisely and to classify them in broad families. Often LBB concepts application imply the knowledge of flaw characteristics such as through wall depth; length at the inner diameter (ID) or outer diameter (OD) surface; orientation or tilt and skew angles; branching; surface roughness; opening or width; crack tip aspect. Besides detection and characterization, LBB evaluations consider important the fact that a crack could be in the weld material or in the base material or in the heat affected zone. Cracks in tee junctions, in homogenous simple welds and in elbows are not considered in the same way. Essential variables of a flaw or defect are illustrated, and examples of flaws found in primary piping as reported by plant operators or service vendors are given. If such flaw variables are important in the applications of LBB concepts, essential is then the knowledge of the performance achievable by NDE techniques, during an ISI, in detecting such flaws, in locating them and in correctly evaluating their characteristics.

  6. Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation.

    PubMed Central

    Burlingame, R P; Wyman, L; Chapman, P J

    1986-01-01

    Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate. Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme activities for the degradation of these compounds and mutants pleiotropically negative for some of these activities. Characterization of these mutants was greatly facilitated by the use of indicator media in which accumulation of 3-(2,3-dihydroxyphenyl)propionate or 2-hydroxy-6-ketononadienedioic acid led to the formation of dark red or bright yellow colors, respectively, in the medium. Assays with wild-type and mutant strains indicated that 3-phenylpropionate (or its dihydrodiol), but none of the hydroxylated derivatives tested, induced the synthesis of enzymes for its conversion to 3-(2,3-dihydroxyphenyl)propionate. The remaining enzymes were induced by the 2- or 3-hydroxy or 2,3-dihydroxy derivatives of 3-phenylpropionate, with the 2-hydroxy compound acting as an apparent gratuitous inducer. Metabolism to nonaromatic intermediates appeared to be unnecessary for full induction of any pathway enzyme. One unusual class of mutants, in which 2-keto-4-pentenoate hydratase appeared to be uninducible, indicated a level of control not previously shown in meta-fission catabolic pathways. PMID:3531186

  7. Isolation and characterization of an olfactory mutant in Drosophila with a chemically specific defect.

    PubMed Central

    Helfand, S L; Carlson, J R

    1989-01-01

    A Drosophila mutant was isolated and shown to exhibit defective response to the chemical odorant benzaldehyde in two distinctly different behavioral assays. The defect exhibited chemical specificity: response to three other chemicals was normal. The mutant also showed abnormalities in pigmentation and fertility. Genetic mapping and complementation analysis provide evidence that the olfactory, pigmentation, and fertility defects arise as a result of a lesion at the pentagon locus. The specificity of the olfactory defect suggests the possibility that the mutation may define a molecule required in reception, transduction, or processing of a specific subset of chemical information in the olfactory system. Images PMID:2495539

  8. Actin: its cumbersome pilgrimage through cellular compartments

    PubMed Central

    Schleicher, Michael

    2008-01-01

    In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days’ knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin. PMID:18438682

  9. F-actin dismantling through a Redox-driven synergy between Mical and cofilin

    PubMed Central

    Grintsevich, Elena E.; Yesilyurt, Hunkar Gizem; Rich, Shannon K.; Hung, Ruei-Jiun; Terman, Jonathan R.; Reisler, Emil

    2016-01-01

    Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF/cofilins/twinstar, sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. Here we uncover a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. We find that Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared to either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodeling, axon guidance, and Semaphorin/Plexin repulsion. Mical and cofilin, therefore, form a Redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties. PMID:27454820

  10. SWAP70 Organizes the Actin Cytoskeleton and Is Essential for Phagocytosis.

    PubMed

    Baranov, Maksim V; Revelo, Natalia H; Dingjan, Ilse; Maraspini, Riccardo; Ter Beest, Martin; Honigmann, Alf; van den Bogaart, Geert

    2016-11-01

    Actin plays a critical role during the early stages of pathogenic microbe internalization by immune cells. In this study, we identified a key mechanism of actin filament tethering and stabilization to the surface of phagosomes in human dendritic cells. We found that the actin-binding protein SWAP70 is specifically recruited to nascent phagosomes by binding to the lipid phosphatidylinositol (3,4)-bisphosphate. Multi-color super-resolution stimulated emission depletion (STED) microscopy revealed that the actin cage surrounding early phagosomes is formed by multiple concentric rings containing SWAP70. SWAP70 colocalized with and stimulated activation of RAC1, a known activator of actin polymerization, on phagosomes. Genetic ablation of SWAP70 impaired actin polymerization around phagosomes and resulted in a phagocytic defect. These data show a key role for SWAP70 as a scaffold for tethering the peripheral actin cage to phagosomes.

  11. 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.

  12. Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ

    PubMed Central

    Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; Zischka, H; Mueller, R; Vollmar, A M

    2014-01-01

    Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as

  13. Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

    PubMed Central

    Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

    2012-01-01

    Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

  14. Characterization of the Optical Properties of Normal and Defective Pickling Cucumbers and Whole Pickles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Internal defect in pickling cucumbers can cause bloater damage during brining, which lowers the quality of final pickled products and results in economic loss for the pickle industry. Hence it is important to have an effective optical inspection system for detection and segregation of defective pick...

  15. Characterization of defect growth structure in ion plated films by scanning electron microscopy

    NASA Technical Reports Server (NTRS)

    Spalvins, T.

    1979-01-01

    Copper and gold films (0.2 to 2 microns) were ion plated onto polished 304-stainless-steel surfaces. These coatings were examined by scanning electron microscopy for coating growth defects. Three types of defects were distinguished: nodular growth, abnormal or runaway growth, and spits. The cause and origin for each type of defect was traced. Nodular growth is primarily due to inherent substrate microdefects, abnormal or runaway growth is due to external surface inclusions, and spits are due to nonuniform evaporation. All these defects have adverse effects on the coatings. They induce stresses and produce porosity in the coatings and thus weaken their mechanical properties. Friction and wear characteristics are affected by coating defects, since the large nodules are pulled out and additional wear debris is generated.

  16. Colloidal Defect-Free Silicalite-1 Single Crystals: Preparation, Structure Characterization, Adsorption, and Separation Properties for Alcohol/Water Mixtures.

    PubMed

    Zhou, Han; Mouzon, Johanne; Farzaneh, Amirfarrokh; Antzutkin, Oleg N; Grahn, Mattias; Hedlund, Jonas

    2015-08-04

    In this work, colloidal silicalite-1 single crystals are for the first time synthesized using fluoride as mineralizing agent at near neutral pH. SEM, TEM, DLS, XRD, solid-state (29)Si MAS NMR, and adsorption/desorption experiments using nitrogen, water, n-butanol, and ethanol as adsorbates were used to characterize the crystals. The single crystals have a platelike habit with a length of less than 170 nm and an aspect ratio (length/width) of about 1.2, and the thickness of the crystals is less than 40 nm. Compared with silicalite-1 crystals grown using hydroxide as mineralizing agent, the amount of structural defects in the lattice is significantly reduced and the hydrophobicity is increased. Membrane separation and adsorption results show that the synthesized defect-free crystals present high selectivity to alcohols from alcohol/water mixtures. The n-butanol/water adsorption selectivities were ca. 165 and 14 for the defect-free crystals and a reference sample containing defects, respectively, illustrating the improvement in n-butanol/water selectivity by eliminating the polar silanol defects.

  17. ACTIN DEPOLYMERIZING FACTOR4 regulates actin dynamics during innate immune signaling in Arabidopsis.

    PubMed

    Henty-Ridilla, Jessica L; Li, Jiejie; Day, Brad; Staiger, Christopher J

    2014-01-01

    Conserved microbe-associated molecular patterns (MAMPs) are sensed by pattern recognition receptors (PRRs) on cells of plants and animals. MAMP perception typically triggers rearrangements to actin cytoskeletal arrays during innate immune signaling. However, the signaling cascades linking PRR activation by MAMPs to cytoskeleton remodeling are not well characterized. Here, we developed a system to dissect, at high spatial and temporal resolution, the regulation of actin dynamics during innate immune signaling in plant cells. Within minutes of MAMP perception, we detected changes to single actin filament turnover in epidermal cells treated with bacterial and fungal MAMPs. These MAMP-induced alterations phenocopied an ACTIN DEPOLYMERIZING FACTOR4 (ADF4) knockout mutant. Moreover, actin arrays in the adf4 mutant were unresponsive to a bacterial MAMP, elf26, but responded normally to the fungal MAMP, chitin. Together, our data provide strong genetic and cytological evidence for the inhibition of ADF activity regulating actin remodeling during innate immune signaling. This work is the first to directly link an ADF/cofilin to the cytoskeletal rearrangements elicited directly after pathogen perception in plant or mammalian cells.

  18. 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

  19. 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.

  20. Development of phased array techniques to improve characterization of defect located in a component of complex geometry.

    PubMed

    Mahaut, Steve; Roy, Olivier; Beroni, Claude; Rotter, Bernhard

    2002-05-01

    Ultrasonic inspection of complex geometry components has to cope with different problems: limited access of the area assumed to be insonified, beam misorientation and distortions, loss of sensitivity. Those harmful effects can lead to inspection performance degradations, especially in terms of defect detection and characterization. Phased array techniques may be used to overcome such difficulties, as they can provide an optimal mastering of the ultrasonic beam radiated through the inspected component. This paper presents some applications of phased array inspections carried out by the French Atomic Energy Commission (CEA) and the French Company of Electricity (EDF) in the framework of R&D studies. Inspections of components with varying profile (of planar and cylindrical parts, misalignment and local depression), and containing artificial reflectors have been carried out with pulse echo immersion techniques, using standard and phased arrays transducers. Optimal delay laws have been applied to preserve the beam characteristics in spite of the varying profile geometry encountered as the phased array transducer was moved over the component. Those delay laws allow to efficiently compensate the beam distortions generated by the profile geometry. They were computed using a specific model and compared to experimental delays obtained using through transmission tests. Experimental and simulation results showed that the defect detection and characterization performances were greatly enhanced using phased array techniques. In the presented examples, with standard transducers, defects located below the irregular parts of the specimen were partially detected, in accurately located or even missed, whereas phased array inspections enabled to detect and locate all of these defects.

  1. Comparison of lock-in and pulse-phase thermography for defect characterization in FRP composites applied to concrete

    NASA Astrophysics Data System (ADS)

    Brown, Jeff; Chittineni, Sai Harsha

    2015-05-01

    Thermal imaging is a well-established technique for the non-destructive evaluation of FRP composites applied to reinforced concrete. Defect characterization using IR thermography, however, remains a topic of on-going research, and there are currently no universally accepted standards for data collection or interpretation. This research involved large scale thermography inspection of two FRP strengthened bridge girders that were removed from service after approximately 10 years of service in a potentially corrosive marine environment. Trial inspections were performed on test areas where defects could be identified using sounding methods. Two procedures showed the most promise for identifying and characterizing defects: sinusoidal (lock-in style) heating with periods ranging from 5 s to 40 s and constant step heating for 30 s followed by 60 s of cooling. Both methods resulted in a series of phase images that provided insight into the depth and general nature of detected defects. This paper presents the findings of a comparison study between these two thermal imaging techniques.

  2. 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

  3. IKKε inhibits PKC to promote Fascin-dependent actin bundling

    PubMed Central

    Ogura, Yosuke; Misaki, Kazuyo; Maeda, Takuya; Kimpara, Akiyo; Yonemura, Shigenobu; Hayashi, Shigeo

    2016-01-01

    Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. We have previously shown that a protein kinase, IKKε, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. Here, we demonstrate that IKKε regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKKε inhibits PKC, thereby protecting Fascin from inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in IKKε-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wild-type bristles, indicating that PKC is repressed by IKKε in wild-type bristle cells. These results suggest that IKKε prevents excess activation of PKC during bristle morphogenesis. PMID:27578797

  4. Characterization of V-shaped defects in 4H-SiC homoepitaxial layers

    SciTech Connect

    Zhang, Lihua; Su, Dong; Kisslinger, Kim; Stach, Eric; Chung, Gil; Zhang, Jie; Thomas, Bernd; Sanchez, Edward K; Mueller, Stephan G.; Hansen, Darren; Loboda, Mark J.; Wu, Fangzhen; Wang, Huanhuan; Raghothamachar, Balaji; Dudley, Michael

    2014-12-04

    Synchrotron white beam x-ray topography images show that faint needle-like surface morphological features observed on the Si-face of 4H-SiC homoepitaxial layers using Nomarski optical microscopy are associated with V shaped stacking faults in the epilayer. KOH etching of the V shaped defect reveals small oval pits connected by a shallow line which corresponding to the surface intersections of two partial dislocations and the stacking fault connecting them. Transmission electron microscopy (TEM) specimens from regions containing the V shaped defects were prepared using focused ion beam milling, and stacking sequences of (85), (50) and (63) are observed at the faulted region with high resolution TEM. In order to study the formation mechanism of V shaped defect, low dislocation density 4H-SiC substrates were chosen for epitaxial growth, and the corresponding regions before and after epitaxy growth are compared in SWBXT images. It is found that no defects in the substrate are directly associated with the formation of the V shaped defect. Simulation results of the contrast from the two partial dislocations associated with V shaped defect in synchrotron monochromatic beam x-ray topography reveals the opposite sign nature of their Burgers vectors. Therefore, a mechanism of 2D nucleation during epitaxy growth is postulated for the formation of the V shaped defect, which requires elimination of non-sequential 1/4[0001] bilayers from the original structure to create the observed faulted stacking sequence.

  5. Characterization of V-shaped defects in 4H-SiC homoepitaxial layers

    DOE PAGES

    Zhang, Lihua; Su, Dong; Kisslinger, Kim; ...

    2014-12-04

    Synchrotron white beam x-ray topography images show that faint needle-like surface morphological features observed on the Si-face of 4H-SiC homoepitaxial layers using Nomarski optical microscopy are associated with V shaped stacking faults in the epilayer. KOH etching of the V shaped defect reveals small oval pits connected by a shallow line which corresponding to the surface intersections of two partial dislocations and the stacking fault connecting them. Transmission electron microscopy (TEM) specimens from regions containing the V shaped defects were prepared using focused ion beam milling, and stacking sequences of (85), (50) and (63) are observed at the faulted regionmore » with high resolution TEM. In order to study the formation mechanism of V shaped defect, low dislocation density 4H-SiC substrates were chosen for epitaxial growth, and the corresponding regions before and after epitaxy growth are compared in SWBXT images. It is found that no defects in the substrate are directly associated with the formation of the V shaped defect. Simulation results of the contrast from the two partial dislocations associated with V shaped defect in synchrotron monochromatic beam x-ray topography reveals the opposite sign nature of their Burgers vectors. Therefore, a mechanism of 2D nucleation during epitaxy growth is postulated for the formation of the V shaped defect, which requires elimination of non-sequential 1/4[0001] bilayers from the original structure to create the observed faulted stacking sequence.« less

  6. CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle.

    PubMed

    Nomura, Kazumi; Ono, Kanako; Ono, Shoichiro

    2012-09-01

    Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.

  7. 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.

  8. Characterization of Vacancy Defects in Carbon Ion Irradiated Graphite Using Positrons

    SciTech Connect

    Anto, C. Varghese; Arunkumar, J.; Rajaraman, R.; Nair, K. G. M.; Amarendra, G.

    2011-07-15

    Highly Oriented Pyrolytic Graphite samples are irradiated with 200 keV Carbon ions to fluences of 10{sup 14} and 10{sup 15} C{sup +} ions/cm{sup 2}. Depth resolved Doppler lineshape S-parameter exhibited large increase in peak damage regions of the sample, indicating the existence of irradiation induced vacancy defects. The depth profile of the defect region has been deduced from the analysis of the experimental data. It is found that divacancies are the dominant defects in the irradiated samples.

  9. 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.

  10. Synchrotron X-ray CT characterization of titanium parts fabricated by additive manufacturing. Part II. Defects.

    PubMed

    Scarlett, Nicola Vivienne Yorke; Tyson, Peter; Fraser, Darren; Mayo, Sheridan; Maksimenko, Anton

    2016-07-01

    Synchrotron X-ray tomography (SXRT) has been applied to the study of defects within three-dimensional printed titanium parts. These parts were made using the Arcam EBM(®) (electron beam melting) process which uses powdered titanium alloy, Ti64 (Ti alloy with approximately 6%Al and 4%V) as the feed and an electron beam for the sintering/welding. The experiment was conducted on the Imaging and Medical Beamline of the Australian Synchrotron. The samples represent a selection of complex shapes with a variety of internal morphologies. Inspection via SXRT has revealed a number of defects which may not otherwise have been seen. The location and nature of such defects combined with detailed knowledge of the process conditions can contribute to understanding the interplay between design and manufacturing strategy. This fundamental understanding may subsequently be incorporated into process modelling, prediction of properties and the development of robust methodologies for the production of defect-free parts.

  11. Characterization of Point Defects in Lithium Aluminate (LiAlO2) Single Crystals

    DTIC Science & Technology

    2015-09-17

    resonance (EPR) and fluorescence spectroscopies, thermoluminescence , and optical absorption. After x ray irradiation, the major hole-trapping defect...22 2.4 Thermoluminescence (TL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27...39 3.3 Thermoluminescence Dosimeter Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.4 Spectrofluorometer

  12. Passivation and characterization of charge defects in ambipolar silicon quantum dots

    PubMed Central

    Spruijtenburg, Paul C.; Amitonov, Sergey V.; Mueller, Filipp; van der Wiel, Wilfred G.; Zwanenburg, Floris A.

    2016-01-01

    In this Report we show the role of charge defects in the context of the formation of electrostatically defined quantum dots. We introduce a barrier array structure to probe defects at multiple locations in a single device. We measure samples both before and after an annealing process which uses an Al2O3 overlayer, grown by atomic layer deposition. After passivation of the majority of charge defects with annealing we can electrostatically define hole quantum dots up to 180 nm in length. Our ambipolar structures reveal amphoteric charge defects that remain after annealing with charging energies of 10 meV in both the positive and negative charge state. PMID:27922048

  13. Mechanics of composite actin networks: in vitro and cellular perspectives

    NASA Astrophysics Data System (ADS)

    Upadhyaya, Arpita

    2014-03-01

    Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

  14. Comprehensive Characterization of Extended Defects in Semiconductor Materials by a Scanning Electron Microscope

    PubMed Central

    Hieckmann, Ellen; Nacke, Markus; Allardt, Matthias; Bodrov, Yury; Chekhonin, Paul; Skrotzki, Werner; Weber, Jörg

    2016-01-01

    Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications of semiconductor materials. However, it is still under debate how the defect structure determines the band structure, and therefore, the recombination behavior of electron-hole pairs responsible for the optical and electrical properties of the extended defects. The present paper is a survey of procedures for the spatially resolved investigation of structural and of physical properties of extended defects in semiconductor materials with a scanning electron microscope (SEM). Representative examples are given for crystalline silicon. The luminescence behavior of extended defects can be investigated by cathodoluminescence (CL) measurements. They are particularly valuable because spectrally and spatially resolved information can be obtained simultaneously. For silicon, with an indirect electronic band structure, CL measurements should be carried out at low temperatures down to 5 K due to the low fraction of radiative recombination processes in comparison to non-radiative transitions at room temperature. For the study of the electrical properties of extended defects, the electron beam induced current (EBIC) technique can be applied. The EBIC image reflects the local distribution of defects due to the increased charge-carrier recombination in their vicinity. The procedure for EBIC investigations is described for measurements at room temperature and at low temperatures. Internal strain fields arising from extended defects can be determined quantitatively by cross-correlation electron backscatter diffraction (ccEBSD). This method is challenging because of the necessary preparation of the sample surface and because of the quality of the diffraction patterns which are recorded during the mapping of the sample. The spatial resolution of the three experimental techniques is compared. PMID:27285177

  15. Characterization of vacancy defects in Cu(In,Ga)Se2 by positron annihilation spectroscopy

    NASA Astrophysics Data System (ADS)

    Elsharkawy, M. R. M.; Kanda, G. S.; Yakushev, M. V.; Abdel-Hady, E. E.; Keeble, D. J.

    2016-12-01

    The photovoltaic performance of Cu(In1-x,Gax)Se2 (CIGS) materials is commonly assumed to be degraded by the presence of vacancy-related defects. However, experimental identification of specific vacancy defects remains challenging. In this work we report positron lifetime measurements on CIGS crystals with x = 0, and x = 0.05, saturation trapping to two dominant vacancy defect types, in both types of crystal, is observed and found to be independent of temperature between 15-300 K. Atomic superposition method calculations of the positron lifetimes for a range of vacancy defects in CIS and CGS are reported. The calculated lifetimes support the assignment of the first experimental lifetime component to monovacancy or divacancy defects, and the second to trivacancies, or possibly the large In-Se divacancy. Further, the calculated positron parameters obtained here provide evidence that positron annihilation spectroscopy has the capability to identify specific vacancy-related defects in the Cu(In1-x,Gax)Se2 chalcogenides.

  16. Characterization and modelling of the boron-oxygen defect activation in compensated n-type silicon

    SciTech Connect

    Schön, J.; Niewelt, T.; Broisch, J.; Warta, W.; Schubert, M. C.

    2015-12-28

    A study of the activation of the light-induced degradation in compensated n-type Czochralski grown silicon is presented. A kinetic model is established that verifies the existence of both the fast and the slow components known from p-type and proves the quadratic dependence of the defect generation rates of both defects on the hole concentration. The model allows for the description of lifetime degradation kinetics in compensated n-type silicon under various intensities and is in accordance with the findings for p-type silicon. We found that the final concentrations of the slow defect component in compensated n-type silicon only depend on the interstitial oxygen concentration and on neither the boron concentration nor the equilibrium electron concentration n{sub 0}. The final concentrations of the fast defect component slightly increase with increasing boron concentration. The results on n-type silicon give new insight to the origin of the BO defect and question the existing models for the defect composition.

  17. The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton

    PubMed Central

    1996-01-01

    Arp2p is an essential yeast actin-related protein. Disruption of the corresponding ARP2 gene leads to a terminal phenotype characterized by the presence of a single large bud. Thus, Arp2p may be important for a late stage of the cell cycle (Schwob, E., and R.P. Martin, 1992. Nature (Lond.). 355:179-182). We have localized Arp2p by indirect immunofluorescence. Specific peptide antibodies revealed punctate staining under the plasma membrane, which partially colocalizes with actin. Temperature-sensitive arp2 mutations were created by PCR mutagenesis and selected by an ade2/SUP11 sectoring screen. One temperature-sensitive mutant that was characterized, arp2-H330L, was osmosensitive and had an altered actin cytoskeleton at a nonpermissive temperature, suggesting a role of Arp2p in the actin cytoskeleton. Random budding patterns were observed in both haploid and diploid arp2- H330L mutant cells. Endocytosis, as judged by Lucifer yellow uptake, was severely reduced in the mutant, at all temperatures. In addition, genetic interaction was observed between temperature-sensitive alleles arp2-H330L and cdc10-1. CDC10 is a gene encoding a neck filament- associated protein that is necessary for polarized growth and cytokinesis. Overall, the immunolocalization, mutant phenotypes, and genetic interaction suggest that the Arp2 protein is an essential component of the actin cytoskeleton that is involved in membrane growth and polarity, as well as in endocytosis. PMID:8698808

  18. 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

  19. Defect characterization of electronic conducting pseudo-perovskite systems. Final report

    SciTech Connect

    Anderson, H.U.; Nasrallah, M.; Sparlin, D.M.; Parris, P.E.

    1994-12-31

    The goal of the program has been to study the interrelationships between electrical conductivity, oxidation-reduction kinetics, defect structure, and composition of n- and p-type binary and ternary transition metal oxides. The stimulus for making these studies was the observation that both conducting n and p type oxides displayed a dependence on oxygen activity that was not predicted by the defect chemistry of their majority defects. The project has focused primarily on the understanding of electronic and ionic conduction in the REBO{sub 3} oxides, where RE is a rare earth ion and B is a transition metal ion. This is being done by studying the interrelationships between the electronic and ionic conductivity, the electronic structure of the B site transition metal ion, and the acceptor concentration. The dependence of these characteristics on the oxygen activity, the temperature, and the defect chemistry of the oxide system is being determined. Theoretical mechanisms and models are being developed from the body of experimental results to provide a predictive tool. The effect of dopants and impurities, processing, electrical and thermal stability as a function of oxygen activity and temperature and their relationship to defect chemistry of the perovskite type oxides have been studied extensively by this research team. As a result of the difference in mobility between electronic and ionic defects, the electrical conductivity changes as the concentration of electronic defects changes. Thus, electrodes or resistors consisting of such oxides are susceptible to instabilities in resistance as they are cycled into temperature regimes where thermodynamic equilibrium may be attained. Many of the new energy conversion systems which use such oxides are encountering difficulties as a result of this instability in resistivity.

  20. 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.

  1. Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs.

    PubMed

    Iskander, Deena; Psaila, Bethan; Gerrard, Gareth; Chaidos, Aristeidis; En Foong, Hui; Harrington, Yvonne; Karnik, Leena C; Roberts, Irene; de la Fuente, Josu; Karadimitris, Anastasios

    2015-04-16

    Diamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin(-)CD34(+)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(-)CD36(-) bone marrow cells as EEP giving rise to BFU-E, and Lin(-)CD34(+/-)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(+)CD36(+) cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis.

  2. Surface defects characterization in a quantum wire by coherent phonons scattering

    SciTech Connect

    Rabia, M. S.

    2015-03-30

    The influence of surface defects on the scattering properties of elastic waves in a quasi-planar crystallographic waveguide is studied in the harmonic approximation using the matching method formalism. The structural model is based on three infinite atomic chains forming a perfect lattice surmounted by an atomic surface defect. Following the Landauer approach, we solve directly the Newton dynamical equation with scattering boundary conditions and taking into account the next nearest neighbour’s interaction. A detailed study of the defect-induced fluctuations in the transmission spectra is presented for different adatom masses. As in the electronic case, the presence of localized defect-induced states leads to Fano-like resonances. In the language of mechanical vibrations, these are called continuum resonances. Numerical results reveal the intimate relation between transmission spectra and localized defect states and provide a basis for the understanding of conductance spectroscopy experiments in disordered mesoscopic systems. The results could be useful for the design of phononic devices.

  3. Defect chemistry and characterization of Hg sub 1x Cd sub x Te

    NASA Technical Reports Server (NTRS)

    Vydyanath, H. R.

    1982-01-01

    Single crystal samples of undoped and doped Hg sub 1-x Cd sub x Te were annealed at varying temperatures and partial pressures of Hg. Hall effect and mobility measurements were carried out on these samples after quenching to room temperature. Based on the variation of the carrier concentration and the carrier mobility as a function of the partial pressure of Hg temperature, and dopant concentration, defect models were established for the doped and the undoped crystals. These models indicate that the native acceptor defects in both Hg0.8Cd0.2Te and Hg0.6Cd0.4Te doubly ionized and the native donor defects are negligible in concentration, implying that p to n conversion in these alloys occurs due only to residual donors. Incorporation mechanism of copper, indium, iodine, and phosphorus were investigated. A large concentration of indium is found to be paired with the native acceptor defects. Results on crystals doped with phosphorus indicate that phosphorus behaves amphoterically, acting as a donor on Hg lattice sites and as an acceptor intersitially on Te lattice sites. A majority of the phosphorus is found to be present as neutral species formed from the pairing reaction between phosphorus on Hg lattice sites and phosphorus in interstitial sites. Equilibrium constants for the intrinsic excitation reaction, as well as for the incorporation of the different dopants and the native acceptor defects were established.

  4. CHARACTERIZATION OF PD IMPURITIES AND TWIN BOUNDARY DEFECTS IN DETECTOR GRADE CDZNTE CRYSTALS

    SciTech Connect

    Duff, M.

    2011-06-22

    Synthetic CdZnTe or ''CZT'' crystals are highly suitable for {gamma}-spectrometers operating at the room temperature. Secondary phases (SP) in CZT are known to inhibit detector performance, particularly when they are present in large numbers or dimensions. These SP may exist as voids or composites of non-cubic phase metallic Te layers with bodies of polycrystalline and amorphous CZT material and voids. Defects associated with crystal twining may also influence detector performance in CZT. Using transmission electron microscopy, we identify two types of defects that are on the nano scale. The first defect consists of 40 nm diameter metallic Pd/Te bodies on the grain boundaries of Te-rich composites. Although the nano-Pd/Te bodies around these composites may be unique to the growth source of this CZT material, noble metal impurities like these may contribute to SP formation in CZT. The second defect type consists of atom-scale grain boundary dislocations. Specifically, these involve inclined ''finite-sized'' planar defects or interfaces between layers of atoms that are associated with twins. Finite-sized twins may be responsible for the subtle but observable striations that can be seen with optical birefringence imaging and synchrotron X-ray topographic imaging.

  5. Comprehensive characterization of C-glycosyl flavones in wheat (Triticum aestivum L.) germ using UPLC-PDA-ESI/HRMSn and mass defect filtering

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comprehensive characterization of C-glycosyl flavones in wheat germ has been conducted using multi-stage high resolution mass spectrometry (HRMS) combined with mass defect filter (MDF). MDF performed the initial search of raw data with defined mass ranges and mass defect windows to generate the n...

  6. Defect characterization of β-Ga2O3 single crystals grown by vertical Bridgman method

    NASA Astrophysics Data System (ADS)

    Ohba, Etsuko; Kobayashi, Takumi; Kado, Motohisa; Hoshikawa, Keigo

    2016-12-01

    The characteristics of structural defects observed on (100) wafers in β-Ga2O3 single crystals grown by directional solidification in a vertical Bridgman furnace were studied in terms of crystal growth conditions. No high-dislocation-density regions near the wafer periphery were observed owing to the lack of adhesion between the as-grown crystal ingot surface and the crucible inner wall, and directional solidification growth in a crucible with a very low temperature gradient resulted in β-Ga2O3 single crystals with a low mean dislocation density of 2.3 × 103 cm-2. Line-shaped defects up to 150 µm long in the [010] direction were detected at a mean density of 0.5 × 102 cm-2, which decreased with decreasing growth rate. The line-shaped defect structure and formation mechanism were discussed.

  7. Characterization of the molecular defect in the ATP7B gene in Wilson disease patients from Yugoslavia.

    PubMed

    Loudianos, Georgios; Kostic, Vladimir; Solinas, Paola; Lovicu, Mario; Dessì, Valeria; Svetel, Marina; Major, Tamara; Cao, Antonio

    2003-01-01

    Wilson disease (WD) is an autosomal recessive disorder of copper metabolism resulting from the absence or dysfunction of a copper transporting P-type ATPase (ATP7B). Approximately 150 mutations of the ATP7B have been identified to date. In this paper, we report the results of molecular characterization and genotype-phenotype analysis, which we have carried out on 35 patients from Yugoslavia affected by WD. Using single-strand conformational polymorphism (SSCP) followed by direct sequencing, we characterized the molecular defect in 80% of WD chromosomes and found 11 different mutations, three of which are novel. The most common mutations that accounted for the molecular defect in 71.3% of WD chromosomes were H1069Q (48.9%), 2304-2305insC (11.4%), R616Q (5.7%), and A1003T (5.7%). The results produced in this paper indicate that the best strategy for mutation detection in Yugoslavian patients with WD is an SSCP analysis of exons 14, 8, 5, and 13, where most of the defects (73.1%) lie, followed by mutation analysis of the remaining exons in ATP7B in patients in whom the mutation was not detected by the finitial screening. These data can be used to develop straightforward genetic testing in this population or in other countries composed of a genetically mixed population like the United States, where a significant number of immigrants came from Central and Eastern Europe.

  8. Evaluating Printability of Buried Native EUV Mask Phase Defects through a Modeling and Simulation Approach

    SciTech Connect

    Upadhyaya, Mihir; Jindal, Vibhu; Basavalingappa, Adarsh; Herbol, Henry; Harris-Jones, Jenah; Jang, Il-Yong; Goldberg, Kenneth A.; Mochi, Iacopo; Marokkey, Sajan; Demmerle, Wolfgang; Pistor, Thomas V.; Denbeaux, Gregory

    2015-03-16

    The availability of defect-free masks is considered to be a critical issue for enabling extreme ultraviolet lithography (EUVL) as the next generation technology. Since completely defect-free masks will be hard to achieve, it is essential to have a good understanding of the printability of the native EUV mask defects. In this work, we performed a systematic study of native mask defects to understand the defect printability caused by them. The multilayer growth over native substrate mask blank defects was correlated to the multilayer growth over regular-shaped defects having similar profiles in terms of their width and height. To model the multilayer growth over the defects, a novel level-set multilayer growth model was used that took into account the tool deposition conditions of the Veeco Nexus ion beam deposition tool. The same tool was used for performing the actual deposition of the multilayer stack over the characterized native defects, thus ensuring a fair comparison between the actual multilayer growth over native defects, and modeled multilayer growth over regular-shaped defects. Further, the printability of the characterized native defects was studied with the SEMATECH-Berkeley Actinic Inspection Tool (AIT), an EUV mask-imaging microscope at Lawrence Berkeley National Laboratory (LBNL). Printability of the modeled regular-shaped defects, which were propagated up the multilayer stack using level-set growth model was studied using defect printability simulations implementing the waveguide algorithm. Good comparison was observed between AIT and the simulation results, thus demonstrating that multilayer growth over a defect is primarily a function of a defect’s width and height, irrespective of its shape. This would allow us to predict printability of the arbitrarily-shaped native EUV mask defects in a systematic and robust manner.

  9. Laser-projected photothermal thermography using thermal wave field interference for subsurface defect characterization

    NASA Astrophysics Data System (ADS)

    Thiel, Erik; Kreutzbruck, Marc; Ziegler, Mathias

    2016-09-01

    The coherent superposition of two anti-phased thermal wave fields creates a zone of destructive interference which is extremely sensitive to the presence of defects without any reference measurements. Combining a high power laser with a spatial light modulator allows modulating phase and amplitude of an illuminated surface that induces spatially and temporally controlled thermal wave fields. The position and depth of defects are reconstructed from analysis of the amplitude and phase of the resulting photothermal signal. The proposed concept is experimentally validated and supported by numerical modeling.

  10. Defect chemistry and characterization of Hg(1-x)Cd(x)Te

    NASA Technical Reports Server (NTRS)

    Vydyanath, H. R.

    1981-01-01

    Undoped mercury cadmium telluride crystals were subjected to high temperature equilibration at temperatures ranging from 400 C to 655 C in various Hg atmospheres. Hall effect and mobility measurements were carried out on the crystals quenched to room temperature subsequent to the high temperature equilibration. The variation of the hole concentration in the cooled crystals at 77 K as a function of the partial pressure of Hg at the equlibration temperatures, together with a comparison of the hole mobility in the undoped samples with that in the copper and phosphorous doped samples yielded a defect model for the undoped crystals, according to which, the undoped crystals are essentially intrinsic at the equilibration temperatures and the native acceptor defects are doubly ionized. Native donor defects appear to be negligible in concentration, implying that the p to n conversion in these alloys is mainly due to residual foreign donor impurities. The thermodynamic constants for the intrinsic excitation process as well as for the incorporation of the doubly ionized native acceptor defects in the undoped crystals were obtained.

  11. Defect chemistry and characterization of Hg(1-x)Cd(x)Te

    NASA Astrophysics Data System (ADS)

    Vydyanath, H. R.

    Undoped mercury cadmium telluride crystals were subjected to high temperature equilibration at temperatures ranging from 400 C to 655 C in various Hg atmospheres. Hall effect and mobility measurements were carried out on the crystals quenched to room temperature subsequent to the high temperature equilibration. The variation of the hole concentration in the cooled crystals at 77 K as a function of the partial pressure of Hg at the equlibration temperatures, together with a comparison of the hole mobility in the undoped samples with that in the copper and phosphorous doped samples yielded a defect model for the undoped crystals, according to which, the undoped crystals are essentially intrinsic at the equilibration temperatures and the native acceptor defects are doubly ionized. Native donor defects appear to be negligible in concentration, implying that the p to n conversion in these alloys is mainly due to residual foreign donor impurities. The thermodynamic constants for the intrinsic excitation process as well as for the incorporation of the doubly ionized native acceptor defects in the undoped crystals were obtained.

  12. Multisensor fusion for 3-D defect characterization using wavelet basis function neural networks

    NASA Astrophysics Data System (ADS)

    Lim, Jaein; Udpa, Satish S.; Udpa, Lalita; Afzal, Muhammad

    2001-04-01

    The primary objective of multi-sensor data fusion, which offers both quantitative and qualitative benefits, has the ability to draw inferences that may not be feasible with data from a single sensor alone. In this paper, data from two sets of sensors are fused to estimate the defect profile from magnetic flux leakage (MFL) inspection data. The two sensors measure the axial and circumferential components of the MFL. Data is fused at the signal level. If the flux is oriented axially, the samples of the axial signal are measured along a direction parallel to the flaw, while the circumferential signal is measured in a direction that is perpendicular to the flaw. The two signals are combined as the real and imaginary components of a complex valued signal. Signals from an array of sensors are arranged in contiguous rows to obtain a complex valued image. A boundary extraction algorithm is used to extract the defect areas in the image. Signals from the defect regions are then processed to minimize noise and the effects of lift-off. Finally, a wavelet basis function (WBF) neural network is employed to map the complex valued image appropriately to obtain the geometrical profile of the defect. The feasibility of the approach was evaluated using the data obtained from the MFL inspection of natural gas transmission pipelines. Results show the effectiveness of the approach.

  13. Bacillus anthracis Edema Toxin Impairs Neutrophil Actin-Based Motility▿

    PubMed Central

    Szarowicz, Sarah E.; During, Russell L.; Li, Wei; Quinn, Conrad P.; Tang, Wei-Jen; Southwick, Frederick S.

    2009-01-01

    Inhalation anthrax results in high-grade bacteremia and is accompanied by a delay in the rise of the peripheral polymorphonuclear neutrophil (PMN) count and a paucity of PMNs in the infected pleural fluid and mediastinum. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of the adenylate cyclase edema factor (EF) and protective antigen (PA). Relatively low concentrations of ET (100 to 500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and ability to polarize. These changes are accompanied by a reduction in chemoattractant-stimulated PMN actin assembly. ET also causes a significant decrease in Listeria monocytogenes intracellular actin-based motility within HeLa cells. These defects in actin assembly are accompanied by a >50-fold increase in intracellular cyclic AMP and a >4-fold increase in the phosphorylation of protein kinase A. We have previously shown that anthrax lethal toxin (LT) also impairs neutrophil actin-based motility (R. L. During, W. Li, B. Hao, J. M. Koenig, D. S. Stephens, C. P. Quinn, and F. S. Southwick, J. Infect. Dis. 192:837-845, 2005), and we now find that LT combined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP (N-formyl-met-leu-phe)-induced actin assembly. We conclude that ET alone or combined with LT impairs PMN actin assembly, resulting in paralysis of PMN chemotaxis. PMID:19349425

  14. Internal dynamics of F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

    SciTech Connect

    Matsuo, Tatsuhito; Arata, Toshiaki; Oda, Toshiro; Nakajima, Kenji; Ohira-Kawamura, Seiko; Kikuchi, Tatsuya; Fujiwara, Satoru

    2015-04-10

    Various biological functions related to cell motility are driven by the interaction between the partner proteins, actin and myosin. To obtain insights into how this interaction occurs, the internal dynamics of F-actin and myosin subfragment-1 (S1) were characterized by the quasielastic neutron scattering measurements on the solution samples of F-actin and S1. Contributions of the internal motions of the proteins to the scattering spectra were separated from those of the global macromolecular diffusion. Analysis of the spectra arising from the internal dynamics showed that the correlation times of the atomic motions were about two times shorter for F-actin than for S1, suggesting that F-actin fluctuates more rapidly than S1. It was also shown that the fraction of the immobile atoms is larger for S1 than for F-actin. These results suggest that F-actin actively facilitates the binding of myosin by utilizing the more frequent conformational fluctuations than those of S1. - Highlights: • We studied the internal dynamics of F-actin and myosin S1 by neutron scattering. • The correlation times of the atomic motions were smaller for F-actin than for S1. • The fraction of the immobile atoms was also smaller for F-actin than for S1. • Our results suggest that mobility of atoms in F-actin is higher than that in S1. • We propose that high flexibility of F-actin facilitates the binding of myosin.

  15. Characterization of the optical properties of normal and defective pickling cucumbers and whole pickles

    NASA Astrophysics Data System (ADS)

    Lu, Renfu; Ariana, Diwan P.; Cen, Haiyan

    2010-04-01

    Internal defect in pickling cucumbers can cause bloater damage during brining, which lowers the quality of final pickled products and results in economic loss for the pickle industry. Hence it is important to have an effective optical inspection system for detection and segregation of defective pickling cucumbers. This research was intended to measure the spectral absorption and scattering properties of normal and internally defective pickling cucumbers and whole pickles, using hyperspectral imaging-based spatially-resolved technique. Spatially-resolved hyperspectral scattering images were acquired from 50 freshly harvested 'Journey' pickling cucumbers in the summer of 2008. The cucumbers were then subjected to rolling under mechanical load to induce internal damage. The damaged cucumbers were imaged again one hour and one day after the mechanical stress treatment. In addition, 20 whole pickles each of normal and defective (bloated) class were also measured by following the same procedure as that for pickling cucumbers. Spectra of the absorption and reduced scattering coefficients for pickling cucumbers and whole pickles were extracted from the spatially-resolved scattering profiles, using an inverse algorithm for a diffusion theory model, for the spectral range of 700-1,000 nm. It was found that within one hour after mechanical damage, changes in the absorption and reduced scattering coefficients for the cucumbers were minimal. One day after mechanical damage, the absorption coefficient for the cucumbers increased noticeably for the wavelengths of 700-920 nm, whereas the reduced scattering coefficient decreased more significantly for the wavelengths of 700-1,000 nm. Overall mechanical damage had greater impact on the scattering properties than on the absorption properties. After brining, pickles became translucent and scattering was greatly diminished. Thus the diffusion theory model was no longer valid for determining the optical properties of whole pickles. This

  16. Regulation of actin catch-slip bonds with a RhoA-formin module

    PubMed Central

    Lee, Cho-yin; Lou, Jizhong; Wen, Kuo-Kuang; McKane, Melissa; Eskin, Suzanne G.; Rubenstein, Peter A.; Chien, Shu; Ono, Shoichiro; Zhu, Cheng; McIntire, Larry V.

    2016-01-01

    The dynamic turnover of the actin cytoskeleton is regulated cooperatively by force and biochemical signaling. We previously demonstrated that actin depolymerization under force is governed by catch-slip bonds mediated by force-induced K113:E195 salt-bridges. Yet, the biochemical regulation as well as the functional significance of actin catch bonds has not been elucidated. Using AFM force-clamp experiments, we show that formin controlled by RhoA switches the actin catch-slip bonds to slip-only bonds. SMD simulations reveal that the force does not induce the K113:E195 interaction when formin binds to actin K118 and E117 residues located at the helical segment extending to K113. Actin catch-slip bonds are suppressed by single residue replacements K113E and E195K that interrupt the force-induced K113:E195 interaction; and this suppression is rescued by a K113E/E195K double mutant (E/K) restoring the interaction in the opposite orientation. These results support the biological significance of actin catch bonds, as they corroborate reported observations that RhoA and formin switch force-induced actin cytoskeleton alignment and that either K113E or E195K induces yeast cell growth defects rescued by E/K. Our study demonstrates how the mechano-regulation of actin dynamics is modulated by biochemical signaling molecules, and suggests that actin catch bonds may be important in cell functions. PMID:27731359

  17. Regulation of actin catch-slip bonds with a RhoA-formin module

    NASA Astrophysics Data System (ADS)

    Lee, Cho-Yin; Lou, Jizhong; Wen, Kuo-Kuang; McKane, Melissa; Eskin, Suzanne G.; Rubenstein, Peter A.; Chien, Shu; Ono, Shoichiro; Zhu, Cheng; McIntire, Larry V.

    2016-10-01

    The dynamic turnover of the actin cytoskeleton is regulated cooperatively by force and biochemical signaling. We previously demonstrated that actin depolymerization under force is governed by catch-slip bonds mediated by force-induced K113:E195 salt-bridges. Yet, the biochemical regulation as well as the functional significance of actin catch bonds has not been elucidated. Using AFM force-clamp experiments, we show that formin controlled by RhoA switches the actin catch-slip bonds to slip-only bonds. SMD simulations reveal that the force does not induce the K113:E195 interaction when formin binds to actin K118 and E117 residues located at the helical segment extending to K113. Actin catch-slip bonds are suppressed by single residue replacements K113E and E195K that interrupt the force-induced K113:E195 interaction; and this suppression is rescued by a K113E/E195K double mutant (E/K) restoring the interaction in the opposite orientation. These results support the biological significance of actin catch bonds, as they corroborate reported observations that RhoA and formin switch force-induced actin cytoskeleton alignment and that either K113E or E195K induces yeast cell growth defects rescued by E/K. Our study demonstrates how the mechano-regulation of actin dynamics is modulated by biochemical signaling molecules, and suggests that actin catch bonds may be important in cell functions.

  18. Characterization of defects and role of molecular flexibility in cyclotrimethyelenetrinitramine (RDX)

    NASA Astrophysics Data System (ADS)

    Pal, Anirban

    Molecular crystals typically comprise irregularly shaped molecules packed in low symmetry crystalline configurations. The anisotropic nature of bonding and low cohesive energies are responsible for their complex and novel mechanical, electronic, vibrational, and optical properties. Owing to their importance in pharmaceutical, electronic, energetic and food materials, this class of crystals has fostered myriad investigations over the last century. This dissertation focuses on a specific molecular crystal, alpha-cyclotrimethylene trinitramine (alpha-RDX), which is a commonly used energetic material. There are three investigation tracks in this work. First, a type of point defect that is specific to molecular crystals, called orientational defect , is presented and studied for alpha-RDX. Secondly, since this molecular crystal comprises flexible molecules, the influence of such flexibility on elastic-plastic properties of alpha-RDX is quantified. Thirdly, the Peierls free energy barriers for the motion of dislocations in alpha-RDX is estimated using a novel umbrella sampling based technique.

  19. Production and characterization of vaccines based on flaviviruses defective in replication

    SciTech Connect

    Mason, Peter W.; Shustov, Alexandr V.; Frolov, Ilya . E-mail: ivfrolov@utmb.edu

    2006-08-01

    To develop new vaccine candidates for flavivirus infections, we have engineered two flaviviruses, yellow fever virus (YFV) and West Nile virus (WNV), that are deficient in replication. These defective pseudoinfectious viruses (PIVs) lack a functional copy of the capsid (C) gene in their genomes and are incapable of causing spreading infection upon infection of cells both in vivo and in vitro. However, they produce extracellular E protein in form of secreted subviral particles (SVPs) that are known to be an effective immunogen. PIVs can be efficiently propagated in trans-complementing cell lines making high levels of C or all three viral structural proteins. PIVs derived from YFV and WNV, demonstrated very high safety and immunization produced high levels of neutralizing antibodies and protective immune response. Such defective flaviviruses can be produced in large scale under low biocontainment conditions and should be useful for diagnostic or vaccine applications.

  20. Analysis of defect structure in silicon. Characterization of samples from UCP ingot 5848-13C

    NASA Technical Reports Server (NTRS)

    Natesh, R.; Guyer, T.; Stringfellow, G. B.

    1982-01-01

    Statistically significant quantitative structural imperfection measurements were made on samples from ubiquitous crystalline process (UCP) Ingot 5848 - 13 C. Important trends were noticed between the measured data, cell efficiency, and diffusion length. Grain boundary substructure appears to have an important effect on the conversion efficiency of solar cells from Semix material. Quantitative microscopy measurements give statistically significant information compared to other microanalytical techniques. A surface preparation technique to obtain proper contrast of structural defects suitable for QTM analysis was perfected.

  1. Mutants of Saccharomyces cerevisiae with defects in acetate metabolism: isolation and characterization of Acn- mutants.

    PubMed

    McCammon, M T

    1996-09-01

    The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn- ("ACetate Nonutilizing") mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism.

  2. Characterization of controlled bone defects using 2D and 3D ultrasound imaging techniques.

    PubMed

    Parmar, Biren J; Longsine, Whitney; Sabonghy, Eric P; Han, Arum; Tasciotti, Ennio; Weiner, Bradley K; Ferrari, Mauro; Righetti, Raffaella

    2010-08-21

    Ultrasound is emerging as an attractive alternative modality to standard x-ray and CT methods for bone assessment applications. As of today, however, there is a lack of systematic studies that investigate the performance of diagnostic ultrasound techniques in bone imaging applications. This study aims at understanding the performance limitations of new ultrasound techniques for imaging bones in controlled experiments in vitro. Experiments are performed on samples of mammalian and non-mammalian bones with controlled defects with size ranging from 400 microm to 5 mm. Ultrasound findings are statistically compared with those obtained from the same samples using standard x-ray imaging modalities and optical microscopy. The results of this study demonstrate that it is feasible to use diagnostic ultrasound imaging techniques to assess sub-millimeter bone defects in real time and with high accuracy and precision. These results also demonstrate that ultrasound imaging techniques perform comparably better than x-ray imaging and optical imaging methods, in the assessment of a wide range of controlled defects both in mammalian and non-mammalian bones. In the future, ultrasound imaging techniques might provide a cost-effective, real-time, safe and portable diagnostic tool for bone imaging applications.

  3. Mutants of Saccharomyces Cerevisiae with Defects in Acetate Metabolism: Isolation and Characterization of Acn(-) Mutants

    PubMed Central

    McCammon, M. T.

    1996-01-01

    The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn(-) (``ACetate Nonutilizing'') mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism. PMID:8878673

  4. Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX.

    PubMed Central

    Thurm, P; Garro, A J

    1975-01-01

    Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since ø105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome. Images PMID:805847

  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. 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

  7. Modeling defect cluster evolution in irradiated structural materials: Focus on comparing to high-resolution experimental characterization studies

    SciTech Connect

    Wirth, Brian D.; Hu, Xunxiang; Kohnert, Aaron; Xu, Donghua

    2015-03-02

    Exposure of metallic structural materials to irradiation environments results in significant microstructural evolution, property changes, and performance degradation, which limits the extended operation of current generation light water reactors and restricts the design of advanced fission and fusion reactors. Further, it is well recognized that these irradiation effects are a classic example of inherently multiscale phenomena and that the mix of radiation-induced features formed and the corresponding property degradation depend on a wide range of material and irradiation variables. This inherently multiscale evolution emphasizes the importance of closely integrating models with high-resolution experimental characterization of the evolving radiation-damaged microstructure. Lastly, this article provides a review of recent models of the defect microstructure evolution in irradiated body-centered cubic materials, which provide good agreement with experimental measurements, and presents some outstanding challenges, which will require coordinated high-resolution characterization and modeling to resolve.

  8. Modeling defect cluster evolution in irradiated structural materials: Focus on comparing to high-resolution experimental characterization studies

    DOE PAGES

    Wirth, Brian D.; Hu, Xunxiang; Kohnert, Aaron; ...

    2015-03-02

    Exposure of metallic structural materials to irradiation environments results in significant microstructural evolution, property changes, and performance degradation, which limits the extended operation of current generation light water reactors and restricts the design of advanced fission and fusion reactors. Further, it is well recognized that these irradiation effects are a classic example of inherently multiscale phenomena and that the mix of radiation-induced features formed and the corresponding property degradation depend on a wide range of material and irradiation variables. This inherently multiscale evolution emphasizes the importance of closely integrating models with high-resolution experimental characterization of the evolving radiation-damaged microstructure. Lastly,more » this article provides a review of recent models of the defect microstructure evolution in irradiated body-centered cubic materials, which provide good agreement with experimental measurements, and presents some outstanding challenges, which will require coordinated high-resolution characterization and modeling to resolve.« less

  9. Nanoscale characterization of local structures and defects in photonic crystals using synchrotron-based transmission soft X-ray microscopy

    PubMed Central

    Nho, Hyun Woo; Kalegowda, Yogesh; Shin, Hyun-Joon; Yoon, Tae Hyun

    2016-01-01

    For the structural characterization of the polystyrene (PS)-based photonic crystals (PCs), fast and direct imaging capabilities of full field transmission X-ray microscopy (TXM) were demonstrated at soft X-ray energy. PS-based PCs were prepared on an O2-plasma treated Si3N4 window and their local structures and defects were investigated using this label-free TXM technique with an image acquisition speed of ~10 sec/frame and marginal radiation damage. Micro-domains of face-centered cubic (FCC (111)) and hexagonal close-packed (HCP (0001)) structures were dominantly found in PS-based PCs, while point and line defects, FCC (100), and 12-fold symmetry structures were also identified as minor components. Additionally, in situ observation capability for hydrated samples and 3D tomographic reconstruction of TXM images were also demonstrated. This soft X-ray full field TXM technique with faster image acquisition speed, in situ observation, and 3D tomography capability can be complementally used with the other X-ray microscopic techniques (i.e., scanning transmission X-ray microscopy, STXM) as well as conventional characterization methods (e.g., electron microscopic and optical/fluorescence microscopic techniques) for clearer structure identification of self-assembled PCs and better understanding of the relationship between their structures and resultant optical properties. PMID:27087141

  10. Ultrasonic Defect Characterization in Heavy Rotor Forgings by Means of the Synthetic Aperture Focusing Technique and Optimization Methods.

    PubMed

    Fendt, Karl T; Mooshofer, Hubert; Rupitsch, Stefan J; Ermert, Helmut

    2016-06-01

    Ultrasonic nondestructive testing of steel forgings aims at the detection and classification of material inhomogeneities to ensure the components fitness for use. Due to the high price and safety critical nature of large forgings for turbomachinery, there is great interest in the application of imaging algorithms to inspection data. However, small flaw indications that cannot be sufficiently resolved have to be characterized using amplitude-based quantification. One such method is the distance gain size method, which converts the maximum echo amplitudes into the diameters of penny-shaped equivalent size reflectors. The approach presented in this contribution combines the synthetic aperture focusing technique (SAFT) with an iterative inversion scheme to locate and quantify small flaws in a more reliable way. Ultrasonic inspection data obtained in a pulse-echo configuration are reconstructed by means of an Synthetic Focusing Technique (SAFT). From the reconstructed data, the amount and approximate location of small flaws are extracted. These predetermined positions, along with the constrained defect model of a penny-shaped crack, provide the initial parametrization for an elastodynamic simulation based on the Kirchhoff approximation. The identification of the optimal parameter set is achieved through an iteratively regularized Gauss-Newton method. By testing the characterization method on a series of flat-bottom holes under laboratory conditions, we demonstrate that the procedure is applicable over a wide range of defect sizes. To show suitability for large forging inspection, we additionally evaluate the inspection data of a large generator shaft forging of 0.6-m diameter.

  11. Nanoscale characterization of local structures and defects in photonic crystals using synchrotron-based transmission soft X-ray microscopy

    NASA Astrophysics Data System (ADS)

    Nho, Hyun Woo; Kalegowda, Yogesh; Shin, Hyun-Joon; Yoon, Tae Hyun

    2016-04-01

    For the structural characterization of the polystyrene (PS)-based photonic crystals (PCs), fast and direct imaging capabilities of full field transmission X-ray microscopy (TXM) were demonstrated at soft X-ray energy. PS-based PCs were prepared on an O2-plasma treated Si3N4 window and their local structures and defects were investigated using this label-free TXM technique with an image acquisition speed of ~10 sec/frame and marginal radiation damage. Micro-domains of face-centered cubic (FCC (111)) and hexagonal close-packed (HCP (0001)) structures were dominantly found in PS-based PCs, while point and line defects, FCC (100), and 12-fold symmetry structures were also identified as minor components. Additionally, in situ observation capability for hydrated samples and 3D tomographic reconstruction of TXM images were also demonstrated. This soft X-ray full field TXM technique with faster image acquisition speed, in situ observation, and 3D tomography capability can be complementally used with the other X-ray microscopic techniques (i.e., scanning transmission X-ray microscopy, STXM) as well as conventional characterization methods (e.g., electron microscopic and optical/fluorescence microscopic techniques) for clearer structure identification of self-assembled PCs and better understanding of the relationship between their structures and resultant optical properties.

  12. Generation of an isogenic collection of yeast actin mutants and identification of three interrelated phenotypes.

    PubMed Central

    Whitacre, J; Davis, D; Toenjes, K; Brower, S; Adams, A

    2001-01-01

    A large collection of yeast actin mutations has been previously isolated and used in numerous studies of actin cytoskeletal function. However, the various mutations have been in congenic, rather than isogenic, backgrounds, making it difficult to compare the subtle phenotypes that are characteristic of these mutants. We have therefore placed 27 mutations in an isogenic background. We used a subset of these mutants to compare the degree to which different actin alleles are defective in sporulation, endocytosis, and growth on NaCl-containing media. We found that the three phenotypes are highly correlated. The correlations are specific and not merely a reflection of general growth defects, because the phenotypes are not correlated with growth rates under normal conditions. Significantly, those actin mutants exhibiting the most severe phenotypes in all three processes have altered residues that cluster to a small region of the actin crystal structure previously defined as the fimbrin (Sac6p)-binding site. We examined the relationship between endocytosis and growth on salt and found that shifting wild-type or actin mutant cells to high salt reduces the rate of alpha-factor internalization. These results suggest that actin mutants may be unable to grow on salt because of additive endocytic defects (due to mutation and salt). PMID:11156976

  13. Characterization of zebrafish mutants with defects in bone calcification during development.

    PubMed

    Xi, Yang; Chen, Dongyan; Sun, Lei; Li, Yuhao; Li, Lei

    2013-10-11

    Using the fluorescent dyes calcein and alcian blue, we stained the F3 generation of chemically (ENU) mutagenized zebrafish embryos and larvae, and screened for mutants with defects in bone development. We identified a mutant line, bone calcification slow (bcs), which showed delayed axial vertebra calcification during development. Before 4-5 days post-fertilization (dpf), the bcs embryos did not display obvious abnormalities in bone development (i.e., normal number, size and shape of cartilage and vertebrae). At 5-6 dpf, when vertebrae calcification starts, bcs embryos began to show defects. At 7 dpf, for example, in most of the bcs embryos examined, calcein staining revealed no signals of vertebrae mineralization, whereas during the same developmental stages, 2-14 mineralized vertebrae were observed in wild-type animals. Decreases in the number of calcified vertebrae were also observed in bcs mutants when examined at 9 and 11 dpf, respectively. Interestingly, by 13 dpf the defects in bcs mutants were no longer evident. There were no significant differences in the number of calcified vertebrae between wild-type and mutant animals. We examined the expression of bone development marker genes (e.g., Sox9b, Bmp2b, and Cyp26b1, which play important roles in bone formation and calcification). In mutant fish, we observed slight increases in Sox9b expression, no alterations in Bmp2b expression, but significant increases in Cyp26b1 expression. Together, the data suggest that bcs delays axial skeletal calcification, but does not affect bone formation and maturation.

  14. 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)

  15. Method for non-referential defect characterization using fractal encoding and active contours

    DOEpatents

    Gleason, Shaun S.; Sari-Sarraf, Hamed

    2007-05-15

    A method for identification of anomalous structures, such as defects, includes the steps of providing a digital image and applying fractal encoding to identify a location of at least one anomalous portion of the image. The method does not require a reference image to identify the location of the anomalous portion. The method can further include the step of initializing an active contour based on the location information obtained from the fractal encoding step and deforming an active contour to enhance the boundary delineation of the anomalous portion.

  16. 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.

  17. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  18. Scanning coherent scattering methods for actinic EUV mask inspection

    NASA Astrophysics Data System (ADS)

    Ekinci, Y.; Helfenstein, P.; Rajeev, R.; Mochi, I.; Mohacsi, I.; Gobrecht, J.; Yoshitake, S.

    2016-10-01

    Actinic mask inspection for EUV lithography with targeted specifications of resolution, sensitivity, and throughput remains a big hurdle for the successful insertion of EUVL into high volume manufacturing and effective solutions are needed to address this. We present a method for actinic mask inspection based on scanning coherent scattering microscopy. In this method, the mask is scanned with an EUV beam of relatively small spot size and the scattered light is recorded with a pixel detector. Customized algorithms reconstruct the aerial image by iteratively solving the phaseproblem using over-determined diffraction data gathered by scanning across the specimen with a finite illumination. This approach provides both phase and amplitude of actinic aerial images of the mask with high resolution without the need to use high NA (numerical aperture) lenses. Futher, we describe a reflective mode EUV mask scanning lensless imaging tool (RESCAN), which was installed at the XIL-II beamline and later at the SIM beamline of the Swiss Light Source and show reconstructed aerial images down to 10 nm (on-wafer) resolution. As a complementary method, the a-priori knowledge of the sample is employed to identify potential defect sites by analyzing the diffraction patterns. In this method, the recorded diffraction patterns are compared with the die or database data (i.e. previously measured or calculated diffraction data from the defect-free mask layout respectively) and their difference is interpreted as the defect signal. Dynamic software filtering helps to suppress the strong diffraction from defect-free structures and allows registration of faint defects with high sensitivity. Here, we discuss the basic principles of these Fourier domain techniques and its potential for actinic mask inspection with high signal-to-noise ratio and high throughput.

  19. Characterization of a Drosophila Alzheimer's Disease Model: Pharmacological Rescue of Cognitive Defects

    PubMed Central

    Mhatre, Siddhita D.; Paddock, Brie E.; Miller, Sean; Michelson, Sarah J.; Delvadia, Radha; Desai, Arkit; Vinokur, Marianna; Melicharek, David J.; Utreja, Suruchi; Khandelwal, Preeti; Ansaloni, Sara; Goldstein, Lee E.; Moir, Robert D.; Lee, Jeremy C.; Tabb, Loni P.; Saunders, Aleister J.; Marenda, Daniel R.

    2011-01-01

    Transgenic models of Alzheimer's disease (AD) have made significant contributions to our understanding of AD pathogenesis, and are useful tools in the development of potential therapeutics. The fruit fly, Drosophila melanogaster, provides a genetically tractable, powerful system to study the biochemical, genetic, environmental, and behavioral aspects of complex human diseases, including AD. In an effort to model AD, we over-expressed human APP and BACE genes in the Drosophila central nervous system. Biochemical, neuroanatomical, and behavioral analyses indicate that these flies exhibit aspects of clinical AD neuropathology and symptomology. These include the generation of Aβ40 and Aβ42, the presence of amyloid aggregates, dramatic neuroanatomical changes, defects in motor reflex behavior, and defects in memory. In addition, these flies exhibit external morphological abnormalities. Treatment with a γ-secretase inhibitor suppressed these phenotypes. Further, all of these phenotypes are present within the first few days of adult fly life. Taken together these data demonstrate that this transgenic AD model can serve as a powerful tool for the identification of AD therapeutic interventions. PMID:21673973

  20. Characterization of fold defects in AZ91D and AE42 magnesium alloy permanent mold castings

    SciTech Connect

    Bichler, L.; Ravindran, C.

    2010-03-15

    Casting premium-quality magnesium alloy components for aerospace and automotive applications poses unique challenges. Magnesium alloys are known to freeze rapidly prior to filling a casting cavity, resulting in misruns and cold shuts. In addition, melt oxidation, solute segregation and turbulent metal flow during casting contribute to the formation of fold defects. In this research, formation of fold defects in AZ91D and AE42 magnesium alloys cast via the permanent mold casting process was investigated. Computer simulations of the casting process predicted the development of a turbulent metal flow in a critical casting region with abrupt geometrical transitions. SEM and light optical microscopy examinations revealed the presence of folds in this region for both alloys. However, each alloy exhibited a unique mechanism responsible for fold formation. In the AZ91D alloy, melt oxidation and velocity gradients in the critical casting region prevented fusion of merging metal front streams. In the AE42 alloy, limited solubility of rare-earth intermetallic compounds in the {alpha}-Mg phase resulted in segregation of Al{sub 2}RE particles at the leading edge of a metal front and created microstructural inhomogeneity across the fold.

  1. Three cotton genes preferentially expressed in flower tissues encode actin-depolymerizing factors which are involved in F-actin dynamics in cells.

    PubMed

    Li, Xue-Bao; Xu, Dan; Wang, Xiu-Lan; Huang, Geng-Qing; Luo, Juan; Li, Deng-Di; Zhang, Ze-Ting; Xu, Wen-Liang

    2010-01-01

    To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination.

  2. 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

  3. 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.

  4. 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.

  5. Human obesity is characterized by defective fat storage and enhanced muscle fatty acid oxidation, and trimetazidine gradually counteracts these abnormalities.

    PubMed

    Bucci, Marco; Borra, Ronald; Någren, Kjell; Maggio, Romina; Tuunanen, Helena; Oikonen, Vesa; Del Ry, Silvia; Viljanen, Tapio; Taittonen, Markku; Rigazio, Sara; Giannessi, Daniela; Parkkola, Riitta; Knuuti, Juhani; Nuutila, Pirjo; Iozzo, Patricia

    2011-07-01

    An impaired ability to store fatty acids (FA) in subcutaneous adipose tissue (SAT) may be implicated in the pathogenesis of obesity-related diseases via overexposure of lean tissues and production of free radicals from FA oxidation (FAO). We studied regional FA metabolism in skeletal muscle and adipose tissue in humans and investigated the long-term effects of the FAO inhibitor trimetazidine on glucose and FA metabolism. Positron emission tomography (PET) and [(11)C]palmitate were used to compare FA metabolism in SAT and skeletal muscle between eight obese and eight nonobese subjects (BMI ≥/< 30 kg/m(2)). A subgroup of nine subjects underwent a 1-mo trimetazidine administration. PET with [(11)C]palmitate and [(18)F]fluorodeoxyglucose, indirect calorimetry, and MRI before and after this period were performed to characterize glucose and FA metabolism, fat masses, skeletal muscle triglyceride, and creatine contents. Obesity was characterized by a 100% elevation in FAO and a defect in the FA esterification rate constant (P < 0.05) in skeletal muscle. FA esterification was reduced by ~70% in SAT (P < 0.001) in obese vs. control subjects. The degrees of obesity and insulin resistance were both negatively associated with esterification-related parameters and positively with FAO (P < 0.05). Trimetazidine increased skeletal muscle FA esterification (P < 0.01) and mildly upregulated glucose phosphorylation (P = 0.066). Our data suggest that human obesity is characterized by a defect in tissue FA storage capability, which is accompanied by a (potentially compensatory) elevation in skeletal muscle FAO; trimetazidine diverted FA from oxidative to nonoxidative pathways and provoked an initial activation of glucose metabolism in skeletal muscle.

  6. Characterization of defects in colloidal CdSe nanocrystals by the modified thermostimulated luminescence technique

    SciTech Connect

    Katsaba, A. V. Fedyanin, V. V.; Ambrozevich, S. A.; Vitukhnovsky, A. G.; Lobanov, A. N.; Selyukov, A. S.; Vasiliev, R. B.; Samatov, I. G.; Brunkov, P. N.

    2013-10-15

    The temperature dependencies of the luminescence spectra of 5-nm-diameter CdSe semiconductor nanocrystals synthesized by colloidal-chemistry methods are investigated. The two bands observed in these spectra around 2.01 and 1.37 eV correspond to band-to-band transitions and luminescence of defect states, respectively. A model explaining the temperature behavior of the luminescence band intensities both upon cooling and heating is put forward. A new modification of spectrally resolved thermostimulated luminescence technique making it possible to determine the activation energies and the character of traps responsible for the temperature dependence of the luminescence intensities is suggested. This technique is used to obtain the activation energies of the emission and capture of electrons at traps (190 and 205 meV, respectively) and to determine the depth of the electron level (57 meV) responsible for luminescence in the 1.37-eV region.

  7. Defect characterization in cadmium telluride and cadmium-zinc-tellurium crystals

    NASA Astrophysics Data System (ADS)

    Awadalla, Salah Abdo

    Intrinsic defects and impurities in undoped CdTe and Cd1-x ZnxTe semiconductor compounds have been investigated using thermally stimulated spectroscopy with the general aim of identifying and then understanding the effects of the defects on the electrical and optical properties of these compounds. In order to identify and understand the effects of these defects (trapping levels), the samples were subjected to room temperature deformation, high temperature annealing, and dopants diffusion. The samples were always analyzed before and after any process. It is found that the trapping levels observed at ˜61 K and ˜114 K with thermal ionization energies of 0.12 +/- 0.01 eV and 0.23 +/- 0.01 eV and trapping cross-sections of 4.7 x 10-16 and 7.8 x 10-17 cm2 are associated with the isolated first and second ionized state of the cadmium vacancy, while trapping levels observed at ˜51 K and ˜94 K with thermal ionization energies of 0.09 +/- 0.01 eV and 0.18 +/- 0.01 eV and trapping cross-sections of 9.3 x 10 -17 and 6.8 x 10-17 cm2 are associated with first and second states of the isoelectronic oxygen-cadmium vacancy complex (VCd-OTe) respectively. In addition, we found that deep level trapping states located near the middle of the band gap (in the region between 230 K and 267 K) in undoped as grown CdTe are related to the tellurium antisite-cadmium vacancy complex (TeCd-V Cd) where the lowest thermal ionization state is 0.78 +/- 0.01 eV. The thermal ionization energies (transition energies) were extracted using variable heating rate and/or initial rise methods. Our results have been reinforced with theoretical calculations using linearized augmented plane wave (LAPW) within the local density approximation (LDA). Also, from our room temperature deformation, we have found evidence of three levels of dislocation defects in CdTe crystals. The first two energy levels, with ionization energies of E1 = 0.06 +/- 0.01 eV and E2 = 0.38 +/- 0.01 eV are due to Cd dislocations. The

  8. Characterization of deep defects responsible for the quenching behavior in undoped GaN layers

    NASA Astrophysics Data System (ADS)

    Witte, H.; Schrenk, E.; Flügge, K.; Krost, A.; Christen, J.; Kuhn, B.; Scholz, F.

    2005-03-01

    In undoped semi-insulating GaN samples grown by metal-organic vapor phase epitaxy on sapphire, the recombination and quenching processes were investigated for the main deep traps responsible for quenching. A comprehensive picture is obtained by using different complementary techniques of thermally stimulated current spectroscopy (TSC). Three traps— Q1 , Q2 , and Q3 —were separated in the temperature region between 200 and 300K . Variations of the excitation and the cleaning temperature suggest a two-step trapping process of the defects Q2 and Q3 . Quenching experiments evidence the involvement of these traps in the quenching process and the existence of two metastable states. These results are summarized in a model involving two metastable states and a complex two-step recharging trapping process.

  9. Defect Characterization for Material Assurance in Metal Additive Manufacturing (FY15-0664)

    SciTech Connect

    Salzbrenner, Bradley; Boyce, Brad; Jared, Bradley Howell; Rodelas, Jeffrey; Laing, John Robert

    2016-02-01

    No industry-wide standards yet exist for minimum properties in additively manufactured (AM) metals. While AM alloys such as 17-4 precipitation hardened stainless steel have been shown to have average properties that can be comparable to wrought or cast product, they suffer from inconsistent performance. Variability in the feedstock powder, feature sizes, thermal history, and laser performance can lead to unpredictable surface finish, chemistry, phase content, and defects. To address this issue, rapid, efficient, high-throughput mechanical testing and data analysis was developed, providing profound statistical insight into the stochastic variability in properties. With this new approach, 1000’s of comprehensive tensile tests can be performed for the cost of 10’s of conventional tests. This new high-throughput approach provides a material qualification pathway that is commensurate with the quick turn-around benefit of AM.

  10. Defect characterization in neodymium doped thallium indium disulfide crystals by thermoluminescence measurements

    NASA Astrophysics Data System (ADS)

    Delice, S.; Gasanly, N. M.

    2016-10-01

    Characteristics of defect centers in neodymium doped TlInS2 single crystals have been investigated in virtue of thermoluminescence measurements carried out at low temperatures (10-300 K) with various heating rates between 0.4 and 1.2 K s-1. One glow peak was detected with peak maximum temperature of 26 K at a rate of 0.4 K s-1. The observed glow peak was analyzed using three points and heating rate methods. The analysis results revealed the presence of one trap level with activation energy of 14 meV. Three points method showed that mixed order of kinetic dominates the trapping level. Shift of peak maximum temperature to higher values and decrease in TL intensity were observed as the heating rate was increased progressively. Distribution of traps was demonstrated using an experimental method based on illumination temperature varying between 10 and 14 K.

  11. Control of nuclear organization by F-actin binding proteins.

    PubMed

    Pfisterer, Karin; Jayo, Asier; Parsons, Maddy

    2017-03-04

    The regulation of nuclear shape and deformability is a key factor in controlling diverse events from embryonic development to cancer cell metastasis, but the mechanisms governing this process are still unclear. Our recent study demonstrated an unexpected role for the F-actin bundling protein fascin in controlling nuclear plasticity through a direct interaction with Nesprin-2. Nesprin-2 is a component of the LINC complex that is known to couple the F-actin cytoskeleton to the nuclear envelope. We demonstrated that fascin, which is predominantly associated with peripheral F-actin rich filopodia, binds directly to Nesprin-2 at the nuclear envelope in a range of cell types. Depleting fascin or specifically blocking the fascin-Nesprin-2 complex leads to defects in nuclear polarization, movement and cell invasion. These studies reveal a novel role for an F-actin bundling protein in control of nuclear plasticity and underline the importance of defining nuclear-associated roles for F-actin binding proteins in future.

  12. Actinic EUV-mask metrology: tools, concepts, components

    NASA Astrophysics Data System (ADS)

    Lebert, Rainer; Farahzadi, Azadeh; Diete, Wolfgang; Schäfer, David; Phiesel, Christoph; Wilhein, Thomas; Herbert, Stefan; Maryasov, Aleksey; Juschkin, Larissa; Esser, Dominik; Hoefer, Marco; Hoffmann, Dieter

    2011-03-01

    There is a strong demand for standalone actinic tools for mask blank and mask metrology. We expect to deliver contributions to key issues for the infrastructure tools such as actinic reflectometer, actinic defect inspection and components like high brightness sources together with our partners. With our EUV-reflectometer EUV-MBR we are ready to fulfill HVM requirements in accurate and sensitive spectral metrology. Migrating from mask blanks to masks is supported with integrated fiducial mark detection and small spot sizes of down to < 0.03 mm2. Hence, the EUV-MBR is able to detect minimal variations on mask blank and can support process monitoring for our partners in European EXEPT project. For actinic blank inspection a proof of concept experiment based on an EUV microscope at BASC's EUV-Lamp allows for comparing actinic signatures with AFM scans. Results allow for extrapolation to sub 30 nm sensitivity and fast full blank scan. For LPP sources we demonstrated a new concept utilizing a laser, with parameters optimized for high brightness EUV generation and a new regenerative target concept for high position stability, gain, repetition rate operation and efficiency in the first proof of concept experiment. Up to 350 W/(mm2 sr) from < 20 μm source size have been demonstrated.

  13. Cortactin promotes exosome secretion by controlling branched actin dynamics

    PubMed Central

    Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Seiki, Motoharu; Tyska, Matthew J.

    2016-01-01

    Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. PMID:27402952

  14. Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins.

    PubMed Central

    Wretlind, B; Pavlovskis, O R

    1984-01-01

    We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase ( Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp -1 near 0', with the gene order cys-59- xcp -1- proB , and loci xcp -2, xcp -3, and xcp -31 at 35', with the gene order trpC , D- xcp -3/ xcp -31- xcp -2- argC . Loci xcp -4 and xcp -41 through xcp -44 were cotransducible with proA at 40'; loci xcp -5, xcp -51, xcp -52, and xcp53 were located at 55', with the gene order leu-10- trpF -met-9010- xcp -53- xcp -5/ xcp -51/ xcp+ ++-52, and xcp -6 was located at 65' to 70', between catA and mtu-9002. Nine mutations ( xcp -2, xcp -3, xcp -31, xcp -4, and xcp -41 through xcp -45) caused decreased production of extracellular enzymes. Six strains with mutations xcp -1, xcp -5, xcp -51, xcp -52, xcp -53, and xcp -6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins , such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme. PMID:6427194

  15. Physicochemical characterization of point defects in fluorine doped tin oxide films

    SciTech Connect

    El Akkad, Fikry; Joseph, Sudeep

    2012-07-15

    The physical and chemical properties of spray deposited FTO films are studied using FESEM, x-ray diffraction (XRD), x-ray photoelectron spectroscopy (XPS), electrical and optical measurements. The results of XRD measurements showed that the films are polycrystalline (grain size 20-50 nm) with Rutile structure and mixed preferred orientation along the (200) and (110) planes. An angular shift of the XRD peaks after F-doping is observed and interpreted as being due to the formation of substitutional fluorine defects (F{sub O}) in presence of high concentration of oxygen vacancies (V{sub O}) that are electrically neutral. The electrical neutrality of oxygen vacancies is supported by the observation that the electron concentration n is two orders of magnitude lower than the V{sub O} concentration calculated from chemical analyses using XPS measurements. It is shown that an agreement between XPS, XRD, and Hall effect results is possible provided that the degree of deviation from stoichiometry is calculated with the assumption that the major part of the bulk carbon content is involved in O-C bonds. High temperature thermal annealing is found to cause an increase in the F{sub O} concentration and a decrease in both n and V{sub O} concentrations with the increase of the annealing temperature. These results could be interpreted in terms of a high temperature chemical exchange reaction between the SnO{sub 2} matrix and a precipitated fluoride phase. In this reaction, fluorine is released to the matrix and Sn is trapped by the fluoride phase, thus creating substitutional fluorine F{sub O} and tin vacancy V{sub Sn} defects. The enthalpy of this reaction is determined to be approximately 2.4 eV while the energy of formation of a V{sub Sn} through the migration of Sn{sub Sn} host atom to the fluoride phase is approximately 0.45 eV.

  16. Isolation and phenotypic characterization of Lotus japonicus mutants specifically defective in arbuscular mycorrhizal formation.

    PubMed

    Kojima, Tomoko; Saito, Katsuharu; Oba, Hirosuke; Yoshida, Yuma; Terasawa, Junya; Umehara, Yosuke; Suganuma, Norio; Kawaguchi, Masayoshi; Ohtomo, Ryo

    2014-05-01

    Several symbiotic mutants of legume plants defective in nodulation have also been shown to be mutants related to arbuscular mycorrhizal (AM) symbiosis. The origin of the AM symbiosis can be traced back to the early land plants. It has therefore been postulated that the older system of AM symbiosis was partially incorporated into the newer system of legume-rhizobium symbiosis. To unravel the genetic basis of the establishment of AM symbiosis, we screened about 34,000 plants derived from ethyl methanesulfonate (EMS)-mutagenized Lotus japonicus seeds by microscopic observation. As a result, three lines (ME778, ME966 and ME2329) were isolated as AM-specific mutants that exhibit clear AM-defective phenotypes but form normal effective root nodules with rhizobial infection. In the ME2329 mutant, AM fungi spread their hyphae into the intercellular space of the cortex and formed trunk hyphae in the cortical cells, but the development of fine branches in the arbuscules was arrested. The ME2329 mutant carried a nonsense mutation in the STR-homolog gene, implying that the line may be an str mutant in L. japonicus. On the ME778 and ME966 mutant roots, the entry of AM fungal hyphae was blocked between two adjacent epidermal cells. Occasionally, hyphal colonization accompanied by arbuscules was observed in the two mutants. The genes responsible for the ME778 and ME966 mutants were independently located on chromosome 2. These results suggest that the ME778 and ME966 lines are symbiotic mutants involved in the early stage of AM formation in L. japonicus.

  17. 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.

  18. 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.

  19. Advanced defect characterization via electron microscopy and its application to cyclically deformed nickel-based superalloy R104

    NASA Astrophysics Data System (ADS)

    Phillips, Patrick J.

    Ni-based superalloys continue to be used in the hot sections of turbine engines due to their superior high temperature properties and retained strength. The present document will focus specifically on the polycrystalline alloy R104, and the deformation substructure observed during and following cyclic mechanical testing. Both low cycle fatigue (LCF) and sustained peak low cycle fatigue (SPLCF) tests are considered. Two chapters on electron microscopy technique development follow a brief introduction on general properties of Nickel superalloys. Almost exclusively, scanning transmission electron microscopy (STEM) was performed for defect characterization. Furthermore, through a systematic study of STEM-based diffraction contrast methods, including experimental and computational results, STEM is presented as a valid means of defect analysis. The second chapter in this set also uses STEM, but in a non-traditional setting: the microscope is configured for high resolution imaging, i.e., the sample is aligned along a low index zone axis and a large convergence angle is used. In this low angle annular dark field (LAADF) mode, an annular detector accepts low-angle scattering, which allows one to obtain atomic resolution images while retaining defect contrast. Both techniques described in these two chapters were used extensively throughout this research. The remaining chapters discuss the application of the microscopy techniques developed in the proceeding chapters to cyclically deformed specimens of R104. Both interrupted and failed samples were deformed in LCF at 427°C and 704°C, and interrupted SPLCF samples were tested at 704 and 760°C. The deformation mechanisms observed will be discussed at length in this document. In general, dislocation activity dominates under LCF conditions while stacking faults and stacking fault ribbons are most prominent under SPLCF conditions. Time and temperature components will be discussed in regards to the operative mechanisms. A point

  20. 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

  1. 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.

  2. Nuclear envelope rupture is induced by actin-based nucleus confinement.

    PubMed

    Hatch, Emily M; Hetzer, Martin W

    2016-10-10

    Repeated rounds of nuclear envelope (NE) rupture and repair have been observed in laminopathy and cancer cells and result in intermittent loss of nucleus compartmentalization. Currently, the causes of NE rupture are unclear. Here, we show that NE rupture in cancer cells relies on the assembly of contractile actin bundles that interact with the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex. We found that the loss of actin bundles or the LINC complex did not rescue nuclear lamina defects, a previously identified determinant of nuclear membrane stability, but did decrease the number and size of chromatin hernias. Finally, NE rupture inhibition could be rescued in cells treated with actin-depolymerizing drugs by mechanically constraining nucleus height. These data suggest a model of NE rupture where weak membrane areas, caused by defects in lamina organization, rupture because of an increase in intranuclear pressure from actin-based nucleus confinement.

  3. NudC regulates actin dynamics and ciliogenesis by stabilizing cofilin 1

    PubMed Central

    Zhang, Cheng; Zhang, Wen; Lu, Yi; Yan, Xiaoyi; Yan, Xiumin; Zhu, Xueliang; Liu, Wei; Yang, Yuehong; Zhou, Tianhua

    2016-01-01

    Emerging data indicate that actin dynamics is associated with ciliogenesis. However, the underlying mechanism remains unclear. Here we find that nuclear distribution gene C (NudC), an Hsp90 co-chaperone, is required for actin organization and dynamics. Depletion of NudC promotes cilia elongation and increases the percentage of ciliated cells. Further results show that NudC binds to and stabilizes cofilin 1, a key regulator of actin dynamics. Knockdown of cofilin 1 also facilitates ciliogenesis. Moreover, depletion of either NudC or cofilin 1 causes similar ciliary defects in zebrafish, including curved body, pericardial edema and defective left-right asymmetry. Ectopic expression of cofilin 1 significantly reverses the phenotypes induced by NudC depletion in both cultured cells and zebrafish. Thus, our data suggest that NudC regulates actin cytoskeleton and ciliogenesis by stabilizing cofilin 1. PMID:26704451

  4. Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

    PubMed Central

    Yehl, Jenna; Kudryashova, Elena; Reisler, Emil; Kudryashov, Dmitri; Polenova, Tatyana

    2017-01-01

    Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin’s involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin. PMID:28303963

  5. 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.

  6. Super-Resolution Defect Characterization Using Microwave Near-Field Resonance Reflectometry and Cross-correlation Image Processing

    NASA Astrophysics Data System (ADS)

    Malyuskin, Oleksandr; Fusco, Vincent

    2017-12-01

    A super-resolution defect characterization technique based on near-field resonance reflectometry and cross-correlation image processing is proposed in this paper. The hardware part of the microwave imaging system employs a novel loaded aperture (LA) probe which allows collimation of the electromagnetic field to approximately λ/10 focal spot(s) at λ/100 to λ/10 stand-off distances, λ being the wavelength of radiation in free space. The characteristic raw image spatial resolution of the LA probe is around λ/10 in one dimension with amplitude contrast/sensitivity exceeding 10-20 dB. It is demonstrated that the LA spatial resolution can be at least two times enhanced in two dimensions in the image plane using basic cross-correlation image processing while retaining a very high level of amplitude contrast of at least 10 dB.

  7. Neuromuscular disease. DOK7 gene therapy benefits mouse models of diseases characterized by defects in the neuromuscular junction.

    PubMed

    Arimura, Sumimasa; Okada, Takashi; Tezuka, Tohru; Chiyo, Tomoko; Kasahara, Yuko; Yoshimura, Toshiro; Motomura, Masakatsu; Yoshida, Nobuaki; Beeson, David; Takeda, Shin'ichi; Yamanashi, Yuji

    2014-09-19

    The neuromuscular junction (NMJ) is the synapse between a motor neuron and skeletal muscle. Defects in NMJ transmission cause muscle weakness, termed myasthenia. The muscle protein Dok-7 is essential for activation of the receptor kinase MuSK, which governs NMJ formation, and DOK7 mutations underlie familial limb-girdle myasthenia (DOK7 myasthenia), a neuromuscular disease characterized by small NMJs. Here, we show in a mouse model of DOK7 myasthenia that therapeutic administration of an adeno-associated virus (AAV) vector encoding the human DOK7 gene resulted in an enlargement of NMJs and substantial increases in muscle strength and life span. When applied to model mice of another neuromuscular disorder, autosomal dominant Emery-Dreifuss muscular dystrophy, DOK7 gene therapy likewise resulted in enlargement of NMJs as well as positive effects on motor activity and life span. These results suggest that therapies aimed at enlarging the NMJ may be useful for a range of neuromuscular disorders.

  8. Integral Characterization of Defective BDNF/TrkB Signalling in Neurological and Psychiatric Disorders Leads the Way to New Therapies

    PubMed Central

    Tejeda, Gonzalo S.; Díaz-Guerra, Margarita

    2017-01-01

    Enhancement of brain-derived neurotrophic factor (BDNF) signalling has great potential in therapy for neurological and psychiatric disorders. This neurotrophin not only attenuates cell death but also promotes neuronal plasticity and function. However, an important challenge to this approach is the persistence of aberrant neurotrophic signalling due to a defective function of the BDNF high-affinity receptor, tropomyosin-related kinase B (TrkB), or downstream effectors. Such changes have been already described in several disorders, but their importance as pathological mechanisms has been frequently underestimated. This review highlights the relevance of an integrative characterization of aberrant BDNF/TrkB pathways for the rational design of therapies that by combining BDNF and TrkB targets could efficiently promote neurotrophic signalling. PMID:28134845

  9. [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.

  10. Structural characterization of polymers by MALDI spiral-TOF mass spectrometry combined with Kendrick mass defect analysis.

    PubMed

    Sato, Hiroaki; Nakamura, Sayaka; Teramoto, Kanae; Sato, Takafumi

    2014-08-01

    High-resolution mass spectrometry (HRMS) continues to play an important role in the compositional characterization of larger organic molecules. In the field of polymer characterization, however, the application of HRMS has made only slow progress because of lower compatibility between matrix-assisted laser desorption/ionization (MALDI) and ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS). In this study, a newly developed type of MALDI high-resolution time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS) was applied to the structural and compositional characterization of polymers. To create a graphical distribution of polymer components on a two-dimensional plot converted from complex mass spectra, we adopted a slightly modified Kendrick mass defect (KMD) analysis based on accurate masses determined using spiral-TOFMS. By setting the Kendrick mass scale based on the mass of the repeating units of a given polymer, components with common repeat units lined up in the horizontal direction on the KMD plot, whereas those components with different structures were shifted vertically. This combination of MALDI spiral-TOFMS measurement and KMD analysis enabled the successful discrimination of the polymer components in a blend of poly(alkylene oxide)s, the compositional analysis of poly(ethylene oxide)/poly(propylene oxide) block copolymers, and profiling of the end-group distribution of poly(ε-caprolactone)s synthesized under different conditions.

  11. New optical approaches to the quantitative characterization of crystal growth, segregation and defect formation

    NASA Technical Reports Server (NTRS)

    Carlson, D. J.; Wargo, M. J.; Cao, X. Z.; Witt, A. F.

    1991-01-01

    Elemental and compound semiconductors were characterized using new optical approach based on NIR microscopy in conjunction with computational image analysis and contrast enhancement. The approach made it possible to perform a quantitative microsegregation analysis of GaAs and InP. NIR dark file illumination in transmission mode makes it possible to detect submicron precipitates in semiinsulating GaAs.

  12. A Laboratory Exercise for Isolation and Characterizing Microbial Mutants with Metabolic Defects.

    ERIC Educational Resources Information Center

    Doe, Frank J.; Leslie, John F.

    1993-01-01

    Describes science experiments for undergraduate biology instruction on the concepts of mutation and characterization of the resulting mutant strains. The filamentous fungi "Fusarium moniliforme" is used to illustrate the induction of mutants (mutagenesis), identification of the mutated gene, construction of a biochemical pathway, and…

  13. 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.

  14. Defect chemistry and characterization Hg(1-x)Cd(x)Te

    NASA Technical Reports Server (NTRS)

    Vydyanath, H. R.; Donovan, J. C.

    1981-01-01

    Iodine doped single crystal samples of mercury cadmium telluride were annealed at temperatures varying from 450 C to 600 C in Hg vapor and quenched to room temperature. Hall effect measurements at 77 K on the crystals cooled to room temperature indicate the samples to be n-type after anneals at high Hg pressures whereas they turn p-type after anneals at low Hg pressures; the electron concentration increases with increase in Hg pressure. The results are explained on the basis that the crystals are saturated with (Hg,Cd)I2, with a fraction of the iodine being present as donor occupying tellurium lattice sites and a fraction being present as acceptors resulting from the iodine on tellurium lattice sites pairing with the doubly ionized native acceptor defects. The solubility of the donor species increases with increase in Hg pressure, whereas that of the acceptor species increases with decrease in Hg pressure. Equilibrium constants for the incorporation of the iodine species as well as the pairing reaction were established.

  15. Defect characterization in ZnGeP2 by time-resolved photoluminescence

    NASA Technical Reports Server (NTRS)

    Dietz, N.; Wood, G.; Bachmann, K. J.; Busse, W.; Gumlich, H. E.; Ruderman, W.; Tsveybak, I.

    1995-01-01

    The native defect-related optical properties of ZnGeP2 are studied by steady-state and time-resolved photoluminescence. ZnGeP2 crystals grown from the vapor phase by high pressure physical vapor transport (HPVT) and from the melt by gradient freezing (GF) show a broad infrared emission with peak position at 1.2 eV with a decay time constant of a few micro-s and a faster recombination center peaked at 1.68 eV. The decay transient for the emission at 1.2 eV exhibits features of classical donor-acceptor recombination and is linked to close-pairs that are tentatively associated with germanium-on-zinc antisite donors and zinc vacancy acceptors. Higher energetic luminescence structures at 1.6eV and 1.7eV are revealed after annealing of the ZnGeP2 crystals and are associated with the formation zinc and phosphorous vacancies in the near surface region. ZnGeP2 crystals grown by HPVT from zinc and phosphorus supersaturated vapor compositions show additional emission structure at 1.8 eV and a sharp donor-acceptor emission at 1.778 eV that is related to the presence of additional donor states.

  16. Characterization of structural defects in GST based nano-PCM devices through resistance drift measurements

    NASA Astrophysics Data System (ADS)

    Cinar, Ibrahim; Cogulu, Egecan; Gokce, Aisha; Stipe, Barry; Katine, Jordan; Aktas, Gulen; Ozatay, Ozhan

    2015-03-01

    Phase change memory (PCM) is a promising nonvolatile data storage technology with its high signal to noise ratio and superior scalability. Resistance drift in amorphous phase of the phase change material poses a crucial reliability problem, especially in multiple-bit-per cell PCM devices. The resistance of the amorphous phase uncontrollably increases with time after a reset operation which alters the read/write conditions of the device. Structural relaxation (SR) through a defect annihilation process is considered to be the underlying physical mechanism for resistance drift. Here, we report on our measurements of the resistance drift in a phase change memory device with a single layer Ge2Sb2Te5 (GST) material not only in the amorphous state but also in the intermediate resistance state in devices with square top contact geometry which enables us to assess the reliability of multiple-bit per cell PCM memory devices. Through an analysis of electrical measurements as a function of time and temperature for increasing annealing times, we estimate a rate of change in trap density for both amorphous and mixed phases of the GST material after a switching operation. Our study allows engineering the phase change materials and optimizing programing conditions for future PCM applications. TUBITAK under Contract Number 113F385, Bogazici University Research Fund, 12B03M1, and European Union FP7 Marie Curie International Re-integration Grant PCM-256281.

  17. Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome.

    PubMed Central

    Shelley, C S; Remold-O'Donnell, E; Davis, A E; Bruns, G A; Rosen, F S; Carroll, M C; Whitehead, A S

    1989-01-01

    Sialophorin (CD43) of leukocytes and platelets is a surface sialoglycoprotein that is phenotypically defective on lymphocytes of patients with the X chromosome-linked immunodeficiency Wiskott-Aldrich syndrome. Previous studies with monoclonal antibodies indicate that sialophorin is a component of a T-lymphocyte activation pathway. Here we describe the cDNA cloning and derived amino acid sequence of human sialophorin. The sequence predicts an integral membrane polypeptide with an N-terminal hydrophobic signal region followed by a mucin-like 235-residue extracellular region with a uniform distribution of 46 serine, 47 threonine, and 24 proline residues. This is followed by a 23-residue transmembrane region and a 123-residue C-terminal intracellular region. These latter regions have been highly conserved during evolution; the intracellular region contains a number of potential phosphorylation sites that might mediate transduction of activation signals. The chromosomal location of the sialophorin gene was determined and the implications of this assignment for the pathogenesis of the Wiskott-Aldrich syndrome are discussed. Images PMID:2784859

  18. Defect chemistry and characterization Hg(1-x)Cd(x)Te

    NASA Astrophysics Data System (ADS)

    Vydyanath, H. R.; Donovan, J. C.

    Iodine doped single crystal samples of mercury cadmium telluride were annealed at temperatures varying from 450 C to 600 C in Hg vapor and quenched to room temperature. Hall effect measurements at 77 K on the crystals cooled to room temperature indicate the samples to be n-type after anneals at high Hg pressures whereas they turn p-type after anneals at low Hg pressures; the electron concentration increases with increase in Hg pressure. The results are explained on the basis that the crystals are saturated with (Hg,Cd)I2, with a fraction of the iodine being present as donor occupying tellurium lattice sites and a fraction being present as acceptors resulting from the iodine on tellurium lattice sites pairing with the doubly ionized native acceptor defects. The solubility of the donor species increases with increase in Hg pressure, whereas that of the acceptor species increases with decrease in Hg pressure. Equilibrium constants for the incorporation of the iodine species as well as the pairing reaction were established.

  19. Optimization of an Optical Test Bench for Tire Properties Measurement and Tread Defects Characterization.

    PubMed

    Castillo Aguilar, Juan Jesús; Cabrera Carrillo, Juan Antonio; Guerra Fernández, Antonio Jesús; Postigo Pozo, Sergio

    2017-03-29

    Tire characteristics and behavior are of great importance in vehicle dynamics since the forces transmitted in the tire-road contact are the main contributors to global vehicle performance. Several research groups have focused on the study and modeling of tires. Some of the most important factors that need to be known are tread characteristics and pressure distribution in the tire-ground contact patch. In this work, a test bench has been used to adequately determine the aforementioned factors. The measurement principle of the test bench is the frustration of total internal reflection (FTIR) of light. It makes use of a laterally illuminated glass on which the tire leans. An interposed plastic interface between them causes the reflection of light. Finally, a video camera captures the bright image formed through the glass. The brightness level in each pixel of the image is related to existing normal pressure. A study of the parameters that affect the test bench calibration such as type of interface material used, diffuse light, hysteresis, creep and transverse light absorption is performed. Experimental tests are conducted to relate tire inflation pressure and camber angle to the pressure distribution. Furthermore, the test bench is used to detect and evaluate the influence of defects in the tire on the contact pressures.

  20. Initial Characterization of Integrase-Defective Lentiviral Vectors for Pancreatic Cancer Gene Therapy.

    PubMed

    Hanoun, Naima; Gayral, Marion; Pointreau, Adeline; Buscail, Louis; Cordelier, Pierre

    2016-02-01

    The vast majority (85%) of pancreatic ductal adenocarcinomas (PDACs) are discovered at too of a late stage to allow curative surgery. In addition, PDAC is highly resistant to conventional methods of chemotherapy and radiotherapy, which only offer a marginal clinical benefit. Consequently, the prognosis of this cancer is devastating, with a 5-year survival rate of less than 5%. In this dismal context, we recently demonstrated that PDAC gene therapy using nonviral vectors is safe and feasible, with early signs of efficacy in selected patients. Our next step is to transfer to the clinic HIV-1-based lentiviral vectors (LVs) that outshine other therapeutic vectors to treat experimental models of PDAC. However, a primary safety issue presented by LVs that may delay their use in patients is the risk of oncogenesis after vector integration in the host's cell DNA. Thus, we developed a novel anticancerous approach based on integrase-defective lentiviral vectors (IDLVs) and demonstrated that IDLVs can be successfully engineered to transiently deliver therapeutic genes to inhibit pancreatic cancer cells proliferation. This work stems for the use of therapeutic IDLVs for the management of PDAC, in forthcoming early phase gene therapy clinical trial for this disease with no cure.

  1. NMR solution structures of actin depolymerizing factor homology domains

    PubMed Central

    Goroncy, Alexander K; Koshiba, Seizo; Tochio, Naoya; Tomizawa, Tadashi; Sato, Manami; Inoue, Makato; Watanabe, Satoru; Hayashizaki, Yoshihide; Tanaka, Akiko; Kigawa, Takanori; Yokoyama, Shigeyuki

    2009-01-01

    Actin is one of the most conserved proteins in nature. Its assembly and disassembly are regulated by many proteins, including the family of actin-depolymerizing factor homology (ADF-H) domains. ADF-H domains can be divided into five classes: ADF/cofilin, glia maturation factor (GMF), coactosin, twinfilin, and Abp1/drebrin. The best-characterized class is ADF/cofilin. The other four classes have drawn much less attention and very few structures have been reported. This study presents the solution NMR structure of the ADF-H domain of human HIP-55-drebrin-like protein, the first published structure of a drebrin-like domain (mammalian), and the first published structure of GMF β (mouse). We also determined the structures of mouse GMF γ, the mouse coactosin-like domain and the C-terminal ADF-H domain of mouse twinfilin 1. Although the overall fold of the five domains is similar, some significant differences provide valuable insights into filamentous actin (F-actin) and globular actin (G-actin) binding, including the identification of binding residues on the long central helix. This long helix is stabilized by three or four residues. Notably, the F-actin binding sites of mouse GMF β and GMF γ contain two additional β-strands not seen in other ADF-H structures. The G-actin binding site of the ADF-H domain of human HIP-55-drebrin-like protein is absent and distorted in mouse GMF β and GMF γ. PMID:19768801

  2. 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

  3. Coupling of the hydration water dynamics and the internal dynamics of actin detected by quasielastic neutron scattering

    SciTech Connect

    Fujiwara, Satoru; Plazanet, Marie; Oda, Toshiro

    2013-02-15

    Highlights: ► Quasielastic neutron scattering spectra of F-actin and G-actin were measured. ► Analysis of the samples in D{sub 2}O and H{sub 2}O provided the spectra of hydration water. ► The first layer hydration water around F-actin is less mobile than around G-actin. ► This difference in hydration water is in concert with the internal dynamics of actin. ► Water outside the first layer behaves bulk-like but influenced by the first layer. -- Abstract: In order to characterize dynamics of water molecules around F-actin and G-actin, quasielastic neutron scattering experiments were performed on powder samples of F-actin and G-actin, hydrated either with D{sub 2}O or H{sub 2}O, at hydration ratios of 0.4 and 1.0. By combined analysis of the quasielastic neutron scattering spectra, the parameter values characterizing the dynamics of the water molecules in the first hydration layer and those of the water molecules outside of the first layer were obtained. The translational diffusion coefficients (D{sub T}) of the hydration water in the first layer were found to be 1.2 × 10{sup −5} cm{sup 2}/s and 1.7 × 10{sup −5} cm{sup 2}/s for F-actin and G-actin, respectively, while that for bulk water was 2.8 × 10{sup −5} cm{sup 2}/s. The residence times were 6.6 ps and 5.0 ps for F-actin and G-actin, respectively, while that for bulk water was 0.62 ps. These differences between F-actin and G-actin, indicating that the hydration water around G-actin is more mobile than that around F-actin, are in concert with the results of the internal dynamics of F-actin and G-actin, showing that G-actin fluctuates more rapidly than F-actin. This implies that the dynamics of the hydration water is coupled to the internal dynamics of the actin molecules. The D{sub T} values of the water molecules outside of the first hydration layer were found to be similar to that of bulk water though the residence times are strongly affected by the first hydration layer. This supports the

  4. The Effect of Crosslinking on the Microscale Stress Response and Molecular Deformations in Actin Networks

    NASA Astrophysics Data System (ADS)

    Gurmessa, Bekele; Fitzpatrick, Robert; Valdivia, Jonathon; Anderson, Rae M. R.

    Actin, the most abundant protein in eukaryotic cells, is a semi-flexible biopolymer in the cytoskeleton that plays a crucial structural and mechanical role in cell stability, motion and replication, as well as muscle contraction. Most of these mechanically driven structural changes in cells stem from the complex viscoelastic nature of entangled actin networks and the presence of a myriad of proteins that cross-link actin filaments. Despite their importance, the mechanical response of actin networks is not yet well understood, particularly at the molecular level. Here, we use optical trapping - coupled with fluorescence microscopy - to characterize the microscale stress response and induced filament deformations in entangled and cross-linked actin networks subject to localized mechanical perturbations. In particular, we actively drive a microsphere 10 microns through an entangled or cross- linked actin network at a constant speed and measure the resistive force that the deformed actin filaments exert on the bead during and following strain. We simultaneously visualize and track individual sparsely-labeled actin filaments to directly link force response to molecular deformations, and map the propagation of the initially localized perturbation field throughout the rest of the network (~100 um). By varying the concentration of actin and cross-linkers we directly determine the role of crosslinking and entanglements on the length and time scales of stress propagation, molecular deformation and relaxation mechanisms in actin networks.

  5. Mechanical detection of a long-range actin network emanating from a biomimetic cortex.

    PubMed

    Bussonnier, Matthias; Carvalho, Kevin; Lemière, Joël; Joanny, Jean-François; Sykes, Cécile; Betz, Timo

    2014-08-19

    Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the "actin cloud" and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.

  6. Ultrasonic Guided-Wave Scan System Used to Characterize Microstructure and Defects in Ceramic Composites

    NASA Technical Reports Server (NTRS)

    Roth, Don J.; Cosgriff, Laura M.; Martin, Richard E.; Verrilli, Michael J.; Bhatt, Ramakrishna T.

    2004-01-01

    Ceramic matrix composites (CMCs) are being developed for advanced aerospace propulsion applications to save weight, improve reuse capability, and increase performance. However, mechanical and environmental loads applied to CMCs can cause discrete flaws and distributed microdamage, significantly reducing desirable physical properties. Such microdamage includes fiber/matrix debonding (interface failure), matrix microcracking, fiber fracture and buckling, oxidation, and second phase formation. A recent study (ref. 1) of the durability of a C/SiC CMC discussed the requirement for improved nondestructive evaluation (NDE) methods for monitoring degradation in these materials. Distributed microdamage in CMCs has proven difficult to characterize nondestructively because of the complex microstructure and macrostructure of these materials. This year, an ultrasonic guided-wave scan system developed at the NASA Glenn Research Center was used to characterize various microstructural and flaw conditions in SiC/SiC (silicon carbide fiber in silicon carbide matrix) and C/SiC (carbon fiber in silicon carbide matrix) CMC samples.

  7. Characterization of diamond thin films: Diamond phase identification, surface morphology, and defect structures

    SciTech Connect

    Williams, B.E.; Glass, J.T.

    1989-03-01

    Thin carbon films grown from a low pressure methane-hydrogen gas mixture by microwave plasma enhanced CVD have been examined by Auger electron spectroscopy, secondary ion mass spectrometry, electron and x-ray diffraction, electron energy loss spectroscopy, and electron microscopy. They were determined to be similar to natural diamond in terms of composition, structure, and bonding. The surface morphology of the diamond films was a function of position on the sample surface and the methane concentration in the feedgas. Well-faceted diamond crystals were observed near the center of the sample whereas a less faceted, cauliflower texture was observed near the edge of the sample, presumably due to variations in temperature across the surface of the sample. Regarding methane concentration effects, threefold /111/ faceted diamond crystals were predominant on a film grown at 0.3% CH/sub 4/ in H/sub 2/ while fourfold /100/ facets were observed on films grown in 1.0% and 2.0% CH/sub 4/ in H/sub 2/. Transmission electron microscopy of the diamond films has shown that the majority of diamond crystals have a very high defect density comprised of /111/ twins, /111/ stacking faults, and dislocations. In addition, cross-sectional TEM has revealed a 50 A epitaxial layer of ..beta..--SiC at the diamond-silicon interface of a film grown with 0.3% CH/sub 4/ in H/sub 2/ while no such layer was observed on a diamond film grown in 2.0% CH/sub 4/ in H/sub 2/.

  8. Defect characterization and stress analysis by white beam synchrotron X-ray topography in single crystal semiconducting materials

    NASA Astrophysics Data System (ADS)

    Sarkar, Vishwanath

    Semiconductor devices are becoming increasingly more complex as the number of transistors increases in the same Integrated Circuit (IC) area. Due to the complexity in design; processing and packaging of the device plays a crucial role in the IC fabrication. Package induced residual stress are not only detrimental to device performance but can also lead to device failure. We propose a non-destructive method to determine the complete stress state at each point on a packaged Silicon device. Surface and edge defect created as a result of various manufacturing steps were characterized using different techniques, primarily X-ray diffraction topography, optical microscopy, SEM and TEM. Residual stress plays an important role in the performance and lifetime of single crystal device material. Here we present a novel technique using white beam synchrotron X-ray diffraction reticulography, Stress Mapping and Analysis via Ray Tracing (SMART) in order to determine residual stress level at an array of points over the entire crystal area. This method has a unique advantage compared with other stress measurement technique in that it can evaluate all six components of the stress tensor. The underlying experimental technique is based on white beam synchrotron X-ray diffraction topography and ray tracing. An array of X-ray micro-beam is illuminated on the single crystal sample and multiple reflections (reticulographs) are recorded simultaneously on a photographic film. Crystallographic plane normal vector at the location of each micro-beam in the crystal is calculated. The variation of the plane normal vector direction is due to residual strain (both sheer and dilatational) present in the crystal. By considering three different diffracting planes and corresponding reticulograph a complete state of stress is calculated. Principle, applications and limitations are discussed. White beam synchrotron reticulography is used in reflection geometry to evaluate complete residual stress tensor

  9. Defect Clustering and Nano-Phase Structure Characterization of Multi-Component Rare Earth Oxide Doped Zirconia-Yttria Thermal Barrier Coatings

    NASA Technical Reports Server (NTRS)

    Zhu, Dongming; Chen, Yuan L.; Miller, Robert A.

    2003-01-01

    Advanced oxide thermal barrier coatings have been developed by incorporating multi-component rare earth oxide dopants into zirconia-yttria to effectively promote the creation of the thermodynamically stable, immobile oxide defect clusters and/or nano-scale phases within the coating systems. The presence of these nano-sized defect clusters has found to significantly reduce the coating intrinsic thermal conductivity, improve sintering resistance, and maintain long-term high temperature stability. In this paper, the defect clusters and nano-structured phases, which were created by the addition of multi-component rare earth dopants to the plasma-sprayed and electron-beam physical vapor deposited thermal barrier coatings, were characterized by high-resolution transmission electron microscopy (TEM). The defect cluster size, distribution, crystallographic and compositional information were investigated using high-resolution TEM lattice imaging, selected area diffraction (SAD), electron energy-loss spectroscopy (EELS) and energy dispersive spectroscopy (EDS) analysis techniques. The results showed that substantial defect clusters were formed in the advanced multi-component rare earth oxide doped zirconia- yttria systems. The size of the oxide defect clusters and the cluster dopant segregation was typically ranging from 5 to 50 nm. These multi-component dopant induced defect clusters are an important factor for the coating long-term high temperature stability and excellent performance.

  10. Defect Clustering and Nano-Phase Structure Characterization of Multi-Component Rare Earth Oxide Doped Zirconia-Yttria Thermal Barrier Coatings

    NASA Technical Reports Server (NTRS)

    Zhu, Dongming; Chen, Yuan L.; Miller, Robert A.

    1990-01-01

    Advanced oxide thermal barrier coatings have been developed by incorporating multi- component rare earth oxide dopants into zirconia-yttria to effectively promote the creation of the thermodynamically stable, immobile oxide defect clusters and/or nano-scale phases within the coating systems. The presence of these nano-sized defect clusters has found to significantly reduce the coating intrinsic thermal conductivity, improve sintering resistance, and maintain long-term high temperature stability. In this paper, the defect clusters and nano-structured phases, which were created by the addition of multi-component rare earth dopants to the plasma- sprayed and electron-beam physical vapor deposited thermal barrier coatings, were characterized by high-resolution transmission electron microscopy (TEM). The defect cluster size, distribution, crystallographic and compositional information were investigated using high-resolution TEM lattice imaging, selected area diffraction (SAD), and energy dispersive spectroscopy (EDS) analysis techniques. The results showed that substantial defect clusters were formed in the advanced multi-component rare earth oxide doped zirconia-yttria systems. The size of the oxide defect clusters and the cluster dopant segregation was typically ranging fiom 5 to 50 nm. These multi-component dopant induced defect clusters are an important factor for the coating long-term high temperature stability and excellent performance.

  11. Characterization of Structural Defects in Wide Band-Gap Compound Materials for Semiconductor and Opto-Electronic Applications

    NASA Astrophysics Data System (ADS)

    Goue, Ouloide Yannick

    Single crystals of binary and ternary compounds are touted to replace silicon for specialized applications in the semiconductor industry. However, the relative high density of structural defects in those crystals has hampered the performance of devices built on them. In order to enhance the performance of those devices, structurally perfect single crystals must be grown. The aim of this thesis is to investigate the interplay between crystal growth process and crystal quality as well as structural defect types and transport property. To this end, the thesis is divided into two parts. The first part provides a general review of the theory of crystal growth (chapter I), an introduction to the materials being investigated (chapter II and III) and the characterization techniques being used (chapter IV). • In chapter I, a brief description of the theory of crystal growth is provided with an eye towards the driving force behind crystal nucleation and growth along with the kinetic factors affecting crystal growth. The case of crystal growth of silicon carbide (SiC) by physical vapor transport (PVT) and chemical vapor deposition (CVD) is discussed. The Bridgman, travelling heater method (THM) and physical transport growth of cadmium zinc telluride (CZT) is also treated. In chapters II and III, we introduce the compound materials being investigated in this study. While a description of their crystal structure and properties is provided, the issues associated with their growth are discussed. In chapter IV, a description of the characterization techniques used in these studies is presented. These techniques are synchrotron X-ray topography (SXRT), transmission electron microscopy, transmission infrared microscopy (TIM), micro-Raman spectroscopy (muRS) and light microscopy. Extensive treatment of SXRT technique is also provided. In the second part, the experimental results obtained in the course of these studies are presented and discussed. These results are divided into

  12. A role for actin polymerization in persistent pulmonary hypertension of the newborn.

    PubMed

    Fediuk, Jena; Dakshinamurti, Shyamala

    2015-03-01

    Persistent pulmonary hypertension of the newborn (PPHN) is defined as the failure of normal pulmonary vascular relaxation at birth. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial myocytes, resulting in elevation of smooth muscle α-actin and γ-actin content in elastic and resistance pulmonary arteries in PPHN compared with age-matched controls. This review examines the original histological characterization of PPHN with attention to cytoskeletal structural remodeling and actin isoform abundance, reviews the existing evidence for understanding the biophysical and biochemical forces at play during neonatal circulatory transition, and specifically addresses the role of the cortical actin architecture, primarily identified as γ-actin, in the transduction of mechanical force in the hypoxic PPHN pulmonary circuit.

  13. Characterization of the Intrinsic Water Wettability of Graphite Using Contact Angle Measurements: Effect of Defects on Static and Dynamic Contact Angles.

    PubMed

    Kozbial, Andrew; Trouba, Charlie; Liu, Haitao; Li, Lei

    2017-01-31

    Elucidating the intrinsic water wettability of the graphitic surface has increasingly attracted research interests, triggered by the recent finding that the well-established hydrophobicity of graphitic surfaces actually results from airborne hydrocarbon contamination. Currently, static water contact angle (WCA) is often used to characterize the intrinsic water wettability of graphitic surfaces. In the current paper, we show that because of the existence of defects, static WCA does not necessarily characterize the intrinsic water wettability. Freshly exfoliated graphite of varying qualities, characterized using atomic force microscopy and Raman spectroscopy, was studied using static, advancing, and receding WCA measurements. The results showed that graphite of different qualities (i.e., defect density) always has a similar advancing WCA, but it could have very different static and receding WCAs. This finding indicates that defects play an important role in contact angle measurements, and the static contact angle does not always represent the intrinsic water wettability of pristine graphite. On the basis of the experimental results, a qualitative model is proposed to explain the effect of defects on static, advancing, and receding contact angles. The model suggests that the advancing WCA reflects the intrinsic water wettability of pristine (defect-free) graphite. Our results showed that the advancing WCA for pristine graphite is 68.6°, which indicates that graphitic carbon is intrinsically mildly hydrophilic.

  14. Hydrogen defects in α-Al2O3 and water weakening of sapphire and alumina ceramics between 600 and 1000°C: I. Infrared characterization of defects

    USGS Publications Warehouse

    Kronenberg, A.K.; Castaing, J.; Mitchell, T.E.; Kirby, S.H.

    2000-01-01

    Hydrogen impurities in materials influence their properties, including flow strength. α-Al2O3 single crystals and polycrystalline ceramics were annealed in supercritical water between 850 and 1025°C, under pressures in the range 1500–2000 MPa. A few specimens were further subjected to plastic deformation. Hydrogen penetration was examined using infrared absorption measurements of O–H bond vibrations, which revealed two kinds of hydrogen defects. In single crystals, defects are characterized by sharp O–H absorption bands assigned to interstitial protons. Hydrogen impurities of hydrothermally annealed ceramics and of all hydrothermally deformed specimens are characterized by broad O–H bands assigned to molecular water. The grain boundaries of hydrothermally annealed ceramics are severely damaged. The kinetics of hydrogen penetration is consistent with diffusion data.

  15. Scanning coherent diffractive imaging methods for actinic extreme ultraviolet mask metrology

    NASA Astrophysics Data System (ADS)

    Helfenstein, Patrick; Mohacsi, Istvan; Rajeev, Rajendran; Ekinci, Yasin

    2016-07-01

    For the successful implementation of extreme ultraviolet (EUV) lithography in the upcoming technology nodes, a major challenge to overcome is the stable and reliable detection and characterization of mask defects. We have recently presented a reflective mode EUV mask scanning lensless imaging tool (RESCAN) which was installed at the XIL-II beamline of the swiss light source and showed reconstructed aerial images of test patterns on EUV masks. RESCAN uses scanning coherent diffractive imaging (SCDI) methods to obtain actinic aerial images of EUV photomasks and was designed for 80 nm onmask resolution. Our SCDI algorithm reconstructs the measured sample by iteratively solving the phase problem using overdetermined diffraction data gathered by scanning across the specimen with a finite illumination. It provides the phase and amplitude aerial images of EUV photomasks with high resolution without the need to use high numerical aperture (NA) lenses. Contrary to scanning microscopy and full-field microscopy, where the resolution is limited by the spot size or NA of the lens, the achievable resolution with our method depends on the detector noise and NA of the detector. To increase the resolution of our tool, we upgraded RESCAN with a detector and algorithms. Here, we present the results obtained with the tool that is capable of up to 40-nm onmask resolution. We believe that the realization of our prototype marks a significant step toward overcoming the limitations imposed by methods relying on imaging optics and shows a viable solution for actinic mask metrology.

  16. Polychromatic X-ray Microdiffraction Characterization of Local Crystallographic Structure and Defect Distributions

    SciTech Connect

    Ice, G.E.; Barabash, R.I.; Pang, J.W. L.

    2007-12-19

    Three-dimensional (3D), nondestructive, spatially resolved characterization of local crystal structure is conveniently made with polychromatic x-ray microdiffraction. In general, polychromatic microdiffraction provides information about the local (subgrain) orientation, unpaired-dislocation density, and elastic strain. This information can be used for direct comparison to theoretical models. Practical microbeams use intense synchrotron x-ray sources and advanced x-ray focusing optics. By employing polychromatic x-ray beams and a virtual pinhole camera method, called differential aperture microscopy, 3D distributions of the local crystalline phase, orientation (texture), and elastic and plastic strain tensors can be measured with submicron 3D resolution. The local elastic strain tensor elements can typically be determined with uncertainties less than 100 ppm. Orientations can be quantified to {approx} 0.01{sup o} and the local unpaired dislocation-density tensor can be simultaneously characterized. The spatial resolution limit for hard x-ray polychromatic microdiffraction is < 40nm and existing instruments operate with {approx} 500 to 1000nm resolution. Because the 3D x-ray crystal microscope is a penetrating nondestructive tool, it is ideal for studies of mesoscale evolution in materials.

  17. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin

    PubMed Central

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-01-01

    ABSTRACT Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation. PMID:25315833

  18. ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays

    PubMed Central

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M.; Lowe, Martin; Vartiainen, Maria K.; Jokitalo, Eija

    2014-01-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. PMID:24523293

  19. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin.

    PubMed

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-12-01

    Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation.

  20. ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays.

    PubMed

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M; Lowe, Martin; Vartiainen, Maria K; Jokitalo, Eija

    2014-04-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

  1. A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.

    PubMed

    Palmer, Sarah E; Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Mishra, Ritu; Johnson, Simeon; Goldberg, Martin W; Ayscough, Kathryn R

    2015-03-30

    Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that--like wild-type Vps1--it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.

  2. A Balance of Capping Protein and Profilin Functions Is Required to Regulate Actin Polymerization in Drosophila Bristle

    PubMed Central

    Hopmann, Roberta; Miller, Kathryn G.

    2003-01-01

    Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell. PMID:12529431

  3. 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.

  4. 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.

  5. Characterization of a defective interfering RNA that contains a mosaic of a plant viral genome. Final report

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1991-12-31

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since then, we have also discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), a virus structurally related to TBSV. We proposed a thorough characterization of this unique class of symptom modulating RNAs with the overall objective of identifying viral RNA nucleotide, sequences involved in such fundamental processes as virus replication and encapsidation as well as the degree of symptom expression resulting from the viral-DI-host interaction. The proposed research focused on the molecular characterization of the DI RNAs and the helper virus. We had demonstrated that the DIs were collinear deletion mutants of the genome of a cherry strain of tomato bushy stunt virus (TBSV). We had also shown that these low molecular weight RNAs interfered with the helper plant virus and modulated disease expression by preventing the development of a lethal necrotic disease in susceptible host plants. We also suggested that by exploring the mechanisms associated with the symptom attenuation effect, we might be able to devise novel strategies useful for engineering viral disease resistance.

  6. Morphology and viscoelasticity of actin networks formed with the mutually interacting crosslinkers: palladin and alpha-actinin.

    PubMed

    Grooman, Brian; Fujiwara, Ikuko; Otey, Carol; Upadhyaya, Arpita

    2012-01-01

    Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not clearly understood. The ABP, palladin, is essential for the maintenance of cell morphology and the regulation of cell movement. Palladin coexists with α-actinin in stress fibers and focal adhesions and binds to both actin and α-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we characterized the micro-structure and mechanics of actin networks crosslinked with palladin and α-actinin. We first showed that palladin crosslinks actin filaments into bundled networks which are viscoelastic in nature. Our studies also showed that composite networks of α-actinin/palladin/actin behave very similar to pure palladin or pure [Formula: see text]-actinin networks. However, we found evidence that palladin and α-actinin synergistically modify network viscoelasticity. To our knowledge, this is the first quantitative characterization of the physical properties of actin networks crosslinked with two mutually interacting crosslinkers.

  7. 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.

  8. Isolation and characterization of Arabidopsis mutants defective in the induction of ethylene biosynthesis by cytokinin

    NASA Technical Reports Server (NTRS)

    Vogel, J. P.; Schuerman, P.; Woeste, K.; Brandstatter, I.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Cytokinins elevate ethylene biosynthesis in etiolated Arabidopsis seedlings via a post-transcriptional modification of one isoform of the key biosynthetic enzyme ACC synthase. In order to begin to dissect the signaling events leading from cytokinin perception to this modification, we have isolated a series of mutants that lack the ethylene-mediated triple response in the presence of cytokinin due to their failure to increase ethylene biosynthesis. Analysis of genetic complementation and mapping revealed that these Cin mutants (cytokinin-insensitive) represent four distinct complementation groups, one of which, cin4, is allelic to the constitutive photomorphogenic mutant fus9/cop10. The Cin mutants have subtle effects on the morphology of adult plants. We further characterized the Cin mutants by analyzing ethylene biosynthesis in response to various other inducers and in adult tissues, as well as by assaying additional cytokinin responses. The cin3 mutant did not disrupt ethylene biosynthesis under any other conditions, nor did it disrupt any other cytokinin responses. Only cin2 disrupted ethylene biosynthesis in multiple circumstances. cin1 and cin2 made less anthocyanin in response to cytokinin. cin1 also displayed reduced shoot initiation in tissue culture in response to cytokinin, suggesting that it affects a cytokinin signaling element.

  9. 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.

  10. Purification and characterization of a mutant DnaB protein specifically defective in ATP hydrolysis.

    PubMed Central

    Shrimankar, P; Stordal, L; Maurer, R

    1992-01-01

    The dnaB gene of Escherichia coli encodes an essential DNA replication enzyme. Fueled by the energy derived from the hydrolysis of ATP to ADP+P(i), this enzyme unwinds double-stranded DNA in advance of the DNA polymerase. While doing so, it intermittently stimulates primase to synthesize an RNA primer for an Okazaki fragment. To better understand the structural basis of these and other aspects of DnaB function, we have initiated a study of mutant DnaB proteins. Here, we report the purification and characterization of a mutant DnaB protein (RC231) containing cysteine in place of arginine at residue 231. The mutant protein attains a stable, properly folded structure that allows association of six promoters to form a hexamer, as is also true for wild-type DnaB. Further, the mutant protein interacts with ATP, the nonhydrolyzable ATP analog adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, and poly(dT), and it stimulates primase action. It is, however, profoundly deficient in ATP hydrolysis, helicase activity, and replication activity at the chromosomal origin of replication. In addition, while general priming reactions with wild-type DnaB and ATP elicited the synthesis of short primers, reactions with DnaB and ATP gamma S or with RC231 and either ATP or ATP gamma S stimulated the synthesis of significantly longer primers. On the basis of these observations, we suggest that primase interacts directly with DnaB throughout primer synthesis during general priming, until dissociation of DnaB from DNA or ATP hydrolysis by DnaB disrupts the interaction and leads to primer termination. Images PMID:1332941

  11. Characterization of genetic defects of hemophilia A in patients of Chinese origin

    SciTech Connect

    Lin, Shu-Wha; Lin, Shu-Rung; Shen, Ming-Ching )

    1993-12-01

    The molecular characterization of hemophilia A of Chinese origin was carried out by the polymerase chain reaction (PCR) and direct sequencing of patient's factor VIII genes. Single-strand conformation polymorphism (SSCP) and dideoxy fingerprinting (ddF) were used as screening methods to detect mutated DNAs. A total of 102 individuals from 87 different families, including 10 patients (10 families) with mild-to-moderate and 92 patients (77 families) with severe hemophilia A, were analyzed by PCR-SSCP and PCR-ddF. Of the 87 independent cases, 40 revealed a single mutation in the coding regions of their factor VIII genes. These mutations include 21 with single base changes resulting in 8 nonsense and 13 missense codons, 16 with deletion or insertion of 1-11 nucleotides, and 3 with deletion of large DNA fragments. The frequency of 8 of the identified factor VIII polymorphisms or silent mutations was also determined among Chinese. The frequencies for codons 1241, 1269, and 2223 (the numbering system follows J. Gitschier et al., 1984, Nature 312: 326-330) were found to be different from those reported for other populations. As for the 47 severe cases whose mutational events were not readily detected by PCR-SSCP and PCR-ddF, the reverse transcriptase PCR method was applied. In 24 such cases analyzed, 17 were found to be of the [open quotes]intron 22 mutations[close quotes] as described by Naylor et al. (1992, The Lancet, 342: 1066-1067), accounting for 39% of Chinese patients with hemophilia A. 31 refs., 2 figs., 6 tabs.

  12. 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.

  13. Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton.

    PubMed

    Crawford, A W; Pino, J D; Beckerle, M C

    1994-01-01

    LIM domains are present in a number of proteins including transcription factors, a proto-oncogene product, and the adhesion plaque protein zyxin. The LIM domain exhibits a characteristic arrangement of cysteine and histidine residues and represents a novel zinc binding sequence (Michelsen et al., 1993). Previously, we reported the identification of a 23-kD protein that interacts with zyxin in vitro (Sadler et al., 1992). In this report, we describe the purification and characterization of this 23-kD zyxin-binding protein from avian smooth muscle. Isolation of a cDNA encoding the 23-kD protein has revealed that it consists of 192 amino acids and exhibits two copies of the LIM motif. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP); therefore we refer to it as the chicken cysteine-rich protein (cCRP). Examination of a number of chick embryonic tissues by Western immunoblot analysis reveals that cCRP exhibits tissue-specific expression. cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. We have performed quantitative Western immunoblot analysis of cCRP, zyxin, and alpha-actinin levels during embryogenesis. By this approach, we have demonstrated that the expression of cCRP is developmentally regulated.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton

    PubMed Central

    1994-01-01

    LIM domains are present in a number of proteins including transcription factors, a proto-oncogene product, and the adhesion plaque protein zyxin. The LIM domain exhibits a characteristic arrangement of cysteine and histidine residues and represents a novel zinc binding sequence (Michelsen et al., 1993). Previously, we reported the identification of a 23-kD protein that interacts with zyxin in vitro (Sadler et al., 1992). In this report, we describe the purification and characterization of this 23-kD zyxin-binding protein from avian smooth muscle. Isolation of a cDNA encoding the 23-kD protein has revealed that it consists of 192 amino acids and exhibits two copies of the LIM motif. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP); therefore we refer to it as the chicken cysteine-rich protein (cCRP). Examination of a number of chick embryonic tissues by Western immunoblot analysis reveals that cCRP exhibits tissue-specific expression. cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. We have performed quantitative Western immunoblot analysis of cCRP, zyxin, and alpha-actinin levels during embryogenesis. By this approach, we have demonstrated that the expression of cCRP is developmentally regulated. PMID:8294495

  19. The anti-proliferative agent jasplakinolide rearranges the actin cytoskeleton of plant cells.

    PubMed

    Sawitzky, H; Liebe, S; Willingale-Theune, J; Menzel, D

    1999-06-01

    In the present study, we have characterized the action of the natural cyclodepsipeptide jasplakinolide (JAS) on the cytoplasmic architecture, actin-based cytoplasmic motility, and the organization of the actin cytoskeleton in selected examples of green algae (Acetabularia, Pseudobryopsis and Nitella) and higher plant cells (Allium bulb scale cells and Sinapis root hairs). JAS was capable of influencing the actin cytoskeleton and inhibiting cytoplasmic streaming in a differential, cell type-specific manner. With the exception of Nitella, two consecutive responses were observed upon incubation with 2.5 microM JAS: In the first phase cytoplasmic streaming increased transiently alongside with minor modifications of the actin cytoskeleton in the form of adventitious actin spots and spikes appearing throughout the cell cortex in addition to the normal actin bundle system typical for each cell type. In the second phase, cytoplasmic streaming stopped and the actin cytoskeleton became heavily reorganized into shorter, straight, more and more randomly oriented bundle segments. JAS exerted severe long-term effects on the actin cytoskeleton when treatments exceeded 30min at a concentration of 2.5 microM. An in situ competition assay using equimolar concentrations of JAS and FITC-phalloidin suggested that JAS has a phalloidin-like action. Effects of JAS were significantly different from those of cytochalasin D with respect to the resulting degree of perturbance of cytoplasmic organization, the distribution of actin filaments and the speed of reversibility.

  20. Actin-crosslinking protein regulation of filament movement in motility assays: a theoretical model.

    PubMed Central

    Janson, L W; Taylor, D L

    1994-01-01

    The interaction of single actin filaments on a myosin-coated coverslip has been modeled by several authors. One model adds a component of "frictional drag" by myosin heads that oppose movement of the actin filaments. We have extended this concept by including the resistive drag from actin crosslinking proteins to understand better the relationship among crosslinking number, actin-myosin force generation, and motility. The validity of this model is supported by agreement with the experimental results from a previous study in which crosslinking proteins were added with myosin molecules under otherwise standard motility assay conditions. The theoretical relationship provides a means to determine many physical parameters that characterize the interaction between a single actin filament and a single actin-crosslinking molecule (various types). In particular, the force constant of a single filamin molecule is calculated as 1.105 pN, approximately 3 times less than a driving myosin head (3.4 pN). Knowledge of this parameter and others derived from this model allows a better understanding of the interaction between myosin and the actin/actin-binding protein cytoskeleton and the role of actin-binding proteins in the regulation and modulation of motility. PMID:7811954

  1. Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction*

    PubMed Central

    Dalghi, Marianela G.; Fernández, Marisa M.; Ferreira-Gomes, Mariela; Mangialavori, Irene C.; Malchiodi, Emilio L.; Strehler, Emanuel E.; Rossi, Juan Pablo F. C.

    2013-01-01

    As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca2+ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca2+-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca2+-ATPase activity was related to an increase in the apparent affinity for Ca2+ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca2+ homeostasis. PMID:23803603

  2. Plasma membrane calcium ATPase activity is regulated by actin oligomers through direct interaction.

    PubMed

    Dalghi, Marianela G; Fernández, Marisa M; Ferreira-Gomes, Mariela; Mangialavori, Irene C; Malchiodi, Emilio L; Strehler, Emanuel E; Rossi, Juan Pablo F C

    2013-08-09

    As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca(2+) with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca(2+)-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca(2+)-ATPase activity was related to an increase in the apparent affinity for Ca(2+) and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca(2+) homeostasis.

  3. Characterization of defects created in Cz and epitaxial Si doped with Ga or B using Laplace-DLTS

    NASA Astrophysics Data System (ADS)

    Nyamhere, Cloud; Deenapanray, P. N. K.; Auret, F. D.; Farlow, F. C.

    2006-04-01

    We have measured the electrical and annealing properties of defects created in epitaxial and Czochralski-grown Si doped with either B or Ga by electron irradiation using both conventional and Laplace deep level transient spectroscopy (L)-DLTS. With L-DLTS, we have been able to resolve several defects that cannot be resolved using conventional DLTS. L-DLTS provides a new avenue to study defect introduction rates and annealing kinetics in B- and Ga-doped Si. The isochronal annealing behaviour of the defects was also investigated.

  4. Structural characterization and novel optical properties of defect chalcopyrite ZnGa{sub 2}Te{sub 4} thin films

    SciTech Connect

    Fouad, S.S.; Sakr, G.B.; Yahia, I.S.; Basset, D.M. Abdel

    2011-11-15

    Highlights: {yields} Preparation and characterization of ZnGa{sub 2}Te{sub 4} in powder and thin film forms. {yields} Structure properties such as XRD and EDX. {yields} Optical constant of the as-deposited ZnGa{sub 2}Te{sub 4} for the first time. {yields} Extraction of the optical parameters of the studied films. -- Abstract: Stoichiometric thin film samples of the ternary ZnGa{sub 2}Te{sub 4} defect chalcopyrite compound were prepared and characterized by X-ray diffraction technique. The elemental chemical composition of the prepared bulk material as well as of the as-deposited film was determined by energy-dispersive X-ray spectrometry. ZnGa{sub 2}Te{sub 4} thin films were deposited, by conventional thermal evaporation technique onto highly cleaned glass substrates. The X-ray and electron diffraction studies revealed that the as-deposited and the annealed ZnGa{sub 2}Te{sub 4} films at annealing temperature t{sub a} {<=} 548 K are amorphous, while those annealed at t{sub a} {>=} 573 K (for 1 h), are polycrystalline. The optical properties of the as-deposited films have been investigated for the first time at normal incidence in the spectral range from 500 to 2500 nm. The refractive index dispersion in the transmission and low absorption region is adequately described by the Wemple-DiDomenico single oscillator model, whereby, the values of the oscillator parameters have been calculated. The analysis of the optical absorption coefficient revealed an in-direct optical transition with energy of 1.33 eV for the as-deposited sample. This work suggested that ZnGa{sub 2}Te{sub 4} is a good candidate in solar cell devices as an absorbing layer.

  5. 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.

  6. 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.

  7. Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

    SciTech Connect

    Cowieson, Nathan P.; King, Gordon; Cookson, David; Ross, Ian; Huber, Thomas; Hume, David A.; Kobe, Bostjan; Martin, Jennifer L.

    2008-08-21

    Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

  8. Cofilin cooperates with fascin to disassemble filopodial actin filaments

    PubMed Central

    Breitsprecher, Dennis; Koestler, Stefan A.; Chizhov, Igor; Nemethova, Maria; Mueller, Jan; Goode, Bruce L.; Small, J. Victor; Rottner, Klemens; Faix, Jan

    2011-01-01

    Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin. PMID:21940796

  9. Formin 1 Regulates Ectoplasmic Specialization in the Rat Testis Through Its Actin Nucleation and Bundling Activity

    PubMed Central

    Li, Nan; Mruk, Dolores D.; Wong, Chris K. C.; Han, Daishu; Lee, Will M.

    2015-01-01

    During spermatogenesis, developing spermatids and preleptotene spermatocytes are transported across the adluminal compartment and the blood-testis barrier (BTB), respectively, so that spermatids line up near the luminal edge to prepare for spermiation, whereas preleptotene spermatocytes enter the adluminal compartment to differentiate into late spermatocytes to prepare for meiosis I/II. These cellular events involve actin microfilament reorganization at the testis-specific, actin-rich Sertoli-spermatid and Sertoli-Sertoli cell junction called apical and basal ectoplasmic specialization (ES). Formin 1, an actin nucleation protein known to promote actin microfilament elongation and bundling, was expressed at the apical ES but limited to stage VII of the epithelial cycle, whereas its expression at the basal ES/BTB stretched from stage III to stage VI, diminished in stage VII, and was undetectable in stage VIII tubules. Using an in vitro model of studying Sertoli cell BTB function by RNA interference and biochemical assays to monitor actin bundling and polymerization activity, a knockdown of formin 1 in Sertoli cells by approximately 70% impeded the tight junction-permeability function. This disruptive effect on the tight junction barrier was mediated by a loss of actin microfilament bundling and actin polymerization capability mediated by changes in the localization of branched actin-inducing protein Arp3 (actin-related protein 3), and actin bundling proteins Eps8 (epidermal growth factor receptor pathway substrate 8) and palladin, thereby disrupting cell adhesion. Formin 1 knockdown in vivo was found to impede spermatid adhesion, transport, and polarity, causing defects in spermiation in which elongated spermatids remained embedded into the epithelium in stage IX tubules, mediated by changes in the spatiotemporal expression of Arp3, Eps8, and palladin. In summary, formin 1 is a regulator of ES dynamics. PMID:25901598

  10. Novel interactions between actin and the proteasome revealed by complex haploinsufficiency.

    PubMed

    Haarer, Brian; Aggeli, Dimitra; Viggiano, Susan; Burke, Daniel J; Amberg, David C

    2011-09-01

    Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous interactions are potentially important to human genetic disease as loss-of-function alleles are common in human genomes. We have been using complex haploinsufficiency (CHI) screening with the actin gene to identify genes related to actin function and as a model to determine the prevalence of CHI interactions in eukaryotic genomes. Previous CHI screening between actin and null alleles for non-essential genes uncovered ∼240 deleterious CHI interactions. In this report, we have extended CHI screening to null alleles for essential genes by mating a query strain to sporulations of heterozygous knock-out strains. Using an act1Δ query, knock-outs of 60 essential genes were found to be CHI with actin. Enriched in this collection were functional categories found in the previous screen against non-essential genes, including genes involved in cytoskeleton function and chaperone complexes that fold actin and tubulin. Novel to this screen was the identification of genes for components of the TFIID transcription complex and for the proteasome. We investigated a potential role for the proteasome in regulating the actin cytoskeleton and found that the proteasome physically associates with actin filaments in vitro and that some conditional mutations in proteasome genes have gross defects in actin organization. Whole-genome screening with actin as a query has confirmed that CHI interactions are important phenotypic drivers. Furthermore, CHI screening is another genetic tool to uncover novel functional connections. Here we report a previously unappreciated role for the proteasome in affecting actin organization and function.

  11. A role for γS-crystallin in the organization of actin and fiber cell maturation in the mouse lens.

    PubMed

    Fan, Jianguo; Dong, Lijin; Mishra, Sanghamitra; Chen, Yingwei; FitzGerald, Paul; Wistow, Graeme

    2012-08-01

    γS-crystallin (γS) is a highly conserved component of the eye lens. To gain insights into the functional role(s) of this protein, the mouse gene (Crygs) was deleted. Although mutations in γS can cause severe cataracts, loss of function of γS in knockout (KO) mice produced no obvious lens opacity, but was associated with focusing defects. Electron microscopy showed no major differences in lens cell organization, suggesting that the optical defects are primarily cytoplasmic in origin. KO lenses were also grossly normal by light microscopy but showed evidence of incomplete clearance of cellular organelles in maturing fiber cells. Phalloidin labeling showed an unusual distribution of F-actin in a band of mature fiber cells in KO lenses, suggesting a defect in the organization or processing of the actin cytoskeleton. Indeed, in wild-type lenses, γS and F-actin colocalize along the fiber cell plasma membrane. Relative levels of F-actin and G-actin in wild-type and KO lenses were estimated from fluorescent staining profiles and from isolation of actin fractions from whole lenses. Both methods showed a two-fold reduction in the F-actin/G-actin ratio in KO lenses, whereas no difference in tubulin organization was detected. In vitro experiments showed that recombinant mouse γS can directly stabilize F-actin. This suggests that γS may have a functional role related to actin, perhaps in 'shepherding' filaments to maintain the optical properties of the lens cytoplasm and normal fiber cell maturation.

  12. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  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. Arg/Abl2 modulates the affinity and stoichiometry of binding of cortactin to F-actin.

    PubMed

    MacGrath, Stacey M; Koleske, Anthony J

    2012-08-21

    The Abl family nonreceptor tyrosine kinase Arg/Abl2 interacts with cortactin, an Arp2/3 complex activator, to promote actin-driven cell edge protrusion. Both Arg and cortactin bind directly to filamentous actin (F-actin). While protein-protein interactions between Arg and cortactin have well-characterized downstream effects on the actin cytoskeleton, it is unclear whether and how Arg and cortactin affect each other's actin binding properties. We employ actin cosedimentation assays to show that Arg increases the stoichiometry of binding of cortactin to F-actin at saturation. Using a series of Arg deletion mutants and fragments, we demonstrate that the Arg C-terminal calponin homology domain is necessary and sufficient to increase the stoichiometry of binding of cortactin to F-actin. We also show that interactions between Arg and cortactin are required for optimal affinity between cortactin and the actin filament. Our data suggest a mechanism for Arg-dependent stimulation of binding of cortactin to F-actin, which may facilitate the recruitment of cortactin to sites of local actin network assembly.

  18. Actin Dosage Lethality Screening in Yeast Mediated by Selective Ploidy Ablation Reveals Links to Urmylation/Wobble Codon Recognition and Chromosome Stability

    PubMed Central

    Haarer, Brian; Mi-Mi, Lei; Cho, Jessica; Cortese, Matthew; Viggiano, Susan; Burke, Daniel; Amberg, David

    2013-01-01

    The actin cytoskeleton exists in a dynamic equilibrium with monomeric and filamentous states of its subunit protein actin. The spatial and temporal regulation of actin dynamics is critical to the many functions of actin. Actin levels are remarkably constant, suggesting that cells have evolved to function within a narrow range of actin concentrations. Here we report the results of screens in which we have increased actin levels in strains deleted for the ~4800 nonessential yeast genes using a technical advance called selective ploidy ablation. We detected 83 synthetic dosage interactions with actin, 78 resulted in reduced growth, whereas in 5 cases overexpression of actin suppressed the growth defects caused by the deleted genes. The genes were highly enriched in several classes, including transfer RNA wobble uridine modification, chromosome stability and segregation, cell growth, and cell division. We show that actin overexpression sequesters a limited pool of eEF1A, a bifunctional protein involved in aminoacyl-transfer RNA recruitment to the ribosome and actin filament cross-linking. Surprisingly, the largest class of genes is involved in chromosome stability and segregation. We show that actin mutants have chromosome segregation defects, suggesting a possible role in chromosome structure and function. Monomeric actin is a core component of the INO80 and SWR chromatin remodeling complexes and the NuA4 histone modification complex, and our results suggest these complexes may be sensitive to actin stoichiometry. We propose that the resulting effects on chromatin structure can lead to synergistic effects on chromosome stability in strains lacking genes important for chromosome maintenance. PMID:23450344

  19. 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.

  20. The yeast dynamin-related GTPase Vps1p functions in the organization of the actin cytoskeleton via interaction with Sla1p.

    PubMed

    Yu, Xianwen; Cai, Mingjie

    2004-08-01

    Recent studies have suggested that the function of the large GTPase dynamin in endocytosis in mammalian cells may comprise a modulation of actin cytoskeleton. The role of dynamin in actin cytoskeleton organization in the yeast Saccharomyces cerevisiae has remained undefined. In this report, we found that one of the yeast dynamin-related proteins, Vps1p, is required for normal actin cytoskeleton organization. At both permissive and non-permissive temperatures, the vps1 mutants exhibited various degrees of phenotypes commonly associated with actin cytoskeleton defects: depolarized and aggregated actin structures, hypersensitivity to the actin cytoskeleton toxin latrunculin-A, randomized bud site selection and chitin deposition, and impaired efficiency in the internalization of membrane receptors. Over-expression of the GTPase mutants of vps1 also led to actin abnormalities. Consistent with these actin-related defects, Vps1p was found to interact physically, and partially co-localize, with the actin-regulatory protein Sla1p. The normal cellular localization of Sla1p required Vps1p and could be altered by over-expression of a region of Vps1p that was involved in the interaction with Sla1p. The same region also promoted mis-sorting of the vacuolar protein carboxypeptidase Y upon over-expression. These findings suggest that the functions of the dynamin-related protein Vps1p in actin cytoskeleton dynamics and vacuolar protein sorting are probably related to each other.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. Structural implications of Ca2+-dependent actin-bundling function of human EFhd2/Swiprosin-1

    PubMed Central

    Park, Kyoung Ryoung; Kwon, Min-Sung; An, Jun Yop; Lee, Jung-Gyu; Youn, Hyung-Seop; Lee, Youngjin; Kang, Jung Youn; Kim, Tae Gyun; Lim, Jia Jia; Park, Jeong Soon; Lee, Sung Haeng; Song, Woo Keun; Cheong, Hae-Kap; Jun, Chang-Duk; Eom, Soo Hyun

    2016-01-01

    EFhd2/Swiprosin-1 is a cytoskeletal Ca2+-binding protein implicated in Ca2+-dependent cell spreading and migration in epithelial cells. EFhd2 domain architecture includes an N-terminal disordered region, a PxxP motif, two EF-hands, a ligand mimic helix and a C-terminal coiled-coil domain. We reported previously that EFhd2 displays F-actin bundling activity in the presence of Ca2+ and this activity depends on the coiled-coil domain and direct interaction of the EFhd2 core region. However, the molecular mechanism for the regulation of F-actin binding and bundling by EFhd2 is unknown. Here, the Ca2+-bound crystal structure of the EFhd2 core region is presented and structures of mutants defective for Ca2+-binding are also described. These structures and biochemical analyses reveal that the F-actin bundling activity of EFhd2 depends on the structural rigidity of F-actin binding sites conferred by binding of the EF-hands to Ca2+. In the absence of Ca2+, the EFhd2 core region exhibits local conformational flexibility around the EF-hand domain and C-terminal linker, which retains F-actin binding activity but loses the ability to bundle F-actin. In addition, we establish that dimerisation of EFhd2 via the C-terminal coiled-coil domain, which is necessary for F-actin bundling, occurs through the parallel coiled-coil interaction. PMID:27974828

  6. 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.

  7. The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

    PubMed Central

    Bane, Kartik S.; Singer, Mirko; Reinig, Miriam; Klug, Dennis; Heiss, Kirsten; Baum, Jake; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics. PMID:27409081

  8. 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

  9. Characterization on defect solids

    NASA Technical Reports Server (NTRS)

    Schlosser, Herbert

    1994-01-01

    The purpose of this research has been to develop new semi-empirical techniques to describe ceramics, alloys, and metal/ceramic interfaces with applications in mind that support the materials aspects of the high speed civil transport program (HSCT). HSCT requires methods that aid in the design of alloys and ceramics for new high strength, high temperature applications. Current theoretical methods are not capable of carrying out this mission. Hence, new accurate and more efficient theoretical techniques are needed to facilitate the design of new materials and the examination of their properties. This program concentrated on modeling ceramics, but also dealt with alloy properties to a lesser degree. The primary accomplishment of this research was the development of a new equation of state (EOS) that models the energy/bond length relationship and includes terms that represent charge transfer between cations and anions. The new EOS has been used by researchers at the Naval Research Laboratory to develop a new semi-empirical method for calculating properties of ceramics and metal ceramic interfaces based on the embedded atom method. The results of the director's discretionary fund (DDF) research describing the binding energy relation was the enabling theoretical basis. Also the workers at Cleveland State and NASA Lewis have detailed an approach based on equivalent crystal theory as part of the DDF which is currently under development. In addition, initial results for using the Harris functional method have been developed. Finally, the techniques developed by us have proved useful for the study of high pressure properties of solids and have been used extensively by researchers in this field.

  10. 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

  11. Holt-Oram syndrome with intermediate atrioventricular canal defect, and aortic coarctation: functional characterization of a de novo TBX5 mutation.

    PubMed

    Baban, Anwar; Pitto, Letizia; Pulignani, Silvia; Cresci, Monica; Mariani, Laura; Gambacciani, Carolina; Digilio, Maria Cristina; Pongiglione, Giacomo; Albanese, Sonia

    2014-06-01

    Holt-Oram syndrome (HOS) is a rare autosomal dominant disorder characterized by upper limb defects and congenital heart defects (CHD), which are often simple septal and conduction defects, less frequently complex CHDs. We report on a 9 year-old boy with clinical and radiologic features of HOS consisting of bilateral asymmetric hypoplastic thumbs, generalized brachydactyly, limited supination due to radioulnar synostosis, and sloping shoulders, and intermediate atrioventricular canal defect (AVCD) with aortic coarctation. A de novo, previously described mutation, (Arg279ter) was identified in the TBX5 gene. Molecular characterization of this mutation was carried out due to the atypical CHD. In order to investigate whether the mutated transcript of TBX5 was able to escape the post-transcriptional surveillance mechanism and to produce a truncated TBX5 protein, we analyzed the TBX5 transcript, and protein pattern in HOS, and WT cardiac tissues. Our results demonstrate that the mutant TBX5 transcript is cleared by the cellular mechanism of surveillance. This data provides some support for the hypothesis that a dominant negative mutation, which strongly impairs the WT allele, might be too hazardous to be maintained. The literature suggests that HOS is relatively common among syndromes associated with AVCD.

  12. 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.

  13. Actinic review of EUV masks: status and recent results of the AIMSTM EUV system

    NASA Astrophysics Data System (ADS)

    Perlitz, Sascha; Peters, Jan Hendrik; Weiss, Markus; Hellweg, Dirk; Capelli, Renzo; Magnusson, Krister; Malloy, Matt; Wurm, Stefan

    2015-10-01

    Key enabler of the successful introduction of EUV lithography into volume production is the EUV mask infrastructure. For the production of defect free masks, actinic review of potential defect sites to decide on the need for repair or compensation is required. Also, the repair or compensation with the ZEISS MERiT electron beam repair tool needs actinic verification in a closed loop mask repair solution. For the realization of actinic mask review, ZEISS and the SEMATECH EUVL Mask Infrastructure consortium started a development program for an EUV aerial image metrology system, the AIMSTM EUV, with realization of a prototype tool. The development and prototype realization of the AIMSTM EUV has entered the tool calibration and qualification phase utilizing the achieved capabilities of EUV aerial image acquisition and EUV mask handling. In this paper, we discuss the current status of the prototype qualification and show recent measurement results.

  14. Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin.

    PubMed

    Tomatis, Vanesa M; Papadopulos, Andreas; Malintan, Nancy T; Martin, Sally; Wallis, Tristan; Gormal, Rachel S; Kendrick-Jones, John; Buss, Folma; Meunier, Frédéric A

    2013-02-04

    Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.

  15. Defect characterization of GaAs/InP layers and MESFETs devices by admittance and photoluminescence spectroscopies

    NASA Astrophysics Data System (ADS)

    Ben Hamida, A.; Bremond, Georges E.; Garcia Perez, M. A.; Guillot, Gerard; Azoulay, Rozette; Chertouk, Mourad; Clei, A.

    1993-11-01

    GaAs layers as well as GaAs MESFET devices on InP are studied by means of DLTS and PL spectroscopies. We correlate the compensation observed on the Si-n-doped GaAs layers to the incorporation of Si that moves from a SiGa donor site to form a complex defect involving Si and As or Ga vacancies. A study of defects on MESFETs grown with various buffer layer thicknesses shows that the thicker this layer is the higher is the defect concentration. This behavior is assumed to be related to the compensation effect.

  16. 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.

  17. 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.

  18. Actinic review of EUV masks: performance data and status of the AIMS EUV System

    NASA Astrophysics Data System (ADS)

    Hellweg, Dirk; Perlitz, Sascha; Magnusson, Krister; Capelli, Renzo; Koch, Markus; Malloy, Matt

    2016-03-01

    The EUV mask infrastructure is of key importance for the successful introduction of EUV lithography into volume production. In particular, for the production of defect free masks an actinic review of potential defect sites is required. ZEISS and the SUNY POLY SEMATECH EUVL Mask Infrastructure consortium started a development program for such an EUV aerial image metrology system, the AIMS EUV. In this paper, we provide measurement data on the system's key specifications and discuss its performance and capability status.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. Ras GTPase-Activating Protein Regulation of Actin Cytoskeleton and Hyphal Polarity in Aspergillus nidulans▿ †

    PubMed Central

    Harispe, Laura; Portela, Cecilia; Scazzocchio, Claudio; Peñalva, Miguel A.; Gorfinkiel, Lisette

    2008-01-01

    Aspergillus nidulans gapA1, a mutation leading to compact, fluffy colonies and delayed polarity establishment, maps to a gene encoding a Ras GTPase-activating protein. Domain organization and phylogenetic analyses strongly indicate that GapA regulates one or more “true” Ras proteins. A gapAΔ strain is viable. gapA colonies are more compact than gapA1 colonies and show reduced conidiation. gapAΔ strains have abnormal conidiophores, characterized by the absence of one of the two layers of sterigmata seen in the wild type. gapA transcript levels are very low in conidia but increase during germination and reach their maximum at a time coincident with germ tube emergence. Elevated levels persist in hyphae. In germinating conidiospores, gapAΔ disrupts the normal coupling of isotropic growth, polarity establishment, and mitosis, resulting in a highly heterogeneous cell population, including malformed germlings and a class of giant cells with no germ tubes and a multitude of nuclei. Unlike wild-type conidia, gapAΔ conidia germinate without a carbon source. Giant multinucleated spores and carbon source-independent germination have been reported in strains carrying a rasA dominant active allele, indicating that GapA downregulates RasA. gapAΔ cells show a polarity maintenance defect characterized by apical swelling and subapical branching. The strongly polarized wild-type F-actin distribution is lost in gapAΔ cells. As GapA-green fluorescent protein shows cortical localization with strong predominance at the hyphal tips, we propose that GapA-mediated downregulation of Ras signaling at the plasma membrane of these tips is involved in the polarization of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis. PMID:18039943

  5. Computational techniques for determining printability of real defects in EUV mask pilot line

    NASA Astrophysics Data System (ADS)

    Morgan, Paul; Rost, Daniel; Price, Daniel; Li, Ying; Peng, Daniel; Chen, Dongxue; Hu, Peter; Corcoran, Noel; Son, Donghwan; Yonenaga, Dean; Tolani, Vikram

    2014-04-01

    With EUV lithography on the ITRS roadmap for sub-2X half-pitch patterning, it has become increasingly essential to ramp up efforts in being able to manufacture defect-free reticles or at least ones with minimal defects initially. For this purpose, much of the focus in recent years has been in finding ways to adequately detect, characterize, and reduce defects on both EUV blanks and patterned masks. For detection purposes, the current high-resolution DUV or e-beam inspection platforms are being extended to inspect EUV blanks and patterned masks but being non-actinic, make it very challenging to assess the real impact of the detected defects on EUV plane. Even with the realization of the EUV beta AIMS™ aerial-image based metrology in 2014-2015, the exact nature of each critical defect needs to be determined in order to be able to come up with an appropriate repair strategy. In this paper, we demonstrate the application of computational techniques to non-actinic supplemental metrology data collected on EUV mask defects to effectively determine the nature and also predict printability of these defects. The fundamental EUV simulation engine used in this approach is the EUV Defect Printability Simulator (DPS), which uses simulation and modeling methods designed specifically for the individual EUV mask components, and achieves runtimes several orders of magnitude faster than rigorous FDTD and RCWA methods while maintaining adequate accuracy. The EUV DPS simulator is then coupled with supplemental inspection and metrology measurements of real defects to effectively predict wafer printability of these defects. Several sources of such supplementary data are explored here, and may sometimes be dependent on the actual nature of defect. These sources include AFM height-profile data, SEM top-down images, and 193nm high-NA inspection images of single or multiple focus plane capture. From each of these supplemental data sources, the mask pattern and defect information is first

  6. Yeast Rsp5 ubiquitin ligase affects the actin cytoskeleton in vivo and in vitro.

    PubMed

    Kaminska, Joanna; Spiess, Matthias; Stawiecka-Mirota, Marta; Monkaityte, Rasa; Haguenauer-Tsapis, Rosine; Urban-Grimal, Daniele; Winsor, Barbara; Zoladek, Teresa

    2011-12-01

    Yeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that the Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Δ lsb2Δ strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization.

  7. The capability of high magnification review function for EUV actinic blank inspection tool

    NASA Astrophysics Data System (ADS)

    Miyai, Hiroki; Suzuki, Tomohiro; Takehisa, Kiwamu; Kusunose, Haruhiko; Yamane, Takeshi; Terasawa, Tsuneo; Watanabe, Hidehiro; Mori, Ichiro

    2013-06-01

    One of the most challenging tasks to make EUVL (Extreme Ultra Violet Lithography) a reality is to achieve zero defects for mask blanks. However, since it is uncertain whether mask blanks can be made completely defect-free, defect mitigation schemes are considered crucial for realization of EUVL. One of the mitigation schemes, pattern shift, covers ML defects under absorber patterns by device pattern adjustment and prevents the defects from being printed onto wafers. This scheme, however, requires accurate defect locations, and blank inspection tools must be able to provide the locations within a margin of the error of tens of nanometers. In this paper we describe a high accuracy defect locating function of the EUV Actinic Blank Inspection (ABI) tool being developed for HVM hp16 nm and 11 nm nodes.

  8. Characterization of electrically-active defects in ultraviolet light-emitting diodes with laser-based failure analysis techniques

    SciTech Connect

    Miller, Mary A.; Tangyunyong, Paiboon; Cole, Edward I.

    2016-01-14

    Laser-based failure analysis techniques demonstrate the ability to quickly and non-intrusively screen deep ultraviolet light-emitting diodes (LEDs) for electrically-active defects. In particular, two laser-based techniques, light-induced voltage alteration and thermally-induced voltage alteration, generate applied voltage maps (AVMs) that provide information on electrically-active defect behavior including turn-on bias, density, and spatial location. Here, multiple commercial LEDs were examined and found to have dark defect signals in the AVM indicating a site of reduced resistance or leakage through the diode. The existence of the dark defect signals in the AVM correlates strongly with an increased forward-bias leakage current. This increased leakage is not present in devices without AVM signals. Transmission electron microscopy analysis of a dark defect signal site revealed a dislocation cluster through the pn junction. The cluster included an open core dislocation. Even though LEDs with few dark AVM defect signals did not correlate strongly with power loss, direct association between increased open core dislocation densities and reduced LED device performance has been presented elsewhere [M. W. Moseley et al., J. Appl. Phys. 117, 095301 (2015)].

  9. Characterization of electrically-active defects in ultraviolet light-emitting diodes with laser-based failure analysis techniques

    SciTech Connect

    Miller, Mary A.; Tangyunyong, Paiboon; Edward I. Cole, Jr.

    2016-01-12

    In this study, laser-based failure analysis techniques demonstrate the ability to quickly and non-intrusively screen deep ultraviolet light-emitting diodes(LEDs) for electrically-active defects. In particular, two laser-based techniques, light-induced voltage alteration and thermally-induced voltage alteration, generate applied voltage maps (AVMs) that provide information on electrically-active defect behavior including turn-on bias, density, and spatial location. Here, multiple commercial LEDs were examined and found to have dark defect signals in the AVM indicating a site of reduced resistance or leakage through the diode. The existence of the dark defect signals in the AVM correlates strongly with an increased forward-bias leakage current. This increased leakage is not present in devices without AVM signals. Transmission electron microscopyanalysis of a dark defect signal site revealed a dislocation cluster through the pn junction. The cluster included an open core dislocation. Even though LEDs with few dark AVM defect signals did not correlate strongly with power loss, direct association between increased open core dislocation densities and reduced LED device performance has been presented elsewhere [M. W. Moseley et al., J. Appl. Phys. 117, 095301 (2015)].

  10. Characterization of electrically-active defects in ultraviolet light-emitting diodes with laser-based failure analysis techniques

    DOE PAGES

    Miller, Mary A.; Tangyunyong, Paiboon; Edward I. Cole, Jr.

    2016-01-12

    In this study, laser-based failure analysis techniques demonstrate the ability to quickly and non-intrusively screen deep ultraviolet light-emitting diodes(LEDs) for electrically-active defects. In particular, two laser-based techniques, light-induced voltage alteration and thermally-induced voltage alteration, generate applied voltage maps (AVMs) that provide information on electrically-active defect behavior including turn-on bias, density, and spatial location. Here, multiple commercial LEDs were examined and found to have dark defect signals in the AVM indicating a site of reduced resistance or leakage through the diode. The existence of the dark defect signals in the AVM correlates strongly with an increased forward-bias leakage current. This increasedmore » leakage is not present in devices without AVM signals. Transmission electron microscopyanalysis of a dark defect signal site revealed a dislocation cluster through the pn junction. The cluster included an open core dislocation. Even though LEDs with few dark AVM defect signals did not correlate strongly with power loss, direct association between increased open core dislocation densities and reduced LED device performance has been presented elsewhere [M. W. Moseley et al., J. Appl. Phys. 117, 095301 (2015)].« less

  11. Characterization of electrically-active defects in ultraviolet light-emitting diodes with laser-based failure analysis techniques

    NASA Astrophysics Data System (ADS)

    Miller, Mary A.; Tangyunyong, Paiboon; Cole, Edward I.

    2016-01-01

    Laser-based failure analysis techniques demonstrate the ability to quickly and non-intrusively screen deep ultraviolet light-emitting diodes (LEDs) for electrically-active defects. In particular, two laser-based techniques, light-induced voltage alteration and thermally-induced voltage alteration, generate applied voltage maps (AVMs) that provide information on electrically-active defect behavior including turn-on bias, density, and spatial location. Here, multiple commercial LEDs were examined and found to have dark defect signals in the AVM indicating a site of reduced resistance or leakage through the diode. The existence of the dark defect signals in the AVM correlates strongly with an increased forward-bias leakage current. This increased leakage is not present in devices without AVM signals. Transmission electron microscopy analysis of a dark defect signal site revealed a dislocation cluster through the pn junction. The cluster included an open core dislocation. Even though LEDs with few dark AVM defect signals did not correlate strongly with power loss, direct association between increased open core dislocation densities and reduced LED device performance has been presented elsewhere [M. W. Moseley et al., J. Appl. Phys. 117, 095301 (2015)].

  12. 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.

  13. Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica.

    PubMed Central

    Edman, U; Meza, I; Agabian, N

    1987-01-01

    Invasiveness of Entamoeba histolytica strains that cause acute amoebiasis is characterized by aggressive behavior associated with cell motility and actin function. Analysis of actin genes from E. histolytica was initiated by devising methods for the isolation of biologically active nucleic acids, which allowed the preparation of cDNA and genomic DNA libraries. E. histolytica actin-encoding cDNAs and genomic clones have been isolated from libraries prepared from the virulent HM1:IMSS strain using a heterologous actin probe. Nucleotide sequence analysis of three independent cDNA clones and one genomic clone reveals a highly unusual codon bias and the absence of intervening sequences in E. histolytica actin. The coding sequence of the genomic clone is identical to that of two of the three cDNA clones. These represent at least two distinct mRNAs differing only by five silent changes in the protein coding sequence. Multiple genomic copies of the actin gene can be detected by Southern hybridization. E. histolytica actin exhibits a higher degree of homology to cytoplasmic than to muscle actin. Although the protein has been shown not to bind DNase I, the inferred amino acid sequence indicates conservation of all residues implied to participate in this binding. Images PMID:2883657

  14. A requirement for polymerized actin in DNA double-strand break repair.

    PubMed

    Andrin, Christi; McDonald, Darin; Attwood, Kathleen M; Rodrigue, Amélie; Ghosh, Sunita; Mirzayans, Razmik; Masson, Jean-Yves; Dellaire, Graham; Hendzel, Michael J

    2012-07-01

    Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.

  15. Spectroscopic study of conformational changes in subdomain 1 of G-actin: influence of divalent cations.

    PubMed

    Nyitrai, M; Hild, G; Belágyi, J; Somogyi, B

    1997-10-01

    Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Ca- and Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34 degrees C. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 5 degrees C, and the difference becomes negligible above 30 degrees C. The semiangle of the cone within which the fluorophore can rotate is larger in Ca-G-actin at all temperatures. Electron paramagnetic resonance measurements on maleimide spin-labeled (on Cys374) monomer actin gave evidence that exchange of Ca2+ for Mg2+ induced a rapid decrease in the mobility of the label immediately after the addition of Mg2+. These results suggest that the C-terminal region of the monomer becomes more rigid as a result of the replacement of Ca2+ by Mg2+. The change can be related to the difference between the polymerization abilities of the two forms of G-actin.

  16. Birth Defects

    MedlinePlus

    ... how the body looks, works or both. Some birth defects like cleft lip or neural tube defects are structural problems that can be easy to see. To find others, like heart defects, doctors use special tests. Birth defects can vary from mild to severe. Some ...

  17. 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

  18. 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

  19. Defect Clustering and Nano-phase Structure Characterization of Multicomponent Rare Earth-Oxide-Doped Zirconia-Yttria Thermal Barrier Coatings

    NASA Technical Reports Server (NTRS)

    Zhu, Dongming; Chen, Yuan L.; Miller, Robert A.

    2004-01-01

    Advanced thermal barrier coatings (TBCs) have been developed by incorporating multicomponent rare earth oxide dopants into zirconia-based thermal barrier coatings to promote the creation of the thermodynamically stable, immobile oxide defect clusters and/or nanophases within the coating systems. In this paper, the defect clusters, induced by Nd, Gd, and Yb rare earth dopants in the zirconia-yttria thermal barrier coatings, were characterized by high-resolution transmission electron microscopy (TEM). The TEM lattice imaging, selected area diffraction (SAD), and electron energy-loss spectroscopy (EELS) analyses demonstrated that the extensive nanoscale rare earth dopant segregation exists in the plasma-sprayed and electron-physical-vapor-deposited (EB PVD) thermal barrier coatings. The nanoscale concentration heterogeneity and the resulting large lattice distortion promoted the formation of parallel and rotational defective lattice clusters in the coating systems. The presence of the 5-to 100-nm-sized defect clusters and nanophases is believed to be responsible for the significant reduction of thermal conductivity, improved sintering resistance, and long-term high temperature stability of the advanced thermal barrier coating systems.

  20. Role of ATP-bound divalent metal ion in the conformation and function of actin. Comparison of Mg-ATP, Ca-ATP, and metal ion-free ATP-actin.

    PubMed

    Valentin-Ranc, C; Carlier, M F

    1991-04-25

    The fluorescence of N-acetyl-N'-(sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently bound to Cys-374 of actin is used as a probe for different conformational states of G-actin according to whether Ca-ATP, Mg-ATP, or unchelated ATP is bound to the nucleotide site. Upon addition of large amounts (greater than 10(2)-fold molar excess) of EDTA to G-actin, metal ion-free ATP-G-actin is obtained with EDTA bound. Metal ion free ATP-G-actin is characterized by a higher AEDANS fluorescence than Mg-ATP-G-actin, which itself has a higher fluorescence than Ca-ATP-G-actin. Evidence for EDTA binding to G-actin is shown using difference spectrophotometry. Upon binding of EDTA, the rate of dissociation of the divalent metal ion from G-actin is increased (2-fold for Ca2+, 10-fold for Mg2+) in a range of pH from 7.0 to 8.0. A model is proposed that quantitatively accounts for the kinetic data. The affinity of ATP is weakened 10(6)-fold upon removal of the metal ion. Metal ion-free ATP-G-actin is in a partially open conformation, as indicated by the greater accessibility of -SH residues, yet it retains functional properties of polymerization and ATP hydrolysis that appear almost identical to those of Ca-ATP-actin, therefore different from those of Mg-ATP-actin. These results are discussed in terms of the role of the ATP-bound metal ion in actin structure and function.

  1. Characterization of Pore Defects and Fatigue Cracks in Die Cast AM60 Using 3D X-ray Computed Tomography

    NASA Astrophysics Data System (ADS)

    Yang, Zhuofei; Kang, Jidong; Wilkinson, David S.

    2015-08-01

    AM60 high pressure die castings have been used in automobile applications to reduce the weight of vehicles. However, the pore defects that are inherent in die casting may negatively affect mechanical properties, especially the fatigue properties. Here we have studied damage ( e.g., pore defects, fatigue cracks) during strained-controlled fatigue using 3-dimensional X-ray computed tomography (XCT). The fatigue test was interrupted every 2000 cycles and the specimen was removed to be scanned using a desktop micro-CT system. XCT reveals pore defects, cracks, and fracture surfaces. The results show that pores can be accurately measured and modeled in 3D. Defect bands are found to be made of pores under 50 µm (based on volume-equivalent sphere diameter). Larger pores are randomly distributed in the region between the defect bands. Observation of fatigue cracks by XCT is performed in three ways such that the 3D model gives the best illustration of crack-porosity interaction while the other two methods, with the cracks being viewed on transverse or longitudinal cross sections, have better detectability on crack initiation and crack tip observation. XCT is also of value in failure analysis on fracture surfaces. By assessing XCT data during fatigue testing and observing fracture surfaces on a 3D model, a better understanding on the crack initiation, crack-porosity interaction, and the morphology of fracture surface is achieved.

  2. On-site implementation of characterization and sizing techniques for outer-wall defects in reactor pressure vessels

    SciTech Connect

    Lasserre, F.; Chapuis, N.

    1994-12-31

    Pressurized reactor vessels in France have been examined from the inside with ultrasonic focused transducers since the very first inspection. The developments carried out to solve the problem of oversizing in the case of defects located near the outer surface in the welds or in the wall thickness and presented in the framework of the 10th and 11th conference of NDE in the nuclear and pressure vessels industries, now have applications through SPARTACUS software work. Indications detected during, the systematic inspection of welds and shells, corresponding to outer wall defects, trigger a digital acquisition of data, the scanning being limited to the area of interest. This acquisition is now followed by analysis through the new system CIVAMIS, which includes the main imaging tools of SPARTACUS, but which has been specifically developed to be implemented on site, for outer wall defects. Characteristics of CIVAMIS in relation with the initial structure of SPARTACUS are discussed on actual results.

  3. Defect recognition by means of light and electron probe techniques for the characterization of mc-Si wafers and solar cells

    NASA Astrophysics Data System (ADS)

    Moralejo, B.; Tejero, A.; Hortelano, V.; Martínez, O.; González, M. A.; Jiménez, J.

    2016-11-01

    Multicristalline Silicon (mc-Si) is the preferred material for current terrestrial photovoltaic applications. However, the high density of defects present in mc-Si deteriorates the material properties, in particular the minority carrier diffusion length. For this reason, a large effort to characterize the mc-Si material is demanded, aiming to visualize the defective areas and to quantify the type of defects, density and its origin. In this work, several complementary light and electron probe techniques are used for the analysis of both mc-Si wafers and solar cells. These techniques comprise both fast and whole-area detection techniques such as Photoluminescence imaging, and highly spatially resolved time consuming techniques, such as light and electron beam induced current techniques and μRaman spectroscopy. These techniques were applied to the characterization of different mc-Si wafers for solar cells, e.g. ribbon wafers, cast mc-Si as well as quasi-monocrystalline material, upgraded metallurgical mc-Si wafers, and finished solar cells.

  4. Enabled Negatively Regulates Diaphanous-Driven Actin Dynamics In Vitro and In Vivo

    PubMed Central

    Bilancia, Colleen G.; Winkelman, Jonathan D.; Tsygankov, Denis; Nowotarski, Stephanie H.; Sees, Jennifer A.; Comber, Kate; Evans, Iwan; Lakhani, Vinal; Wood, Will; Elston, Timothy C.; Kovar, David R.; Peifer, Mark

    2014-01-01

    Summary Actin regulators facilitate cell migration by controlling cell protrusion architecture and dynamics. As the behavior of individual actin regulators becomes clear, we must address why cells require multiple regulators with similar functions and how they cooperate to create diverse protrusions. We characterized Diaphanous (Dia) and Enabled (Ena) as a model, using complementary approaches: cell culture, biophysical analysis, and Drosophila morphogenesis. We found that Dia and Ena have distinct biochemical properties that contribute to the different protrusion morphologies each induces. Dia is a more processive, faster elongator, paralleling the long, stable filopodia it induces in vivo, while Ena promotes filopodia with more dynamic changes in number, length, and lifetime. Acting together, Ena and Dia induce protrusions distinct from those induced by either alone, with Ena reducing Dia-driven protrusion length and number. Consistent with this, EnaEVH1 binds Dia directly and inhibits DiaFH1FH2-mediated nucleation in vitro. Finally, Ena rescues hemocyte migration defects caused by activated Dia. PMID:24576424

  5. Computational spatiotemporal analysis identifies WAVE2 and Cofilin as joint regulators of costimulation-mediated T cell actin dynamics

    PubMed Central

    Roybal, Kole T.; Buck, Taráz E.; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J.; Ambler, Rachel; Tunbridge, Helen M.; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F.

    2016-01-01

    Fluorescence microscopy is one of the most important tools in cell biology research and it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells; however, given extensive cell-to-cell variation, methods do not currently exist to assemble these data into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. Here, we have developed one such method and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28 and we have determined how CD28 modulates actin dynamics. We imaged actin and eight core actin regulators under conditions where CD28 in the context of a strong TCR signal was engaged or blocked to yield over a thousand movies. Our computational analysis identified diminished recruitment of the activator of actin nucleation WAVE2 and the actin severing protein cofilin to F-actin as the dominant difference upon costimulation blockade. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics upon costimulation blockade. Thus we have developed and validated an approach to quantify protein distributions in time and space for analysis of complex regulatory systems. PMID:27095595

  6. 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.

  7. 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

  8. 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

  9. Fascin links Btl/FGFR signalling to the actin cytoskeleton during Drosophila tracheal morphogenesis.

    PubMed

    Okenve-Ramos, Pilar; Llimargas, Marta

    2014-02-01

    A key challenge in normal development and in disease is to elucidate the mechanisms of cell migration. Here we approach this question using the tracheal system of Drosophila as a model. Tracheal cell migration requires the Breathless/FGFR pathway; however, how the pathway induces migration remains poorly understood. We find that the Breathless pathway upregulates singed at the tip of tracheal branches, and that this regulation is functionally relevant. singed encodes Drosophila Fascin, which belongs to a conserved family of actin-bundling proteins involved in cancer progression and metastasis upon misregulation. We show that singed is required for filopodia stiffness and proper morphology of tracheal tip cells, defects that correlate with an abnormal actin organisation. We propose that singed-regulated filopodia and cell fronts are required for timely and guided branch migration and for terminal branching and branch fusion. We find that singed requirements rely on its actin-bundling activity controlled by phosphorylation, and that active Singed can promote tip cell features. Furthermore, we find that singed acts in concert with forked, another actin cross-linker. The absence of both cross-linkers further stresses the relevance of tip cell morphology and filopodia for tracheal development. In summary, our results on the one hand reveal a previously undescribed role for forked in the organisation of transient actin structures such as filopodia, and on the other hand identify singed as a new target of Breathless signal, establishing a link between guidance cues, the actin cytoskeleton and tracheal morphogenesis.

  10. Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

    PubMed

    Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

    2014-02-01

    During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

  11. Fission yeast profilin is tailored to facilitate actin assembly by the cytokinesis formin Cdc12.

    PubMed

    Bestul, Andrew J; Christensen, Jenna R; Grzegorzewska, Agnieszka P; Burke, Thomas A; Sees, Jennifer A; Carroll, Robert T; Sirotkin, Vladimir; Keenan, Robert J; Kovar, David R

    2015-01-15

    The evolutionarily conserved small actin-monomer binding protein profilin is believed to be a housekeeping factor that maintains a general pool of unassembled actin. However, despite similar primary sequences, structural folds, and affinities for G-actin and poly-L-proline, budding yeast profilin ScPFY fails to complement fission yeast profilin SpPRF temperature-sensitive mutant cdc3-124 cells. To identify profilin's essential properties, we built a combinatorial library of ScPFY variants containing either WT or SpPRF residues at multiple positions and carried out a genetic selection to isolate variants that support life in fission yeast. We subsequently engineered ScPFY(9-Mut), a variant containing nine substitutions in the actin-binding region, which complements cdc3-124 cells. ScPFY(9-Mut), but not WT ScPFY, suppresses severe cytokinesis defects in cdc3-124 cells. Furthermore, the major activity rescued by ScPFY(9-Mut) is the ability to enhance cytokinesis formin Cdc12-mediated actin assembly in vitro, which allows cells to assemble functional contractile rings. Therefore an essential role of profilin is to specifically facilitate formin-mediated actin assembly for cytokinesis in fission yeast.

  12. Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction.

    PubMed

    Blunk, Aline D; Akbergenova, Yulia; Cho, Richard W; Lee, Jihye; Walldorf, Uwe; Xu, Ke; Zhong, Guisheng; Zhuang, Xiaowei; Littleton, J Troy

    2014-07-01

    Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act(E84K), harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act(E84K) mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act(E84K) mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act(E84K) mutants. Genetic interactions between act(E84K) and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.

  13. Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction

    PubMed Central

    Blunk, Aline D.; Akbergenova, Yulia; Cho, Richard W.; Lee, Jihye; Walldorf, Uwe; Xu, Ke; Zhong, Guisheng; Zhuang, Xiaowei; Littleton, J. Troy

    2014-01-01

    Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, actE84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous actE84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. actE84K mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Disc-Large are all mislocalized in actE84K mutants. Genetic interactions between actE84K and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization. PMID:25066865

  14. Aerial Image Microscopes for the Inspection of Defects in EUV Masks

    SciTech Connect

    Barty, A; Taylor, J S; Hudyma, R; Spiller, E; Sweeney, D W; Shelden, G; Urbach, J-P

    2002-10-22

    The high volume inspection equipment currently available to support development of EUV blanks is non-actinic. The same is anticipated for patterned EUV mask inspection. Once potential defects are identified and located by such non-actinic inspection techniques, it is essential to have instrumentation to perform detailed characterization, and if repairs are performed, re-evaluation. The ultimate metric for the acceptance or rejection of a mask due to a defect, is the wafer level impact. Thus measuring the aerial image for the site under question is required. An EUV Aerial Image Microscope (''AIM'') similar to the current AIM tools for 248nm and 193nm exposure wavelength is the natural solution for this task. Due to the complicated manufacturing process of EUV blanks, AIM measurements might also be beneficial to accurately assessing the severity of a blank defect. This is an additional application for an EUV AIM as compared to today's use In recognition of the critical role of an EUV AIM for the successful implementation of EUV blank and mask supply, International SEMATECH initiated this design study with the purpose to define the technical requirements for accurately simulating EUV scanner performance, demonstrating the feasibility to meet these requirements and to explore various technical approaches to building an EUV AIM tool.

  15. 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.

  16. 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.

  17. Burkholderia pseudomallei type III secreted protein BipC: role in actin modulation and translocation activities required for the bacterial intracellular lifecycle

    PubMed Central

    Kang, Wen Tyng; Vellasamy, Kumutha Malar; Rajamani, Lakshminarayanan; Beuerman, Roger W.

    2016-01-01

    Melioidosis, an infection caused by the facultative intracellular pathogen Burkholderia pseudomallei, has been classified as an emerging disease with the number of patients steadily increasing at an alarming rate. B. pseudomalleipossess various virulence determinants that allow them to invade the host and evade the host immune response, such as the type III secretion systems (TTSS). The products of this specialized secretion system are particularly important for the B. pseudomallei infection. Lacking in one or more components of the TTSS demonstrated different degrees of defects in the intracellular lifecycle of B. pseudomallei. Further understanding the functional roles of proteins involved in B. pseudomallei TTSS will enable us to dissect the enigma of B. pseudomallei-host cell interaction. In this study, BipC (a translocator), which was previously reported to be involved in the pathogenesis of B. pseudomallei, was further characterized using the bioinformatics and molecular approaches. The bipCgene, coding for a putative invasive protein, was first PCR amplified from B. pseudomallei K96243 genomic DNA and cloned into an expression vector for overexpression in Escherichia coli. The soluble protein was subsequently purified and assayed for actin polymerization and depolymerization. BipC was verified to subvert the host actin dynamics as demonstrated by the capability to polymerize actin in vitro. Homology modeling was also attempted to predict the structure of BipC. Overall, our findings identified that the protein encoded by the bipC gene plays a role as an effector involved in the actin binding activity to facilitate internalization of B. pseudomalleiinto the host cells. PMID:28028452

  18. The ability of an attaching and effacing pathogen to trigger localized actin assembly contributes to virulence by promoting mucosal attachment

    PubMed Central

    Mallick, Emily M.; Garber, John J.; Vanguri, Vijay K.; Balasubramanian, Sowmya; Blood, Timothy; Clark, Stacie; Vingadassalom, Didier; Louissaint, Christopher; McCormick, Beth; Snapper, Scott B.; Leong, John M.

    2014-01-01

    Enterohemorrhagic Escherichia coli (EHEC) adheres to intestinal epithelial cells, then stimulates the actin nucleation promoting factor N-WASP to induce localized actin assembly resulting in an actin “pedestal”, the function of which is poorly understood. EHEC also produces Shiga toxin (Stx), which penetrates the intestinal epithelium to cause a life-threatening renal and systemic disease. To assess the role of pedestal formation in colonization and disease, we utilized the murine pathogen Citrobacter rodentium, which also forms actin pedestals, and the genetically engineered C. rodentium (Φstx2dact), which additionally triggers Stx-mediated systemic disease. We found that an intestine-specific N-WASP-deficient (iNWKO) mouse suffered dramatically less colonization and disease than N-WASP-proficient littermate controls when infected with either strain. In addition, upon infection of wild type mice, mutants of C. rodentium or C. rodentium (Φstx2dact) that are specifically defective in pedestal formation demonstrated a relatively modest defect in cecal colonization and fecal shedding, but a more severe defect in colonization of the colonic mucosa. The C. rodentium (Φstx2dact) pedestal-defective mutant did not cause renal disease and, after normalizing for fecal bacterial load, was associated with a 16-fold lower risk of lethality. These findings suggest that the ability of an attaching and effacing pathogen to promote localized actin assembly contributes to virulence by promoting mucosal attachment. PMID:24780054

  19. 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.

  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. Screening and Characterization of Spontaneous Porcine Congenital Heart Defects for Gene Identification and Models of Human Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Rodent models of human congenital birth defects have been instrumental for gene discovery and investigation of mechanisms of disease. However, these models are limited by their small size making practiced intervention or detailed anatomic evaluation difficult. Swine have similar anato...

  4. 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.

  5. 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.

  6. Arabidopsis CAP regulates the actin cytoskeleton necessary for plant cell elongation and division.

    PubMed

    Barrero, Roberto A; Umeda, Masaaki; Yamamura, Saburo; Uchimiya, Hirofumi

    2002-01-01

    An Arabidopsis cDNA (AtCAP1) that encodes a predicted protein of 476 amino acids highly homologous with the yeast cyclase-associated protein (CAP) was isolated. Expression of AtCAP1 in the budding yeast CAP mutant was able to rescue defects such as abnormal cell morphology and random budding pattern. The C-terminal domain, 158 amino acids of AtCAP1 possessing in vitro actin binding activity, was needed for the regulation of cytoskeleton-related defects of yeast. Transgenic plants overexpressing AtCAP1 under the regulation of a glucocorticoid-inducible promoter showed different levels of AtCAP1 accumulation related to the extent of growth abnormalities, in particular size reduction of leaves as well as petioles. Morphological alterations in leaves were attributable to decreased cell size and cell number in both epidermal and mesophyll cells. Tobacco suspension-cultured cells (Bright Yellow 2) overexpressing AtCAP1 exhibited defects in actin filaments and were unable to undergo mitosis. Furthermore, an immunoprecipitation experiment suggested that AtCAP1 interacted with actin in vivo. Therefore, AtCAP1 may play a functional role in actin cytoskeleton networking that is essential for proper cell elongation and division.

  7. A Role for Nuclear F-Actin Induction in Human Cytomegalovirus Nuclear Egress

    PubMed Central

    Wilkie, Adrian R.; Lawler, Jessica L.

    2016-01-01

    ABSTRACT Herpesviruses, which include important pathogens, remodel the host cell nucleus to facilitate infection. This remodeling includes the formation of structures called replication compartments (RCs) in which herpesviruses replicate their DNA. During infection with the betaherpesvirus, human cytomegalovirus (HCMV), viral DNA synthesis occurs at the periphery of RCs within the nuclear interior, after which assembled capsids must reach the inner nuclear membrane (INM) for translocation to the cytoplasm (nuclear egress). The processes that facilitate movement of HCMV capsids to the INM during nuclear egress are unknown. Although an actin-based mechanism of alphaherpesvirus capsid trafficking to the INM has been proposed, it is controversial. Here, using a fluorescently-tagged, nucleus-localized actin-binding peptide, we show that HCMV, but not herpes simplex virus 1, strongly induced nuclear actin filaments (F-actin) in human fibroblasts. Based on studies using UV inactivation and inhibitors, this induction depended on viral gene expression. Interestingly, by 24 h postinfection, nuclear F-actin formed thicker structures that appeared by super-resolution microscopy to be bundles of filaments. Later in infection, nuclear F-actin primarily localized along the RC periphery and between the RC periphery and the nuclear rim. Importantly, a drug that depolymerized nuclear F-actin caused defects in production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization near the nuclear rim, without decreasing capsid accumulation in the nucleus. Thus, our results suggest that for at least one herpesvirus, nuclear F-actin promotes capsid movement to the nuclear periphery and nuclear egress. We discuss our results in terms of competing models for these processes. PMID:27555312

  8. Determining the critical size of EUV mask substrate defects

    SciTech Connect

    Goldberg, Kenneth A.; Gullikson, Eric M.; Han, Hakseung; Cho, Wonil; Jeon, Chan-Uk; Wurm, Stefan

    2008-05-26

    Determining the printability of substrate defects beneath the extreme ultraviolet (EUV) reflecting multilayer stack is an important issue in EUVL lithography. Several simulation studies have been performed in the past to determine the tolerable defect size on EUV mask blank substrates but the industry still has no exact specification based on real printability tests. Therefore, it is imperative to experimentally determine the printability of small defects on a mask blanks that are caused by substrate defects using direct printing of programmed substrate defect in an EUV exposure tools. SEMATECH fabricated bump type program defect masks using standard electron beam lithography and performed printing tests with the masks using an EUV exposure tool. Defect images were also captured using SEMATECH's Berkeley Actinic Imaging Tool in order to compare aerial defect images with secondary electron microscope images from exposed wafers. In this paper, a comprehensive understanding of substrate defect printability will be presented and printability specifications of EUV mask substrate defects will be discussed.

  9. Determining the Critcial Size of EUV Mask Substrate Defects

    SciTech Connect

    Mccall, Monnikue M; Han, Hakseung; Cho, Wonil; Goldberg, Kenneth; Gullikson, Eric; Jeon, Chan-Uk; Wurm, Stefan

    2008-02-28

    Determining the printability of substrate defects beneath the extreme ultraviolet (EUV) reflecting multilayer stack is an important issue in EUVL lithography. Several simulation studies have been performed in the past to determine the tolerable defect size on EUV mask blank substrates but the industry still has no exact specification based on real printability tests. Therefore, it is imperative to experimentally determine the printability of small defects on a mask blanks that are caused by substrate defects using direct printing of programmed substrate defect in an EUV exposure tool. SEMATECH fabricated bump type program defect masks using standard electron beam lithography and performed printing tests with the masks using an EUV exposure tool. Defect images were also captured using SEMATECH's Berkeley Actinic Imaging Tool in order to compare aerial defect images with secondary electron microscope images from exposed wafers. In this paper, a comprehensive understanding of substrate defect printability will be presented and printability specifications of EUV mask substrate defects will be discussed.

  10. 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.

  11. 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.

  12. 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

  13. Study of actin and its interactions with heavy meromyosin and the regulatory proteins by the pulse fluorimetry in polarized light of a fluorescent probe attached to an actin cysteine.

    PubMed

    Tawada, K; Wahl, P; Auchet, J C

    1978-08-01

    The decay of anisotropy of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine fluorescence attached to cysteine-373 of actin can be characterized by two correlation times theta1 and theta2. theta1 has a value of several nanoseconds and is thought to represent some local protein motion. theta2 is of the order of several hundreds of nanoseconds. Its value increases with actin concentration. It represents an average of the G and F actin correlation times. When actin interacts with heavy meromyosin, theta2 increases and becomes infinite at a molar ratio of one heavy meromyosin molecule per four actin protomers. It is concluded that a definite complex is then formed between F actin and heavy meromyosin. In the same time, G actin concentration becomes equal to zero. Finally, when F actin forms a complex with the regulatory proteins tropomyosin and troponin, the value of theta2 is greater in the absence than in the presence of Ca2+. This result indicates that micromolar concentrations of Ca2+ induces a conformation change of the complex of F actin with the regulatory proteins.

  14. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria

    PubMed Central

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-01-01

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  15. The toxofilin-actin-PP2C complex of Toxoplasma: identification of interacting domains.

    PubMed

    Jan, Gaelle; Delorme, Violaine; David, Violaine; Revenu, Celine; Rebollo, Angelita; Cayla, Xavier; Tardieux, Isabelle

    2007-02-01

    Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure-function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein-protein interactions that are important to parasite motility.

  16. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    PubMed

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

  17. Atrial septal defect combined with partial anomalous pulmonary venous return: complete anatomic and functional characterization by cardiac magnetic resonance.

    PubMed

    Dellegrottaglie, Santo; Pedrotti, Patrizia; Pedretti, Stefano; Mauri, Francesco; Roghi, Alberto

    2008-11-01

    The presented case regards a 17-year-old male with new-onset right bundle branch block and significantly enlarged right-heart sections as the only pathologic finding on transthoracic echocardiography. Cardiac magnetic resonance (CMR) revealed the presence of a superior sinus venosus atrial septal defect associated with a partial anomalous pulmonary venous return, with the right upper lobe pulmonary vein draining into the superior vena cava. CMR has developed in recent years into an accurate modality for non-invasive evaluation of patients with congenital heart disease, especially through improvements in quality and speed of image acquisition. With echocardiography, sinus venosus defects and anomalous pulmonary vein drainage may be more easily detected by a transoesophageal approach because of the proximity of the transducer to the atrial septum. CMR may be specifically recommended as an alternative to transoesophageal echocardiography in any patient with an unexplained dilatation of the right ventricle.

  18. Growth and Defect Characterization of Quantum Dot-Embedded III-V Semiconductors for Advanced Space Photovoltaics

    DTIC Science & Technology

    2014-05-15

    provide, which could be useful in the future development of intermediate band solar cell (IBSC) devices. Defect spectroscopy was also performed on OMVPE...grown InAs/GaAs QD-embedded solar cells . A large increase in mid-gap trap density surrounding the embedded QDs was found and points to a potentially... cell calibration, high altitude solar cell calibration, high altitude balloon solar cell calibration, III-V compound semiconductors, solar cells

  19. Characterization of the skeletal fusion with sterility (sks) mouse showing axial skeleton abnormalities caused by defects of embryonic skeletal development.

    PubMed

    Akiyama, Kouyou; Katayama, Kentaro; Tsuji, Takehito; Kunieda, Tetsuo

    2014-01-01

    The development of the axial skeleton is a complex process, consisting of segmentation and differentiation of somites and ossification of the vertebrae. The autosomal recessive skeletal fusion with sterility (sks) mutation of the mouse causes skeletal malformations due to fusion of the vertebrae and ribs, but the underlying defects of vertebral formation during embryonic development have not yet been elucidated. For the present study, we examined the skeletal phenotypes of sks/sks mice during embryonic development and the chromosomal localization of the sks locus. Multiple defects of the axial skeleton, including fusion of vertebrae and fusion and bifurcation of ribs, were observed in adult and neonatal sks/sks mice. In addition, we also found polydactyly and delayed skull ossification in the sks/sks mice. Morphological defects, including disorganized vertebral arches and fusions and bifurcations of the axial skeletal elements, were observed during embryonic development at embryonic day 12.5 (E12.5) and E14.5. However, no morphological abnormality was observed at E11.5, indicating that defects of the axial skeleton are caused by malformation of the cartilaginous vertebra and ribs at an early developmental stage after formation and segmentation of the somites. By linkage analysis, the sks locus was mapped to an 8-Mb region of chromosome 4 between D4Mit331 and D4Mit199. Since no gene has already been identified as a cause of malformation of the vertebra and ribs in this region, the gene responsible for sks is suggested to be a novel gene essential for the cartilaginous vertebra and ribs.

  20. Characterization of Three nef-Defective Human Immunodeficiency Virus Type 1 Strains Associated with Long-Term Nonprogression

    PubMed Central

    Rhodes, David I.; Ashton, Lesley; Solomon, Ajantha; Carr, Andrew; Cooper, David; Kaldor, John; Deacon, Nicholas

    2000-01-01

    Long-term survivors (LTS) of human immunodeficiency virus type 1 (HIV-1) infection provide an opportunity to investigate both viral and host factors that influence the rate of disease progression. We have identified three HIV-1-infected individuals in Australia who have been infected for over 11 years with viruses that contain deletions in the nef and nef-long terminal repeat (nef/LTR) overlap regions. These viruses differ from each other and from other nef-defective strains of HIV-1 previously identified in Australia. One individual, LTS 3, is infected with a virus containing a nef gene with a deletion of 29 bp from the nef/LTR overlap region, resulting in a truncated Nef open reading frame. In addition to the Nef defect, only viruses containing truncated Vif open reading frames of 37 or 69 amino acids could be detected in peripheral blood mononuclear cells isolated from this patient. LTS 3 had a viral load of less than 20 copies of RNA/ml of plasma. The other two long-term survivors, LTS 9 and LTS 11, had loads of less than 200 copies of RNA/ml of plasma and are infected with viruses with larger deletions in both the nef alone and nef/LTR overlap regions. These viruses contain wild-type vif, vpu, and vpr accessory genes. All three strains of virus had envelope sequences characteristic of macrophagetropic viruses. These findings further indicate the reduced pathogenic potential of nef-defective viruses. PMID:11044102

  1. Arabidopsis ACT11 modifies actin turnover to promote pollen germination and maintain the normal rate of tube growth.

    PubMed

    Chang, Ming; Huang, Shanjin

    2015-08-01

    Actin is an ancient conserved protein that is encoded by multiple isovariants in multicellular organisms. There are eight functional actin genes in the Arabidopsis genome, and the precise function and mechanism of action of each isovariant remain poorly understood. Here, we report the characterization of ACT11, a reproductive actin isovariant. Our studies reveal that loss of function of ACT11 causes a delay in pollen germination, but enhances pollen tube growth. Cytological analysis revealed that the amount of filamentous actin decreased, and the rate of actin turnover increased in act11 pollen. Convergence of actin filaments upon the germination aperture was impaired in act11 pollen, consistent with the observed delay of germination. Reduction of actin dynamics with jasplakinolide suppressed the germination and tube growth phenotypes in act11 pollen, suggesting that the underlying mechanisms involve an increase in actin dynamics. Thus, we demonstrate that ACT11 is required to maintain the rate of actin turnover in order to promote pollen germination and maintain the normal rate of pollen tube growth.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses

    NASA Astrophysics Data System (ADS)

    Orange, Jordan S.; Ramesh, Narayanaswamy; Remold-O'Donnell, Eileen; Sasahara, Yoji; Koopman, Louise; Byrne, Michael; Bonilla, Francisco A.; Rosen, Fred S.; Geha, Raif S.; Strominger, Jack L.

    2002-08-01

    The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.

  13. 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

  14. 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.

  15. 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.

  16. Myosin III-mediated cross-linking and stimulation of actin bundling activity of Espin.

    PubMed

    Liu, Haiyang; Li, Jianchao; Raval, Manmeet H; Yao, Ningning; Deng, Xiaoying; Lu, Qing; Nie, Si; Feng, Wei; Wan, Jun; Yengo, Christopher M; Liu, Wei; Zhang, Mingjie

    2016-01-19

    Class III myosins (Myo3) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells, and their defects cause hereditary hearing impairments. Myo3 interacts with Espin1 through its tail homology I motif (THDI), however it is not clear how Myo3 specifically acts through Espin1 to regulate the actin bundle assembly and stabilization. Here we discover that Myo3 THDI contains a pair of repeat sequences capable of independently and strongly binding to the ankyrin repeats of Espin1, revealing an unexpected Myo3-mediated cross-linking mechanism of Espin1. The structures of Myo3 in complex with Espin1 not only elucidate the mechanism of the binding, but also reveal a Myo3-induced release of Espin1 auto-inhibition mechanism. We also provide evidence that Myo3-mediated cross-linking can further promote actin fiber bundling activity of Espin1.

  17. 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.

  18. The myosin X motor is optimized for movement on actin bundles

    PubMed Central

    Ropars, Virginie; Yang, Zhaohui; Isabet, Tatiana; Blanc, Florian; Zhou, Kaifeng; Lin, Tianming; Liu, Xiaoyan; Hissier, Pascale; Samazan, Frédéric; Amigues, Béatrice; Yang, Eric D.; Park, Hyokeun; Pylypenko, Olena; Cecchini, Marco; Sindelar, Charles V.; Sweeney, H. Lee; Houdusse, Anne

    2016-01-01

    Myosin X has features not found in other myosins. Its structure must underlie its unique ability to generate filopodia, which are essential for neuritogenesis, wound healing, cancer metastasis and some pathogenic infections. By determining high-resolution structures of key components of this motor, and characterizing the in vitro behaviour of the native dimer, we identify the features that explain the myosin X dimer behaviour. Single-molecule studies demonstrate that a native myosin X dimer moves on actin bundles with higher velocities and takes larger steps than on single actin filaments. The largest steps on actin bundles are larger than previously reported for artificially dimerized myosin X constructs or any other myosin. Our model and kinetic data explain why these large steps and high velocities can only occur on bundled filaments. Thus, myosin X functions as an antiparallel dimer in cells with a unique geometry optimized for movement on actin bundles. PMID:27580874

  19. Comprehensive characterization of C-glycosyl flavones in wheat (Triticum aestivum L.) germ using UPLC-PDA-ESI/HRMS(n) and mass defect filtering.

    PubMed

    Geng, Ping; Sun, Jianghao; Zhang, Mengliang; Li, Xingnuo; Harnly, James M; Chen, Pei

    2016-10-01

    A comprehensive characterization of C-glycosyl flavones in wheat germ has been conducted using multi-stage high resolution mass spectrometry (HRMS(n) ) in combination with a mass defect filtering (MDF) technique. MDF performed the initial search of raw data with defined C-glycosyl flavone mass windows and mass defect windows to generate the noise-reduced data focusing on targeted flavonoids. The high specificity of the exact mass measurement permits the unambiguous discrimination of acyl groups (nominal masses of 146, 162 and 176.) from sugar moieties (rhamnose, glucose or galactose and glucuronic acid). A total of 72 flavone C-glycosyl derivatives, including 2 mono-C-glycosides, 34 di-C-glycosides, 15 tri-glycosides, 14 acyl di-C-glycosides and 7 acyl tri-glycosides, were characterized in wheat germ, some of which were considered to be important marker compounds for differentiation of whole grain and refined wheat products. The 7 acylated mono-O-glycosyl-di-C-glycosyl flavones and some acylated di-C-glycosyl flavones are reported in wheat for the first time. The frequent occurrence of numerous isomers is a remarkable feature of wheat germ flavones. Both UV and mass spectra are needed to maximize the structure information obtained for data interpretation. Copyright © 2016 John Wiley & Sons, Ltd.

  20. Characterization of deep level defects present in mono-like, quasi-mono and multicrystalline silicon solar substrates

    NASA Astrophysics Data System (ADS)

    Pérez, E.; García, H.; Castán, H.; Dueñas, S.

    2015-03-01

    Defects on mono-like (ml-Si), quasi-mono (qm-Si) and multicrystalline silicon solar cell substrates are studied in depth. Using the thermal admittance spectroscopy technique we found a single deep level with an activation energy between 213 and 224 meV and a capture cross section in the order of 10-15-10-14 cm2, in the case of ml-Si samples. The 271, 291 and 373 meV levels were found in qm-Si samples. The first one is associated with a capture cross section in the order of 10-16 cm2, the second one in the order of 10-14, while the third one is associated, for the same magnitude, with a value in the order of 10-12 cm2. Multicrystalline samples showed two tendencies in the Arrhenius plot fit associated with a deep level in each one. The activation energy of the first one ranges from 336 meV to 342 meV, and the capture cross sections are in the order of 10-13-10-11 cm2. The values obtained for the second one are 251 and 171 meV, with the capture cross section values in the order of 10-15 and 10-18 cm2, respectively. The nature of these defects is probably due to iron-based impurities in different complexes. Segregation into extended defects of Fei or Fei-V is the most probable cause of the deep levels with higher capture cross section value. Punctual complexes such as Fei or Fei-V2 are probably the reason for the deep levels with lower capture cross section value.

  1. Observations of secondary defects and vacancies in CZ silicon crystals detached from melt using four different types of characterization technique

    NASA Astrophysics Data System (ADS)

    Abe, T.; Takahashi, T.; Shirai, K.

    2016-02-01

    The crystals were grown by a gradually decreased pulling rate method, a special crystal growing method, and detached from a melt during the growth so as to rapidly cool the grown crystal and then to observe the appearance and disappearance of point defects at the moment of the detachment. This observation - nearly in situ observation, as it were - revealed that vacancies (Vs) were introduced through a growth interface, and interstitials (Is) were generated at an interstitial generation area, an area at which the thermal stress was increased through the increased thermal gradient, above the growth interface. In the beginning of the gradually decreased pulling rate method, since the pulling rate was high, the Vs introduced through the growth interface remained in the crystal; as the pulling rate was decreased, the generation of the Is began from the interstitial generation area, and these interstitials were recombined with the Vs introduced through the growth interface, thereby forming a first recombination area. As the concentration of the Is increased due to a lower pulling rate, a dislocation loop region began to be formed. On the growth interface side of this dislocation loop region, a V region from the growth interface and a second recombination area were similarly formed. The formation of these two recombination areas proves that the growth interface was the V region. In this paper, the point defects and secondary defects thereof were observed by our three new observation methods and the etch-pit method used in product inspection. The results of these methods were consistent with all the above phenomenon.

  2. Characterization of defectiveness in endogenous antigen presentation of novel murine cells established from methylcholanthrene-induced fibrosarcomas.

    PubMed Central

    Kuroda, K; Yamashina, K; Kitatani, N; Kagishima, A; Hamaoka, T; Hosaka, Y

    1995-01-01

    Three cell lines (4A1, 4C2 and 6D1 cells) derived from fibrosarcoma induced by the inoculation of 3-methylcholanthrene into C3H/HeN (H-2k) mice were examined for their ability to present antigens to CD8+ cytotoxic T lymphocytes (CTL). 6D1 and 4C2 cells were deficient in presenting endogenously synthesized influenza virus antigens to CTL, but they were able to present antigens when they were sensitized with a synthetic epitope peptide. The expression of the H-2 Kk gene in 4C2 and 6D1 cells was much reduced and was detectable only with Northern blot hybridization. The expression of two transporter genes (TAP1 and TAP2), examined by Northern hybridization, was also reduced in both cells, and negligible particularly in 4C2 cells. Interferon-gamma (IFN-gamma) treatment of these cells induced expression of Kk, TAP1 and TAP2 genes and rescued the defect of class I-restricted antigen presentation in 4C2 and 6D1 cells. Even after this treatment, however, antigen-presentation capability of 4C2 cells was still much lower than that of normal 4A1 cells. This finding suggests that 4C2 cells might have an additional defective gene(s), whose products are involved in the processing of class I-restricted antigen, besides the Kk and TAP genes, and this may explain the difficulty of 4C2 cells to induce tumour-specific immunity, as described previously. To our knowledge, the 4C2 cell is the first tumour cell postulated to have more than three defective genes involved in class I-restricted antigen presentation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:7890298

  3. Coiled-Coil–Mediated Dimerization Is Not Required for Myosin VI to Stabilize Actin during Spermatid Individualization in Drosophila melanogaster

    PubMed Central

    Noguchi, Tatsuhiko; Frank, Deborah J.; Isaji, Mamiko

    2009-01-01

    Myosin VI is a pointed-end–directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo. PMID:19005209

  4. Abundance of actin filaments in the preprophase band and mitotic spindle of brick1 Zea mays mutant.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Tzioutziou, Nickoleta A

    2009-07-01

    The preprophase band and mitotic spindle of dividing protodermal cells of wild-type Zea mays leaves include few actin filaments. Surprisingly, abundant actin filaments were observed in the above arrays, in dividing protodermal cells in the leaves of the brick1 mutant. The same abundance was observed in the spindle of Taxol-treated brick1 mitotic protodermal cells. Apart from the above difference, the relevant arrays displayed normal microtubule organization in both wild type and mutant cells, as far as can be discerned by immunofluorescence microscopy. Accordingly, the abundance of actin filaments in the preprophase band and spindle of brick1 mitotic cells seems not to influence the structure of the above arrays and might be a non-functional "side-effect" of defective F-actin organization in this mutant.

  5. Self-Organized Gels in DNA/F-Actin Mixtures without Crosslinkers: Networks of Induced Nematic Domains with Tunable Density

    NASA Astrophysics Data System (ADS)

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

    2008-11-01

    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 dactin∝ρDNA-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.

  6. Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Weaver, K E; Tabita, F R

    1983-11-01

    Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.

  7. Synthesis and characterization of magnesium oxide nanocrystallites and probing the vacancy-type defects through positron annihilation studies

    NASA Astrophysics Data System (ADS)

    Das, Anjan; Mandal, Atis Chandra; Roy, Soma; Prashanth, Pendem; Ahamed, Sk Izaz; Kar, Subhrasmita; Prasad, Mithun S.; Nambissan, P. M. G.

    2016-09-01

    Magnesium oxide nanocrystallites exhibit certain abnormal characteristics when compared to those of other wide band gap oxide semiconductors in the sense they are most prone to water absorption and formation of a hydroxide layer on the surface. The problem can be rectified by heating and pure nanocrystallites can be synthesized with controllable sizes. Inevitably the defect properties are distinctly divided between two stages, the one with the hydroxide layer (region I) and the other after the removal of the layer by annealing (region II). The lattice parameters, the optical band gap and even the positron annihilation characteristics are conspicuous by their distinct behavior in the two stages of the surface configurations of nanoparticles. While region I was specific with the formation of positronium-hydrogen complexes that drastically altered the defect-specific positron lifetimes, pick-off annihilation of orthopositronium atoms marked region II. The vacancy clusters within the nanocrystallites also trapped positrons. They agglomerated due to the effect of the higher temperatures and resulted in the growth of the nanocrystallites. The coincidence Doppler broadening spectroscopic measurements supported these findings and all the more indicated the trapping of positrons additionally into the neutral divacancies and negatively charged trivacancies. This is apart from the Mg2+ monovacancies which acted as the dominant trapping centers for positrons.

  8. Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

    PubMed Central

    Gao, Ying; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES. PMID:27358069

  9. 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

  10. 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

  11. 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.

  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. 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.

  14. Disruption of the Rickettsia rickettsii Sca2 autotransporter inhibits actin-based motility.

    PubMed

    Kleba, Betsy; Clark, Tina R; Lutter, Erika I; Ellison, Damon W; Hackstadt, Ted

    2010-05-01

    Rickettsii rickettsii, the etiologic agent of Rocky Mountain spotted fever, replicates within the cytosol of infected cells and uses actin-based motility to spread inter- and intracellularly. Although the ultrastructure of the actin tail and host proteins associated with it are distinct from those of Listeria or Shigella, comparatively little is known regarding the rickettsial proteins involved in its organization. Here, we have used random transposon mutagenesis of R. rickettsii to generate a small-plaque mutant that is defective in actin-based motility and does not spread directly from cell to cell as is characteristic of spotted fever group rickettsiae. The transposon insertion site of this mutant strain was within Sca2, a member of a family of large autotransporter proteins. Sca2 exhibits several features suggestive of its apparent role in actin-based motility. It displays an N-terminal secretory signal peptide, a C-terminal predicted autotransporter domain, up to four predicted Wasp homology 2 (WH2) domains, and two proline-rich domains, one with similarity to eukaryotic formins. In a guinea pig model of infection, the Sca2 mutant did not elicit fever, suggesting that Sca2 and actin-based motility are virulence factors of spotted fever group rickettsiae.

  15. Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

    PubMed Central

    Baranwal, Somesh

    2015-01-01

    Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. PMID:25792565

  16. 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

  17. Actin-based propulsion of a microswimmer.

    PubMed

    Leshansky, A M

    2006-07-01

    A simple hydrodynamic model of actin-based propulsion of microparticles in dilute cell-free cytoplasmic extracts is presented. Under the basic assumption that actin polymerization at the particle surface acts as a force dipole, pushing apart the load and the free (nonanchored) actin tail, the propulsive velocity of the microparticle is determined as a function of the tail length, porosity, and particle shape. The anticipated velocities of the cargo displacement and the rearward motion of the tail are in good agreement with recently reported results of biomimetic experiments. A more detailed analysis of the particle-tail hydrodynamic interaction is presented and compared to the prediction of the simplified model.

  18. MALDI SpiralTOF high‐resolution mass spectrometry and Kendrick mass defect analysis applied to the characterization of poly(ethylene‐co‐vinyl acetate) copolymers

    PubMed Central

    Nakamura, Sayaka

    2016-01-01

    Rationale Poly(ethylene‐co‐vinyl acetate) copolymers – usually referred to as EVA – are first class industrial polymers used for applications ranging from padding to photovoltaics as encapsulant for the silicon solar cells. Various techniques have been used for their characterization but the analysis of intact EVA chains using mass spectrometry (MS) has not been reported so far. Methods Three copolymers containing 18, 25 and 40 wt% vinyl acetate (VA) have been characterized using an off‐line coupling of size‐exclusion chromatography (SEC) and matrix‐assisted laser desorption/ionization (MALDI) spiral‐time‐of‐flight (TOF) high‐resolution mass spectrometry (HRMS). The representativeness of those results for the entire samples has been checked using 13C NMR spectroscopy. Lastly, Kendrick mass defect analysis has been proposed as an alternative and user‐friendly data treatment method. Results The shortest chains isolated by SEC fractionation and mass‐analyzed by HRMS have been thoroughly described in terms of end‐groups (found to be hydrogens) and co‐monomeric composition. The VA content was successfully derived from the peak assignments in MS spectra for the EVA 40 wt% and 25 wt% while it tended to be overestimated for the latest EVA 18 wt% (increasing poly(ethylene) character). Similar results have been found using a faster data treatment method relying on the Kendrick mass defect analysis of the MS data. Conclusions EVA low molecular weight intact oligomers have been extensively characterized by MS for the first time and the structural features confidently extended to the full sample according to NMR data. The Kendrick mass analysis finally constituted an efficient method for a fast evaluation of their VA content with no need for manual assignment. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd. PMID:26969940

  19. The rearrangement of filamentous actin in mossy fiber synapses in pentylenetetrazol-kindled C57BL/6 mice.

    PubMed

    Zhang, Yan-Feng; Li, Shu-Lei; Xiong, Tian-Qing; Yang, Li-Bin; Li, Yong-Nan; Tan, Bai-Hong; Liu, Qun; Li, Yan-Chao

    2014-01-01

    Chemical kindling, as an experimental model of epileptogenesis, is induced by repetitive administration of subconvulsive amount of excitatory drugs. Kindled mice do not typically display spontaneous recurrent seizures, but are instead characterized by enhanced seizure susceptibility to convulsive stimulations. In order to provide insights into the aberrant synaptic plasticity during kindling, this study investigated the effect of pentylenetetrazol (PTZ) kindling on filamentous actin (F-actin) in mossy fiber synapses in C57BL/6 mice. Phalloidin labeling of F-actin showed that F-actin puncta were increased in number in the stratum lucidum of CA3 region in the hippocampus after kindling. The rearrangement of F-actin seemed to occur presynaptically, since synapsin I, a specific marker for mossy fiber terminals, was also up-regulated. Such subtle structural modifications occurring in the synapses are thought to contribute to the long-lasting increased sensitivity in the PTZ-kindled C57BL/6 mice.

  20. Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion.

    PubMed

    Mouneimne, Ghassan; Hansen, Scott D; Selfors, Laura M; Petrak, Lara; Hickey, Michele M; Gallegos, Lisa L; Simpson, Kaylene J; Lim, James; Gertler, Frank B; Hartwig, John H; Mullins, R Dyche; Brugge, Joan S

    2012-11-13

    Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.

  1. Structural and defect characterization of Gd-doped GaN films by X-ray diffraction and positron annihilation

    NASA Astrophysics Data System (ADS)

    Yabuuchi, A.; Oshima, N.; O'Rourke, B. E.; Suzuki, R.; Ito, K.; Sano, S.; Higashi, K.; Zhou, Y.-K.; Hasegawa, S.

    2014-04-01

    Molecular-beam-epitaxy-grown Ga1-xGdxN films were investigated by X-ray diffraction and slow positron beams. From the positron lifetime results, N-vacancy-related defects may be expected in the Ga0.9Gd0.1N film grown under Ga-rich conditions which exhibits a lattice expansion in the c-axis direction. In contrast, Ga vacancies more than 1019 cm-3 were detected in the Ga0.9Gd0.1N film grown under N-rich conditions which does not exhibit the lattice expansion, implying that the highly-concentrated Ga vacancies contribute to a relaxation of the lattice distortion caused by incorporating oversized Gd atoms.

  2. Identification and characterization of novel rare mutations in the planar cell polarity gene PRICKLE1 in human neural tube defects.

    PubMed

    Bosoi, Ciprian M; Capra, Valeria; Allache, Redouane; Trinh, Vincent Quoc-Huy; De Marco, Patrizia; Merello, Elisa; Drapeau, Pierre; Bassuk, Alexander G; Kibar, Zoha

    2011-12-01

    The planar cell polarity (PCP) pathway controls the process of convergent extension (CE) during gastrulation and neural tube closure, and has been implicated in the pathogenesis of neural tube defects (NTDs) in animal models and human cohorts. In this study, we analyzed the role of one core PCP gene PRICKLE1 in these malformations. We screened this gene in 810 unrelated NTD patients and identified seven rare missense heterozygous mutations that were absent in all controls analyzed and predicted to be functionally deleterious using bioinformatics. Functional validation of five PRICKLE1 variants in a zebrafish model demonstrated that one variant, p.Arg682Cys, antagonized the CE phenotype induced by the wild-type zebrafish prickle1a (zpk1a) in a dominant fashion. Our study demonstrates that PRICKLE1 could act as a predisposing factor to human NTDs and further expands our knowledge of the role of PCP genes in the pathogenesis of these malformations.

  3. Structural characterization of new defective molecules in poly(amidoamide) dendrimers by combining mass spectrometry and nuclear magnetic resonance.

    PubMed

    Tintaru, Aura; Ungaro, Rémi; Liu, Xiaoxiuan; Chen, Chao; Giordano, Laurent; Peng, Ling; Charles, Laurence

    2015-01-01

    A new side-reaction occurring during divergent synthesis of PAMAM dendrimers (generations G0-G2) was revealed by mass spectrometric detection of defective molecules with a net gain of a single carbon atom as compared to expected compounds. Combining MS/MS experiments performed on different electrosprayed precursor ions (protonated molecules and lithiated adducts) with NMR analyses allowed the origin of these by-products to be elucidated. Modification of one ethylenediamine end-group of perfect dendrimers into a cyclic imidazolidine moiety was induced by formaldehyde present at trace level in the methanol solvent used as the synthesis medium. Dendrimers studied here were purposely constructed from a triethanolamine core to make them more flexible, as compared to NH3- or ethylenediamine-core PAMAM, and hence improve their interaction with DNA. Occurrence of this side-reaction would be favored by the particular flexibility of the dendrimer branches.

  4. 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

  5. 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.

  6. 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

  7. F-actin staining of Drosophila testes.

    PubMed

    Bonaccorsi, Silvia; Giansanti, Maria G; Cenci, Giovanni; Gatti, Maurizio

    2012-01-01

    Preparations of Drosophila testes fixed with paraformaldehyde can be stained for F-actin according to the protocol described here. This staining procedure is particularly suitable for staining the male fusome and the cytokinetic contractile ring.

  8. [Actin in the wound healing process].

    PubMed

    Nowak, Dorota; Popow-Woźniak, Agnieszka; Raźnikiewicz, Linda; Malicka-Błaszkiewicz, Maria

    2009-01-01

    Wound healing is an important biological process of crucial value for organisms survival and retention of its proper functions. The recognition of molecular mechanisms of these phenomenon is still under investigation. The transition of mesenchymal fibroblasts to myofibroblasts is a key point in wound healing. The contraction ability of myofibroblast enables the shrinkage of a wound and closes its edges. Alpha smooth muscle actin (alpha-SMA), one of six actin isoforms, is a marker of compeletely differentiated myofibroblast. The regulation of differentiation process depends on many growth factors (especially TGF beta 1), the level of active thymosin beta 4, extracellular matrix proteins--including fibronectin, and also on specificity of microenvironment. Thymosin beta 4 is responsible for maintenance of pool of monomeric actin and actin filaments depolymerization. It can also act as a transcription factor, migration stimulator and immunomodulator, so this protein deserves for more attention in wound healing research field.

  9. Mechanics model for actin-based motility

    NASA Astrophysics Data System (ADS)

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

  10. Mechanics model for actin-based motility.

    PubMed

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment ob